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Regulation of NADPH Oxidase Activity in Phagocytes
The X+-linked chronic granulomatous disease (X+-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X+-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X+-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, ?507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.
Debeurme, Franck; Picciocchi, Antoine; Dagher, Marie-Claire; Grunwald, Didier; Beaumel, Sylvain; Fieschi, Franck; Stasia, Marie-Jose
NADPH dehydrogenase activity of p67PHOX, a cytosolic subunit of the leukocyte NADPH oxidase.
The leukocyte NADPH oxidase catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH. It is a multicomponent enzyme comprising a membrane-bound flavocytochrome (cytochrome b558) and at least four cytosolic components: p47PHOX, p67PHOX, p40PHOX, and Rac, a small GTPase. All the oxidase components except p40PHOX are required for enzyme activity. Many aspects of their function, however, are unclear. Using the electron acceptor ferricyanide, we found that recombinant p67PHOX from baculovirus-infected Sf9 cells could mediate the dehydrogenation of NADPH. NADPH dehydrogenation was not dependent on FAD and was insensitive to superoxide dismutase. Several control experiments showed that NADPH dehydrogenation was accomplished by p67PHOX, not by a trace contaminant in the p67PHOX preparation. The NADPH dehydrogenase activity of p67PHOX was proportional to enzyme concentration, and showed saturation kinetics with NADPH (Km 92 +/- 5 microM), but was inhibited at high concentrations of ferricyanide. NADH was also used as a substrate by p67PHOX (Km 123 +/- 38 microM). Taken together, these results show that p67PHOX is able to mediate pyridine nucleotide dehydrogenation. These findings raise the possibility that p67PHOX might participate directly in electron transfer between NADPH and the oxidase flavin. PMID:10231525
Dang, P M; Babior, B M; Smith, R M
Coupling of 6-phosphogluconate dehydrogenase with NADPH oxidase in neutrophils: Nox2 activity regulation by NADPH availability.
It is well known that activation of the phagocyte NADPH oxidase requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac) with the membrane cytochrome b(558), leading to its conformation change. Recently, the phagocyte NADPH oxidase complex was isolated in a constitutively active form. In this complex, 6-phosphogluconate dehydrogenase (6PGDH), an enzyme involved in the production of intracellular NADPH, was identified. This protein was absent from the oxidase complex isolated from B lymphocytes, suggesting a specific interaction with the neutrophil NADPH oxidase. To clarify the implication of 6PGDH in the NADPH oxidase activity, a siRNA approach was conducted in neutrophil-like PLB985 cells. NADPH oxidase activity of siRNA-transfected cells was shown to be decreased. Similar results were obtained in vitro, after reconstitution of oxidase activity with subcellular fractions isolated from siRNA-transfected cells. Interestingly, the Michaelis constant (K(m)) of Nox2 for NADPH increases in 6PGDH-depleted cells. Moreover, 6PGDH coimmunoprecipitated with oxidase cytosolic factors from cytosol of stimulated cells. Data suggested that the affinity of Nox2 for NADPH is increased in the presence of 6PGDH on cell stimulation. The present work proposes a new way of NADPH oxidase activity regulation by modulating Nox2 affinity for NADPH. PMID:21444627
Baillet, Athan; Xu, Rang; Grichine, Alexei; Berthier, Sylvie; Morel, Fran�oise; Paclet, Marie-H�l�ne
Activation and assembly of the NADPH oxidase: a structural perspective
The NADPH oxidase of professional phagocytes is a crucial component of the innate immune response due to its fundamental role in the production of reactive oxygen species that act as powerful microbicidal agents. The activity of this multi-protein enzyme is dependent on the regulated assembly of the six enzyme subunits at the membrane where oxygen is reduced to superoxide anions. In the resting state, four of the enzyme subunits are maintained in the cytosol, either through auto-inhibitory interactions or through complex formation with accessory proteins that are not part of the active enzyme complex. Multiple inputs are required to disrupt these inhibitory interactions and allow translocation to the membrane and association with the integral membrane components. Protein interaction modules are key regulators of NADPH oxidase assembly, and the protein�protein interactions mediated via these domains have been the target of numerous studies. Many models have been put forward to describe the intricate network of reversible protein interactions that regulate the activity of this enzyme, but an all-encompassing model has so far been elusive. An important step towards an understanding of the molecular basis of NADPH oxidase assembly and activity has been the recent solution of the three-dimensional structures of some of the oxidase components. We will discuss these structures in the present review and attempt to reconcile some of the conflicting models on the basis of the structural information available.
Increased Myocardial NADPH Oxidase Activity in Human Heart Failure
OBJECTIVES This study was designed to investigate whether nicotinamide adenine dinucleotide 3-phosphate (reduced form) (NADPH) oxidase is expressed in the human heart and whether it contributes to reactive oxygen species (ROS) production in heart failure. BACKGROUND A phagocyte-type NADPH oxidase complex is a major source of ROS in the vasculature and is implicated in the pathophysiology of hypertension and atherosclerosis.
Christophe Heymes; Jennifer K. Bendall; Philippe Ratajczak; Alison C. Cave; Jane-Lise Samuel; Gerd Hasenfuss; Ajay M. Shah
Cell-free NADPH oxidase activation assays: "in vitro veritas".
The superoxide (O2 (?-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (?-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (?-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (?-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (?-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (?-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount role in the identification and characterization of the components of the NADPH oxidase complex, the deciphering of the mechanisms of assembly, the search for inhibitory drugs, and the diagnosis of various forms of chronic granulomatous disease (CGD). PMID:24504963
Pick, Edgar
Activation of NADPH oxidase 1 in tumour colon epithelial cells.
In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells. PMID:18518859
Nisimoto, Yukio; Tsubouchi, Ryoko; Diebold, Becky A; Qiao, Shanlou; Ogawa, Hisamitsu; Ohara, Takuya; Tamura, Minoru
Fungal Metabolite Gliotoxin Targets Flavocytochrome b558 in the Activation of the Human Neutrophil NADPH Oxidase
Fungal gliotoxin (GT) is a potent inhibitor of the O2 -generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47phox, a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C II with p47phox on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here,
Satoshi Nishida; Lucia S. Yoshida; Takashi Shimoyama; Hiroyuki Nunoi; Toshihiro Kobayashi; Shohko Tsunawaki
Inhibition of NADPH oxidase promotes alternative and anti-inflammatory microglial activation during neuroinflammation
Like macrophages, microglia are functionally polarized into different phenotypic activation states, referred as classical and alternative. The balance of the two phenotypes may be critical to ensure proper brain homeostasis, and may be altered in brain pathological states, such as Alzheimer�s disease. We investigated the role of NADPH oxidase in microglial activation state using p47phox and gp91phox-deficient mice as well as apocynin, a NADPH oxidase inhibitor during neuroinflammation induced by an intracerebroventricular injection of LPS or A?1�42. We showed that NADPH oxidase plays a critical role in the modulation of microglial phenotype and subsequent inflammatory response. We demonstrated that inhibition of NADPH oxidase or gene deletion of its functional p47phox subunit switched microglial activation from a classical to an alternative state in response to an inflammatory challenge. Moreover, we showed a shift in redox state towards an oxidized milieu and that subpopulations of microglia retain their detrimental phenotype in Alzheimer�s disease brains. Microglia can change their activation phenotype depending on NADPH oxidase-dependent redox state of microenvironment. Inhibition of NADPH oxidase represents a promising neuroprotective approach to reduce oxidative stress and modulate microglial phenotype towards an alternative state.
Choi, Sang-Ho; Aid, Saba; Kim, Hyung-Wook; Jackson, Sharon H.; Bosetti, Francesca
Association of phagocytic NADPH oxidase activity with hypertensive heart disease: a role for cardiotrophin-1?
Left ventricular hypertrophy (LVH) is an independent marker of mortality in hypertension. Although the mechanisms contributing to LVH are complex, inflammation and oxidative stress may favor its development. We analyzed the association of the phagocytic NADPH oxidase-mediated superoxide anion release and LVH in patients with essential hypertension and the role of cardiotrophin-1 (CT-1) and interleukin-6 (IL-6), cytokines implicated in cardiac growth. Blood pressure, echocardiography data, and serum CT-1 and IL-6 levels were obtained in 140 subjects: 18 normotensives without LVH, 42 hypertensives without LVH, and 80 hypertensives with LVH. The NADPH oxidase-dependent superoxide production was assessed by chemiluminescence in peripheral blood mononuclear cells. Peripheral blood mononuclear cells were stimulated with CT-1 in vitro. Superoxide anion production by peripheral blood mononuclear cells associated with LVH and correlated with the left ventricular mass index. Serum CT-1 and IL-6 levels, which associated with the left ventricular mass index, correlated with superoxide production. Serum CT-1 and IL-6 levels were correlated. CT-1 stimulated NADPH oxidase superoxide production in peripheral blood mononuclear cells, which resulted in an increased release of IL-6. Our results show that superoxide anion production by the phagocytic NADPH oxidase associates with hypertensive heart disease, being significantly enhanced in hypertensive patients with LVH. This may be attributable to the activation of the NADPH oxidase by CT-1 and the subsequent release of IL-6. The phagocytic NADPH oxidase may be a therapeutic target in hypertensive heart disease. PMID:24324051
Moreno, Mar�a U; San Jos�, Gorka; Pejenaute, �lvaro; Landecho, Manuel F; D�ez, Javier; Beloqui, �scar; Fortu�o, Ana; Zalba, Guillermo
NADPH Oxidase and Neurodegeneration
NADPH oxidase (Nox) is a unique, multi-protein, electron transport system that produces large amounts of superoxide via the reduction of molecular oxygen. Nox-derived reactive oxygen species (ROS) are known to be involved in a variety of physiological processes, including host defense and signal transduction. However, over the past decade, the involvement of (Nox)-dependent oxidative stress in the pathophysiology of several neurodegenerative diseases has been increasingly recognized. ROS produced by Nox proteins contribute to neurodegenerative diseases through distinct mechanisms, such as oxidation of DNA, proteins, lipids, amino acids and metals, in addition to activation of redox-sensitive signaling pathways. In this review, we discuss the recent literature on Nox involvement in neurodegeneration, focusing on Parkinson and Alzheimer diseases.
Hernandes, Marina S; Britto, Luiz R G
NADPH oxidase activity of cytochrome P-450 BM3 and its constituent reductase domain.
Cytochrome P-450 BM3 from Bacillus megaterium catalyses NADPH oxidation in the absence of added substrate. This activity is also associated with the independently expressed flavin-containing reductase domain of the protein. The rates of these activities are more than two orders of magnitude lower than those in the presence of fatty acid P-450 substrates or artificial electron acceptors. Electrons derived from NADPH in this fashion are transferred onto oxygen, generating superoxide (O2-) anions. The formation of these active oxygen species is detectable by luminometry and the chemiluminescence can be inhibited through the addition of superoxide dismutase (but not catalase). This activity is reminiscent of the microbicidal NADPH oxidase activity associated with neutrophils and other leukocyte blood cell types. Diphenyliodonium, a potent inhibitor of the neutrophil NADPH oxidase, effectively inhibits fatty acid hydroxylase and electron transferase activities catalysed by P-450 BM3 and its reductase domain. CD studies on the native and NADPH-reduced P-450 BM3 and BM3 reductase indicate that no secondary structural alteration is caused by pre-incubation with the reductant. Therefore, the previously recognised reversible time-dependent inactivation of P-450 BM3 by NADPH may be attributed to the NADPH oxidase activity associated with the reductase domain of the enzyme. PMID:7578214
Munro, A W; Lindsay, J G; Coggins, J R; Kelly, S M; Price, N C
NADPH Oxidases in Vascular Pathology
Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of �kindling radicals,� which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794�2814.
Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta
NADPH oxidases in vascular pathology.
Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of "kindling radicals," which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794-2814. PMID:24180474
Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta; Guzik, Tomasz J
TK1299, a highly thermostable NAD(P)H oxidase from Thermococcus kodakaraensis exhibiting higher enzymatic activity with NADPH.
Seven nicotinamide adenine dinucleotide oxidase homologs have been found in the genome of Thermococcus kodakaraensis. The gene encoding one of them, TK1299, consisted of 1326 nucleotides, corresponding to a polypeptide of 442 amino acids. To examine the molecular properties of TK1299, the structural gene was cloned, expressed in Escherichia coli and the gene product was characterized. Molecular weight of the recombinant protein was 49,375 Da when determined by matrix-assisted laser desorption/ionization time-of-flight and 300 kDa when analyzed by gel filtration chromatography indicating that it existed in a hexameric form. The enzyme was highly thermostable even in boiling water where it exhibited more than 95% of the enzyme activity after incubation of 150 min. TK1299 catalyzed the oxidation of NADH as well as NADPH and predominantly converted O? to H?O (more than 75%). K(m) value of the enzyme towards NADH and NADPH was almost same (24 � 2 ?M) where as specific activity was higher with NADPH compared to NADH. To our knowledge this is the most thermostable and unique NAD(P)H oxidase displaying higher enzyme activity with NADPH. PMID:23453203
Nisar, Muhammad Atif; Rashid, Naeem; Bashir, Qamar; Gardner, Qurra-tul-Ann Afza; Shafiq, Muhammad Hassan; Akhtar, Muhammad
In vitro NADH-oxidase, NADPH-oxidase and myeloperoxidase activity of macrophages after Tinospora cordifolia (guduchi) treatment.
It is believed that the enhanced microbicidal and tumoricidal capability of activated macrophages is related to the remarkable increase in the production of oxygen metabolites. Both the production of H2O2 and the oxidation of NAD(P)H are directly dependent upon NAD(P)H-oxidase. It has been established that the respiratory burst is due to activation of NAD(P)H-oxidase localised in the plasmalemma. Myeloperoxidase is believed to be involved in augmenting the cytotoxic activity of H2O2. It was observed that the macrophage cell line J774A.1 when treated with Tinospora cordifolia (guduchi) and LPS showed enhanced NADH-oxidase, NADPH-oxidase and myeloperoxidase production as compared to macrophages treated with medium alone. The direct drug treatment to J774A cells showed activation as assessed by biochemical assays. These results suggest that high NADH-oxidase, NADPH-oxidase and myeloperoxidase activities may account for tumoricidal and microbicidal properties via macrophage activation. PMID:22295977
More, Priti; Pai, Kalpana
Rotenone induces neurotoxicity through Rac1-dependent activation of NADPH oxidase in SHSY-5Y cells.
Neurodegenerative diseases are attributed to impairment of the ubiquitin-proteasome system (UPS). Oxidative stress has been considered a contributing factor in the pathology of impaired UPS by promoting protein misfolding and subsequent protein aggregate formation. Increasing evidence suggests that NADPH oxidase is a likely source of excessive oxidative stress in neurodegenerative disorders. However, the mechanism of activation and its role in impaired UPS is not understood. We show that activation of NADPH oxidase in a neuroblastoma cell line (SHSY-5Y) resulted in increased oxidative and nitrosative stress, elevated cytosolic calcium, ER-stress, impaired UPS, and apoptosis. Rac1 inhibition mitigated the oxidative/nitrosative stress, prevented calcium-dependent ER-stress, and partially rescued UPS function. These findings demonstrate that Rac1 and NADPH oxidase play an important role in rotenone neurotoxicity. PMID:24374334
Pal, Rituraj; Monroe, Tanner O; Palmieri, Michela; Sardiello, Marco; Rodney, George G
Riboflavin kinase couples TNF receptor 1 to NADPH oxidase
Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a
Benjamin Yazdanpanah; Katja Wiegmann; Vladimir Tchikov; Oleg Krut; Carola Pongratz; Michael Schramm; Andre Kleinridders; Thomas Wunderlich; Hamid Kashkar; Olaf Uterm�hlen; Jens C. Br�ning; Stefan Sch�tze; Martin Kr�nke
Activation of the NADPH oxidase in a cell-free system from human neutrophils stimulated by phorbol myristate acetate
Kinetics of activation of the NADPH oxidase in a fully soluble cell-free system from phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system in which Mg{sup 2+} and sodium dodecyl sulfate, an anionic detergent required for the activation of NADPH oxidase are contained, cytosol prepared from PMA-stimulated neutrophils failed to activate PMA-stimulated neutrophil oxidase. However, cytosol prepared
S UMEKI; Shigenobu
Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils.
We have analysed the involvement of the beta isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-beta is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcgamma-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-beta specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-beta, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-beta also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-beta isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcgamma receptors in neutrophils.
Dekker, L V; Leitges, M; Altschuler, G; Mistry, N; McDermott, A; Roes, J; Segal, A W
Attenuation of NADPH Oxidase Activation and Glomerular Filtration Barrier Remodeling With Statin Treatment
Activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase by angiotensin II is integral to the formation of oxidative stress in the vasculature and the kidney. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition is associated with reductions of oxidative stress in the vasculature and kidney and associated decreases in albuminuria. Effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition on oxidative stress in the kidney and filtration barrier integrity are poorly understood. To investigate, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and renin-angiotensin system activation, and an immortalized murine podocyte cell line. We treated young, male Ren2 and Sprague-Dawley rats with rosuvastatin (20 mg/kg IP) or placebo for 21 days. Compared with controls, we observed increases in systolic blood pressure, albuminuria, renal NADPH oxidase activity, and 3-nitrotryosine staining, with reductions in the rosuvastatin-treated Ren2. Structural changes on light and transmission electron microscopy, consistent with periarteriolar fibrosis and podocyte foot-process effacement, were attenuated with statin treatment. Nephrin expression was diminished in the Ren2 kidney and trended to normalize with statin treatment. Angiotensin II�dependent increases in podocyte NADPH oxidase activity and subunit expression (NOX2, NOX4, Rac, and p22phox) and reactive oxygen species generation were decreased after in vitro statin treatment. These data support a role for increased NADPH oxidase activity and subunit expression with resultant reactive oxygen species formation in the kidney and podocyte. Furthermore, statin attenuation of NADPH oxidase activation and reactive oxygen species formation in the kidney/podocyte seems to play roles in the abrogation of oxidative stress-induced filtration barrier injury and consequent albuminuria.
Whaley-Connell, Adam; Habibi, Javad; Nistala, Ravi; Cooper, Shawna A.; Karuparthi, Poorna R.; Hayden, Melvin R.; Rehmer, Nathan; DeMarco, Vincent G.; Andresen, Bradley T.; Wei, Yongzhong; Ferrario, Carlos; Sowers, James R.
NADPH-oxidase expression and in situ production of superoxide by osteoclasts actively resorbing bone
Recent reports have suggested that production of superoxide or other reactive oxygen species by activated osteoclasts may play a role in the complex process of bone resorption; however, the enzyme responsible for production of superoxide by osteoclasts has not been characterized. To determine if osteoclasts express NADPH-oxidase, a superoxide-generating enzyme found in phagocytic leukocytes, immunohistochemical studies were performed on tibia from 1-5-d-old rats using mAbs 449 and 48 and an antiserum specific for p47-phox. These antibodies recognize epitopes on the alpha and beta subunits of cytochrome b558, respectively, and the p47 cytosolic component of NADPH-oxidase. We found that osteoclasts attached to bone surfaces in tibia expressed all three components, as did mature polymorphonuclear and some mononuclear leukocytes in the bone marrow. In many adherent osteoclasts, the cytochrome b558 subunits were localized to the ruffled-border and bone interfaces. Studies were also performed on mature rat tibia that had undergone controlled fracture. By two weeks the healing fractures develop a callus rich in actively resorbing osteoclasts. Osteoclasts within the calluses, and attached to bone surface, also expressed the cytochrome b558 proteins. In addition to demonstrating the expression of NADPH-oxidase, the active production of superoxide by osteoclasts was also demonstrated in situ in freshly isolated tibia using 3,3'-diaminobenzidine (DAB)-Mn2+, a histochemical method specific for superoxide localization. Osteoclasts attached to bone surfaces contained deposits of oxidized DAB which were observed by light microscopy. Nonstimulated polymorphonuclear and mononuclear leukocytes in the bone marrow did not contain DAB deposits unless stimulated with phorbol myristate acetate, a known activator of NADPH-oxidase. These findings indicate that osteoclasts contain NADPH-oxidase, and during the process of resorbing bone, are actively producing superoxide.
Crosstalk between mitochondria and NADPH oxidases
Reactive oxygen species (ROS) play an important role in physiological and pathological processes. In recent years, a feed-forward regulation of the ROS sources has been reported. The interaction between main cellular sources of ROS, such as mitochondria and NADPH oxidases, however, remain obscure. This work summarizes the latest findings on the role of crosstalk between mitochondria and NADPH oxidases in pathophysiological processes. Mitochondria have the highest levels of antioxidants in the cell and play an important role in the maintenance of cellular redox status, thereby acting as an ROS and redox sink and limiting NADPH oxidase activity. Mitochondria, however, are not only a target for ROS produced by NADPH oxidase but also a significant source of ROS, which under certain condition may stimulate NADPH oxidases. This crosstalk between mitochondria and NADPH oxidases, therefore, may represent a feed-forward vicious cycle of ROS production which can be pharmacologically targeted under conditions of oxidative stress. It has been demonstrated that mitochondria-targeted antioxidants break this vicious cycle, inhibiting ROS production by mitochondria and reducing NADPH oxidase activity. This may provide a novel strategy for treatment of many pathological conditions including aging, atherosclerosis, diabetes, hypertension and degenerative neurological disorders in which mitochondrial oxidative stress seems to play a role. It is conceivable that the use of mitochondria-targeted treatments would be effective in these conditions.
Dikalov, Sergey
Activation of the NADPH oxidase in a cell-free system from human neutrophils stimulated by phorbol myristate acetate
Kinetics of activation of the NADPH oxidase in a fully soluble cell-free system from phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system in which Mg{sup 2+} and sodium dodecyl sulfate, an anionic detergent required for the activation of NADPH oxidase are contained, cytosol prepared from PMA-stimulated neutrophils failed to activate PMA-stimulated neutrophil oxidase. However, cytosol prepared from resting (control) neutrophils was capable of activating PMA-stimulated neutrophil oxidase in a cell-free system in which its Km for NADPH was almost similar to that of control neutrophil oxidase. Cytosol from PMA-stimulated neutrophils could not activate control neutrophil oxidase, although it did not contain any inhibitors of NADPH oxidase activation. These results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted, and that the affinity for NADPH of PMA-stimulated neutrophil oxidase may be the same as that of control neutrophil oxidase.
Umeki, Shigenobu (Kawasaki Medical School, Okayama (Japan))
Adenosine A3 Receptor Suppresses Prostate Cancer Metastasis by Inhibiting NADPH Oxidase Activity12
Prostate cancer is the most commonly diagnosed and second most lethal malignancy in men, due mainly to a lack of effective treatment for the metastatic disease. A number of recent studies have shown that activation of the purine nucleoside receptor, adenosine A3 receptor (A3AR), attenuates proliferation of melanoma, colon, and prostate cancer cells. In the present study, we determined whether activation of the A3AR reduces the ability of prostate cancer cells to migrate in vitro and metastasize in vivo. Using severe combined immunodeficient mice, we show that proliferation and metastasis of AT6.1 rat prostate cancer cells were decreased by the administration of A3AR agonist N6-(3-iodobenzyl) adenosine-5?-N-methyluronamide. In vitro studies show that activation of A3AR decreased high basal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity present in these cells, along with the expression of Rac1 and p47phox subunits of this enzyme. Inhibition of NADPH oxidase activity by the dominant-negative RacN17 or short interfering (si)RNA against p47phox reduced both the generation of reactive oxygen species and the invasion of these cells on Matrigel. In addition, we show that membrane association of p47phox and activation of NADPH oxidase is dependent on the activity of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase pathway. We also provide evidence that A3AR inhibits ERK1/2 activity in prostate cancer cells through inhibition of adenylyl cyclase and protein kinase A. We conclude that activation of the A3AR in prostate cancer cells reduces protein kinase A-mediated stimulation of ERK1/2, leading to reduced NADPH oxidase activity and cancer cell invasiveness.
Jajoo, Sarvesh; Mukherjea, Debashree; Watabe, Kounosuke; Ramkumar, Vickram
The NADPH oxidase diaphorase activity in permeabilized human neutrophils and granulocytic like PLB-985 cells.
The phagocyte NADPH oxidase is a multicomponent transport chain that generates superoxide, a precursor of microbicidal oxidants, important for host defense. This transport chain is contained mainly in the large membrane subunit of the oxidase (gp91phox), and transfers electrons from cytosolic NADPH, through FAD binding and heme centers, to molecular oxygen (Babior, 1999; Fujii and Kakinuma, 1991; Rotrosen et al., 1992; Segal and Abo, 1993). Cross et al. have recently described a novel NADPH oxidase diaphorase activity present in the membrane fraction of activated neutrophils, using a cell free model (Cross et al., 1994). This diaphorase activity is measured by the artificial electron acceptor 4-iodonitrotetrazolium violet (INT) and is attributed to the reduction of the flavin center of the flavocytochrome (Cross et al., 1994; Li and Guillory, 1997). In the present study we establish a system for detecting diaphorase activity in intact cells. Neutrophils and PLB-985 cells, that were differentiated using 1.25% dimethyl sulfoxide (DMSO) to granulocyte phenotype, were permeabilized by electroporation, and diaphorase activity was determined using INT. Neutrophils and differentiated PLB-985 cells stimulated by PMA or GTP gamma S showed a diaphorase activity that was not present in unstimulated differentiated cells. The diaphorase activity could not be detected in undifferentiated cells and was developed during differentiation. The pattern of diaphorase activity in stimulated parent differentiated PLB cells was similar to that observed in stimulated human neutrophils. The permeabilized-INT cell system offers a unique tool for the evaluation of NADPH oxidase diaphorase activity, in whole cells. PMID:10897413
Pessach, I; Levy, R
Phagocytosis-Coupled Activation of the Superoxide-Producing Phagocyte Oxidase, a Member of the NADPH Oxidase (Nox) Family
The phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a crucial role in host defense by neutrophils\\u000a and macrophages. When cells ingest invading microbes, this enzyme becomes activated to reduce molecular oxygen to superoxide,\\u000a a precursor of microbicidal oxidants, in the phagosome. The catalytic core of the oxidase is membrane-bound cytochrome b\\u000a 558, which comprises gp91\\u000a phox\\u000a and p22\\u000a phox
Reiko Minakami; Hideki Sumimotoa
Restraint of spreading-dependent activation of polymorphonuclear leukocyte NADPH oxidase in an acidified environment.
Elucidation of the mechanisms by which environmental pH affects or regulates the functions of polymorphonuclear leukocytes (PMNs) is important because severe acidification of the microenvironment often prevails at sites of inflammation where they act in host defense. In the present study, we investigated the effect of an acidic environment on spreading-dependent activation of O2- -producing NADPH oxidase in PMNs. We found that PMNs underwent spreading spontaneously over type I collagen and plastic surfaces at both neutral and acidic pH, although spreading over fibrinogen surfaces, for which cellular stimulation with H2O2 is required, was inhibited by acidic pH. At acidic pH, however, PMNs were unable to undergo spreading-dependent production of O2-. Pharmacological experiments showed that p38 mitogen-activated protein kinase (MAPK) was involved in the signaling pathways mediating the spreading-dependent activation of NADPH oxidase, and that its spreading-dependent phosphorylation of Thr-180 and Tyr-182, a hallmark of activation, was impaired at acidic pH. Furthermore, the inhibition by acidic pH of O2- production as well as p38 MAPK phosphorylation subsequent to spreading induction was reversible; environmental neutralization and acidification after induction of spreading at acidic and neutral pH, respectively, up- and down-regulated the two phenomena. Acidic pH did not affect the O2- production activity of NADPH oxidase pre-activated by phorbol 12-myristate 13-acetate (PMA). These results suggest that, in PMNs, the p38 MAPK-mediated signaling pathway functions as a pH-sensing regulator of spreading-dependent NADPH oxidase activation. PMID:22371970
Suzuki, Kingo; Namiki, Hideo
Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization.
Activation of Rap1 GTPase can improve the integrity of the barrier of the retina pigment epithelium (RPE) and reduce choroidal neovascularization (CNV). Inhibition of NADPH oxidase activation also reduces CNV. We hypothesize that Rap1 inhibits NADPH oxidase-generated ROS and thereby reduces CNV formation. Using a murine model of laser-induced CNV, we determined that reduced Rap1 activity in RPE/choroid occurred with CNV formation and that activation of Rap1 by 2'-O-Me-cAMP (8CPT)-reduced laser-induced CNV via inhibiting NADPH oxidase-generated ROS. In RPE, inhibition of Rap1 by Rap1 GTPase-activating protein (Rap1GAP) increased ROS generation, whereas activation of Rap1 by 8CPT reduced ROS by interfering with the assembly of NADPH oxidase membrane subunit p22phox with NOX4 or cytoplasmic subunit p47phox. Activation of NADPH oxidase with Rap1GAP reduced RPE barrier integrity via cadherin phosphorylation and facilitated choroidal EC migration across the RPE monolayer. Rap1GAP-induced ROS generation was inhibited by active Rap1a, but not Rap1b, and activation of Rap1a by 8CPT in Rap1b(-/-) mice reduced laser-induced CNV, in correlation with decreased ROS generation in RPE/choroid. These findings provide evidence that active Rap1 reduces CNV by interfering with the assembly of NADPH oxidase subunits and increasing the integrity of the RPE barrier. PMID:24043260
Wang, Haibo; Jiang, Yanchao; Shi, Dallas; Quilliam, Lawrence A; Chrzanowska-Wodnicka, Magdalena; Wittchen, Erika S; Li, Dean Y; Hartnett, M Elizabeth
Fungal metabolite gliotoxin targets flavocytochrome b558 in the activation of the human neutrophil NADPH oxidase.
Fungal gliotoxin (GT) is a potent inhibitor of the O(2)(-)-generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47(phox), a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C betaII with p47(phox) on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O(2)(-)-generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome b(558) is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome b(558) and cytosolic components (native cytosol or recombinant p67(phox), p47(phox), and Rac2) causes a striking inhibition (50% inhibitory concentration = 3.3 microM) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67(phox), p47(phox), and Rac2 with flavocytochrome b(558) is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome b(558)'s ability to reconstitute O(2)(-) generation, supporting the conclusion at the cellular level. The flavocytochrome b(558) heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome b(558), which is accessible only before oxidase activation. PMID:15618159
Nishida, Satoshi; Yoshida, Lucia S; Shimoyama, Takashi; Nunoi, Hiroyuki; Kobayashi, Toshihiro; Tsunawaki, Shohko
Fungal Metabolite Gliotoxin Targets Flavocytochrome b558 in the Activation of the Human Neutrophil NADPH Oxidase
Fungal gliotoxin (GT) is a potent inhibitor of the O2?-generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47phox, a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C ?II with p47phox on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O2?-generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome b558 is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome b558 and cytosolic components (native cytosol or recombinant p67phox, p47phox, and Rac2) causes a striking inhibition (50% inhibitory concentration = 3.3 ?M) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67phox, p47phox, and Rac2 with flavocytochrome b558 is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome b558's ability to reconstitute O2? generation, supporting the conclusion at the cellular level. The flavocytochrome b558 heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome b558, which is accessible only before oxidase activation.
Nishida, Satoshi; Yoshida, Lucia S.; Shimoyama, Takashi; Nunoi, Hiroyuki; Kobayashi, Toshihiro; Tsunawaki, Shohko
Ultrastructural localization of NADPH-oxidase activity in murine peritoneal macrophages during phagocytosis of Brucella
Summary��The localization of the enzyme NADPH oxidase in mouse peritoneal macrophages unstimulated or after phagocytosis ofBruceila suis was investigated by electron microscopy in normal mice and mice immunized againstB. suis. The enzyme was clearly visualized on mitochondrial cristae, smooth endoplasmic reticulum, and the plasma membrane; its activity\\u000a correlated mainly with the state of the endoplasmic reticulum which itself reflected macrophage
Bernard Gay; Simone Sanchez-Teff; Ren� Caravano
20-HETE increases Superoxide production and activates NADPH Oxidase in Pulmonary Artery Endothelial Cells
Reactive oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time- and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47phox association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47phox and tyrosine phosphorylation of p47phox in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid.
Medhora, Meetha; Chen, Yuenmu; Gruenloh, Stephanie; Harland, Daniel; Bodiga, Sreedhar; Zielonka, Jacek; Gebremedhin, Debebe; Gao, Ying; Falck, John R.; Anjaiah, Siddam; Jacobs, Elizabeth R.
Aryl hydrocarbon receptor modulates NADPH oxidase activity via direct transcriptional regulation of p40phox expression.
A member of the NADPH oxidase subunits, p40(phox) plays an important role in the regulation of NADPH oxidase activity and the subsequent production of reactive oxygen species (ROS). In this study, we show that mouse p40(phox) is a novel transcriptional target of the aryl hydrocarbon receptor (AhR), known as a dioxin receptor or xenobiotic receptor, in the liver. Treatment of mice with 3-methylcholanthrene (3MC) increased p40(phox) gene expression in the liver, but this induction of p40(phox) gene expression was diminished by the deletion of the AhR gene in the liver. Consistent with the in vivo results, the expression of the p40(phox) gene was increased in 3MC-treated Hepa1c1c7 cells in an AhR-dependent manner. In addition, promoter analysis established p40(phox) as a transcriptional target of AhR. Studies using the RNA-interference technique revealed that p40(phox) is involved in the increase of NADPH oxidase activity and the subsequent ROS production in AhR-activated Hepa1c1c7 cells. Consequently, the results obtained here may provide a novel molecular mechanism for ROS production after exposure to dioxins. PMID:23478803
Wada, Taira; Sunaga, Hiroshi; Ohkawara, Reiko; Shimba, Shigeki
Effects of IFN-? on intracellular trafficking and activity of macrophage NADPH oxidase flavocytochrome b558
Flavocytochrome b558, the catalytic core of the phagocyte NADPH oxidase (NOX2), mediates electron transfer from NADPH to molecular oxygen to generate superoxide, the precursor of highly ROS for host defense. Flavocytochrome b558 is an integral membrane heterodimer consisting of a large glycosylated subunit, gp91phox, and a smaller subunit, p22phox. We recently showed in murine macrophages that flavocytochrome b558 localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91phox and p22phox reside in the PM and the ER. The antimicrobial activity of macrophages, including ROS production, is greatly enhanced by IFN-?, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-? enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b558 expression and localization, along with NADPH oxidase activity, in IFN-? stimulated RAW 264.7 cells and primary murine BMDMs and hMDMs. We found that enhanced capacity for ROS production is, in part, a result of increased protein expression of gp91phox and p22phox but also demonstrate that IFN-? induced a shift in the predominant localization of gp91phox and p22phox from intracellular membrane compartments to the PM. Our results are the first to show that a cytokine can change the distribution of macrophage flavocytochrome b558 and provide a potential, new mechanism by which IFN-? modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-? regulates antimicrobial activity of macrophages are more complex than previously appreciated.
Casbon, Amy-Jo; Long, Matthew E.; Dunn, Kenneth W.; Allen, Lee-Ann H.; Dinauer, Mary C.
Mechanisms for the activation\\/electron transfer of neutrophil NADPH-oxidase complex and molecular pathology of chronic granulomatous disease
Summary Professional phagocytes, neutrophils, possess a unique membrane-associated NADPH-oxidase system, dormant in resting cells, which becomes activated upon exposure to the appropriate stimuli and catalyzes the one-electron reduction of molecular oxygen to superoxide, O2-. Oxidase activation involves the assembly, in the plasma membrane, of membrane-bound and cytosolic constituents of the oxidase system, which are disassembled in the resting state. The
S. Umeki
Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity
Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination.
Liu, Jian; Zhou, Jun; Xing, Da
Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation
The effect of oleic, linoleic and ?-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and ?-linolenic acids. ROS production was dependent on the addition of ?-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and ?-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: ?-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and ?-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts.
Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurelio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui
Intermittent Hypoxia-Induced Cognitive Deficits Are Mediated by NADPH Oxidase Activity in a Murine Model of Sleep Apnea
Background In rodents, exposure to intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Excessive NADPH oxidase activity may play a role in IH-induced CNS dysfunction. Methods and Findings The effect of IH during light period on two forms of spatial learning in the water maze and well as markers of oxidative stress was assessed in mice lacking NADPH oxidase activity (gp91phox_/Y) and wild-type littermates. On a standard place training task, gp91phox_/Y displayed normal learning, and were protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to IH as compared to room air (RA) controls, while no changes emerged in gp91phox_/Y mice. Additionally, wild-type mice, but not gp91phox_/Y mice had significantly elevated levels of NADPH oxidase expression and activity, as well as MDA and 8-OHDG in cortical and hippocampal lysates following IH exposures. Conclusions The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by excessive NADPH oxidase activity, and thus pharmacological agents targeting NADPH oxidase may provide a therapeutic strategy in sleep-disordered breathing.
Nair, Deepti; Dayyat, Ehab A.; Zhang, Shelley X.; Wang, Yang; Gozal, David
Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia
Background Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton. Results Here, we in addition to toxins use conditional expression of the major actin regulatory protein LIM kinase-1 (LIMK1), and shRNA knock-down of cofilin to modulate the cellular F/G-actin ratio in the Ra2 microglia cell line, and we use Fluorescence Recovery after Photobleaching (FRAP) in ?-actin-YFP-transduced cells to obtain a dynamic measure of actin recovery rates (actin turn-over rates) in different F/G-actin states of the actin cytoskeleton. Our data demonstrate that stimulated NADPH oxidase function was severely impaired only at extreme actin recovery rates and F/G-actin ratios, and surprisingly, that any moderate changes of these parameters of the actin cytoskeleton invariably resulted in an increased NADPH oxidase activity. Conclusion moderate actin polymerization and depolymerization both increase the FMLP and PMA-stimulated NADPH oxidase activity of microglia, which is directly correlated with neither actin recovery rate nor F/G- actin ratio. Our results indicate that NADPH oxidase functions in an enhanced state of activity in stimulated phagocytes despite widely different states of the actin cytoskeleton.
Chloride channels activated by swell can regulate the NADPH oxidase generated membrane depolarisation in activated human neutrophils
Chloride channels activated by swell have important functions in many physiological processes. The phagocyte NADPH oxidase is essential for host defence and it generates superoxide by transferring electrons from the donor NADPH to the acceptor O{sub 2}. This electron current, induces a depolarisation of the plasma membrane. In this study, I report that chloride channels activated by swell can counteract the depolarisation induced by the NADPH oxidase. When a chloride conductance was activated by swelling, its inhibition by either 50 {mu}M NPPB or removing external chloride, depolarised the plasma membrane potential to +26 mV {+-} 3.1 (n = 4) and +40 {+-} 1 mV (n = 4), respectively. These channels were partially inhibited by the NADPH oxidase inhibitor AEBSF (1 mM) and potently inhibited by ZnCl{sub 2} (3 mM). These currents were not activated by a phosphorylation step and elevations in intracellular calcium did not appear to activate chloride currents similar to those activated by swell.
Ahluwalia, Jatinder [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom)], E-mail: firstname.lastname@example.com
Kinase-dependent activation of the leukocyte NADPH oxidase in a cell-free system. Phosphorylation of membranes and p47(PHOX) during oxidase activation.
The leukocyte NADPH oxidase catalyzes the 1-electron reduction of oxygen to O2- at the expense of NADPH: 2 O2 + NADPH --> 2 O2- + NADP+ + H+. The oxidase is dormant in resting cells but acquires activity when the cells are stimulated with a suitable agent. Activation in whole cells is accompanied by extensive phosphorylation of p47(PHOX), an oxidase subunit located in the cytosol of resting cells that during oxidase activation migrates to the plasma membrane to complex with cytochrome b558, an oxidase-specific flavohemoprotein. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile as activating agent. We now report a cell-free system in which the oxidase can be activated in two stages using phosphorylated p47(PHOX). The first stage, which effects a change in the membrane, requires ATP and GTP and is blocked by the protein kinase inhibitor GF-109203X, suggesting a protein kinase requirement. The second stage requires phosphorylated p47(PHOX) and GTP, but no ATP, and is unaffected by GF-109203X; assembly of the oxidase may take place during this stage. Activation is accomplished by p47(PHOX) phosphorylated by protein kinase C but not protein kinase A or mitogen-activated protein kinase. We believe that activation by phosphorylated p47(PHOX) is more physiological than activation by amphiphiles, because the mutant p47(PHOX) S379A, which is inactive in whole cells, is also inactive in this system but works in systems activated by amphiphiles. PMID:9110996
Park, J W; Hoyal, C R; Benna, J E; Babior, B M
NADPH oxidases: progress and opportunities.
Abstract From the initial discovery in 1999 that NADPH oxidases comprise a family of enzymes to our current focus on drug development to treat multiple pathologies related to this enzyme family, progress has been swift and impressive. We have expanded our understanding of the extent of the family, the basic enzymatic biochemistry, the multiple cellular functions controlled by NADPH oxidases, and their varied roles in physiology and diseases. We have developed numerous cell culture tools, animal models, and human databases that have allowed us to delve deeply into the various roles of these enzymes. However, it is clear that much remains to be learned and that there are many opportunities for new tools and new research directions to more fully understand these critical enzymes. With this Antioxidants and Redox Signaling Forum, we explore in detail the progress, challenges, and opportunities in Nox biology. Progress so far has clearly shown that NADPH oxidases are integral to fully functioning organisms and that the dysregulation of Nox enzymes contributes to a wide variety of pathologies. We have the opportunity to develop new tools and small molecules that will not only help us to better understand the molecular underpinnings of NADPH oxidases but also to develop treatments for diverse human diseases. Antioxid. Redox Signal. 20, 2692-2694. PMID:24730700
San Martin, Alejandra; Griendling, Kathy K
Targeting NADPH oxidases in vascular pharmacology
Oxidative stress is a molecular dysregulation in reactive oxygen species (ROS) metabolism, which plays a key role in the pathogenesis of atherosclerosis, vascular inflammation and endothelial dysfunction. It is characterized by a loss of nitric oxide (NO) bioavailability. Large clinical trials such as HOPE and HPS have not shown a clinical benefit of antioxidant vitamin C or vitamin E treatment, putting into question the role of oxidative stress in cardiovascular disease. A change in the understanding of the molecular nature of oxidative stress has been driven by the results of these trials. Oxidative stress is no longer perceived as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS production. NADPH oxidases are at the center of these events, underlying the dysfunction of other oxidases including eNOS uncoupling, xanthine oxidase and mitochondrial dysfunction. Thus NADPH oxidases are important therapeutic targets. Indeed, HMG-CoA reductase inhibitors (statins) as well as drugs interfering with the renin-angiotensin-aldosterone system inhibit NADPH oxidase activation and expression. Angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists (sartans) and aliskiren, as well as spironolactone or eplerenone, have been discussed. Molecular aspects of NADPH oxidase regulation must be considered, while thinking about novel pharmacological targeting of this family of enzymes consisting of several homologs Nox1, Nox2, Nox3, Nox4 and Nox5 in humans. In order to properly design trials of antioxidant therapies, we must develop reliable techniques for the assessment of local and systemic oxidative stress. Classical antioxidants could be combined with novel oxidase inhibitors. In this review, we discuss NADPH oxidase inhibitors such as VAS2870, VAS3947, GK-136901, S17834 or plumbagin. Therefore, our efforts must focus on generating small molecular weight inhibitors of NADPH oxidases, allowing the selective inhibition of dysfunctional NADPH oxidase homologs. This appears to be the most reasonable approach, potentially much more efficient than non-selective scavenging of all ROS by the administration of antioxidants.
Schramm, Agata; Matusik, Pawel; Osmenda, Grzegorz; Guzik, Tomasz J
Targeting NADPH oxidases in vascular pharmacology.
Oxidative stress is a molecular dysregulation in reactive oxygen species (ROS) metabolism, which plays a key role in the pathogenesis of atherosclerosis, vascular inflammation and endothelial dysfunction. It is characterized by a loss of nitric oxide (NO) bioavailability. Large clinical trials such as HOPE and HPS have not shown a clinical benefit of antioxidant vitamin C or vitamin E treatment, putting into question the role of oxidative stress in cardiovascular disease. A change in the understanding of the molecular nature of oxidative stress has been driven by the results of these trials. Oxidative stress is no longer perceived as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS production. NADPH oxidases are at the center of these events, underlying the dysfunction of other oxidases including eNOS uncoupling, xanthine oxidase and mitochondrial dysfunction. Thus NADPH oxidases are important therapeutic targets. Indeed, HMG-CoA reductase inhibitors (statins) as well as drugs interfering with the renin-angiotensin-aldosterone system inhibit NADPH oxidase activation and expression. Angiotensin-converting enzyme (ACE) inhibitors, AT1 receptor antagonists (sartans) and aliskiren, as well as spironolactone or eplerenone, have been discussed. Molecular aspects of NADPH oxidase regulation must be considered, while thinking about novel pharmacological targeting of this family of enzymes consisting of several homologs Nox1, Nox2, Nox3, Nox4 and Nox5 in humans. In order to properly design trials of antioxidant therapies, we must develop reliable techniques for the assessment of local and systemic oxidative stress. Classical antioxidants could be combined with novel oxidase inhibitors. In this review, we discuss NADPH oxidase inhibitors such as VAS2870, VAS3947, GK-136901, S17834 or plumbagin. Therefore, our efforts must focus on generating small molecular weight inhibitors of NADPH oxidases, allowing the selective inhibition of dysfunctional NADPH oxidase homologs. This appears to be the most reasonable approach, potentially much more efficient than non-selective scavenging of all ROS by the administration of antioxidants. PMID:22405985
Schramm, Agata; Matusik, Pawe?; Osmenda, Grzegorz; Guzik, Tomasz J
Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity.
VCAM-1 (vascular cell adhesion molecule-1) plays an important role in the regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease and transplantation. VCAM-1 activates endothelial cell NADPH oxidase, and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We reported previously that a mouse microvascular endothelial cell line promotes lymphocyte migration that is dependent on VCAM-1, but not on other known adhesion molecules. Here we have investigated the signalling mechanisms underlying VCAM-1 function. Lymphocyte binding to VCAM-1 on the endothelial cell surface activated an endothelial cell calcium flux that could be inhibited with anti-alpha4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an inhibitor of intracellular calcium mobilization, a calcium channel inhibitor or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker suppression of VCAM-1-stimulated NADPH oxidase activity, but could not reverse the inhibitory effect imposed by intracellular calcium blockage, indicating that both intracellular and extracellular calcium mobilization are required for VCAM-1-mediated activation of NADPH oxidase. Furthermore, VCAM-1 specifically activated the Rho-family GTPase Rac1, and VCAM-1 activation of NADPH oxidase was blocked by a dominant negative Rac1. Thus VCAM-1 stimulates the mobilization of intracellular and extracellular calcium and Rac1 activity that are required for the activation of NADPH oxidase.
Cook-Mills, Joan M; Johnson, Jacob D; Deem, Tracy L; Ochi, Atsuo; Wang, Lei; Zheng, Yi
Involvement of Rac1 in Activation of Multicomponent Nox1- and Nox3-Based NADPH Oxidases
Several Nox family NADPH oxidases function as multicomponent enzyme systems. We explored determinants of assembly of the multicomponent oxidases Nox1 and Nox3 and examined the involvement of Rac1 in their regulation. Both enzymes are supported by p47phox and p67phox or homologous regulators called Noxo1 and Noxa1, although Nox3 is less dependent on these cofactors for activity. Plasma membrane targeting of Noxa1 depends on Noxo1, through tail-to-tail interactions between these proteins. Noxa1 can support Nox1 without Noxo1, when targeted to the plasma membrane by fusing membrane-binding sequences from Rac1 (amino acids 183 to 192) to the C terminus of Noxa1. However, membrane targeting of Noxa1 is not sufficient for activation of Nox1. Both the Noxo1-independent and -dependent Nox1 systems involve Rac1, since they are affected by Rac1 mutants or Noxa1 mutants defective in Rac binding or short interfering RNA-mediated Rac1 silencing. Nox1 or Nox3 expression promotes p22phox transport to the plasma membrane, and both oxidases are inhibited by mutations in the p22phox binding sites (SH3 domains) of the Nox organizers (p47phox or Noxo1). Regulation of Nox3 by Rac1 was also evident from the effects of mutant Rac1 or mutant Nox3 activators (p67phox or Noxa1) or Rac1 silencing. In the absence of Nox organizers, the Nox activators (p67phox or Noxa1) colocalize with Rac1 within ruffling membranes, independently of their ability to bind Rac1. Thus, Rac1 regulates both oxidases through the Nox activators, although it does not appear to direct the subcellular localization of these activators.
Ueyama, Takehiko; Geiszt, Miklos; Leto, Thomas L.
Protein kinase C-dependent and -independent activation of the NADPH oxidase of human neutrophils.
The protein kinase C inhibitor, staurosporine, inhibited NADPH oxidase activity of human neutrophils activated by phorbol myristate acetate. However, this inhibitor had no effect on either the initiation or the maximal rate of O2- secretion activated by the chemotactic peptide, fMet-Leu-Phe, but resulted in a more rapid termination of oxidant production. Similarly, staurosporine had no effect on the rapid (1 min) increase in luminol-dependent chemiluminescence activated by fMet-Leu-Phe, but the second (intracellular) phase of oxidant production was inhibited. The initial burst of oxidant production during phagocytosis was similarly protein kinase C-independent, but again the later phases of oxidase activity were staurosporine-sensitive. Neutrophils loaded with Quin-2 at concentrations sufficient to act as a Ca2+ buffer could not secrete O2- in response to fMet-Leu-Phe; although the initial (protein kinase C-independent) burst of luminol chemiluminescence was not observed in fMet-Leu-Phe-stimulated Ca2(+)-buffered cells, the second phase of (protein kinase C-dependent) oxidant production was largely unaffected. Hence, the initial burst of oxidant production activated by fMet-Leu-Phe, opsonized zymosan, and latex beads is independent of the activity of protein kinase C-dependent intracellular activation processes, but the activity of this kinase is required to extend or sustain the duration of oxidant production. PMID:1850407
Watson, F; Robinson, J; Edwards, S W
NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury
Members of the NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O2 to reactive oxygen species, have increased in number during eukaryotic evolution. Seven isoforms of the NOX gene family have been identified in mammals; however, specific roles of NOX enzymes in mammalian physiology and pathophysiology have not been fully elucidated. The best established physiological role of
Louise Hecker; Ragini Vittal; Tamara Jones; Rajesh Jagirdar; Tracy R Luckhardt; Jeffrey C Horowitz; Subramaniam Pennathur; Fernando J Martinez; Victor J Thannickal
The complex roles of NADPH oxidases in fungal infection.
NADPH oxidases play key roles in immunity and inflammation that go beyond the production of microbicidal reactive oxygen species (ROS). The past decade has brought a new appreciation for the diversity of roles played by ROS in signalling associated with inflammation and immunity. NADPH oxidase activity affects disease outcome during infections by human pathogenic fungi, an important group of emerging and opportunistic pathogens that includes Candida, Aspergillus and Cryptococcus species. Here we review how alternative roles of NADPH oxidase activity impact fungal infection and how ROS signalling affects fungal physiology. Particular attention is paid to roles for NADPH oxidase in immune migration, immunoregulation in pulmonary infection, neutrophil extracellular trap formation, autophagy and inflammasome activity. These recent advances highlight the power and versatility of spatiotemporally controlled redox regulation in the context of infection, and point to a need to understand the molecular consequences of NADPH oxidase activity in the cell. PMID:24905433
Hogan, Deborah; Wheeler, Robert T
NADPH oxidase promotes neutrophil extracellular trap formation in pulmonary aspergillosis.
NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47(phox-/-)) mice which had resolved in wild-type mice by day 5 but progressed in p47(phox-/-) mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47(phox-/-) mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323
R�hm, Marc; Grimm, Melissa J; D'Auria, Anthony C; Almyroudis, Nikolaos G; Segal, Brahm H; Urban, Constantin F
Inhibition of NADPH oxidase activation by peptides mapping within the dehydrogenase region of Nox2-A "peptide walking" study.
In this study, the "peptide walking" approach was applied to the DH region of Nox2 (residues 288-570) with the purpose of identifying domains of functional importance in the assembly and/or catalytic function of the NADPH oxidase complex of phagocytes. Ninety-one overlapping 15-mer peptides were synthesized to cover the full length of the Nox2 DH region, and these were tested for the ability to interfere with the activation of the oxidase in vitro in two semi-recombinant cell-free systems. The first consisted of phagocyte membranes p47(phox), p67(phox), and Rac1 and an amphiphile; the second was p47(phox)- and amphiphile-free and contained prenylated Rac1. We identified 10 clusters of inhibitory peptides with IC(50) values of 10 ?M, all of which were inhibitory, also in the absence of p47(phox). Based on the identification of residues shared by peptides in a particular cluster, we defined 10 functional domains in the Nox2 DH region. One domain corresponded to one FAD-binding subdomain, and four domains overlapped parts of three NADPH-binding subdomains. As expected, most inhibitory peptides acted only when added prior to the completion of oxidase assembly, but peptides associated with two NADPH-binding subdomains were also active after assembly. Kinetic analysis demonstrated that inhibition by peptides was not explained by competition for substrates (FAD, NADPH) but was of a more complex nature: noncompetitive with respect to FAD and uncompetitive with respect to NADPH. We conclude that oxidase-inhibitory peptides, in five out of 10 clusters identified, act by interfering with FAD- and NADPH-related redox reactions. PMID:22184755
Dahan, Iris; Molshanski-Mor, Shahar; Pick, Edgar
NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A*S?
Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation.
Frasch, S. Courtney; Berry, Karin Zemski; Fernandez-Boyanapalli, Ruby; Jin, Hyun-Sun; Leslie, Christina; Henson, Peter M.; Murphy, Robert C.; Bratton, Donna L.
NMDA Receptor-Mediated Activation of NADPH Oxidase and Glomerulosclerosis in Hyperhomocysteinemic Rats
Abstract This study investigated the role of NMDA receptor in hyperhomocyteinemia (hHcys)-induced NADPH oxidase (Nox) activation and glomerulosclerosis. Sprague�Dawley rats were fed a folate-free (FF) diet to produce hHcys, and a NMDA receptor antagonist, MK-801, was administrated. Rats fed the FF diet exhibited significantly increased plasma homocysteine levels, upregulated NMDA receptor expression, enhanced Nox activity and Nox-dependent O2.? production in the glomeruli, which were accompanied by remarkable glomerulosclerosis. MK-801 treatment significantly inhibited Nox-dependent O2.? production induced by hHcys and reduced glomerular damage index as compared with vehicle-treated hHcys rats. Correspondingly, glomerular deposition of extracellular matrix components in hHcys rats was ameliorated by the administration of MK-801. Additionally, hHcys induced an increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and a decrease in matrix metalloproteinase (MMP)-1 and MMP-9 activities, all of which were abolished by MK-801 treatment. In vitro studies showed that homocysteine increased Nox-dependent O2.? generation in rat mesangial cells, which was blocked by MK-801. Pretreatment with MK-801 also reversed homocysteine-induced decrease in MMP-1 activity and increase in TIMP-1 expression. These results support the view that the NMDA receptor may mediate Nox activation in the kidney during hHcys and thereby play a critical role in the development of hHcys-induced glomerulosclerosis. Antioxid. Redox Signal. 13, 975�986.
Zhang, Chun; Yi, Fan; Xia, Min; Boini, Krishna M.; Zhu, Qing; Laperle, Laura A.; Abais, Justine M.; Brimson, Christopher A.
Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor
NASA Technical Reports Server (NTRS)
The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.
Morre, D. James
Natural Compounds as Modulators of NADPH Oxidases
Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols.
NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development.
A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2(-/-)/p19(ARF-/-), Stat3(?hc)/Mdr2(-/-)) and a model of experimental induced fibrosis (CCl(4)) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-? pathway activation. In vitro TGF-?-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-? is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-?-induced epithelial-mesenchymal transition (EMT), but was required for TGF-?-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-?-induced death of hepatocytes. PMID:23049784
Sancho, Patricia; Mainez, J�ssica; Crosas-Molist, Eva; Roncero, C�sar; Fern�ndez-Rodriguez, Conrado M; Pinedo, Fernando; Huber, Heidemarie; Eferl, Robert; Mikulits, Wolfgang; Fabregat, Isabel
Novel p47(phox)-related organizers regulate localized NADPH oxidase 1 (Nox1) activity.
The mechanisms that determine localized formation of reactive oxygen species (ROS) through NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox) family members in nonphagocytic cells are unknown. We show that the c-Src substrate proteins Tks4 (tyrosine kinase substrate with four SH3 domains) and Tks5 are functional members of a p47(phox)-related organizer superfamily. Tks proteins selectively support Nox1 and Nox3 (and not Nox2 and Nox4) activity in reconstituted cellular systems and interact with the NoxA1 activator protein through an Src homology 3 domain-mediated interaction. Endogenous Tks4 is required for Rac guanosine triphosphatase- and Nox1-dependent ROS production by DLD1 colon cancer cells. Our results are consistent with the Tks-mediated recruitment of Nox1 to invadopodia that form in DLD1 cells in a Tks- and Nox-dependent fashion. We propose that Tks organizers represent previously unrecognized members of an organizer superfamily that link Nox to localized ROS formation. PMID:19755710
Gianni, Davide; Diaz, Bego�a; Taulet, Nicolas; Fowler, Bruce; Courtneidge, Sara A; Bokoch, Gary M
NADPH oxidase limits lipopolysaccharide-induced lung inflammation and injury in mice through redox regulation of NF-?B activity
Although reactive oxygen species (ROS) produced by NADPH oxidase are known to regulate inflammatory responses, the impact of ROS on intracellular signaling pathways is incompletely understood. In these studies, we treated wild-type and p47phox deficient mice with LPS to investigate mechanisms by which NADPH oxidase regulates signaling through the NF-?B pathway. After intratracheal instillation of LPS, ROS generation was impaired in p47phox-/- mice while these mice had increased neutrophilic alveolitis and greater lung injury compared to wild-type controls. In mice interbred with transgenic NF-?B reporters (HLL), we found exaggerated LPS-induced NF-?B activation and increased expression of pro-inflammatory cytokines in lungs of p47phox-/-/HLL mice compared to controls. Both lung macrophages and bone marrow-derived macrophages (BMDMs) isolated from p47phox-/-/HLL mice showed enhanced LPS-stimulated NF-?B activity compared to controls. While nuclear translocation of NF-?B proteins was similar between genotypes, electrophoretic mobility shift assays under non-reducing conditions showed increased DNA binding in p47phox-/-/HLL BMDMs, suggesting that ROS production reduces NF-?B binding to DNA without affecting nuclear translocation. Increased intracellular GSH/GSSG ratio and greater nuclear redox factor 1 (Ref-1) levels were present in p47phox-/-/HLL compared to wild-type BMDMs, pointing to NADPH oxidase modulating intracellular redox status in macrophages. Treatment with the Ref-1 specific inhibitor E3330 or hydrogen peroxide inhibited LPS-induced NF-?B activation in p47phox-/-/HLL BMDMs but not in wild-type/HLL cells. Consistent with these findings, siRNA against Ref-1 selectively reduced NF-?B activity in LPS-treated p47phox-/-/HLL BMDMs. Together, these results indicate that NADPH oxidase limits LPS-induced NF-?B transcriptional activity through regulation of intracellular redox state.
Han, Wei; Li, Hui; Cai, Jiyang; Gleaves, Linda A.; Polosukhin, Vasiliy V.; Segal, Brahm H.; Yull, Fiona E.; Blackwell, Timothy S.
Protein kinase C-alpha and -delta are required for NADPH oxidase activation in WKYMVm-stimulated IMR90 human fibroblasts.
The regulation of the activation of non phagocytic NADPH oxidase is poorly understood. Previously we demonstrated that in fibroblasts the exposure to WKYMVm induced p47(phox) phosphorylation and translocation and that these effects were mediated by ERKs activation. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in polymorphonucleate cells stimulated via FPRL1 receptor, but its involvement in fibroblasts was not demonstrated. Therefore, we investigated in IMR90 cells exposed to WKYMVm the role of PKC isoenzymes in the activation of NADPH oxidase-like enzyme. Preincubation with general pharmacological inhibitors of PKC, before stimulation with WKYMVm, prevented the ERKs activation, p47(phox) phosphorylation and translocation. The analysis of cellular partitioning of PKC isoenzymes demonstrated that PKCalpha and PKCdelta translocated from the cytosolic to the membrane fraction upon stimulation with WKYMVm. Preincubation with G�6976 or with rottlerin prevented the phosphorylation and translocation of NADPH oxidase regulatory subunit. PMID:17166481
Iaccio, Annalisa; Collinet, Claudio; Gesualdi, Nicola Montesano; Ammendola, Rosario
Induction of genes encoding NADPH oxidase components and activation of IFN regulatory factor-1 by prolactin in fish macrophages.
The role played by prolactin (PRL) in fish immunity is scant. We report here that stimulation of the Atlantic salmon monocytic cell line SHK-1 with native salmon PRL resulted in activation of the respiratory burst and induction of the expression of the genes encoding the phagocyte NADPH oxidase components p47phox, p67phox and gp91phox, and the transcription factor IFN regulatory factor-1 (IRF-1). Interestingly, the pharmacologic inhibition of the Jak/Stat signaling pathway with AG490 blocked reactive oxygen species (ROS) production, and the induction of genes encoding the NADPH oxidase components and IRF-1 in PRL-activated SHK-1 cells. In addition, PRL promoted the phosphorylation of Stat and induced the DNA binding activity of IRF-1. These results, together with the presence of several consensus target motifs for Stat and IRF-1 in the promoter of the tilapia p47phox gene, suggest that PRL regulates p47phox gene expression in fish through the activation of these two key transcription factors. Taken together, our results demonstrate that PRL induces the expression of the genes encoding the major phagocyte NADPH oxidase components and ROS production in fish macrophages via the JAK2/Stat/IRF-1 signaling pathway. PMID:23548829
Olavarr�a, V�ctor H; Figueroa, Jaime E; Mulero, Victoriano
Contribution of NADPH Oxidase to Membrane CD38 Internalization and Activation in Coronary Arterial Myocytes
The CD38-ADP-ribosylcyclase-mediated Ca2+ signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2�?) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2�? serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2�? production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2�? significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2�? production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells.
Xu, Ming; Li, Xiao-Xue; Ritter, Joseph K.; Abais, Justine M.; Zhang, Yang; Li, Pin-Lan
Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase
The neutrophil NADPH-oxidase can be activated by protein kinase C (PKC) agonists such as phorbol myristate acetate (PMA), resulting in superoxide anion release. This superoxide release is independent of phosphatidylinositol 3-kinase (PI 3-kinase) because the inhibitor wortmannin does not affect the response. In this study, PMA is shown to also induce a wortmannin-sensitive NADPH- oxidase activation, however, not resulting in
Anna Karlsson; Jennifer B. Nixon; Linda C. McPhail
Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavocytochrome b558 during phagocytic NADPH oxidase assembly
The role of Leu505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mim- icking the phenotype of a previously published X91- CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) re- ductase activities were partially maintained concom- itantly with a partial
Xing Jun Li; Franck Fieschi; Marie-Helene Paclet; Didier Grunwald; Yannick Campion; Philippe Gaudin; Francoise Morel; M.-J. Stasia
Current status of NADPH oxidase research in cardiovascular pharmacology
The implications of reactive oxygen species in cardiovascular disease have been known for some decades. Rationally, therapeutic antioxidant strategies combating oxidative stress have been developed, but the results of clinical trials have not been as good as expected. Therefore, to move forward in the design of new therapeutic strategies for cardiovascular disease based on prevention of production of reactive oxygen species, steps must be taken on two fronts, ie, comprehension of reduction-oxidation signaling pathways and the pathophysiologic roles of reactive oxygen species, and development of new, less toxic, and more selective nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, to clarify both the role of each NADPH oxidase isoform and their utility in clinical practice. In this review, we analyze the value of NADPH oxidase as a therapeutic target for cardiovascular disease and the old and new pharmacologic agents or strategies to prevent NADPH oxidase activity. Some inhibitors and different direct or indirect approaches are available. Regarding direct NADPH oxidase inhibition, the specificity of NADPH oxidase is the focus of current investigations, whereas the chemical structure-activity relationship studies of known inhibitors have provided pharmacophore models with which to search for new molecules. From a general point of view, small-molecule inhibitors are preferred because of their hydrosolubility and oral bioavailability. However, other possibilities are not closed, with peptide inhibitors or monoclonal antibodies against NADPH oxidase isoforms continuing to be under investigation as well as the ongoing search for naturally occurring compounds. Likewise, some different approaches include inhibition of assembly of the NADPH oxidase complex, subcellular translocation, post-transductional modifications, calcium entry/release, electron transfer, and genetic expression. High-throughput screens for any of these activities could provide new inhibitors. All this knowledge and the research presently underway will likely result in development of new drugs for inhibition of NADPH oxidase and application of therapeutic approaches based on their action, for the treatment of cardiovascular disease in the next few years.
Rodino-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Castineiras-Landeira, Maria Isabel; Raposeiras-Roubin, Sergio; Gonzalez-Juanatey, Jose R; Alvarez, Ezequiel
Elevated NADPH oxidase activity contributes to oxidative stress and cell death in Huntington's disease
A mutation in the huntingtin (Htt) gene produces mutant Htt and Huntington's disease (HD), a neurodegenerative disorder. HD patients have oxidative damage in the brain, but the causes are unclear. Compared with controls, we found brain levels of NADPH oxidase (NOX) activity, which produces reactive oxygen species (ROS), elevated in human HD postmortem cortex and striatum and highest in striatum of presymptomatic individuals. Synaptosome fractions from cortex and striatum of HD140Q/140Q mice had elevated NOX activity at 3 months of age and a further rise at 6 and 12 months compared with synaptosomes of age-matched wild-type (WT) mice. High NOX activity in primary cortical and striatal neurons of HD140Q/140Q mice correlated with more ROS and neurite swellings. These features and neuronal cell death were markedly reduced by treatment with NOX inhibitors such as diphenyleneiodonium (DPI), apocynin (APO) and VAS2870. The rise in ROS levels in mitochondria of HD140Q/140Q neurons followed the rise in NOX activity and inhibiting only mitochondrial ROS was not neuroprotective. Mutant Htt colocalized at plasma membrane lipid rafts with gp91-phox, a catalytic subunit for the NOX2 isoform. Assembly of NOX2 components at lipid rafts requires activation of Rac1 which was also elevated in HD140Q/140Q neurons. HD140Q/140Q mice bred to gp91-phox knock-out mice had lower NOX activity in the brain and in primary neurons, and neurons had normal ROS levels and significantly improved survival. These findings suggest that increased NOX2 activity at lipid rafts is an early and major source of oxidative stress and cell death in HD140Q/140Q neurons.
Valencia, Antonio; Sapp, Ellen; Kimm, Jeffrey S.; McClory, Hollis; Reeves, Patrick B.; Alexander, Jonathan; Ansong, Kwadwo A.; Masso, Nicholas; Frosch, Matthew P.; Kegel, Kimberly B.; Li, Xueyi; DiFiglia, Marian
Activation of NAD(P)H Oxidase by tryptophan-derived 3-hydroxykynurenine Accelerates Endothelial Apoptosis and Dysfunction In Vivo
Rationale The kynurenine (Kyn) pathway is the major route for tryptophan (Trp) metabolism in mammals. The Trp-Kyn pathway is reported to regulate several fundamental biological processes, including cell death. Objective The aim of this study is to elucidate the contributions and molecular mechanism of Trp-Kyn pathway to endothelial cell death. Methods and Results Endogenous reactive oxygen species (ROS), endothelial cell apoptosis, and endothelium-dependent and -independent vasorelaxation were measured in aortas of wild-type mice or mice deficient for nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase subunits (p47phox or gp91phox) or indoleamine 2,3-dioxygenase 1 (IDO1) with or without angiotensin (Ang) II infusion. As expected, AngII increased plasma levels of Kyn and 3-hydroxykynurenine (3-OHKyn)-modified proteins in endothelial cells in vivo. Consistent with this, AngII markedly increased the expression of IDO in parallel with increased expression of interferon-gamma (IFN-?). Further, in wild-type mice, AngII significantly increased oxidative stress, endothelial cell apoptosis, and endothelial dysfunction. These effects of AngII infusion were significantly suppressed in mice deficient for p47phox, gp91phox or IDO1, suggesting that AngII-induced enhancement of Kynurenines via NAD(P)H oxidase-derived oxidants causes endothelial cell apoptosis and dysfunction in vivo. Furthermore, IFN-? neutralization eliminates AngII-increased superoxide products and endothelial apoptosis by inhibiting AngII-induced Kynurenines generation, suggesting that AngII-activated Kyn pathway is IFN-? dependent. Mechanistically, we found that AngII-enhanced 3-OHKyn promoted the generation of NAD(P)H oxidase-mediated superoxide anions by increasing the translocation and membrane assembly of NAD(P)H oxidase subunits in endothelial cells (ECs), resulting in accelerated apoptosis and consequent endothelial dysfunction. Conclusions Kyn pathway activation accelerates apoptosis and dysfunction of the endothelium by up-regulating NAD(P)H-derived superoxide.
Wang, Qiongxin; Zhang, Miao; Ding, Ye; Wang, Qilong; Zhang, Wencheng; Song, Ping; Zou, Ming-Hui
Epithelial-to-mesenchymal transition in podocytes mediated by activation of NADPH oxidase in hyperhomocysteinemia.
The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1), in conditionally immortalized mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the abundance of mesenchymal markers, such as fibroblast specific protein-1 (FSP-1) and ?-smooth muscle actin (?-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide (O??) production, which was substantially suppressed by inhibition of Nox activity. Functionally, Hcys significantly enhanced the permeability of the podocyte monolayer coupled with increased EMT, and this EMT-related increase in cell permeability could be restored by Nox inhibitors. In mice lacking gp91( phox ) (gp91(-/-)), an essential Nox subunit gene, hHcys-enhanced podocyte EMT and consequent glomerular injury were examined. In wild-type (gp91(+/+)) mice, hHcys induced by a folate-free diet markedly enhanced expression of mesenchymal markers (FSP-1 and ?-SMA) but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in gp91(-/-) mouse glomeruli. Podocyte injury, glomerular sclerotic pathology, and marked albuminuria observed in gp91(+/+) mice with hHcys were all significantly attenuated in gp91(-/-) mice. These results suggest that hHcys induces EMT of podocytes through activation of Nox, which represents a novel mechanism of hHcys-associated podocyte injury. PMID:21647593
Zhang, Chun; Xia, Min; Boini, Krishna M; Li, Cai-Xia; Abais, Justine M; Li, Xiao-Xue; Laperle, Laura A; Li, Pin-Lan
Kinetic study of the activation of the neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and phenylarsine oxide.
The O(2)(-) generating NADPH oxidase complex of neutrophils comprises two sets of components, namely a membrane-bound heterodimeric flavocytochrome b which contains the redox centers of the oxidase and water-soluble proteins of cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils in the presence of GTPgammaS and arachidonic acid. NADPH oxidase activation is inhibited by phenylarsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol groups. The site of action of PAO was localized by photolabeling in the beta-subunit of flavocytochrome b [Doussi�re, J., Poinas, A, Blais, C., and Vignais, P. V. (1998) Eur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocytochrome b [Doussi�re, J., Gaillard, J., and Vignais, P. V. (1996) Biochemistry 35, 13400-13410]. Here we report that the promoting effect of arachidonic acid on the activation of NADPH oxidase is due to specific binding of arachidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of flavocytochrome b for O(2), an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affected by arachidonic acid. We further demonstrate that PAO antagonizes the effect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O(2), but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flavocytochrome b at two different interacting sites. PMID:10587465
Doussiere, J; Bouzidi, F; Poinas, A; Gaillard, J; Vignais, P V
Characterization of the expression and inflammatory activity of NADPH oxidase after spinal cord injury.
Abstract Reactive oxygen species (ROS) and the NADPH oxidase (NOX) enzyme are both up-regulated after spinal cord injury (SCI) and play significant roles in promoting post-injury inflammation. However, the cellular and temporal expression profile of NOX isotypes, including NOX2, 3, and 4, after SCI is currently unclear. The purpose of this study was to resolve this expression profile and examine the effect of inhibition of NOX on inflammation after SCI. Briefly, adult male rats were subjected to moderate contusion SCI. Double immunofluorescence for NOX isotypes and CNS cellular types was performed at 24 h, 7 days, and 28 days post-injury. NOX isotypes were found to be expressed in neurons, astrocytes, and microglia, and this expression was dependent on injury status. NOX2 and 4 were found in all cell types assessed, while NOX3 was positively identified in neurons only. NOX2 was the most responsive to injury, increasing in both microglia and astrocytes. The biggest increases in expression were observed at 7 days post-injury and increased expression was maintained through 28 days. NOX2 inhibition by systemic administration of gp91ds-tat at 15 min, 6 h or 7 days after injury reduced both pro-inflammatory cytokine expression and evidence of oxidative stress in the injured spinal cord. This study therefore illustrates the regional and temporal influence on NOX isotype expression and the importance of NOX activation in SCI. This information will be useful in future studies of understanding ROS production after injury and therapeutic potentials. PMID:24866054
Cooney, S J; Zhao, Y; Byrnes, K R
NADPH oxidase and reactive oxygen species contribute to alcohol-induced microglial activation and neurodegeneration
Background Activation of microglia causes the production of proinflammatory factors and upregulation of NADPH oxidase (NOX) that form reactive oxygen species (ROS) that lead to neurodegeneration. Previously, we reported that 10 daily doses of ethanol treatment induced innate immune genes in brain. In the present study, we investigate the effects of chronic ethanol on activation of NOX and release of ROS, and their contribution to ethanol neurotoxicity. Methods Male C57BL/6 and NF-?B enhanced GFP mice were treated intragastrically with water or ethanol (5 g/kg, i.g., 25% ethanol w/v) daily for 10 days. The effects of chronic ethanol on cell death markers (activated caspase-3 and Fluoro-Jade B), microglial morphology, NOX, ROS and NF-?B were examined using real-time PCR, immunohistochemistry and hydroethidine histochemistry. Also, Fluoro-Jade B staining and NOX gp91phox immunohistochemistry were performed in the orbitofrontal cortex (OFC) of human postmortem alcoholic brain and human moderate drinking control brain. Results Ethanol treatment of C57BL/6 mice showed increased markers of neuronal death: activated caspase-3 and Fluoro-Jade B positive staining with Neu-N (a neuronal marker) labeling in cortex and dentate gyrus. The OFC of human post-mortem alcoholic brain also showed significantly more Fluoro-Jade B positive cells colocalized with Neu-N, a neuronal marker, compared to the OFC of human moderate drinking control brain, suggesting increased neuronal death in the OFC of human alcoholic brain. Iba1 and GFAP immunohistochemistry showed activated morphology of microglia and astrocytes in ethanol-treated mouse brain. Ethanol treatment increased NF-?B transcription and increased NOX gp91phox at 24 hr after the last ethanol treatment that remained elevated at 1 week. The OFC of human postmortem alcoholic brain also had significant increases in the number of gp91phox + immunoreactive (IR) cells that are colocalized with neuronal, microglial and astrocyte markers. In mouse brain ethanol increased gp91phox expression coincided with increased production of O2- and O2- - derived oxidants. Diphenyleneiodonium (DPI), a NOX inhibitor, reduced markers of neurodegeneration, ROS and microglial activation. Conclusions Ethanol activation of microglia and astrocytes, induction of NOX and production of ROS contribute to chronic ethanol-induced neurotoxicity. NOX-ROS and NF-?B signaling pathways play important roles in chronic ethanol-induced neuroinflammation and neurodegeneration.
Inhibition of NADPH Oxidase by Apocynin Attenuates Progression of Atherosclerosis
Of the multiple sources of reactive oxygen species (ROS) in the blood vessel, NADPH oxidases are the primary source. Whereas several studies have implicated NADPH oxidases in the initiation of atherosclerosis, their roles in disease progression are incompletely understood. Our objective was to determine the potential clinical relevance of inhibiting NADPH oxidase in established atherosclerosis. Using a hypercholesteremic murine model of atherosclerosis (ApoE?/?/LDLR?/? (AS) mice on normal chow diet), we first established a time-dependent relationship between superoxide levels and lesion size in AS mice. Next, we identified NADPH oxidase as the primary source of ROS in atherosclerotic lesions. Treatment of aortic segments from AS mice with apocynin, which interferes with NADPH oxidase activation in part by preventing translocation of the subunit p47phox, significantly reduced superoxide levels. Moreover, addition of apocynin to the drinking water of AS mice produced a decrease in lesion size as compared to untreated AS mice, with the effect most pronounced in the thoracoabdominal aorta but absent from the aortic arch. Granulocyte function in AS+apocynin mice was suppressed, confirming efficacy of apocynin treatment. We conclude that apocynin attenuates the progression of atherosclerosis in hypercholesterolemic mice, potentially by its ability to inhibit generation of superoxide by NADPH oxidase.
Kinkade, Kara; Streeter, Jennifer; Miller, Francis J.
NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats
Oxidative stress is one of the causes of cardiomyopathy. In the present study, NecroXs, novel class of mitochondrial ROS/RNS scavengers, were evaluated for cardioprotection in in vitro and in vivo model, and the putative mechanism of the cardioprotection of NecroX-7 was investigated by global gene expression profiling and subsequent biochemical analysis. NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced death of H9C2 rat cardiomyocytes at EC{sub 50} = 0.057 ?M. In doxorubicin (DOX)-induced cardiomyopathy in rats, NecroX-7 significantly reduced the plasma levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) which were increased by DOX treatment (p < 0.05). Microarray analysis revealed that 21 genes differentially expressed in tBHP-treated H9C2 cells were involved in �Production of reactive oxygen species� (p = 0.022), and they were resolved by concurrent NecroX-7 treatment. Gene-to-gene networking also identified that NecroX-7 relieved cell death through Ncf1/p47phox and Rac2 modulation. In subsequent biochemical analysis, NecroX-7 inhibited NADPH oxidase (NOX) activity by 53.3% (p < 0.001). These findings demonstrate that NecroX-7, in part, provides substantial protection of cardiomyopathy induced by tBHP or DOX via NOX-mediated cell death. -- Highlights: ? NecroX-7 prevented tert-butyl hydroperoxide-induced in vitro cardiac cell death. ? NecroX-7 ameliorated doxorubicin-induced in vivo cardiomyopathy. ? NecroX-7 prevented oxidative stress and necrosis-enriched transcriptional changes. ? NecroX-7 effectively inhibited NADPH oxidase activation. ? Cardioprotection of Necro-7 was brought on by modulation of NADPH oxidase activity.
Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of)] [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Park, Ji-hoon [Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of)] [Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of)] [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Kim, Soon Ha, E-mail: firstname.lastname@example.com [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of)
Nox NADPH oxidases and the endoplasmic reticulum.
Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between Noxes and the ER may provide relevant insights in Nox-related (patho)physiology. Antioxid. Redox Signal. 20, 2755-2775. PMID:24386930
Laurindo, Francisco R M; Araujo, Tha�s L S; Abrah�o, Thalita B
Kaempferol modulates pro-inflammatory NF-?B activation by suppressing advanced glycation endproducts-induced NADPH oxidase
Advanced glycation endproducts (AGE) are oxidative products formed from the reaction between carbohydrates and a free amino group of proteins that are provoked by reactive species (RS). It is also known that AGE enhance the generation of RS and that the binding of AGE to a specific AGE receptor (RAGE) induces the activation of the redox-sensitive, pro-inflammatory transcription factor, nuclear factor-kappa B (NF-?B). In this current study, we investigated the anti-oxidative effects of short-term kaempferol supplementation on the age-related formation of AGE and the binding activity of RAGE in aged rat kidney. We further investigated the suppressive action of kaempferol against AGE's ability to stimulate activation of pro-inflammatory NF-?B and its molecular mechanisms. For this study, we utilized young (6�months old), old (24�months old), and kaempferol-fed (2 and 4�mg/kg/day for 10�days) old rats. In addition, for the molecular work, the rat endothelial cell line, YPEN-1 was used. The results show that AGE and RAGE were increased during aging and that these increases were blunted by kaempferol. In addition, dietary kaempferol reduced age-related increases in NF-?B activity and NF-?B-dependant pro-inflammatory gene activity. The most significant new finding from this study is that kaempferol supplementation prevented age-related NF-?B activation by suppressing AGE-induced nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). Taken together, our results demonstrated that dietary kaempferol exerts its anti-oxidative and anti-inflammatory actions by modulating the age-related NF-?B signaling cascade and its pro-inflammatory genes by suppressing AGE-induced NADPH oxidase activation. Based on these data, dietary kaempferol is proposed as a possible anti-AGE agent that may have the potential for use in anti-inflammation therapies.
Kim, Ji Min; Lee, Eun Kyeong; Kim, Dae Hyun; Yu, Byung Pal
Siglec-E Promotes ?2-Integrin-dependent NADPH Oxidase Activation to Suppress Neutrophil Recruitment to the Lung*
Siglec-E is a sialic acid-binding Ig-like lectin expressed on murine myeloid cells. It has recently been shown to function as a negative regulator of ?2-integrin-dependent neutrophil recruitment to the lung following exposure to lipopolysaccharide (LPS). Here, we demonstrate that siglec-E promoted neutrophil production of reactive oxygen species (ROS) following CD11b ?2-integrin ligation with fibrinogen in a sialic acid-dependent manner, but it had no effect on ROS triggered by a variety of other stimulants. Siglec-E promotion of ROS was likely mediated via Akt activation, because siglec-E-deficient neutrophils plated on fibrinogen exhibited reduced phosphorylation of Akt, and the Akt inhibitor, MK2206, blocked fibrinogen-induced ROS. In vivo imaging showed that siglec-E also promoted ROS in acutely inflamed lungs following exposure of mice to LPS. Importantly, siglec-E-promoted ROS were required for its inhibitory function, as the NADPH oxidase inhibitor, apocynin, reversed the siglec-E-mediated suppression of neutrophil recruitment and blocked neutrophil ROS production in vitro. Taken together, these results demonstrate that siglec-E functions as an inhibitory receptor of neutrophils via positive regulation of NADPH oxidase activation and ROS production. Our findings have implications for the inhibitory role of siglec-9 on human neutrophils in sepsis and acute lung injury.
McMillan, Sarah J.; Sharma, Ritu S.; Richards, Hannah E.; Hegde, Vikas; Crocker, Paul R.
Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease.
Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease (SCD). One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle red blood cells (RBC). Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. In this study, we demonstrate that a significant part of ROS production in sickle cells is mediated enzymatically by NADPH oxidase, which is regulated by protein kinase C, Rac GTPase, and intracellular Ca(2+) signaling within the sickle RBC. Moreover, plasma from patients with SCD and isolated cytokines, such as transforming growth factor ?1 and endothelin-1, enhance RBC NADPH oxidase activity and increase ROS generation. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte rigidity and fragility in SCD. Erythrocyte ROS generation, hemolysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive-feedback loop to drive the pathophysiology of sickle cell disease. These findings suggest a novel pathogenic mechanism in SCD and may offer new therapeutic targets to counteract inflammation and RBC rigidity and fragility in SCD. PMID:23349388
George, Alex; Pushkaran, Suvarnamala; Konstantinidis, Diamantis G; Koochaki, Sebastian; Malik, Punam; Mohandas, Narla; Zheng, Yi; Joiner, Clinton H; Kalfa, Theodosia A
The cytosolic subunit p67phox of the NADPH-oxidase complex does not bind NADPH.
The NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b558 and four cytosolic proteins; p67phox, p47phox, p40phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b558. However, a number of studies have suggested the presence of another site on the p67phox subunit which is the key activating component. Using several approaches like tryptophan quenching fluorescence measurement, inhibition by 2',3'-dialdehyde NADPH, and free/bound NADPH concentration measurements, we demonstrate that no NADPH binds on p67phox, thus definitively solving the controversy on the number and location of the NADPH-binding sites on this complex. PMID:19751728
Baciou, Laura; Erard, Marie; Dagher, Marie-Claire; Bizouarn, Tania
p67phox terminates the phospholipase A2-derived signal for activation of NADPH oxidase (NOX2)
The phospholipase A2 (PLA2)activity of phosphorylated peroxiredoxin 6 (Prdx6) is required for activation of NADPH oxidase (NOX2). We investigated the interaction of Prdx6 with p67phox and its effect on NOX2 activity. With the use of specific antibodies, coimmunoprecipitation of p67phox and phosphorylated Prdx6 was demonstrated with lysates of mouse pulmonary microvascular endothelial cells (MPMVECs) that were stimulated with angiotensin II; the interaction of p67phox with nonphosphorylated Prdx6 was relatively weak. Association of p67phox and phosphoPrdx6 in intact MPMVECs after angiotensin II stimulation was demonstrated by proximity ligation assay and was abolished by U0126, a MAP kinase inhibitor. By isothermal titration calorimetry, p67phox bound strongly to phosphoPrdx6 but bound poorly to Prdx6; phosphorylated p67phox did not bind to either Prdx6 or phosphoPrdx6. PLA2 activity of recombinant phosphoPrdx6 was decreased by >98% in the presence of p67phox; the calculated dissociation constant (Kd) of the p67phox: phosphoPrdx6 complex was 65 nM. PLA2 activity (MJ33 sensitive) in cell lysates following angiotensin II treatment of MPMVECs was increased by 85% following knockdown of p67phox with siRNA. These data indicate that p67phox binds to phosphoPrdx6 and inhibits its PLA2 activity, an interaction that could function to terminate the PLA2-mediated NOX2 activation signal.�Krishnaiah, S. Y., Dodia, C., Feinstein, S. I., and Fisher, A. B. p67phox terminates the phospholipase A2-derived signal for activation of NADPH oxidase (NOX2).
Krishnaiah, Saikumari Y.; Dodia, Chandra; Feinstein, Sheldon I.; Fisher, Aron B.
Reactive Oxygen Intermediate-Dependent NF-kB Activation by Interleukin1b Requires 5Lipoxygenase or NADPH Oxidase Activity
We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells.
p40(phox) Participates in the activation of NADPH oxidase by increasing the affinity of p47(phox) for flavocytochrome b(558).
NADPH oxidase is one of the major components of the innate immune system and is used by phagocytes to generate microbicidal reactive oxygen species. Activation of the enzyme requires the participation of a minimum of five proteins, p22(phox), gp91(phox) (together forming flavocytochrome b(558)), p47(phox), p67(phox) and the GTP-binding protein, Rac2. A sixth protein, p40(phox), has been implicated in the control of the activity of NADPH oxidase principally based on its sequence homology to, and physical association with, other phox components, and also the observation that it is phosphorylated during neutrophil activation. However, to date its role in regulating the activity of the enzyme has remained obscure, with evidence for both positive and negative influences on oxidase activity having being reported. Data are presented here using the cell-free system for NADPH oxidase activation that shows that p40(phox) can function to promote oxidase activation by increasing the affinity of p47(phox) for the enzyme approx. 3-fold. PMID:10861218
Cross, A R
Adverse Cognitive Effects of High-Fat Diet in a Murine Model of Sleep Apnea Are Mediated by NADPH Oxidase Activity
Intermittent hypoxia (IH) during sleep, such as occurs in sleep apnea (SA), induces increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and deficits in hippocampal learning and memory. Similar to IH, high fat-refined carbohydrate diet (HFD), a frequent occurrence in patients with SA, can also induce similar oxidative stress and cognitive deficits under normoxic conditions, suggesting that excessive NADPH oxidase activity may underlie central nervous system (CNS) dysfunction in both conditions. The effect of HFD and IH during the light period on two forms of spatial learning in the water maze as well as on markers of oxidative stress was assessed in male mice lacking NADPH oxidase activity (gp91phox-/Y) and wild-type littermates fed HFD. On a standard place training task, gp91phox_/Y displayed normal learning, and was protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to HFD and IH as compared to controls, while no changes emerged in gp91phox_/Y mice. Additionally, wild-type mice, but not gp91phox_/Y mice, had significantly elevated levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampal lysates following IH-HFD exposures. The cognitive deficits of obesity and westernized diets and those of sleep disorders that are characterized by IH during sleep are both mediated, at least in part, by excessive NADPH oxidase activity.
Nair, D.; Ramesh, V.; Gozal, D.
NADPH oxidase and apoptosis in cerulein-stimulated pancreatic acinar AR42J cells
Apoptosis linked to oxidative stress has been implicated in pancreatitis. We investigated whether NADPH oxidase mediates apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. We report here that cerulein treatment resulted in the activation of NADPH oxidase, as determined by ROS production, translocation of cytosolic subunits p47phox and p67phox to the membrane, and interaction between NADPH oxidase subunits. Cerulein induced Ca2+
Ji Hoon Yu; Joo Weon Lim; Kyung Hwan Kim; Tomohiro Morio; Hyeyoung Kim
NADPH oxidases: novel therapeutic targets for neurodegenerative diseases
Oxidative stress is a key pathologic factor in neurodegenerative diseases, such as Alzheimer�s and Parkinson�s diseases. The failure of free-radical-scavenging antioxidants in clinical trials pinpoints an urgent need to identify and to block major sources of oxidative stress in neurodegenerative diseases. As a major superoxide-producing enzyme complex in activated phagocytes, phagocytic NADPH oxidase (PHOX) is essential for host defense. However, recent preclinical evidence has underscored a pivotal role of over-activated PHOX in chronic neuroinflammation and progressive neurodegeneration. Deficiency in PHOX subunits mitigates neuronal damage induced by diverse insults/stresses relevant to neurodegenerative diseases. More importantly, the suppression of PHOX activity correlates with less neuronal impairment in models of neurodegenerative diseases. The discovery of PHOX and non-phagocytic NADPH oxidases in astroglia and neurons further reinforces the critical role of NADPH oxidases in oxidative stress-mediated chronic neurodegeneration. Thus, proper modulation of NADPH oxidase activity might hold therapeutic potential for currently incurable neurodegenerative diseases.
Gao, Hui-Ming; Zhou, Hui; Hong, Jau-Shyong
Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer's Disease
Alzheimer�s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (A?) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by A?. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of A? in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A2 (PLA2) and secretory PLA2. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving A? NADPH oxidase and PLA2 can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.
Simonyi, Agnes; He, Yan; Sheng, Wenwen; Sun, Albert Y.; Wood, W. Gibson; Weisman, Gary A.
A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase*
The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91phox requires the cytosolic proteins p67phox, p47phox, and Rac (a small GTPase). p67phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47phox and there interacts with Rac; these processes are prerequisite for gp91phox activation. Here we show that a region of p67phox (amino acids 190�200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67phox to support superoxide production by gp91phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67phox interaction with the gp91phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67phox (amino acids 190�210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91phox.
Maehara, Yuichi; Miyano, Kei; Yuzawa, Satoru; Akimoto, Risa; Takeya, Ryu; Sumimoto, Hideki
The mechanism of activation of NADPH oxidase in the cell-free system: the activation process is primarily catalytic and not through the formation of a stoichiometric complex.
It is commonly assumed that activation of the superoxide-generating NADPH oxidase requires the formation of a stable complex between flavocytochrome b-245 (the gp91phox/p22phox heterodimer) and the cytosolic cofactors p47phox, p67phox and Rac2. This association is thought to convert flavocytochrome b-245, which contains the NADPH-binding site, flavin and haem centres, from an inactive into an active state. Here we provide evidence that, in the cell-free system, this activation process does not necessarily require the formation of a stable stoichiometric complex between the phox proteins. To explain this data we propose the hypothesis that p67phox (and possibly Rac2), are capable of activating flavocytochrome b-245 in a catalytic fashion, where a single molecule of p67phox (or Rac2) is capable of activating multiple flavocytochrome b-245 molecules. PMID:10393079
Cross, A R; Erickson, R W; Curnutte, J T
Characterization of membrane-localized and cytosolic Rac-GTPase-activating proteins in human neutrophil granulocytes: contribution to the regulation of NADPH oxidase.
We have investigated the intracellular localization and molecular identity of Rac-GTPase-activating proteins (Rac-GAPs) in human neutrophils. Immunoblot analysis detected the presence of both p190RhoGAP and Bcr mainly in the cytosol. An overlay assay performed with [gamma-(32)P]GTP-bound Rac revealed dominant GAP activity related to a 50 kDa protein both in the membrane and cytosol. This activity could be identified by Western blotting and immunoprecipitation with specific antibody directed against the GAP domain of p50RhoGAP. Using a semirecombinant or fully purified cell-free activation assay of the Rac-activated enzyme NADPH oxidase, we demonstrated the regulatory effect of both the membrane-localized and soluble GAPs. We suggest that in neutrophil granulocytes Rac-GAPs have redundant function and represent suitable targets for both the up-regulation and down-regulation of the NADPH oxidase.
Geiszt, M; Dagher, M C; Molnar, G; Havasi, A; Faure, J; Paclet, M H; Morel, F; Ligeti, E
Perturbation of actin dynamics induces NF-?B activation in myelomonocytic cells through an NADPH oxidase-dependent pathway
Although several reports showed the effect of compounds disrupting microtubules on NF-?B (nuclear factor ?B) activation, nothing is known about agents perturbing actin dynamics. In the present study, we have shown that actin cytoskeleton disruption induced by actin-depolymerizing agents such as cytochalasin D and latrunculin B and actin-polymerizing compounds such as jasplakinolide induced NF-?B activation in myelomonocytic cells. The transduction pathway involved the I?B (inhibitory ?B) kinase complex and a degradation of I?B?. We have shown that NF-?B activation in response to the perturbation of actin dynamics required reactive oxygen species, as demonstrated by the effect of antioxidants. Actin cytoskeleton disruption by cytochalasin D induced O2? release from human monocytes, through the activation of the NADPH oxidase, as confirmed by the phosphorylation and by the membrane translocation of p47phox. NF-?B activation after actin cytoskeleton disruption could be physiologically relevant during monocyte activation and/or recruitment into injured tissues, where cellular attachment, migration and phagocytosis result in cyclic shifts in cytoskeletal organization and disorganization.
Noxa1 as a moderate activator of Nox2-based NADPH oxidase.
Noxa1 was discovered as an activating factor for Nox1, an O(2)(-)-generating enzyme. Subsequent studies have shown that Noxa1 is colocalized with Nox2 in several cell types, including vascular cells. Nox2 activation by Noxa1 has been examined in reconstituted model cells. However, little is known about the kinetic properties of Noxa1 in Nox2 activation. In the present study, we used purified cyt.b(558) (Nox2 plus p22(phox)), Rac(Q61L), and Noxo1 to examine the ability of Noxa1 to activate Nox2. In the pure reconstitution system, Noxa1 activated Nox2 with lower efficiency than p67(phox), a canonical activator of Nox2. The EC(50) value of Noxa1 was considerably higher than that of p67(phox). The V(max) value with Noxa1 and Noxo1 was one-third of that with p67(phox) and p47(phox). The EC(50) value of Noxo1 or Rac(Q61L) was also higher when Noxa1 was used. The affinity of FAD for the oxidase and the stability of the active complex were remarkably low when Noxa1 and Noxo1 were used compared with p67(phox) and p47(phox). The stability was not improved by fusion of Noxa1 with Rac(Q61L). These findings show that Noxa1 has quite different kinetic properties from p67(phox) and suggest that Noxa1 may function as a moderate activator of Nox2. PMID:22244833
Kawano, Masahito; Miyamoto, Kazuhiro; Kaito, Yuki; Sumimoto, Hideki; Tamura, Minoru
Neutral Sphingomyelinase Activation Precedes NADPH Oxidase-Dependent Damage in Neurons Exposed to the Proinflammatory Cytokine TNF?
Inflammation accompanied by severe oxidative stress plays a vital role in the orchestration and progression of neurodegeneration prevalent in chronic and acute CNS pathologies as well as in aging. The proinflammatory cytokine tumor necrosis factor alpha (TNF?) elicits the formation of the bioactive ceramide by stimulating the hydrolysis of the membrane lipid sphingomyelin by sphingomyelinase activities. Ceramide stimulates the formation of reactive oxygen species (ROS) and apoptotic mechanisms in both neurons and non-neuronal cells establishing a link between sphingolipid metabolism and oxidative stress. We demonstrated in SH-SY5Y human neuroblastoma cells and primary cortical neurons, that TNF? is a potent stimulator of Mg2+-dependent neutral sphingomyelinase (Mg2+-nSMase) activity and sphingomyelin hydrolysis rather than de novo synthesis, was the predominant source of ceramide increases. Mg2+-nSMase activity preceded an accumulation of ROS by a neuronal NADPH oxidase (NOX). Notably, TNF? provoked a NOX-dependent oxidative damage to sphingosine kinase-1, which generates sphingosine-1-phosphate, a ceramide metabolite associated with neurite outgrowth. Indeed, ceramide and ROS inhibited neurite outgrowth of DRG neurons by disrupting growth cone motility. Blunting ceramide and ROS formation both rescued sphingosine kinase-1 activity and neurite outgrowth. Our studies suggest that TNF?-mediated activation of Mg2+-nSMase and NOX in neuronal cells not only produced the neurotoxic intermediates ceramide and ROS but also directly antagonized neuronal survival mechanisms thus accelerating neurodegeneration.
Barth, Brian M.; Gustafson, Sally J.; Kuhn, Thomas B.
NADPH oxidase mediates interleukin-6 expression in cerulein-stimulated pancreatic acinar cells
NADPH oxidase produces a large amount of reactive oxygen species (ROS) mainly in phagocytic cells. ROS are involved in NF-?B activation, cytokine expression and thus, pathogenesis of pancreatitis. However, the source of ROS in pancreatic acinar cells has not been clarified. Cerulein rapidly induces acute and edematous form of pancreatitis. We investigated whether pancreatic acinar cells contain NADPH oxidase, and
Ji Hoon Yu; Joo Weon Lim; Hyeyoung Kim; Kyung Hwan Kim
Reduction of NADPH-Oxidase Activity Ameliorates the Cardiovascular Phenotype in a Mouse Model of Williams-Beuren Syndrome
A hallmark feature of Williams-Beuren Syndrome (WBS) is a generalized arteriopathy due to elastin deficiency, presenting as stenoses of medium and large arteries and leading to hypertension and other cardiovascular complications. Deletion of a functional NCF1 gene copy has been shown to protect a proportion of WBS patients against hypertension, likely through reduced NADPH-oxidase (NOX)�mediated oxidative stress. DD mice, carrying
Victoria Campuzano; Maria Segura-Puimedon; Verena Terrado; Carolina S�nchez-Rodr�guez; Mathilde Coustets; Mauricio Menacho-M�rquez; Juli�n Nevado; Xos� R. Bustelo; Uta Francke; Luis A. P�rez-Jurado
Modulation of cellular redox status by thiamine-activated NADPH oxidase confers Arabidopsis resistance to Sclerotinia sclerotiorum.
Sclerotinia sclerotiorum can initially suppress host oxidative burst to aid infection establishment, but later promotes reactive oxygen species (ROS) generation as proliferation advances. Here, it was shown that the cellular redox status can be modulated by thiamine to protect Arabidopsis thaliana against Sclerotinia at the early stages of infection. The initial inhibition of host ROS generation by Sclerotinia-secreted oxalate could effectively be alleviated by thiamine. Thiamine pre-treatment and subsequent wild-type Sclerotinia invasion induced an increase of ascorbate peroxidase activity concomitant with decreased ascorbate/dehydroascorbate ratios, which led to the cellular transition towards oxidative status in infected tissues. Particularly, it was observed that wild-type Sclerotinia, but not oxalate-deficient A2 mutant, could suppress the activity of NADPH oxidase (NOX), which might be an important mechanism underlying the early inhibition of ROS burst. Nevertheless, thiamine pre-treatment followed by wild-type Sclerotinia infection promoted NOX-derived ROS accumulation. Further studies showed that cytosolic Ca(2+) and staurosporine-sensitive protein kinase(s) participated in thiamine-induced activation of NOX. Moreover, thiamine-induced tissue defence responses including callose/lignin deposition and stomatal closure were closely correlated with NOX-derived ROS generation. Additionally, studies with Brassica species indicated that the regulation of thiamine is largely conserved upon Sclerotinia infection. Collectively, it was concluded that thiamine reverses the initial reducing status through activating NOX-dependent ROS signalling to perturb the disease progress of Sclerotinia. PMID:23814275
Zhou, Jun; Sun, Aizhen; Xing, Da
Real-time imaging of NADPH oxidase activity in living cells using a novel fluorescent protein reporter.
Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47(phox), we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47(phox) deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors. PMID:23704967
Pal, Rituraj; Basu Thakur, Poulami; Li, Shumin; Minard, Charles; Rodney, George G
Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter
Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47phox, we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47phox deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors.
Pal, Rituraj; Basu Thakur, Poulami; Li, Shumin; Minard, Charles; Rodney, George G.
NADPH oxidase in stroke and cerebrovascular disease.
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) was originally identified in immune cells as playing an important microbicidal role. In stroke and cerebrovascular disease, inflammation is increasingly being recognized as contributing negatively to neurological outcome, with NOX as an important source of superoxide. Several labs have now shown that blocking or deleting NOX in the experimental stroke models protects from brain ischemia. Recent work has implicated glucose as an important NOX substrate leading to reperfusion injury, and that NOX inhibition can improve the detrimental effects of hyperglycemia on stroke. NOX inhibition also appears to ameliorate complications of thrombolytic therapy by reducing blood-brain barrier disruption, edema formation, and hemorrhage. Further, NOX from circulating inflammatory cells seems to contribute more to ischemic injury more than NOX generated from endogenous brain residential cells. Several pharmacological inhibitors of NOX are now available. Thus, blocking NOX activation may prove to be a promising treatment for stroke as well as an adjunctive agent to prevent its secondary complications. PMID:22643077
Tang, Xian Nan; Cairns, Belinda; Kim, Jong Youl; Yenari, Midori A
HaCaT Keratinocytes Overexpressing the S100 Proteins S100A8 and S100A9 Show Increased NADPH Oxidase and NF-?B Activities
The calcium- and arachidonic acid (AA)-binding proteins S100A8 and S100A9 are involved in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in phagocytes. They are specifically expressed in myeloid cells, and are also found in epithelial cells in various (patho)physiological conditions. We have investigated the consequences of S100A8\\/A9 overexpression in epithelial cell lines on reactive oxygen species (ROS) generation and downstream
Malgorzata Benedyk; Claudia Sopalla; Wolfgang Nacken; G�nther Bode; Harut Melkonyan; Botond Banfi; Claus Kerkhoff
Antioxidant Effects of Vitamins C and E Are Associated With Altered Activation of Vascular NADPH Oxidase and Superoxide Dismutase in Stroke-Prone SHR
Ascorbic acid (vitamin C) and a-tocopherol (vitamin E) have antioxidant properties that could improve redox-sensitive vascular changes associated with hypertension. We determined whether vitamins C and E influence vascular function and structure in hypertension by modulating activity of NADPH oxidase and superoxide dismutase (SOD). Adult stroke-prone spontaneously hypertensive rats (SHRSP) were divided into 3 groups: control (C; n56), vitamin C-treated
Xin Chen; Rhian M. Touyz; Jeong Bae Park; Ernesto L. Schiffrin
p21-Activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes.
Ischemic conditions reduce the activity of the p21-activated kinase (Pak1) resulting in increased arrhythmic activity. Triggered arrhythmic activity during ischemia is based on changes in cellular ionic balance and the cells Ca(2+) handling properties. In the current study we used isolated mouse ventricular myocytes (VMs) deficient for the expression of Pak1 (Pak1(-/-)) to determine the mechanism by which Pak1 influences the generation of arrhythmic activity during simulated ischemia. The Ca(2+) transient amplitude and kinetics did not significantly change in wild type (WT) and Pak1(-/-) VMs during 15 min of simulated ischemia. However, Pak1(-/-) VMs exhibited an exaggerated increase in [Ca(2+)]i, which resulted in spontaneous Ca(2+) release events and waves. The Ca(2+) overload in Pak1(-/-) VMs could be suppressed with a reverse mode blocker (KB-R7943) of the sodium calcium exchanger (NCX), a cytoplasmic scavenger of reactive oxygen species (ROS; TEMPOL) or a RAC1 inhibitor (NSC23766). Measurements of the cytoplasmic ROS levels revealed that decreased Pak1 activity in Pak1(-/-) VMs or VMs treated with the Pak1 inhibitor (IPA3) enhanced cellular ROS production. The Pak1 dependent increase in ROS was attenuated in VMs deficient for NADPH oxidase 2 (NOX2; p47(phox-/-)) or in VMs where NOX2 was inhibited (gp91ds-tat). Voltage clamp recordings showed increased NCX activity in Pak1(-/-) VMs that depended on enhanced NOX2 induced ROS production. The exaggerated Ca(2+) overload in Pak1(-/-) VMs could be mimicked by low concentrations of ouabain. Overall our data show that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose VMs to Ca(2+) overload under conditions where no significant changes in excitation-contraction coupling are yet evident. PMID:24380729
DeSantiago, Jaime; Bare, Dan J; Xiao, Lei; Ke, Yunbo; Solaro, R John; Banach, Kathrin
Khz (fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating JNK and NADPH oxidase-dependent generation of reactive oxygen species.
Khz is a compound derived from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia that inhibits the growth of cancer cells. The results of the present study show that Khz induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz induced apoptosis by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)) and activating JNK to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-induced apoptosis was caspase-dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the translocation of regulatory subunits p47(phox) and p67(phox) to the cell membrane and was necessary for ROS generation by Khz. Khz triggered a rapid and sustained increase in [Ca(2+)](i), which activated JNK. JNK plays a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47(phox) and p67(phox) subunits and ROS generation. In summary, these data indicate that Khz preferentially induces apoptosis in cancer cells, and the signaling mechanisms involve an increase in [Ca(2+)](i), JNK activation, and ROS generation via NADPH oxidase and mitochondria. PMID:23056263
Kim, Tae Hwan; Kim, Ju sung; Kim, Zoo haye; Huang, Ren Bin; Wang, Ren Sheng
A Novel Nontoxic Inhibitor of the Activation of NADPH Oxidase Reduces Reactive Oxygen Species Production in Mouse LungS?
1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) is a fluorinated phospholipid analog that inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6). Prdx6 PLA2 activity is required for activation of NADPH oxidase 2 and subsequent generation of reactive oxygen species (ROS). In vitro, MJ33 inhibited agonist-stimulated production of ROS by the isolated perfused mouse lung, lung microvascular endothelial cells, and polymorphonuclear leukocytes. MJ33 (0.02�0.5 �mol MJ33/kg body weight) in mixed unilamellar liposomes was administered to C57BL/6 mice by either intratracheal (i.t.) or i.v. routes. Lung MJ33 content, measured by liquid chromatography/mass spectroscopy, showed uptake of 67�87% of the injected dose for i.t. and 23�42% for i.v. administration at 4 hours postinjection. PLA2 activity of lung homogenates was markedly inhibited (>85%) at 4 hours postadministration. Both MJ33 content and PLA2 activity gradually returned to near control levels over the subsequent 24�72 hours. Mice treated with MJ33 at 12.5�25 �mol/kg did not show changes (compared with control) in clinical symptomatology, body weight, hematocrit, and histology of lung, liver, and kidney during a 30- to 50-day observation period. Thus, the toxic dose of MJ33 was >25 �mol/kg, whereas the PLA2 inhibitory dose was approximately 0.02 �mol/kg, indicating a high margin of safety. MJ33 administered to mice prior to lung isolation markedly reduced ROS production and tissue lipid and protein oxidation during ischemia followed by reperfusion. Thus, MJ33 could be useful as a therapeutic agent to prevent ROS-mediated tissue injury associated with lung inflammation or in harvested lungs prior to transplantation.
Lee, Intae; Dodia, Chandra; Chatterjee, Shampa; Zagorski, John; Mesaros, Clementina; Blair, Ian A.; Feinstein, Sheldon I.; Jain, Mahendra
A novel nontoxic inhibitor of the activation of NADPH oxidase reduces reactive oxygen species production in mouse lung.
1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) is a fluorinated phospholipid analog that inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6). Prdx6 PLA2 activity is required for activation of NADPH oxidase 2 and subsequent generation of reactive oxygen species (ROS). In vitro, MJ33 inhibited agonist-stimulated production of ROS by the isolated perfused mouse lung, lung microvascular endothelial cells, and polymorphonuclear leukocytes. MJ33 (0.02-0.5 �mol MJ33/kg body weight) in mixed unilamellar liposomes was administered to C57BL/6 mice by either intratracheal (i.t.) or i.v. routes. Lung MJ33 content, measured by liquid chromatography/mass spectroscopy, showed uptake of 67-87% of the injected dose for i.t. and 23-42% for i.v. administration at 4 hours postinjection. PLA2 activity of lung homogenates was markedly inhibited (>85%) at 4 hours postadministration. Both MJ33 content and PLA2 activity gradually returned to near control levels over the subsequent 24-72 hours. Mice treated with MJ33 at 12.5-25 �mol/kg did not show changes (compared with control) in clinical symptomatology, body weight, hematocrit, and histology of lung, liver, and kidney during a 30- to 50-day observation period. Thus, the toxic dose of MJ33 was >25 �mol/kg, whereas the PLA2 inhibitory dose was approximately 0.02 �mol/kg, indicating a high margin of safety. MJ33 administered to mice prior to lung isolation markedly reduced ROS production and tissue lipid and protein oxidation during ischemia followed by reperfusion. Thus, MJ33 could be useful as a therapeutic agent to prevent ROS-mediated tissue injury associated with lung inflammation or in harvested lungs prior to transplantation. PMID:23475902
Lee, Intae; Dodia, Chandra; Chatterjee, Shampa; Zagorski, John; Mesaros, Clementina; Blair, Ian A; Feinstein, Sheldon I; Jain, Mahendra; Fisher, Aron B
The cytosolic subunit p67phox contains an NADPH-binding site that participates in catalysis by the leukocyte NADPH oxidase.
The NADPH-dependent respiratory burst oxidase of human neutrophils catalyzes the reduction of oxygen to superoxide using NADPH as the electron donor and is essential for normal host defenses. To gain insight into the function of the various oxidase subunits that are required for the full expression of catalytic activity, we studied the interactions between the 2',3'-dialdehyde derivative of NADPH (NADPH dialdehyde) and neutrophil cytosol. NADPH dialdehyde treatment of cytosol resulted in the loss of the ability of the cytosol to participate in cell-free oxidase activation; this inactivation was blocked by NADPH but not by NAD, NADP, or GTP. Partial purification of neutrophil cytosol yielded a single peak which could restore the activity lost in cytosol treated with NADPH dialdehyde. This peak contained p67phox but not p47phox or Rac2. Purified recombinant p67phox was similarly able to restore the activity lost in NADPH dialdehyde-treated cytosol and bound [32P]NADPH dialdehyde in a specific fashion. The activity of recombinant p67phox in cell-free oxidase assays was lost on treatment with NADPH dialdehyde. Together, these data suggest p67phox contains the catalytic NADPH-binding site of the leukocyte NADPH oxidase. PMID:8770870
Smith, R M; Connor, J A; Chen, L M; Babior, B M
Regulation of swelling-activated Cl? current by angiotensin II signalling and NADPH oxidase in rabbit ventricle
Aims We assessed whether hypoosmotic swelling of cardiac myocytes activates volume-sensitive Cl? current (ICl,swell) via the angiotensin II (AngII)-reactive oxygen species (ROS) signalling cascade. The AngII-ROS pathway previously was shown to elicit ICl,swell upon mechanical stretch of ?1D integrin. Integrin stretch and osmotic swelling are, however, distinct stimuli. For example, blocking Src kinases stimulates swelling-induced but inhibits stretch-induced ICl,swell. Methods and results ICl,swell was measured in rabbit ventricular myocytes by whole-cell voltage clamp. Swelling-induced ICl,swell was completely blocked by losartan and eprosartan, AngII type I receptor (AT1) antagonists. AT1 stimulation transactivates epidermal growth factor receptor (EGFR) kinase. Blockade of EGFR kinase with AG1478 abolished both ICl,swell and AngII-induced Cl? current, whereas exogenous EGF evoked a Cl? current that was suppressed by osmotic shrinkage. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase, and PI-3K inhibitors LY294002 and wortmannin blocked ICl,swell. Ultimately, AngII signals via NADPH oxidase (NOX) and superoxide anion, �O2?. NOX inhibitors, diphenyleneiodonium, apocynin and gp91ds-tat, eliminated ICl,swell, whereas scramb-tat, an inactive gp91ds-tat analogue, was ineffective. �O2? rapidly dismutates to H2O2. Consistent with H2O2 being a downstream effector, catalase inhibited ICl,swell, and exogenous H2O2 overcame suppression of ICl,swell by AT1 receptor, EGFR kinase, and PI-3K blockers. H2O2-induced current was not blocked by osmotic shrinkage, however. Conclusion Activation of ICl,swell by osmotic swelling is controlled by the AngII-ROS cascade, the same pathway previously implicated in ICl,swell activation by integrin stretch. This in part explains why ICl,swell is persistently activated in several models of cardiac disease.
Ren, Zuojun; Raucci, Frank J.; Browe, David M.; Baumgarten, Clive M.
NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-?-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells.
TNF-? plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-? in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-?-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-? induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-?-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-l-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-?B (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47(phox), p42, p38, JNK1, p65, or PYK2. Moreover, TNF-? markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-?-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-?B (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-?-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-?-stimulated MAPKs and NF-?B activation. Thus, in H9c2 cells, we are the first to show that TNF-?-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-?B cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-?-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-? on H9c2 cells may provide potential therapeutic targets of chronic heart failure. PMID:23774252
Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der
Inhibition of thyroid NADPH-oxidase by 2-iodohexadecanal in a cell-free system.
The major nonpolar iodolipid formed in horse thyroid cells has recently been identified as 2-iodohexadecanal (2-IHDA). We have investigated in vitro the effect of 2-IHDA on the NADPH-oxidase, NADPH-cytochrome c reductase, and thyroid peroxidase (TPO) activities of a porcine thyroid plasma membrane preparation. 2-IHDA inhibited NADPH-oxidase activity, with half-inhibition at 3-5 microM, but it had no effect on NADPH-cytochrome c reductase. It inhibited the TPO-catalyzed iodination of protein, but not iodide oxidation. Hexadecanal also inhibited NADPH-oxidase. Inhibition by the non-iodinated lipid aldehydes depended on the length of their aliphatic chain: dodecanal and tridecanal gave maximal inhibition. Free iodide, 2-iodohexadecanol and palmitic acid all had no inhibitory effect. Washing treated membranes showed that the inhibition of NADPH-oxidase by hexadecanal was fully reversible, whereas that of 2-IHDA and other iodinated or brominated alkanals was irreversible. Thus the interaction between some residues of the thyroid NADPH-oxidase and the lipid aldehyde groups was favored or stabilized by the iodine atom. Modification of primary amine and thiol groups of NADPH-oxidase inhibited its activity. These groups could also be the target of lipid aldehydes. We suggest that 2-IHDA, because it inhibits TPO and more profoundly the H2O2-generating system in thyroid plasma membrane, modulates iodide metabolism in the thyrocyte and may mediate the Wolff-Chaikoff effect. PMID:8187956
Ohayon, R; Boeynaems, J M; Braekman, J C; Van den Bergen, H; Gorin, Y; Virion, A
NADPH Oxidase Activation Is Required in Reactive Oxygen Species Generation and Cell Transformation Induced by Hexavalent Chromium
Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Although overproduction of reactive oxygen species (ROS) has been suggested to play a major role in its carcinogenicity, the mechanisms of Cr(VI)-induced ROS production remain unclear. In this study, we investigated the role of NADPH oxidase (NOX), one of the major sources of cellular ROS, in Cr(VI)-induced oxidative stress and carcinogenesis. We found that short-term exposure to Cr(VI) (2?M) resulted in a rapid increase in ROS generation in Beas-2B cells, and concomitantly increased NOX activity and expression of NOX members (NOX1�3 and NOX5) and subunits (p22phox, p47phox, p40phox, and p67phox). Cr(VI) also induced phosphorylation of p47phox and membrane translocation of p47phox and p67phox, further confirming NOX activation. Knockdown of p47phox with a short hairpin RNA attenuated the ROS production induced by Cr(VI). Chronic exposure (up to 3 months) to low doses of Cr(VI) (0.125, 0.25, and 0.5?M) also promoted ROS generation and the expression of NOX subunits, such as p47phox and p67phox, but inhibited the expression of main antioxidant enzymes, such as superoxidase dismutase (SOD) and glutathione peroxidase (GPx). Chronic Cr(VI) exposure resulted in transformation of Beas-2B cells, increasing cell proliferation, anchorage independent growth in soft agar, and forming aggressive tumors in nude mice. Stable knockdown of p47phox or overexpression of SOD1, SOD2, or catalase (CAT) eliminated Cr(VI)-induced malignant transformation. Our results suggest that NOX plays an important role in Cr(VI)-induced ROS generation and carcinogenesis.
Wang, Xin; Son, Young-Ok; Chang, Qingshan; Sun, Lijuan; Hitron, J.Andrew; Budhraja, Amit; Zhang, Zhuo; Ke, Zunji; Chen, Fei; Luo, Jia; Shi, Xianglin
Nadph oxidase regulates alveolar epithelial sodium channel activity and lung fluid balance in vivo via O2? signaling
To define roles for reactive oxygen species (ROS) and epithelial sodium channel (ENaC) in maintaining lung fluid balance in vivo, we used two novel whole animal imaging approaches. Live X-ray fluoroscopy enabled quantification of air space fluid content of C57BL/6J mouse lungs challenged by intratracheal (IT) instillation of saline; results were confirmed by using conventional lung wet-to-dry weight ratios and Evans blue as measures of pulmonary edema. Visualization and quantification of ROS produced in lungs was performed in mice that had been administered a redox-sensitive dye, hydro-Cy7, by IT instillation. We found that inhibition of NADPH oxidase with a Rac-1 inhibitor, NSC23766, resulted in alveolar flooding, which correlated with a decrease in lung ROS production in vivo. Consistent with a role for Nox2 in alveolar fluid balance, Nox2?/? mice showed increased retention of air space fluid compared with wild-type controls. Interestingly, fluoroscopic analysis of C57BL/6J lungs IT instilled with LPS showed an acute stimulation of lung fluid clearance and ROS production in vivo that was abrogated by the ROS scavenger tetramethylpiperidine-N-oxyl (TEMPO). Acute application of LPS increased the activity of 20 pS nonselective ENaC channels in rat type 1 cells; the average number of channel and single-channel open probability (NPo) increased from 0.14 � 0.04 to 0.62 � 0.23. Application of TEMPO to the same cell-attached recording caused an immediate significant decrease in ENaC NPo to 0.04 � 0.03. These data demonstrate that, in vivo, ROS has the capacity to stimulate lung fluid clearance by increasing ENaC activity.
Goodson, Preston; Kumar, Amrita; Jain, Lucky; Kundu, Kousik; Murthy, Niren; Koval, Michael
Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice.
Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate ?-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation. PMID:23509361
Grimm, Melissa J; Vethanayagam, R Robert; Almyroudis, Nikolaos G; Dennis, Carly G; Khan, A Nazmul H; D'Auria, Anthony C; Singel, Kelly L; Davidson, Bruce A; Knight, Paul R; Blackwell, Timothy S; Hohl, Tobias M; Mansour, Michael K; Vyas, Jatin M; R�hm, Marc; Urban, Constantin F; Kelkka, Tiina; Holmdahl, Rikard; Segal, Brahm H
Monocyte and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice
Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was more than 100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and pro-inflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate beta-glucans, whereas inflammation in transgenic and wildtype mice was mild and transient. Together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation.
Grimm, Melissa J.; Vethanayagam, R. Robert; Almyroudis, Nikolaos G.; Dennis, Carly G.; Khan, A. Nazmul H.; D'Auria, Anthony; Singel, Kelly L.; Davidson, Bruce A.; Knight, Paul R.; Blackwell, Timothy S.; Hohl, Tobias M.; Mansour, Michael K.; Vyas, Jatin M.; Rohm, Marc; Urban, Constantin F.; Kelkka, Tiina; Holmdahl, Rikard; Segal, Brahm H.
A newly synthesized molecule derived from ruthenium cation, with antitumour activity, activates NADPH oxidase in human neutrophils.
To determine the nature of the mechanism by which certain derived ruthenium (Ru) complexes induce regression in tumour growth, we have investigated the possibility that this mechanism was associated with an increase of superoxide anion (O2-. production by phagocytic cells, which are usually found in tumour nodes. Here we present evidence that a newly synthesized complex, Ru3+-propylene-1, 2-diaminotetra-acetic acid (Ru-PDTA), derived from Ru and the sequestering ligand (PDTA), specifically stimulates O2-. production. This increase was associated with the translocation of cytosolic factors p47(phox) and p67(phox) of NADPH oxidase to the plasma membrane. The Ru-PDTA-complex-dependent O2-. production was abrogated by staurosporine, partially inhibited by diphenylene iodonium, and it was insensitive to pertussis toxin or dibutyryl cyclic AMP pretreatment. An increase of cytosolic Ca2+ levels were also detected in neutrophils treated with the Ru-PDTA complex. Also, Ru-PDTA complex induced the phosphorylation of tyrosine residues of several proteins as assessed by Western blotting. Present data are consistent with the possibility that Ru-PDTA-dependent antitumour effects are due in part to the complex's ability to stimulate the release of toxic oxygen metabolites from phagocytic cells infiltrating tumour masses.
Carballo, M; Vilaplana, R; Marquez, G; Conde, M; Bedoya, F J; Gonzalez-Vilchez, F; Sobrino, F
Inhibition of the human neutrophil NADPH oxidase by Coxiella burnetii.
Coxiella burnetii is an obligate intracellular Gram-negative pathogen. A notable feature of C. burnetii is its ability to replicate within acidic phagolysosomes; however, the mechanisms utilized in evading host defenses are not well defined. Here, we investigated human neutrophil phagocytosis of C. burnetii (Nine Mile, phase II; NMII) and the effect of phagocytosed organisms on neutrophil reactive oxygen species (ROS) production. We found that opsonization with immune serum substantially enhanced phagocytosis of NMII. Human neutrophils phagocytosing opsonized NMII generated very little ROS compared to cells phagocytosing opsonized Staphylococcus aureus, Escherichia coli, or zymosan. However, phagocytosis of NMII did not affect the subsequent ROS response to a soluble agonist, indicating inhibition was localized to the phagolysosome and was not a global effect. Indeed, analysis of NADPH oxidase assembly in neutrophils after phagocytosis showed that translocation of cytosolic NADPH oxidase proteins, p47(phox) and p67(phox), to the membrane was absent in cells phagocytosing NMII, as compared to cells phagocytosing S. aureus or activated by phorbol myristate acetate. Thus, phagocytosed NMII is able to disrupt assembly of the human neutrophil NADPH oxidase, which represents a novel virulence mechanism for this organism and appears to be a common mechanism of virulence for many intracellular pathogens. PMID:19379824
Siemsen, Daniel W; Kirpotina, Liliya N; Jutila, Mark A; Quinn, Mark T
Synergistic Activation of Glucose-6-Phosphate Dehydrogenase and NAD(P)H oxidase by Src kinase Elevates Superoxide in Type 2 Diabetic, Zucker fa/fa, Rat Liver
Glucose metabolism through glycolysis and hexosamine pathway has been shown to be altered in type 2 diabetes. However, its fate through the pentose phosphate pathway (PPP) is currently unclear. In this study, we determined whether the activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the PPP, is modulated in the liver of Zucker obese fa/fa rats (9�11 weeks old). We found that G6PD expression and activity, NADPH levels and 6-phosphogluconate generation was significantly increased in liver of fa/fa rats. Inhibition of PI3 kinase and Src kinases decreased (P<0.05) G6PD activity in the fa/fa but not in the lean rat liver, suggesting that G6PD activity is regulated by PI3/Src kinase signaling pathways. G6PD-derived NADPH increased (P<0.05) superoxide anion levels by 70�90% in the fa/fa vs the lean rat liver, which was inhibited by NADPH oxidase inhibitor, gp91ds-tat (50 ?M), and G6PD inhibitors, 6-aminonicotinamide (1 mM) and dihydroepiandrosterone (100 ?M); therefore, indicating that elevated G6PD activity may be responsible for mediating superoxide generation. Interestingly, we also found a positive correlation between liver hypertrophy/increased G6PD activity (r2=0.77; P=0.0009) and liver hypertrophy/superoxide production (r2=0.51; P=0.0091) in fa/fa rats. Increased G6PD and NADPH oxidase expression and activity, in young hyper-glycemic and -insulinemic rats prior to the development of diabetes, appear to be contributing factors for the induction of oxidative stress. Since inhibition of G6PD activity decreases oxidative stress, we conclude that it behaves as a pro-oxidant in the fa/fa rat liver, in type 2 diabetes.
Gupte, Rakhee S.; Floyd, Beverly C.; Kozicky, Mark; George, Shimran; Ungvari, Zoltan I.; Neito, Vanessa; Wolin, Michael S.; Gupte, Sachin A
Rac1-mediated NADPH oxidase release of O2- regulates epithelial sodium channel activity in the alveolar epithelium.
We examine whether alveolar cells can control release of O(2)(-) through regulated NADPH oxidase (NOX) 2 (NOX2) activity to maintain lung fluid homeostasis. Using FACS to purify alveolar epithelial cells, we show that type 1 cells robustly express each of the critical NOX components that catalyze the production of O(2)(-) (NOX2 or gp91(phox), p22(phox), p67(phox), p47(phox), and p40(phox) subunits) as well as Rac1 at substantially higher levels than type 2 cells. Immunohistochemical labeling of lung tissue shows that Rac1 expression is cytoplasmic and resides near the apical surface of type 1 cells, whereas NOX2 coimmunoprecipitates with epithelial sodium channel (ENaC). Since Rac1 is a known regulator of NOX2, and hence O(2)(-) release, we tested whether inhibition or activation of Rac1 influenced ENaC activity. Indeed, 1 microM NSC23766 inhibition of Rac1 decreased O(2)(-) output in lung cells and significantly decreased ENaC activity from 0.87 +/- 0.16 to 0.52 +/- 0.16 [mean number of channels (N) and single-channel open probability (P(o)) (NP(o)) +/- SE, n = 6; P < 0.05] in type 2 cells. NSC23766 (10 microM) decreased ENaC NP(o) from 1.16 +/- 0.27 to 0.38 +/- 0.10 (n = 6 in type 1 cells). Conversely, 10 ng/ml EGF (a known stimulator of both Rac1 and O(2)(-) release) increased ENaC NP(o) values in both type 1 and 2 cells. NP(o) values increased from 0.48 +/- 0.21 to 0.91 +/- 0.28 in type 2 cells (P < 0.05; n = 10). In type 1 cells, ENaC activity also significantly increased from 0.40 +/- 0.15 to 0.60 +/- 0.23 following EGF treatment (n = 7). Sequestering O(2)(-) using 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) compound prevented EGF activation of ENaC in both type 1 and 2 cells. In conclusion, we report that Rac1-mediated NOX2 activity is an important component in O(2)(-) regulation of ENaC. PMID:20097736
Takemura, Yoshizumi; Goodson, Preston; Bao, Hui Fang; Jain, Lucky; Helms, My N
Skeletal Muscle NADPH Oxidase Is Increased and Triggers Stretch-Induced Damage in the mdx Mouse
Recent studies have shown that oxidative stress contributes to the pathogenesis of muscle damage in dystrophic (mdx) mice. In this study we have investigated the role of NADPH oxidase as a source of the oxidative stress in these mice. The NADPH oxidase subunits gp91phox, p67phox and rac 1 were increased 2�3 fold in tibilais anterior muscles from mdx mice compared to wild type. Importantly, this increase occurred in 19 day old mice, before the onset of muscle necrosis and inflammation, suggesting that NADPH oxidase is an important source of oxidative stress in mdx muscle. In muscles from 9 week old mdx mice, gp91phox and p67phox were increased 3�4 fold and NADPH oxidase superoxide production was 2 times greater than wild type. In single fibers from mdx muscle NADPH oxidase subunits were all located on or near the sarcolemma, except for p67phox,which was expressed in the cytosol. Pharmacological inhibition of NADPH oxidase significantly reduced the intracellular Ca2+ rise following stretched contractions in mdx single fibers, and also attenuated the loss of muscle force. These results suggest that NADPH oxidase is a major source of reactive oxygen species in dystrophic muscle and its enhanced activity has a stimulatory effect on stretch-induced Ca2+ entry, a key mechanism for muscle damage and functional impairment.
Whitehead, Nicholas P.; Yeung, Ella W.; Froehner, Stanley C.; Allen, David G.
Nitric oxide inactivates NADPH oxidase in pig neutrophils by inhibiting its assembling process.
The effects of nitric oxide (NO) on superoxide (O-2) generation of the NADPH oxidase in pig neutrophils were studied. NO dose-dependently suppressed O-2 generation of both neutrophil NADPH oxidase and reconstituted NADPH oxidase. Effects of NO on NADPH-binding site and the redox centers including FAD and low spin heme in cytochrome b558 and the electron transfer rates from NADPH to heme via FAD were examined under anaerobic conditions. Both reaction rates and the Km value for NADPH were unchanged by NO. Visible and EPR spectra of cytochrome b558 showed that the structure of heme was unchanged by NO, indicating that NO does not affect the redox centers of the oxidase. In reconstituted NADPH oxidase system, NO did not inhibit O-2 generation of the oxidase when added after activation. The addition of NO to the membrane component or the cytosol component inhibited the activity by 24.0 +/- 5.3 or 37.4 +/- 7.1%, respectively. The addition of NO during the activation process or to the cytosol component simultaneously with myristate inhibited the activity by 74.0 +/- 5.2 or 70.0 +/- 8.3%, respectively, suggesting that cytosol protein(s) treated with myristate becomes susceptible to NO. Peroxynitrite did not interfere with O-2 generation. PMID:9407051
Fujii, H; Ichimori, K; Hoshiai, K; Nakazawa, H
NADPH oxidases in lung health and disease.
Abstract Significance: The evolution of the lungs and circulatory systems in vertebrates ensured the availability of molecular oxygen (O2; dioxygen) for aerobic cellular metabolism of internal organs in large animals. O2 serves as the physiologic terminal acceptor of mitochondrial electron transfer and of the NADPH oxidase (Nox) family of oxidoreductases to generate primarily water and reactive oxygen species (ROS), respectively. Recent advances: The purposeful generation of ROS by Nox family enzymes suggests important roles in normal physiology and adaptation, most notably in host defense against invading pathogens and in cellular signaling. Critical issues: However, there is emerging evidence that, in the context of chronic stress and/or aging, Nox enzymes contribute to the pathogenesis of a number of lung diseases. Future Directions: Here, we review evolving functions of Nox enzymes in normal lung physiology and emerging pathophysiologic roles in lung disease. Antioxid. Redox Signal. 20, 2838-2853. PMID:24093231
Bernard, Karen; Hecker, Louise; Luckhardt, Tracy R; Cheng, Guangjie; Thannickal, Victor J
IL-6 Mediated Degeneration of Forebrain GABAergic Interneurons and Cognitive Impairment in Aged Mice through Activation of Neuronal NADPH Oxidase
Background Multiple studies have shown that plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) are elevated in patients with important and prevalent adverse health conditions, including atherosclerosis, diabetes, obesity, obstructive sleep apnea, hypertension, and frailty. Higher plasma levels of IL-6, in turn, increase the risk of many conditions associated with aging including age-related cognitive decline. However, the mechanisms underlying this association between IL-6 and cognitive vulnerability remain unclear. Methods and Findings We investigated the role of IL-6 in brain aging in young (4 mo) and aged (24 mo) wild-type C57BL6 and genetically-matched IL-6?/? mice, and determined that IL-6 was necessary and sufficient for increased neuronal expression of the superoxide-producing immune enzyme, NADPH-oxidase, and this was mediated by non-canonical NF?B signaling. Furthermore, superoxide production by NADPH-oxidase was directly responsible for age-related loss of parvalbumin (PV)-expressing GABAergic interneurons, neurons essential for normal information processing, encoding, and retrieval in hippocampus and cortex. Targeted deletion of IL-6 or elimination of superoxide by chronic treatment with a superoxide-dismutase mimetic prevented age-related loss of PV-interneurons and reversed age-related cognitive deficits on three standard tests of spatial learning and recall. Conclusions Present results indicate that IL-6 mediates age-related loss of critical PV-expressing GABAergic interneurons through increased neuronal NADPH-oxidase-derived superoxide production, and that rescue of these interneurons preserves cognitive performance in aging mice, suggesting that elevated peripheral IL-6 levels may be directly and mechanistically linked to long-lasting cognitive deficits in even normal older individuals. Further, because PV-interneurons are also selectively affected by commonly used anesthetic agents and drugs, our findings imply that IL-6 levels may predict adverse CNS effects in older patients exposed to these compounds through specific derangements in inhibitory interneurons, and that therapies directed at lowering IL-6 may have cognitive benefits clinically.
Dugan, Laura L.; Ali, Sameh S.; Shekhtman, Grigoriy; Roberts, Amanda J.; Lucero, Jacinta; Quick, Kevin L.; Behrens, M. Margarita
Hypohalous acid-modified human serum albumin induces neutrophil NADPH oxidase activation, degranulation, and shape change.
Halogenated lipids, proteins, and lipoproteins formed in reactions with myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) and hypobromous acid (HOBr) can contribute to the regulation of functional activity of cells and serve as mediators of inflammation. Human serum albumin (HSA) is the major plasma protein target of hypohalous acids. This study was performed to assess the potency of HSA modified by HOCl (HSA-Cl) and HOBr (HSA-Br) to elicit selected neutrophil responses. HSA-Cl/Br were found to induce neutrophil degranulation, generation of reactive oxygen intermediates, shape change, and actin cytoskeleton reorganization. Thus HSA-Cl/Br can initially act as a switch and then as a feeder of the "inflammatory loop" under oxidative stress. In HSA-Cl/Br-treated neutrophils, monoclonal antibodies against CD18, the ? subunit of ?2 integrins, reduced the production of superoxide anion radicals and hydrogen peroxide as well as MPO exocytosis, suggesting that CD18 contributed to neutrophil activation. HSA-Cl/Br-induced neutrophil responses were also inhibited by genistein, a broad-specificity tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, supporting the notion that activation of both tyrosine kinase and PI3K may play a role in neutrophil activation by HSA modified in MPO-dependent reactions. These results confirm the hypothesis that halogenated molecules formed in vivo via MPO-dependent reactions can be considered as a new class of biologically active substances potentially able to contribute to activation of myeloid cells in sites of inflammation and serve as inflammatory response modulators. PMID:24384524
Gorudko, Irina V; Grigorieva, Daria V; Shamova, Ekaterina V; Kostevich, Valeria A; Sokolov, Alexey V; Mikhalchik, Elena V; Cherenkevich, Sergey N; Arnhold, J�rgen; Panasenko, Oleg M
Activation of the granule pool of the NADPH oxidase accelerates apoptosis in human neutrophils.
Oxidative stress induces apoptosis in many types of cells, including human neutrophils. Our objective was to determine whether reactive oxygen species (ROS) produced by activated neutrophils are associated with accelerated apoptosis. Exposing neutrophils to ionomycin or phorbol myristate acetate (PMA) induced intracellular H2O2 production and rapid onset of apoptosis, measured as condensed chromatin, cellular shrinkage, and DNA fragmentation. Neutrophils activated with formyl-methionyl-leucyl-phenylalanine (fMLP) generated mainly extracellular H2O2 and did not undergo apoptosis. Exogenously added H2O2, together with the catalase blocker sodium azide, induced apoptosis to the same extent and with similar kinetics as PMA and ionomycin. Adenosine inhibited ionomycin-induced intracellular H2O2 production and apoptosis. Neither PMA nor ionomycin caused apoptosis in dimethyl sulfoxide-differentiated HL-60 cells, which are incapable of intracellular H2O2 production, whereas H2O2 induced apoptosis more efficiently in these cells than in neutrophils. We propose that activated neutrophils use intracellularly formed H2O2 to commit suicide. PMID:10088602
Lundqvist-Gustafsson, H; Bengtsson, T
Role of TLR4/NADPH oxidase/ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysaccharide in human renal mesangial cells
Background In bacteria-induced glomerulonephritis, Toll-like receptor 4 (TLR4) activation by lipopolysaccharide (LPS, a key component of the outer membranes of Gram-negative bacteria) can increase oxidative stress and the expression of vascular cell adhesion molecule-1 (VCAM-1), which recruits leukocytes to the glomerular mesangium. However, the mechanisms underlying VCAM-1 expression induced by LPS are still unclear in human renal mesangial cells (HRMCs). Results We demonstrated that LPS induced VCAM-1 mRNA and protein levels associated with an increase in the promoter activity of VCAM-1, determined by Western blot, RT-PCR, and promoter assay. LPS-induced responses were inhibited by transfection with siRNAs of TLR4, myeloid differentiation factor 88 (MyD88), Nox2, Nox4, p47phox, c-Src, p38 MAPK, activating transcription factor 2 (ATF2), and p300 or pretreatment with the inhibitors of reactive oxygen species (ROS, edaravone), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], c-Src (PP1), p38 MAPK (SB202190), and p300 (GR343). LPS induced NADPH oxidase activation, ROS production, and p47phox translocation from the cytosol to the membrane, which were reduced by PP1 or c-Src siRNA. We observed that LPS induced TLR4, MyD88, c-Src, and p47phox complex formation determined by co-immunoprecipitation and Western blot. We further demonstrated that LPS stimulated ATF2 and p300 phosphorylation and complex formation via a c-Src/NADPH oxidase/ROS/p38 MAPK pathway. Up-regulation of VCAM-1 led to enhancing monocyte adhesion to HRMCs challenged with LPS, which was inhibited by siRNAs of c-Src, p47phox, p38 MAPK, ATF2, and p300 or pretreatment with an anti-VCAM-1 neutralizing antibody. Conclusions In HRMCs, LPS-induced VCAM-1 expression was, at least in part, mediated through a TLR4/MyD88/ c-Src/NADPH oxidase/ROS/p38 MAPK-dependent p300 and ATF2 pathway associated with recruitment of monocyte adhesion to kidney. Blockade of these pathways may reduce monocyte adhesion via VCAM-1 suppression and attenuation of the inflammatory responses in renal diseases.
Carbon ion beams induce hepatoma cell death by NADPH oxidase-mediated mitochondrial damage.
Mitochondria are a major source of reactive oxygen species (ROS) and are also the target of cellular ROS. ROS damage to mitochondria leads to dysfunction that further enhances the production of mitochondrial ROS. This feed-forward vicious cycle between mitochondria and ROS induces cell death. Within a few minutes of radiation exposure, NADPH oxidase is activated to elevate the ROS level. Activated NADPH oxidase might induce the feed-forward cycle of mitochondria and this is a possible mechanism for cancer cell death induced by heavy ion irradiation. We found that after 4?Gy of (12) C(6+) ion radiation of HepG2 cells, the NADPH oxidase membrane subunit gp91(phox) was not involved in enzyme activation through increased expression; however, the subunit p47(phox) was involved in activation by being translocated to the membrane. (12) C(6+) ion radiation clearly decreased the ??m of HepG2 cells, increasing mitochondrial DNA damage and inducing cell death. Pretreatment with apocynin (APO, an NADPH oxidase inhibitor) effectively prevented the ??m decrease, mitochondrial DNA damage, and cell death induced by radiation. However, these protective effects were not observed with APO treatment after irradiation exposure. These data demonstrated that NADPH oxidase activation was an initiator in mitochondrial damage. Once mitochondria entered the feed-forward cycle, cell fate was no longer controlled by NADPH oxidase. Only antioxidants that targeted mitochondria such as MitoQ could break the cycle and release cells from death. PMID:23804302
Sun, Chao; Wang, Zhenhua; Liu, Yang; Liu, Yuanyuan; Li, Hongyan; Di, Cuixia; Wu, Zhenhua; Gan, Lu; Zhang, Hong
Ca2+-activated K+ channels of small and intermediate conductance control eNOS activation through NAD(P)H oxidase.
Ca(2+)-activated K(+) channels (K(Ca)) and NO play a central role in the endothelium-dependent control of vasomotor tone. We evaluated the interaction of K(Ca) with NO production in isolated arterial mesenteric beds of the rat. In phenylephrine-contracted mesenteries, acetylcholine (ACh)-induced vasodilation was reduced by NO synthase (NOS) inhibition with N(?)-nitro-L-arginine (L-NA), but in the presence of tetraethylammonium, L-NA did not further affect the response. In KCl-contracted mesenteries, the relaxation elicited by 100 nM ACh or 1 ?M ionomycin was abolished by L-NA, tetraethylammonium, or simultaneous blockade of small-conductance K(Ca) (SK(Ca)) channels with apamin and intermediate-conductance K(Ca) (IK(Ca)) channels with triarylmethane-34 (TRAM-34). Apamin-TRAM-34 treatment also abolished 100 nM ACh-activated NO production, which was associated with an increase in superoxide formation. Endothelial cell Ca(2+) buffering with BAPTA elicited a similar increment in superoxide. Apamin-TRAM-34 treatment increased endothelial NOS phosphorylation at threonine 495 (P-eNOS(Thr495)). Blockade of NAD(P)H oxidase with apocynin or superoxide dismutation with PEG-SOD prevented the increment in superoxide and changes in P-eNOS(Thr495) observed during apamin and TRAM-34 application. Our results indicate that blockade of SK(Ca) and IK(Ca) activates NAD(P)H oxidase-dependent superoxide formation, which leads to inhibition of NO release through P-eNOS(Thr495). These findings disclose a novel mechanism involved in the control of NO production. PMID:22210378
Gaete, Pablo S; Lillo, Mauricio A; Ardiles, Nicol�s M; P�rez, Francisco R; Figueroa, Xavier F
Influence of rosuvastatin on the NAD(P)H oxidase activity in the retina and electroretinographic response of spontaneously hypertensive rats
Background and purpose: Retinal complications may be encountered during the development of hypertension as a response to oxidative stress. Statins may reduce the risk of developing hypertension and ocular diseases. We evaluate the effects of rosuvastatin (ROSU) on retinal functionality and oxidative stress levels in normotensive and spontaneously hypertensive rats (SHR). Experimental approach: Wistar Kyoto (WKY) and SHR were treated for 3 weeks with rosuvastatin (10?mg?kg?1?day?1). Electroretinograms (ERG) were recorded before and after rosuvastatin treatment. Reactive oxygen species (ROS) were determined in the retina with dihydroethidium staining and NAD(P)H oxidase activity was evaluated. Key results: Retinal ganglion cell ROS and retinal NAD(P)H oxidase activity were higher in SHR than in WKY rats, respectively (17.1�1.1 vs 10.2�1.2?AU, P<0.01; 38095�8900 vs 14081�5820?RLU?mg?1; P<0.05). The ERG b-wave amplitude in SHR was significantly lower than that in WKY rats. Rosuvastatin reduced SBP in SHR but did not change plasma lipid levels. Rosuvastatin treatment in SHR significantly decreased ROS levels (11.2�1.3, P<0.01), NAD(P)H activity in retinal ganglion cells (9889�4290; P<0.05), and increased retinal plasmalogen content in SHR, but did not modify the ERG response. Conclusions and implications: Rosuvastatin, beyond lowering cholesterol levels, was able to lower ROS in the retina induced by hypertension, but without improving retinal function in SHR. These findings point to a complex relationship between ROS in the pathogenesis of retinal disease and hypertension.
Sicard, P; Acar, N; Gregoire, S; Lauzier, B; Bron, A M; Creuzot-Garcher, C; Bretillon, L; Vergely, C; Rochette, L
Inhaled birch pollen extract induces airway hyperresponsiveness via oxidative stress but independently of pollen-intrinsic NADPH oxidase activity, or the TLR4-TRIF pathway.
Oxidative stress in allergic asthma may result from oxidase activity or proinflammatory molecules in pollens. Signaling via TLR4 and its adaptor Toll-IL-1R domain-containing adapter inducing IFN-? (TRIF) has been implicated in reactive oxygen species-mediated acute lung injury and in Th2 immune responses. We investigated the contributions of oxidative stress and TLR4/TRIF signaling to experimental asthma induced by birch pollen exposure exclusively via the airways. Mice were exposed to native or heat-inactivated white birch pollen extract (BPEx) intratracheally and injected with the antioxidants, N-acetyl-L-cysteine or dimethylthiourea, prior to sensitization, challenge, or all allergen exposures, to assess the role of oxidative stress and pollen-intrinsic NADPH oxidase activity in allergic sensitization, inflammation, and airway hyperresponsiveness (AHR). Additionally, TLR4 signaling was antagonized concomitantly with allergen exposure, or the development of allergic airway disease was evaluated in TLR4 or TRIF knockout mice. N-acetyl-L-cysteine inhibited BPEx-induced eosinophilic airway inflammation and AHR except when given exclusively during sensitization, whereas dimethylthiourea was inhibitory even when administered with the sensitization alone. Heat inactivation of BPEx had no effect on the development of allergic airway disease. Oxidative stress-mediated AHR was also TLR4 and TRIF independent; however, TLR4 deficiency decreased, whereas TRIF deficiency increased BPEx-induced airway inflammation. In conclusion, oxidative stress plays a significant role in allergic sensitization to pollen via the airway mucosa, but the pollen-intrinsic NADPH oxidase activity and TLR4 or TRIF signaling are unnecessary for the induction of allergic airway disease and AHR. Pollen extract does, however, activate TLR4, thereby enhancing airway inflammation, which is restrained by the TRIF-dependent pathway. PMID:23776177
Shalaby, Karim H; Allard-Coutu, Alexandra; O'Sullivan, Michael J; Nakada, Emily; Qureshi, Salman T; Day, Brian J; Martin, James G
Inhaled birch pollen extract induces airway hyperresponsiveness via oxidative stress but independently of pollen-intrinsic NADPH oxidase activity, or the Toll-like receptor 4-TRIF pathway
Oxidative stress in allergic asthma may result from oxidase activity or pro-inflammatory molecules in pollens. Signaling via TLR4 and its adaptor TRIF has been implicated in reactive oxygen species (ROS)-mediated acute lung injury and in T helper 2 immune responses. We investigated the contributions of oxidative stress and TLR4/TRIF signaling to experimental asthma induced by birch pollen exposure exclusively via the airways. Mice were exposed to native or heat-inactivated white birch pollen extract (BPEx) intratracheally and injected with the antioxidants, N-acetyl-L-cysteine (NAC) or dimethylthiourea (DMTU) prior to sensitization, challenge, or all allergen exposures, to assess the role of oxidative stress and pollen-intrinsic NADPH oxidase activity in allergic sensitization, inflammation and airway hyperresponsiveness (AHR). Additionally, TLR4 signaling was antagonized concomitantly with allergen exposure, or the development of allergic airway disease was evaluated in TLR4 or TRIF knockout mice. NAC inhibited BPEx-induced eosinophilic airway inflammation and AHR except when given exclusively during sensitization, whereas DMTU was inhibitory even when administered with the sensitization alone. Heat-inactivation of BPEx had no effect on the development of allergic airway disease. Oxidative stress-mediated AHR was also TLR4- and TRIF-independent, however, TLR4 deficiency decreased, while TRIF deficiency increased BPEx-induced airway inflammation. In conclusion, oxidative stress plays a significant role in allergic sensitization to pollen via the airway mucosa, but the pollen-intrinsic NADPH oxidase activity and TLR4 or TRIF signaling are unnecessary for the induction of allergic airway disease and AHR. Pollen extract does, however, activate TLR4, thereby enhancing airway inflammation which is restrained by the TRIF-dependent pathway.
Shalaby, Karim H.; Allard-Coutu, Alexandra; O'Sullivan, Michael; Nakada, Emily; Qureshi, Salman T.; Day, Brian J.; Martin, James G.
Phorbol myristate acetate stimulates the formation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human neutrophils by activating NADPH oxidase.
We have shown previously that human neutrophil microsomes contain a highly specific dehydrogenase which, in the presence of NADP+, converts 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5S-HETE) to its 5-oxo metabolite, 5-oxo-ETE, a potent agonist of these cells. However, intact neutrophils convert 5S-HETE principally to its omega-oxidation product, 5,20-diHETE, and to only small amounts of 5-oxo-ETE. Phorbol myristate acetate (PMA) dramatically shifts the metabolism of 5S-HETE by intact cells so that 5-oxo-ETE is the major metabolite. The objective of this investigation was to determine the mechanism for the stimulatory effect of PMA on 5-oxo-ETE formation. The possibility that oxidants released in response to PMA nonenzymatically oxidized 5S-HETE was ruled out, since PMA did not appreciably stimulate the formation of 5-oxo-ETE from 5R-HETE. On the other hand, inhibition of NADPH oxidase either by diphenylene iodonium or by mild heating nearly completely prevented the stimulatory effect of PMA on the formation of 5-oxo-ETE. The possibility that this effect was mediated by superoxide seems unlikely, since it was still observed, although somewhat attenuated, in the presence of superoxide dismutase. Moreover, superoxide generated by another mechanism (xanthine/xanthine oxidase) did not appreciably affect the formation of 5-oxo-ETE by neutrophils. However, phenazine methosulfate, which can nonenzymatically convert NADPH to NADP+, mimicked the effect of PMA on 5-oxo-ETE formation by intact neutrophils. It is concluded that PMA acts by activating NADPH oxidase, resulting in conversion of NADPH to NADP+, which enhances the formation of 5-oxo-ETE and reduces the formation of 5,20-diHETE. Serum-treated zymosan has an effect on the metabolism of 5S-HETE similar to that of PMA in that it also stimulates the formation of 5-oxo-ETE and inhibits that of 5,20-diHETE. PMID:7929234
Powell, W S; Gravelle, F; Gravel, S
Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells.
Reactive oxygen species (ROS) produced by different NADPH oxidases (NOX) play a role in cardiomyocyte hypertrophy induced by different stimuli, such as angiotensin II and pressure overload. However, the role of the specific NOX isoforms in phenylephrine (PE)-induced cardiomyocyte hypertrophy is unknown. Therefore we aimed to determine the involvement of the NOX isoforms NOX1, NOX2 and NOX4 in PE-induced cardiomyocyte hypertrophy. Hereto rat neonatal cardiomyoblasts (H9c2 cells) were incubated with 100?M PE to induce hypertrophy after 24 and 48h as determined via cell and nuclear size measurements using digital imaging microscopy, electron microscopy and an automated cell counter. Digital-imaging microscopy further revealed that in contrast to NOX1 and NOX4, NOX2 expression increased significantly up to 4h after PE stimulation, coinciding and co-localizing with ROS production in the cytoplasm as well as the nucleus. Furthermore, inhibition of NOX-mediated ROS production with apocynin, diphenylene iodonium (DPI) or NOX2 docking sequence (Nox2ds)-tat peptide during these first 4h of PE stimulation significantly inhibited PE-induced hypertrophy of H9c2 cells, both after 24 and 48h of PE stimulation. These data show that early NOX2-mediated ROS production is crucial in PE-induced hypertrophy of H9c2 cells. PMID:24794531
Hahn, Nynke E; Musters, Ren� J P; Fritz, Jan M; Pagano, Patrick J; Vonk, Alexander B A; Paulus, Walter J; van Rossum, Albert C; Meischl, Christof; Niessen, Hans W M; Krijnen, Paul A J
A Model of Interaction between Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase and Apocynin Analogues by Docking Method
Some apocynin analogues have exhibited outstanding inhibition to NADPH oxidase. In this study, the key interactions between apocynin analogues and NADPH oxidase were analyzed by the docking method. The potential active site was first identified by the SiteID program combining with the key residue CYS378. Afterwards, the compounds in the training set were docked into NADPH oxidase (1K4U) under specific docking constraints to discuss the key interactions between ligands and the receptor. These key interactions were then validated by the consistence between the docking result and the experimental result of the test set. The result reveals that the Pi interaction between apocynin analogues and NADPH oxidase has a direct contribution to inhibition activities, except for H-bond formation and docking score. The key interactions might be valuable to discover and screen apocynin analogues as potent inhibitors of NADPH oxidase.
Jiang, Jie; Kang, Hongjun; Song, Xiaoliang; Huang, Sichao; Li, Sha; Xu, Jun
Role of NADPH oxidases in liver fibrosis.
Abstract Significance: Hepatic fibrosis is the common pathophysiologic process resulting from chronic liver injury, characterized by the accumulation of an excessive extracellular matrix. Multiple lines of evidence indicate that oxidative stress plays a pivotal role in the pathogenesis of liver fibrosis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. In addition to phagocytic NOX2, there are six nonphagocytic NOX proteins. Recent Advances: In the liver, NOX is functionally expressed both in the phagocytic form and in the nonphagocytic form. NOX-derived ROS contributes to various kinds of liver disease caused by alcohol, hepatitis C virus, and toxic bile acids. Recent evidence indicates that both phagocytic NOX2 and nonphagocytic NOX isoforms, including NOX1 and NOX4, mediate distinct profibrogenic actions in hepatic stellate cells, the main fibrogenic cell type in the liver. The critical role of NOX in hepatic fibrogenesis provides a rationale to assess pharmacological NOX inhibitors that treat hepatic fibrosis in patients with chronic liver disease. Critical Issues: Although there is compelling evidence indicating a crucial role for NOX-mediated ROS generation in hepatic fibrogenesis, little is known about the expression, subcellular localization, regulation, and redox signaling of NOX isoforms in specific cell types in the liver. Moreover, the exact mechanism of NOX-mediated fibrogenic signaling is still largely unknown. Future Directions: A better understanding through further research about NOX-mediated fibrogenic signaling may enable the development of novel anti-fibrotic therapy using NOX inhibition strategy. Antioxid. Redox Signal. 20, 2854-2872. PMID:24040957
Paik, Yong-Han; Kim, Jonghwa; Aoyama, Tomonori; De Minicis, Samuele; Bataller, Ramon; Brenner, David A
NADPH Oxidase 4 Regulates Cardiomyocyte Differentiation via Redox Activation of c-Jun Protein and the cis-Regulation of GATA-4 Gene Transcription*
NADPH oxidase 4 (Nox4) generates reactive oxygen species (ROS) that can modulate cellular phenotype and function in part through the redox modulation of the activity of transcription factors. We demonstrate here the potential of Nox4 to drive cardiomyocyte differentiation in pluripotent embryonal carcinoma cells, and we show that this involves the redox activation of c-Jun. This in turn acts to up-regulate GATA-4 expression, one of the earliest markers of cardiotypic differentiation, through a defined and highly conserved cis-acting motif within the GATA-4 promoter. These data therefore suggest a mechanism whereby ROS act in pluripotential cells in vivo to regulate the initial transcription of critical tissue-restricted determinant(s) of the cardiomyocyte phenotype, including GATA-4. The ROS-dependent activation, mediated by Nox4, of widely expressed redox-regulated transcription factors, such as c-Jun, is fundamental to this process.
Murray, Thomas V. A.; Smyrnias, Ioannis; Shah, Ajay M.; Brewer, Alison C.
Interferon-alpha2b (IFN-alpha2b)-induced apoptosis is mediated by p38 MAPK in hepatocytes from rat preneoplastic liver via activation of NADPH oxidase.
It is still unclear how Interferon-alfa (IFN-alpha) acts on preventing the appearance of hepatocarcinogenesis. We have demonstrated that IFN-alpha2b induces hepatocytic transforming growth factor-beta1 (TGF-beta(1)) production and secretion by inducing reactive oxygen species (ROS) formation through the activation of NADPH oxidase. This TGF-beta(1), alters antioxidant defences and induces programmed cell death. Since it was demonstrated that IFN-alpha induces apoptosis through the activation of p38 mitogen-activated protein kinase (p38 MAPK), this study was aimed to assess the role of this kinase in the IFN-alpha2b-induced apoptosis in rat liver preneoplasia; and to further evaluate the participation of NADPH oxidase. p38 MAPK pathway was activated during the IFN-alpha2b-induced apoptosis in rat liver preneoplasia. This activation was accompanied with phosphorylation of different transcription factors, depending on the time of IFN-alpha2b stimulus. Our data suggest that NADPH oxidase is activated by IFN-alpha2b through p38 MAPK. p38 MAPK-induced activation of NADPH oxidase is accomplished by a two-step pathway: first, ROS-independent and second ROS- and TGF-beta(1)-dependent. PMID:19455458
Quiroga, Ariel D; de Lujan Alvarez, Maria; Parody, Juan P; Ronco, Maria T; Carnovale, Cristina E; Carrillo, Maria Cristina
Heme Oxygenase-1 Regulates Matrix Metalloproteinase MMP-1 Secretion and Chondrocyte Cell Death via Nox4 NADPH Oxidase Activity in Chondrocytes
Interleukin-1? (IL-1?) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1? stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1? signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx� Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22phox heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1?. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis.
Rousset, Francis; Nguyen, Minh Vu Chuong; Grange, Laurent; Morel, Francoise; Lardy, Bernard
A functional polymorphism in the NAD(P)H oxidase subunit CYBA is related to gene expression, enzyme activity, and outcome in non-Hodgkin lymphoma.
NAD(P)H oxidase is a major endogenous source of reactive oxygen species (ROS). ROS may not only be involved in carcinogenesis but also in efficacy of chemotherapeutic agents like doxorubicin. By a comprehensive genotyping approach covering 48 genetic polymorphisms (single-nucleotide polymorphisms) in five subunits of phagocytic NAD(P)H oxidase, we asked whether they affect gene expression, enzymatic activity, and outcome of CHO(E)P chemotherapy. A highly consistent effect was observed for the CYBA 640A>G variant. In peripheral blood granulocytes of 125 healthy volunteers, the G allele of 640A>G was associated with lower NAD(P)H oxidase activity (P = 0.006). Moreover, the G allele was associated with lower mRNA and protein expression (both P = 0.02). Of clinical importance, the outcome of patients suffering from non-Hodgkin lymphoma and treated with CHO(E)P regimen was dependent on the CYBA 640A>G polymorphism. In an exploratory study (n = 401), carriers of 640GG had an event-free survival (EFS) risk ratio of 1.95 [95% confidence interval (95% CI), 1.31-2.90; P = 0.001] compared with 640AA. In a confirmatory set (n = 477), the risk ratios were 1.53 (1.04-2.25, P = 0.03). The complete set of 878 patients showed a relative risk of 1.72 (1.30-2.26) and 1.59 (1.14-2.21) for EFS and overall survival, respectively. Further molecular-biological experiments showed lower expression and reduced stability of transcripts with the G allele in lymphoblastoid cell lines. Transfection of allele-specific plasmids into HEK293 cells elicited lower activity for the G allele in a luciferase reporter gene construct. Thus, CYBA 640A>G was shown to be a functional polymorphism with possible consequences for patients receiving CHO(E)P chemotherapy and might have further implications for other ROS-mediated modalities. PMID:20215507
Hoffmann, Marion; Schirmer, Markus A; Tzvetkov, Mladen V; Kreuz, Markus; Ziepert, Marita; Wojnowski, Leszek; Kube, Dieter; Pfreundschuh, Michael; Tr�mper, Lorenz; Loeffler, Markus; Brockm�ller, J�rgen
NADPH-binding protein of the neutrophil superoxide-generating oxidase of guinea pigs.
Polymorphonuclear neutrophil membranes, activated in a cell-free system, contain all of the essential components required for superoxide formation including the NADPH-binding component. Arylazido-beta-alanyl- [32P]NADPH--3'-O-(3-[N-(4-azido-2-nitrophenyl)amino] propionyl)-[32P]NADPH--an NADPH analogue and photoaffinity probe, has been used to identify the specific NADPH binding component of the oxidase in activated membranes. A protein of about 52 kDa was photodependently labeled in the activated membranes by arylazido-beta-alanyl-[32P]NADPH. Specificity of labeling was indicated by the absence of such labeling in nonactivated membranes. The 52-kDa-labeled protein was the only isotopically labeled protein extracted from the labeled membranes with the chaotrope sodium perchlorate. Sodium perchlorate extraction of the 52-kDa protein from activated membranes correlates with the loss of the membranes' superoxide-generating capability. Reconstitution of the lost activity for sodium perchlorate-extracted membranes was accomplished by reincubating the extracted membranes with cytosol. It is proposed that the arylazido- beta-alanyl-[32P]NADPH-labeled protein of 52- to 57-kDa present on the activated membranes is the NADPH-binding protein of the neutrophil superoxide-generating oxidase. PMID:8078933
Ge, F; Guillory, R J
Endothelin-1-mediated increase in reactive oxygen species and NADPH Oxidase activity in hearts of aryl hydrocarbon receptor (AhR) null mice.
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor characterized to play a role in detection and adaptation to environmental stimuli. Genetic deletion of the AhR results in cardiac hypertrophy that is mediated primarily by endothelin-1 (ET-1); ET-1 has been implicated in the elevation of reactive oxygen species (ROS) in the heart, which are thought to contribute to several cardiovascular disorders, including cardiac hypertrophy. Thus, we tested the novel hypothesis that ET-1 induces ROS in AhR null mice via ET(A) receptor activation. We first confirmed the presence of ROS in the hearts of AhR null mice by measuring superoxide (O2*-)-dependent oxidation of dihydroethidium. Ethidium fluorescence was increased 10-fold in the hearts of AhR null mice, compared to the wild type. Then, to elucidate whether ET-1 mediated the increase in ROS, mice were chronically treated with 100 ng/kg/day of the ET(A) receptor antagonist BQ-123. In AhR null mice, BQ-123 significantly reduced elevated plasma 8-isoprostane, a systemic end product of phospholipid oxidation by ROS, and cardiac thiobarbituric acid reactive substances (TBARS), a nonspecific assessment of ROS production. Furthermore, BQ-123 reduced both cardiac lucigenin chemiluminescence and cardiac mRNA expression of NAD(P)H oxidase subunits gp91phox, p47phox, and p67phox in AhR null mice below the levels observed in wild-type mice. These findings demonstrate that ET-1 activation of ET(A) receptors mediates an increase in ROS that is associated with cardiac hypertrophy in AhR null mice. In addition, the ET-1-mediated increase in ROS appears to be initiated via increased NAD(P)H oxidase activity. PMID:16107552
Lund, Amie K; Peterson, Steven L; Timmins, Graham S; Walker, Mary K
TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase
Interactions of polymorphonuclear neutrophils (PMNs) with endothelial cells may contribute to the activation of endothelial cell responses involved in innate immunity. We explored a novel function of PMN NADPH oxidase in the mechanism of Toll-like receptor-2 (TLR2) upregulation induced by LPS-TLR4 signaling in endothelial cells. We showed that LPS induced TLR2 up-regulation through TLR4- and MyD88-dependent signaling. In neutropenic mice, the LPS-induced NF-kB activation and TLR2 expression were significantly reduced, and both responses were restored upon repletion by PMN obtained from WT mice but not by PMNs from NADPH oxidase gp91phox�/� mice. These findings were recapitulated in mouse lung vascular endothelial cells cocultured with PMNs, indicating that the augmented NF-kB activation and the resultant TLR2 upregulation in endothelial cells were secondary to oxidant signaling generated by PMN NADPH oxidase. The functional relevance of NADPH oxidase in mediating TLR4-induced TLR2 expression in endothelial cells was evident by markedly elevated and stable ICAM-1 expression as well as augmented PMN migration in response to sequential challenge with LPS and peptidoglycan. Thus, PMN NADPH oxidase�derived oxidant signaling is an important determinant of the cross talk between TLR4 and TLR2 and the control of endothelial cell activation.
Fan, Jie; Frey, Randall S.; Malik, Asrar B.
Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase.
The hypothesis that a high salt (HS) intake increases oxidative stress was investigated and was related to renal cortical expression of NAD(P)H oxidase and superoxide dismutase (SOD). 8-Isoprostane PGF(2alpha) and malonyldialdehyde were measured in groups (n = 6 to 8) of conscious rats during low-salt, normal-salt, or HS diets. NADPH- and NADH-stimulated superoxide anion (O(2)(.-)) generation was assessed by chemiluminescence, and expression of NAD(P)H oxidase and SOD were assessed with real-time PCR. Excretion of 8-isoprostane and malonyldialdehyde increased incrementally two- to threefold with salt intake (P < 0.001), whereas prostaglandin E(2) was unchanged. Renal cortical NADH- and NADPH-stimulable O(2)(.-) generation increased (P < 0.05) 30 to 40% with salt intake. Compared with low-salt diet, HS significantly (P < 0.005) increased renal cortical mRNA expression of gp91(phox) and p47(phox) and decreased expression of intracellular CuZn (IC)-SOD and mitochondrial (Mn)-SOD. Despite suppression of the renin-angiotensin system, salt loading enhances oxidative stress. This is accompanied by increased renal cortical NADH and NADPH oxidase activity and increased expression of gp91(phox) and p47(phox) and decreased IC- and Mn-SOD. Thus, salt intake enhances generation of O(2)(.-) accompanied by enhanced renal expression and activity of NAD(P)H oxidase with diminished renal expression of IC- and Mn-SOD. PMID:14569087
Kitiyakara, Chagriya; Chabrashvili, Tina; Chen, Yifan; Blau, Jonathan; Karber, Alex; Aslam, Shakil; Welch, William J; Wilcox, Christopher S
Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: role of NADPH oxidase.
The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers ?-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers ?-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide (O2(-)) production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, ?-SMA, collagen I, and TIMP-1 by inhibition of O2(-) production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on O2(-) production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF. PMID:24657416
Wang, Mi; Abais, Justine M; Meng, Nan; Zhang, Yang; Ritter, Joseph K; Li, Pin-Lan; Tang, Wang-Xian
NADPH oxidase is internalized by clathrin-coated pits and localizes to a Rab27A/B GTPase-regulated secretory compartment in activated macrophages.
Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b(558)) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91(phox) and CeCl(3) cytochemistry showed the presence of gp91(phox) and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b(558) is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b(558)-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5'-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b(558) under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNF?, and CD40L as physiological inducers of cyt b(558) exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b(558), which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b(558) did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b(558) depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell. PMID:22157766
Ejlerskov, Patrick; Christensen, Dan Ploug; Beyaie, David; Burritt, James B; Paclet, Marie-Helene; Gorlach, Agnes; van Deurs, Bo; Vilhardt, Frederik
Angiotensin II type 1 receptor blocker attenuates the activation of ERK and NADPH oxidase by mechanical strain in mesangial cells in the absence of angiotensin II
It has been reported that mechanical strain activates extracellular signal-regulated protein kinases (ERK) without the involvement of angiotensin II (Ang II) in cardiomyocytes. We examined the effects of mechanical strain on ERK phosphorylation levels in the absence of Ang II using rat mesangial cells. The ratio of phosphorylated ERK (p-ERK) to total ERK expression was increased by cyclic mechanical strain in a time- and elongation strength-dependent manner. With olmesartan [Ang II type 1 receptor (AT1R) antagonist] pretreatment, p-ERK plateau levels decreased in a dose-dependent manner (EC50 = 1.3 � 10?8 M, maximal inhibition 50.6 � 11.0% at 10?5 M); a similar effect was observed with RNA interference against Ang II type 1A receptor (AT1AR) and Tempol, a superoxide dismutase mimetic. In addition to the inhibition of p-ERK levels, olmesartan blocked the increase in cell surface and phosphorylated p47phox induced by mechanical strain and also lowered the mRNA expression levels of NADPH oxidase subunits. These results demonstrate that mechanical strain stimulates AT1R to phosphorylate ERK in mesangial cells in the absence of Ang II. This mechanotransduction mechanism is involved in the oxidative stress caused by NADPH oxidase and is blocked by olmesartan. The inverse agonistic activity of this AT1R blocker may be useful for the prevention of mesangial proliferation and renal damage caused by mechanical strain/oxidative stress regardless of circulating or tissue Ang II levels.
Yatabe, Junichi; Sanada, Hironobu; Yatabe, Midori Sasaki; Hashimoto, Shigeatsu; Yoneda, Minoru; Felder, Robin A.; Jose, Pedro A.; Watanabe, Tsuyoshi
NADPH Oxidase Is Internalized by Clathrin-coated Pits and Localizes to a Rab27A/B GTPase-regulated Secretory Compartment in Activated Macrophages*
Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b558) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91phox and CeCl3 cytochemistry showed the presence of gp91phox and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b558 is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b558-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5?-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b558 under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNF?, and CD40L as physiological inducers of cyt b558 exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b558, which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b558 did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b558 depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.
Ejlerskov, Patrick; Christensen, Dan Ploug; Beyaie, David; Burritt, James B.; Paclet, Marie-Helene; Gorlach, Agnes; van Deurs, Bo; Vilhardt, Frederik
Characterization of mitochondrial and extra-mitochondrial oxygen consuming reactions in human hematopoietic stem cells. Novel evidence of the occurrence of NAD(P)H oxidase activity.
This study was aimed to characterize the mitochondrial and extra-mitochondrial oxygen consuming reactions in human CD34+ hematopoietic stem cells. Cell samples were collected by apheresis following pre-conditioning by granulocyte colony-stimulating factor and isolated by anti-CD34 positive immunoselection. Polarographic analysis of the CN-sensitive endogenous cell respiration revealed a low mitochondrial oxygen consumption rate. Differential absorbance spectrometry on whole cell lysate and two-dimensional blue native-PAGE analysis of mitoplast proteins confirmed a low amount of mitochondrial respiratory chain complexes thus qualifying the hematopoietic stem cell as a poor oxidative phosphorylating cell type. Confocal microscopy imaging showed, however, that the intracellular content of mitochondria was not homogeneously distributed in the CD34+ hematopoietic stem cell sample displaying a clear inverse correlation of their density with the expression of the CD34 commitment marker. About half of the endogenous oxygen consumption was extra-mitochondrial and completely inhibitable by enzymatic scavengers of reactive oxygen species and by diphenylene iodinium. By spectral analysis, flow cytometry, reverse transcriptase-PCR, immunocytochemistry, and immunoprecipitation it was shown that the extra-mitochondrial oxygen consumption was contributed by the NOX2 and NOX4 isoforms of the O2-*. producer plasma membrane NAD(P)H oxidase with low constitutive activity. A model is proposed suggesting for the NAD(P)H oxidase a role of O2 sensor and/or ROS source serving as redox messengers in the activation of intracellular signaling pathways leading (or contributing) to mitochondriogenesis, cell survival, and differentiation in hematopoietic stem cells. PMID:15883163
Piccoli, Claudia; Ria, Roberto; Scrima, Rosella; Cela, Olga; D'Aprile, Annamaria; Boffoli, Domenico; Falzetti, Franca; Tabilio, Antonio; Capitanio, Nazzareno
Expression of NADPH oxidases and enhanced H 2O 2-generating activity in human coronary artery endothelial cells upon induction with tumor necrosis factor-?
Tumor necrosis factor (TNF)-?, which potentiates reactive oxygen species (ROS) generation, is crucial for the development of coronary arteritis and aneurysm in Kawasaki disease. We hypothesized that vascular NADPH oxidase (Nox) enzymes participate in the TNF-?-triggered endothelial damage through elevating ROS generation. Thus, we herein examine the expression of Nox enzymes in human coronary artery endothelial cells (HCAEC) and the
Lucia S. Yoshida; Shohko Tsunawaki
[NADPH oxidase controls EGF-induced proliferation via the ERK1/2-independent mechanism].
Using HyPer, a ratiometric GFP-based biosensor, the dynamics of H2O2 in living cells has been studied. It was found that activation of the receptor of the epidermal growth factor (EGF) in epithelial cells leads to sustained generation of intracellular H2O2, which is blocked by apocynin, an inhibitor of the assembly of plasma membrane NADPH oxidase. Apocynin also blocked HeLa cell proliferation induced by EGF, indicating that NADPH oxidase should be involved in the process. However, apocynin failed to alter the kinetics of the EGF-stimulated ERK1/2 activation. It was concluded that NADPH oxidase and intracellular H2O2 are the important downstream targets of the EGF receptor, which mediate the proliferation response by mechanisms distinct from the activation of the classical ERK1/2 MAP-kinase pathway. PMID:21268349
Tiurin-Kuz'min, P A; Agaronian, K M; Morozov, Ia I; Mishina, N M; Belousov, V V; Vorotnikov, A V
Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation
Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.
Pescatore, Luciana A.; Bonatto, Diego; Forti, Fabio L.; Sadok, Amine; Kovacic, Herve; Laurindo, Francisco R. M.
Marinobufagenin and cyclic strain may activate endothelial NADPH oxidase, contributing to the adverse impact of salty diets on vascular and cerebral health.
Limited but provocative ecologic epidemiology suggests that dietary salt may play a central role in the genesis of not only of stroke, but also dementia, including Alzheimer's disease. Impairment of nitric oxide bioactivity in the cerebral microvasculature is a likely mediator of this effect. Salted diets evoke increased adrenal secretion of the natriuretic steroid marinobufagenin (MBG), which promotes natriuresis via inhibition of renal tubular Na+/K+-ATPase; this effect is notably robust in salt-sensitive rodent strains in which other compensatory natriuretic mechanisms are subnormally efficient. MBG-mediated inhibition of sodium pumps in vascular smooth muscle likely plays a role in the hypertension induced by salty diets in these rodents. However, salt sensitivity in humans is associated with increased vascular mortality and ventricular hypertrophy independent of blood pressure; this suggests that MBG may be pathogenic via mechanisms unrelated to blood pressure control. Indeed, recent evidence indicates that MBG, via interaction with alpha1 isoforms of the sodium pump, can activate various intracellular signaling pathways at physiological concentrations too low to notably inhibit pump activity. An overview of current evidence suggests the hypothesis that MBG - as well as the cyclic strain induced by hypertension per se - may induce endothelial oxidative stress by activating NADPH oxidase. If so, this could rationalize the increase in vascular and systemic oxidative stress observed in salt-sensitive rodents fed salty diets, or in rodents infused with MBG; moreover, if this effect is a particularly prominent determinant of oxidative stress in cerebrovascular endothelium, it might help to explain the virtual absence of stroke and dementia in low-salt societies. As a corollary of this hypothesis, it can be predicted that spirulina-derived phycobilins, which appear to mimic the physiological role of bilirubin as an inhibitor of NAPDH oxidase complexes, may have potential for ameliorating the adverse health impacts of MBG and of salty diets. Potassium-rich diets are also likely to be protective in this regard, as they should suppress MBG production via their natriuretic impact, while their stimulatory effect on sodium pump activity may exert a hyperpolarizing effect on plasma membranes that suppresses NADPH oxidase activity. PMID:21968275
McCarty, Mark F
NADPH Oxidase-Dependent Signaling in Endothelial Cells: Role in Physiology and Pathophysiology
Abstract Reactive oxygen species (ROS) including superoxide (O2�?) and hydrogen peroxide (H2O2) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, �oxidant signaling,� has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47phox, p67phox and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91phox (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets. Antioxid. Redox Signal. 11, 791�810.
Ushio-Fukai, Masuko; Malik, Asrar B.
Role of NADPH Oxidase and Stat3 in Statin-Mediated Protection against Diabetic Retinopathy
PURPOSE Inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (statins) reduce signs of diabetic retinopathy in diabetic patients and animals. Indirect clinical evidence supports the actions of statins in improving cardiovascular function, but the mechanisms of their protective actions in the retina are not understood. Prior studies have implicated oxidative stress and NADPH oxidase�mediated activation of signal transducer and activator of transcription 3 (STAT3) in diabetes-induced increases in expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1 and breakdown of the blood�retinal barrier (BRB). Because statins are known to be potent antioxidants, the hypothesis for the current study was that the protective effects of statins in preventing diabetic retinopathy involve blockade of diabetes-induced activation of NADPH oxidase and STAT3. METHODS The hypothesis was tested by experiments in which rats with streptozotocin (STZ)-induced diabetes and retinal endothelial cells maintained in high-glucose medium were treated with simvastatin. Blood�retinal barrier (BRB) function was assayed by determining extravasation of albumin. Oxidative stress was assayed by measuring lipid peroxidation, protein nitration of tyrosine, dihydroethidine oxidation, and chemiluminescence. Immunoprobe techniques were used to determine the levels of NADPH oxidase subunit expression and STAT3 activation. RESULTS These studies showed that simvastatin blocks diabetes or high-glucose�induced increases in VEGF and ICAM-1 and preserves the BRB by a process involving blockade of diabetes/high-glucose�induced activation of STAT3 and NADPH oxidase. Statin treatment also prevents diabetes-induced increases in expression of the NADPH oxidase catalytic and subunit NOX2. CONCLUSIONS These results suggest that simvastatin protects against the early signs of diabetic retinopathy by preventing NADPH oxidase-mediated activation of STAT3.
Al-Shabrawey, Mohamed; Bartoli, Manuela; El-Remessy, Azza B.; Ma, Guochuan; Matragoon, Suraporn; Lemtalsi, Tahira; Caldwell, R. William; Caldwell, Ruth B.
Physiological roles of NOX/NADPH oxidase, the superoxide-generating enzyme
NADPH oxidase is a superoxide (O2�?)-generating enzyme first identified in phagocytes, essential for their bactericidal activities. Later, in non-phagocytes, production of O2�? was also demonstrated in an NADPH-dependent manner. In the last decade, several non-phagocyte-type NADPH oxidases have been identified. The catalytic subunit of these oxidases, NOX, constitutes the NOX family. There are five homologs in the family, NOX1 to NOX5, and two related enzymes, DUOX1 and DUOX2. Transgenic or gene-disrupted mice of the NOX family have also been established. NOX/DUOX proteins possess distinct features in the dependency on other components for their enzymatic activities, tissue distributions, and physiological functions. This review summarized the characteristics of the NOX family proteins, especially focused on their functions clarified through studies using gene-modified mice.
Katsuyama, Masato; Matsuno, Kuniharu; Yabe-Nishimura, Chihiro
A 29-kDa protein associated with p67phox expresses both peroxiredoxin and phospholipase A2 activity and enhances superoxide anion production by a cell-free system of NADPH oxidase activity.
Production of toxic oxygen metabolites provides a mechanism for microbicidal activity of the neutrophil. The NADPH oxidase enzyme system initiates the production of oxygen metabolites by reducing oxygen to form superoxide anion (O(2)()). With stimulation of the respiratory burst, cytosolic oxidase components, p47(phox), p67(phox), and Rac, translocate to the phagolysomal and plasma membranes where they form a complex with cytochrome b(558) and express enzyme activity. A 29-kDa neutrophil protein (p29) was identified by co-immunoprecipitation with p67(phox). N-terminal sequence analysis of p29 revealed homology to an open reading frame gene described in a myeloid leukemia cell line. A cDNA for p29 identical to the open reading frame protein was amplified from RNA of neutrophils. Significant interaction between p29 and p67(phox) was demonstrated using a yeast two-hybrid system. A recombinant (rh) p29 was expressed in Sf9 cells resulting in a protein with an apparent molecular weight of 34,000. The rh-p29 showed immunoreactivity with the original rabbit antiserum that detected p47(phox) and p67(phox). In addition, rh-p29 exhibited PLA(2) activity, which was Ca(2+) independent, optimal at low pH, and preferential for phosphatidylcholine substrates. The recombinant protein protected glutathione synthetase and directly inactivated H(2)O(2). By activity and sequence homology, rh-p29 can be classified as a peroxiredoxin. Finally, O(2)() production by plasma membrane and recombinant cytosolic oxidase components in the SDS-activated, cell-free NADPH oxidase system were enhanced by rh-p29. This effect was not inhibited by PLA(2) inhibitors. Thus, p29 is a novel protein that associates with p67 and has peroxiredoxin activity. This protein has a potential role in protecting the NADPH oxidase by inactivating H(2)O(2) or altering signaling pathways affected by H(2)O(2). PMID:12121978
Leavey, Patrick J; Gonzalez-Aller, Carolina; Thurman, Gail; Kleinberg, Michael; Rinckel, Lori; Ambruso, Daniel W; Freeman, Stefanie; Kuypers, Frans A; Ambruso, Daniel R
Mitochondrial regulation of NADPH oxidase in hindlimb unweighting rat cerebral arteries.
Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation. PMID:24759683
Zhang, Ran; Ran, Hai-hong; Peng, Liang; Xu, Fei; Sun, Jun-fang; Zhang, Lan-ning; Fan, Yong-yan; Peng, Li; Cui, Geng
Mitochondrial Regulation of NADPH Oxidase in Hindlimb Unweighting Rat Cerebral Arteries
Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation.
Xu, Fei; Sun, Jun-fang; Zhang, Lan-ning; Fan, Yong-yan; Peng, Li; Cui, Geng
Regulation of the NADPH oxidase and associated ion fluxes during phagocytosis.
The production of reactive oxygen species (ROS) within immune cell phagosomes is critical for antimicrobial activity and for correct antigen processing, and influences signaling pathways that direct host responses to infection and inflammation. Because excess oxidants can cause tissue damage and oxidative stress, phagocytes must precisely control both the location and timing of NADPH oxidase activity. How differential regulation is achieved at phagosomes is not well understood. Recent studies have revealed that the PI(3)P phosphoinositide plays an important role in locally boosting phagosomal NADPH oxidase activity through its binding to the p40(phox) NADPH oxidase subunit. Furthermore, phox subunit dynamics at phagosomes may regulate the timing of the oxidative burst. Novel elements regulating catalytic core trafficking include Rab27 and SNAP-23. In addition to trafficking events, the activity of the electrogenic oxidase is also governed by ionic fluxes, which are constrained at phagosomes owing to low intraphagosomal volume and dynamic display of channels, transporters, and pumps. New insights on the interdependence of phagosomal pH and ROS have been recently elucidated, and chloride channels important for microbicidal functions, including CFTR, and CLIC family channels, have been identified. Finally, periphagosomal calcium microdomains and calcium-dependent S100A8/9 protein recruitment may help fine-tune spatiotemporal regulation of NADPH oxidase activation for an effective immune response. PMID:23980663
Nunes, Paula; Demaurex, Nicolas; Dinauer, Mary C
Electroacupuncture pretreatment inhibits NADPH oxidase-mediated oxidative stress in diabetic mice with cerebral ischemia.
We investigated the protective effect of electroacupuncture (EA) on cerebral ischemic injury in diabetic mice, and explored the role of NADPH oxidase-mediated oxidative stress. Male C57BL/6 mice were injected streptozotocin to induce diabetes. The mice were pretreated with EA at acupoint "Baihui" for 30min. Two hours after the end of EA pretreatment, focal cerebral ischemia was induced following 24h reperfusion. The neurobehavioral scores and infarction volumes, malondialdehyde (MDA), reactive oxygen species (ROS), and activation of NADPH oxidase were determined in the presence or absence of the NADPH oxidase inhibitor apocynin or activator tetrabromocinnamic acid (TBCA). EA pretreatment reduced infarct size and improved neurological outcomes 24h after reperfusion in the diabetic mice. EA also decreased cerebral MDA and ROS levels compared with the control group, and inhibited the NADPH oxidase activation. The beneficial effects were abolished by TBCA while pretreatment with apocynin mimicked the neuroprotective and anti-oxidative effects of EA. Our results demonstrated that EA attenuated cerebral ischemic injury by inhibiting NAPDH oxidase-mediated oxidative damage in diabetic mice. These results suggest a novel mechanism of EA pretreatment-induced tolerance in diabetic cerebral ischemia. PMID:24854123
Guo, Fan; Song, Wenying; Jiang, Tao; Liu, Lixin; Wang, Feng; Zhong, Haixing; Yin, Hong; Wang, Qiang; Xiong, Lize
Increased NAD(P)H Oxidase and Reactive Oxygen Species in Coronary Arteries After Balloon Injury
Reactive oxygen species (ROS), produced by cellular constituents of the arterial wall, provide a signaling mechanism involved in vascular remodeling. Because adventitial fibroblasts are actively involved in coronary remodeling, we examined whether the changes in the redox state affect their phenotypic characteristics. To this end, superoxide anion production and NAD(P)H oxidase activity were measured in porcine coronary arteries in vivo,
Yi Shi; Rodica Niculescu; Dian Wang; Sachin Patel; Kelly L. Davenpeck; Andrew Zalewski
Superoxide production and expression of NAD(P)H oxidases by transformed and primary human colonic epithelial cells
Background: Superoxide (O2?) generation through the activity of reduced nicotinamide dinucleotide (NADH) or reduced nicotinamide dinucleotide phosphate (NADPH) oxidases has been demonstrated in a variety of cell types, but not in human colonic epithelial cells.Aims: To measure O2? production and effects of modulators of NAD(P)H oxidase activity and inhibitors of potential O2? generating enzymes in cultures of human colonic epithelial
A Perner; L Andresen; G Pedersen; J Rask-Madsen
Identification of the NADPH-binding protein of the neutrophil superoxide-generating oxidase of guinea pigs.
A cell-free system prepared from polymorphonuclear neutrophils is capable of NADPH-dependent generation of superoxide anion, but requires the simultaneous presence of plasma membranes, cytosol, arachidonate and guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The isolated membranes from such a preparation are able to catalyse NADPH-dependent superoxide formation independently of added cytosol and activators. Such activated membranes, activated in the cell-free system, must consequently contain all of the essential components required by the oxidase for superoxide formation, including the NADPH-binding component. Arylazido-beta-alanyl-[32P]NADPH (3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino] propionyl)-[32P]NADPH), an NADPH analogue and photoaffinity probe, is shown to act in the dark as a substrate for the oxidase activity in the activated membranes and an irreversible photodependent inhibitor following photoirradiation. The photoaffinity probe has been used to identify the specific NADPH-binding component of the oxidase in the activated membranes. In contrast with the sensitivity of the activated membranes, photoirradiation of arylazido-beta-alanyl-[32P]-NADPH under identical conditions, but with non-activated membranes, did not prevent subsequent activation of the treated membranes by cytosol, arachidonate and GTP[S]. However, photoirradiation of the cytosolic fraction in the presence of arylazido-beta-alanyl-[32P]NADPH resulted in an inhibition of the cytosol's ability to activate superoxide generation upon subsequent incubation with plasma membranes in the presence of arachidonate and GTP[S]. These observations are taken as a strong indication that the NADPH-binding protein of the oxidase is a cytosolic factor which associates with the plasma membrane upon activation. The superoxide-generating activity of the activated membranes was inhibited irreversibly in a concentration- and photo-dependent manner by arylazido-beta-alanyl-NADPH. The arylazido-beta-alanyl-NADPH-photodependent inhibition of superoxide generation in the activated membranes correlated with the photodependent labelling of a protein of 55.6 kDa by the arylazido-beta-alanyl-NADPH. Specificity of labelling was indicated by a lack of labelling of the 55.6 kDa region in the non-activated membranes and protection of the photodependent inhibition and labelling of the 55.6 kDa protein by NADPH. It is proposed that the arylazido-beta-alanyl-NADPH-labelled 55.6 kDa protein present on the activated membranes is the NADPH-binding protein of the neutrophil superoxide-generating oxidase. PMID:8136076
Ge, F; Guillory, R J
NAD(P)H oxidase-stimulating activity of serum from type 2 diabetic patients with retinopathy mediates enhanced endothelial expression of E-selectin
Endothelial expression of E-selectin is enhanced in diabetic patients with retinopathy, however, the underlying mechanisms are unclear. Therefore, this study was aimed to determine if endothelial expression of E-selectin is stimulated with serum from type 2 diabetic patients with retinopathy, and whether this process is related to NAD(P)H oxidase-derived oxidative stress. Serum was obtained from type 2 diabetic patients with
Mi Ran Yun; Dong Sun Im; Jong-Soo Lee; Seok Man Son; Sang-Min Sung; Sun Sik Bae; Chi Dae Kim
NADPH oxidase mediates radiation-induced oxidative stress in rat brain microvascular endothelial cells
The need to both understand and minimize the side effects of brain irradiation is heightened by the ever-increasing number of patients with brain metastases that require treatment with whole brain irradiation (WBI); some 200,000 cancer patients/year receive partial or WBI. At the present time, there are no successful treatments for radiation-induced brain injury, nor are there any known effective preventive strategies. Data support a role for chronic oxidative stress in radiation-induced late effects. However, the pathogenic mechanism(s) involved remains unknown. One candidate source of reactive oxygen species (ROS) is nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase, which converts molecular oxygen (O2) to the superoxide anion (O2.?) upon activation. We hypothesize that brain irradiation leads to activation of NADPH oxidase. We report that irradiating rat brain microvascular endothelial cells in vitro leads to increased i] intracellular ROS generation, ii] activation of the transcription factor NF?B, iii] expression of ICAM-1 and PAI-1, and iv] expression of Nox4, p22phox, and p47phox. Pharmacologic and genetic inhibition of NADPH oxidase blocked the radiation-mediated upregulation of intracellular ROS, activation of NF?B, and upregulation of ICAM-1 and PAI-1. These results suggest that activation of NADPH oxidase may play a role in radiation-induced oxidative stress.
Collins-Underwood, J. Racquel; Zhao, Weiling; Sharpe, Jessica G.; Robbins, Mike E
Activation of the neutrophil NADPH-oxidase by free fatty acids requires the ionized carboxyl group and partitioning into membrane lipid.
To investigate NADPH-oxidase activation, we studied the effects of free fatty acid (FFA), their uncharged derivatives, and calcium on membrane lipid structure and superoxide anion (O2-) release from intact neutrophils and in cell-free O2(-)-generating systems. This study determined that in calcium-free phosphate-buffered saline (PBS), cis-unsaturated FFA (cis FFA), trans-unsaturated FFA (trans FFA), and to a limited extent saturated FFA decreased the polarization of the membrane lipid structure probe 1,6-diphenyl-1,3,5-hexatriene (DPH), and these decreases correlated with partitioning of the FFA into the plasmalemma and the release of O2- from intact neutrophils and in cell-free preparations. Although a decrease in DPH polarization was always observed under conditions that resulted in the release of O2-, there was not a direct correlation between the amount of decrease in DPH polarization and the release of O2-. Trans FFA did not induce a dose-dependent decrease or as dramatic a decrease in DPH polarization compared with cis FFA, yet the trans FFA stimulated a greater release of O2- at 2.5, 5, and 10 microM concentrations. In addition, responses of the neutrophil to trans FFA, but not cis FFA, were differentially affected by the presence of calcium. When 0.1 mM calcium was added to the PBS decreases in DPH polarization in response to trans FFA were reduced by greater than 60%, whereas O2- was reduced by only 25-36%. The addition of 0.1 mM calcium 3 min after the trans FFA had partitioned into the membrane also reversed by 50-65% decreases in DPH polarization but did not affect the release of O2-. In the presence of 0.9 mM calcium, only the cis FFA decreased DPH polarization or stimulated the release of O2-. Calcium is known to interact more readily with the ionized carboxyl group of trans FFA, neutralizing the anionic charge through an electrostatic interaction. In support of the requirement for the ionized carboxyl group, structurally similar uncharged cis, trans, and saturated fatty alcohols; methyl esters; and aldehydes decreased DPH polarization in the absence and presence of 0.9 mM calcium; however, none of these compounds stimulated the release of O2-. These results indicate that, in addition to the partitioning of FFA into the plasmalemma, which results in calcium-modulatable decreases in DPH polarization, activation or assembly of the NADPH-oxidase requires the ionized carboxyl group. PMID:1848271
Steinbeck, M J; Robinson, J M; Karnovsky, M J
Inhibition by alkylamines of NADPH oxidase through blocking the assembly of enzyme components.
Alkylamines inhibit NADPH oxidase both in intact neutrophils and in a cell-free system. The aim of this study was to examine the mechanism underlying this inhibitory effect. Among alkylamines with different chain lengths, the C12 compound (laurylamine) showed the greatest inhibitory effect on the cell-free NADPH oxidase activity induced by arachidonic acid (AA) in the presence of GTPgammaS. The inhibition was overcome by further addition of AA, and it was observed irrespective of whether laurylamine was added before or after the enzyme activation by AA. When added prior to the enzyme activation, laurylamine blocked translocation to the membrane of all three cytosolic components (p47-phox, p67-phox and rac) in a cell-free translocation assay. When added after the activation, laurylamine released only rac from the membrane. Laurylamine did not inhibit the reduction of cytochrome c by xanthine oxidase, suggesting that it does not have superoxide-scavenging activity. These results indicate that laurylamine inhibits both the activation process of NADPH oxidase and the activated enzyme itself by blocking the assembly of the oxidase components. PMID:10461769
Sawai, T; Asada, M; Nishizawa, Y; Nunoi, H; Katayama, K
Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase
Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin (GT), and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically
Shohko Tsunawaki; Lucia S. Yoshida; Satoshi Nishida; Toshihiro Kobayashi; Takashi Shimoyama
Functional analysis of the Trichoderma harzianum nox1 gene, encoding an NADPH oxidase, relates production of reactive oxygen species to specific biocontrol activity against Pythium ultimum.
The synthesis of reactive oxygen species (ROS) is one of the first events following pathogenic interactions in eukaryotic cells, and NADPH oxidases are involved in the formation of such ROS. The nox1 gene of Trichoderma harzianum was cloned, and its role in antagonism against phytopathogens was analyzed in nox1-overexpressed transformants. The increased levels of nox1 expression in these transformants were accompanied by an increase in ROS production during their direct confrontation with Pythium ultimum. The transformants displayed an increased hydrolytic pattern, as determined by comparing protease, cellulase, and chitinase activities with those for the wild type. In confrontation assays against P. ultimum the nox1-overexpressed transformants were more effective than the wild type, but not in assays against Botrytis cinerea or Rhizoctonia solani. A transcriptomic analysis using a Trichoderma high-density oligonucleotide (HDO) microarray also showed that, compared to gene expression for the interaction of wild-type T. harzianum and P. ultimum, genes related to protease, cellulase, and chitinase activities were differentially upregulated in the interaction of a nox1-overexpressed transformant with this pathogen. Our results show that nox1 is involved in T. harzianum ROS production and antagonism against P. ultimum. PMID:21421791
Montero-Barrientos, M; Hermosa, R; Cardoza, R E; Guti�rrez, S; Monte, E
HaCaT keratinocytes overexpressing the S100 proteins S100A8 and S100A9 show increased NADPH oxidase and NF-kappaB activities.
The calcium- and arachidonic acid (AA)-binding proteins S100A8 and S100A9 are involved in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in phagocytes. They are specifically expressed in myeloid cells, and are also found in epithelial cells in various (patho)physiological conditions. We have investigated the consequences of S100A8/A9 overexpression in epithelial cell lines on reactive oxygen species (ROS) generation and downstream signaling. Epithelial carcinoma HeLa cells, which exclusively express Nox2, showed dramatically increased activation of NADPH oxidase by phorbol 12-myristate 13-acetate after S100A8/A9 gene transfection. HaCaT keratinocytes overexpressing S100A8/A9 showed enhanced, transient ROS generation in response to the calcium ionophore A23187 compared to mock-transfected cells. Polymerase chain reaction analysis revealed mRNA transcripts for Nox1, Nox2, and Nox5 in HaCaT keratinocytes. Detailed transfection studies confirmed that NADPH oxidase activities in Nox1- and Nox5-transfected HeLa cells were enhanced after S100A8/A9 gene complementation. Furthermore, mutational analysis revealed that AA binding and Thr113 phosphorylation are important for S100A8/A9-enhanced activation of NADPH oxidase. Nuclear factor-kappaB (NF-kappaB) activation and interleukin-8 mRNA levels were increased in S100A8/A9-HaCaT keratinocytes, consistent with the view that NF-kappaB is a redox-sensitive transcription factor. Because they are expressed in epithelia under specific conditions, S100A8 and S100A9 might be involved in skin pathogenesis by modulating aspects of downstream signaling. PMID:17429438
Benedyk, Malgorzata; Sopalla, Claudia; Nacken, Wolfgang; Bode, G�nther; Melkonyan, Harut; Banfi, Botond; Kerkhoff, Claus
Hace1 controls ROS generation of vertebrate Rac1-dependent NADPH oxidase complexes
The Hace1-HECT E3 ligase is a tumor suppressor that ubiquitylates the activated GTP-bound form of the Rho family GTPase Rac1, leading to Rac1 proteasomal degradation. Here we show that, in vertebrates, Hace1 targets Rac1 for degradation when Rac1 is localized to the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase holoenzyme. This event blocks de novo reactive oxygen species generation by Rac1-dependent NADPH oxidases, and thereby confers cellular protection from reactive oxygen species-induced DNA damage and cyclin D1-driven hyper-proliferation. Genetic inactivation of Hace1 in mice or zebrafish, as well as Hace1 loss in human tumor cell lines or primary murine or human tumors, leads to chronic NADPH oxidase-dependent reactive oxygen species elevation, DNA damage responses and enhanced cyclin D1 expression. Our data reveal a conserved ubiquitin-dependent molecular mechanism that controls the activity of Rac1-dependent NADPH oxidase complexes, and thus constitutes the first known example of a tumor suppressor protein that directly regulates reactive oxygen species production in vertebrates.
Daugaard, Mads; Nitsch, Roberto; Razaghi, Babak; McDonald, Lindsay; Jarrar, Ameer; Torrino, Stephanie; Castillo-Lluva, Sonia; Rotblat, Barak; Li, Liheng; Malliri, Angeliki; Lemichez, Emmanuel; Mettouchi, Amel; Berman, Jason N.; Penninger, Josef M.; Sorensen, Poul H.
Hypoxia activates NADPH oxidase to increase [ROS]i and [Ca2+]i through mitochondrial ROS-PKC? signaling axis in pulmonary artery smooth muscle cells
The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells including pulmonary artery smooth muscle cells (PASMCs) remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22phox, p47phox, and p67phox were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47phox protein to the plasma membrane in pulmonary, but not mesenteric arteries. The Nox inhibitor apocynin and p47phox gene deletion attenuated hypoxic increase in intracellular reactive oxygen species and Ca2+ concentration ([ROS]i and [Ca2+]i) as well as contraction in mouse PASMCs, and abolished hypoxic activation of Nox in pulmonary arteries. The conventional/novel protein kinase C (PKC) inhibitor chelerythrine, specific PKC? translocation peptide inhibitor and PKC? gene deletion, but not the conventional PKC inhibitor G�6976, prevented hypoxic increase in Nox activity in pulmonary arteries and in [ROS]i in PASMCs. The PKC activator phorbol-12-myristate-13-acetate could increase Nox activity in pulmonary and mesenteric arteries. Inhibition of mitochondrial ROS generation with rotenone or myxothiazol prevented hypoxic activation of Nox. Glutathione peroxidase-1 (Gpx1) gene overexpression to enhance H2O2 removal significantly inhibited hypoxic activation of Nox, whereas Gpx1 gene deletion had an opposite effect. Exogenous H2O2 increased Nox activity in pulmonary and mesenteric arteries. These findings suggest that acute hypoxia may distinctively activate Nox to increase [ROS]i through mitochondrial ROS�PKC? signaling axis, providing a positive feedback mechanism to contribute to hypoxic increase in [ROS]i and [Ca2+]i as well as contraction in PASMCs.
Rathore, Rakesh; Zheng, Yun-Min; Niu, Chun-Feng; Liu, Qing-Hua; Korde, Amit; Ho, Ye-Shih; Wang, Yong-Xiao
NADPH oxidases regulate CD44 and hyaluronic acid expression in thrombin-treated vascular smooth muscle cells and in atherosclerosis.
The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE(-/-) mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE(-/-) mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47(phox-/-) VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE(-/-) compared with apoE(-/-)/p47(phox-/-) mice. HA-regulated pro-inflammatory gene expression was higher in apoE(-/-) than apoE(-/-)/p47(phox-/-) mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation. PMID:20558727
Vendrov, Aleksandr E; Madamanchi, Nageswara R; Niu, Xi-Lin; Molnar, Kimberly C; Runge, Mason; Szyndralewiez, C�dric; Page, Patrick; Runge, Marschall S
NADPH Oxidases Regulate CD44 and Hyaluronic Acid Expression in Thrombin-treated Vascular Smooth Muscle Cells and in Atherosclerosis*
The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE?/? mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE?/? mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47phox?/? VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE?/? compared with apoE?/?/p47phox?/? mice. HA-regulated pro-inflammatory gene expression was higher in apoE?/? than apoE?/?/p47phox?/? mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation.
Vendrov, Aleksandr E.; Madamanchi, Nageswara R.; Niu, Xi-Lin; Molnar, Kimberly C.; Runge, Mason; Szyndralewiez, Cedric; Page, Patrick; Runge, Marschall S.
Peroxisomal polyamine oxidase and NADPH-oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana.
Homeostasis of reactive oxygen species (ROS) in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA) are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd) and spermine to putrescine and Spd, respectively, is catalyzed by two peroxisomal PA oxidases (AtPAO). However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI). Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions ([Formula: see text] ), but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX). On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and [Formula: see text] . These results suggest that the ratio of [Formula: see text] /H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of [Formula: see text] by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed. PMID:24765099
Andronis, Efthimios A; Moschou, Panagiotis N; Toumi, Imene; Roubelakis-Angelakis, Kalliopi A
Peroxisomal polyamine oxidase and NADPH-oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana
Homeostasis of reactive oxygen species (ROS) in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA) are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd) and spermine to putrescine and Spd, respectively, is catalyzed by two peroxisomal PA oxidases (AtPAO). However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI). Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions (O2�? ), but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX). On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and O2�? . These results suggest that the ratio of O2�? /H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of O2�? by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed.
Andronis, Efthimios A.; Moschou, Panagiotis N.; Toumi, Imene; Roubelakis-Angelakis, Kalliopi A.
Multiple mechanisms of NADPH oxidase inhibition by type A and type B Francisella tularensis.
Ft is a facultative intracellular pathogen that infects many cell types, including neutrophils. In previous work, we demonstrated that the type B Ft strain LVS disrupts NADPH oxidase activity throughout human neutrophils, but how this is achieved is incompletely defined. Here, we used several type A and type B strains to demonstrate that Ft-mediated NADPH oxidase inhibition is more complex than appreciated previously. We confirm that phagosomes containing Ft opsonized with AS exclude flavocytochrome b(558) and extend previous results to show that soluble phox proteins were also affected, as indicated by diminished phosphorylation of p47(phox) and other PKC substrates. However, a different mechanism accounts for the ability of Ft to inhibit neutrophil activation by formyl peptides, Staphylococcus aureus, OpZ, and phorbol esters. In this case, enzyme targeting and assembly were normal, and impaired superoxide production was characterized by sustained membrane accumulation of dysfunctional NADPH oxidase complexes. A similar post-assembly inhibition mechanism also diminished the ability of anti-Ft IS to confer neutrophil activation and bacterial killing, consistent with the limited role for antibodies in host defense during tularemia. Studies of mutants that we generated in the type A Ft strain Schu S4 demonstrate that the regulatory factor fevR is essential for NADPH oxidase inhibition, whereas iglI and iglJ, candidate secretion system effectors, and the acid phosphatase acpA are not. As Ft uses multiple mechanisms to block neutrophil NADPH oxidase activity, our data strongly suggest that this is a central aspect of virulence. PMID:20610796
McCaffrey, Ramona L; Schwartz, Justin T; Lindemann, Stephen R; Moreland, Jessica G; Buchan, Blake W; Jones, Bradley D; Allen, Lee-Ann H
Multiple mechanisms of NADPH oxidase inhibition by type A and type B Francisella tularensis
Ft is a facultative intracellular pathogen that infects many cell types, including neutrophils. In previous work, we demonstrated that the type B Ft strain LVS disrupts NADPH oxidase activity throughout human neutrophils, but how this is achieved is incompletely defined. Here, we used several type A and type B strains to demonstrate that Ft-mediated NADPH oxidase inhibition is more complex than appreciated previously. We confirm that phagosomes containing Ft opsonized with AS exclude flavocytochrome b558 and extend previous results to show that soluble phox proteins were also affected, as indicated by diminished phosphorylation of p47phox and other PKC substrates. However, a different mechanism accounts for the ability of Ft to inhibit neutrophil activation by formyl peptides, Staphylococcus aureus, OpZ, and phorbol esters. In this case, enzyme targeting and assembly were normal, and impaired superoxide production was characterized by sustained membrane accumulation of dysfunctional NADPH oxidase complexes. A similar post-assembly inhibition mechanism also diminished the ability of anti-Ft IS to confer neutrophil activation and bacterial killing, consistent with the limited role for antibodies in host defense during tularemia. Studies of mutants that we generated in the type A Ft strain Schu S4 demonstrate that the regulatory factor fevR is essential for NADPH oxidase inhibition, whereas iglI and iglJ, candidate secretion system effectors, and the acid phosphatase acpA are not. As Ft uses multiple mechanisms to block neutrophil NADPH oxidase activity, our data strongly suggest that this is a central aspect of virulence.
McCaffrey, Ramona L.; Schwartz, Justin T.; Lindemann, Stephen R.; Moreland, Jessica G.; Buchan, Blake W.; Jones, Bradley D.; Allen, Lee-Ann H.
NADPH oxidase 4 limits bone mass by promoting osteoclastogenesis
ROS are implicated in bone diseases. NADPH oxidase 4 (NOX4), a constitutively active enzymatic source of ROS, may contribute to the development of such disorders. Therefore, we studied the role of NOX4 in bone homeostasis. Nox4�/� mice displayed higher bone density and reduced numbers and markers of osteoclasts. Ex vivo, differentiation of monocytes into osteoclasts with RANKL and M-CSF induced Nox4 expression. Loss of NOX4 activity attenuated osteoclastogenesis, which was accompanied by impaired activation of RANKL-induced NFATc1 and c-JUN. In an in vivo model of murine ovariectomy�induced osteoporosis, pharmacological inhibition or acute genetic knockdown of Nox4 mitigated loss of trabecular bone. Human bone obtained from patients with increased osteoclast activity exhibited increased NOX4 expression. Moreover, a SNP of NOX4 was associated with elevated circulating markers of bone turnover and reduced bone density in women. Thus, NOX4 is involved in bone loss and represents a potential therapeutic target for the treatment of osteoporosis.
Goettsch, Claudia; Babelova, Andrea; Trummer, Olivia; Erben, Reinhold G.; Rauner, Martina; Rammelt, Stefan; Weissmann, Norbert; Weinberger, Valeska; Benkhoff, Sebastian; Kampschulte, Marian; Obermayer-Pietsch, Barbara; Hofbauer, Lorenz C.; Brandes, Ralf P.; Schroder, Katrin
NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy
Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and N?-(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25?mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy.
Mustapha, Nik M.; Tarr, Joanna M.; Kohner, Eva M.; Chibber, Rakesh
NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy.
Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and Nepsilon-(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25 mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy. PMID:20652059
Mustapha, Nik M; Tarr, Joanna M; Kohner, Eva M; Chibber, Rakesh
Role of NADPH Oxidase-Mediated Reactive Oxygen Species in Podocyte Injury
Proteinuria is an independent risk factor for end-stage renal disease (ESRD) (Shankland, 2006). Recent studies highlighted the mechanisms of podocyte injury and implications for potential treatment strategies in proteinuric kidney diseases (Zhang et al., 2012). Reactive oxygen species (ROS) are cellular signals which are closely associated with the development and progression of glomerular sclerosis. NADPH oxidase is a district enzymatic source of cellular ROS production and prominently expressed in podocytes (Zhang et al., 2010). In the last decade, it has become evident that NADPH oxidase-derived ROS overproduction is a key trigger of podocyte injury, such as renin-angiotensin-aldosterone system activation (Whaley-Connell et al., 2006), epithelial-to-mesenchymal transition (Zhang et al., 2011), and inflammatory priming (Abais et al., 2013). This review focuses on the mechanism of NADPH oxidase-mediated ROS in podocyte injury under different pathophysiological conditions. In addition, we also reviewed the therapeutic perspectives of NADPH oxidase in kidney diseases related to podocyte injury.
Chen, Shan; Meng, Xian-Fang; Zhang, Chun
Respiratory burst and NAD(P)H oxidase activity are involved in capacitation of cryopreserved bovine spermatozoa
Heparin (a glycosaminoglycan) and quercetin (a calcium-ATPase plasma membrane specific inhibitor) induce bovine sperm capacitation. Mitochondria from frozen semen are capable of generating oxidative energy. The aim of the study was to determine oxygen uptake variation and the participation of diphenileneiodonium (DPI)-sensitive oxidases from spermatozoa capacitated with heparin or quercetin. Oxygen uptake was measured polarographically and 2?M diphenileneiodonium (DPI) was
M. C�rdoba; N. Mora; M. T. Beconi
Impact of Cyanidin-3-Glucoside on Glycated LDL-Induced NADPH Oxidase Activation, Mitochondrial Dysfunction and Cell Viability in Cultured Vascular Endothelial Cells
Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC.
Xie, Xueping; Zhao, Ruozhi; Shen, Garry X.
Activation of microglia with zymosan promotes excitatory amino acid release via volume-regulated anion channels: the role of NADPH oxidases.
Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study, we used an electrophysiologic approach and a D-[(3)H]aspartate (glutamate) release assay to explore the ROS-dependent regulation of glutamate-permeable volume-regulated anion channels (VRACs). Exposure of rat microglia to hypo-osmotic media stimulated Cl(-) currents and D-[(3)H]aspartate release, both of which were inhibited by the selective VRAC blocker, DCPIB. Exogenously applied H(2)O(2) potently increased swelling-activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger, MnTMPyP, and by two inhibitors of NADPH oxidase (NOX), diphenyliodonium and thioridazine. However, zymosan-stimulated glutamate release was insensitive to other NOX blockers, apocynin and HEBSF. This pharmacologic profile pointed to the potential involvement of apocynin-insensitive NOX4. Using RT-PCR we confirmed that NOX4 is expressed in rat microglial cells along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2, we stimulated this isoform using protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate or inhibited it with the broad spectrum PKC blocker, G�6983. Both agents potently modulated endogenous ROS production by NOX2 but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release and that its activity is regulated by endogenous ROS originating from NOX4. PMID:18624925
Harrigan, Timothy J; Abdullaev, Iskandar F; Jourd'heuil, David; Mongin, Alexander A
Activation of microglia with zymosan promotes excitatory amino acid release via volume-regulated anion channels: the role of NADPH oxidases
Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study we used an electrophysiological approach and a D-[3H]aspartate (glutamate) release assay to explore the ROS-dependent regulation of glutamate-permeable volume-regulated anion channels (VRACs). Exposure of rat microglia to hypoosmotic media stimulated Cl? currents and D-[3H]aspartate release, both of which were inhibited by the selective VRAC blocker DCPIB. Exogenously applied H2O2 potently increased swelling-activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger MnTMPyP, and by two inhibitors of NADPH oxidase (NOX) diphenyliodonium and thioridazine. However, zymosan-stimulated glutamate release was insensitive to other NOX blockers, apocynin and AEBSF. This pharmacological profile pointed to the potential involvement of apocynin-insensitive NOX4. Using RT-PCR we confirmed that NOX4 is expressed in rat microglial cells, along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2 we stimulated this isoform using protein kinase C (PKC) activator PMA, or inhibited it with the broad spectrum PKC blocker G�6983. Both agents potently modulated endogenous ROS production by NOX2, but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release, and that its activity is regulated by endogenous ROS originating from NOX4.
Harrigan, Timothy J.; Abdullaev, Iskandar F.; Jourd'heuil, David; Mongin, Alexander A.
Correction of Aberrant NADPH Oxidase Activity in Blood-Derived Mononuclear Cells from Type II Diabetes Mellitus Patients by a Naturally Fermented Papaya Preparation
Abstract Supplementation of standardized fermented papaya preparation (FPP) to adult diabetic mice improves dermal wound healing outcomes. Peripheral blood mononuclear cells (PBMC) from type II diabetes mellitus (T2DM) patients elicit a compromised respiratory burst activity resulting in increased risk of infections for the diabetic patients. Aims: The objectives of the current study were to determine the effect of FPP supplementation on human diabetic PBMC respiratory burst activity and to understand underlying mechanisms of such action of FPP. Results: When stimulated with phorbol 12-myristate 13-acetate, the production of reactive oxygen species by T2DM PBMC was markedly compromised compared to that of the PBMC from non-DM donors. FPP treated ex vivo improved respiratory burst outcomes in T2DM PBMC. FPP treatment significantly increased phosphorylation of the p47phox subunit of NADPH oxidase. In addition, the protein and mRNA expression of Rac2 was potently upregulated after FPP supplemention. The proximal human Rac2 gene promoter is G�C rich and contains consensus binding sites for Sp1 and AP-1. While FPP had no significant effect on the AP-1 DNA binding activity, the Sp1 DNA binding activity was significantly upregulated in PBMC after treatment of the cells with FPP. Innovation: This work provided first evidence that compromised respiratory burst performance of T2DM PBMC may be corrected by a nutritional supplement. Conclusion: FPP can correct respiratory burst performance of T2DM PBMC via an Sp-1-dependant pathway. Studies testing the outcome of FPP supplementation in diabetic patients are warranted. Antioxid. Redox Signal. 17, 485�491.
Dickerson, Ryan; Deshpande, Bhakthi; Gnyawali, Urmila; Lynch, Debbie; Gordillo, Gayle M.; Schuster, Dara; Osei, Kwame
Molecular quality control machinery contributes to the leukocyte NADPH oxidase deficiency in chronic granulomatous disease
Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease caused by defects in leukocyte NADPH oxidase. Various inherited defects in one of the membrane-bound components of NADPH oxidase, gp91-phox, cause X-linked (X91) CGD. Analysis of three patients with X91 CGD revealed that different mechanisms of molecular quality control lead to the common phenotype of absence of mature membrane-bound NADPH oxidase
Shio-Jean Lin; Ya-Fang Huang; Jing-Yi Chen; Paul G Heyworth; Deborah Noack; Ji-Yao Wang; Ching-Yuan Lin; Bor-Luen Chiang; Chin-Mu Yang; Ching-Chuan Liu; Chi-Chang Shieh
Does NAD(P)H oxidase-derived H2O2 participate in hypotonicity-induced insulin release by activating VRAC in ?-cells?
NAD(P)H oxidase (NOX)-derived H(2)O(2) was recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion channels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 and rat ?-cells. Exogenous H(2)O(2) (100 to 200�?M) at basal glucose concentration (1.1 to 2.8�mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H(2)O(2). In patch clamp experiments, exogenous H(2)O(2) was observed to stimulate NPPB-sensitive anion channel activity, which induced cell membrane depolarization. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenyleneiodonium chloride (DPI), a universal NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by exposure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl-L-cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by H(2)O(2). Taken together, these data suggest that NOX-derived H(2)O(2) plays a key role in the insulin secretory response of BRIN-BD11 and native ?-cells to extracellular hypotonicity. PMID:22089811
Crutzen, R; Shlyonsky, V; Louchami, K; Virreira, M; Hupkens, E; Boom, A; Sener, A; Malaisse, W J; Beauwens, R
Amyloid-? and Proinflammatory Cytokines Utilize a Prion Protein-Dependent Pathway to Activate NADPH Oxidase and Induce Cofilin-Actin Rods in Hippocampal Neurons
Neurites of neurons under acute or chronic stress form bundles of filaments (rods) containing 1?1 cofilin?actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (5�30 min) in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% of maximum response in ?6 h) occurs in a subpopulation (?20%) of hippocampal neurons upon exposure to soluble human amyloid-? dimer/trimer (A?d/t) at subnanomolar concentrations. Here we show that proinflammatory cytokines (TNF?, IL-1?, IL-6) also induce rods at the same rate and within the same neuronal population as A?d/t. Neurons from prion (PrPC)-null mice form rods in response to glutamate or antimycin A, but not in response to proinflammatory cytokines or A?d/t. Two pathways inducing rod formation were confirmed by demonstrating that NADPH-oxidase (NOX) activity is required for prion-dependent rod formation, but not for rods induced by glutamate or energy depletion. Surprisingly, overexpression of PrPC is by itself sufficient to induce rods in over 40% of hippocampal neurons through the NOX-dependent pathway. Persistence of PrPC-dependent rods requires the continuous activity of NOX. Removing inducers or inhibiting NOX activity in cells containing PrPC-dependent rods causes rod disappearance with a half-life of about 36 min. Cofilin-actin rods provide a mechanism for synapse loss bridging the amyloid and cytokine hypotheses for Alzheimer disease, and may explain how functionally diverse A?-binding membrane proteins induce synaptic dysfunction.
Kane, Sarah J.; Shaw, Alisa E.; Brown, David R.; Pulford, Bruce; Zabel, Mark D.; Lambeth, J. David; Bamburg, James R.
The NADPH oxidase inhibitor apocynin (acetovanillone) induces oxidative stress
Apocynin (acetovanillone) is often used as a specific inhibitor of NADPH oxidase. In N11 glial cells, apocynin induced, in a dose-dependent way, a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect). Apocynin evoked also, in a significant way, an increase of H{sub 2}O{sub 2} concentration and a decrease of the intracellular glutathione/glutathione disulfide ratio, accompanied by augmented efflux of glutathione and glutathione disulfide. Apocynin induced the activation of both pentose phosphate pathway and tricarboxylic acid cycle, which was blocked when the cells were incubated with glutathione together with apocynin. The cell incubation with glutathione prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate dehydrogenase leakage. Apocynin exerted an oxidant effect also in a cell-free system: indeed, in aqueous solution, it evoked a faster oxidation of the thiols glutathione and dithiothreitol, and elicited the generation of reactive oxygen species, mainly superoxide anions. Our results suggest that apocynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, and that some effects of apocynin in in vitro and in vivo experimental models should be interpreted with caution.
Riganti, Chiara [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Turin (Italy); Costamagna, Costanzo [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Turin (Italy); Bosia, Amalia [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Turin (Italy); Ghigo, Dario [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Turin (Italy)]. E-mail: email@example.com
Phagocyte NADPH oxidase, chronic granulomatous disease and mycobacterial infections.
Infection of humans with Mycobacterium tuberculosis remains frequent and may still lead to death. After primary infection, the immune system is often able to control M. tuberculosis infection over a prolonged latency period, but a decrease in immune function (from HIV to immunosenescence) leads to active disease. Available vaccines against tuberculosis are restricted to BCG, a live vaccine with an attenuated strain of M. bovis. Immunodeficiency may not only be associated with an increased risk of tuberculosis, but also with local or disseminated BCG infection. Genetic deficiency in the reactive oxygen species (ROS)-producing phagocyte NADPH oxidase NOX2 is called chronic granulomatous disease (CGD). CGD is among the most common primary immune deficiencies. Here we review our knowledge on the importance of NOX2-derived ROS in mycobacterial infection. A literature review suggests that human CGD patient frequently have an increased susceptibility to BCG and to M. tuberculosis. In vitro studies and experiments with CGD mice are incomplete and yielded - at least in part - contradictory results. Thus, although observations in human CGD patients leave little doubt about the role of NOX2 in the control of mycobacteria, further studies will be necessary to unequivocally define and understand the role of ROS. PMID:24916152
Deffert, Christine; Cachat, Julien; Krause, Karl-Heinz
Biphasic Regulation of the NADPH Oxidase by HGF/c-Met Signaling Pathway in Primary Mouse Hepatocytes
Redox signaling is emerging as an essential mechanism in the regulation of biological activities of the cell. The HGF/c-Met signaling pathway has been implicated as a key regulator of the cellular redox homeostasis and oxidative stress. We previously demonstrated that genetic deletion of c-met in hepatocytes disrupts redox homeostasis by a mechanism involving NADPH oxidase. Here, we were focused to address the mechanism of NADPH oxidase regulation by HGF/c-Met signaling in primary mouse hepatocytes and its relevance. HGF induced a biphasic mechanism of NADPH oxidase regulation. The first phase employed the rapid increase in production of ROS as signaling effectors to activate the Nrf2-mediated protective response resulting in up-regulation of the antioxidant proteins, such as NAD(P)H quinone oxidoreductase and ?-glutamylcysteine synthetase. The second phase operated under a prolonged HGF exposure, caused a suppression of the NADPH oxidase components, including NOX2, NOX4, p22 and p67, and was able to abrogate the TGF?-induced ROS production and improve cell viability. In conclusion, HGF/c-Met induces a Nrf2-mediated protective response by a double mechanism driven by NADPH oxidase.
Clavijo-Cornejo, Denise; Enriquez-Cortina, Cristina; Lopez-Reyes, Alberto; Dominguez-Perez, Mayra; Nuno, Natalia; Dominguez-Meraz, Marcela; Bucio, Leticia; Souza, Veronica; Factor, Valentina M.; Thorgeirsson, Snorri S.; Gutierrez-Ruiz, Maria Concepcion; Gomez-Quiroz, Luis E.
Enhanced Depolarization-Induced Pulmonary Vasoconstriction Following Chronic Hypoxia Requires EGFR-Dependent Activation of NAD(P)H Oxidase 2
Abstract Aims: Chronic hypoxia (CH) enhances depolarization-induced myofilament Ca2+ sensitization and resultant pulmonary arterial constriction through superoxide (O2?)-dependent stimulation of RhoA. Because NAD(P)H oxidase (NOX) has been implicated in the development of pulmonary hypertension, we hypothesized that vascular smooth muscle (VSM) depolarization increases NOX-derived O2? production leading to myofilament Ca2+ sensitization and augmented vasoconstrictor reactivity following CH. As epidermal growth factor receptor (EGFR) mediates Rac1-dependent NOX activation in renal mesangial cells, we further sought to examine the role EGFR plays in this response. Results: Vasoconstrictor responses to depolarizing concentrations of KCl were greater in lungs isolated from CH (4 wk, 0.5 atm) rats compared to normoxic controls, and this effect of CH was abolished by the general NOX inhibitor, apocynin. CH similarly augmented KCl-induced vasoconstriction and O2? generation (assessed using the fluorescent indicator, dihydroethidium) in Ca2+-permeabilized, pressurized small pulmonary arteries. These latter responses to CH were prevented by general inhibition of NOX isoforms (apocynin, diphenylene iodonium), and by selective inhibition of NOX 2 (gp91ds-tat), Rac1 (NSC 23766), and EGFR (AG 1478). Consistent with these observations, CH increased KCl-induced EGFR phosphorylation, and augmented depolarization-induced Rac1 activation in an EGFR-dependent manner. Innovation: This study establishes a novel signaling axis in VSM linking membrane depolarization to contraction that is independent of Ca2+ influx, and which mediates myofilament Ca2+ sensitization in the hypertensive pulmonary circulation. Conclusion: CH augments membrane depolarization-induced pulmonary VSM Ca2+ sensitization and vasoconstriction through EGFR-dependent stimulation of Rac1 and NOX 2. Antioxid. Redox Signal. 18, 1777�1788.
Norton, Charles E.; Broughton, Brad R.S.; Jernigan, Nikki L.; Walker, Benjimen R.
Expression of NADPH oxidases and enhanced H(2)O(2)-generating activity in human coronary artery endothelial cells upon induction with tumor necrosis factor-alpha.
Tumor necrosis factor (TNF)-alpha, which potentiates reactive oxygen species (ROS) generation, is crucial for the development of coronary arteritis and aneurysm in Kawasaki disease. We hypothesized that vascular NADPH oxidase (Nox) enzymes participate in the TNF-alpha-triggered endothelial damage through elevating ROS generation. Thus, we herein examine the expression of Nox enzymes in human coronary artery endothelial cells (HCAEC) and the effects of TNF-alpha on Nox-mediated ROS generation. We show that HCAEC in culture spontaneously generate H(2)O(2) at basal level (0.53 nmol/min/mg protein). In searching for Nox components responsible for the H(2)O(2) generation, two distinct isoforms of Nox4 are found expressed in HCAEC: the prototype Nox4A and the shorter Nox4B, respectively in the postnuclear supernatant and the nuclear fractions. Other expressed Nox family components are: as mRNAs, Nox4C, Nox4D, Nox1, p51(nox), and Racs; as mRNAs and proteins, Nox2, p22(phox), p47(phox), and p67(phox). The H(2)O(2)-generating activity increases up to three-fold upon inclusion of TNF-alpha in culture, concomitantly with augmented expressions of Nox4A, p22(phox), p47(phox) and p67(phox) proteins. Together, these results suggest that Nox2 and Nox4A enzymes are induced by TNF-alpha endowing HCAEC with enhanced ROS-generating activity, which may play a role in the initial endothelial dysfunction through oxidative stress. PMID:18687299
Yoshida, Lucia S; Tsunawaki, Shohko
Oxidative innate immune defenses by Nox/Duox family NADPH Oxidases
The importance of reactive oxygen species (ROS) in innate immunity was first recognized in professional phagocytes undergoing a �respiratory burst� upon activation. This robust oxygen consumption is related to a superoxide-generating enzyme, the phagocytic NADPH oxidase (Nox2 or phox). The oxidase is essential for microbial killing, since patients lacking a functional oxidase suffer from enhanced susceptibility to microbial infections. ROS derived from superoxide attack bacteria in the isolated niche of the neutrophil phagosome. The oxidase is electrogenic, alters ion currents across membranes, induces apoptosis, regulates cytokine production, influences gene expression, and promotes formation of extracellular traps. Recently, new homologues of Nox2 were discovered establishing the Nox family of NADPH oxidases that encompasses seven members. Nox1 is highly expressed in the colon epithelium, and can be induced by LPS or IFN-?. Nox4 was implicated in innate immunity since LPS induces Nox4-dependent ROS generation. Duox1 and Duox2 localize to the apical plasma membrane of epithelial cells in major airways, salivary glands, and the gastrointestinal tract, and provide extracellular hydrogen peroxide to lactoperoxidase to produce antimicrobial hypothiocyanite ions. Th1 and Th2 cytokines regulate expression of Dual oxidases in human airways and may thereby act in host defense or in proinflammatory responses.
Rada, Balazs; Leto, Thomas L.
Subtractive screening reveals up-regulation of NADPH oxidase expression in Crohn's disease intestinal macrophages
Macrophages play a central role during the pathogenesis of inflammation. In normal intestinal mucosa surface expression of typical macrophage markers such as CD14, CD16, CD11b or T-cell co-stimulatory molecules such as CD80 or CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a population of macrophages displaying these markers, secreting higher cytokine levels and representing an activated cell population. CD33+ cells (macrophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed subtracting mRNA from normal macrophages from those of Crohn's disease macrophages. Oxidative burst activity was determined by flow cytometry. Seventy clones were obtained by the subtractive mRNA screening. Sequencing showed >99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed >99% homology to mRNA of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed by RT-PCR and �virtual� Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0�9 � 0�16 in control macrophages indicating a lack of oxidative burst activity. In Crohn's disease this ratio was significantly increased to 1�80 � 0�8 (P = 0�004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macrophages from normal mucosa correlating with a lack of oxidative burst activity. In IBD macrophage-oxidative burst activity is increased and NADPH oxidase mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical target in IBD and reduce mucosal tissue damage in active IBD.
Hausmann, M; Spottl, T; Andus, T; Rothe, G; Falk, W; Scholmerich, J; Herfarth, H; Rogler, G
Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent ?-helix of p22phox
Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151�160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161�164), adopting an ?-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.
Nobuhisa, Ikuo; Takeya, Ryu; Ogura, Kenji; Ueno, Noriko; Kohda, Daisuke; Inagaki, Fuyuhiko; Sumimoto, Hideki
NAD(P)H oxidase and eNOS play differential roles in cytomegalovirus infection-induced microvascular dysfunction.
Primary cytomegalovirus (CMV) infection promotes oxidative stress and reduces nitric oxide (NO) bioavailability in endothelial cells. These events are among the earliest vascular responses to cardiovascular risk factors. We assessed the roles of NAD(P)H oxidase and NO bioavailability in microvascular responses to persistent CMV infection alone or with hypercholesterolemia. Wild-type (WT) or gp91(phox) (NAD(P)H oxidase subunit) knockout mice received mock inoculum or 3�10(4) PFU murine CMV (mCMV) ip 5 weeks before placement on a normal or high-cholesterol diet (HC) for 4 weeks before assessment of arteriolar function and venular blood cell recruitment using intravital microscopy. Some WT groups received sepiapterin (a precursor of the nitric oxide synthase cofactor tetrahydrobiopterin) or apocynin (NAD(P)H oxidase inhibitor/antioxidant). Endothelium-dependent vasodilation was impaired in mCMV vs mock WT, regardless of diet. This was not affected by sepiapterin, and pharmacological inhibition of nitric oxide synthase reduced dilation similarly in mock and mCMV mice. Apocynin or deficiency of total, but not blood cell or vascular wall only (tested using bone marrow chimeras), gp91(phox) protected against arteriolar dysfunction. Blood cell recruitment was induced by mCMV-HC. Sepiapterin, but not NAD(P)H oxidase deficiency/apocynin, reduced leukocyte accumulation, whereas platelet adhesion was reduced by sepiapterin, apocynin, or total, platelet-specific, or vascular wall gp91(phox) deficiency. These data implicate activation of both hematopoietic and vessel wall NAD(P)H oxidase in mCMV-induced arteriolar dysfunction and platelet and vascular NAD(P)H oxidase in the thrombogenic phenotype induced by mCMV-HC. In contrast, findings with sepiapterin suggest that eNOS dysfunction, perhaps uncoupling, mediates venular, but not arteriolar, responses to mCMV-HC, thus indicating that NAD(P)H oxidase and eNOS differentially regulate microvascular responses to mCMV. PMID:22033010
Leskov, Igor L; Whitsett, Jennifer; Vasquez-Vivar, Jeannette; Stokes, Karen Y
NAD(P)H Oxidase and eNOS Play Differential Roles in Cytomegalovirus Infection-Induced Microvascular Dysfunction
Primary cytomegalovirus (CMV) infection promotes oxidative stress and reduces nitric oxide (NO) bioavailability in endothelial cells. These events are among the earliest vascular responses to cardiovascular risk factors. We assessed the roles of NAD(P)H oxidase and NO bioavailability in microvascular responses to persistent CMV infection alone or with hypercholesterolemia. Wildtype (WT) or gp91phox (NAD(P)H oxidase subunit) knockout mice received mock-inoculum or 3�104PFU murine CMV (mCMV) IP 5wks before placement on normal or high cholesterol diet (?HC) for 4wks before assessment of arteriolar function and venular blood cell recruitment using intravital microscopy. Some WT groups received sepiapterin (a precursor of the nitric oxide synthase co-factor, tetrahydrobiopterin), or apocynin (NAD(P)H oxidase inhibitor/antioxidant). Endothelium-dependent vasodilation was impaired in mCMV vs. mock WT, regardless of diet. This was not affected by sepiapterin, and pharmacological inhibition of nitric oxide synthase reduced dilation similarly in Mock and mCMV mice. Apocynin or deficiency of total, but not only blood cell or vascular wall (tested using bone marrow chimeras), gp91phox protected against arteriolar dysfunction. Blood cell recruitment was induced by mCMV-HC. Sepiapterin, but not NAD(P)H oxidase deficiency/apocynin, reduced leukocyte accumulation, whereas platelet adhesion was reduced by sepiapterin, apocynin or total, platelet-specific or vascular wall gp91phox-deficiency. These data implicate activation of both hematopoietic and vessel wall NAD(P)H oxidase in mCMV-induced arteriolar dysfunction, and platelet and vascular NAD(P)H oxidase in the thrombogenic phenotype induced by mCMV-HC. In contrast, findings with sepiapterin suggest eNOS dysfunction, perhaps uncoupling, mediates venular, but not arteriolar, responses to mCMV-HC, thus indicating that NAD(P)H oxidase and eNOS differentially regulate microvascular responses to mCMV.
Leskov, Igor L.; Whitsett, Jennifer; Vasquez-Vivar, Jeannette; Stokes, Karen Y.
NADPH oxidase inhibits the pathogenesis of systemic lupus erythematosus.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by loss of tolerance to self nucleic acids. The source of autoantigen that drives disease onset and progression is unclear. A candidate source of autoantigen is the neutrophil extracellular trap (NET), which releases nucleic acids into the extracellular environment, generating a structure composed of DNA coated with antimicrobial proteins. On the basis of in vitro and patient correlative studies, several groups have suggested that NETs may provide lupus autoantigens. The observation that NET release (NETosis) relies on activity of the phagocyte NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox2) in neutrophils of both humans and mice provided a genetic strategy to test this hypothesis in vivo. Therefore, we crossed an X-linked nox2 null allele onto the lupus-prone MRL.Fas(lpr) genetic background and assessed immune activation, autoantibody generation, and SLE pathology. Counter to the prevailing hypothesis, Nox2-deficient lupus-prone mice had markedly exacerbated lupus, including increased spleen weight, increased renal disease, and elevated and altered autoantibody profiles. Moreover, heterozygous female mice, which have Nox2 deficiency in 50% of neutrophils, also had exacerbated lupus and altered autoantibody patterns, suggesting that failure to undergo normal Nox2-dependent cell death may result in release of immunogenic self-constituents that stimulate lupus. Our results indicate that NETosis does not contribute to SLE in vivo; instead, Nox2 acts to inhibit disease pathogenesis, making this enzyme an important target for further study and a candidate for therapeutic intervention. PMID:23100627
Campbell, Allison M; Kashgarian, Michael; Shlomchik, Mark J
Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase.
Diphenyleneiodonium (DPI) and its analogues have been previously shown to react via a radical mechanism whereby an electron is abstracted from a nucleophile to form a radical, which then adds back to the nucleophile to form covalent adducts [Banks (1966) Chem. Rev. 66, 243-266]. We propose that the inhibition of neutrophil NADPH oxidase by DPI occurs via a similar mechanism. A reduced redox centre in the oxidase could serve as electron donor to DPI, and inhibition would occur after direct phenylation of the redox cofactor, or of adjacent amino acid groups by the DPI radical. In the absence of an activatory stimulus, human neutrophil NADPH-oxidase was not inhibited by DPI. The Ki for time-dependent inhibition by DPI of human neutrophil membrane NADPH oxidase was found to be 5.6 microM. Inhibitory potency of DPI was shown to be directly related to rate of enzyme turnover, indicating the need for a reduced redox centre. Adducts were formed between photoreduced flavin (FAD or FMN) and inhibitor (DPI or diphenyliodonium). These were separated by h.p.l.c. and characterized by absorbance spectroscopy, 1H-n.m.r. and fast-atom-bombardment m.s. and found to have properties consistent with substituted 4a,5-dihydroflavins. After incubation of pig neutrophil membranes with DPI, the quantity of recoverable intact flavin was greatly diminished when NADPH was present to initiate oxidase turnover, indicating that the flavin may be the site of DPI activation. These results may provide a common mechanism of action for iodonium compounds as inhibitors of other flavoenzymes. Images Figure 9
O'Donnell, B V; Tew, D G; Jones, O T; England, P J
Release of mitochondrial apoptogenic factors and cell death are mediated by CK2 and NADPH oxidase
Activation of the NADPH oxidase subunit, NOX2, and increased oxidative stress are associated with neuronal death after cerebral ischemia and reperfusion. Inhibition of NOX2 by casein kinase 2 (CK2) leads to neuronal survival, but the mechanism is unknown. In this study, we show that in copper\\/zinc-superoxide dismutase transgenic (SOD1 Tg) mice, degradation of CK2? and CK2?? and dephosphorylation of CK2?
Gab Seok Kim; Joo Eun Jung; Purnima Narasimhan; Hiroyuki Sakata; Hideyuki Yoshioka; Yun Seon Song; Nobuya Okami; Pak H Chan
Bradykininstimulates phosphoinositideturnover and phospholipase C but not phospholipaseD and NADPH oxidase in human neutrophils
In response to formyl-Met-Leu-Phe (ThILP), human neutrophils (PMN) generate super- oxide anion (02) by the enzyme complex NADPH oxidase. The modulation of phosphomositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of IMLP-stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its
Sergio D. Catz; Norma B. Sterin-Speziale
Absence of Proton Channels in COS7 Cells Expressing Functional NADPH Oxidase Components
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O 2 ? ) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the Hefflux was thought to be contained within the gp91 phox subunit of NADPH oxidase, but recent data do not
Deri Morgan; Vladimir V. Cherny; Marianne O. Price; Mary C. Dinauer; Thomas E. DeCoursey
Differential roles of NADPH oxidases in vascular physiology and pathophysiology
Reactive oxygen species (ROS) are produced by all vascular cells and regulate the major physiological functions of the vasculature. Production and removal of ROS are tightly controlled and occur in discrete subcellular locations, allowing for specific, compartmentalized signaling. Among the many sources of ROS in the vessel wall, NADPH oxidases are implicated in physiological functions such as control of vasomotor tone, regulation of extracellular matrix and phenotypic modulation of vascular smooth muscle cells. They are involved in the response to injury, whether as an oxygen sensor during hypoxia, as a regulator of protein processing, as an angiogenic stimulus, or as a mechanism of wound healing. These enzymes have also been linked to processes leading to disease development, including migration, proliferation, hypertrophy, apoptosis and autophagy. As a result, NADPH oxidases participate in atherogenesis, systemic and pulmonary hypertension and diabetic vascular disease. The role of ROS in each of these processes and diseases is complex, and a more full understanding of the sources, targets, cell-specific responses and counterbalancing mechanisms is critical for the rational development of future therapeutics.
Amanso, Angelica M.; Griendling, Kathy K.
Role of apoptosis-inducing factor, proline dehydrogenase, and NADPH oxidase in apoptosis and oxidative stress
Flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. The oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. Here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (AIF), proline dehydrogenase, and NADPH oxidase. These proteins have diverse biochemical functions and influence apoptotic signaling by unique mechanisms. The role of AIF in apoptotic signaling is two-fold, with AIF changing intracellular location from the inner mitochondrial membrane space to the nucleus upon exposure of cells to apoptotic stimuli. In the mitochondria, AIF enhances mitochondrial bioenergetics and complex I activity/assembly to help maintain proper cellular redox homeostasis. After translocating to the nucleus, AIF forms a chromatin degrading complex with other proteins, such as cyclophilin A. AIF translocation from the mitochondria to the nucleus is triggered by oxidative stress, implicating AIF as a mitochondrial redox sensor. Proline dehydrogenase is a membrane-associated flavoenzyme in the mitochondrion that catalyzes the rate-limiting step of proline oxidation. Upregulation of proline dehydrogenase by the tumor suppressor, p53, leads to enhanced mitochondrial reactive oxygen species that induce the intrinsic apoptotic pathway. NADPH oxidases are a group of enzymes that generate reactive oxygen species for oxidative stress and signaling purposes. Upon activation, NADPH oxidase 2 generates a burst of superoxide in neutrophils that leads to killing of microbes during phagocytosis. NADPH oxidases also participate in redox signaling that involves hydrogen peroxide-mediated activation of different pathways regulating cell proliferation and cell death. Potential therapeutic strategies for each enzyme are also highlighted.
Natarajan, Sathish Kumar; Becker, Donald F
Post-treatment of an NADPH oxidase inhibitor prevents seizure-induced neuronal death.
The present study sought to evaluate the neuroprotective effects of apocynin, an NADPH oxidase assembly inhibitor, on seizure-induced neuronal death. Apocynin, also known as acetovanillone, is a natural organic compound isolated from the root of Canadian hemp (Apocynum cannabium). It has been extensively studied to determine its disease-fighting capabilities and application in several brain insults, such as traumatic brain injury and stroke. Here we tested the hypothesis that post-treatment of apocynin may prevent seizure-induced neuronal death by suppression of NADPH oxidase-mediated superoxide production. Temporal lobe epilepsy (TLE) was induced by intraperitoneal injection of pilocarpine (25mg/kg) in male rats. Apocynin (30mg/kg, i.p.) was injected into the intraperitoneal space two hours after seizure onset. A second injection was performed 24h after seizure. To test whether apocynin inhibits NADPH oxidase activation-induced reactive oxygen species (ROS) production, dihydroethidium (dHEt, 5mg/kg, i.p.) was injected before onset of seizure and ROS production was detected five hours after seizure onset. Neuronal oxidative injury (4HNE), neuronal death (Fluoro Jade-B), blood brain barrier (BBB) disruption (IgG leak), neurotrophil infiltration (MPO) and microglia activation (CD11b) in the hippocampus was evaluated at three days after status epilepticus (SE). Pilocarpine-induced seizure increased p47 immunofluorescence in the plasma membrane of hippocampal neurons at 12h post-insult and apocynin treatment prevented this increase. The present study found that apocynin post-treatment decreased ROS production and lipid peroxidation after seizure and decreased the number of degenerating hippocampal neurons. Apocynin also reduced seizure-induced BBB disruption, neurotrophil infiltration and microglial activation. Taken together, the present results suggest that inhibition of NADPH oxidase by apocynin may have a high therapeutic potential to reduce seizure-induced neuronal dysfunction. PMID:23313582
Kim, Jin Hee; Jang, Bong Geom; Choi, Bo Young; Kim, Hyeong Seop; Sohn, Min; Chung, Tae Nyoung; Choi, Hui Chul; Song, Hong Ki; Suh, Sang Won
Interleukin-6 Mediates the Increase in NADPH-Oxidase in the Ketamine Model of Schizophrenia
Adult exposure to NMDA receptor antagonists, such as ketamine, produces psychosis in humans, and exacerbates symptoms in schizophrenic patients. We recently showed that ketamine activates the innate immune enzyme NADPH-oxidase in brain, and that the superoxide produced leads to dysfunction of a subset of fast-spiking inhibitory interneurons expressing the calcium-binding protein parvalbumin (PV). Here we show that neuronal production of interleukin-6 (IL-6) is necessary and sufficient for ketamine-mediated activation of NADPH-oxidase in brain. Removal of IL-6 in neuronal cultures by anti-IL-6 blocking antibodies, or in vivo by use of IL-6-deficient mice, prevented the increase in superoxide by ketamine and rescued the interneurons. Accumulating evidence suggests that schizophrenia patients suffer from diminished antioxidant defenses, and a recent clinical trial showed that enhancing these defenses may ameliorate symptoms of the disease. Our results showing that ketamine-induced IL-6 is responsible for the activation of NADPH-oxidase in brain suggest that reducing brain levels of this cytokine may protect the GABAergic phenotype of fast-spiking PV-interneurons and thus attenuate the propsychotic effects of ketamine.
Behrens, M. Margarita; Ali, Sameh S.; Dugan, Laura L.
The deliberate production of reactive oxygen species (ROS) by phagocyte NADPH oxidase is widely appreciated as a critical component of antimicrobial host defense. Recently, additional homologs of NADPH oxidase (NOX) have been discovered throughout the animal and plant kingdoms, which appear to possess diverse functions in addition to host defense, including cell proliferation, differentiation, and regulation of gene expression. Several of these NOX homologs are also expressed within the respiratory tract, where they participate in innate host defense as well as in epithelial and inflammatory cell signaling and gene expression, and fibroblast and smooth muscle cell proliferation, in response to bacterial or viral infection and environmental stress. Inappropriate expression or activation of NOX/DUOX during various lung pathologies suggests their specific involvement in respiratory disease. This review summarizes the current state of knowledge regarding the general functional properties of mammalian NOX enzymes, and their specific importance in respiratory tract physiology and pathology.
van der Vliet, Albert
Pulmonary Surfactant Protein A Inhibits Macrophage Reactive Oxygen Intermediate Production in Response to Stimuli by Reducing NADPH Oxidase Activity1
Alveolar macrophages are important host defense cells in the human lung that continuously phagocytose environmental and infectious particles that invade the alveolar space. Alveolar macrophages are prototypical alternatively activated macrophages, with up-regulated innate immune receptor expression, down-regulated costimulatory molecule expression, and limited production of reactive oxygen intermediates (ROI) in response to stimuli. Surfactant protein A (SP-A) is an abundant protein
Joy E. Crowther; Vijay Kumar Kutala; Periannan Kuppusamy; J. Scott Ferguson; Alison A. Beharka; Jay L. Zweier; Francis X. McCormack; Larry S. Schlesinger
NADPH Oxidase 1 and Its Derived Reactive Oxygen Species Mediated Tissue Injury and Repair
Reactive oxygen species are mostly viewed to cause oxidative damage to various cells and induce organ dysfunction after ischemia-reperfusion injury. However, they are also considered as crucial molecules for cellular signal transduction in biology. NADPH oxidase, whose only function is reactive oxygen species production, has been extensively investigated in many cell types especially phagocytes. The deficiency of NADPH oxidase extends the process of inflammation and delays tissue repair, which causes chronic granulomatous disease in patients. NADPH oxidase 1, one member of the NADPH oxidase family, is not only constitutively expressed in a variety of tissues, but also induced to increase expression in both mRNA and protein levels under many circumstances. NADPH oxidase 1 and its derived reactive oxygen species are suggested to be able to regulate inflammation reaction, cell proliferation and migration, and extracellular matrix synthesis, which contribute to the processes of tissue injury and repair.
Fu, Xiu-Jun; Peng, Ying-Bo; Hu, Yi-Ping; Shi, You-Zhen; Yao, Min; Zhang, Xiong
The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels
The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp
Thomas E. DeCoursey; Deri Morgan; Vladimir V. Cherny
Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells.
The phagocyte-type NADPH oxidase expressed in endothelial cells differs from the neutrophil enzyme in that it exhibits low level activity even in the absence of agonist stimulation, and it generates intracellular reactive oxygen species. The mechanisms underlying these differences are unknown. We studied the subcellular location of (a) oxidase subunits and (b) functionally active enzyme in unstimulated endothelial cells. Confocal microscopy revealed co-localization of the major oxidase subunits, i.e. gp91(phox), p22(phox), p47(phox), and p67(phox), in a mainly perinuclear distribution. Plasma membrane biotinylation experiments confirmed the predominantly (>90%) intracellular distribution of gp91(phox) and p22(phox). After subcellular protein fractionation, approximately 50% of the gp91(phox) (91-kDa band), p22(phox), p67(phox), and p40(phox) pools and approximately 30% of the p47(phox) were present in the 1475 x g ("nucleus-rich") fraction. Likewise, approximately 50% of total NADPH-dependent O(2)() production (assessed by lucigenin (5 microm) chemiluminescence) was found in the 1475 x g fraction. Co-immunoprecipitation studies and measurement of NADPH-dependent reactive oxygen species production (cytochrome c reduction assay) demonstrated that p22(phox), gp91(phox), p47(phox), p67(phox), and p40(phox) existed as a functional complex in the cytoskeletal fraction. These results indicate that, in contrast to the neutrophil enzyme, a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as a preassembled intracellular complex associated with the cytoskeleton. PMID:11893732
Li, Jian-Mei; Shah, Ajay M
Unique role of NADPH oxidase 5 in oxidative stress in human renal proximal tubule cells
NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5) gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs) are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT) and hypertensive subjects (HT). We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2�0.7-fold, 5.2�0.7-fold, and 2.8�0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5�0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2�0.2-fold greater in HT than NT (P=<0.001, n=6�14/group). Ionomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA), effects that were abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (?45.1�3.2% vs. mock-siRNA, n=6�8) in HT. D1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT (?32.5�1.8%) than HT (?14.8�1.8). In contrast to the marked increase in expression and activity of NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system.
Yu, Peiying; Han, Weixing; Villar, Van Anthony M.; Yang, Yu; Lu, Quansheng; Lee, Hewang; Li, Fengmin; Quinn, Mark T.; Gildea, John J.; Felder, Robin A.; Jose, Pedro A.
Regulation of NADPH oxidase gene expression with PKA and cytokine IL-4 in neurons and microglia.
Neuronal excitation is mediated by the activation of NMDA receptor and associated with the formation of reactive oxygen species due to the activation of NADPH oxidase complex proteins. The activation of Gs protein coupled receptors (GPCRs) induces neuronal activation in the cAMP-dependent protein kinase A (PKA)-mediated signal cascade and regulates NADPH oxidase activity. However, it is unknown whether PKA regulates NADPH oxidase gene expression in neurons and microglia. In the present research, the NADPH oxidase gene expression was studied in rat cortical neurons and microglia in vitro. Purified microglial cells were identified with OX-42 antibody and they also expressed apolipoprotein E (ApoE). The time-dependent effect of cytokine interleukin-4 (IL-4) (20�ng/ml) in NADPH oxidase gene expression was studied in microglial cells. The levels of mRNA were determined by quantitative RT-PCR. The expression of NOX1, NOX2, and NCF2 was upregulated after IL-4 treatment for 4�h, but it was downregulated after 8-24�h. The expression of NCF1 was suppressed during any time of cytokine effect. IL-4 upregulated arginase1 (Arg1) and serine racemase1 (SRR1) gene expressions in microglia. Amyloid beta (Ab) suppressed NOX2, NCF1, and NCF2 gene expressions and upregulated glutamate cystine transporter (xCT), although IL-4 attenuated the effect of Ab (500�?M) in the upregulation of xCT gene expression. The activation of PKA with agonist dibutyryl cAMP (dbcAMP) (100�?M) induced the upregulation of Arg1 gene expression in microglia involving in the process of microglial activation. The transcription of NOX1, NOX2, and NCF1 was suppressed in microglial cells after dbcAMP treatment within 24�h. Neurons were identified with the microtubule-associated protein tau. The uniform distribution of tau along axons was established in normal neurons. Tau protein was redistributed after PKA agonist dbcAMP treatment for 24�h. L-glutamate (50�?M) caused the apoptotic processes and the accumulation of tau in the soma of neurons and along axons. The activation of PKA for 24�h induced the transcriptional upregulation of NOX1 and NCF1 in cortical neurons. However, L-glutamate suppressed NOX1 gene expression in neurons. These data demonstrate that the effects of IL-4 and dbcAMP are similar in the regulation of SRR1, Arg1, and NADPH oxidase complex gene expressions in neurons and microglia. IL-4 prevents glutamate release from microglia suppressing xCT expression induced by Ab. These findings suggest that the activation of GPCR in PKA-mediated pathway leads to transcriptional regulation of NADPH oxidase complex. The modulation of GPCR activation may inhibit the oxidative stress in neurons. PMID:22565378
Savchenko, Valentina L
Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen.
Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O(3)) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O(3) fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O(3) fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O(3), determined from the mRNA levels of the major allergens. We conclude that O(3) can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. PMID:21605929
Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D'Amato, Gennaro; Ederli, Luisa
Reactive Oxygen Species and Inhibitors of Inflammatory Enzymes, NADPH Oxidase, and iNOS in Experimental Models of Parkinson's Disease
Reactive oxygen species (ROSs) are emerging as important players in the etiology of neurodegenerative disorders including Parkinson's disease (PD). Out of several ROS-generating systems, the inflammatory enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and inducible nitric oxide synthase (iNOS) were believed to play major roles. Mounting evidence suggests that activation of NADPH oxidase and the expression of iNOS are directly linked to the generation of highly reactive ROS which affects various cellular components and preferentially damage midbrain dopaminergic neurons in PD. Therefore, appropriate management or inhibition of ROS generated by these enzymes may represent a therapeutic target to reduce neuronal degeneration seen in PD. Here, we have summarized recently developed agents and patents claimed as inhibitors of NADPH oxidase and iNOS enzymes in experimental models of PD.
Koppula, Sushruta; Kumar, Hemant; Kim, In Su; Choi, Dong-Kug
Role of PKC-? in NADPH oxidase-PKC?-Gi? axis dependent inhibition of ?-adrenergic response by U46619 in pulmonary artery smooth muscle cells.
Treatment of bovine pulmonary artery smooth muscle cells (BPASMCs) with U46619 attenuated isoproterenol caused stimulation of adenyl cyclase activity and cAMP production. Pretreatment with SQ29548 (Tp receptor antagonist), apocynin (NADPH oxidase inhibitor) and Go6976 (PKC-? inhibitor) eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity. Pretreatment with SQ29548 and apocynin prevented U46619 induced increase in NADPH oxidase activity, PKC-? activity and Gi? phosphorylation. However, pretreatment with CZI, a PKC-? inhibitor, markedly, but not completely, inhibited U46619 induced increase in NADPH oxidase activity, PKC-? activity, Gi? phosphorylation and also significantly eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976 inhibited U46619 induced increase in Gi? phosphorylation, but not PKC-? activity and NADPH oxidase activity. Pretreatment with pertussis toxin eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity without any discernible change in PKC-?, NADPH oxidase and PKC-? activities. Transfection of the cells with Tp, PKC-? and PKC-? siRNA duplexes corroborate the findings observed with their respective pharmacological inhibitors on the responses produced by U46619. Taken together, we suggest involvement of PKC-? in U46619 caused attenuation of isoproterenol stimulated ?-adrenergic response, which is regulated by NADPH oxidase-PKC?-Gi? axis in pulmonary artery smooth muscle cells. PMID:24184446
Chakraborti, Sajal; Roy, Soumitra; Mandal, Amritlal; Chowdhury, Animesh; Chakraborti, Tapati
Apocynin: chemical and biophysical properties of a NADPH oxidase inhibitor.
Apocynin is the most employed inhibitor of NADPH oxidase (NOX), a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V), the hydrophobicity index was calculated (logP = 0.83) and the molar absorption coefficient was determined (e275nm = 1.1 � 104 M-1 cm-1). Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays) when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA) with a binding affinity of 2.19 � 104 M-1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin. PMID:23455672
Petr�nio, Maicon S; Zeraik, Maria Luiza; Fonseca, Luiz Marcos da; Ximenes, Valdecir F
The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels
NASA Astrophysics Data System (ADS)
The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100mV to >0mV, but is steeply inhibited by further depolarization, and is abolished at about +190mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.
DeCoursey, Thomas E.; Morgan, Deri; Cherny, Vladimir V.
NADPH Oxidase-derived ROS Contributes to Upregulation of TRPC6 Expression in Puromycin Aminonucleoside-induced Podocyte Injury
Recent studies have demonstrated upregulation of transient receptor potential cation channel 6 (TRPC6) contributes to podocyte injury in acquired forms of proteinuric kidney diseases, such as focal segmental glomerulosclerosis (FSGS). However, under these pathophysiological conditions, the mechanisms of regulation of TRPC6 expression and activity remain unknown. The present study tested the hypothesis that NADPH oxidase-mediated redox signaling importantly participates in
Ziying Wang; Xinbing Wei; Yan Zhang; Xuelian Ma; Boqin Li; Shuya Zhang; Pengchao Du; Xiumei Zhang; Fan Yi
Nox1Based NADPH Oxidase Is the Major Source of UVA-Induced Reactive Oxygen Species in Human Keratinocytes
UVA radiation is a major environmental stress on skin, causing acute and chronic photodamage. These responses are mediated by reactive oxygen species (ROS), although the cellular source of these ROS is unknown. We tested the hypotheses that UVA-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is required for ROS generation in human keratinocytes (HK) and that these ROS initiate
Antonio Valencia; Irene E Kochevar
ANXUR Receptor-Like Kinases Coordinate Cell Wall Integrity with Growth at the Pollen Tube Tip Via NADPH Oxidases
It has become increasingly apparent that the extracellular matrix (ECM), which in plants corresponds to the cell wall, can influence intracellular activities in ways that go far beyond their supposedly passive mechanical support. In plants, growing cells use mechanisms sensing cell wall integrity to coordinate cell wall performance with the internal growth machinery to avoid growth cessation or loss of integrity. How this coordination precisely works is unknown. Previously, we reported that in the tip-growing pollen tube the ANXUR receptor-like kinases (RLKs) of the CrRLK1L subfamily are essential to sustain growth without loss of cell wall integrity in Arabidopsis. Here, we show that over-expression of the ANXUR RLKs inhibits growth by over-activating exocytosis and the over-accumulation of secreted cell wall material. Moreover, the characterization of mutations in two partially redundant pollen-expressed NADPH oxidases coupled with genetic interaction studies demonstrate that the ANXUR RLKs function upstream of these NADPH oxidases. Using the H2O2-sensitive HyPer and the Ca2+-sensitive YC3.60 sensors in NADPH oxidase-deficient mutants, we reveal that NADPH oxidases generate tip-localized, pulsating H2O2 production that functions, possibly through Ca2+ channel activation, to maintain a steady tip-focused Ca2+ gradient during growth. Our findings support a model where ECM-sensing receptors regulate reactive oxygen species production, Ca2+ homeostasis, and exocytosis to coordinate ECM-performance with the internal growth machinery.
Boisson-Dernier, Aurelien; Lituiev, Dmytro S.; Nestorova, Anna; Franck, Christina Maria; Thirugnanarajah, Sharme; Grossniklaus, Ueli
Angiotensin II (AT1) Receptors and NADPH Oxidase Regulate Cl? Current Elicited by ?1 Integrin Stretch in Rabbit Ventricular Myocytes
Direct stretch of ?1 integrin activates an outwardly rectifying, tamoxifen-sensitive Cl? current (Cl? SAC) via focal adhesion kinase (FAK) and/or Src. The characteristics of Cl? SAC resemble those of the volume-sensitive Cl? current, ICl,swell. Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their downstream signaling cascade participate in mechanotransduction. Paramagnetic beads coated with mAb for ?1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 ?M), an AT1R competitive antagonist, blocked Cl? SAC but did not significantly alter the background Cl? current in the absence of integrin stretch. AT1R signaling is mediated largely by H2O2 produced from superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 ?M), a potent NADPH oxidase inhibitor, rapidly and completely blocked both Cl? SAC elicited by stretch and the background Cl? current. A structurally unrelated NADPH oxidase inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, 0.5 and 2 mM), also rapidly and completely blocked Cl? SAC as well as a large fraction of the background Cl? current. With continuing integrin stretch, Cl? SAC recovered upon washout of AEBSF (2 mM). In the absence of stretch, exogenous AngII (5 nM) activated an outwardly rectifying Cl? current that was rapidly and completely blocked by DPI (60 ?M). Moreover, exogenous H2O2 (10, 100, and 500 ?M), the eventual product of NADPH oxidase activity, also activated Cl? SAC in the absence of stretch, whereas catalase (1,000 U/ml), an H2O2 scavenger, attenuated the response to stretch. Application of H2O2 during NADPH oxidase inhibition by either DPI (60 ?M) or AEBSF (0.5 mM) did not fully reactivate Cl? SAC, however. These results suggest that stretch of ?1-integrin in cardiac myocytes elicits Cl? SAC by activating AT1R and NADPH oxidase and, thereby, producing reactive oxygen species. In addition, NADPH oxidase may be intimately coupled to the channel responsible for Cl? SAC, providing a second regulatory pathway.
Browe, David M.; Baumgarten, Clive M.
Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency
Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency.BackgroundChronic renal failure (CRF) is associated with oxidative stress, the mechanism of which remains uncertain. Superoxide is the primary oxygen free radical produced in the body, NAD(P)H oxidase is the major source of superoxide production and superoxide dismutase (SOD) is responsible for removal of superoxide. We hypothesized that
Nosratola D Vaziri; Michael Dicus; Nathan D Ho; Laleh Boroujerdi-Rad; Ram K Sindhu
NADPH Oxidase as a Therapeutic Target for Neuroprotection against Ischaemic Stroke: Future Perspectives
Oxidative stress caused by an excess of reactive oxygen species (ROS) is known to contribute to stroke injury, particularly during reperfusion, and antioxidants targeting this process have resulted in improved outcomes experimentally. Unfortunately these improvements have not been successfully translated to the clinical setting. Targeting the source of oxidative stress may provide a superior therapeutic approach. The NADPH oxidases are a family of enzymes dedicated solely to ROS production and pre-clinical animal studies targeting NADPH oxidases have shown promising results. However there are multiple factors that need to be considered for future drug development: There are several homologues of the catalytic subunit of NADPH oxidase. All have differing physiological roles and may contribute differentially to oxidative damage after stroke. Additionally, the role of ROS in brain repair is largely unexplored, which should be taken into consideration when developing drugs that inhibit specific NADPH oxidases after injury. This article focuses on the current knowledge regarding NADPH oxidase after stroke including in vivo genetic and inhibitor studies. The caution required when interpreting reports of positive outcomes after NADPH oxidase inhibition is also discussed, as effects on long term recovery are yet to be investigated and are likely to affect successful clinical translation.
McCann, Sarah K.; Roulston, Carli L.
Suppression of NADPH oxidase- and mitochondrion-derived superoxide by Notoginsenoside R1 protects against cerebral ischemia-reperfusion injury through estrogen receptor-dependent activation of Akt/Nrf2 pathways.
Abstract Notoginsenoside R1 (NGR1) is a novel phytoestrogen that is isolated from Panax notoginseng. We have recently found that NGR1 showed neuroprotection in vitro against oxidative stress through estrogen receptor (ER)-dependent activation of Akt/Nrf2 pathways. However, whether NGR1 has neuroprotective effect against cerebral ischemia-reperfusion (I/R) injury in vivo is unknown. In this study, we used in vivo and in vitro models of cerebral I/R injury that demonstrate middle cerebral artery occlusion and reperfusion in rats, as well as oxygen-glucose deprivation followed by reoxygenation (OGD/R) in primary cortical neurons. These models were used to evaluate NGR1 neuroprotection. Three-day pretreatment with NGR1 (20 mg/kg; i.p.) significantly improved neurologic outcomes and reduced cerebral infarct volume. Pretreatment of primary cortical neurons with NGR1 (25 ?M) for 24 h prevented apoptosis and oxidative stress induced by OGD/R. NGR1 inhibited apoptosis by inhibiting mitochondrial membrane potential disruption, caspase-3 activation, and DNA fragmentation. NGR1 prevented oxidative stress by suppressing NADPH oxidase- and mitochondrion-derived superoxide and inhibiting production of malondialdehyde, protein carbonyl, and 8-hydroxydeoxyguanosine in vivo and in vitro. NGR1 induced ER-dependent activation of Akt/Nrf2 pathways by increasing ER?, ER?, phospho-Akt, phospho-GSK3?, nuclear Nrf2, and HO-1 expression in vivo and in vitro. Pretreatment with ICI-182780, LY294002, or Snpp abolished NGR1-mediated neuroprotection against oxidative stress and apoptosis in vitro. In conclusion, NGR1 showed neuroprotection against cerebral I/R injury in vivo and in vitro. The mechanism of NGR1 neuroprotection involves inhibition of NADPH oxidase activity and mitochondrial dysfunction via ER-dependent activation of Akt/Nrf2 pathways. PMID:24720662
Meng, X; Wang, M; Wang, X; Sun, G; Ye, J; Xu, H; Sun, X
Involvement of NOX1/NADPH oxidase in morphine-induced analgesia and tolerance.
The involvement of reactive oxygen species (ROS) in morphine-induced analgesia and tolerance has been suggested, yet how and where ROS take part in these processes remains largely unknown. Here, we report a novel role for the superoxide-generating enzyme NOX1/NADPH oxidase in the regulation of analgesia and acute analgesic tolerance. In mice lacking Nox1 (Nox1(-/Y)), the magnitude of the analgesia induced by morphine was significantly augmented. More importantly, analgesic tolerance induced by repeated administration of morphine was significantly suppressed compared with that in the littermates, wild-type Nox1(+/Y). In a membrane fraction obtained from the dorsal spinal cord, no difference was observed in morphine-induced [(35)S]GTP?S-binding between the genotypes, whereas morphine-stimulated GTPase activity was significantly attenuated in Nox1(-/Y). At 2 h after morphine administration, a significant decline in [(35)S]GTP?S-binding was observed in Nox1(+/Y) but not in Nox1(-/Y). No difference in the maximal binding and affinity of [(3)H]DAMGO was observed between the genotypes, but the translocation of protein kinase C isoforms to the membrane fraction following morphine administration was almost completely abolished in Nox1(-/Y). Finally, the phosphorylation of RGS9-2 and formation of a complex by G?i2/RGS9-2 with 14-3-3 found in morphine-treated Nox1(+/Y) were significantly suppressed in Nox1(-/Y). Together, these results suggest that NOX1/NADPH oxidase attenuates the pharmacological effects of opioids by regulating GTPase activity and the phosphorylation of RGS9-2 by protein kinase C. NOX1/NADPH oxidase may thus be a novel target for the development of adjuvant therapy to retain the beneficial effects of morphine. PMID:22159121
Ibi, Masakazu; Matsuno, Kuniharu; Matsumoto, Misaki; Sasaki, Mika; Nakagawa, Takayuki; Katsuyama, Masato; Iwata, Kazumi; Zhang, Jia; Kaneko, Shuji; Yabe-Nishimura, Chihiro
Genetic Ablation of NADPH Oxidase Enhances Susceptibility to Cigarette Smoke-Induced Lung Inflammation and Emphysema in Mice
Cigarette smoke (CS) induces recruitment of inflammatory cells in the lungs leading to the generation of reactive oxygen species (ROS), which are involved in lung inflammation and injury. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a multimeric system that is responsible for ROS production in mammalian cells. We hypothesized that NADPH oxidase-derived ROS play an important role in lung inflammation and injury and that targeted ablation of components of NADPH oxidase (p47phox and gp91phox) would protect lungs against the detrimental effects of CS. To test this hypothesis, we exposed p47phox?/? and gp91phox?/? mice to CS and examined inflammatory response and injury in the lung. Surprisingly, although CS-induced ROS production was decreased in the lungs of p47phox?/? and gp91phox?/? mice compared with wild-type mice, the inflammatory response was significantly increased and was accompanied by development of distal airspace enlargement and alveolar destruction. This pathological abnormality was associated with enhanced activation of the TLR4-nuclear factor-?B pathway in response to CS exposure in p47phox?/? and gp91phox?/? mice. This phenomenon was confirmed by in vitro studies in which treatment of peritoneal macrophages with a nuclear factor-?B inhibitor reversed the CS-induced release of proinflammatory mediators. Thus, these data suggest that genetic ablation of components of NADPH oxidase enhances susceptibility to the proinflammatory effects of CS leading to airspace enlargement and alveolar damage.
Yao, Hongwei; Edirisinghe, Indika; Yang, Se-Ran; Rajendrasozhan, Saravanan; Kode, Aruna; Caito, Samuel; Adenuga, David; Rahman, Irfan
The NADPH oxidase of phagocytic cells is an electron pump that alkalinises the phagocytic vacuole
Summary Phagocytic cells of the immune system contain an oxidase that is important for the killing and digestion of engulfed microbes. This is an electron transport chain that transfers electrons from NADPH in the cytosol to oxygen to form superoxide and hydrogen peroxide in the phagocytic vacuole. Absence or abnormality of this oxidase results in the syndrome of CGD, characterised
A. W. Segal
Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation
Protein�phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47phox, a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47phox with phosphoinositides. Although the isolated phox homology (PX) domain of p47phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47phox is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47phox, because protein kinase C treatment of the wild-type p47phox but not of a mutant protein with the S303/304/328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47phox nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47phox enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.
Ago, Tetsuro; Kuribayashi, Futoshi; Hiroaki, Hidekazu; Takeya, Ryu; Ito, Takashi; Kohda, Daisuke; Sumimoto, Hideki
Generation of reactive oxygen species in 1-methyl-4-phenylpyridinium (MPP+) treated dopaminergic neurons occurs as an NADPH oxidase-dependent two-wave cascade
Background Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H2O2), are necessary for appropriate responses to immune challenges. In the brain, excess superoxide production predicts neuronal cell loss, suggesting that Parkinson's disease (PD) with its wholesale death of dopaminergic neurons in substantia nigra pars compacta (nigra) may be a case in point. Although microglial NADPH oxidase-produced superoxide contributes to dopaminergic neuron death in an MPTP mouse model of PD, this is secondary to an initial die off of such neurons, suggesting that the initial MPTP-induced death of neurons may be via activation of NADPH oxidase in neurons themselves, thus providing an early therapeutic target. Methods NADPH oxidase subunits were visualized in adult mouse nigra neurons and in N27 rat dopaminergic cells by immunofluorescence. NADPH oxidase subunits in N27 cell cultures were detected by immunoblots and RT-PCR. Superoxide was measured by flow cytometric detection of H2O2-induced carboxy-H2-DCFDA fluorescence. Cells were treated with MPP+ (MPTP metabolite) following siRNA silencing of the Nox2-stabilizing subunit p22phox, or simultaneously with NADPH oxidase pharmacological inhibitors or with losartan to antagonize angiotensin II type 1 receptor-induced NADPH oxidase activation. Results Nigral dopaminergic neurons in situ expressed three subunits necessary for NADPH oxidase activation, and these as well as several other NADPH oxidase subunits and their encoding mRNAs were detected in unstimulated N27 cells. Overnight MPP+ treatment of N27 cells induced Nox2 protein and superoxide generation, which was counteracted by NADPH oxidase inhibitors, by siRNA silencing of p22phox, or losartan. A two-wave ROS cascade was identified: 1) as a first wave, mitochondrial H2O2 production was first noted at three hours of MPP+ treatment; and 2) as a second wave, H2O2 levels were further increased by 24 hours. This second wave was eliminated by pharmacological inhibitors and a blocker of protein synthesis. Conclusions A two-wave cascade of ROS production is active in nigral dopaminergic neurons in response to neurotoxicity-induced superoxide. Our findings allow us to conclude that superoxide generated by NADPH oxidase present in nigral neurons contributes to the loss of such neurons in PD. Losartan suppression of nigral-cell superoxide production suggests that angiotensin receptor blockers have potential as PD preventatives.
SUMO1 Negatively Regulates Reactive Oxygen Species Production From NADPH Oxidases
Objective Increased protein SUMOylation provides protection from cellular stress including oxidative stress, but the mechanisms involved are incompletely understood. The NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS) and oxidative stress and thus our goal was to determine whether SUMO regulates NADPH oxidase activity. Methods and Results Increased expression of SUMO1 potently inhibited the activity of Nox1-5. In contrast, inhibition of endogenous SUMOylation with siRNA to SUMO1 or UBC9 or with the inhibitor, anacardic acid, increased ROS production from HEK-Nox5 cells, human vascular smooth muscle cells and neutrophils. The suppression of ROS production was unique to SUMO1, required a C-terminal di-glycine and the SUMO-specific conjugating enzyme, UBC9. SUMO1 did not modify intracellular calcium or Nox5 phosphorylation but reduced ROS output in an isolated enzyme assay suggesting direct effects of SUMOylation on enzyme activity. However, we could not detect the presence of SUMO1-conjugation on Nox5 using a variety of approaches. Moreover the mutation of over 17 predicted and conserved lysine residues on Nox5 did not alter the inhibitory actions of SUMO1. Conclusion Together, these results suggest that SUMO is an important regulatory mechanism that indirectly represses the production of reactive oxygen species to ameliorate cellular stress.
Pandey, D; Chen, F; Patel, A; Wang, CY; Dimitropoulou, C.; Patel, V.S; Rudic, R.D.; Stepp, DW; Fulton, DJR.
Temporal changes in the expression of mRNA of NADPH oxidase subunits in renal epithelial cells exposed to oxalate or calcium oxalate crystals
Background. Exposure of renal epithelial cells to oxalate (Ox) or calcium oxalate (CaOx) crystals leads to the production of reactive oxygen species and cell injury. We have hypothesized that Ox and CaOx crystals activate NADPH oxidase through upregulation of its various subunits. Methods. Human renal epithelial-derived cell line, HK-2, was exposed to 100�?mol Ox or 66.7�?g/cm2 CaOx monohydrate crystals for 6, 12, 24 or 48�h. After exposure, the cells and media were processed to determine activation of NADPH oxidase, production of superoxide and 8-isoprostane (8IP), and release of lactate dehydrogenase (LDH). RT-PCR was performed to determine mRNA expression of NADPH subunits p22phox, p40phox, p47phox, p67phox and gp91phox as well as Rac-GTPase. Results. Exposure to Ox and CaOx crystals resulted in increase in LDH release, production of 8-IP, NADPH oxidase activity and production of superoxide. Exposure to CaOx crystals resulted in significantly higher NADPH oxidase activity, production of superoxide and LDH release than Ox exposure. Exposure to Ox and CaOx crystals altered the expression of various subunits of NADPH oxidase. More consistent were increases in the expression of membrane-bound p22phox and cytosolic p47phox. Significant and strong correlations were seen between NADPH oxidase activity, the expression of p22phox and p47phox, production of superoxide and release of LDH when cells were exposed to CaOx crystals. The expressions of neither p22phox nor p47phox were significantly correlated with increased NADPH oxidase activity after the Ox exposure. Conclusions. As hypothesized, exposure to Ox or CaOx crystals leads to significant increases in the expression of p22phox and p47phox, leading to activation of NADPH oxidase. Increased NADPH oxidase activity is associated with increased superoxide production and lipid peroxidation. Different pathways appear to be involved in the stimulation of renal epithelial cells by exposure to Ox and CaOx crystals.
Khan, Saeed R.; Khan, Aslam; Byer, Karen J.
The Anorexigenic Effect of Serotonin Is Mediated by the Generation of NADPH Oxidase-Dependent ROS
Serotonin (5-HT) is a central inhibitor of food intake in mammals. Thus far, the intracellular mechanisms for the effect of serotonin on appetite regulation remain unclear. It has been recently demonstrated that reactive oxygen species (ROS) in the hypothalamus are a crucial integrative target for the regulation of food intake. To investigate the role of ROS in the serotonin-induced anorexigenic effects, conscious mice were treated with 5-HT alone or combination with Trolox (a ROS scavenger) or Apocynin (an NADPH oxidase inhibitor) by acute intracerebroventricular injection. Both Trolox and Apocynin reversed the anorexigenic action of 5-HT and the 5-HT-induced hypothalamic ROS elevation. The mRNA and protein expression levels of pro-opiomelanocortin (POMC) were dramatically increased after ICV injection with 5-HT. The anorexigenic action of 5-HT was accompanied by markedly elevated hypothalamic MDA levels and GSH-Px activity, while the SOD activity was decreased. Moreover, 5-HT significantly increased the mRNA expression of UCP-2 but reduced the levels of UCP-3. Both Trolox and Apocynin could block the 5-HT-induced changes in UCP-2 and UCP-3 gene expression. Our study demonstrates for the first time that the anorexigenic effect of 5-HT is mediated by the generation of ROS in the hypothalamus through an NADPH oxidase-dependent pathway.
Wang, Li-Na; Yang, Jing; Zeng, Qing-Jie; Cheng, Xiao; Zhang, Zhi-Qi; Wang, Song-Bo; Gao, Ping; Zhu, Xiao-Tong; Xi, Qian-Yun; Zhang, Yong-Liang; Jiang, Qing-Yan
NADPH oxidase mediates glucolipotoxicity-induced beta cell dysfunction--clinical implications.
An impairment of glucose-stimulated insulin secretion--reflecting decreased glucokinase expression--and a moderate decrease in beta cell mass attributable to increased apoptosis, constitute the key features of beta cell failure in type 2 diabetes. Oxidative stress, provoked by prolonged exposure to excessive levels of glucose and/or fatty acids (glucolipotoxicity), appears to be a key mediator of these defects. Oxidant-provoked JNK activation induces nuclear export of the PDX-1 transcription factor, required for expression of glucokinase and other beta cell proteins. Conversely, increases in cAMP induced by incretin hormones promote the nuclear importation of PDX-1, counteracting the diabetogenic impact of oxidant stress; this may explain the utility of measures that slow dietary carbohydrate absorption for diabetes prevention. The ability of oxidative stress to boost apoptosis in beta cells is poorly understood, but may also entail JNK activation. Recent work establishes a phagocyte-type NADPH oxidase as the chief source of glucotoxicity-mediated oxidative stress in beta cells. Since bilirubin is now known to function physiologically as an inhibitor of NADPH oxidase, and phycocyanobilin (PCB) derived from spirulina likewise can inhibit this enzyme complex, supplemental PCB may have utility in the prevention and control of diabetes, and Gilbert syndrome, associated with chronically elevated free bilirubin, may be associated with decreased diabetes risk. PMID:19576699
McCarty, Mark F; Barroso-Aranda, Jorge; Contreras, Francisco
NADPH Oxidase, NOX1, Mediates Vascular Injury in Ischemic Retinopathy.
Abstract Aims: Ischemic retinal diseases such as retinopathy of prematurity are major causes of blindness due to damage to the retinal microvasculature. Despite this clinical situation, retinopathy of prematurity is mechanistically poorly understood. Therefore, effective preventative therapies are not available. However, hypoxic-induced increases in reactive oxygen species (ROS) have been suggested to be involved with NADPH oxidases (NOX), the only known dedicated enzymatic source of ROS. Our major aim was to determine the contribution of NOX isoforms (1, 2, and 4) to a rodent model of retinopathy of prematurity. Results: Using a genetic approach, we determined that only mice with a deletion of NOX1, but not NOX2 or NOX4, were protected from retinal neovascularization and vaso-obliteration, adhesion of leukocytes, microglial accumulation, and the increased generation of proangiogenic and proinflammatory factors and ROS. We complemented these studies by showing that the specific NOX inhibitor, GKT137831, reduced vasculopathy and ROS levels in retina. The source of NOX isoforms was evaluated in retinal vascular cells and neuro-glial elements. Microglia, the immune cells of the retina, expressed NOX1, 2, and 4 and responded to hypoxia with increased ROS formation, which was reduced by GKT137831. Innovation: Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity. Conclusions: Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathies. Antioxid. Redox Signal. 20, 2726-2740. PMID:24053718
Wilkinson-Berka, Jennifer L; Deliyanti, Devy; Rana, Indrajeetsinh; Miller, Antonia G; Agrotis, Alex; Armani, Roksana; Szyndralewiez, C�dric; Wingler, Kirstin; Touyz, Rhian M; Cooper, Mark E; Jandeleit-Dahm, Karin A; Schmidt, Harald H H W
The Importance of Myeloperoxidase in Apocynin-Mediated NADPH Oxidase Inhibition
Apocynin is widely used as an inhibitor of the NADPH oxidase. Since myeloperoxidase (MPO) has been considered as essential for the mechanism of action of apocynin, here we used cells with different levels of MPO and compared their sensitivity to apocynin. HL-60 cells were differentiated with DMSO or IFN?/TNF? and compared with peripheral mononuclear (PBMC) and polymorphonuclear cells (PMN). The relative MPO activity was PBMC = HL60 DMSO < HL60 IFN? < PMN. Apocynin inhibited the intracellular reactive oxygen species production by PMN (80%) and IFN?/TNF?-differentiated HL-60 cells (45%) but showed a minor effect in PBMC and DMSO differentiated HL-60 cells (20%). The addition of azide decreased the efficiency of apocynin in PMN and the addition of peroxidase increased the inhibition in PBMC. We also determined the gene expression of the components gp91phox, p47phox, p22phox and p67phox in the resting cells. Apocynin did not change gp91phox, p47phox or p22phox gene expression in nonstimulated PBMC, HL60 DMSO, HL60 IFN?/TNF?, and PMN and has a subtle increase in p67phox in HL60 IFN?/TNF?. The results from this work suggest that a rational search for better inhibitors of NADPH oxidase in leukocytes should include a correlation with their affinity as substrates for MPO.
de Almeida, Ana Carolina; dos Santos Vilela, Maria Marluce; Condino-Neto, Antonio; Ximenes, Valdecir F.
NADPH Oxidase Biology and the Regulation of Tyrosine Kinase Receptor Signaling and Cancer Drug Cytotoxicity
The outdated idea that reactive oxygen species (ROS) are only dangerous products of cellular metabolism, causing toxic and mutagenic effects on cellular components, is being replaced by the view that ROS have several important functions in cell signaling. In aerobic organisms, ROS can be generated from different sources, including the mitochondrial electron transport chain, xanthine oxidase, myeloperoxidase, and lipoxygenase, but the only enzyme family that produces ROS as its main product is the NADPH oxidase family (NOX enzymes). These transfer electrons from NADPH (converting it to NADP?) to oxygen to make O2�?. Due to their stability, the products of NADPH oxidase, hydrogen peroxide, and superoxide are considered the most favorable ROS to act as signaling molecules. Transcription factors that regulate gene expression involved in carcinogenesis are modulated by NADPH oxidase, and it has emerged as a promising target for cancer therapies. The present review discusses the mechanisms by which NADPH oxidase regulates signal transduction pathways in view of tyrosine kinase receptors, which are pivotal to regulating the hallmarks of cancer, and how ROS mediate the cytotoxicity of several cancer drugs employed in clinical practice.
Paletta-Silva, Rafael; Rocco-Machado, Nathalia; Meyer-Fernandes, Jose Roberto
NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality
Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.
Brothers, Kimberly M.; Gratacap, Remi L.; Barker, Sarah E.; Newman, Zachary R.; Norum, Ashley; Wheeler, Robert T.
Non-steroidal anti-inflammatory drugs activate NADPH oxidase in adipocytes and raise the H2O2 pool to prevent cAMP-stimulated protein kinase a activation and inhibit lipolysis
Background Non-steroidal anti-inflammatory drugs (NSAIDs) �aspirin, naproxen, nimesulide, and piroxicam� lowered activation of type II cAMP-dependent protein kinase A (PKA-II) in isolated rat adipocytes, decreasing adrenaline- and dibutyryl cAMP (Bt2cAMP)-stimulated lipolysis. The molecular bases of insulin-like actions of NSAID were studied. Results Based on the reported inhibition of lipolysis by H2O2, catalase was successfully used to block NSAID inhibitory action on Bt2cAMP-stimulated lipolysis. NSAID, at (sub)micromolar range, induced an H2O2 burst in rat adipocyte plasma membranes and in whole adipocytes. NSAID-mediated rise of H2O2 was abrogated in adipocyte plasma membranes by: diphenyleneiodonium, an inhibitor of NADPH oxidase (NOX); the NOX4 antibody; and cytochrome c, trapping the NOX-formed superoxide. These three compounds prevented the inhibition of Bt2cAMP-stimulated lipolysis by NSAIDs. Inhibition of aquaporin-mediated H2O2 transport with AgNO3 in adipocytes allowed NOX activation but prevented the lipolysis inhibition promoted by NSAID: i.e., once synthesized, H2O2 must reach the lipolytic machinery. Since insulin inhibits adrenaline-stimulated lipolysis, the effect of aspirin on isoproterenol-stimulated lipolysis in rat adipocytes was studied. As expected, isoproterenol-mediated lipolysis was blunted by both insulin and aspirin. Conclusions NSAIDs activate NOX4 in adipocytes to produce H2O2, which impairs cAMP-dependent PKA-II activation, thus preventing isoproterenol-activated lipolysis. H2O2 signaling in adipocytes is a novel and important cyclooxygenase-independent effect of NSAID.
NADPH oxidase and extracellular regulated kinases 1/2 are targets of prion protein signaling in neuronal and nonneuronal cells
Putative functions of the cellular prion protein, PrPC, include resistance to oxidative stress, copper uptake, cell adhesion, and cell signaling. Here, we report NADPH oxidase-dependent reactive oxygen species (ROS) production and extracellular regulated kinases 1/2 (ERK1/2) phosphorylation on PrPC stimulation in the 1C11 neuroectodermal precursor, in its neuronal differentiated progenies, and in GT1-7 neurohypothalamic and BW5147 lymphoid cells. In neuroprogenitor, hypothalamic, and lymphoid cells, ERK1/2 activation is fully controlled by the NADPH oxidase-dependent ROS production. In 1C11-derived bioaminergic cells, ROS signaling and ERK1/2 phosphorylation are both controlled by Fyn kinase activation, introducing some specificity in PrPC transduction associated with this neuronal context. These data argue for an ubiquitous function of PrPC in cell-redox homeostasis through ROS production.
Schneider, Benoit; Mutel, Vincent; Pietri, Mathea; Ermonval, Myriam; Mouillet-Richard, Sophie; Kellermann, Odile
Induction of gp91-phox, a component of the phagocyte NADPH oxidase, in microglial cells during central nervous system inflammation.
Gp91-phox is an integral component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex that generates reactive oxygen species (ROS) in activated circulating phagocytes. The authors previously demonstrated that gp91-phox knockout (KO) mice show significant protection from neuronal injury after cerebral ischemia--reperfusion injury, suggesting a pivotal role for this enzyme. Moreover, results from chimeric mice suggested that elimination of gp91-phox from both circulating phagocytes and a putative central nervous system (CNS) source were required to confer neuroprotection. In the current study, the authors demonstrated gp91-phox-specific immunostaining of perivascular cells in the CNS of control rats. However, after transient cerebral ischemia, gp91-phox-positive phagocytes were observed within the core ischemic region and activated microglial cells were positive in the penumbra. Such activated microglial cells were also gp91-phox-positive in the CNS of a chimpanzee with mild meningitis. Finally, in humans, both normal adult CNS tissues and isolated fetal microglial cells expressed gp91-phox mRNA. These microglia also expressed mRNA for the five other known components that comprise the NADPH oxidase complex. These data strongly suggest that microglial cells may contain a functionally active NADPH oxidase capable of generating ROS during CNS inflammation. PMID:11323523
Green, S P; Cairns, B; Rae, J; Errett-Baroncini, C; Hongo, J A; Erickson, R W; Curnutte, J T
Increased epidermal growth factor-like ligands are associated with elevated vascular NADPH oxidase in a primate model of atherosclerosis
Objective To characterize the relationship between expression of EGF-like ligands and vascular NADPH oxidase expression and activity in a primate model of atherosclerosis. Methods and Results Adult male Cynomolgus monkeys were fed a normal or atherogenic (AS) diet for 45 months, after which animals from the AS group were placed on a normal diet for 8 months (regression). Expression of membrane-associated EGF-like ligands was increased in arteries from animals on the AS diet and normalized in the regression group. EGF-like ligands were distributed throughout atherosclerotic vessels but predominantly co-localized with macrophages. Consistent with ligand shedding, circulating heparin bound-EGF was elevated in plasma of AS monkeys but not in those on regression diet. Atherosclerosis was associated with activation of EGF receptor (EGFR) signaling. Expression of NADPH oxidase subunits Nox1 and Nox2 but not Nox4 or Nox5 was increased in arteries from monkeys on the AS diet and returned to normal with regression. Levels of Nox1 and Nox2 positively correlated with EGF-like ligands. In cultured monkey smooth muscle cells, treatment with EGF-like ligands increased Nox1 expression and activity. Conclusions These data identify EGF-like ligands as potential modulators of atherogenesis due in part to increased vascular NADPH oxidase activity.
Stanic, Bojana; Pandey, Deepesh; Fulton, David J.; Miller, Francis J.
FFA-induced hepatic insulin resistance in vivo is mediated by PKC?, NADPH oxidase, and oxidative stress.
Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKC? and oxidative stress play causal roles in this process and whether this occurs via IKK?- and JNK-dependent pathways. Rats received a 7-h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2 h of the infusion, a hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-l-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased I?B? content, increased JNK phosphorylation (markers of IKK? and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver without affecting IH-induced hepatic PKC? activation. Furthermore, an antisense oligonucleotide against PKC? prevented IH-induced phosphorylation of p47(phox) (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similarly to NAC. These results demonstrate that PKC?, NADPH oxidase, and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA ? PKC? ? NADPH oxidase and oxidative stress ? IKK?/JNK ? impaired hepatic insulin signaling. PMID:24824652
Pereira, Sandra; Park, Edward; Mori, Yusaku; Haber, C Andrew; Han, Ping; Uchida, Toyoyoshi; Stavar, Laura; Oprescu, Andrei I; Koulajian, Khajag; Ivovic, Alexander; Yu, Zhiwen; Li, Deling; Bowman, Thomas A; Dewald, Jay; El-Benna, Jamel; Brindley, David N; Gutierrez-Juarez, Roger; Lam, Tony K T; Najjar, Sonia M; McKay, Robert A; Bhanot, Sanjay; Fantus, I George; Giacca, Adria
Inhibition of NADPH Oxidase Mediates Protective Effect of Cardiotonic Pills against Rat Heart Ischemia/Reperfusion Injury
Cardiotonic pill (CP) is a compound Chinese medicine currently used in China for treatment of ischemic angina pectoris. Our previous results indicated that a single dosing of CP pretreatment at 0.8?g/kg attenuates ischemia/reperfusion- (I/R-) induced myocardial injury and cardiac microcirculatory disturbance. The present study aimed to investigate the effect of CP at low dosage in a multiple dosing manner and to uncover the mechanism of antioxidative activity of CP. Male Sprague-Dawley rats were subjected to left anterior descending artery occlusion for 30?min followed by 60?min reperfusion. CP was administrated daily by gavage for six days at 0.1, 0.4, and 0.8?g/kg/day before I/R. Results showed that multiple dosing of CP at three doses significantly reduced I/R-induced myocardial injury, microcirculatory disturbance, and oxidative stress. CP dramatically inhibited I/R-induced nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase subunit gp91phox expression and p67phox and p47phox translocation from cytosol to cell membrane. Translocation of cytosolic subunits to membrane is required for the activation of NADPH oxidase. These data suggested that multiple dosing of CP at doses ranging from 0.1 to 0.8?g/kg/day reduced I/R-induced rat myocardial injury and microcirculatory disturbance, which was mediated by inhibition of NADPH oxidase activation.
Yang, Xiao-Yuan; Zhao, Na; Liu, Yu-Ying; Hu, Bai-He; Sun, Kai; Chang, Xin; Wei, Xiao-Hong; Fan, Jing-Yu; Han, Jing-Yan
Arthritis suppression by NADPH activation operates through an interferon-? pathway
Background A polymorphism in the activating component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, neutrophil cytosolic factor 1 (NCF1), has previously been identified as a regulator of arthritis severity in mice and rats. This discovery resulted in a search for NADPH oxidase-activating substances as a potential new approach to treat autoimmune disorders such as rheumatoid arthritis (RA). We have recently shown that compounds inducing NCF1-dependent oxidative burst, e.g. phytol, have a strong ameliorating effect on arthritis in rats. However, the underlying molecular mechanism is still not clearly understood. The aim of this study was to use gene-expression profiling to understand the protective effect against arthritis of activation of NADPH oxidase in the immune system. Results Subcutaneous administration of phytol leads to an accumulation of the compound in the inguinal lymph nodes, with peak levels being reached approximately 10 days after administration. Hence, global gene-expression profiling on inguinal lymph nodes was performed 10 days after the induction of pristane-induced arthritis (PIA) and phytol administration. The differentially expressed genes could be divided into two pathways, consisting of genes regulated by different interferons. IFN-? regulated the pathway associated with arthritis development, whereas IFN-? regulated the pathway associated with disease protection through phytol. Importantly, these two molecular pathways were also confirmed to differentiate between the arthritis-susceptible dark agouti (DA) rat, (with an Ncf-1DA allele that allows only low oxidative burst), and the arthritis-protected DA.Ncf-1E3 rat (with an Ncf1E3 allele that allows a stronger oxidative burst). Conclusion Naturally occurring genetic polymorphisms in the Ncf-1 gene modulate the activity of the NADPH oxidase complex, which strongly regulates the severity of arthritis. We now show that the Ncf-1 allele that enhances oxidative burst and protects against arthritis is operating through an IFN-?-associated pathway, whereas the arthritis-driving allele operates through an IFN-?-associated pathway. Treatment of arthritis-susceptible rats with an NADPH oxidase-activating substance, phytol, protects against arthritis. Interestingly, the treatment led to a restoration of the oxidative-burst effect and induction of a strikingly similar IFN-?-dependent pathway, as seen with the disease-protective Ncf1 polymorphism.
Olofsson, Peter; Nerstedt, Annika; Hultqvist, Malin; Nilsson, Elisabeth C; Andersson, Sofia; Bergelin, Anna; Holmdahl, Rikard
Expression of NADPH Oxidase (NOX) 5 in Rabbit Corneal Stromal Cells
Purpose To determine whether NOX 5 is expressed in rabbit corneal stromal cells (RCSC). NADPH oxidases (NOXes) are enzymes that preferentially use NADPH as a substrate and generate superoxide. Several isoforms of NOXes function as multi-protein complexes while NOX5 and DUOXs do not require the accessory proteins for their activity and possess calcium binding EF hands. Methods Human NOX5 primers were used to amplify the rabbit NOX5 by RT-PCR. Amplified product was sequenced to confirm its identity. The protein encoded by the NOX5 was identified by western blot analysis. NOX5 siRNA was used to reduce transcript, protein, and calcium stimulated activity. In silico analyses were performed to establish the putative structure, functions, and evolution of rabbit NOX5. Results NOX activity was measured in RCSC with NADPH rather than NADH as a substrate. RT-PCR with NOX5 primers amplified 288 bp product using RCSC cDNA, which, when sequenced, confirmed its identity to human NOX5 mRNA. This sequence was used to predict the rabbit (Oryctolagus cuniculus) NOX5 gene. NOX5 siRNA reduced amounts of NOX5 mRNA in RCSC and reduced ionomycin stimulated superoxide production. A protein of about 65 to 70 kDa encoded by the NOX5 was detected by western blot analysis. In silico analysis predicted a putative rabbit NOX5 protein containing 801 amino acids. Motif searches predicted the presence of at least 3 putative EF-hands in N-terminus and a NOX domain in C terminal region. Conclusions The data document that the NOX5 gene was expressed in cells of lagomorphs unlike rodents, making the rabbit an interesting model to study NOX5 functions. The activity of the rabbit NOX5 was calcium stimulated, a trait of NOX5 in general. NOX5 may also prove to be a useful genetic marker for studying the taxonomic position of lagomorphs and the Glires classification.
Rizvi, Farhan; Heimann, Tom; O'Brien, William J.
Deficiency of NADPH Oxidase Components p47phox and gp91phox Caused Granulomatous Synovitis and Increased Connective Tissue Destruction in Experimental Arthritis Models
Recent studies indicated that the nicotinamide dinucleotide phosphate oxidase (NADPH) oxidase-derived oxygen radicals plays a deleterious role in arthritis. To study this in more detail, gonarthritis was induced in NADPH oxidase-deficient mice. Mice received an intraarticular injection of either zymosan, to elicit an irritant-induced inflammation, or poly-l-lysine coupled lysozyme, to evoke an immune-complex mediated inflammation in passively immunized mice. In contrast to wild-type mice, arthritis elicited in both p47phox?/? and gp91?/? mice showed more severe joint inflammation, which developed into a granulomatous synovitis. Treatment with either Zileuton or cobra venom factor showed that the chemokines LTB4 and complement C3 were not the driving force behind the aggravated inflammation in these mice. Arthritic NADPH oxidase-deficient mice showed irreversible cartilage damage as judged by the enhanced aggrecan VDIPEN expression, and chondrocyte death. Furthermore, only in the absence of NADPH oxidase-derived oxygen radicals, the arthritic joints showed osteoclast-like cells, tartrate-resistant acid phosphatase (TRAP)-positive/multinucleated cells, extensive bone erosion, and osteolysis. The enhanced synovial gene expression of tumor necrosis factor-?, interleukin-1?, matrix metalloproteinase (MMP)-3, MMP-9 and receptor activator of NF-?B ligand (RANKL) might contribute to the aggravated arthritis in the NADPH oxidase-deficient mice. This showed that the involvement of NADPH oxidase in arthritis is probably far more complex and that oxygen radicals might also be important in controlling disease severity, and reducing joint inflammation and connective tissue damage.
van de Loo, Fons A. J.; Bennink, Miranda B.; Arntz, Onno J.; Smeets, Ruben L.; Lubberts, Erik; Joosten, Leo A. B.; van Lent, Peter L. E. M.; Coenen-de Roo, Christina J. J.; Cuzzocrea, Salvatore; Segal, Brahm H.; Holland, Steven M.; van den Berg, Wim B.
NADPH Oxidase and Angiogenesis Following Endothelin-1 Induced Stroke in Rats: Role for Nox2 in Brain Repair
NADPH oxidases contribute to brain injury, yet they may also have a role in brain repair, particularly in vascular signaling and angiogenesis. This study determined the temporal and spatial profile of NADPH oxidase subunit expression/activity concurrently with angiogenesis in the brain following transient ischemic stroke induced by prolonged constriction of the middle cerebral artery by perivascular injection of endothelin-1 in conscious Hooded Wistar rats (n = 47). VEGF mRNA expression was increased in the ipsilateral cortex and striatum between 6 h and 28 days post-stroke concurrently with a marked increase in Nox2 mRNA expression up to 7 days, and increased Nox4 mRNA expression detected between 7 and 28 days. Point counting of blood vessels using Metamorph imaging software showed increased vascular sprouting between 3 and 7 days after stroke with new vascular networks detected in the core infarct region by 14 days. Angiogenic blood vessels 3 and 7 days post-stroke were observed to co-localise with both Nox2 antibody and dihydroethidium fluorescence suggesting a role for Nox2 generated superoxide during the phase of vascular remodeling, whilst Nox4 expression was detected once new cerebral vessels had formed. These results indicate for the first time that ROS signaling through a cerebrovascular Nox2 NADPH oxidase may be important in initiating brain angiogenesis.
Taylor, Caroline J.; Weston, Robert M.; Dusting, Gregory J.; Roulston, Carli L.
Apocynin-Treatment Reverses Hyperoxaluria Induced Changes in NADPH Oxidase System Expression in Rat Kidneys: A Transcriptional Study
Purpose We have previously shown that production of reactive oxygen species (ROS) is an important contributor to renal injury and inflammation following exposure to oxalate (Ox) or calcium-oxalate (CaOx) crystals. The present study was conducted, utilizing global transcriptome analyses, to determine the effect of Apocynin on changes in the NADPH oxidase system activated in kidneys of rats fed a diet leading to hyperoxaluria and CaOx crystal deposition. Approach Age-, sex- and weight-matched rats were either fed regular rat chow or regular rat chow supplemented with 5% w/w hydroxy-L-proline (HLP). Half of the rats on the HLP diet were also placed on Apocynin-supplemented H2O. After 28 days, each rat was euthanized, their kidneys freshly explanted and dissected to obtain both cortex and medulla tissues. Total RNA was extracted from each tissue and subjected to genomic microarrays to obtain global transcriptome data. KEGG was used to identify gene clusters with differentially expressed genes. Immunohistochemistry was used to confirm protein expressions of selected genes. Results Genes encoding both membrane- and cytosolic-NADPH oxidase complex-associated proteins, together with p21rac and Rap1a, were coordinately up-regulated significantly in both renal medulla and cortex tissues in the HLP-fed rats compared to normal healthy untreated controls. Activation of NADPH oxidase appears to occur via the angiotensin-II/angiotensin-II receptor-2 pathway, although the DAG-PKC pathway of neutrophils may also contribute. Immuno histochemical staining confirmed up-regulated gene expressions. Simultaneously, genes encoding ROS scavenger proteins were down-regulated. HLP-fed rats receiving Apocynin had a complete reversal in the differential-expression of the NADPH oxidase system genes, despite showing similar levels of hyperoxaluria. Conclusions A strong up-regulation of an oxidative/respiratory burst involving the NADPH oxidase system, activated via the angiotensin-II and most likely the DAG-PKC pathways, occurs in kidneys of hyperoxaluric rats. Apocynin treatment reversed this activation without affecting the levels of hyperoxaluria.
Joshi, Sunil; Saylor, Benjamin T.; Wang, Wei; Peck, Ammon B.; Khan, Saeed R.
NADPH Oxidases in Heart Failure: Poachers or Gamekeepers?
Abstract Significance: Oxidative stress is involved in the pathogenesis of heart failure but clinical antioxidant trials have been unsuccessful. This may be because effects of reactive oxygen species (ROS) depend upon their source, location, and concentration. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) proteins generate ROS in a highly regulated fashion and modulate several components of the heart failure phenotype. Recent Advances: Two Nox isoforms, Nox2 and Nox4, are expressed in the heart. Studies using gene-modified mice deficient in Nox2 activity indicate that Nox2 activation contributes to angiotensin II�induced cardiomyocyte hypertrophy, atrial fibrillation, and the development of interstitial fibrosis but may also positively modulate physiological excitation-contraction coupling. Nox2 contributes to myocyte death under stress situations and plays important roles in postmyocardial infarction remodeling, in part by modulating matrix metalloprotease activity. In contrast to Nox2, Nox4 is constitutively active at a low level and induces protective effects in the heart under chronic stress, for example, by maintaining myocardial capillary density. However, high levels of Nox4 could have detrimental effects. Critical Issues: The effects of Nox proteins during the development of heart failure likely depend upon the isoform, activation level, and cellular distribution, and may include beneficial as well as detrimental effects. More needs to be learnt about the precise regulation of abundance and biochemical activity of these proteins in the heart as well as the downstream signaling pathways that they regulate. Future Directions: The development of specific approaches to target individual Nox isoforms and/or specific cell types may be important for the achievement of therapeutic efficacy in heart failure. Antioxid. Redox Signal. 18, 1024�1041.
Zhang, Min; Perino, Alessia; Ghigo, Alessandra; Hirsch, Emilio
The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress
We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-{kappa}B and decreased intracellular level of its inhibitor IkB{alpha}. These effects, accompanied by increased production of H{sub 2}O{sub 2}, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-{kappa}B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.
Riganti, Chiara [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Torino (Italy)], E-mail: email@example.com; Costamagna, Costanzo; Doublier, Sophie; Miraglia, Erica; Polimeni, Manuela; Bosia, Amalia; Ghigo, Dario [Dipartimento di Genetica, Biologia e Biochimica, Universita di Torino, Via Santena 5/bis, 10126 Torino (Italy)
Ethanol preconditioning protects against ischemia/reperfusion-induced brain damage: Role of NADPH oxidase-derived ROS
Ethanol preconditioning (EtOH-PC) refers to a phenomenon in which tissues are protected from the deleterious effects of ischemia/reperfusion (I/R) by prior ingestion of ethanol at low to moderate levels. In this study, we tested whether prior (24 hrs) administration of ethanol as a single bolus that produced a peak plasma concentration of 42-46 mg/dl in gerbils would offer protective effects on neuronal damage due to cerebral I/R. In addition, we also tested whether reactive oxygen species (ROS)-derived from NADPH oxidase played a role as an initiator of these putative protective effects. Groups of gerbils were administered either ethanol or the same volume of water by gavage 24 hrs prior to transient global cerebral ischemia induced by occlusion of both common carotid arteries (CCA) for 5 min. In some experiments, apocynin, a specific inhibitor of NADPH oxidase, was administered (5 mg/kg body wt, i.p.) 10 min before ethanol administration. EtOH-PC ameliorated behavioral deficit induced by cerebral I/R and protected the brain against I/R-induced delayed neuronal death (DND), neuronal and dendritic degeneration, oxidative DNA damage, and glial cell activation. These beneficial effects were attenuated by apocynin treatment coincident with ethanol administration. Ethanol ingestion was associated with translocation of the NADPH oxidase subunit, p67phox, from hippocampal cytosol fraction to membrane, increased NADPH oxidase activity in hippocampus within the first hour after gavage, and increased lipid peroxidation (4-hydroxy-2-nonenal, HNE) in plasma and hippocampus within the first 2 hrs after gavage. These effects were also inhibited by concomitant apocynin treatment. Our data are consistent with the hypothesis that antecedent ethanol ingestion at socially-relevant levels induces neuroprotective effects in I/R by a mechanism that is triggered by ROS produced through NADPH oxidase. Our results further suggest the possibility that preconditioning with other pharmacological agents that induce a mild oxidative stress may have similar therapeutic value for suppressing stroke-mediated damage in brain.
Wang, Qun; Sun, Albert Y.; Simonyi, Agnes; Kalogeris, Theodore J.; Miller, Dennis K.; Sun, Grace Y.; Korthuis, Ronald J.
Assembly of the phagocyte NADPH oxidase complex: chimeric constructs derived from the cytosolic components as tools for exploring structure-function relationships.
Phagocytes generate superoxide (O2*-) by an enzyme complex known as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Its catalytic component, responsible for the NADPH-driven reduction of oxygen to O2*-, is flavocytochrome b559, located in the membrane and consisting of gp91phox and p22phox subunits. NADPH oxidase activation is initiated by the translocation to the membrane of the cytosolic components p47phox, p67phox, and the GTPase Rac. Cytochrome b559 is converted to an active form by the interaction of gp91phox with p67phox, leading to a conformational change in gp91phox and the induction of electron flow. We designed a new family of NADPH oxidase activators, represented by chimeras comprising various segments of p67phox and Rac1. The prototype chimera p67phox (1-212)-Rac1 (1-192) is a potent activator in a cell-free system, also containing membrane p47phox and an anionic amphiphile. Chimeras behave like bona fide GTPases and can be prenylated, and prenylated (p67phox -Rac1) chimeras activate the oxidase in the absence of p47phox and amphiphile. Experiments involving truncations, mutagenesis, and supplementation with Rac1 demonstrated that the presence of intrachimeric bonds between the p67phox and Rac1 moieties is an absolute requirement for the ability to activate the oxidase. The presence or absence of intrachimeric bonds has a major impact on the conformation of the chimeras, as demonstrated by fluorescence resonance energy transfer, small angle X-ray scattering, and gel filtration. Based on this, a "propagated wave" model of NADPH oxidase activation is proposed in which a conformational change initiated in Rac is propagated to p67phox and from p67phox to gp91phox. PMID:16641134
Mizrahi, Ariel; Berdichevsky, Yevgeny; Ugolev, Yelena; Molshanski-Mor, Shahar; Nakash, Yael; Dahan, Iris; Alloul, Nathalie; Gorzalczany, Yara; Sarfstein, Rive; Hirshberg, Miriam; Pick, Edgar
Annexin Peptide Ac2-26 Suppresses TNF?-Induced Inflammatory Responses via Inhibition of Rac1-Dependent NADPH Oxidase in Human Endothelial Cells
The anti-inflammatory peptide annexin-1 binds to formyl peptide receptors (FPR) but little is known about its mechanism of action in the vasculature. Here we investigate the effect of annexin peptide Ac2-26 on NADPH oxidase activity induced by tumour necrosis factor alpha (TNF?) in human endothelial cells. Superoxide release and intracellular reactive oxygen species (ROS) production from NADPH oxidase was measured with lucigenin-enhanced chemiluminescence and 2?,7?-dichlorodihydrofluorescein diacetate, respectively. Expression of NADPH oxidase subunits and intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were determined by real-time PCR and Western blot analysis. Promoter activity of nuclear factor kappa B (NF?B) was measured by luciferase activity assay. TNF? stimulated NADPH-dependent superoxide release, total ROS formation and expression of ICAM-1and VCAM-1. Pre-treatment with N-terminal peptide of annexin-1 (Ac2-26, 0.5�1.5 �M) reduced all these effects, and the inhibition was blocked by the FPRL-1 antagonist WRW4. Furthermore, TNF?-induced NF?B promoter activity was attenuated by both Ac2-26 and NADPH oxidase inhibitor diphenyliodonium (DPI). Surprisingly, Nox4 gene expression was reduced by TNF? whilst expression of Nox2, p22phox and p67phox remained unchanged. Inhibition of NADPH oxidase activity by either dominant negative Rac1 (N17Rac1) or DPI significantly attenuated TNF?-induced ICAM-1and VCAM-1 expression. Ac2-26 failed to suppress further TNF?-induced expression of ICAM-1 and VCAM-1 in N17Rac1-transfected cells. Thus, Ac2-26 peptide inhibits TNF?-activated, Rac1-dependent NADPH oxidase derived ROS formation, attenuates NF?B pathways and ICAM-1 and VCAM-1 expression in endothelial cells. This suggests that Ac2-26 peptide blocks NADPH oxidase activity and has anti-inflammatory properties in the vasculature which contributes to modulate in reperfusion injury inflammation and vascular disease.
Goh, Celeste; Liu, Guei-Sheung; Jiang, Fan; Chan, Elsa C.; Dusting, Gregory J.
Association of Anthracycline-Related Cardiac Histological Lesions With NADPH Oxidase Functional Polymorphisms
Objective. Treatment with anthracyclines may cause cardiac dysfunction, but the sequence of anthracycline-induced heart lesions has been incompletely characterized. NADPH oxidase, a key mediator of oxidative cardiac damage and remodeling, modulates anthracycline clinical cardiotoxicity. Our aim was to determine which cardiac histological lesions are specifically induced by anthracycline treatment and to investigate the role of NADPH functional genetic polymorphisms in their development. Patients and Methods. Using a retrospective case�control design, we evaluated cardiac histological lesions and NADPH genotype (polymorphisms rs1883112, rs4673, and rs13058338) in 97 consecutive decedents with a cancer diagnosis (48 treated with anthracyclines). Results. Myocytolysis (60%), patched myocardial necrosis (19%), and myocardial fibrosis (diffuse and patched; 62% and 23%, respectively) were associated with anthracycline treatment. In patients receiving anthracyclines, NADPH oxidase polymorphism rs4673 protected against focal myocardial necrosis (odds ratio [OR], 0.11; 95% confidence interval [CI], 0.20�0.63) whereas rs1883112 was strongly associated with cardiac fibrosis (OR, 5.11; 95% CI, 1.59�16.43), which was present in all homozygotes. Conclusion. Anthracyclines induce a cardiac remodeling pattern characterized by interstitial or patched fibrosis. The contribution of the functionally relevant NADPH polymorphisms rs1883112 and rs4673 to anthracycline-related heart lesions provides a plausible explanation for their modulation of cardiotoxicity. If confirmed, these findings may lead to better individualized strategies for early detection and prevention of anthracycline cardiotoxicity.
Cascales, Almudena; Pastor-Quirante, Francisco; Sanchez-Vega, Beatriz; Luengo-Gil, Gines; Corral, Javier; Ortuno-Pacheco, Guzman; Vicente, Vicente
Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress
NASA Technical Reports Server (NTRS)
Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.
McNally, J. Scott; Davis, Michael E.; Giddens, Don P.; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G.
Hu-Lu-Ba-Wan Attenuates Diabetic Nephropathy in Type 2 Diabetic Rats through PKC-?/NADPH Oxidase Signaling Pathway
Hu-Lu-Ba-Wan (HLBW) is a Chinese herbal prescription used to treat kidney deficiency. The aim of this study was to explore the effect and mechanism of HLBW on diabetic nephropathy (DN) in type 2 diabetic rats. The rat model of DN was established by being fed a high-fat diet and intravenous injection of streptozotocin. Then, HLBW decoction was administered for 16 weeks. Blood glucose level, lipid profile, renal function, 24-hour total urinary protein, and albumin content were examined. Renal morphology and superoxide anion levels were evaluated. The activity of nicotinamide-adenine dinucleotide phosphate (NADPH) and protein kinase C-alpha (PKC-?) related genes expression in renal tissue were also determined. Our data demonstrated that HLBW significantly improved hyperglycemia, hyperlipidemia, and proteinuria in diabetic rats compared with those of control group. HLBW also alleviated glomerular expansion and fibrosis, extracellular matrix accumulation and effacement of the foot processes. Additionally, HLBW reduced superoxide anion level, NADPH oxidase activity, the protein and mRNA expressions of p47phox, and the protein expression of phosphorylated PKC-? in renal tissue. These results suggest that HLBW is effective in the treatment of DN in rats. The underlying mechanism may be related to the attenuation of renal oxidative stress via PKC-?/NADPH oxidase signaling pathway.
Zhou, Lishan; Dong, Hui; Huang, Yi; Xu, Lijun; Zou, Xin; Wang, Kaifu; Chen, Guang; Lu, Fuer
NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action
BACKGROUND Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood. EXPERIMENTAL APPROACH Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins. KEY RESULTS Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration�response curves exhibiting higher Hill coefficients and lower IC50 values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC50 of 1.24 and 8.4 �M for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47phox translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47phox and disrupted the binding of the proline rich region of p22phox to the tandem SH3 domain of p47phox and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively. CONCLUSIONS AND IMPLICATIONS These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome.
Jaquet, Vincent; Marcoux, Julien; Forest, Eric; Leidal, Kevin G; McCormick, Sally; Westermaier, Yvonne; Perozzo, Remo; Plastre, Olivier; Fioraso-Cartier, Laetitia; Diebold, Becky; Scapozza, Leonardo; Nauseef, William M; Fieschi, Franck; Krause, Karl-Heinz; Bedard, Karen
Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ssI and prooxidant enzyme NADPH oxidase
Blood�brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25�mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100�nM) significantly increased NADPH oxidase activity, O2�- generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-� or PKC-�I via their specific inhibitors and neutralisation of O2�- by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-�I gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2�- production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-�I and NADPH oxidase.
Shao, Beili; Bayraktutan, Ulvi
Loss of cytochrome c oxidase promotes RAS-dependent ROS production from the ER resident NADPH oxidase, Yno1p, in yeast.
Many disease states, including the aging process, are associated with the accumulation of mitochondria harboring respiratory dysfunction. Mitochondrial dysfunction is often accompanied by increased ROS levels that can contribute to cellular dysfunction and disease etiology. Here we use the model eukaryote S. cerevisiae to investigate whether reduced cytochrome c oxidase (COX) activity, commonly reported in aging organisms and associated with neurodegenerative disorders, leads to ROS production from mitochondria. We provide evidence that although reduced COX complex activity correlates with ROS accumulation, mitochondria are not the major production center. Instead we show that COX-deficient mitochondria activate Ras upon their outer membrane that establishes a pro-ROS accumulation environment by suppressing antioxidant defenses and the ERAD-mediated turnover of the ER-localized NADPH oxidase Yno1p. Our data suggest that dysfunctional mitochondria can serve as a signaling platform to promote the loss of redox homeostasis, ROS accumulation, and accelerate aging in yeast. PMID:23931758
Leadsham, Jane E; Sanders, Geraldine; Giannaki, Samantha; Bastow, Emma L; Hutton, Rachael; Naeimi, Wesley R; Breitenbach, Michael; Gourlay, Campbell W
Pharmacological Inhibition of Neuronal NADPH Oxidase Protects against 1-Methyl-4-Phenylpyridinium (MPP+)-Induced Oxidative Stress and Apoptosis in Mesencephalic Dopaminergic Neuronal Cells
Oxidative stress is widely recognized as a key mediator of degenerative processes in Parkinson�s disease (PD). Recently, we demonstrated that the dopaminergic toxin MPP+ initiates oxidative stress to cause caspase-3-dependent apoptotic cell death in mesencephalic dopaminergic neuronal (N27) cells. In this study, we determined the source of reactive oxygen species (ROS) produced during MPP+-induced apoptotic cell death. In addition to mitochondria, plasma membrane NADPH oxidase is considered a major producer of ROS inside the cell. Here, we show that N27 cells express key NADPH oxidase subunits gp91phox and p67phox. We used structurally diverse NADPH oxidase inhibitors, aminoethyl-benzenesulfonylfluoride (AEBSF, 100-1000 ?M), apocynin (100-1000 ?M), and diphenylene iodonium (DPI, 3-30 ?M), to inhibit intrinsic NADPH oxidase activity in N27 cells. Flow cytometric analysis using the ROS-sensitive dye hydroethidine revealed that AEBSF blocked 300 ?M MPP+-induced ROS production for over 45 min in N27 cells, in a dose-dependent manner. Further treatment with DPI, apocynin, and SOD also blocked MPP+-induced ROS production. In Sytox cell death assays, co-treatment with AEBSF, apocynin, or DPI for 24 hr significantly suppressed MPP+-induced cytotoxic cell death. Similarly, co-treatment with these inhibitors also significantly attenuated MPP+-induced increases in caspase-3 enzymatic activity. Furthermore, quantitative DNA fragmentation ELISA assays revealed that AEBSF, DPI, and apocynin rescue N27 cells from MPP+-induced apoptotic cell death. Together, these results indicate for the first time that intracellular ROS generated by NAPDH oxidase are present within the mesencephalic neuronal cells, and are a key determinant of MPP+-mediated dopaminergic degeneration in in vitro models of dopaminergic degeneration. This study supports a critical role of NADPH oxidase in the oxidative damage in PD; targeting this enzyme may lead to novel therapies for PD.
Anantharam, Vellareddy; Kaul, Siddharth; Song, Chunjuan; Kanthasamy, Arthi; Kanthasamy, Anumantha G.
Polymorphism of the NADH\\/NADPH Oxidase p22 phox Gene in Patients With Coronary Artery Disease
Background�Oxidative stress in the vasculature has been implicated in the pathogenesis of coronary artery disease (CAD). NADH\\/NADPH oxidase is a key enzyme of superoxide production in the vasculature. p22 phox, an essential component of NADH\\/NADPH oxidase, has four types of polymorphism. The C242T polymorphism changes histidine-72 to tyrosine, located in the potential heme-binding sites, whereas A640G polymorphism is located in
Nobutaka Inoue; Seinosuke Kawashima; Kenji Kanazawa; Shinichiro Yamada; Hozuka Akita; Mitsuhiro Yokoyama
ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence.
Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22(phox) that produces H(2)O(2). Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by ?-H2A.X foci analysis. Using HyPer, a specific probe for H(2)O(2), we detected an increase in H(2)O(2) in the nucleus correlated with NOX4-p22(phox) perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1? foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence. PMID:21841825
Weyemi, U; Lagente-Chevallier, O; Boufraqech, M; Prenois, F; Courtin, F; Caillou, B; Talbot, M; Dardalhon, M; Al Ghuzlan, A; Bidart, J-M; Schlumberger, M; Dupuy, C
NADPH oxidase inhibition reduces tubular sodium transport and improves kidney oxygenation in diabetes
Sustained hyperglycemia is associated with increased oxidative stress resulting in decreased intrarenal oxygen tension (Po2) due to increased oxygen consumption (Qo2). Chronic blockade of the main superoxide radicals producing system, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalizes Qo2 by isolated proximal tubular cells (PTC) and reduces proteinuria in diabetes. The aim was to investigate the effects of acute NADPH oxidase inhibition on tubular Na+ transport and kidney Po2 in vivo. Glomerular filtration rate (GFR), renal blood flow (RBF), filtration fraction (FF), Na+ excretion, fractional Li+ excretion, and intrarenal Po2 was measured in control and streptozotocin-diabetic rats during baseline and after acute NADPH oxidase inhibition using apocynin. The effects on tubular transporters were investigated using freshly isolated PTC. GFR was increased in diabetics compared with controls (2.2 � 0.3 vs. 1.4 � 0.1 ml�min?1�kidney?1). RBF was similar in both groups, resulting in increased FF in diabetics. Po2 was reduced in cortex and medulla in diabetic kidneys compared with controls (34.4 � 0.7 vs. 42.5 � 1.2 mmHg and 15.7 � 1.2 vs. 25.5 � 2.3 mmHg, respectively). Na+ excretion was increased in diabetics compared with controls (24.0 � 4.7 vs. 9.0 � 2.0 ?m�min?1�kidney?1). In controls, all parameters were unaffected. However, apocynin increased Na+ excretion (+112%) and decreased fractional lithium reabsorption (?10%) in diabetics, resulting in improved cortical (+14%) and medullary (+28%) Po2. Qo2 was higher in PTC isolated from diabetic rats compared with control. Apocynin, dimethylamiloride, and ouabain reduced Qo2, but the effects of combining apocynin with either dimethylamiloride or ouabain were not additive. In conclusion, NADPH oxidase inhibition reduces tubular Na+ transport and improves intrarenal Po2 in diabetes.
Persson, Patrik; Hansell, Peter
NADPH oxidase inhibition improves neurological outcomes in surgically-induced brain injury
Neurosurgical procedures can result in brain injury by various means including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically-induced brain injury (SBI) can cause post-operative complications such as brain edema. By creating a mouse model of SBI, we tested whether NADPH oxidase, an important reactive oxygen species producing enzyme, is involved in SBI using transgenic mice lacking gp91phox subunit
Wendy Lo; Thomas Bravo; Vikram Jadhav; Elena Titova; John H. Zhang; Jiping Tang
Diphenyleneiodonium, an NAD(P)H Oxidase Inhibitor, also Potently Inhibits Mitochondrial Reactive Oxygen Species Production
Diphenyleneiodonium (DPI) has frequently been used to inhibit reactive oxygen species (ROS) production mediated by flavoenzymes, particularly NAD(P)H oxidase. This study was undertaken to examine if DPI could also inhibit production of superoxide and H2O2by mitochondria, the major source of cellular ROS. Detection of mitochondrial superoxide by lucigenin-derived chemiluminescence (CL) with unstimulated monocytes\\/macrophages showed that DPI at concentrations that inhibit
Yunbo Li; Michael A. Trush
Methamphetamine (METH) is a drug of abuse with neurotoxic and vascular effects that may be mediated by reactive oxygen species (ROS). However, potential sources of METH-induced generation of ROS are not fully understood. The present study is focused on the role of NAD(P)H oxidase (NOX) in METH-induced dysfunction of brain endothelial cells. Treatment with METH induced a time-dependent increase in phosphorylation of NOX subunit p47, followed by its binding with gp91 and p22, and the formation of an active NOX complex. An increase in NOX activity was associated with elevated production of ROS, alterations of occludin levels, and increased transendothelial migration of monocytes. Inhibition of NOX by NSC 23766 attenuated METH-induced ROS generation, changes in occludin protein levels, and monocyte migration. Because an active NOX complex is localized to caveolae, we next evaluated the role of caveolae in METH-mediated toxicity to brain endothelial cells. Treatment with METH induced phosphorylation of ERK1/2 and caveolin-1 protein. Inhibition of ERK1/2 activity or caveolin-1 silencing protected against METH-induced alterations of occludin levels. These findings indicate an important role of NOX and functional caveolae in METH-induced oxidative stress in brain endothelial cells that contribute to the subsequent alterations of occludin levels and transendothelial migration of inflammatory cells.
Park, Minseon; Hennig, Bernhard; Toborek, Michal
Relative contributions of mitochondria and NADPH oxidase to deoxycorticosterone acetate-salt hypertension in mice.
We assessed the relative contribution of the mitochondrial respiratory chain and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase to deoxycorticosterone acetate (DOCA)-salt hypertension in mice. The daily mean arterial pressure was monitored by radiotelemetry in DOCA-salt-treated mice given vehicle or the mitochondrial respiratory chain complex I inhibitor rotenone. This treatment produced remarkable attenuation of DOCA-salt hypertension. Similar results were obtained with other inhibitors of mitochondrial function, including 5-hydroxydecanoate (specific for mitochondrial potassium-ATP channels), benzylguanidine (complexes I and III), and the cell-permeable manganese tetrakis (4-benzoic acid) porphyrin (a mimic of mitochondrial superoxide dismutase). In parallel with the blood pressure-lowering effect of rotenone, the DOCA-salt-induced increases in urinary 8-isoprostane excretion and in reactive oxygen species production of isolated kidney mitochondria were both significantly attenuated. Conversely, the DOCA-salt-induced reduction of urinary nitrate/nitrite excretion was significantly elevated. Following DOCA-salt treatment, mice deficient in NADPH oxidase subunits gp91(phox) or p47(phox) exhibited a partial attenuation of the hypertensive response at early but not later time points. Thus, the mitochondrial respiratory chain is a major source of oxidative stress in DOCA-salt hypertension, whereas NADPH oxidase may have a relatively minor role during the early stage of hypertension. PMID:21368743
Zhang, Aihua; Jia, Zhanjun; Wang, Ningning; Tidwell, Tyson J; Yang, Tianxin
Astragaloside IV prevents damage to human mesangial cells through the inhibition of the NADPH oxidase/ROS/Akt/NF??B pathway under high glucose conditions.
Glomerular hypertrophy and hyperfiltration are the two major pathological characteristics of the early stages of diabetic nephropathy�(DN), which are respectively related to mesangial cell�(MC) proliferation and a decrease in calcium influx conducted by canonical transient receptor potential cation channel�6�(TRPC6). The marked increase in the production of reactive oxygen species�(ROS) induced by hyperglycemia is the main sponsor of multiple pathological pathways in DN. Nicotinamide adenine dinucleotide phosphate�(NADPH) oxidase is an important source of ROS production in MCs. Astragaloside�IV�(AS?IV) is an active ingredient of Radix Astragali which has a potent antioxidative effect. In this study, we aimed to investigate whether high glucose�(HG)?induced NADPH oxidase activation and ROS production contribute to MC proliferation and the downregulation of TRPC6 expression; we also wished to determine the effects of AS?IV on MCs under HG conditions. Using a human glomerular mesangial cell line, we found that treatment with AS?IV for 48�h markedly attenuated HG?induced proliferation and the hypertrophy of MCs in a dose?dependent manner. The intracellular ROS level was also markedly reduced following treatment with AS?IV. In addition, the enhanced activity of NADPH oxidase and the expression level of NADPH oxidase�4 (Nox4) protein were decreased. Treatment with AS?IV also inhibited the phosphorylation level of Akt and I?B? in the MCs. In addition, TRPC6 protein expression and the intracellular free calcium concentration were also markedly reduced following treatment with AS?IV under HG conditions. These results suggest that AS?IV inhibits HG?induced mesangial cell proliferation and glomerular contractile dysfunction through the NADPH oxidase/ROS/Akt/nuclear factor??B�(NF??B) pathway, providing a new perspective for the clinical treatment of DN. PMID:24718766
Sun, Li; Li, Weiping; Li, Weizu; Xiong, Li; Li, Guiping; Ma, Rong
Anti-inflammatory effects of apocynin, an inhibitor of NADPH oxidase, in airway inflammation.
Oxidative stress is implicated in the pathogenesis of allergic asthma and remains an attractive target for the prevention of the disease. Herein, we investigated the anti-inflammatory effects of apocynin, a NADPH oxidase (NOX) inhibitor, in both in vitro and in vivo allergen-induced experimental asthma mediated by Th2 hyperresponsiveness. Apocynin showed potential antioxidant activities and inhibitory effects on the activation of redox-sensitive transcription factors, such as NF-?B and AP-1, induced by pro-inflammatory stimuli, such as TNF-?, lipopolysaccharide and Poly I:C, and that inhibited the production of pro-inflammatory cytokines, such as TNF-?, IL-1? and IL-6. In in vivo experimental asthma model, moreover, apocynin significantly attenuated ovalbumin-induced airway hyperresponsiveness and inflammation, as shown by the attenuation of total inflammatory cell and soluble product influx into bronchoalveolar lavage fluid, such as macrophages, eosinophils, IL-4, IL-5, IL-12, IL-13 and TNF-?. Apocynin also significantly reduced lung inflammation in the tissues. Altogether, these results suggest that apocynin may be useful in the treatment of inflammatory diseases induced by oxidative stress through NOX activity. PMID:21709687
Kim, So Yong; Moon, Keun-Ai; Jo, Hee-Yeon; Jeong, Sookyung; Seon, Seung-Han; Jung, Eunyu; Cho, You Sook; Chun, Eunyoung; Lee, Ki-Young
NADPH Oxidase and the Degeneration of Dopaminergic Neurons in Parkinsonian Mice
Several lines of investigation have implicated oxidative stress in Parkinson's disease (PD) pathogenesis, but the mechanisms involved are still unclear. In this study, we characterized the involvement of NADPH oxidase (Nox), a multisubunit enzyme that catalyzes the reduction of oxygen, in the 6-hydroxydopamine- (6-OHDA-) induced PD mice model and compared for the first time the effects of this neurotoxin in mice lacking gp91phox?/?, the catalytic subunit of Nox2, and pharmacological inhibition of Nox with apocynin. Six-OHDA induced increased protein expression of p47phox, a Nox subunit, in striatum. gp91phox?/? mice appear to be completely protected from dopaminergic cell loss, whereas the apocynin treatment conferred only a limited neuroprotection. Wt mice treated with apocynin and gp91phox?/? mice both exhibited ameliorated apomorphine-induced rotational behavior. The microglial activation observed within the striatum and the substantia nigra pars compacta (SNpc) of 6-OHDA-injected Wt mice was prevented by apocynin treatment and was not detected in gp91phox?/? mice. Apocynin was not able to attenuate astrocyte activation in SN. The results support a role for Nox2 in the 6-OHDA-induced degeneration of dopaminergic neurons and glial cell activation in the nigrostriatal pathway and reveal that no comparable 6-OHDA effects were observed between apocynin-treated and gp91phox?/? mice groups.
Hernandes, Marina S.; Cafe-Mendes, Cecilia C.; Britto, Luiz R. G.
Pressor Effect of Apelin-13 in the Rostral Ventrolateral Medulla: Role of NAD(P)H Oxidase-Derived Superoxide
Microinjection of apelin-13 into the rostral ventrolateral medulla (RVLM) in the brainstem increases blood pressure in rats. In the present study, we tested the hypotheses that apelin-13 directly stimulates neuronal activity in neurons cultured from the brainstem and that NAD(P)H oxidase-derived reactive oxygen species are involved in this action of apelin-13. Microinjection of apelin-13 into the RVLM resulted in increases in arterial pressure and in renal sympathetic nerve activity in Sprague-Dawley rats. The pressor effect of apelin-13 was attenuated by the specific NAD(P)H-oxidase inhibitor gp91ds-tat. In neurons cultured from the ventral brainstem, spontaneous action potentials were recorded using current-clamp recording. Superfusion of neurons with apelin-13 (100 nM) increased the neuronal firing rate from 0.79 � 0.14 to 1.45 � 0.26 Hz (n = 7, P < 0.01) in angiotensin II receptor-like 1-positive neurons, identified with single-cell reverse transcriptase-polymerase chain reaction. Neither the angiotensin II type 1 receptor antagonist losartan nor the angiotensin II type 2 receptor antagonist 1-[[4-(dimethylamino)-3-methylphenyl[methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate (PD123319) altered the positive chronotropic effect of apelin-13. Pretreatment of cells with either the reactive oxygen species scavenger superoxide dismutase [polyethylene glycol-superoxide dismutase (PEG-SOD), 25 U/ml] or with gp91ds-tat significantly attenuated the chronotropic action of apelin-13. PEG-SOD and gp91ds-tat alone had no effect on basal neuronal firing. In addition, apelin-13 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in neuronal cultures. The superoxide generator xanthine-xanthine oxidase also increased neuronal activity in neurons, mimicking the neuronal response to apelin-13. These observations provide the first evidence that apelin-13 directly increases neuronal activity via stimulation of NAD(P)H oxidase-derived superoxide, a cellular signaling mechanism that may be involved in the pressor effect of apelin-13 in the RVLM.
Yao, Fanrong; Modgil, Amit; Zhang, Qi; Pingili, Ajeeth; Singh, Neha; O'Rourke, Stephen T.
Hydrogen peroxide generated by NADPH oxidase is involved in high blue-light-induced chloroplast avoidance movements in Arabidopsis
NASA Astrophysics Data System (ADS)
One of the most important functions of blue light is to induce chloroplast movements by reducing the damage to photosynthetic machinery under excess light. Hydrogen peroxide (H2O2), generated by various environmental stimuli, can act as a signaling molecule that regulates a number of developmental processes and environmental responses. To investigate whether H2O2 is involved in high blue light-induced chloroplast avoidance movements, we use luminescence spectrometer to observe H2O2 generation with the assistance of the fluorescence probe dichlorofluorescin diacetate (H2DCF-DA). After treatment with high blue light, a large quantity of H2O2 indicated by the fluorescence intensity of DCF is produced in a dose-dependent manner in leaf strip of Arabidopsis. Enzymatic assay shows that the activity of NADPH oxidase, which is a major site for H2O2 generation, also rapidly increases in treated strips. Exogenously applied H2O2 can promote the high blue light-induced chloroplast movements. Moreover, high blue light-induced H2O2 generation can be abolished completely by addition of exogenous catalase (CAT), and partly by diphenylene iodonium (DPI) and dichlorophenyl dimethylurea (DCMU), which are an NADPH oxidase inhibitor and a blocker of electron transport chain. And subsequent chloroplast movements can be abolished by CAT and DPI, but not by DCMU. These results presented here suggested that high blue light can induce oxidative burst, and NADPH oxidase as a major producer for H2O2 is involved in blue light-induced chloroplast avoidance movements.
Wen, Feng; Xing, Da; Zhang, Lingrui
Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation.
Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-?B activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia. PMID:24326071
Arai, Yasuyuki; Nishinaka, Yoko; Arai, Toshiyuki; Morita, Makiko; Mizugishi, Kiyomi; Adachi, Souichi; Takaori-Kondo, Akifumi; Watanabe, Tomohiro; Yamashita, Kouhei
Protection against LPS-induced acute lung injury by a mechanism-based inhibitor of NADPH oxidase (type 2).
The phospholipase A2 activity of peroxiredoxin 6 is inhibited by the transition state analog, 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol (MJ33). This activity is required for the activation of NADPH oxidase, type 2. The present study evaluated the effect of MJ33 on manifestations of acute lung injury. Mice were injected intratracheally (IT) with LPS from Escherichia coli 0111:B4 (LPS, 1 or 5 mg/kg), either concurrently with LPS or 2 h later, and evaluated for lung injury 24 h later. MJ33 inhibited reactive oxygen species (ROS) generation by lungs when measured at 24 h after LPS. LPS at either a low or high dose significantly increased lung infiltration with inflammatory cells, secretion of proinflammatory cytokines (IL-6, TNF-?, and the chemokine macrophage inflammatory protein-2), expression of lung vascular cell adhesion molecule, lung permeability (protein in bronchoalveolar lavage fluid, leakage of FITC-dextran, lung wet-to-dry weight ratio), tissue lipid peroxidation (thiobarbituric acid reactive substances, 8-isoprostanes), tissue protein oxidation (protein carbonyls), and activation of NF-?B. MJ33, given either concurrently or 2 h subsequent to LPS, significantly reduced all of these measured parameters. Previous studies of toxicity showed a high margin of safety for MJ33 in the intact mouse. Thus we have identified MJ33 as a potent, nontoxic, and specific mechanism-based inhibitor of NADPH oxidase type 2-mediated ROS generation that protects mice against lung injury associated with inflammation. PMID:24487388
Lee, Intae; Dodia, Chandra; Chatterjee, Shampa; Feinstein, Sheldon I; Fisher, Aron B
Urotensin II Inhibits Skeletal Muscle Glucose Transport Signaling Pathways via the NADPH Oxidase Pathway
Our previous studies have demonstrated that the urotensin (UII) and its receptor are up-regulated in the skeletal muscle of mice with type II diabetes mellitus (T2DM), but the significance of UII in skeletal muscle insulin resistance remains unknown. The purpose of this study was to investigate the effect of UII on NADPH oxidase and glucose transport signaling pathways in the skeletal muscle of mice with T2DM and in C2C12 mouse myotube cells. KK/upj-AY/J mice (KK) mice were divided into the following groups: KK group, with saline treatment for 2 weeks; KK+ urantide group, with daily 30 �g/kg body weight injections over the same time period of urantide, a potent urotensin II antagonist peptide; Non-diabetic C57BL/6J mice were used as normal controls. After urantide treatment, mice were subjected to an intraperitoneal glucose tolerance test, in addition to measurements of the levels of ROS, NADPH oxidase and the phosphorylated AKT, PKC and ERK. C2C12 cells were incubated with serum-free DMEM for 24 hours before conducting the experiments, and then administrated with 100 nM UII for 2 hours or 24 hours. Urantide treatment improved glucose tolerance, decreased the translocation of the NADPH subunits p40-phox and p47-phox, and increased levels of the phosphorylated PKC, AKT and ERK. In contrast, UII treatment increased ROS production and p47-phox and p67-phox translocation, and decreased the phosphorylated AKT, ERK1/2 and p38MAPK; Apocynin abrogated this effect. In conclusion, UII increased ROS production by NADPH oxidase, leading to the inhibition of signaling pathways involving glucose transport, such as AKT/PKC/ERK. Our data imply a role for UII at the molecular level in glucose homeostasis, and possibly in skeletal muscle insulin resistance in T2DM.
Wang, Hong-Xia; Wu, Xin-Rui; Yang, Hui; Yin, Chun-Lin; Shi, Li-Jin; Wang, Xue-Jiang
Lipopolysaccharide induces hypoxia-inducible factor-1 alpha mRNA expression and activation via NADPH oxidase and Sp1-dependent pathway in BV2 murine microglial cells
Hypoxia-inducible factor-1 (HIF-1), the key transcription factor of hypoxia-inducible genes, is known to be involved in inflammation and immune response, but little is known about the regulation of HIF-1 during microglial activation. Thus, we examined effect of lipopolysaccharide (LPS) on HIF-1 activation and its signaling mechanism in BV2 microglial cells. LPS induced HIF-1? mRNA and protein expression as well as
Young Taek Oh; Jung Yeon Lee; Hana Yoon; Eunjoo H. Lee; Hyung Hwan Baik; Sung Soo Kim; Joohun Ha; Kyung-Sik Yoon; Wonchae Choe; Insug Kang
Critical role of Nox4-based NADPH oxidase in glucose-induced oxidative stress in the kidney: implications in type 2 diabetic nephropathy.
Molecular mechanisms underlying renal complications of diabetes remain unclear. We tested whether renal NADPH oxidase (Nox) 4 contributes to increased reactive oxygen species (ROS) generation and hyperactivation of redox-sensitive signaling pathways in diabetic nephropathy. Diabetic mice (db/db) (20 wk) and cultured mouse proximal tubule (MPT) cells exposed to high glucose (25 mmol/l, D-glucose) were studied. Expression (gene and protein) of Nox4, p22(phox), and p47(phox), but not Nox1 or Nox2, was increased in kidney cortex, but not medulla, from db/db vs. control mice (db/m) (P < 0.05). ROS generation, p38 mitogen-activated protein (MAP) kinase phosphorylation, and content of fibronectin and transforming growth factor (TGF)-?1/2 were increased in db/db vs. db/m (P < 0.01). High glucose increased expression of Nox4, but not other Noxes vs. normal glucose (P < 0.05). This was associated with increased NADPH oxidase activation and enhanced ROS production. Nox4 downregulation by small-interfering RNA and inhibition of Nox4 activity by GK-136901 (Nox1/4 inhibitor) attenuated d-glucose-induced NADPH oxidase-derived ROS generation. High d-glucose, but not l-glucose, stimulated phosphorylation of p38MAP kinase and increased expression of TGF-?1/2 and fibronectin, effects that were inhibited by SB-203580 (p38MAP kinase inhibitor). GK-136901 inhibited d-glucose-induced actions. Our data indicate that, in diabetic conditions: 1) renal Nox4 is upregulated in a cortex-specific manner, 2) MPT cells possess functionally active Nox4-based NADPH, 3) Nox4 is a major source of renal ROS, and 4) activation of profibrotic processes is mediated via Nox4-sensitive, p38MAP kinase-dependent pathways. These findings implicate Nox4-based NADPH oxidase in molecular mechanisms underlying fibrosis in type 2 diabetic nephropathy. PMID:20630933
Sedeek, M; Callera, G; Montezano, A; Gutsol, A; Heitz, F; Szyndralewiez, C; Page, P; Kennedy, C R J; Burns, K D; Touyz, R M; H�bert, R L
NADPH oxidase elevations in pyramidal neurons drive psychosocial stress-induced neuropathology
Oxidative stress is thought to be involved in the development of behavioral and histopathological alterations in animal models of psychosis. Here we investigate the causal contribution of reactive oxygen species generation by the phagocyte NADPH oxidase NOX2 to neuropathological alterations in a rat model of chronic psychosocial stress. In rats exposed to social isolation, the earliest neuropathological alterations were signs of oxidative stress and appearance of NOX2. Alterations in behavior, increase in glutamate levels and loss of parvalbumin were detectable after 4 weeks of social isolation. The expression of the NOX2 subunit p47phox was markedly increased in pyramidal neurons of isolated rats, but below detection threshold in GABAergic neurons, astrocytes and microglia. Rats with a loss of function mutation in the NOX2 subunit p47phox were protected from behavioral and neuropathological alterations induced by social isolation. To test reversibility, we applied the antioxidant/NOX inhibitor apocynin after initiation of social isolation for a time period of 3 weeks. Apocynin reversed behavioral alterations fully when applied after 4 weeks of social isolation, but only partially after 7 weeks. Our results demonstrate that social isolation induces rapid elevations of the NOX2 complex in the brain. Expression of the enzyme complex was strongest in pyramidal neurons and a loss of function mutation prevented neuropathology induced by social isolation. Finally, at least at early stages, pharmacological targeting of NOX2 activity might reverse behavioral alterations.
Schiavone, S; Jaquet, V; Sorce, S; Dubois-Dauphin, M; Hultqvist, M; Backdahl, L; Holmdahl, R; Colaianna, M; Cuomo, V; Trabace, L; Krause, K-H
Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane
Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation,\\u000a neutrophils produce superoxide anion ($${\\\\text{O}}_2 ^{ - .} $$), which generates other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical ($${\\\\text{OH}}^{ \\\\bullet } $$) and hypochlorous acid (HOCl), together with microbicidal peptides and proteases. The enzyme responsible for $${\\\\text{O}}_2
Jamel El-Benna; Pham My-Chan Dang; Marie-Anne Gougerot-Pocidalo
Endothelin1-Mediated Increase in Reactive Oxygen Species and NADPH Oxidase Activity in Hearts of Aryl Hydrocarbon Receptor (AhR) Null Mice
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor characterized to play a role in detection and adaptation to environmental stimuli. Genetic deletion of the AhR results in cardiac hypertrophy that is mediated primarily by endothelin-1 (ET-1); ET-1 has been implicated in the elevation of reactive oxygen species (ROS) in the heart, which are thought to contribute to several
Amie K. Lund; Steven L. Peterson; Graham S. Timmins; Mary K. Walker
Panaxydol induces apoptosis through an increased intracellular calcium level, activation of JNK and p38 MAPK and NADPH oxidase-dependent generation of reactive oxygen species
Panaxydol, a polyacetylenic compound derived from Panax ginseng roots, has been shown to inhibit the growth of cancer cells. In this study, we demonstrated that panaxydol induced apoptosis\\u000a preferentially in transformed cells with a minimal effect on non-transformed cells. Furthermore, panaxydol was shown to induce\\u000a apoptosis through an increase in intracellular Ca2+ concentration ([Ca2+]i), activation of JNK and p38 MAPK,
Joo Young Kim; Su-Jin Yu; Hyun Ju Oh; Ji Young Lee; Yongjin Kim; Jeongwon Sohn
The role of NADPH oxidase 1-derived reactive oxygen species in paraquat-mediated dopaminergic cell death.
Oxidative stress is the common downstream effect of a variety of environmental neurotoxins that are strongly implicated in the pathogenesis of Parkinson's disease. We demonstrate here that the activation of NADPH oxidase 1 (Nox1), a specialized superoxide-generating enzyme complex, plays a key role in the oxidative stress and subsequent dopaminergic cell death elicited by paraquat. Paraquat increased the expression of Nox1 in a concentration-dependent manner in rat dopaminergic N27 cells. Rac1, a key component necessary for Nox1-mediated superoxide generation, also was activated by paraquat. Paraquat-induced reactive oxygen species generation and dopaminergic cell death were significantly reduced after pretreatment with apocynin, a putative NADPH oxidase inhibitor, and Nox1 knockdown with siRNA. Male C57BL/6 mice received intraperitoneal (IP) injections of paraquat (10 mg/kg) once every 3 days and showed increased Nox1 levels in the substantia nigra as well as a 35% reduction in tyrosine hydroxylase-positive dopaminergic neurons 5 days after the last injection. Preadministration of apocynin (200 mg/kg, IP) led to a significant decrease in dopaminergic neuronal loss. Our results suggest that Nox1-generated superoxide is implicated in the oxidative stress elicited by paraquat in DA cells, and it can serve as a novel target for pharmacologic intervention. PMID:19450058
Crist�v�o, Ana Clara; Choi, Dong-Hee; Baltazar, Gra�a; Beal, M Flint; Kim, Yoon-Seong
The Effect of Orexin-A on Cardiac Dysfunction Mediated by NADPH Oxidase-Derived Superoxide Anion in Ventrolateral Medulla
Hypocretin/orexin-producing neurons, located in the perifornical region of the lateral hypothalamus area (LHA) and projecting to the brain sites of rostral ventrolateral medulla (RVLM), involve in the increase of sympathetic activity, thereby regulating cardiovascular function. The current study was designed to test the hypothesis that the central orexin-A (OXA) could be involved in the cardiovascular dysfunction of acute myocardial infarction (AMI) by releasing NAD(P)H oxidase-derived superoxide anion (O2?) generation in RVLM, AMI rat model established by ligating the left anterior descending (LAD) coronary artery to induce manifestation of cardiac dysfunction, monitored by the indicators as heart rate (HR), heart rate variability (HRV), mean arterial pressure (MAP) and left intraventricular pressure. The results showed that the expressions of OXA in LHA and orexin 1 receptor (OX1R) increased in RVLM of AMI rats. The double immunofluorescent staining indicated that OX1R positive cells and NAD(P)H oxidative subunit gp91phox or p47phox-immunoreactive (IR) cells were co-localized in RVLM. Microinjection of OXA into the cerebral ventricle significantly increased O2? production and mRNA expression of NAD(P)H oxidase subunits when compared with aCSF-treated ones. Exogenous OXA administration in RVLM produced pressor and tachycardiac effects. Furthermore, the antagonist of OX1R and OX2R (SB-408124 and TCS OX2 29, respectively) or apocynin (APO), an inhibitor of NAD(P)H oxidase, partly abolished those cardiovascular responses of OXA. HRV power spectral analysis showed that exogenous OXA led to decreased HF component of HRV and increased LF/HF ratio in comparison with aCSF, which suggested that OXA might be related to sympathovagal imbalance. As indicated by the results, OXA might participate in the central regulation of cardiovascular activities by disturbing the sympathovagal balance in AMI, which could be explained by the possibility that OXR and NAD(P)H-derived O2? in RVLM mediates OXA-induced cardiovascular responses.
Chen, Jun; Xia, Chunmei; Wang, Jin; Jiang, Meiyan; Zhang, Huanhuan; Zhang, Chengrong; Zhu, Minxia; Shen, Linlin; Zhu, Danian
Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4.
Physiological sensing of O(2) tension (partial O(2) pressure, pO(2)) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO(2) to altered calcium release through the ryanodine receptor-Ca(2+)-release channel (RyR1). Reactive oxygen species are generated in proportion to pO(2) by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca(2+) release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O(2) sensor in skeletal muscle, and O(2)-coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O(2)-based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1. PMID:21896730
Sun, Qi-An; Hess, Douglas T; Nogueira, Leonardo; Yong, Sandro; Bowles, Dawn E; Eu, Jerry; Laurita, Kenneth R; Meissner, Gerhard; Stamler, Jonathan S
Role of NADPH oxidase in arsenic-induced reactive oxygen species formation and cytotoxicity in myeloid leukemia cells
Arsenic has played a key medicinal role against a variety of ailments for several millennia, but during the past century its prominence has been displaced by modern therapeutics. Recently, attention has been drawn to arsenic by its dramatic clinical efficacy against acute promyelocytic leukemia. Although toxic reactive oxygen species (ROS) induced in cancer cells exposed to arsenic could mediate cancer cell death, how arsenic induces ROS remains undefined. Through the use of gene expression profiling, interference RNA, and genetically engineered cells, we report here that NADPH oxidase, an enzyme complex required for the normal antibacterial function of white blood cells, is the main target of arsenic-induced ROS production. Because NADPH oxidase enzyme activity can also be stimulated by phorbol myristate acetate, a synergism between arsenic and the clinically used phorbol myristate acetate analog, bryostatin 1, through enhanced ROS production can be expected. We show that this synergism exists, and that the use of very low doses of both arsenic and bryostatin 1 can effectively kill leukemic cells. Our findings pinpoint the arsenic target of ROS production and provide a conceptual basis for an anticancer regimen.
Chou, Wen-Chien; Jie, Chunfa; Kenedy, Andrew A.; Jones, Richard J.; Trush, Michael A.; Dang, Chi V.
Role of Rac1-dependent NADPH oxidase in the growth of pancreatic cancer.
K-ras mutations occur in as high as 95% of patients with pancreatic cancer. K-ras activates Rac1-dependent NADPH oxidase, a key source of superoxide. Superoxide has an important function in pancreatic cancer cell proliferation, and scavenging or decreasing the levels of superoxide inhibits pancreatic cancer cell growth both in vitro and in vivo. DNA microarray analysis and RT-PCR has demonstrated that Rac1 is also upregulated in pancreatic cancer. The aim of this study was to determine whether inhibiting Rac1 would alter pancreatic tumor cell behavior. Human pancreatic cancer cells with mutant K-ras (MIA PaCa-2), wild-type K-ras (BxPC-3) and the immortal H6c7 cell line (pancreatic ductal epithelium) expressing K-ras oncogene (H6c7eR-KrasT) that is tumorigenic, were infected with a dominant/negative Rac1 construct (AdN17Rac1). In cells with mutant K-ras, AdN17Rac1 decreased rac activity, decreased superoxide levels and inhibited in vitro growth. However, in the BxPC-3 cell line, AdN17Rac1 did not change rac activity, superoxide levels or in vitro cell growth. Additionally, AdN17Rac1 decreased superoxide levels and inhibited in vitro growth in the KrasT tumorigenic cell line, but had no effect in the immortalized H6c7 cell line. In human pancreatic tumor xenografts, intratumoral injections of AdN17Rac1 inhibited tumor growth. These results suggest that activation of Rac1-dependent superoxide generation leads to pancreatic cancer cell proliferation. In pancreatic cancer, inhibition of Rac1 may be a potential therapeutic target. PMID:21037555
Du, J; Liu, J; Smith, B J; Tsao, M S; Cullen, J J
Endothelin signalling regulates volume-sensitive Cl? current via NADPH oxidase and mitochondrial reactive oxygen species
Aims We assessed regulation of volume-sensitive Cl? current (ICl,swell) by endothelin-1 (ET-1) and characterized the signalling pathway responsible for its activation in rabbit atrial and ventricular myocytes. Methods and results ET-1 elicited ICl,swell under isosmotic conditions. Outwardly rectified Cl? current was blocked by the ICl,swell-selective inhibitor DCPIB or osmotic shrinkage and involved ETA but not ETB receptors. ET-1-induced current was abolished by inhibiting epidermal growth factor receptor (EGFR) kinase or phosphoinositide-3-kinase (PI-3K), indicating that these kinases were downstream. Regarding upstream events, activation of ICl,swell by osmotic swelling or angiotensin II (AngII) was suppressed by ETA blockade, whereas AngII AT1 receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate ICl,swell. As expected, blockade of NOX suppressed ET-1-induced ICl,swell, but blockade of mitochondrial ROS production with rotenone also suppressed ICl,swell. ICl,swell was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, ICl,swell was insensitive to NOX inhibitors, indicating that mitochondria were downstream from NOX. ROS generation in HL-1 cardiomyocytes measured by flow cytometry confirmed the electrophysiological findings. ET-1-induced ROS production was inhibited by blocking either NOX or mitochondrial complex I, whereas complex III-induced ROS production was insensitive to NOX blockade. Conclusion ET-1�ETA signalling activated ICl,swell via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ETA receptors were downstream effectors when ICl,swell was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent ICl,swell is likely to participate in multiple physiological and pathophysiological processes.
Deng, Wu; Baki, Lia; Baumgarten, Clive M.
Neuroprotection from Retinal Ischemia/Reperfusion Injury by NOX2 NADPH Oxidase Deletion
Purpose. The aim of this study was to determine whether NOX2, one of the homologs of NADPH oxidase, plays a role in neuronal cell death during retinal ischemia. Methods. Ischemia reperfusion (I/R) injury was generated in C57/BL6 and NOX2?/? mice by increasing the intraocular pressure (IOP) to 110 mm Hg for 40 minutes followed by reperfusion. Quantitative PCR and Western blot analysis were performed to measure NOX2 expression. Reactive oxygen species (ROS) formation was assessed by dihydroethidium imaging of superoxide formation and Western blot analysis for tyrosine nitration. TUNEL assay was performed to determine cell death at 3 days after I/R. Survival of neurons within the ganglion cell layer (GCL) was assessed at 7 days after I/R by confocal morphometric imaging of retinal wholemounts immunostained with NeuN antibody. Activation of mitogen-activated protein kinases and nuclear factor ?B (NF-??) was measured by Western blot analysis. Results. NOX2 mRNA and protein and ROS were significantly increased in wild-type I/R retinas. This effect was associated with a 60% decrease in the number of GCL neurons and a 10-fold increase in TUNEL-positive cells compared with the fellow sham control eyes. Phosphorylation of ERK and NF-?B was significantly increased in wild-type I/R retinas. Each of these effects was markedly attenuated in the NOX2?/? retina (P < 0.01). Conclusions. These data demonstrate that the deletion of NOX2 can reduce I/R-induced cell death and preserve retinal GCL neurons after I/R injury. The neuronal cell injury caused by I/R is associated with the activation of ERK and NF-?B signaling mechanisms.
Yokota, Harumasa; Narayanan, Subhadra P.; Zhang, Wenbo; Liu, Hua; Rojas, Modesto; Xu, Zhimin; Lemtalsi, Tahira; Nagaoka, Taiji; Yoshida, Akitoshi; Brooks, Steven E.; Caldwell, Robert W.
Molecular evolution of Phox-related regulatory subunits for NADPH oxidase enzymes
Background The reactive oxygen-generating NADPH oxidases (Noxes) function in a variety of biological roles, and can be broadly classified into those that are regulated by subunit interactions and those that are regulated by calcium. The prototypical subunit-regulated Nox, Nox2, is the membrane-associated catalytic subunit of the phagocyte NADPH-oxidase. Nox2 forms a heterodimer with the integral membrane protein, p22phox, and this heterodimer binds to the regulatory subunits p47phox, p67phox, p40phox and the small GTPase Rac, triggering superoxide generation. Nox-organizer protein 1 (NOXO1) and Nox-activator 1 (NOXA1), respective homologs of p47phox and p67phox, together with p22phox and Rac, activate Nox1, a non-phagocytic homolog of Nox2. NOXO1 and p22phox also regulate Nox3, whereas Nox4 requires only p22phox. In this study, we have assembled and analyzed amino acid sequences of Nox regulatory subunit orthologs from vertebrates, a urochordate, an echinoderm, a mollusc, a cnidarian, a choanoflagellate, fungi and a slime mold amoeba to investigate the evolutionary history of these subunits. Results Ancestral p47phox, p67phox, and p22phox genes are broadly seen in the metazoa, except for the ecdysozoans. The choanoflagellate Monosiga brevicollis, the unicellular organism that is the closest relatives of multicellular animals, encodes early prototypes of p22phox, p47phox as well as the earliest known Nox2-like ancestor of the Nox1-3 subfamily. p67phox- and p47phox-like genes are seen in the sea urchin Strongylocentrotus purpuratus and the limpet Lottia gigantea that also possess Nox2-like co-orthologs of vertebrate Nox1-3. Duplication of primordial p47phox and p67phox genes occurred in vertebrates, with the duplicated branches evolving into NOXO1 and NOXA1. Analysis of characteristic domains of regulatory subunits suggests a novel view of the evolution of Nox: in fish, p40phox participated in regulating both Nox1 and Nox2, but after the appearance of mammals, Nox1 (but not Nox2) became independent of p40phox. In the fish Oryzias latipes, a NOXO1 ortholog retains an autoinhibitory region that is characteristic of mammalian p47phox, and this was subsequently lost from NOXO1 in later vertebrates. Detailed amino acid sequence comparisons identified both putative key residues conserved in characteristic domains and previously unidentified conserved regions. Also, candidate organizer/activator proteins in fungi and amoeba are identified and hypothetical activation models are suggested. Conclusion This is the first report to provide the comprehensive view of the molecular evolution of regulatory subunits for Nox enzymes. This approach provides clues for understanding the evolution of biochemical and physiological functions for regulatory-subunit-dependent Nox enzymes.
Kawahara, Tsukasa; Lambeth, J David
Ebselen and congeners inhibit NADPH-oxidase 2 (Nox2)-dependent superoxide generation by interrupting the binding of regulatory subunits
Summary NADPH-oxidases are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits.
Smith, Susan M.E.; Min, Jaeki; Ganesh, Thota; Diebold, Becky; Kawahara, Tsukasa; Zhu, Yerun; McCoy, James; Sun, Aiming; Snyder, James P.; Fu, Haian; Du, Yuhong; Lewis, Iestyn; Lambeth, J. David
Ebselen and congeners inhibit NADPH oxidase 2-dependent superoxide generation by interrupting the binding of regulatory subunits.
NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits. PMID:22726689
Smith, Susan M E; Min, Jaeki; Ganesh, Thota; Diebold, Becky; Kawahara, Tsukasa; Zhu, Yerun; McCoy, James; Sun, Aiming; Snyder, James P; Fu, Haian; Du, Yuhong; Lewis, Iestyn; Lambeth, J David
NADPH Oxidase 1-Mediated Oxidative Stress Leads to Dopamine Neuron Death in Parkinson's Disease
Abstract Aim: Oxidative stress has long been considered as a major contributing factor in the pathogenesis of Parkinson's disease. However, molecular sources for reactive oxygen species in Parkinson's disease have not been clearly elucidated. Herein, we sought to investigate whether a superoxide-producing NADPH oxidases (NOXs) are implicated in oxidative stress-mediated dopaminergic neuronal degeneration. Results: Expression of various Nox isoforms and cytoplasmic components were investigated in N27, rat dopaminergic cells. While most of Nox isoforms were constitutively expressed, Nox1 expression was significantly increased after treatment with 6-hydroxydopamine. Rac1, a key regulator in the Nox1 system, was also activated. Striatal injection of 6-hydroxydopamine increased Nox1 expression in dopaminergic neurons in the rat substantia nigra. Interestingly, it was localized into the nucleus, and immunostaining for DNA oxidative stress marker, 8-oxo-dG, was increased. Nox1expression was also found in the nucleus of dopaminergic neurons in the substantia nigra of Parkinson's disease patients. Adeno-associated virus-mediated Nox1 knockdown or Rac1 inhibition reduced 6-hydroxydopamine-induced oxidative DNA damage and dopaminergic neuronal degeneration significantly. Innovation: Nox1/Rac1 could serve as a potential therapeutic target for Parkinson's disease. Conclusion: We provide evidence that dopaminergic neurons are equipped with the Nox1/Rac1 superoxide-generating system. Stress-induced Nox1/Rac1 activation causes oxidative DNA damage and neurodegeneration. Reduced dopaminergic neuronal death achieved by targeting Nox1/Rac1, emphasizes the impact of oxidative stress caused by this system on the pathogenesis and therapy in Parkinson's disease. Antioxid. Redox Signal. 16, 1033�1045.
Choi, Dong-Hee; Cristovao, Ana Clara; Guhathakurta, Subhrangshu; Lee, Jongmin; Joh, Tong H.; Beal, M. Flint
Cellular and temporal expression of NADPH oxidase (NOX) isotypes after brain injury
Background Brain injury results in an increase in the activity of the reactive oxygen species generating NADPH oxidase (NOX) enzymes. Preliminary studies have shown that NOX2, NOX3, and NOX4 are the most prominently expressed NOX isotypes in the brain. However, the cellular and temporal expression profile of these isotypes in the injured and non-injured brain is currently unclear. Methods Double immunofluorescence for NOX isotypes and brain cell types was performed at acute (24 hours), sub-acute (7 days), and chronic (28 days) time points after controlled cortical impact-induced brain injury or sham-injury in rats. Results NOX2, NOX3, and NOX4 isotypes were found to be expressed in neurons, astrocytes, and microglia, and this expression was dependent on both cellular source and post-injury time. NOX4 was found in all cell types assessed, while NOX3 was positively identified in neurons only, and NOX2 was identified in microglia and neurons. NOX2 was the most responsive to injury, increasing primarily in microglia in response to injury. Quantitation of this isotype showed a significant increase in NOX2 expression at 24 hours, with reduced expression at 7 days and 28 days post-injury, although expression remained above sham levels at later time points. Cellular confirmation using purified primary or cell line culture demonstrated similar patterns in microglia, astrocytes, and neurons. Further, inhibition of NOX, and more specifically NOX2, reduced pro-inflammatory activity in microglia, demonstrating that NOX is not only up-regulated after stimulation, but may also play a significant role in post-injury neuroinflammation. Conclusions This study illustrates the expression profiles of NOX isotypes in the brain after injury, and demonstrates that NOX2, and to a lesser extent, NOX4, may be responsible for the majority of oxidative stress observed acutely after traumatic brain injury. These data may provide insight into the design of future therapeutic approaches.
Prostacyclin signaling boosts NADPH oxidase 4 in the endothelium promoting cytoprotection and angiogenesis.
Abstract Aims: Prostacyclin (PGI2) that is released from the vascular endothelium plays an important role in vasodilatation and thrombo-resistance, and it has long been suspected to protect cell survival. How it does so has never been clear. Recently, it has been shown that the NADPH oxidase 4 (Nox4) improves endothelial cell functions and promotes angiogenesis in vivo, but it was not known how to boost Nox4 therapeutically to exploit its protective functions in the vasculature. Here, we identified such a stimulus. Results: The selective and stable prostacyclin receptor (IP-R) agonist cicaprost increases the expression of Nox4 in human endothelial cells of several types, including endothelial progenitor cells. The elevation of cellular cyclic-AMP increased Nox4 expression and H2O2 production and prevented endothelial cell apoptosis. We delineate the intracellular signaling that promotes cytoprotection: Cicaprost acts via the IP-R/protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein pathway. Importantly, the up-regulation of Nox4 by cicaprost also enhanced endothelial cell proliferation, migration, and angiogenesis, with all effects being substantially decreased by Nox4 gene silencing. Finally, cicaprost enhanced the growth of blood vessels into subcutaneous sponges implanted in mice, an effect that was also blocked by Nox4 gene silencing. Innovation: The prostacyclin analogue cicaprost induces Nox4 via IP receptor-cAMP/PKA/CREB pathway. The activation of this pathway protects endothelial cells and enhances pro-angiogenic activity both in vitro and in vivo. Conclusion: Prostacyclin promotes the up-regulation of Nox4 in endothelial cells, which opens up a novel strategy that protects and enhances endothelial cell functions in cardiovascular disease, such as repair after myocardial infarction or other ischemic conditions. Antioxid. Redox Signal. 20, 2710-2725. PMID:24450852
Peshavariya, Hitesh M; Liu, Guei-Sheung; Chang, Catherine W T; Jiang, Fan; Chan, Elsa C; Dusting, Gregory J
NADPH oxidase inhibitor, apocynin, improves renal glutathione status in Zucker diabetic fatty rats: A comparison with melatonin.
Apocynin (4'-hydroxy-3'-methoxyacetophenone) is the most commonly used NADPH oxidase (Nox) inhibitor. However, its application raises serious controversies, as the compound has been reported to reveal some prooxidative effects. The aim of this study was to elucidate apocynin action on glutathione, the main intracellular antioxidant, metabolism in kidneys of Zucker diabetic fatty (ZDF) rat, a well established model of diabetes type 2. Additionally, apocynin effects were compared with those of melatonin. The experiments were performed on five groups of animals: (1) untreated lean (?/+) ZDF rats, (2) ZDF ?/+ rats treated with apocynin (2g/l) in drinking water, (3) untreated obese diabetic (fa/fa) ZDF rats, (4) ZDF fa/fa rats treated with apocynin (2g/l) in drinking water, and (5) ZDF fa/fa rats treated with melatonin (20mg/l) in drinking water. After 8weeks of the treatment, the following parameters were measured in kidneys: NADPH oxidase activity, the rate of hydroxyl free radicals (HFR) production, GSH and GSSG content and the activities of the enzymes of glutathione metabolism: ?-glutamylcysteine synthetase (GCS), glutathione reductase (GR) and glutathione peroxidase (GPx). Compared to ?/+ controls, ZDF fa/fa rats exhibited increased Nox activity, accelerated HFR generation and dramatically lowered GSH/GSSG ratio accompanied by increased GPx and diminished GCS activities. In case of diabetic animals, apocynin treatment resulted in attenuation of both Nox activity and HFR production, restoration of control GSH/GSSG ratio (due to both an increase in GSH and a decline in GSSG content), normalization of GPx activity and a slight increase in GCS activity. Similar observations were made upon melatonin application to ZDF fa/fa rats. Thus, it is concluded that, in the diabetic model studied, apocynin extends a beneficial effect on renal glutathione homeostasis. The mechanism of this phenomenon involves attenuation of glutathione peroxidase activity, which is overstimulated under conditions of oxidative stress accompanying diabetes. PMID:24797087
Winiarska, Katarzyna; Focht, Dorota; Sierakowski, Bartosz; Lewandowski, Krystian; Orlowska, Marta; Usarek, Michal
NADPH oxidase plays a central role in cone cell death in retinitis pigmentosa.
Retinitis pigmentosa (RP) is a collection of diseases in which rod photoreceptors die from a variety of mutations. After rods die, the level of tissue oxygen in the outer retina becomes elevated and there is progressive oxidative damage to cones that ultimately triggers apoptosis. In this study, we investigated the hypothesis that NADPH oxidase (Nox) and/or xanthine oxidase serve as critical intermediaries between increased tissue oxygen and the generation of excessive reactive oxygen species that cause oxidative damage to cones. Apocynin, a blocker of Nox, but not allopurinol, a blocker of xanthine oxidase, markedly reduced the superoxide radicals visualized by hydroethidine in the outer retina in the retinal degeneration-1 (rd1(+/+)) model of RP. Compared to rd1(+/+) mice treated with vehicle, those treated with apocynin, but not those treated with allopurinol, had significantly less oxidative damage in the retina measured by ELISA for carbonyl adducts. Apocynin-treated, but not allopurinol-treated, rd1(+/+) mice had preservation of cone cell density, increased mRNA levels for m- and s-cone opsin, and increased mean photopic b-wave amplitude. In Q344ter mice, a model of dominant RP in which mutant rhodopsin is expressed, apocynin treatment preserved photopic electroretinogram b-wave amplitude compared to vehicle-treated controls. These data indicate that Nox, but not xanthine oxidase, plays a critical role in generation of the oxidative stress that leads to cone cell death in RP and inhibition of Nox provides a new treatment strategy. PMID:19493169
Usui, Shinichi; Oveson, Brian C; Lee, Sun Young; Jo, Young-Joon; Yoshida, Tsunehiko; Miki, Akiko; Miki, Katsuaki; Iwase, Takeshi; Lu, Lili; Campochiaro, Peter A
Intracellular Ca2+ can compensate for the lack of NADPH oxidase-derived ROS in endothelial cells.
The aim of the present study is to determine the role of intracellular Ca(2+) in VEGF signaling. We demonstrate that reduction in Ca(2+) by chelating compound BAPTA-AM or by IP(3)-endoplasmic reticulum blocker 2-APB selectively inhibited VEGF-induced activation of c-Src-PI3K-Akt but not ERK1/2 in human coronary artery endothelial cells (HCAEC). We also show that the selective inhibitory effects of NADPH oxidase knockdown on VEGF-mediated activation of c-Src-PI3K-Akt signaling and cell proliferation in HCAEC can be reversed by increase in intracellular Ca(2+). These results suggest an essential role for Ca(2+) in redox-dependent selective activation of c-Src-PI3K-Akt and endothelial cell proliferation. PMID:20621840
Lee, Monica; Spokes, Katherine C; Aird, William C; Abid, Md Ruhul
NADPH Oxidase 1, a Novel Molecular Source of ROS in Hippocampal Neuronal Death in Vascular Dementia.
Abstract Aims: Chronic cerebral hypoperfusion (CCH) is a common pathological factor that contributes to neurodegenerative diseases such as vascular dementia (VaD). Although oxidative stress has been strongly implicated in the pathogenesis of VaD, the molecular mechanism underlying the selective vulnerability of hippocampal neurons to oxidative damage remains unknown. We assessed whether the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, a specialized superoxide generation system, plays a role in VaD by permanent ligation of bilateral common carotid arteries in rats. Results: Male Wistar rats (10 weeks of age) were subjected to bilateral occlusion of the common carotid arteries (two-vessel occlusion [2VO]). Nox1 expression gradually increased in hippocampal neurons, starting at 1 week after 2VO and for approximately 15 weeks after 2VO. The levels of superoxide, DNA oxidation, and neuronal death in the CA1 subfield of the hippocampus, as well as consequential cognitive impairment, were increased in 2VO rats. Both inhibition of Nox by apocynin, a putative Nox inhibitor, and adeno-associated virus-mediated Nox1 knockdown significantly reduced 2VO-induced reactive oxygen species generation, oxidative DNA damage, hippocampal neuronal degeneration, and cognitive impairment. Innovation and Conclusion: We provided evidence that neuronal Nox1 is activated in the hippocampus under CCH, causing oxidative stress and consequential hippocampal neuronal death and cognitive impairment. This evidence implies that Nox1-mediated oxidative stress plays an important role in neuronal cell death and cognitive dysfunction in VaD. Nox1 may serve as a potential therapeutic target for VaD. Antioxid. Redox Signal. 21, 533-550. PMID:24294978
Choi, Dong-Hee; Lee, Kyoung-Hee; Kim, Ji-Hye; Seo, Ju-Ha; Kim, Hahn Young; Shin, Chan Young; Han, Jung-Soo; Han, Seol-Heui; Kim, Yoon-Seong; Lee, Jongmin
NADPH oxidase 4 promotes cardiac microvascular angiogenesis after hypoxia/reoxygenation in vitro.
Microvascular endothelial cell dysfunction plays a key role in myocardial ischemia/reperfusion (I/R) injury, wherein reactive oxygen species (ROS)-dependent signaling is intensively involved. However, the roles of the various ROS sources remain unclear. This study sought to investigate the role of NADPH oxidase 4 (Nox4) in the cardiac microvascular endothelium in response to I/R injury. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and subjected to hypoxia/reoxygenation (H/R). Our results showed that Nox4 was highly expressed in CMECs, was significantly increased at both mRNA and protein levels after H/R injury, and contributed to H/R-stimulated increase in Nox activity and ROS generation. Downregulation of Nox4 by small interfering RNA transfection did not affect cell viability or ROS production under normoxia, but exacerbated H/R injury as evidenced by increased apoptosis and inhibited cell survival, migration, and angiogenesis after H/R. Nox4 inhibition also increased prolyl hydroxylase 2 (PHD2) expression and blocked H/R-induced increases in HIF-1? and VEGF expression. Pretreatment with DMOG, a specific competitive PHD inhibitor, upregulated HIF-1? and VEGF expression and significantly reversed Nox4 knockdown-induced injury. However, Nox2 was scarcely expressed and played a minimal role in CMEC survival and angiogenesis after H/R, though a modest upregulation of Nox2 was observed. In conclusion, this study demonstrated a previously unrecognized protective role of Nox4, a ROS-generating enzyme and the major Nox isoform in CMECs, against H/R injury by inhibiting apoptosis and promoting migration and angiogenesis via a PHD2-dependent upregulation of HIF-1/VEGF proangiogenic signaling. PMID:24480752
Wang, Jinyi; Hong, Zhibo; Zeng, Chao; Yu, Qiujun; Wang, Haichang
NADPH Oxidase 1, a Novel Molecular Source of ROS in Hippocampal Neuronal Death in Vascular Dementia
Abstract Aims: Chronic cerebral hypoperfusion (CCH) is a common pathological factor that contributes to neurodegenerative diseases such as vascular dementia (VaD). Although oxidative stress has been strongly implicated in the pathogenesis of VaD, the molecular mechanism underlying the selective vulnerability of hippocampal neurons to oxidative damage remains unknown. We assessed whether the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, a specialized superoxide generation system, plays a role in VaD by permanent ligation of bilateral common carotid arteries in rats. Results: Male Wistar rats (10 weeks of age) were subjected to bilateral occlusion of the common carotid arteries (two-vessel occlusion [2VO]). Nox1 expression gradually increased in hippocampal neurons, starting at 1 week after 2VO and for approximately 15 weeks after 2VO. The levels of superoxide, DNA oxidation, and neuronal death in the CA1 subfield of the hippocampus, as well as consequential cognitive impairment, were increased in 2VO rats. Both inhibition of Nox by apocynin, a putative Nox inhibitor, and adeno-associated virus-mediated Nox1 knockdown significantly reduced 2VO-induced reactive oxygen species generation, oxidative DNA damage, hippocampal neuronal degeneration, and cognitive impairment. Innovation and Conclusion: We provided evidence that neuronal Nox1 is activated in the hippocampus under CCH, causing oxidative stress and consequential hippocampal neuronal death and cognitive impairment. This evidence implies that Nox1-mediated oxidative stress plays an important role in neuronal cell death and cognitive dysfunction in VaD. Nox1 may serve as a potential therapeutic target for VaD. Antioxid. Redox Signal. 21, 533�550.
Choi, Dong-Hee; Lee, Kyoung-Hee; Kim, Ji-Hye; Seo, Ju-Ha; Kim, Hahn Young; Shin, Chan Young; Han, Jung-Soo; Han, Seol-Heui; Kim, Yoon-Seong
NADPH oxidases: a perspective on reactive oxygen species production in tumor biology.
Abstract Significance: Reactive oxygen species (ROS) promote genomic instability, altered signal transduction, and an environment that can sustain tumor formation and growth. The NOX family of NADPH oxidases, membrane-bound epithelial superoxide and hydrogen peroxide producers, plays a critical role in the maintenance of immune function, cell growth, and apoptosis. The impact of NOX enzymes in carcinogenesis is currently being defined and may directly link chronic inflammation and NOX ROS-mediated tumor formation. Recent Advances: Increased interest in the function of NOX enzymes in tumor biology has spurred a surge of investigative effort to understand the variability of NOX expression levels in tumors and the effect of NOX activity on tumor cell proliferation. These initial efforts have demonstrated a wide variance in NOX distribution and expression levels across numerous cancers as well as in common tumor cell lines, suggesting that much remains to be discovered about the unique role of NOX-related ROS production within each system. Progression from in vitro cell line studies toward in vivo tumor tissue screening and xenograft models has begun to provide evidence supporting the importance of NOX expression in carcinogenesis. Critical Issues: A lack of universally available, isoform-specific antibodies and animal tumor models of inducible knockout or over-expression of NOX isoforms has hindered progress toward the completion of in vivo studies. Future Directions: In vivo validation experiments and the use of large, existing gene expression data sets should help define the best model systems for studying the NOX homologues in the context of cancer. Antioxid. Redox Signal. 20, 2873-2889. PMID:24156355
Meitzler, Jennifer L; Antony, Smitha; Wu, Yongzhong; Juhasz, Agnes; Liu, Han; Jiang, Guojian; Lu, Jiamo; Roy, Krishnendu; Doroshow, James H
Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase.
All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 microM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 +/- 6% and Nox4 siRNA 83 +/- 7% of control) and lucigenin chemiluminescence (Nox2; 54 +/- 6% and Nox4 74 +/- 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase. PMID:17516243
Peshavariya, Hitesh M; Dusting, Gregory James; Selemidis, Stavros
NADPH oxidases regulate cell growth and migration in myeloid cells transformed by oncogenic tyrosine kinases
Transformation by tyrosine kinase oncogenes in myeloid malignancies, including BCR-ABL in chronic myeloid leukemia, FLT3ITD in acute myeloid leukemia (AML) or JAK2V617F in myeloproliferative neoplasms (MPN), is associated with increased growth and cytoskeletal abnormalities. Using targeted approaches against components of the superoxide-producing NADPH-oxidases, including NOX2, NOX4 and the common p22phox subunit of NOX1-4, myeloid cells were found to display reduced cell growth and spontaneous migration. Consistent with a role of NOX as regulators of membrane proximal signaling events in non-phagocytic cells, NOX2 and NOX4 were not involved in the excess production of intracellular reactive oxygen species and did not significantly increase oxygen consumption. All NOX family members are controlled in part through levels of the rate-limiting substrate NADPH, which was found to be significantly elevated in tyrosine kinase oncogene transformed cells. Also, reduced phosphorylation of the actin filament crosslinking protein MARCKS in response to suppression of p22phox hints at a novel effector of NOX signaling. MARCKS was also found to be required for increased migration. Overall, these data suggest a model whereby NOX links metabolic NADPH production to cellular events that directly contribute to transformation.
Reddy, Mamatha M.; Fernandes, Margret S.; Salgia, Ravi; Levine, Ross L.; Griffin, James D.; Sattler, Martin
Thioredoxin-interacting Protein Mediates High Glucose-induced Reactive Oxygen Species Generation by Mitochondria and the NADPH Oxidase, Nox4, in Mesangial Cells*
Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced ?-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN.
Shah, Anu; Xia, Ling; Goldberg, Howard; Lee, Ken W.; Quaggin, Susan E.; Fantus, I. George
NADPH oxidase contributes to coronary endothelial dysfunction in the failing heart
Increased reactive oxygen species (ROS) produced by the failing heart can react with nitric oxide (NO), thereby decreasing NO bioavailability. This study tested the hypothesis that increased ROS generation contributes to coronary endothelial dysfunction in the failing heart. Congestive heart failure (CHF) was produced in six dogs by ventricular pacing at 240 beats/min for 4 wk. Studies were performed at rest and during treadmill exercise under control conditions and after treatment with the NADPH oxidase inhibitor and antioxidant apocynin (4 mg/kg iv). Apocynin caused no significant changes in heart rate, aortic pressure, left ventricular (LV) systolic pressure, LV end-diastolic pressure, or maximum rate of LV pressure increase at rest or during exercise in normal or CHF dogs. Apocynin caused no change in coronary blood flow (CBF) in normal dogs but increased CBF at rest and during exercise in animals with CHF (P < 0.05). Intracoronary ACh caused dose-dependent increases of CBF that were blunted in CHF. Apocynin had no effect on the response to ACh in normal dogs but augmented the response to ACh in CHF dogs (P < 0.05). The oxidative stress markers nitrotyrosine and 4-hydroxy-2-nonenal were significantly greater in failing than in normal myocardium. Furthermore, coelenterazine chemiluminescence for O2? was more than twice normal in failing myocardium, and this difference was abolished by apocynin. Western blot analysis of myocardial lysates demonstrated that the p47phox and p22phox subunits of NADPH were significantly increased in the failing hearts, while real-time PCR demonstrated that Nox2 mRNA was significantly increased. The data indicate that increased ROS generation in the failing heart is associated with coronary endothelial dysfunction and suggest that NADPH oxidase may contribute to this abnormality.
Zhang, Ping; Hou, Mingxiao; Li, Yunfang; Xu, Xin; Barsoum, Michel; Chen, YingJie; Bache, Robert J.
The FRE1 ferric reductase of Saccharomyces cerevisiae is a cytochrome b similar to that of NADPH oxidase.
Plasma membrane preparations from strains of the yeast Saccharomyces cerevisiae gave a reduced minus oxidized spectrum characteristic of a b-type cytochrome and very similar to the spectrum of flavocytochrome b558 of human neutrophils. The magnitude of the signal correlated with the level of ferric reductase activity and the copy number of the FRE1 gene, indicating that the FRE1 protein is a cytochrome b. Sequence similarities with the flavin binding site of flavocytochrome b558 and other members of the ferredoxin-NADP reductase family, together with increased levels of noncovalently bound FAD and iodonitrotetrazolium violet reductase activity in membranes from a yeast strain overexpressing ferric reductase, suggested that the FRE1 protein may also carry a flavin group. Potentiometric titrations indicated that FRE1, like neutrophil NADPH oxidase, has an unusually low redox potential, in the region of -250 mV, and binds CO. PMID:8662973
Shatwell, K P; Dancis, A; Cross, A R; Klausner, R D; Segal, A W
Escherichia Coli Mutant with Elevated Nicotinamide Adenine Dinucleotide Phosphate (Reduced) Oxidase Activity.
National Technical Information Service (NTIS)
A slow-growing mutant of Escherichia coli with greatly elevated nicotinamide adenine dinucleotide phosphate (reduced; NADPH) oxidase activity has been isolated. The oxidase activity of the wild-type organism, normally low at pH 7.5, was increased when the...
L. M. Corwin G. R. Fanning
Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells.
The effect of race-specific elicitors on NADPH oxidase was examined in vivo by treating tomato cells with elicitor-containing intercellular fluids prepared from infected tomato leaves inoculated with specific Cladosporium fulvum races. Treatment of Cf-4 or Cf-5 cells with intercellular fluids from incompatible but not from compatible races of C. fulvum increased oxidase activity and the amount of p67-phox, p47-phox, and rac2 in the plasma membrane. Comparison of these three components in the cytosol and plasma membrane indicated that elicitors promoted the translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells carrying the appropriate resistance gene. Protein kinase C activators and inhibitors did not affect enzyme activity or the binding of these three components to the plasma membrane. In contrast, staurosporine, calmodulin antagonists, and EGTA inhibited elicitor-induced oxidase activity and the translocation of the cytosolic components. The assembly process involves a Ca(2+)-dependent protein kinase that catalyzes the phosphorylation of p67-phox and p47-phox, facilitating their translocation to the plasma membrane. Our data suggest that although both plants and animals share common elements in eukaryotic signal transduction, the involvement of different protein kinases mediating the activation of phosphorylation of p67-phox and p47-phox may reflect the unique spatial and temporal distribution of signal transduction pathways in plants. PMID:9061955
Xing, T; Higgins, V J; Blumwald, E
In vitro screening of NADPH oxidase inhibitors and in vivo effects of L-leucinethiol on experimental autoimmune encephalomyelitis-induced mice.
Experimental autoimmune encephalomyelitis (EAE), a Th1 polarized demyelinating disease of the central nervous system, shares many pathological and clinical similarities with multiple sclerosis (MS). The objectives of this study were i) to evaluate the suppressive effects of L-leucinethiol (LeuSH), a metalloprotease inhibitor on EAE-induced mice and ii) to study the effects of LeuSH on matrix metalloproteinase-9 (MMP-9), NADPH oxidase and cytokines (IFN-?, IL-5 and IL-10) in tissues and plasma of EAE mice as a measure of potential markers associated with EAE disease. C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG35-55) peptide in complete Freund's adjuvant to induce EAE. A significant difference was observed in body weights and clinical signs of LeuSH (8 mg/kg) administered EAE-induced mice compared to control mice. The findings of this study include alterations in the enzymatic expression of MMP-9, NADPH oxidase and cytokine levels in the brain, spinal cord, spleen, thymus and plasma of inhibitor-treated EAE mice as well as EAE-induced mice. The enzyme activities of NADPH oxidase were inhibited by LeuSH. From these results, it can be considered that LeuSH acts as one of the antigen candidates in ameliorating the clinical symptoms of EAE disease in mice. PMID:22554692
Kandagaddala, Lakshmi Devi; Kang, Min-Jung; Haque, Md Mamunul; Im, Hye-Yeon; Seo, Ji-Eun; Chung, Bong Chul; Jung, Byung Hwa; Patterson, Tucker A; Kwon, Oh-Seung
Practical strategies for targeting NF-kappaB and NADPH oxidase may improve survival during lethal influenza epidemics.
The most foolproof way to promote survival in epidemics of potentially lethal influenza is to target, not highly mutable viral proteins, but rather intracellular signaling pathways which promote viral propagation or lung inflammation. NF-kappaB, activated in influenza-infected lung epithelial cells and macrophages, is one likely target in this regard, as it plays a role both in viral replication and in the excessive lung inflammation often evoked by influenza infection. Indeed, salicylates, which suppress NF-kappaB activation, have been shown to reduce the lethality of H5N1 avian-type influenza in mice. Another potential target is NADPH oxidase, as this may be a major source of influenza-evoked oxidant stress in lung epithelial cells as well as in phagocytes attracted to lung parenchyma. A number of studies demonstrate that oxidant stress contributes to overexuberant lung inflammation and lethality in influenza-infected mice. The documented utility of N-acetylcysteine, a glutathione precursor, for promoting survival in influenza-infected mice, and diminishing the severity of influenza-like infections in elderly humans, presumably reflects a key role for oxidative stress in influenza. The lethality of influenza is also reduced in mice pretreated with adenovirus carrying the gene for heme oxygenase-1; this benefit may be mediated, at least in part, by the ability of bilirubin to inhibit NADPH oxidase. It may be feasible to replicate this benefit clinically by administering biliverdin or its homolog phycocyanobilin, richly supplied by spirulina. If this latter speculation can be confirmed in rodent studies, a practical and inexpensive regimen consisting of high-dose salicylates, spirulina, and N-acetylcysteine, initiated at the earliest feasible time, may prove to have life-saving efficacy when the next killer influenza pandemic strikes. PMID:19573997
McCarty, Mark F; Barroso-Aranda, Jorge; Contreras, Francisco
NADPH oxidase-derived reactive oxygen species increases expression of monocyte chemotactic factor genes in cultured adipocytes.
Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 ?M) in either 5 or 25 mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors. PMID:22287546
Han, Chang Yeop; Umemoto, Tomio; Omer, Mohamed; Den Hartigh, Laura J; Chiba, Tsuyoshi; LeBoeuf, Renee; Buller, Carolyn L; Sweet, Ian R; Pennathur, Subramaniam; Abel, E Dale; Chait, Alan
Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine induces vascular endothelial superoxide production: Implication of NADPH oxidase
Modified low-density lipoprotein (LDL) induces reactive oxygen species (ROS) production by vascular cells. It is unknown if specific oxidized components in these LDL particles such as oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) can stimulate ROS production. Bovine aortic endothelial cells (BAEC) were incubated with ox-PAPC (50 ?g/ml). At 4 h, ox-PAPC significantly enhanced the rate of O2?� production. Pretreatment of BAEC in glucose-free Dulbecco�s modified Eagle�s medium plus 10 mM 2-deoxyglucose (2-DOG), the latter being an antimetabolite that blocks NADPH production by the pentose shunt, significantly reduced the rate of O2?� production. The intensity of NAD(P)H autofluorescence decreased by 28 � 12% in BAEC incubated with ox-PAPC compared to untreated cells, with a further decrease in the presence of 2-DOG. Ox-PAPC also increased Nox4 mRNA expression by 2.4-fold � 0.1 while pretreatment of BAEC with the small interfering RNA (siNox4) attenuated Nox4 RNA expression. Ox-PAPC further reduced the level of glutathione while pretreatment with apocynin (100 ?M) restored the GSH level (control = 22.54 � 0.23, GSH = 18.06 � 0.98, apocynin = 22.55 � 0.60, ox-PAPC + apocynin = 21.17 � 0.36 nmol/106 cells). Treatment with ox-PAPC also increased MMP-2 mRNA expression accompanied by a 1.5-fold increase in MMP-2 activity. Ox-PAPC induced vascular endothelial O2?� production that appears to be mediated largely by NADPH oxidase activity.
Rouhanizadeh, Mahsa; Hwang, Juliana; Clempus, Roza E.; Marcu, Laura; Lassegue, Bernard; Sevanian, Alex; Hsiai, Tzung K.
NADPH Oxidase-derived Reactive Oxygen Species Increases Expression of Monocyte Chemotactic Factor Genes in Cultured Adipocytes*
Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 ?m) in either 5 or 25 mm glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.
Han, Chang Yeop; Umemoto, Tomio; Omer, Mohamed; Den Hartigh, Laura J.; Chiba, Tsuyoshi; LeBoeuf, Renee; Buller, Carolyn L.; Sweet, Ian R.; Pennathur, Subramaniam; Abel, E. Dale; Chait, Alan
Superoxide Production in Galleria mellonella Hemocytes: Identification of Proteins Homologous to the NADPH Oxidase Complex of Human Neutrophils
The insect immune response has a number of structural and functional similarities to the innate immune response of mammals. The objective of the work presented here was to establish the mechanism by which insect hemocytes produce superoxide and to ascertain whether the proteins involved in superoxide production are similar to those involved in the NADPH oxidase-induced superoxide production in human
David Bergin; Emer P. Reeves; Julie Renwick; Frans B. Wientjes; Kevin Kavanagh
A Myocardial Nox2 Containing NAD(P)H Oxidase Contributes to Oxidative Stress in Human Atrial Fibrillation
Human atrial fibrillation (AF) has been associated with increased atrial oxidative stress. In animal models, inhibition of reactive oxygen species prevents atrial remodeling induced by rapid pacing, suggesting that oxidative stress may play an important role in the pathophysiology of AF. NAD(P)H oxidase is a major source of superoxide in the cardiovascular system; however, whether this enzyme contributes to atrial
Young M. Kim; Tomasz J. Guzik; Yin Hua Zhang; Mei Hua Zhang; Hassan Kattach; Chandi Ratnatunga; Ravi Pillai; Keith M. Channon; Barbara Casadei
The effect of inorganic arsenic on endothelium-dependent relaxation: Role of NADPH oxidase and hydrogen peroxide
Chronic arsenic ingestion predisposes to vascular disease, but underlying mechanisms are poorly understood. In the present study we have analyzed the effects of short-term arsenite exposure on vascular function and endothelium-dependent relaxation. Endothelium-dependent relaxations, nitric oxide (NO) and endothelium derived hyperpolarizing factor (EDHF)-type, were studied in rabbit iliac artery and aortic rings using the G protein-coupled receptor agonist acetylcholine (ACh) and by cyclopiazonic acid (CPA), which promotes store-operated Ca2+ entry by inhibiting the endothelial SERCA pump. Production of reactive oxygen species (ROS) in the endothelium of rabbit aortic valve leaflets and endothelium-denuded RIA and aortic rings was assessed by imaging of dihydroethidium. In the iliac artery, exposure to 100�?M arsenite for 30�min potentiated EDHF-type relaxations evoked by both CPA and ACh. Potentiation was prevented by catalase, the catalase/superoxide dismutase mimetic manganese porphyrin and the NADPH oxidase inhibitor apocynin. By contrast in aortic rings, that exhibited negligible EDHF-type responses, endothelium-dependent NO-mediated relaxations evoked by CPA and ACh were unaffected by arsenite. Arsenite induced apocynin-sensitive increases in ROS production in the aortic valve endothelium, but not in the media and adventitia of the iliac artery and aorta. Our results suggest that arsenite can potentiate EDHF-type relaxations via a mechanism that is dependent on hydrogen peroxide, thus demonstrating that dismutation of the superoxide anion generated by NADPH oxidase can potentially offset loss of NO bioavailability under conditions of reduced eNOS activity. By contrast, selective increases in endothelial ROS production following exposure to arsenite failed to modify relaxations mediated by endogenous NO.
Edwards, David H.; Li, Yiwen; Ellinsworth, David C.; Griffith, Tudor M.
Reciprocal regulation of endothelial nitric-oxide synthase and NADPH oxidase by betulinic acid in human endothelial cells.
Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is a protective principle in the vasculature. Many cardiovascular diseases are associated with reduced NO bioactivity and eNOS uncoupling due to oxidative stress. Compounds that reverse eNOS uncoupling and increase eNOS expression are of therapeutic interest. Zizyphi Spinosi semen (ZSS) is one of the most widely used traditional Chinese herbs with protective effects on the cardiovascular system. In human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells, an extract of ZSS increased eNOS promoter activity, eNOS mRNA and protein expression, and NO production in a concentration- and time-dependent manner. Major ZSS constituents include saponins, such as jujuboside A and B, and pentacyclic triterpenes, such as betulin and betulinic acid. Jujuboside A, jujuboside B, or betulin had no significant effect on eNOS expression, whereas betulinic acid increased eNOS mRNA and protein expression in HUVEC and EA.hy 926 cells. Interestingly, betulinic acid also attenuated the expression of NADPH oxidase subunits Nox4 and p22phox, thereby reducing oxidative stress and improving eNOS function. Consequently, betulinic acid-treated endothelial cells showed an increased production of bioactive NO (as indicated by a higher efficacy in stimulating cGMP generation in RFL-6 reporter cells). Thus, betulinic acid possesses combined properties of eNOS up-regulation and NADPH oxidase down-regulation. Compounds such as betulinic acid may have a therapeutic potential in cardiovascular disease. PMID:17496167
Steinkamp-Fenske, Katja; Bollinger, Larissa; Xu, Hui; Yao, Ying; Horke, Sven; F�rstermann, Ulrich; Li, Huige
Biochemistry, Physiology and Pathophysiology of NADPH Oxidases in the Cardiovascular System
The NADPH oxidase (Nox) enzymes are critical mediators of cardiovascular physiology and pathophysiology. These proteins are expressed in virtually all cardiovascular cells, and regulate such diverse functions as differentiation, proliferation, apoptosis, senescence, inflammatory responses and oxygen sensing. They target a number of important signaling molecules, including kinases, phosphatases, transcription factors, ion channels and proteins that regulate the cytoskeleton. Nox enzymes have been implicated in many different cardiovascular pathologies: atherosclerosis, hypertension, cardiac hypertrophy and remodeling, angiogenesis and collateral formation, stroke and heart failure. In this review, we discuss in detail the biochemistry of Nox enzymes expressed in the cardiovascular system (Nox1, 2, 4 and 5), their roles in cardiovascular cell biology, and their contributions to disease development.
Lassegue, Bernard; San Martin, Alejandra; Griendling, Kathy K.
NADPH oxidase-derived ROS and the regulation of pulmonary vessel tone
Pulmonary vessel constriction results from an imbalance between vasodilator and vasoconstrictor factors released by the endothelium including nitric oxide, endothelin, prostanoids, and reactive oxygen species (ROS). ROS, generated by a variety of enzymatic sources (such as mitochondria and NADPH oxidases, a.k.a. Nox), appear to play a pivotal role in vascular homeostasis, whereas elevated levels effect vascular disease. The pulmonary circulation is very sensitive to changes in the partial pressure of oxygen and differs from the systemic circulation in its response to this change. In fact, the pulmonary vessels contract in response to low oxygen tension, whereas systemic vessels dilate. Growing evidence suggests that ROS production and ROS-related pathways may be key factors that underlie this differential response to oxygen tension. A major emphasis of our laboratory is the role of Nox isozymes in cardiovascular disease. In this review, we will focus our attention on the role of Nox-derived ROS in the control of pulmonary vascular tone.
Frazziano, G.; Champion, H. C.
NADPH oxidases as a source of oxidative stress and molecular target in ischemia/reperfusion injury.
Ischemia/reperfusion injury (IRI) is crucial in the pathology of major cardiovascular diseases, such as stroke and myocardial infarction. Paradoxically, both the lack of oxygen during ischemia and the replenishment of oxygen during reperfusion can cause tissue injury. Clinical outcome is also determined by a third, post-reperfusion phase characterized by tissue remodeling and adaptation. Increased levels of reactive oxygen species (ROS) have been suggested to be key players in all three phases. As a second paradox, ROS seem to play a double-edged role in IRI, with both detrimental and beneficial effects. These Janus-faced effects of ROS may be linked to the different sources of ROS or to the different types of ROS that exist and may also depend on the phase of IRI. With respect to therapeutic implications, an untargeted application of antioxidants may not differentiate between detrimental and beneficial ROS, which might explain why this approach is clinically ineffective in lowering cardiovascular mortality. Under some conditions, antioxidants even appear to be harmful. In this review, we discuss recent breakthroughs regarding a more targeted and promising approach to therapeutically modulate ROS in IRI. We will focus on NADPH oxidases and their catalytic subunits, NOX, as they represent the only known enzyme family with the sole function to produce ROS. Similar to ROS, NADPH oxidases may play a dual role as different NOX isoforms may mediate detrimental or protective processes. Unraveling the precise sequence of events, i.e., determining which role the individual NOX isoforms play in the various phases of IRI, may provide the crucial molecular and mechanistic understanding to finally effectively target oxidative stress. PMID:23090009
Kleikers, Pamela W M; Wingler, K; Hermans, J J R; Diebold, I; Altenh�fer, S; Radermacher, K A; Janssen, B; G�rlach, A; Schmidt, H H H W
Nox4 NADPH oxidase contributes to smooth muscle cell phenotypes associated with unstable atherosclerotic plaques
Plaque instability associated with acute coronary syndromes results in part from apoptosis and senescence of cells within the atherosclerotic (AS) lesion. Increased cellular oxidative stress has been proposed to contribute to plaque progression and changes in composition, leading to plaque instability. Our objective was to examine the role of NADPH oxidase in smooth muscle cell (SMC) phenotypes associated with an unstable plaque. Aortae were isolated from pre-lesion (8�weeks of age) and post-lesion (35�weeks of age) hypercholesterolemic mice (ApoE-/-/LDLR-/-, AS), and age-matched normal C57BL/6J mice. We observed an age-dependent increase in reactive oxygen species (ROS) in aorta from AS mice, with evidence for elevated ROS prior to lesion development. Whereas macrophage infiltration was restricted to the lesion, oxidized lipids extended beyond the plaque and into the vessel wall. Consistent with these findings, we observed dynamic changes in the expression of NADPH oxidases in AS vessels. Specifically, Nox1 expression was increased early and decreased with lesion progression, while induction of Nox4 was a late event. Nox2 and p22phox were elevated throughout lesion development. Similar to observations in aortae, SMCs isolated from the lesion of AS aortae had decreased Nox1 and increased Nox4 levels as compared to SMCs from normal mice. AS SMCs demonstrated increased generation of ROS, cell cycle arrest, evidence of senescence, and increased susceptibility to apoptosis. Overexpression of Nox4 in normal SMCs recapitulated the phenotypes of the AS SMCs. We conclude that increased expression of Nox4 in AS may drive SMC phenotypes that lead to the plaque instability and rupture responsible for myocardial infarction and stroke.
Xu, Shaoping; Chamseddine, Ali H.; Carrell, Samuel; Miller, Francis J.
Which NADPH Oxidase Isoform Is Relevant for Ischemic Stroke? The Case for Nox 2
Abstract Significance and Recent Advances: Ischemic stroke is the leading cause of disability and third in mortality in industrialized nations. Immediate restoration of cerebral blood flow is crucial to salvage brain tissue, but only few patients are eligible for recanalization therapy. Thus, the need for alternative neuroprotective strategies is huge, and antioxidant interventions have long been studied in this context. Reactive oxygen species (ROS) physiologically serve as signaling molecules, but excessive amounts of ROS, as generated during ischemia/reperfusion (I/R), contribute to tissue injury. Critical Issues: Nevertheless and despite a strong rational of ROS being a pharmacological target, all antioxidant interventions failed to improve functional outcome in human clinical trials. Antioxidants may interfere with physiological functions of ROS or do not reach the crucial target structures of ROS-induced injury effectively. Future Directions: Thus, a potentially more promising approach is the inhibition of the source of disease-promoting ROS. Within recent years, NADPH oxidases (Nox) of the Nox family have been identified as mediators of neuronal pathology. As, however, several Nox homologs are expressed in neuronal tissue, and as many of the pharmacological inhibitors employed are rather unspecific, the concept of Nox as mediators of brain damage is far from being settled. In this review, we will discuss the contribution of Nox homologs to I/R injury at large as well as to neuronal damage in particular. We will illustrate that the current data provide evidence for Nox2 as the most important NADPH oxidase mediating cerebral injury. Antioxid. Redox Signal. 18, 1400�1417.
Kahles, Timo
NADPH oxidase pathway is involved in aortic contraction induced by A3 adenosine receptor in mice.
The NADPH oxidase (Nox) subunits 1, 2 (gp91 phox), and 4 are the major sources for reactive oxygen species (ROS) in vascular tissues. In conditions such as ischemia-reperfusion and hypoxia, both ROS and adenosine are released, suggesting a possible interaction. Our aim in this study was to examine the A(3) adenosine receptor (A(3)AR)-induced vascular effects and its relation to ROS and Nox1, 2, and 4 using aortic tissues from wild-type (WT) and A(3)AR knockout (A(3)KO) mice. The selective A(3)AR agonist 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IBMECA) (10(-10)-10(-5) M) induced contraction of the aorta from WT but not from A(3)KO mice, and this contraction was inhibited by the Nox inhibitor apocynin (10(-5) M) and the ROS scavengers superoxide dismutase-polyethylene glycol and catalase-polyethylene glycol (100 U/ml each). Cl-IBMECA-induced contraction was not affected by the mast cell degranulator compound 48/80 (100 ?g/ml) or the stabilizer cromolyn sodium (10(-4) M). In addition, Cl-IBMECA (10(-7) M) increased intracellular ROS generation by 35 � 14% in WT but not in A(3)KO aorta, and this increase was inhibited by apocynin (10(-5) M), diphenyleneiodonium chloride (10(-5) M), and the A(3)AR antagonist 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate (MRS1523) (10(-5) M). Furthermore, Cl-IBMECA selectively increased the protein expression of the Nox2 subunit by 150 � 15% in WT but not in A(3)KO mice without affecting either Nox1 or 4, and this increase was inhibited by apocynin. The mRNA of Nox2 was unchanged by Cl-IBMECA in either WT or A(3)KO aortas. In conclusion, A(3)AR enhances ROS generation, possibly through activation of Nox2, with subsequent contraction of the mouse aorta. PMID:21606175
El-Awady, Mohammed S; Ansari, Habib R; Fil, Daniel; Tilley, Stephen L; Mustafa, S Jamal
Riboflavin (vitamin B2 ) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes.
Riboflavin, also known as vitamin B2 , is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte-specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK-deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF-induced priming of Nox2, which is needed for oxidative burst, was defective in RFK-deficient phagocytes. Lack of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity. Vice versa, ROS production by riboflavin-deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo. PMID:24272050
Schramm, Michael; Wiegmann, Katja; Schramm, Sandra; Gluschko, Alexander; Herb, Marc; Uterm�hlen, Olaf; Kr�nke, Martin
Stoichiometry of the subunits of flavocytochrome b558 of the NADPH oxidase of phagocytes.
Flavocytochrome b558, the membrane-spanning component of the NADPH oxidase system of phagocytic cells, is composed of two subunits, p22phox and gp91phox (where phox stands for phagocyte oxidase). The stoichiometry of the subunits has been determined for purified flavocytochrome b556 by: (1) densitometry of Coomassie Blue-stained proteins separated by SDS/PAGE, (2) aromatic absorbance at 280 mm by the subunits after separation by gel filtration under denaturing conditions, (3) crosslinking studies with bis[sulphosuccinimidyl]suberate, where the molecular mass of the cross-linked complex was determined by Western blotting, and (4) radiolabelling of pure flavocytochrome b556 on lysine residues with 125I-labelled Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate), followed by SDS/PAGE and determination of the radioactivity on each subunit. The ratio of p22phox to gp91phox in the purified flavocytochrome b556 was related back to that in the neutrophil membrane by quantitative Western and dot-blotting to ensure that the stoichiometry was maintained during purification. These measurements showed that the two subunits were present in neutrophil membranes in a molar ratio of 1:1.
Wallach, T M; Segal, A W
Tempol attenuates atherosclerosis associated with metabolic syndrome via decreased vascular inflammation and NADPH-2 oxidase expression.
Oxidative stress is an important factor in the generation of vascular injury in atherosclerosis. Chronic administration of fructose in rodents is able to facilitate oxidative damage. In the present study we evaluated the role of Tempol, a superoxide dismutase mimetic, on the effect of high fructose intake in apolipoprotein E-deficient (ApoE-KO) mice. Rodents were fed with fructose overload (FF, 10% w/v) for 8 weeks and treated with Tempol 1 mg/kg/day the latest 4 weeks. Tempol revert the pro-oxidant effects caused by FF, diminished lipid peroxidation and impaired vascular NADPH oxidase system through the downregulation of p47phox expression in the vascular wall. Tempol inhibited the expression of vascular adhesion molecule 1 (VCAM-1) in aorta and reduced the development of atheroma plaques. Our results indicate that tempol attenuates oxidative stress by interfering with the correct assembly of Nox2 oxidase complex in the vascular wall and is able to reduce atherosclerosis. Thus tempol represents a potential therapeutic target for preventing risk factors associated with metabolic syndrome. PMID:24490696
Cannizzo, B; Quesada, I; Militello, R; Amaya, C; Miatello, R; Cruzado, M; Castro, C
NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death
Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91{sup phox} was dose-dependent. Meanwhile, the cytoplasmic subunit p47{sup phox} was translocated to the cell membrane and localized with p22{sup phox} and gp91{sup phox} to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.
Liu Qing [Department of Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Rd., Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Medicine of Gansu Province, 509 Nanchang Rd., Lanzhou 730000 (China); He Xiaoqing [First Affiliated Hospital of Lanzhou University, 1 Donggang West Rd., Lanzhou 730000 (China); Liu Yongsheng [Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural, 1 Xujiaping Rd., Lanzhou 730046 (China); Du Bingbing; Wang Xiaoyan [College of Medicine, Lanzhou University, 222, South Tianshui Rd., Lanzhou 730000 (China); Zhang Weisheng [Gansu Province People's Hospital, 160 Donggang West Rd., Lanzhou 730000 (China); Jia Pengfei [Institute of Cell Biology, School of Life Science, Lanzhou University, 222, South Tianshui Rd., Lanzhou 730000 (China); Dong Jingmei [Institute of Sports Medicine, Lanzhou City University, Jiefang Rd., Lanzhou 730070 (China); Ma Jianxiu [College of Medicine, Northwest University for Nationalities, 1 Northwest New Village, Lanzhou 730030 (China); Wang Xiaohu [Department of Radiology, Gansu Province Cancer Hospital, 2 Xiaoxihu East Rd., Lanzhou 730050 (China); Li Sha [Department of Radiology, Lanzhou General Hospital of PLA, 98 Xiaoxihu West Rd., 730050 (China); Zhang Hong [Department of Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Rd., Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Medicine of Gansu Province, 509 Nanchang Rd., Lanzhou 730000 (China)], E-mail: email@example.com
NADPH Oxidase 2 Mediates Intermittent Hypoxia-Induced Mitochondrial Complex I Inhibition: Relevance to Blood Pressure Changes in Rats
Abstract Previous studies identified NADPH oxidases (Nox) and mitochondrial electron transport chain at complex I as major cellular sources of reactive oxygen species (ROS) mediating systemic and cellular responses to intermittent hypoxia (IH). In the present study, we investigated potential interactions between Nox and the mitochondrial complex I and assessed the contribution of mitochondrial ROS in IH-evoked elevation in blood pressure. IH treatment led to stimulus-dependent activation of Nox and inhibition of complex I activity in rat pheochromocytoma (PC)12 cells. After re-oxygenation, Nox activity returned to baseline values within 3?h, whereas the complex I activity remained downregulated even after 24?h. IH-induced complex I inhibition was prevented by Nox inhibitors, Nox2 but not Nox 4 siRNA, in cell cultures and was absent in gp91phox-/Y (Nox2 knock-out; KO) mice. Using pharmacological inhibitors, we show that ROS generated by Nox activation mobilizes Ca2+ flux from the cytosol to mitochondria, leading to S-glutathionylation of 75- and 50-kDa proteins of the complex I and inhibition of complex I activity, which results in elevated mitochondrial ROS. Systemic administration of mito-tempol prevented the sustained but not the acute elevations of blood pressure in IH-treated rats, suggesting that mitochondrial-derived ROS contribute to sustained elevation of blood pressure. Antioxid. Redox Signal. 14, 533�542.
Khan, Shakil A.; Nanduri, Jayasri; Yuan, Guoxiang; Kinsman, Brian; Kumar, Ganesh K.; Joseph, Joy; Kalyanaraman, Balaraman
NADPH oxidase-derived reactive oxygen species contribute to impaired cutaneous microvascular function in chronic kidney disease.
Oxidative stress promotes vascular dysfunction in chronic kidney disease (CKD). We utilized the cutaneous circulation to test the hypothesis that reactive oxygen species derived from NADPH oxidase and xanthine oxidase impair nitric oxide (NO)-dependent cutaneous vasodilation in CKD. Twenty subjects, 10 stage 3 and 4 patients with CKD (61 � 4 yr; 5 men/5 women; eGFR: 39 � 4 ml�min(-1)�1.73 m(-2)) and 10 healthy controls (55 � 2 yr; 4 men/6 women; eGFR: >60 ml�min(-1)�1.73 m(-2)) were instrumented with 4 intradermal microdialysis fibers for the delivery of 1) Ringer solution (Control), 2) 10 ?M tempol (scavenge superoxide), 3) 100 ?M apocynin (NAD(P)H oxidase inhibition), and 4) 10 ?M allopurinol (xanthine oxidase inhibition). Skin blood flow was measured via laser-Doppler flowmetry during standardized local heating (42�C). N(g)-nitro-l-arginine methyl ester (l-NAME; 10 mM) was infused to quantify the NO-dependent portion of the response. Cutaneous vascular conductance (CVC) was calculated as a percentage of the maximum CVC achieved during sodium nitroprusside infusion at 43�C. Cutaneous vasodilation was attenuated in patients with CKD (77 � 3 vs. 88 � 3%, P = 0.01), but augmented with tempol and apocynin (tempol: 88 � 2 (P = 0.03), apocynin: 91 � 2% (P = 0.001). The NO-dependent portion of the response was reduced in patients with CKD (41 � 4 vs. 58 � 2%, P = 0.04), but improved with tempol and apocynin (tempol: 58 � 3 (P = 0.03), apocynin: 58 � 4% (P = 0.03). Inhibition of xanthine oxidase did not alter cutaneous vasodilation in either group (P > 0.05). These data suggest that NAD(P)H oxidase is a source of reactive oxygen species and contributes to microvascular dysfunction in patients with CKD. PMID:24761000
DuPont, Jennifer J; Ramick, Meghan G; Farquhar, William B; Townsend, Raymond R; Edwards, David G
EGFR Kinase Regulates Volume-sensitive Chloride Current Elicited by Integrin Stretch via PI-3K and NADPH Oxidase in Ventricular Myocytes
Stretch of ?1 integrins activates an outwardly rectifying, tamoxifen-sensitive Cl? current (Cl? SAC) via AT1 receptors, NADPH oxidase, and reactive oxygen species, and Cl? SAC resembles the volume-sensitive Cl? current (ICl,swell). Epidermal growth factor receptor (EGFR) kinase undergoes transactivation upon stretch, integrin engagement, and AT1 receptor activation and, in turn, stimulates NADPH oxidase. Therefore, we tested whether Cl? SAC is regulated by EGFR kinase signaling and is volume sensitive. Paramagnetic beads coated with mAb for ?1 integrin were attached to myocytes and pulled with an electromagnet. Stretch activated a Cl? SAC that was 1.13 � 0.10 pA/pF at +40 mV. AG1478 (10 ?M), an EGFR kinase blocker, inhibited 93 � 13% of Cl? SAC, and intracellular pretreatment with 1 ?M AG1478 markedly suppressed Cl? SAC activation. EGF (3.3 nM) directly activated an outwardly rectifying Cl? current (0.81 � 0.05 pA/pF at +40 mV) that was fully blocked by 10 ?M tamoxifen, an ICl,swell blocker. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase. Wortmannin (500 nM) and LY294002 (100 ?M), blockers of PI-3K, inhibited Cl? SAC by 67 � 6% and 91 � 25% respectively, and the EGF-induced Cl? current also was fully blocked by LY294002. Furthermore, gp91ds-tat (500 nM), a cell-permeable, chimeric peptide that specifically blocks NADPH oxidase assembly, profoundly inhibited the EGF-induced Cl? current. Inactive permeant and active impermeant control peptides had no effect. Myocyte shrinkage with hyperosmotic bathing media inhibited the Cl? SAC and EGF-induced Cl? current by 88 � 9% and 127 � 11%, respectively. These results suggest that ?1 integrin stretch activates Cl? SAC via EGFR, PI-3K, and NADPH oxidase, and that both the Cl? SAC and the EGF-induced Cl? currents are likely to be the volume-sensitive Cl? current, ICl,swell.
Browe, David M.; Baumgarten, Clive M.
Involvement of myeloperoxidase and NADPH oxidase in the covalent binding of amodiaquine and clozapine to neutrophils: implications for drug-induced agranulocytosis.
Amodiaquine (AQ) and clozapine (CLZ) are associated with a relatively high incidence of idiosyncratic agranulocytosis, a reaction that is suspected to involve covalent binding of reactive metabolites to neutrophils. Previous studies have shown that both AQ and CLZ are oxidized to reactive intermediates in vitro by activated neutrophils or by the combination of hydrogen peroxide and myeloperoxidase (MPO). Neutrophil activation leads to an oxidative burst with activation of NADPH oxidase and the production of hydrogen peroxide. However, the importance of this pathway in covalent binding in vivo has not been examined. In this study, we found that the binding of both AQ and CLZ to neutrophils from MPO knockout mice ex vivo decreased approximately 2-fold compared to neutrophils from wild-type mice, whereas binding to activated neutrophils from gp91 knockout (NADPH oxidase null) mice decreased 6-7-fold. When the AQ studies were performed in vivo, again the binding was decreased in MPO knockout mice to about 50% of the binding in wild-type mice; however, covalent binding was significant in the absence of MPO. Surprisingly, there was no significant decrease in covalent binding of AQ to neutrophils in vivo in gp91 knockout mice. In addition, there was extensive binding of AQ to many types of bone marrow cells and to peripheral lymphocytes. These results indicate that MPO is not the only neutrophil enzyme involved in the oxidation of AQ and that NADPH oxidase is not the major source of peroxide. There was also no decrease in AQ binding to neutrophils in COX-1 or COX-2 knockout mice. We were not able to readily reproduce the AQ in vivo studies with CLZ because of its acute toxicity in mice. These are the first studies to examine the enzymes involved in the bioactivation of AQ by neutrophils in vivo. PMID:24588327
Lobach, Alexandra R; Uetrecht, Jack
Regulation of the NADPH-oxidase complex of phagocytic leukocytes. Recent insights from structural biology, molecular genetics, and microscopy
The NADPH-oxidase complex is a multisubunit enzyme complex that catalyzes the formation of superoxide (O 2 ?) by phagocytic leukocytes. This paper reviews some of the major advances in understanding the assembly and regulation of this enzyme system that have occurred during the past decade. For example, novel domains\\/motifs have been identified in p47- phox (PX and super SH3 domains)
John M. Robinson; Taisuke Ohira; John A. Badwey
Genetic and Genomic Analysis of Rhizoctonia solani Interactions with Arabidopsis; Evidence of Resistance Mediated through NADPH Oxidases
Rhizoctonia solani is an important soil-borne necrotrophic fungal pathogen, with a broad host range and little effective resista