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Obesity is one of the most prevalent chronic diseases worldwide, and dysregulated adipocyte function plays an important role in obesity-associated metabolic disorder. Plasma plasminogen activator inhibitor-1 (PAI-1) level is increased in obese subjects and PAI-1 null mice show improved insulin sensitivity under high-fat and high-sucrose diet-induced metabolic stress, suggesting that best-in-class PAI-1 inhibitor may become a novel therapeutic agent for obesity-associated metabolic syndrome. TM5441 (5-chloro-2{[(2-{[3-(furan-3-yl)phenyl]amino}-2-oxoethoxy) acethyl]amino}benzoic acid) is a novel orally active PAI-1 inhibitor that does not cause bleeding episodes. The present study, thus, examined the preventive effect of TM5441 on high fat diet (HFD)-induced adipocyte dysfunction.
10-week-old C57BL/6 J mice were fed a normal diet (ND; 18% of total calories from fat) or HFD (60% of total calories from fat) for 10 weeks, and TM5441 (20 mg kg−1 oral gavage) was daily administered with the initiation of HFD.
TM5441 prevented HFD-induced body weight gain and systemic insulin resistance. TM5441 normalized HFD-induced dysregulated JNK and Akt phosphorylation, suggesting prevention of adipocyte insulin resistance. TM5441 also attenuated HFD-induced macrophage infiltration along with decreased expression of proinflammatory cytokines, such as inducible nitric oxide synthase. In addition, TM5441 prevented HFD-induced downregulation of genes involved in mitochondrial biogenesis and function, suggesting that TM5441 may prevent adipocyte inflammation and dysregulation through maintaining mitochondrial fitness.
Our data suggest that TM5441 may become a novel therapeutic agent for obesity and obesity-related metabolic disorders.
α- and β-melanocyte-stimulating hormone (MSH) are derived from pro-opiomelanocortin (POMC) and are the natural agonist ligands of the melanocortin 4 receptor (MC4 receptor), a key regulator of energy homeostasis. Recent rodent and human data have implicated the MAGEL2 gene, which may regulate activation of POMC neurons, as a significant contributor to the metabolic symptoms observed in Prader-Willi Syndrome (PWS). Firstly, patients with protein truncating mutations in MAGEL2 exhibit numerous clinical characteristics of PWS. Secondly, Magel2-null mice may not normally activate MC4 receptors, as they are defective in the activation of their POMC neurons and hence may fail to normally release the POMC-derived MC4 receptor agonist ligands α- and β-MSH. Magel2-null mice represent a tractable animal model for the metabolic and appetitive imbalance seen in patients with PWS.
We tested a dose titration of the MC4 receptor agonist setmelanotide, in development for rare monogenic forms of obesity, in Magel2-null mice.
We show that Magel2-null mice are hypersensitive to the appetite suppressing and metabolic effects of setmelanotide.
Setmelanotide may be a useful investigational hormone/neuropeptide replacement therapy for PWS and rare monogenic forms of obesity exhibiting impaired POMC neuronal function.
Cytoplasmic retention of β-catenin will lead to its nuclear translocation and subsequent interaction with the transcription factor TCF/LEF that regulates target gene expression. We've previously demonstrated abberant expression of β-catenin in a toluene diisocyanate (TDI)-induced asthma model. The aim of this study was to examine whether the receptor for advanced glycation end products (RAGE) can regulate β-catenin expression in TDI-induced asthma.
Male BALB/c mice were sensitized and challenged with TDI to generate a chemical-induced asthma model. Inhibitors of RAGE, FPS-ZM1 and RAGE antagonist peptide (RAP), were respectively given to the mice via intraperitoneal injection after each challenge.
Increased expression of RAGE as well as its ligands HMGB1, S100A12, S100B, HSP70 was detected in TDI-exposed lungs, and was then inhibited by FPS-ZM1/RAP. Mice treated with either antagonist showed significantly blunted airway reactivity, airway inflammation and goblet cell metaplasia, as well as decreased release of Th2 cytokines. TDI exposure decreases level of membrane β-catenin, phosphorylates Akt (Ser473), inactivates GSK3β (Ser9), dephosphorylates β-catenin at Ser33/37/Thr41 which controls its cytoplasmic degradation, increases phosphorylated β-catenin at Ser552, raises cytoplasmic and nuclear levels of β-catenin, and upregulates its targeted gene expression (MMP2, MMP7, MMP9, VEGF, cyclin D1, fibronectin), all of which were rescued by RAGE inhibition.
Our results showed that RAGE is required for β-catenin stabilization in TDI-induced asthma, identifying protective effects of RAGE blockade in TDI-induced asthma.
Signalling through phospholipase C (PLC) controls many cellular processes. Much information on the relevance of this important pathway has been derived from pharmacological inhibition of the enzymatic activity of PLC. We find that the most frequently employed PLC inhibitor, U73122, activates endogenous ionic currents in widely used cell lines. Given the extensive use of U73122 in research, we set out to identify the U73122-sensitive ion channels.
We performed detailed biophysical analysis of the U73122-induced currents in frequently used cell lines.
We find that at concentrations required to inhibit PLC U73122 modulates the activity of transient receptor potential melastatin (TRPM) channels through covalent modification. U73122 is a potent agonist of ubiquitously expressed TRPM4 channels that activates endogenous TRPM4 in Chinese hamster ovary (CHO) cells with an EC50 of approximately 440 nM independently of PLC and of the downstream second messengers PI(4,5)P2 and Ca2+. U73122 also potentiates Ca2+-dependent TRPM4 currents in human Jurkat T-cells, endogenous TRPM4 in HEK293T cells, and recombinant human TRPM4. In contrast to TRPM4, TRPM3 channels are inhibited whereas the closely related TRPM5 channels are insensitive to U73122, showing that U73122 exhibits high specificity within the TRPM channel family.
Given the wide spread expression of TRPM4 and TRPM3 channels, these actions of U73122 must be considered when interpreting its effects on cell function. U73122 may also be useful to identify and characterise TRPM channels in native tissue thus facilitating the analysis of their physiology.
Nitidine chloride (NC), a benzophenanthridine alkaloid, has various biological properties including anti-cancer and analgesic activities. The aim of the present study was to evaluate the role of organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1) in renal disposition and nephrotoxicity of NC.
Madin-Darby canine kidney (MDCK) cells stably expressing human OCT2 or/and hMATE1 were used to investigate the OCT2 or/and MATE1-mediated NC transport; meanwhile, NC accumulation and toxicity were also studied in rat primary-cultured proximal tubular (rPCPT) cells and rats.
Our study demonstrated that NC was a high affinity substrate of OCT2 and MATE1 with the Km of 0.97 and 0.90 μM, and the LC50 of NC-induced cytotoxicity in MDCK-hOCT2/hMATE1, MDCK-hOCT2 and mock cells were 1.60, 0.47 and 29.1 μM, respectively. OCT2 inhibitors, cimetidine and (+)-tetrahydropalmatine ((+)-THP), significantly reduced NC accumulation and cytotoxicity in MDCK-hOCT2, MDCK-hOCT2/hMATE1 and rPCPT cells. Severe kidney injury with high level of blood urea nitrogen (BUN) and lactate dehydrogenase (LDH), lower level of alkaline phosphatase (ALP), and pathological changes was found in rats after 20-day successively intravenous doses of 5 mg · kg-1 · day-1 NC. Concomitantly, the concentration of NC in kidney reached as high as 55.7 and 40.4 µg · g-1 at 2 h after the last dose and a single dose of 5 mg · kg-1, respectively.
Our data indicated NC-induced nephrotoxicity might be mainly attributed to OCT2-mediated extensive renal uptake and weak tubular secretion by MATE1.
Melatonin receptors are seven transmembrane-spanning proteins belonging to the G protein-coupled receptor super-family. In mammals, two melatonin receptor subtypes exit MT1 and MT2 encoded by the MTNR1A and MTNR1B genes, respectively. The current review provides an update on melatonin receptors by the corresponding sub-committee of the International Union of Basic and Clinical Pharmacology. We will highlight recent developments of melatonin receptor ligands, including radioligands and give an update on the latest phenotyping results of melatonin receptor knockout mice. The current status and perspectives of the structure of melatonin receptor structures will be summarized. The physiological importance of melatonin receptor dimers and biologically important and type 2 diabetes-associated genetic variants of melatonin receptors will be discussed. The role of melatonin receptors in physiology and disease will be further exemplified by its functions in the immune system and the central nervous system. Finally, antioxidant and free radical scavenger properties of melatonin and its relation to melatonin receptors will be critically addressed.
IL-33 is a novel cytokine that is believed to be involved in inflammation and carcinogenesis; however, its source, its production and its secretion process remain unclear. Recently we have reported that interleukin-33(IL-33) is up regulated in dextran sulfate sodium (DSS) colitis in mice.
We examined how IL-33 is produced from intestinal tissue using the murine azoxymethane (AOM)/DSS-induced cancer model and IEC-6 epithelial cells.
Mice with AOM/DSS-induced colitis expressed all the characteristic symptoms of colon cancer pathology. Immunohistochemical analysis demonstrated epithelial derived IL-33 in the colon tissues in AOM/DSS colitis mice. RT-PCR and quantitative PCR analysis of colon tissues revealed that AOM/DSS colitis tissues expressed upregulated IL-1β, IL-33, TGF-β, and EGF mRNA. Gefitinib, an EGFR inhibitor, inhibited IL-33 mRNA expression in AOM/DSS colitis mice. The pathophysiological role of IL-33 in rat IEC-6 epithelial cells was then investigated. We found that EGF, but not TGF-β1 or PDGF, greatly enhanced mRNA expression of IL-33 and its receptor ST2. In accordance with the gene expression and immunohistochemical analysis of IL-33 levels, ELISA based analysis of cytoplasmic and nuclear extracts showed increased IL-33 protein levels in IEC-6 cells after treatment with EGF.
Our results suggest that EGF is a key growth factor that triggers IL-33 production and ST2 receptor expression during intestinal inflammation and carcinogenesis. The EGF/IL-33/ST2 axis represents a novel therapeutic target of colon cancer.
Clinical studies have reported that pan-cannabinoid receptor agonists may have efficacy in neuropathic pain states and that this might be enhanced by co-administration with opioids. While cannabinoid-opioid analgesic synergy has been demonstrated in animal models of acute pain, it has not been examined in neuropathic pain models. We examined the effect of combination treatment with cannabinoid and opioid receptor agonists on allodynia and side-effects in a nerve injury induced neuropathic pain model.
C57BL/6 mice were subjected to chronic constriction injury (CCI) of the sciatic nerve. The effect of systemic administration of morphine and the pan-cannabinoid receptor agonist WIN55212 and allodynia and side effects was examined at 7 – 10 days post-CCI surgery. Isobolographic analysis was used to determine whether combination effects were synergistic.
The opioid agonist morphine reduced CCI-induced mechanical and cold allodynia, and produced motor incoordination, in a dose dependent manner. WIN55212 reduced CCI-induced allodynia, and produced motor incoordination, catalepsy and sedation, in a dose dependent manner, as we have observed previously. When administered together, WIN55212 and morphine reduced allodynia in a synergistic manner, but had only an additive effect on motor incoordination.
These findings indicate that combination administration of non-selective opioid and cannabinoid receptor agonists synergistically reduces nerve injury induced allodynia, while producing side-effects in an additive manner. This suggests that combination treatment has an improved anti-allodynic potency and therapeutic index in a neuropathic pain model.
Acid-sensing ion channels (ASICs) and the Epithelial Na+ channel (ENaC) are both members of the ENaC/DEG (degenerin) family of amiloride-sensitive Na+ channels. ASICs act as proton sensors and in the nervous system where they contribute, besides other roles, to fear behavior, learning and pain sensation. ENaC mediates Na+ reabsorption across epithelia of the distal kidney and colon and of the airways. ENaC is a clinically used drug target in the context of hypertension and cystic fibrosis, while ASIC is an interesting potential target. Following a brief introduction we will review here selected aspects of ASIC and ENaC function. We discuss the origin and nature of pH changes in the brain and the involvement of ASICs in synaptic signaling. We expose how in the peripheral nervous system, ASICs cover together with other ion channels a wide pH range as proton sensors. We introduce the mechanisms of aldosterone-dependent ENaC regulation and the evidence for an aldosterone-independent control of ENaC activity, such as regulation by dietary K+. We then provide an overview of the regulation of ENaC by proteases, a topic with increasing interest over the last years. In spite of the profound differences in the physiological and pathological roles of ASICs and ENaC, these channels share many basic functional and structural properties. It is likely that further research will identify physiological contexts in which ASICs and ENaC have similar or overlapping roles.
The NADPH oxidase (NOX) family of enzymes produces reactive oxygen species (ROS) as their sole function and are becoming recognized as key modulators of signal transduction pathways with a physiological role under acute stress and a pathological role after excessive activation under chronic stress. The 7 isoforms differ in their regulation, tissue and subcellular localization and ROS products. The most studied are NOX1, 2 and 4. Genetic deletion of NOX1 and 4, in contrast to NOX2, has revealed no significant spontaneous pathologies and a pathogenic relevance of both NOX1 and 4 across multiple organs in a wide range of diseases and in particular inflammatory and fibrotic diseases. This has stimulated interest in NOX inhibitors for therapeutic application. GKT136901 and GKT137831 are two structurally-related compounds demonstrating a preferential inhibition of NOX1 and 4 that have suitable properties for in vivo studies and have consequently been evaluated across a range of disease models and compared with gene deletion. In contrast to gene deletion, these inhibitors do not completely suppress ROS production, maintaining some basal level of ROS. Despite this and consistent with most gene deletion studies, these inhibitors are well tolerated and slow or prevent disease progression in a range of models of chronic inflammatory and fibrotic diseases by modulating common signal transduction pathways. Clinical trials in patients with GKT137831 have demonstrated excellent tolerability and reduction of various markers of chronic inflammation. NOX1/4 inhibition may provide a safe and effective therapeutic strategy for a range of inflammatory and fibrotic diseases.
Agmatine, a putative neurotransmitter plays a vital role in learning and memory. Although, it is considered as endogenous ligand of imidazoline receptors, exhibits high affinity towards α-adrenoceptors (α-ARs), nitric oxide synthase (NOS), and N-methyl-D-aspartic acid (NMDA) receptors. These substrates within locus coeruleus (LC) are critically involved in learning and memory processes.
Male Wistar rat were stereotaxicaly cannulated into LC and hippocampus. Different groups were administered with agmatine (i.p./i-LC) to observe its effect on acquisition, consolidation and retrieval of IA memory. NO donor (SNG), non-specific (L-NAME) and specific NOS inhibitors (L-NIL, 7-NI, L-NIO), α2-AR antagonist (yohimbine), agonist (clonidine) were administered before agmatine in LC. Moreover, influence of intrahippocampal NMDA antagonist, MK-801 was observed on memory enhancing effect of agmatine, SNG and yohimbine. In addition, immunohistochemical expression of TH and eNOS were observed in LC.
Agmatine (i-LC/ i.p.) facilitates the retrieval of memory in IA test. S-Nitrosoglutathione potentiated while L-NAME and L-NIO decreased the agmatine's effect in IA test. In contrast, L-NIL and 7-NI does not alter the effect of agmatine. Further, yohimbine potentiated whereas clonidine attenuated effects of agmatine within LC. Moreover, effect of agmatine, SNG and yohimbine was blocked by intrahippocampal administration of NMDA antagonist, MK-801. In addition, agmatine treatment significantly increased the population of TH- as well as eNOS-immunoreactive elements in the LC.
Thus results of present study have demonstrated that the facilitation of memory retrieval in IA task by agmatine is probably mediated by eNOS-NO-NE pathway in LC.
Fibrosis refers to the hardening or scarring of tissues that usually results from aberrant wound healing in response to organ injury, and its manifestations in various organs have collectively been estimated to contribute to around 45-50% of deaths in the Western world. Despite this, there is currently no effective cure for the tissue structural and functional damage induced by fibrosis-related disorders. Relaxin meets several criteria of an effective anti-fibrotic based on its specific ability to inhibit pro-fibrotic cytokine and/or growth factor-mediated, but not normal/unstimulated fibroblast proliferation, differentiation and matrix production. Furthermore, relaxin augments matrix degradation through its ability to up-regulate the release and activation of various matrix-degrading matrix metalloproteinases and/or being able to down-regulate tissue inhibitor of metalloproteinase activity. Relaxin can also indirectly suppress fibrosis through its other well-known (anti-inflammatory, anti-oxidant, anti-hypertrophic, anti-apoptotic, angiogenic, wound-healing and vasodilatory) properties. This review will outline the organ-specific and general anti-fibrotic significance of exogenously-administered relaxin, and its mechanisms of action, that have been documented in various non-reproductive organs such as the cardiovascular system, kidney, lung, liver, skin and tendons. In addition, it will outline the influence of sex on relaxin's anti-fibrotic actions, highlighting its potential as an emerging anti-fibrotic therapeutic.
Relaxin-3 (RLN3) is a neuropeptide that is strongly expressed in the pontine nucleus incertus (NI) and binds with high affinity to its cognate receptor RXFP3. Central administration of RLN3 in rats increases food intake and adiposity. In humans, RLN3 polymorphism was associated with obesity and hypercholesterolemia. Emerging evidence suggests that the effects of RLN3 may have sex-specific aspects. Thus, the RLN3 knockout female but not male mice are hypoactive. RLN3 produced stronger orexigenic and obesogenic effects in female rats compared to male rats. In addition, female rats demonstrated higher sensitivity to lower doses of RLN3. Repeated cycles of food restriction and stress were accompanied by an increase in RLN3 expression and hyperphagia in female but not in male rats. Furthermore, stress-induced binge eating in female rats was blocked by an RXFP3 antagonist. RLN3 increased the expression of corticotropin releasing factor in the paraventricular hypothalamic nucleus in male but not in female rats. Conversely, in female rats, RLN3 increased the expression of orexin in the lateral hypothalamus. There is evidence that orexin directly activates the RLN3 neurons in the NI. The positive reinforcement of the RLN3 effects by orexin may intensify behavioural activation and feeding in females. Sex-specific effects of RLN3 may also depend on differential expression of RXFP3 in the brain. Given the higher sensitivity of females to the orexigenic effects of RLN3 and the stress-induced activation of RLN3, the overall data suggest a possible role of RLN3 in eating disorders that show a higher propensity in women.
Orthosteric agonists and positive allosteric modulators (PAMs) of the α7 nicotinic acetylcholine receptor (nAChR) represent novel therapeutic approaches for pain modulation. Moreover, compounds with dual function as allosteric agonists and PAMs, known as ago-PAMs add further regulation of receptor function.
Initial studies examined the α7 ago-PAM, GAT107, in the formalin, complete Freund's adjuvant (CFA), lipopolysaccharide inflammatory pain models, the chronic constriction injury neuropathic pain model, the tail flick and hot plate acute thermal nociceptive assays. Additional studies examined the locus of action of GAT107, and immunohistochemical markers in the dorsal horn of the spinal cord in the CFA model.
Complementary pharmacological and genetic approaches confirmed that the dose-dependent antinociceptive effects of GAT107 were mediated through α7 nAChR. However, GAT107 was inactive in the tail-flick and hot-plate assays. In addition, GAT107 blocked conditioned place aversion elicited by acetic acid injection. Furthermore, intrathecal, but not intraplantar, injections of GAT107 reversed nociception in the CFA model, suggesting a spinal component of action. Immunohistochemical evaluation revealed an increase in the expression of astrocyte-specific glial fibrillary acidic protein and phosphorylated p38-mitogen-activated-kinase within the spinal cords of mice treated with CFA, which was attenuated by intrathecal GAT107 treatment. Importantly, GAT107 did not elicit motor impairment and continued to produce antinociceptive effects after subchronic administration in both phases of the formalin test.
Collectively, these results provide the first proof-of-principle that α7 ago-PAMs represent an effective pharmacological strategy for treating inflammatory and neuropathic pain.
Insulin-like peptide 5 (INSL5) is a two-chain, three-disulphide bonded peptide of the insulin/relaxin superfamily, uniquely expressed in enteroendocrine L-cells of the colon. It is the cognate ligand of relaxin family peptide receptor 4 (RXFP4) that is mainly expressed in the colorectum and enteric nervous system. This study identifies new signalling pathways activated by INSL5 acting on RXFP4.
INSL5/RXFP4 signalling was investigated using AlphaScreen® proximity assays. RXFP4 recruitment of Gαi/o proteins was determined by rescue of pertussis toxin (PTX)-inhibited cAMP and ERK1/2 responses following transient transfection of PTX-insensitive Gαi/o C351I mutants. Cell proliferation was studied using bromodeoxyuridine cell proliferation ELISA. Real-time BRET was used to examine RXFP4 interactions with β-arrestins, G protein-coupled receptor kinase 2 (GRK2), KRas and Rab5a. Gene expression was investigated using real-time quantitative PCR. Insulin release was measured using HTRF and intracellular Ca2+ flux monitored in a Flexstation® using Fluo-4-AM.
INSL5 inhibited forskolin-stimulated cAMP accumulation and increased phosphorylation of ERK1/2, p38MAPK, Akt-Ser473, Akt-Thr308 and S6 ribosomal protein (S6RP). cAMP and ERK1/2 responses were abolished by PTX and rescued by mGαoA, mGαoB and mGαi2 and to a lesser extent mGαi1 and mGαi3. RXFP4 interacted with GRK2 and β-arrestins, moved towards Rab5a and away from KRas, indicating internalisation following receptor activation. INSL5 treatment inhibited glucose-stimulated insulin secretion and Ca2+ mobilisation in MIN6 insulinoma cells, and forskolin-stimulated cAMP accumulation in NCI-H716 enteroendocrine cells.
The insulin-like peptide relaxin, originally identified as a hormone of pregnancy, is now known to exert a range of pleiotropic effects including vasodilatory, anti-fibrotic, angiogenic, anti-apoptotic and anti-inflammatory effects in both males and females. Relaxin produces these effects by binding to a cognate receptor RXFP1 and activating a variety of signalling pathways including cAMP, cGMP and MAP kinases as well as by altering gene expression of TGFβ, matrix metalloproteinases, angiogenic growth factors and endothelin receptors. The peptide has been shown to be effective in halting or reversing many of the adverse effects including fibrosis in animal models of cardiovascular disease including ischaemia/reperfusion injury, myocardial infarction, hypertensive heart disease and cardiomyopathy. Relaxin given to humans is safe and produces favourable haemodynamic changes. Serelaxin, the recombinant form of relaxin, is now in extended phase III clinical trials for the treatment of acute heart failure. Previous clinical studies indicated that a 48 hr infusion of relaxin improved 180 day mortality yet the mechanism underlying this effect is not clear. This article provides an overview of the cellular mechanism of effects of relaxin, summarises its beneficial actions in animal models and in the clinic. We also hypothesize potential mechanisms for the clinical efficacy of relaxin, identify current knowledge gaps and suggest new ways in which relaxin could be useful therapeutically.
Elevated serum uric acid (UA) is a risk factor for the development of kidney disease. Inhibitors of xanthine oxidase (XOi), an enzyme involved in UA synthesis, have protective effects at early stages of experimental diabetic nephropathy (DN). However, long-term effects of XOi in models of DN remain to be determined.
Development of albuminuria, renal structure and molecular markers of DN were studied in Type 2 diabetic Zucker obese rats (ZO) treated for 18 weeks with XOi febuxostat (FBX) and compared to vehicle-treated ZO rats (ZO-VE), ZO rats treated with enalapril (ZO-ENL) or a combination of both agents (ZO-FBX + ENL), and lean Zucker rats without metabolic defects (ZL-VE).
FBX normalized serum UA and attenuated the development of albuminuria, renal structural changes, with no significant effects on blood pressure, metabolic control or systemic markers of oxidative stress (OS). Most of these actions were comparable to ZO-ENL. Combination treatment lead to marked decreases in BP, and was more effective in amelioration of structural changes, expression of profibrotic genes and systemic OS than both monotherapies. FBX attenuated renal protein expression of TGF-ß, CTGF, collagen 4, mesenchymal markers (FSP1, vimentin), and a tissue marker of OS nitrotyrosine. Moreover, FBX attenuated TGF-ß- or s100B-induced increases in expression of fibrogenic molecules in renal tubular cells in vitro in UA-free media in Akt kinase-dependent manner.
FBX is protective and enhances actions of ENL in experimental DN. Multiple mechanisms might contribute, such as reduction of UA, renal OS and inhibition of profibrotic signaling.
The opioid antagonist nalmefene (selincro®) was approved for alcohol-related disorders by the European Medicines Agency in 2013. However, there have been no studies regarding the effectiveness of nalmefene in the combined use of alcohol and cocaine.
Using operant alcohol self-administration in Wistar rats and qRT-PCR, we evaluated (a) the dose-response curve for subcutaneous and oral nalmefene; (b) the effects of nalmefene with increasing concentrations of alcohol; (c) the efficacy of nalmefene on cocaine-induced increases in alcohol responses; and (d) the gene expression profiles of histone deacetylases (Hdac1-11) in peripheral blood in vivo and in the prefrontal cortex, heart, liver and kidney post mortem.
Subcutaneous (0.01, 0.05, 0.1 mg/kg) and oral (10, 20, 40 mg/kg) nalmefene reduced dose-dependently the operant responses for alcohol up to 50.3%. Nalmefene systematically reduced alcohol self-administration independently from alcohol concentration (10%, 15%, 20%). While cocaine caused an increase of approximately 40% in alcohol responses, this result was fully reversed with a low dose of nalmefene (0.05 mg/kg). Additionally, alcohol caused a general increase in Hdac gene expression in blood, which was reduced in Hdac3, 8, 5, 7, 9, 6, 10 by nalmefene. In the other tissues, alcohol and nalmefene either did not alter the gene expression of Hdacs, as in the prefrontal cortex, or the gene expression showed a tissue-Hdac-specific effect.
Nalmefene might be effective in the treatment of the combined use of alcohol and cocaine, and Hdacs gene expression in peripheral blood might be used as a biomarker of alcohol use and drug response.
5-HT increases force, L-type Ca2+ current (ICa,L) and causes arrhythmias through 5-HT4 receptors in human atrium. In permanent atrial fibrillation (peAF), atrial force responses to 5-HT are blunted, arrhythmias abolished, but ICa,L responses only moderately attenuated. We investigated whether in peAF this could be due to an increased function of phosphodiesterases PDE3 and/or PDE4, using the inhibitors cilostamide (300 nM) and rolipram (1 μM) respectively.
Contractile force, arrhythmic contractions and ICa,L were assessed in right atrial trabeculae and myocytes, obtained from patients with sinus rhythm (SR), paroxysmal atrial fibrillation (pAF) and peAF.
The maximum force response to 5-HT was reduced to 15% in peAF, but not in pAF. Cilostamide, but not rolipram, increased both the blunted force responses to 5-HT in peAF and the inotropic potency of 5-HT 4-7-fold in trabeculae of patients with SR, pAF and peAF. Lusitropic responses to 5-HT were not decreased in peAF. Responses of ICa,L to 5-HT did not differ and were unaffected by cilostamide or rolipram in myocytes from patients with SR or peAF. Concurrent cilostamide and rolipram increased 5-HT's propensity to elicit arrhythmias in trabeculae from patients with SR but not peAF.
PDE3, but not PDE4, reduces the inotropic responses to 5-HT in peAF, independently of lusitropy and ICa,L, but the activity of PDE3 appears not greater than in patients with SR and pAF. Atrial remodelling in peAF abolishes the facilitation of 5-HT to induce arrhythmias by inhibition of PDE3 plus PDE4.
Control of nematode parasite infections relies largely on anthelminthic drugs, several of which act on nicotinic acetylcholine receptors (nAChRs) and there are concerns about the development of resistance. There is an urgent need for development of new resistance-busting drugs and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of the homomeric nAChR, ACR-16, from a nematode parasite.
Using RT-PCR, molecular cloning, and two-electrode voltage-clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum, then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of the receptor to cholinergic anthelmintics and a range of nicotinic agonists was then tested.
Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from A. suum somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, we observed that existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, bephenium, tribendimidine & pyrantel) did not activate Asu-ACR-16 (except for small currents to oxantel). Other nAChR agonists: nicotine, acetylcholine, cytisine, 3-bromocytisine and epibatidine produced robust current responses which desensitized at a rate that varied with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin, and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had a lower calcium permeability than α7 receptors.
We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
Flecainide is a use-dependent blocker of cardiac Na+ channels. The mechanisms involved in block were elucidated using fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide, establishing that Na+ channel block requires the cationic form of the molecule to enter the cytosolic vestibule of the open channel. Flecainide is also effective in the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), however its mechanism of action is contentious. We investigated how flecainide derivatives influence RyR2-mediated Ca2+-release from the sarcoplasmic reticulum and whether this correlates with their effectiveness as blockers of Na+ channels and/or RyR2.
We compared the ability of QX-FL and NU-FL to block individual recombinant human RyR2 channels incorporated into planar phospholipid bilayers, and their effects on the properties of Ca2+ sparks in intact adult rat cardiac myocytes.
Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal flux of cations through RyR2 but were significantly less effective than flecainide. None of the compounds influenced the physiologically relevant luminal to cytosol cation flux through RyR2. Intracellular flecainide or QX-FL reduced Ca2+ spark frequency whereas NU-FL didn't.
Conclusions and implications
Given its inability to block physiologically-relevant cation flux through RyR2, and its lack of efficacy in blocking the cytosolic-to-luminal current, the effect of QX-FL on Ca2+ sparks is likely, by analogy with flecainide, to result from Na+ channel block. Our data reveal important differences in the nature of flecainide interaction with sites in the cytosolic vestibules of Na+ and RyR2 channels.
Cardiovascular diseases are major contributors to global deaths and disability-adjusted life years, with hypertension as a significant risk factor for all cause death. The endothelium that lines the inner wall of the vasculature regulates essential haemostatic functions such as regulation of the vascular tone, circulation of blood cells, inflammation and platelet activity. Endothelial dysfunction is an early predictor of atherosclerosis and future cardiovascular events. We review the prognostic value of measurements of endothelial function, the clinical techniques for its determination, mechanisms leading to endothelial dysfunction and the therapeutic treatment of endothelial dysfunction. Since vascular oxidative stress and inflammation are major determinants of endothelial function, we also address current antioxidant and anti-inflammatory therapies. In light of recent data that speak against the prognostic value of endothelial function in healthy human cohorts, we also discuss alternative diagnostic parameters such as vascular stiffness index and intima/media thickness ratio. We also suggest that investigation of vascular function, including that of the smooth muscle and even perivascular adipose tissue, may be appropriate parameters for clinical investigation.
Endoplasmic reticulum (ER) stress is increasingly recognized as an important causal factor of many diseases. Targeting ER stress has now emerged as a new therapeutic strategy for treating cardiovascular diseases. Here we investigated the effects and the underlying mechanism of Ginkgolide K (1,10-dihydroxy-3,14-didehydroginkgolide, GK) on cardiac ER stress.
Cell death, apoptosis, and ER stress-related signalling pathways were measured in cultured neonatal rat cardiomyocytes (NRCMs), treated with the ER stress inducers tunicamycin, hydrogen peroxide, and thapsigargin. Acute myocardial infarction was established using left coronary artery occlusion in mice, and infarct size was measured by triphenyltetrazolium chloride (TTC) staining. Echocardiography was used to assess heart function and transmission electron microscopy for evaluating ER expansion.
GK significantly decreased ER stress-induced cell death in both in vitro and in vivo models. In ischemic injured mice, GK treatment reduced infarct size, rescued heart dysfunction and ameliorated ER dilation. Mechanistic studies revealed that the beneficial effects of GK occurred through enhancement of inositol-requiring enzyme 1α (IRE1α)/ X box-binding protein-1 (XBP1) activity, which in turn led to increased ER-associated degradation (ERAD)-mediated clearance of misfolded proteins and autophagy. In addition, GK was also able to partially repress the pro-apoptotic action of regulated IRE1-dependent decay (RIDD) and JNK pathway.
In conclusion, GK acts through selective activation of the IRE1α/XBP1 pathway to limit ER stress injury. GK is revealed as a promising therapeutic agent to ameliorate ER stress for treating cardiovascular diseases.
Neutrophil serine proteases (NSPs) are activated by dipeptidyl peptidase 1 (DPP1) during neutrophil maturation. The impact of neutrophil turnover rate on NSP activity following DPP1 inhibition was studied in a rat pharmacokinetic/pharmacodynamic (PKPD) model.
Rats were dosed with a DPP1 inhibitor twice daily for up to 14 days, NSP activity was measured in onset or recovery studies and an indirect response model was fitted to the data to estimate the turnover rate of the response.
Maximum NSP inhibition was achieved after 8 days of dosing and a reduction of around 75% NSP activity was achieved at 75% in vitro DPP1 inhibition. Both the rate of inhibition and recovery of NSP activity were consistent with a neutrophil turnover rate of between 4-6 days. Using human neutrophil turnover rate it is predicted that maximum NSP inhibition following DPP1 inhibition takes around 20 days in human.
Following inhibition of DPP1 in the rat the NSP activity was determined by the amount of DPP1 inhibition and the turnover of neutrophils and is thus supportive of the role of neutrophil maturation in the activation of NSPs. Clinical trials to monitor the effect of a DPP1 inhibitor on NSPs should take into account the delay in maximal response on the one hand as well as the potential delay in a return to baseline NSP levels following treatment cessation.
Aging is an important risk factor of nonalcoholic fatty liver disease (NAFLD). Here, we investigated whether the deficiency of nicotinamide adenine dinucleotide (NAD+), a ubiquitous coenzyme, links aging with NAFLD.
Hepatic NAD+ concentration, together with the protein levels of nicotinamide phosphoribosyltransferase (NAMPT) and several other critical enzymes regulating NAD+ biosynthesis, were compared between middle-aged and aged mice or patients. The influences of NAD+ decline on the steatosis and steatohepatitis was evaluated in wild-type (WT) and H247A dominant-negative enzymatic-dead NAMPT transgenic mice (DN-NAMPT) under normal and high-fat diet (HFD).
Hepatic NAD+ level decreased in aged mice and people. NAMPT-controlled NAD+ salvage, but not de novo biosynthesis pathway, was compromised in liver of elderly mice and human. Under normal chow, middle-age DN-NAMPT mice displayed systemic NAD+ reduction, and had moderate NAFLD phenotypes, including lipid accumulation, enhanced oxidative stress, triggered inflammation and impaired insulin sensitivity in liver. All these NAFLD phenotypes, especially the pro-inflammatory factors release, Kupffer cell accumulation, monocytes infiltration, NLRP3 inflammasome pathway, and hepatic fibrosis (Masson's staining and α-SMA staining), were further deteriorated under HFD challenge. Orally administration of nicotinamide riboside, a natural NAD+ precursor, completely corrected these NAFLD phenotypes induced by NAD+ deficiency alone or HFD, whereas adenovirus-mediated SIRT1 overexpression only partially rescued these phenotypes.
These results provide the first evidence that aging-associated NAD+ deficiency is a critical risk factor for NAFLD, and suggest that supplement of NAD+ substrates may be a promising therapeutic strategy to prevent and treat NAFLD.
We investigated the effectiveness of RPL554 a dual phosphodiesterase 3 and 4 enzyme inhibitor, on airway smooth muscle relaxation and compared it to that induced by salbutamol, ipratropium bromide, glycopyrrolate or their combination on bronchomotor tone induced by different spasmogenic agents.
Guinea-pig tracheal preparations were suspended under 1 g tension in Krebs Henseleit solution maintained at 37 °C and aerated with 95% O2/5% CO2 and incubated in the presence of indomethacin (5 μM). Relaxation induced by cumulative concentrations of muscarinic receptor antagonists (ipratropium bromide or glycopyrrolate), beta2-agonists (salbutamol or formoterol), a PDE3 inhibitor (cilostamide, cilostazol or siguazodan) or a PDE4 inhibitor (roflumilast) was evaluated in comparison with RPL554. Maximal relaxation was calculated (% Emax papaverine) and expressed as mean ± sem.
Bronchomotor tone induced by the various spasmogens was reduced by the different bronchodilators to varying degrees. RPL554 (10-300 M) caused near maximum relaxation irrespective of the spasmogen examined, whereas the efficacy of the other relaxant agents varied according to the contractile stimulus used. In further studies to evaluate potential synergistic interaction between bronchodilators, RPL554 proved superior to salbutamol when either was combined with muscarinic receptor antagonists.
RPL554 produces near maximal relaxation of highly contracted respiratory smooth muscle and provides additional relaxation compared with that produced by other clinically used bronchodilator drugs. This suggests that RPL554 has the potential to produce additional beneficial bronchodilation over and above that of maximal clinical doses of standard bronchodilators in highly constricted airways of patients.
Optimal drug therapy often requires continuing high levels of target occupancy. Besides the traditional pharmacokinetic contribution thereto, target binding kinetics is increasingly considered to play an important role as well. While most attention has been focused on the dissociation rate of the complex, recent reports expressed doubt about the unreserved translatability of this pharmacodynamic property into clinical efficacy. "Micro"- pharmacokinetic mechanisms like drug rebinding and partitioning into the cell membrane may constitute a potential fix.
Simulations based on solving differential equations.
Based on a selected range of association and dissociation rate constants, kon and koff, and rebinding potencies of the drugs as variables, we have presently simulated their impact on the temporal in vivo occupancy profile of their targets after one or multiple repetitive dosings.
Most strikingly, the simulations show that, when rebinding is also taken into account, increasing kon may produce closely the same outcome as decreasing koff when dosing is performed in accordance with the therapeutically most relevant constant [Lmax]/KD ratio paradigm. Also, under certain conditions, rebinding may produce closely the same outcome as invoking slow diffusion of the drug between the plasma- and a target-containing "effect" compartments.
Although the present simulations should only be regarded to constitute "proof of principle", the findings may help pharmacologists and medicinal chemists to device ex-vivo and in vitro binding kinetic assays that are more relevant and translatable to in vivo settings. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Leptin, an important regulator of the energy balance, acts on the brain to inhibit feeding. However, the mechanisms involved in leptin signalling have not yet been fully elucidated. Heat shock protein 90 (HSP90) is a molecular chaperone that is involved in regulating cellular homeostasis. In the present study, we investigated the possible involvement of HSP90 in leptin signal transduction.
HEK293 and SH-SY5Y cell lines stably transfected with the Ob-Rb leptin receptor (HEK293 Ob-Rb, SH-SY5Y Ob-Rb) were used in the present study. Phosphorylation of JAK2 and STAT3 was analysed by western blotting. An HSP90 inhibitor was administered i.c.v. into rats and their food intake was analysed.
The knock-down of HSP90 in the HEK293 Ob-Rb cell line attenuated leptin-induced JAK2 and STAT3 signalling. Moreover, leptin-induced JAK2/STAT3 phosphorylation was markedly attenuated by the HSP90 inhibitors geldanamycin, radicicol and novobiocin. However, these effects were not mediated through previously known factors, which are known to be involved in the development of leptin resistance, such as suppressor of cytokine signalling 3 or endoplasmic reticulum stress. The infusion of an HSP90 inhibitor into the CNS blunted the anorexigenic actions of leptin in rats (male Wister rat).
HSP90 may be a novel factor involved in leptin-mediated signalling that is linked to anorexia.
Mitochondria are dynamic organelles, which couple the various cellular processes that regulate metabolism, cell proliferation and survival. Environmental stress can cause mitochondrial dysfunction and dynamic changes including reduced mitochondrial biogenesis, oxidative phosphorylation and ATP production, as well as mitophagy impairment, which leads to increased ROS, inflammatory responses and cellular senescence. Oxidative stress, inflammation and cellular senescence all have important roles in the pathogenesis of chronic lung diseases, such as chronic obstructive pulmonary disease, pulmonary fibrosis and bronchopulmonary dysplasia. In this review, we discuss the current state on how mitochondrial dysfunction affects inflammatory responses and cellular senescence, the mechanisms of mitochondrial dysfunction underlying the pathogenesis of chronic lung diseases and the potential of mitochondrial transfer and replacement as treatments for these diseases.]]>
Transient receptor potential ankyrin-1 (TRPA1) activation is known to mediate neurogenic vasodilatation. We investigated the mechanisms involved in TRPA1-mediated peripheral vasodilatation in vivo using the TRPA1 agonist cinnamaldehyde.
Changes in vascular ear blood flow were measured in anaesthetized mice using laser Doppler flowmetry.
Topical application of cinnamaldehyde to the mouse ear caused a significant increase in blood flow in the skin of anaesthetized wild-type (WT) mice but not in TRPA1 knockout (KO) mice. Cinnamaldehyde-induced vasodilatation was inhibited by the pharmacological blockade of the potent microvascular vasodilator neuropeptide CGRP and neuronal NOS-derived NO pathways. Cinnamaldehyde-mediated vasodilatation was significantly reduced by treatment with reactive oxygen nitrogen species (RONS) scavenger such as catalase and the SOD mimetic TEMPOL, supporting a role of RONS in the downstream vasodilator TRPA1-mediated response. Co-treatment with a non-selective NOS inhibitor L-NAME and antioxidant apocynin further inhibited the TRPA1-mediated vasodilatation. Cinnamaldehyde treatment induced the generation of peroxynitrite that was blocked by the peroxynitrite scavenger FeTPPS and shown to be dependent on TRPA1, as reflected by an increase in protein tyrosine nitration in the skin of WT, but not in TRPA1 KO mice.
This study provides in vivo evidence that TRPA1-induced vasodilatation mediated by cinnamaldehyde requires neuronal NOS-derived NO, in addition to the traditional neuropeptide component. A novel role of peroxynitrite is revealed, which is generated downstream of TRPA1 activation by cinnamaldehyde. This mechanistic pathway underlying TRPA1-mediated vasodilatation may be important in understanding the role of TRPA1 in pathophysiological situations.
The growing epidemic of obesity and metabolic diseases necessitates the development of novel strategies to prevent and treat such diseases. Current research suggests that browning of white adipose tissue (WAT) promotes energy expenditure to counter obesity. Recent research suggests that activation of the TRPV1 channels counters obesity. However, the mechanism by which activation of TRPV1 channels counters obesity still remains unclear.
We evaluated the effect of dietary capsaicin to induce a browning program in WAT by activating TRPV1 channels to prevent diet-induced obesity using wild-type and TRPV1−/− mouse models. We performed experiments using preadipocytes and fat pads from these mice.
Capsaicin stimulated the expression of brown fat-specific thermogenic uncoupling protein-1 and bone morphogenetic protein-8b in WAT. Capsaicin triggered browning of WAT by promoting sirtuin-1 expression and activity via TRPV1 channel-dependent elevation of intracellular Ca2+ and phosphorylation of Ca2+/calmodulin-activated protein kinase II and AMP-activated kinase. Capsaicin increased the expression of PPARγ 1 coactivator α and enhanced metabolic and ambulatory activity. Further, capsaicin stimulated sirtuin-1-dependent deacetylation of PPARγ and the transcription factor PRDM-16 and facilitated PPARγ–PRDM-16 interaction to induce browning of WAT. Dietary capsaicin did not protect TRPV1−/− mice from obesity.
Our results show for the first time that activation of TRPV1 channels by dietary capsaicin triggers browning of WAT to counteract obesity. Our results suggest that activation of TRPV1 channels is a promising strategy to counter obesity.
Insulin sensitivity and metabolic homeostasis depend on the capacity of adipose tissue to take up and utilize excess glucose and fatty acids. The key aspects that determine the fuel-buffering capacity of adipose tissue depend on the physiological levels of the small redox molecule, nitric oxide (NO). In addition to impairment of NO synthesis, excessive formation of the superoxide anion (О2•–) in adipose tissue may be an important interfering factor diverting the signalling of NO and other reactive oxygen and nitrogen species in obesity, resulting in metabolic dysfunction of adipose tissue over time. Besides its role in relief from superoxide burst, enhanced NO signalling may be responsible for the therapeutic benefits of different superoxide dismutase mimetics, in obesity and experimental diabetes models. This review summarizes the role of NO in adipose tissue and highlights the effects of NO/О2•– ratio 'teetering' as a promising pharmacological target in the metabolic syndrome.]]>
The antioxidant effects of resveratrol (3,5,4'-trihydroxy-trans-stilbene) contribute substantially to the health benefits of this compound. Resveratrol has been shown to be a scavenger of a number of free radicals. However, the direct scavenging activities of resveratrol are relatively poor. The antioxidant properties of resveratrol in vivo are more likely to be attributable to its effect as a gene regulator. Resveratrol inhibits NADPH oxidase-mediated production of ROS by down-regulating the expression and activity of the oxidase. This polyphenolic compound reduces mitochondrial superoxide generation by stimulating mitochondria biogenesis. Resveratrol prevents superoxide production from uncoupled endothelial nitric oxide synthase by up-regulating the tetrahydrobiopterin-synthesizing enzyme GTP cyclohydrolase I. In addition, resveratrol increases the expression of various antioxidant enzymes. Some of the gene-regulating effects of resveratrol are mediated by the histone/protein deacetylase sirtuin 1 or by the nuclear factor-E2-related factor-2. In this review article, we have also summarized the cardiovascular effects of resveratrol observed in clinical trials.]]>
Skeletal muscles and bones form a joined functional unit sharing a complex mechanical, biochemical and hormonal crosstalk. A number of factors, including sex hormones, physiologically regulate the musculoskeletal system. Striking gender differences in muscle and bone mass, and function are mainly caused by distinct actions exerted by oestrogens and androgens. However, relaxin and relaxin-related peptides, such as insulin-like peptide 3 (INSL3), might contribute to these sex-associated differences in physiological and pathological conditions (such as osteoporosis and sarcopenia). Relaxin is a 'pregnancy' hormone, but it is also produced from the prostate gland, and has recently attracted attention as a potential drug for cardiovascular disorders and fibrosis. In contrast, INSL3 is a male-specific hormone produced by the Leydig cells of the testis with a fundamental role in testicular descent during fetal life. Recent evidence suggests that both hormones have interesting roles in the musculoskeletal system. Relaxin and INSL3, by finely tuning bone formation and resorption, are involved in bone remodelling processes, and relaxin contributes to the healing of injured ligaments and promotes skeletal muscle regeneration. Here, we review the most recent findings on the effects of relaxin and INSL3 on skeletal muscle and the cell components of bone. In the light of the experimental evidence available and animal models, their clinical implications are also discussed.]]>
Forkhead box, class O (FOXO) transcription factors are major regulators of diverse cellular processes, including fuel metabolism, oxidative stress response and redox signalling, cell cycle progression and apoptosis. Their activities are controlled by multiple posttranslational modifications and nuclear-cytoplasmic shuttling. Recently, post-transcriptional regulation of FOXO synthesis has emerged as a new regulatory level of their functions. Accumulating evidence suggests that this post-transcriptional mode of regulation of FOXO activity operates in response to stressful stimuli, including oxidative stress. Here, we give a brief overview on post-transcriptional regulation of FOXO synthesis by microRNAs (miRNAs) and by RNA-binding regulatory proteins, human antigen R (HuR) and quaking (QKI). Aberrant post-transcriptional regulation of FOXOs is frequently connected with various disease states. We therefore discuss characteristic examples of FOXO regulation at the post-transcriptional level under various physiological and pathophysiological conditions, including oxidative stress and cancer. The picture emerging from this summary points to a diversity of interactions between miRNAs/miRNA-induced silencing complexes and RNA-binding regulatory proteins. Better insight into these complexities of post-transcriptional regulatory interactions will add to our understanding of the mechanisms of pathological processes and the role of FOXO proteins.]]>
Although reactive oxygen species (ROS) act as crucial factors in the onset and progression of a wide array of diseases, they are also involved in numerous signalling pathways related to cell metabolism, growth and survival. ROS are produced at various cellular sites, and it is generally agreed that mitochondria generate the largest amount, especially those in cardiomyocytes. However, the identification of the most relevant sites within mitochondria, the interaction among the various sources, and the events responsible for the increase in ROS formation under pathological conditions are still highly debated, and far from being clarified. Here, we review the information linking the adaptor protein p66Shc with cardiac injury induced by ischaemia and reperfusion (I/R), including the contribution of risk factors, such as metabolic syndrome and ageing. In response to several stimuli, p66Shc migrates into mitochondria where it catalyses electron transfer from cytochrome c to oxygen resulting in hydrogen peroxide formation. Deletion of p66Shc has been shown to reduce I/R injury as well as vascular abnormalities associated with diabetes and ageing. However, p66Shc-induced ROS formation is also involved in insulin signalling and might contribute to self-endogenous defenses against mild I/R injury. In addition to its role in physiological and pathological conditions, we discuss compounds and conditions that can modulate the expression and activity of p66Shc.]]>
Protease-activated receptors are a family of four GPCRs (PAR1–PAR4) with a number of unique attributes. Nearly two and a half decades after the discovery of the first PAR, an antagonist targeting this receptor has been approved for human use. The first-in-class PAR1 antagonist, vorapaxar, was approved for use in the USA in 2014 for the prevention of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. These recent developments indicate the clinical potential of manipulating PAR function. While much work has been aimed at uncovering the function of PAR1 and, to a lesser extent, PAR2, comparatively little is known regarding the pharmacology and physiology of PAR3 and PAR4. Recent studies have begun to develop the pharmacological and genetic tools required to study PAR4 function in detail, and there is now emerging evidence for the function of PAR4 in disease settings. In this review, we detail the discovery, structure, pharmacology, physiological significance and therapeutic potential of PAR4.]]>
GPCRs are the most successful pharmaceutical targets in history. Nevertheless, the pharmacology of many GPCRs remains inaccessible as their endogenous or exogenous modulators have not been discovered. Tools that explore the physiological functions and pharmacological potential of these 'orphan' GPCRs, whether they are endogenous and/or surrogate ligands, are therefore of paramount importance. Rates of receptor deorphanization determined by traditional reverse pharmacology methods have slowed, indicating a need for the development of more sophisticated and efficient ligand screening approaches. Here, we discuss the use of structure-based ligand discovery approaches to identify small molecule modulators for exploring the function of orphan GPCRs. These studies have been buoyed by the growing number of GPCR crystal structures solved in the past decade, providing a broad range of template structures for homology modelling of orphans. This review discusses the methods used to establish the appropriate signalling assays to test orphan receptor activity and provides current examples of structure-based methods used to identify ligands of orphan GPCRs.]]>
The intestine is composed of many distinct cell types that respond to commensal microbiota or pathogens with immune tolerance and proinflammatory signals respectively. ROS produced by mucosa-resident cells or by newly recruited innate immune cells are essential for antimicrobial responses and regulation of signalling pathways including processes involved in wound healing. Impaired ROS production due to inactivating patient variants in genes encoding NADPH oxidases as ROS source has been associated with Crohn's disease and pancolitis, whereas overproduction of ROS due to up-regulation of oxidases or altered mitochondrial function was linked to ileitis and ulcerative colitis. Here, we discuss recent advances in our understanding of how maintaining a redox balance is crucial to preserve gut homeostasis.]]>
For many years, microglia, the resident CNS macrophages, have been considered only in the context of pathology, but microglia are also glial cells with important physiological functions. Microglia-derived oxidant production by NADPH oxidase (NOX2) is implicated in many CNS disorders. Oxidants do not stand alone, however, and are not always pernicious. We discuss in general terms, and where available in microglia, GSH synthesis and relation to cystine import and glutamate export, and the thioredoxin system as the most important antioxidative defence mechanism, and further, we discuss in the context of protein thiolation of target redox proteins the necessity for tightly localized, timed and confined oxidant production to work in concert with antioxidant proteins to promote redox signalling. NOX2-mediated redox signalling modulates the acquisition of the classical or alternative microglia activation phenotypes by regulating major transcriptional programs mediated through NF-κB and Nrf2, major regulators of the inflammatory and antioxidant response respectively. As both antioxidants and NOX-derived oxidants are co-secreted, in some instances redox signalling may extend to neighboring cells through modification of surface or cytosolic target proteins. We consider a role for microglia NOX-derived oxidants in paracrine modification of synaptic function through long term depression and in the communication with the adaptive immune system. There is little doubt that a continued foray into the functions of the antioxidant response in microglia will reveal antioxidant proteins as dynamic players in redox signalling, which in concert with NOX-derived oxidants fulfil important roles in the autocrine or paracrine regulation of essential enzymes or transcriptional programs.]]>
Microglia are the resident immune cells of the CNS and constitute a self-sustaining population of CNS-adapted tissue macrophages. As mononuclear phagocytic cells, they express high levels of superoxide-producing NADPH oxidases (NOX). The sole function of the members of the NOX family is to generate reactive oxygen species (ROS) that are believed to be important in CNS host defence and in the redox signalling circuits that shape the different activation phenotypes of microglia. NOX are also important in pathological conditions, where over-generation of ROS contributes to neuronal loss via direct oxidative tissue damage or disruption of redox signalling circuits. In this review, we assess the evidence for involvement of NOX in CNS physiopathology, with particular emphasis on the most important surface receptors that lead to generation of NOX-derived ROS. We evaluate the potential significance of the subcellular distribution of NOX isoforms for redox signalling or release of ROS to the extracellular medium. Inhibitory mechanisms that have been reported to restrain NOX activity in microglia and macrophages in vivo are also discussed. We provide a critical appraisal of frequently used and recently developed NOX inhibitors. Finally, we review the recent literature on NOX and other sources of ROS that are involved in activation of the inflammasome and discuss the potential influence of microglia-derived oxidants on neurogenesis, neural differentiation and culling of surplus progenitor cells. The degree to which excessive, badly timed or misplaced NOX activation in microglia may affect neuronal homeostasis in physiological or pathological conditions certainly merits further investigation.
Classically, receptor-mediated signalling was conceived as a linear process involving one agonist, a variety of potential targets within a receptor family (e.g. α- and β-adrenoceptors) and a second messenger (e.g. cAMP)-triggered response. If distinct responses were stimulated by the same receptor in different tissues (e.g. lipolysis in adipocytes vs. increased beating rate in the heart caused by adrenaline), the differences were attributed to different second messenger targets in the different tissues. It is now realized that an individual receptor can couple to multiple effectors (different G proteins and different β-arrestins), even in the same cell, to drive very distinct responses. Furthermore, tailored agonists can mould the receptor conformation to activate one signal pathway versus another by a process termed 'biased signalling'. Complicating issues further, we now know that activating one receptor can rapidly trigger the local release of agonists for a second receptor via a process termed 'transactivation'. Thus, the end response can represent a cooperative signalling process involving two or more receptors linked by transactivation. This overview, with a focus on the GPCR, protease-activated receptor-1, integrates both of these processes to predict the complex array of responses that can arise when biased receptor signalling also involves the receptor transactivation process. The therapeutic implications of this signalling matrix are also briefly discussed.]]>
Cardiovascular diseases are associated with and/or caused by oxidative stress. This concept has been proven by using the approach of genetic deletion of reactive species producing (pro-oxidant) enzymes as well as by the overexpression of reactive species detoxifying (antioxidant) enzymes leading to a marked reduction of reactive oxygen and nitrogen species (RONS) and in parallel to an amelioration of the severity of diseases. Likewise, the development and progression of cardiovascular diseases is aggravated by overexpression of RONS producing enzymes as well as deletion of antioxidant RONS detoxifying enzymes. Thus, the consequences of the interaction (redox crosstalk) of superoxide/hydrogen peroxide produced by mitochondria with other ROS producing enzymes such as NADPH oxidases (Nox) are of outstanding importance and will be discussed including the consequences for endothelial nitric oxide synthase (eNOS) uncoupling as well as the redox regulation of the vascular function/tone in general (soluble guanylyl cyclase, endothelin-1, prostanoid synthesis). Pathways and potential mechanisms leading to this crosstalk will be analysed in detail and highlighted by selected examples from the current literature including hypoxia, angiotensin II-induced hypertension, nitrate tolerance, aging and others. The general concept of redox-based activation of RONS sources via "kindling radicals" and enzyme-specific "redox switches" will be discussed providing evidence that mitochondria represent key players and amplifiers of the burden of oxidative stress.
The metabotropic glutamate receptor subtype 5 (mGlu5) is a family C GPCR that has been implicated in various neuronal processes and, consequently, in several CNS disorders. Over the past few decades, GPCR-based drug discovery, including that for mGlu5 receptors, has turned considerable attention to targeting allosteric binding sites. Modulation of endogenous agonists by allosteric ligands offers the advantages of spatial and temporal fine-tuning of receptor activity, increased selectivity and reduced adverse effects with the potential to elicit improved clinical outcomes. Further, with greater appreciation of the multifaceted nature of the transduction of mGlu5 receptor signalling, it is increasingly apparent that drug discovery must take into consideration unique receptor conformations and the potential for stimulus-bias. This novel paradigm proposes that different ligands may differentially modulate distinct signalling pathways arising from the same receptor. We review our current understanding of the complexities of mGlu5 receptor signalling and regulation, and how these relate to allosteric ligands. Ultimately, a deeper appreciation of these relationships will provide the foundation for targeted drug design of compounds with increased selectivity, not only for the desired receptor but also for the desired signalling outcome from the receptor.]]>
Ligand binding is a vital component of any pharmacologist's toolbox and allows the detailed investigation of how a molecule binds to its receptor. These studies enable the experimental determination of binding affinity of labelled and unlabelled compounds through kinetic, saturation (Kd) and competition (Ki) binding assays. Traditionally, these studies have used molecules labelled with radioisotopes; however, more recently, fluorescent ligands have been developed for this purpose. This review will briefly cover receptor ligand binding theory and then discuss the use of fluorescent ligands with some of the different technologies currently employed to examine ligand binding. Fluorescent ligands can be used for direct measurement of receptor-associated fluorescence using confocal microscopy and flow cytometry as well as in assays such as fluorescence polarization, where ligand binding is monitored by changes in the free rotation when a fluorescent ligand is bound to a receptor. Additionally, fluorescent ligands can act as donors or acceptors for fluorescence resonance energy transfer (FRET) with the development of assays based on FRET and time-resolved FRET (TR-FRET). Finally, we have recently developed a novel bioluminescence resonance energy transfer (BRET) ligand binding assay utilizing a small (19 kDa), super-bright luciferase subunit (NanoLuc) from a deep sea shrimp. In combination with fluorescent ligands, measurement of RET now provides an array of methodologies to study ligand binding. While each method has its own advantages and drawbacks, binding studies using fluorescent ligands are now a viable alternative to the use of radioligands.]]>
Ligands targeting GPCRs can be categorized according to their intrinsic efficacy to trigger a specific, receptor-mediated response. A ligand endowed with the same level of efficacy as the endogenous agonist can be classified as a full agonist, whereas a compound that displays greater efficacy, that is, higher receptor signalling output than the endogenous agonist, can be called a superagonist. Subsequent to GPCR activation, an intracellular signalling cascade is set in motion, which may generate substantial amplification of the signal. This may obscure superagonism in pharmacological assays and, therefore, the definition of superagonism necessitates a combination of operational approaches, reduction of spare receptors or estimation of receptor activation close to the receptor level to quantify relative agonist efficacies in a particular system.
The first part of this review will compare GPCR superagonism with superagonism in the field of immunology, where this term is well established. In the second part, known GPCR superagonists will be reviewed. Then, the experimental and analytical challenges in the deconvolution of GPCR superagonism will be addressed. Finally, the potential benefit of superagonism is discussed.
The molecular mechanisms behind GPCR superagonism are not completely understood. However, crystallography shows that agonist binding alone is not sufficient for a fully active receptor state and that binding of the G protein is at least equally important. Accordingly, the emerging number of reported superagonists implies that ligand-induced receptor conformations more active than the ones stabilized by the endogenous agonist are indeed feasible. Superagonists may have therapeutic potential when receptor function is impaired or to induce negative feedback mechanisms.
Obestatin is a 23-amino acid C-terminally amidated gastrointestinal peptide derived from preproghrelin and which forms an α helix. Although obestatin has a short biological half-life and is rapidly degraded, it is proposed to exert wide-ranging pathophysiological actions. Whilst the precise nature of many of its effects is unclear, accumulating evidence supports positive actions on both metabolism and cardiovascular function. For example, obestatin has been reported to inhibit food and water intake, body weight gain and gastrointestinal motility and also to mediate promotion of cell survival and prevention of apoptosis. Obestatin-induced increases in beta cell mass, enhanced adipogenesis and improved lipid metabolism have been noted along with up-regulation of genes associated with beta cell regeneration, insulin production and adipogenesis. Furthermore, human circulating obestatin levels generally demonstrate an inverse association with obesity and diabetes, whilst the peptide has been shown to confer protective metabolic effects in experimental diabetes, suggesting that it may hold therapeutic potential in this setting. Obestatin also appears to be involved in blood pressure regulation and to exert beneficial effects on endothelial function, with experimental studies indicating that it may also promote cardioprotective actions against, for example, ischaemia–reperfusion injury. This review will present a critical appraisal of the expanding obestatin research area and discuss the emerging therapeutic potential of this peptide for both metabolic and cardiovascular complications of diabetes.]]>
In this review, we focus on the secreted form of nicotinamide phosphoribosyltransferase (NAMPT); extracellular NAMPT (eNAMPT), also known as pre-B cell colony-enhancing factor or visfatin. Although intracellular NAMPT is a key enzyme in controlling NAD metabolism, eNAMPT has been reported to function as a cytokine, with many roles in physiology and pathology. Circulating eNAMPT has been associated with several metabolic and inflammatory disorders, including cancer. Because cytokines produced in the tumour micro-environment play an important role in cancer pathogenesis, in part by reprogramming cellular metabolism, future improvements in cancer immunotherapy will require a better understanding of the crosstalk between cytokine action and tumour biology. In this review, the knowledge of eNAMPT in cancer will be discussed, focusing on its immunometabolic function as a metabokine, its secretion, its mechanism of action and possible roles in the cancer micro-environment.
GPCRs make up the largest family of human membrane proteins and of drug targets. Recent advances in GPCR pharmacology and crystallography have shed new light on signal transduction, allosteric modulation and biased signalling, translating into new mechanisms and principles for drug design. The GPCR database, GPCRdb, has served the community for over 20 years and has recently been extended to include a more multidisciplinary audience. This review is intended to introduce new users to the services in GPCRdb, which meets three overall purposes: firstly, to provide reference data in an integrated, annotated and structured fashion, with a focus on sequences, structures, single-point mutations and ligand interactions. Secondly, to equip the community with a suite of web tools for swift analysis of structures, sequence similarities, receptor relationships, and ligand target profiles. Thirdly, to facilitate dissemination through interactive diagrams of, for example, receptor residue topologies, phylogenetic relationships and crystal structure statistics. Herein, these services are described for the first time; visitors and guides are provided with good practices for their utilization. Finally, we describe complementary databases cross-referenced by GPCRdb and web servers with corresponding functionality.
Chemical conjugation of therapeutic proteins with polyethylene glycol (PEG) is an established strategy to extend their biological half-life (t1/2) to a clinically useful range. We developed a novel uncharged and unstructured recombinant polypeptide composed of five amino acids (P, S, T, A and G), named PsTag, as another approach to extend the t1/2 of human FGF21, with increased hydrodynamic radius.
Human FGF21 was fused with PsTag polymers of differing lengths (200 - 600 residues). Three fusion proteins and native FGF21 were produced in Escherichia coli. The biophysical characteristics, metabolic stability, immunogenicity and pharmacokinetics in were assessed in first. In lean and diet-induced obese (DIO) mice, effects on body weight, oral glucose tolerance tests and levels of relevant hormones and metabolites were studied.
Fusion proteins were solubly expressed in E. coli and prolonged the t1/2 from 0.34h up to 12.9 h in mice. Fusion proteins were also biodegradable, thus avoiding vacuole formation, while lacking immunogenicity in mice. In DIO mice, administration of PsTag fused to FGF21 reduced body weight, blood glucose and lipids levels and reversed hepatic steatosis.
The novel recombinant polypeptide, PsTag, should be useful in the development of biological drugs with properties comparable to those achievable by PEGylation, but with potentially less side effects. In mice, fusion of FGF21 to PsTag prolonged and potentiated pharmacological effects of native FGF21, and may offer greater therapeutic effects in treatment of obesity.
The aims of the present study were to characterize the role of PAR1 in rat bladder under inflammatory conditions and determine whether a selective PAR1 antagonist, F16357, can prevent the pathophysiological symptoms of cyclophosphamide-induced interstitial cystitis (IC).
Immunohistochemistry, contractile activity in isolated bladder and urodynamics were determined before and after cyclophosphamide treatment. F16357 was administered intravesically during the acute phase of inflammation, and effects on PAR1 and PAR1-related bladder contraction evaluated 24 h after cyclophosphamide injection. Urodynamics and associated voided volumes were recorded 7 and 24 h after cyclophosphamide.
In control conditions, PAR1 was present only in some umbrella cells. Cyclophosphamide disrupted the urothelium and expression of PAR1 by all remaining urothelial cells. After F16357 treatment, urothelial damage was absent and PAR1 immunoreactivity similar to control tissues. Thrombin and TFLLR-NH2 induced bladder contractions. These were increased in inflammatory conditions and antagonized by F16357 in a concentration-dependent manner. In telemetric experiments, furosemide increased urine production and voiding frequency for 60 min, 7 h after cyclophosphamide injection. Intravesical administration of F16357 blocked these changes with a return to a physiological profile; 24 h after cyclophosphamide, the volume of micturition was still lower with no increase in number of micturitions. F16357 30 μM reduced the number of micturitions and improved bladder capacity, but did not affect diuresis. Under similar experimental conditions, lidocaine 2% induced comparable effects.
PAR1 is expressed in rat bladder, overactivated in inflammatory conditions and involved in bladder function and sensation. F16357 could represent an interesting candidate for IC treatment.
Previous findings suggest that the growth and differentiation factor midkine (Mdk) is a negative regulator of osteoblast activity and bone formation, thereby raising the possibility that a specific Mdk antagonist might improve bone formation during fracture healing.
In the present study, we investigated the effects of a monoclonal anti-Mdk antibody (Mdk-Ab) on bone healing using a standardized femur osteotomy model in mice. Additional in vitro experiments using chondroprogenitor and preosteoblastic cells were conducted to analyse the effects of recombinant Mdk and Mdk-Ab on differentiation markers and potential binding partners in these cells.
We demonstrated that treatment with Mdk-Ab accelerated bone healing in mice based on increased bone formation in the fracture callus. In vitro experiments using preosteoblastic cells showed that Mdk-Ab treatment abolished the Mdk-induced negative effects on the expression of osteogenic markers and Wnt/β-catenin target proteins, whereas the differentiation of chondroprogenitor cells was unaffected. Phosphorylation analyses revealed an important role for the low-density lipoproteinLDL receptor-related protein 6 in Mdk signalling in osteoblasts.
We conclude that Mdk-Ab treatment may be a potential novel therapeutic strategy to enhance fracture healing in patients with orthopaedic complications such as delayed healing or non-union formation.
Prenatal exposure to dexamethasone slows down fetal linear growth and bone mineralization but the regulatory mechanism remains unknown. Here we assessed how dexamethasone regulates bone development in the fetus.
Dexamethasone (1 mg·kg−1·day−1) was injected subcutaneously every morning in pregnant rats from gestational day (GD)9 to GD20. Fetal femurs and tibias were harvested at GD20 for histological and gene expression analysis. Femurs of 12-week-old female offspring were harvested for microCT (μCT) measurement. Primary chondrocytes were treated with dexamethasone (10, 50, 250 and 1000 nM).
Prenatal dexamethasone exposure resulted in accumulation of hypertrophic chondrocytes and delayed formation of the primary ossification centre in fetal long bone. The retardation was accompanied by reduced maturation of hypertrophic chondrocytes, decreased osteoclast number and down-regulated expression of osteocalcin and bone sialoprotein in long bone. In addition, the mitogen-inducible gene-6 (Mig6) and osteoprotegerin (OPG) expression were stimulated, and the receptor activator of NF-κB ligand (RANKL) expression was repressed. Moreover, dexamethasone activated OPG and repressed RANKL expression in both primary chondrocytes and primary osteoblasts, and the knockdown of Mig6 abolished the effect of dexamethasone on OPG expression. Further, μCT measurement showed loss of bone mass in femur of 12-week-old offspring with prenatal dexamethasone exposure.
Prenatal dexamethasone exposure delays endochondral ossification by suppressing chondrocyte maturation and osteoclast differentiation, which may be partly mediated by Mig6 activation in bone. Bone development retardation in the fetus may be associated with reduced bone mass in later life.
Gelsemine is one of the principal alkaloids produced by the Gelsemium genus of plants belonging to the Loganiaceae family. The extracts of these plants have been used for many years, for a variety of medicinal purposes. Coincidentally, recent studies have shown that gelsemine exerts anxiolytic and analgesic effects on behavioural models. Several lines of evidence have suggested that these beneficial actions were dependent on glycine receptors, which are inhibitory neurotransmitter-gated ion channels of the CNS. However, it is currently unknown whether gelsemine can directly modulate the function of glycine receptors.
We examined the functional effects of gelsemine on glycine receptors expressed in transfected HEK293 cells and in cultured spinal neurons by electrophysiological techniques.
Gelsemine directly modulated recombinant and native glycine receptors and exerted conformation-specific and subunit-selective effects. Gelsemine modulation was voltage-independent and was associated with differential changes in the apparent affinity for glycine and in the open probability of the ion channel. In addition, the alkaloid preferentially targeted glycine receptors in spinal neurons and showed only minor effects on GABAA and AMPA receptors. Furthermore, gelsemine significantly diminished the frequency of glycinergic and glutamatergic synaptic events without altering the amplitude.
Our results provide a pharmacological basis to explain, at least in part, the glycine receptor-dependent, beneficial and toxic effects of gelsemine in animals and humans. In addition, the pharmacological profile of gelsemine may open new approaches to the development of subunit-selective modulators of glycine receptors.
Phosphate imbalance is often present in chronic kidney disease (CKD), and it contributes to a higher cardiovascular mortality rate. A phosphate binder is typically part of a treatment strategy for controlling phosphate imbalance. However, safety concerns and low compliance are two well-recognized disadvantages of on-market phosphate binders. This report describes the preclinical studies of VS-505, a non-absorbable, calcium- and aluminum-free, plant-derived polymer currently being evaluated in haemodialysis patients in Australia.
Normal Sprague Dawley (SD) rats or uraemic SD rats induced by 5/6 nephrectomy fed a high-phosphate diet were treated with VS-505 or sevelamer (0.05–10% in food) for 5 and 28 days respectively.
Urinary and serum phosphate levels were significantly elevated in untreated rats, and were decreased by VS-505 and sevelamer. VS-505 increased faecal phosphate levels in a dose-dependent manner. High-phosphate diet also caused an increase in serum FGF-23 and parathyroid hormone in nephrectomized (NX) rats, effects prevented by VS-505 or sevelamer. Significant aortic calcification was observed in NX rats treated with 5% sevelamer, whereas VS-505 at all doses tested did not show effects. VS-505 had no effects on small intestine histomorphology and intestinal sodium-dependent phosphate cotransporter gene expression. In vitro characterizations showed that VS-505 has a relatively high density and low expansion volume when exposed to simulated gastric fluid.
VS-505 is a safe and effective phosphate binder and may offer the advantage of having a reduced pill burden and minimal GI side effects for CKD patients.
NO deficiency and oxidative stress are crucially involved in the development or progression of cardiovascular disease, including hypertension and stroke. We have previously demonstrated that acute treatment with the newly discovered organic nitrate, 2-nitrate-1,3-dibuthoxypropan (NDBP), is associated with NO-like effects in the vasculature. This study aimed to further characterize the mechanism(s) and to elucidate the therapeutic potential in a model of hypertension and oxidative stress.
A combination of ex vivo, in vitro and in vivo approaches was used to assess the effects of NDBP on vascular reactivity, NO release, NADPH oxidase activity and in a model of hypertension.
Ex vivo vascular studies demonstrated NDBP-mediated vasorelaxation in mesenteric resistance arteries, which was devoid of tolerance. In vitro studies using liver and kidney homogenates revealed dose-dependent and sustained NO generation by NDBP, which was attenuated by the xanthine oxidase inhibitor febuxostat. In addition, NDBP reduced NADPH oxidase activity in the liver and prevented angiotensin II-induced activation of NADPH oxidase in the kidney. In vivo studies showed that NDBP halted the progression of hypertension in mice with chronic angiotensin II infusion. This was associated with attenuated cardiac hypertrophy, and reduced NADPH oxidase-derived oxidative stress and fibrosis in the kidney and heart.
The novel organic nitrate NDBP halts the progression of angiotensin II-mediated hypertension. Mechanistically, our findings suggest that NDBP treatment is associated with sustained NO release and attenuated activity of NADPH oxidase, which to some extent requires functional xanthine oxidase.


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Search by BreedSearch Breeders by Washington Gun Dog Classifieds
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Lacey Mountain Brittanys - Washington American Brittany Breeders
Want to list your puppies for sale? Post a classified today!
About Lacey Mountain Brittanys
Litter out of Leslie & Colt planned to be born around August 10th.
Lacey Mountian Brittanys is a small kennel dedicated to the commitment of breeding the highest quality of Brittany's. Lacey Mtn's Frosty Freckled Ruffian "Leslie" is CH Barb's Miss Lacey Girl's daughter. Their bloodlines come from: FC/AFC Hi-Scor Jac D Ruffian, FC/AFC Rimarada's Trademark, NFC/DC/NGDC/AFC Winchester of Hanging Jaze. The sire is CH Mustang's Sterling Edition JH. His bloodlines come from CH Sandbar's Call When Redi, DC AFC Masked Shoshone War Chief. The combination of these bloodlines will produce excellent dual dogs.
Hunting Dog Breeds Raised
American Brittany
Additional Services
Stud Services
Breeder Contact Info
Barbara Stevens
52677 Railroad Ave.
Rockport, Washington 98283
Breeding quality gun dogs for over 9 years.
360-770-7211Send Email to Lacey Mountain BrittanysClick here to visit Lacey Mountain Brittanys website
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Testimonials (2)
Submitted by: Holly Hatter on Jan 25, 2012This last summer we adopted a beautiful O/W female Brittany from Lacey Mountain Brittanys. And we had the best experience finding ourselves such a well behaved, smart, and beautiful puppy. She does so well with her litters that it is so easy to puppy train. Her puppies are already used to the crate, so crate training was not a problem at all, and when her puppies are able to go outside they use the doggie door and go in and out to use the potty. It only took our Mya two weeks after being home to get the whole potty training down. I truly believe that if we didn't have Barbara raise the puppies as she does, it would have been a lot harder. I am so satisfied with my whole experience with Barbara as a breeder that if we decide to get a Brittany again I am definitely only going through Barbara at Lacey Mountain Brittany's. Thank you so much for much an amazing dog and a wonderful experience.
Submitted by: tracey glaub on Jul 27, 2011Last summer my husband and I decided to add another brittany to our family. We already had two rescue brittanys and this time we choose to adopt from a breeder. I researched and met with several breeders. I contacted Barbara via email and she responded to us immediately. She was very open to adopting to us even though we do not officially hunt our brittanys. We just love the breed and they fit well with our active lifestyle. We decided on a puppy based on her description of the personalities of the puppies. We live in Phoenix so we decided to fly to washington to bring him back.Barbara was very helpful in making sure he was ready to travel and I even shipped my crate to her. I flew into seattle and drove to Rockport where I met my little boy Cooper. He was and is the cutest little boy ever. He was so well behaved in the car and at the airport. Barbara had made sure he has his health certification and shot records to make flying with him a breeze. He was four months old when I picked him up and he was crate trained and basically housebroken. he was so easy to travel with. We flew back home the next day and he fit immediately into our family. He is very smart, social, healthy and adorable. I can not imagine life without him. I will adopt again from Barbara.


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NC Lawmakers Consider HB2 Revisions Twitch's Andy Swanson Dishes On E-Sports Executive Transactions Names In The News Nike's Battle For CEO An Internal Affair T'Wolves Welcome First Chinese Minority Owner Executive Transactions Names In The News Blue Jackets Minority Owner Wolfe Passes Executive Transactions
Upcoming Conferences and Events
SBD/11/Sports Industrialists
Published May 11, 1998
MAGIC JOHNSON was profiled by Leslie Stahl on "60 Minutes" and discussed his battle against HIV, his feelings about today's NBA and his burgeoning business empire. Johnson, on MICHAEL JORDAN's domination of today's younger players: "We love to play. We love to compete. Most of these guys, they'd rather have a shoe commercial on TV than win a championship." Stahl: "Magic worked to make a bundle from basketball and endorsements, which he has now parlayed into a business empire and a personal fortune estimated at $100 million. His latest project is to bring upscale stores and services to the inner-city." Johnson, on joining with Sony to open a 15-screen multiplex movie theater in South Central L.A.: "Minorities used to [be], or still are, the number one moviegoers of any group of people, but they don't have any in their neighborhoods." With the movie theaters, Johnson said he and Sony have "changed Hollywood, I think, a little bit, and their mindset." Stahl reported that other companies "are clamoring" to be Johnson's partner, from TGI Friday's to Starbucks. Stahl: "Magic says he's showing that black customers are eager to support businesses owned by blacks and staffed by blacks" ("60 Minutes," CBS, 5/10).


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Kids Klub Pasadena has been in our family for a few years now, and we LOVE it! Our daughter transfered in when she was just over two. Within a few weeks of moving she began speaking in full sentences and her vocabulary completely expanded. Could be that it was just a matter of putting her in a different environment but she has consistently learned a great deal over the past few years in all her classes. My son started at 12 weeks, and has been in the program for the past year and a half. He loves going to school each day, and loves his teachers. The Evening / Weekend program is great for working parents, who can extend the hours and even get their kids dinner with just a call should a work emergency come up. Many times the kids are hanging with their school buddies, so it turns into an impromptu party that the kids really enjoy. Also great for working parents is the extracurricular offerings that are integrated during and just after the school day - instead of running all over town you get access to ballet, karate, and gymnastics in one easy location. They're even testing out some additional programs - theatre and art. We've tried all the programs and have really liked each one - the hard part is picking which ones NOT to do. The faculty and staff at Kids Klub are just like part of the family now, and we're so happy they're in our lives!
Pasadena Pumpkin Patch
1. Pasadena Pumpkin Patch
900 North Lake AvenuePasadena, CA 91104
2. Pumpkin Patch
1106 Glendale GalleriaGlendale, CA 91210
3. Pumpkin Patch - CLOSED
400 S Baldwin AveArcadia, CA 91007
4. Tahoe Scottys Pumpkin Patch & Christmas Trees - CLOSED
926 Weimar StAltadena, CA 91001
5. Mr Bones Pumpkin Patch
702 North Doheny DriveWest Hollywood, CA 90069
6. The Pumpkin Patch
14006 Riverside Dr Ste 111Sherman Oaks, CA 91423
7. Shawn's Pumpkin Patch
This is a fantastic pumpkin patch it really had so many things going on my family and I loved it. They have beautiful pumpkins and all kinds of coo…
8. Shawn's Pumpkin Patch
6815 S La Cienega BlvdLos Angeles, CA 90045
9. Pumpkin Patch
322 Los Cerritos MallCerritos, CA 90703
10. Sonshine Pumpkin Patch
4100 N Harbor BlvdFullerton, CA 92835
11. Pumpkin Patch
837 15th St Apt DSanta Monica, CA 90403
12. Pa's Pumpkin Patch
6701 East Pacific Coast HwyLong Beach, CA 90803
13. Pumpkin Patch - CLOSED
10800 W Pico Blvd # 369Los Angeles, CA 90064
14. Sonshine Pumpkin Patch - CLOSED
1200 N Harbor BlvdAnaheim, CA 92801
15. Pumpkin Patch - CLOSED
6600 Topanga Canyon Blvd #1006Canoga Park, CA 91303
16. Pumpkin Patch Inc - CLOSED
2800 N Main St Ste 540ASanta Ana, CA 92705
17. Cabbage PatchMenu
520 W 6th StLos Angeles, CA 90014
18. Cabbage PatchMenu
I have beeen to the Cabbage Patch 6 times already and it just opened! Everytime I am in the area I make sure to stop in. The burger - AMAZING. The …
19. Pumkins Patch Preschool
11240 Victory BlvdNorth Hollywood, CA 91606
20. Cabbage PatchMenu
12531 Beatrice StLos Angeles, CA 90066
21. Sunny Patch Child Care
3837 W 110th StInglewood, CA 90303
22. Tomato PatchMenu
the tomato patch is itsy bitsy but the food is bursting with flavor. the tuna is amazing!!! try it, sooooo good!!
23. A Little Patch Of Earth Preschool
24436 Valley StNewhall, CA 91321
24. Cactus Patch Cafe - CLOSED
My mom was a waitress for Ray in the 1920's where my dad met her and the rest is *HISTORY*.. So we five kids literally grew up on Ray's cooking some…
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I went for an orientation today. Took about 20 minutes. The gal I talked to there was really nice and seems really sincere about the education for the kids. I was blown away by the food options. She really does feed those kids better than any place I've come across. Most family child care places in this area are above the 210 and kind of ghetto. She is located at the mall in downtown and the place is really nice. They have the cutest puppy for the kids too! She said they are having the grand opening soon so I added myself to the grand opening list and she gave me a killer discount. She explained everything in detail which I appreciated and I am really excited to see my daughter's progress. Seriously, I am just blown away. Will update after the first art show.
I started buying my pearl inlay, rosewood, Chinese style furniture here in 2002 when this store was known as "Chineart". The prices were extremely reasonable and the service was fantastic. I just added more furniture which all matches the pieces I previously purchased. I consider this furniture pure art and love living with it. They now carry more American style sofas and sectionals and are not featuring as much rosewood as they used to as the wood is getting quite scarce and governments are prohibiting export to China. If you love pearl inlay, carved rosewood pieces, you must shop here first. Patty Wang is the manager and wonderful to work with - - she really wants to please her customers. I highly recommend shopping here!!