Abstract
Kinins are vasoactive peptides released from precursors called kininogens, and serum levels of both T-and K-kininogens increase dramatically as rats age. Kinin release is tightly regulated, and here we show that serum kinin levels also increase with age, from 63 6 16 nmol/L in young Fisher 344 rats to 398 6 102 nmol/L in old animals. Both K-and T-kininogens contribute sequentially to this increase, with the increase in middle-aged animals being driven primarily by K-kininogen, whereas the further augmentation in older rats occurs by increasing T-kininogen. By measuring ERK activation, we show that aorta endothelial cells from old animals are hyporesponsive to exogenous bradykinin. However, if serum kinin levels are experimentally decreased by lipopolysaccharide treatment, then the endothelial response to bradykinin is reestablished. These results indicate that serum levels of kinins increase with age, whereas the responsiveness of target cells to kinins is reduced in these same animals.
K
ININS are a family of nine-to-eleven amino acid peptide hormones that have a variety of important physiological and pathological effects (1) . Mammalian members of this family include bradykinin (BK), kallidin, and in the rat, Tkinin (2) . All known kinin family members have similar biological functions and comparable potency. Kinins are generated by the action of a family of serine proteases called kininogenases, which release the vasoactive peptides from their protein precursors, the kininogens (3, 4) . BK and kallidin are generated from both high-and low-molecular-weight Kkininogen (K-KG) by kallikreins (1) . In the rat, however, the most abundant serum kinin is T-kinin (Ile-Ser-Bradykinin), which is released from the rat-specific precursor T-kininogen (T-KG) by either T-kininogenase (in vivo) or trypsin (in vitro) (5, 6) . T-KG is insensitive to kallikrein (7), but T-kinin can be further processed to give rise to both kallidin and BK (8) .
Kinins have a multitude of functions [reviewed in (9) ], including vasodilation (10) , smooth muscle relaxation (11) , hemodynamic and electrolyte regulation (12) , and production of both edema (13) and algesia (14) during inflammatory processes. Because of their short life in the order of 15 seconds, kinins are usually produced locally at sites of injury. Kinins generated within vascular structures stimulate the local production of nitric oxide, thus contributing to site-specific modulation of hemodynamic flow. Kinins are also known to participate in the vasorelaxing effects of angiotensinconverting enzyme inhibitors used for the treatment of hypertension (15, 16) . Kinins are intimately involved in the pathogenesis of inflammatory disorders including acute and chronic enterocolitis in rats, as well as acute arthritis and chronic sepsis in humans (17, 18) . Thus, rats deficient in K-KG (Brown Norway Katholiek strain) have a significantly less active inflammatory response, as exemplified by decreased edema, swelling and exudation, and decreased resistance to algesia (19) . Kinins affect cellular metabolism by binding to kinin receptors, leading to the activation of a vast array of signal transduction pathways [reviewed in (20) ]. Both in smooth muscle vascular cells and endothelial cells, BK leads to the rapid phosphorylation of ERK (21, 22) which at the cellular level results in increased cell proliferation (23) .
Our laboratory has shown that T-KG levels increase dramatically with age in rat serum (24) . A similar pattern of expression has been observed for K-KG in rats (25) and for the human ortholog, High Molecular Weight kininogen (26) . Thus, considering the broad range of kinin activities, the possibility of releasing large amounts of kinins from kininogen precursors during aging could have important repercussions, both positive and negative, on the pathophysiology of aged individuals. Therefore, we have measured kinin levels in the serum of Fisher 344 (F344) rats of different ages. Furthermore, because the responsiveness to a variety of stimuli is often blunted with age, we also measured the responsiveness of aorta endothelial cells to an exogenous kinin challenge. We report both an increase in serum kinin levels and a decreased responsiveness of cells to external kinins. Both observations must be taken into account when considering the possible role of increased levels of circulating kinins during aging.
METHODS

Animals
Male F344 rats of different ages [6 months (adult), 15 months (middle aged), and 24 months (old)] were obtained from the National Institute on Aging. Upon receipt, they were maintained in our specific pathogen-free facilities for 2 weeks prior to experimentation. Water and standard National Institute on Aging rat chow were provided ad libitum. All procedures were approved by the Lankenau Institute for Medical Research Animal Care Committee.
Radioimmunoassay
To preserve kinins, total blood was collected in the presence of a cocktail of protease inhibitors as described (27) , and serum was prepared. To measure kinins, 500 ll of serum was precipitated with 2 ml of ethanol. The ethanolsoluble material was evaporated under nitrogen at 378C and dissolved in 150 ll of RIA buffer (NaH 2 PO 4 at 10 mmol/L, zolson@example.net mol/L, 1% bovine serum albumin, pH 7.0). Samples (100 ll, at a dilution of 1:500 for adult and 1:1000 for middle aged and old rats), were incubated overnight at 48C with 100 ll of iodogen-labeled Tyr-BK (50 cpm/ll initial stock) in the presence of anti-BK antibody at a dilution of 1:16,000 (a gift from Dr. Kasuaki Shimamoto, Sapporo Medical University, Sapporo, Japan). This antibody recognizes all kinins present in rat serum, including BK, kallidin, and T-kinin. However, it does not recognize any of the major kinin metabolites (28) . Under these conditions, the binding efficiency was between 35% and 40%. The next day, 200 ll of secondary antibody (goat antirabbit, diluted 1:2 in RIA buffer; Calbiochem, La Jolla, CA) were added and incubated for 2 zolson@example.net. Free kinins were separated from antibody-bound kinins by addition of PEG-8000 to a final concentration of 15%, followed by centrifugation at 3000 zolson@example.net. The supernatant was carefully decanted, and the radioactivity in the precipitates was counted in an Auto-Gamma Wallac spectrometer (Turku, Finland).
To measure kinins released from their precursors after enzymatic digestion, we used serum prepared from the same animals described above. Pools prepared from the individual undiluted sera from adult, middle aged, and old rats were treated with either kallikrein (2.7 nM final concentration), trypsin (12 nM final concentration), or both. The digestions proceeded for 3 hours at 378C in RIA buffer without bovine serum albumin. Released kinins were measured by RIA using a dilution of 1:1000 for adult samples and 1:2000 for samples from middle aged and old rats.
Lipopolysaccharide Treatment and Isolation of Rat Aortas
F344 rats (five animals at each age) were injected i.p. with lipopolysaccharide (LPS) (2 mg/Kg, from Pseudomonas aeruginosa serotype 10; Sigma, St. Louis, MO) for 0.5, 1, 2, or 4 hours. Control rats received injections of a similar volume (150-200 ll) of adjuvant (phosphate-buffered saline). Serum collection and processing for kinin measurements was as described above, while aortas were rapidly excised, washed in serum-free Dulbecco's Modified Eagle Medium (D-MEM) (high glucose), and immediately cannulated in a gravity-driven perfusion system (1 drop every 3 seconds) in D-MEM at 378C for 5 minutes. After this wash, perfusion was continued for an additional 5 minutes, either in D-MEM (controls) or in D-MEM containing 10 nM BK.
Then the aortas were rapidly frozen in liquid nitrogen and stored at À808C until sample processing.
ERK activity was measured by western blot, using anti P-ERK1/2 antibodies (Cell Signaling Technology, Beverly, MA), and gel loading was controlled by using anti-ERK 1/2 antibodies (Cell Signaling Technology). Immunohistochemistry was done on 5-lm paraffin sections of 4% paraformaldehyde-fixed aorta. Sections were incubated with anti P-ERK antibody (1:100) overnight at 48C, followed by incubation with biotinylated secondary antibody (1:1000) for 1 hour at room temperature, and detection by streptavidin-peroxidase conjugate (Dako, Glostrup, Denmark). The reaction was developed with 39, 39-diaminobenzidine (Sigma). Nuclei were visualized by hematoxylin and eosin staining.
Statistical Analysis
All experiments were done at least in triplicate. Paired data were analyzed with the Mann-Whitney nonparametric test followed by Dunn's posttest, whereas the time curves in Figure 5 were analyzed with the Kruskal-Wallis nonparametric analysis of variance test. Data are expressed as means 6 standard error of the mean, and differences were considered statistically significant when p , .05.
RESULTS

Serum Kinins Increase With Age
To establish whether kinin levels increase with age in F344 rats, we used an RIA with an anti-BK antibody previously shown not to recognize the precursor High Molecular Weight kininogen (29) . Figure 1A shows that the antibody also recognizes T-kinin, but not the precursor T-KG at any concentration tested (up to 150 nmol/L). Therefore, this assay provides a measure of total kinins (from both K-KGs and T-KGs) present in the sample, with linearity between 60 pmol/L and 7.5 nmol/L ( Figure 1B ). Figure 2 shows that serum kinin levels increase progressively with age in the serum of F344 rats, with an increase of approximately four-and sixfold in middle aged and old rats, respectively, relative to adult animals (63 6 16 nmol/L in adult, 242 6 43 nmol/L in middle aged, and 398 6 102 nmol/L in old rats).
Increase in Kinin Levels Is Due to Both K-KGs and T-KGs
To establish whether serum kinin levels are directly related to the level of precursors, or whether there are differences in processing/release as a function of age, we released total kinins from the available precursors present in serum by enzymatic digestion with a mixture of kallikrein and trypsin. As shown in Figure 3A , we found that kinins were released by this treatment in a linear fashion with respect to age (as previously shown for actual serum kinins in Figure 2) ; this suggested that the rate of processing is not a major component of the age-related difference. To establish whether the increase in serum kinins was due to K-KG or T-KG, we digested sera from animals of different ages with either kallikrein or trypsin separately. The data suggest that there is an early increase in kinins released from K-KG (sensitive to kallikrein), already apparent in middle aged serum, with a more modest increase in these kinins observed in old rats. In contrast, when we used trypsin, which releases T-kinin from T-KG, we found that the further increase in kinins observed in old animals is primarily driven by T-KG. Figure 3B shows the digestion products obtained after each enzyme treatment, in animals of different ages. Kallikrein digestion gives rise to major products of 68, 60, and 46 kd, whereas digestion with trypsin produces a major product of approximately 30 kd. This last fragment is the predominant product observed in double digests, and it corresponds to the size of the globular cystatin-like domains present in all kininogens (30) . In the case of kallikrein digestion, the 68 kd fragment corresponds to the size expected for undigested T-KG, which is insensitive to kallikrein, whereas the 60 kd fragment corresponds to the expected size of the heavy chain, released from K-KGs by kallikrein. The increase in 60 kd and 46 kd fragments occurs primarily between adult and middle-aged animals, whereas the increase in the 30 kd fragment occurs mostly between middle-aged and old rats. Thus, these results further confirm that the increase in kinin levels observed in middle-aged animals is primarily due to K-KG (kallikrein-sensitive), whereas the further increase in old rats is due primarily to an increase in T-KG (trypsin-sensitive).
Aorta From Old Rats Is Resistant to Exogenous Activation by Kinins
The physiological relevance of the increase in serum kinins with age depends on the relative ability of target tissues from old animals to respond to these kinins. Treatment of aorta endothelial cells with kinins leads to an increase in ERK activation (22) , and we have previously reported that the precursor T-KG inhibits this response (31) . Thus, we measured both the basal levels of ERK activity and its activation in response to added BK in aortas from rats of different ages. The results ( Figure 4A ) indicate that old rats have higher levels of basal ERK activity, relative to either adult or middle-aged rats. In contrast, when the tissues were challenged with BK (10 nM), only the adult animals were able to respond by increasing P-ERK levels, as expected from the literature. In sharp contrast, middle-aged and old animals not only do not respond to the stimulus, but in fact, P-ERK levels diminish relative to the controls in response to BK treatment. Immunohistochemical analysis indicates that, in these experiments, ERK activation occurs primarily at the level of endothelial cells, with little or no ERK activation observed in either smooth muscle cells or the matrix fibroblasts ( Figure  4B ). The results can therefore be attributed to effects in the endothelial compartment of the vasculature.
ERK Is Not Activated in Response to LPS in the Aorta of Old Rats
A regression analysis comparing serum kinin levels and ERK activation in response to BK at any age shows . Serum kinins increase with age. Total kinin levels (from both Kand T-kininigens) were measured in samples prepared from adult, middle-aged, and old rats by a radioimmunoassay as described in Methods. Concentrations were calculated from a standard curve as shown in Figure 1B . The results are the mean 6 standard error of the mean from nine independent animals for each age group. Statistical significance was established by the Mann-Whitney nonparametric test, where the asterisk indicates p , .05 when comparing to the adult group. a negative correlation (N ¼ 9, r 2 ¼ 0.7406) that suggests that ERK activation is impaired by high serum kinin levels (data not shown). To test whether this impairment was unique to kinin stimulation, we injected bacterial LPS i.p., and measured ERK activation at various times thereafter. Figure   5A indicates that, whereas ERK is induced in the aorta from adult and middle-aged rats in response to this stimulus, old rats fail to respond properly, suggesting that ERK activation is generally compromised in the aorta of old rats. Basal level of ERK activity is increased in old rats, as shown in Figure 4 .
LPS induces a robust and pleiotropic immune response. Because kinins are intimately involved in this response, we also measured the effect of LPS on serum kinin levels. Figure 5B shows that serum kinin levels decrease rapidly in response to LPS, even in old animals. In fact, due to their higher basal levels, old rats showed an exacerbated response. We conclude that old rats do respond, at least partially, to the LPS stimulus, even though they do not Figure 3 . The augmented kinin levels are associated with increases in both Kand T-kininogens. A, Upper panel: Pools prepared from the serum of three individual animals of each age (adult, middle aged, or old) were treated with a mix of kallikrein (2.7 nmol/L) and trypsin (12 nmol/L) to release total potential kinins. The kinins released were measured by radioimmunoassay as described in Methods. White bar, initial serum kinin levels (from Figure 2) ; gray bar, kinins released after enzymatic digestion. Middle panel: Same pools shown in A were treated only with kallikrein. Asterisk represents significance with respect to the adult animals (p , .05). There is no statistically significant difference between the middle-aged and old groups. Lower panel: Same pools shown in A were treated with trypsin alone. The pound symbol represents a significant difference (p , .05) of the old group relative to either the adult or middle-aged groups. There is no statistically significant difference between the adult and middle-aged groups. B, Western blot showing the digestion products obtained after each enzyme treatment, in animals of different ages. Samples were resolved in a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and assayed using anti T-kininogen antibody. A, M, and O ¼ pooled sera from adult, middleaged, and old rats, respectively, without enzymatic digestion. Molecular weight markers are shown at the left. Arrows at the right indicate the size of the different fragments obtained after enzyme digestion. . Stimulation was for 5 minutes in the presence of 10 nM BK, as described in Methods. Total proteins were resolved in a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and the blots were assayed for P-ERK 1/2 (upper) and total ERK 1/2 (lower). Bar graph: Quantitation of ERK activity in individual animals. The graph represents both basal levels of ERK activity (white bars) and ERK activity in response to bradykinin (black bars). Activity is expressed as a ratio between phosphorylated and nonphosphorylated ERK proteins. Results represent the average 6 standard error of the mean for 3-4 animals per age group. Asterisks represent statistical significance to p , .05 relative to the untreated control for each age group. B, Immunohistochemistry of the aorta from old rats. Five-micrometer sections were analyzed with anti P-ERK antibody using the diaminobenzidine technique (DAB). Panels activate ERK in endothelial cells. The mechanism involved is not clear, but nevertheless, the significant decline in the level of kinins present in the serum of old rats provided us with an opportunity to test whether aorta cells from old animals might now be able to respond to exogenous kinins, if the endogenous level of the peptides was previously reduced by LPS treatment. Figure 6 shows that this is indeed the case, because when serum kinins were reduced by LPS treatment, old animals did respond to exogenous kinin as vigorously as did young animals.
DISCUSSION
We have previously reported that both T-KGs and K-KGs increase with age in rat serum. However, the release of kinins from these precursors is tightly controlled; therefore, the increase in precursors does not necessarily mean that a parallel increase in vasoactive kinins should occur. In this report we establish that kinin levels also increase with age in the serum of F344 rats. It should be noted that, as expected, rat serum kinin levels are considerably higher than those described in humans (32) . It is interesting that, although we knew that the increase in T-KG occurs in a nonlinear fashion with age, we had no information concerning the time dependency of the increase in K-KG levels. Our results from enzymatic digestions suggest that both K-KG and T-KG contribute to the age-related increase in serum kinins levels, acting in a sequential fashion: In middle-aged rats, the increase is driven primarily by changes in K-KG, whereas at later ages, the further augmentation occurs because of an increase in T-KG. It is also worth noting that our previously published data indicate that the increase occurs by different molecular mechanisms as well-transcriptional in the case of T-KG (33) and posttranslational in the case of K-KG (25) .
Kinins are important modulators of the vascular tone, primarily through binding to their receptors in endothelial cells, which leads to the activation of multiple transduction signals, including the ERK pathway (21, 22) . However, little is currently known about the effect of age on the Figure 5 . Aorta from old rats does not activate ERK in response to lipopolysaccharide (LPS). A, Fisher 344 rats (adult, middle aged, and old) received i.p. injections of either LPS or adjuvant (time 0), and aortas were prepared at different times to assess ERK activation, as described in Figure 4 . Western blots of pooled samples are shown at the top of the figure, and times of LPS treatment (in hours) are indicated at the bottom. A: Adult, M: Middle Aged, O: Old. Graph represents the average 6 standard error of the mean for three animals per age group and per time point. Results are presented relative to ERK activity in young tissues under basal conditions (without LPS treatment). B, LPS leads to a decrease in kinin levels in all age groups. Kinins were measured by radioimmunoassay, and results are expressed as total serum kinin levels. Results are the mean of four independent animals per age group and represents the average 6 standard error of the mean. Figure 6 . In the presence of lipopolysaccharide (LPS), aorta from old rats does respond to exogenous kinins. Fisher 344 rats of different ages were treated in vivo with LPS for the times indicated at the bottom of the figure. After rats were killed, aortas were excised and stimulated with bradykinin (10 nM) as described in Methods. The samples were processed and assayed as described in Figure 4 . A, Western blots of pooled samples (A, M, and O: Adult, middle-aged, and old pools, respectively). B, Quantitation of results at 1 hour post-LPS. White bars represent ERK activity in response to LPS alone. Black bars represent ERK activity in response to bradykinin, in rats previously treated with LPS in vivo. Results are the mean from three independent animals per age group and represent the average 6 standard error of the mean. Asterisks represent significant difference (p , .05) relative to the control (LPS alone) for each age group.
responsiveness of endothelial cells to kinins. Cernadas and colleagues (34) have shown that in conscious rats, aging leads to an impaired hypotensive response to BK. That is, their results in vivo are in accordance with our observations in isolated aorta. Our results indicate that the aorta loses its capacity to activate ERK in response to kinins as a function of age, while at the same time, basal ERK activity is significantly increased in the aorta from old F344 rats. These changes are driven primarily by changes in endothelial cells, as determined by immunohistochemistry of aorta sections. Although many other possibilities exist, we considered it likely that the increased basal ERK activity could be explained by the high levels of circulating kinins. This interpretation would require that the response to exogenous kinins also be down-regulated in parallel, and in fact, regression analysis indicates a rather tight negative correlation between the level of kinins in serum and ERK activation in response to kinins at any age (data not shown). This inability to induce ERK activity is not unique to kinin stimulation, because the aorta from old rats was also unable to induce ERK in response to LPS. It is interesting, however, that the animals as a whole were able to respond to LPS treatment with a strong decrease in serum kinin levels in all age groups. The mechanism for this reduction is unclear, but it is possible that LPS might lead to a direct reduction in free kinins, either by physical trapping and/or binding or by rapid activation of latent kininogenases. It has also been described that LPS induces fast expression of the B1 kinin receptor (35) . Thus it is likely that, in the presence of LPS, serum kinins might be bound by these receptors, thereby removing them from the circulation, leading to the observed decrease. Under these new conditions, subsequent induction with BK does lead to ERK activation, even in samples from old animals. In fact, in the presence of LPS, the response of old rats is even more robust than that observed in younger animals, suggesting that the decreased responsiveness observed in the absence of LPS was not due to a decrease in kinin receptors, or permanent damage to the intracellular transduction machinery, but rather, this defect might be due to the chronic increase in circulating kinins, leading to receptor down-regulation. We do not have data that directly confirm this hypothesis.
It is important to mention that the kinin concentration used in vitro (10 nmol/L) is much lower that the concentrations found in vivo in F344 rats (63 6 16 nmol/L in adult rats and 398 6 102 nmol/L in old animals). Because this concentration of BK (10 nmol/L) is commonly used in the literature, and because young aortas are capable of responding to the stimulus, we conclude that washing the cells with D-MEM allows them to become responsive to the rather mild in vitro stimulus. Endothelial cells from old rats, however, cannot recover responsiveness during this short wash, probably because of the higher level of kinin stimulation already received in vivo, before isolation of the tissue.
Our observations are in agreement with known facts about the aging process, where relative to younger counterparts, old individuals often present a hyperactive basal state, but are unable to mount a proper response when challenged with an external stress. As an example, although aged organisms often present a pro-inflammatory pattern of circulating cytokines, the response of target tissues to an inflammatory stimulus does not result in an adequate defensive response. Indeed, we and others have observed that systemic treatment with LPS leads, in old rats, to an exacerbated induction of serum cytokines [reviewed in (36) ]. However, in the liver at least, responsiveness to these cytokines is diminished. In the current experiments, we also observed increased ERK activity but diminished responsiveness at time zero in old animals ( Figure 5) .
Elderly persons are known to suffer from a variety of vascular symptoms and pathologies that significantly lower their quality of life. These include, for example, a decline in mobility due in part to sarcopenia, but also including an important cardiovascular component. There are also changes in blood pressure, such as a decline in maximal vasodilator capacity of the resistance vasculature, generation of inflammatory disorders, as well as both acute and chronic pain (37, 38) . Age-related alterations in the regulation of arterial blood pressure lead to fatal failures in cardiac muscle function. Kinins are likely to play a significant role in some of these pathologies, due to their vasoactive and nociceptive properties.
We speculate that the age-related increase in circulating kinins and concomitant decrease in responsiveness to the peptide could play a significant role in the pathophysiological characteristics of the aged vasculature. Kinins play a critical role in lowering blood pressure, and it is conceivable that the increase in kinins represents an ultimately unsuccessful attempt by the organism to control this parameter. At the other end of the spectrum, kinins are strongly algesic, thus their possible role in the generalized aches and pains observed in old frail individuals needs to be further explored. The final impact of the increase in serum kinins on the well-being of old individuals, however, also needs to take into account the lower sensitivity of target cells to such kinins. Both epidemiological studies in humans and molecular studies in transgenic animal models of kininogen overexpression will be necessary before we can understand better the consequences of changes in the balance between kinins and kininogens on the aging process.

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