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HIGHLIGHTED TOPICS
Physiology of Aging
Departments of Biomedical Sciences and Physiology and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211
Submitted 22 November 2002 ; accepted in final form 26 July 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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-nitro-L-arginine (L-NNA; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), and L-NNA + Indo. In the presence of L-NNA, Indo, or L-NNA + Indo, flow-induced dilation was inhibited in young SFA, resulting in a response to flow that was no longer greater than old SFA. In the presence of L-NNA or Indo, ACh-induced dilation was not significantly inhibited in young or old SFA; however, double blockade with L-NNA + Indo inhibited ACh-induced dilation in young SFA such that the response to ACh was no longer greater than old SFA. Collectively, these data indicate that aging impairs vasodilator responses in SFA by attenuating NO- and prostacyclin-mediated, endothelium-dependent, dilation. endothelial nitric oxide synthase; cyclooxygenase; endothelium-derived hyperpolarizing factor; acetylcholine; flow-induced dilation
The mechanism for the detrimental effects of age on endothelium-dependent dilation to ACh in SFA is not known. In addition, the effect of age on vasodilator responses to other endothelium-dependent agonists has not been studied in these arteries. One mechanism that may contribute to the detrimental effects of aging on endothelial function is decreased production and/or release of endothelial cell-derived nitric oxide (NO). This speculation is supported by experimental evidence indicating that expression of endothelial NO synthase (eNOS) and Cu/Zn-dependent superoxide dismutase (SOD-1) are reduced in SFA of senescent rats (30). An age-associated reduction in eNOS expression could contribute to endothelial dysfunction in old SFA by impairing the ability to produce NO, whereas an age-related decrease in SOD-1 expression may impair the ability to scavenge superoxide anion (O2-·), reducing the biological half-life of NO. Importantly, Muller-Delp et al. (21) reported an age-associated decrease in NO-mediated, endothelium-dependent, dilation in first-order arterioles from soleus muscle. In addition, Csiszar et al. (6) reported an age-related decrease in NO-mediated, endothelium-dependent, dilation of coronary arteries that was associated with increased vascular O2-· production.
Although impaired NO mediation of endothelium-dependent vasodilation has been demonstrated in some arteries from senescent humans and animals (2-7, 12, 18, 21, 27, 28), the effect of age on NO-mediated, endothelium-dependent, dilation in feed arteries that perfuse skeletal muscle is not known. Given that feed arteries are an important site in regulating skeletal muscle blood flow during exercise (29), and the ability to increase soleus muscle blood flow during exercise is impaired in senescent rats (13), it is important that the effects of aging on endothelium-dependent dilation be determined in these arteries. Consequently, the purpose of this study was to test the hypothesis that aging decreases NO-mediated, endothelium-dependent, vasodilator responses in SFA. To test this hypothesis, we examined the effects of age on endothelium-dependent vasodilator responses in SFA to step increases in intraluminal flow and ACh. In addition, we used pharmacological inhibitors of NO synthase (NOS) and cyclooxygenase to determine whether NO and/or prostacyclin (PGI2) mediation of endothelium-dependent dilation is impaired by aging.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Before initiation of this study, approval was received from the Institutional Animal Care and Use Committee at the University of Missouri. Male Fischer 344 rats (age 4 and 24 mo; n = 9/age group) were purchased from a commercial dealer (Harlan Sprague Dawley, Indianapolis, IN) and housed in the College of Veterinary Medicine's Animal Care Facility. The facility was maintained at 24°C with a 12:12-h light-dark cycle. Animals were provided food and water ad libitum, and the rats were examined daily by the investigators and by veterinarians affiliated with the University of Missouri's College of Veterinary Medicine.
Isolation of Feed Arteries
Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight ip). Soleus muscles from the left and right hindlimb were removed and placed in MOPS-buffered physiological saline solution (PSS) containing (in mM) 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 25.0 MOPS, at pH 7.4. SFA were dissected free of paired veins and connective tissue, cut on both ends, and transferred to a Lucite chamber containing MOPS-PSS (4°C) for cannulation.
Determination of Vasodilator Responses
Preparation of arteries. SFA were prepared for functional analysis as described previously (14, 30). Specifically, arteries were cannulated with two resistance-matched glass micropipettes and secured with 11-0 surgical silk. The micropipettes were subsequently attached to separate pressure reservoirs filled with MOPS-PSS supplemented with albumin (1 g/100 ml). The height of each reservoir was initially adjusted to set intraluminal pressure in each SFA to 60 cmH2O (1 mmHg = 1.36 cmH2O) for 20 min. After 20 min, intraluminal pressure was raised to 90 cmH2O, and the feed arteries were allowed to equilibrate for an additional 40 min at 37°C. At the end of the 60-min equilibration period, feed arteries that did not develop at least 25% spontaneous tone were constricted with phenylephrine. All experimental protocols were subsequently conducted at an intraluminal pressure of 90 cmH2O to approximate in vivo intraluminal pressure (29).
Endothelium-dependent dilation. Endothelium-dependent dilation was assessed in SFA by measuring vasodilator responses to increases in intraluminal flow or increasing doses of ACh. Intraluminal flow was established in feed arteries by changing proximal and distal pressures in equal but opposite directions while maintaining constant pressure at the midpoint of the artery (19). Vasodilator responses were assessed at pressure gradients of 0, 2, 4, 6, 8, 10, 15, 20, 30, and 40 cmH2O corresponding to flow rates ranging from 0 to 62 µl/min. Each flow rate was maintained for 5 min to ensure that the feed artery reached a steady diameter. ACh-induced dilation was assessed in SFA by adding increasing doses of ACh to the bath solution in cumulative doses over the range of 10-9 to 10-4 M in whole-log increments. Because preliminary experiments indicated that SFA exhibited tachyphylaxis to ACh, individual drug treatments had to be performed on separate SFA. Thus a total of four SFA were studied in parallel from each rat. In SFA 1, flow- and ACh-induced dilation was assessed in the absence of enzyme inhibitors (control). In SFA 2, the role of NO in vasodilator responses was assessed in the presence of N-nitro-L-arginine (L-NNA; 300 µM) to block NOS. In SFA 3, the contribution of prostacyclin (PGI2) to endothelium-dependent dilation was assessed by measuring dilator responses in the presence of indomethacin (Indo; 5 µM) to block COX. In SFA 4, combined treatment with L-NNA + Indo was used to assess the importance of NOS- and COX-independent mechanisms of dilation. The experimental protocol was designed such that intraluminal flow was always administered first followed by ACh.
Endothelium-independent dilation. Endothelium-independent dilation was assessed by adding increasing doses of sodium nitroprusside (SNP) to the bath solution in cumulative doses over the range of 10-9 to 10-4 M in whole-log increments.
Passive diameter. At the end of each experiment, feed arteries were incubated for 30 min in Ca2+-free PSS to determine passive diameter at an intraluminal pressure of 90 cmH2O.
Solutions and Drugs
All reagents used in dose-response experiments were obtained from Sigma Chemical (St. Louis, MO). Reagents were prepared on the day of the experiment.
Statistical Analysis
All values are means ± SE. Between-group differences in body mass and passive diameter were assessed by using Student's t-tests for unpaired observations. Two-way repeated-measures ANOVA with repeated measures on one factor (flow rate or dose) was used to determine whether dilation to intraluminal flow, ACh, and SNP differed by age. To determine the mechanism(s) by which aging affected relaxation to flow and ACh, the two-way repeated-measures ANOVA was used under the following four conditions: in the absence of enzyme inhibitors, in the presence of L-NNA, in the presence of Indo, and in the presence of L-NNA + Indo. Concentration-response data were expressed as a percentage of maximal possible dilation. Percent possible dilation was calculated as [(Ddose - DB)/(DP - DB)] x 100, where Ddose is measured diameter for a given dose (or flow rate), DB is baseline diameter before a given dose (or flow rate), and DP is maximal passive diameter. A total of 72 SFA were used in the present study. Seventeen SFA (6 young, 11 old) required phenylephrine to achieve 25% tone. Deleting these arteries from the statistical analyses did not alter interpretation of the results; therefore, all 72 SFA were included in the final analyses. Statistical significance was set at the P
0.05 probability level.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Body weights were similar in young (360 ±7 g) and old rats (369 ±8 g).Similarly, maximal passive diameter of SFA was similar in SFA from young and old rats (Table 1).
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Flow-Induced Dilation
Before dose-response curves were initiated, percent tone was similar in young and old SFA in all treatments groups (Table 1). In the absence of enzyme inhibitors, step increases in intraluminal flow elicited a flow rate-dependent dilation of young SFA but not old SFA (Fig. 1). In the presence of L-NNA (Fig. 2), Indo (Fig. 3), or L-NNA + Indo (Fig. 4), flow-induced dilation was inhibited in young SFA, resulting in a response to flow that was similar to old SFA.
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ACh-Induced Dilation
Before ACh dose-response curves were initiated, percent tone was similar in young and old SFA in all treatments groups except the L-NNA + Indo group where percent tone was greater in young SFA than in old SFA (Table 1). In the absence of enzyme inhibitors, ACh elicited a dose-dependent dilation of young and old SFA; however, the vasodilator response to ACh was significantly less in SFA from the old rats (Fig. 5). In the presence of L-NNA (Fig. 6) or Indo (Fig. 7), ACh-induced dilation was not significantly inhibited in young or old SFA. In the presence of L-NNA + Indo (Fig. 8), ACh-induced dilation was significantly inhibited in young SFA (not old SFA), resulting in a response to ACh that was similar to old SFA.
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Endothelium-Independent Vasodilator Responses
SNP elicited a concentration-dependent dilation of young and old SFA (Fig. 9). Statistical analysis revealed no significant between-group differences.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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In rat skeletal muscle resistance arteries and arterioles, intraluminal flow is a signal producing endothelium-dependent dilation mediated in part by the production of endothelial cell-derived NO (17, 25). In the present study, step increases in intraluminal flow elicited a flow rate-dependent vasodilation of young SFA but not in old SFA (Fig. 1). The finding that flow-induced dilation was impaired in old SFA suggested that age-related decrements in endothelial function were due in part to decreased release of NO. Therefore, to test the hypothesis that age-related decrements in flow-induced dilation were associated with an impaired ability to release NO, flow-induced dilation was assessed in the presence of L-NNA to inhibit NOS. The finding that L-NNA inhibited flow-induced dilation in young SFA (Fig. 2), such that the response to flow was no longer greater than in old SFA, is consistent with the interpretation that age-related decrements in flow-induced dilation were due in part to decreased production or release of NO. This finding is in accord with previous work demonstrating an age-related reduction in NO mediation of flow-induced dilation in coronary arteries (6) and soleus muscle first-order arterioles (21) and indicates that age-related decrements in the ability to produce NO also occur in SFA.
On the basis of experimental evidence that flow-induced dilation can be mediated in part by the production of PGI2 (16, 17, 25), we examined vasodilator responses to intraluminal flow in the presence of Indo to inhibit COX. Importantly, Indo inhibited flow-induced dilation in young SFA, whereas flow-induced dilation tended to be enhanced in the old SFA (Fig. 3). Consequently, the difference in flow-induced dilation between young and old SFA was no longer apparent. Directionally opposite shifts in the flow-diameter relationship in the presence of Indo have been reported previously in soleus first order arterioles from young and old rats (21). Collectively, these data indicate that age-related decrements in flow-induced dilation were due in part to decreased production of PGI2. In addition, these data suggest that aging may be associated with enhanced production of a prostanoid constrictor in SFA. Importantly, enhanced COX-2-mediated vasoconstriction has been reported in aorta and mesenteric arteries of senescent rats (10, 24). In addition, an age-associated increase in the production of a COX-derived vasoconstrictor has been reported in hypertensive human subjects (26). Further study will be needed to determine whether aging is associated with enhanced production of a prostanoid constrictor in response to intraluminal flow in senescent SFA.
To assess whether blunted dilator responses to intraluminal flow in old SFA were associated with impairment of a NOS- and COX-independent vasodilator mechanism, flow-induced dilation was assessed in the presence of L-NNA + Indo (double blockade). In the presence of double blockade, residual dilation to increases in intraluminal flow can be attributed to a vasodilator molecule other than NO and PGI2, such as endothelium-derived hyperpolarizing factor (EDHF). Importantly, flow-induced dilation was eliminated in the presence of double blockade in young SFA such that the response was no longer greater than old SFA (Fig. 4). These data indicate that NO and PGI2 completely accounted for flow-induced dilation in young SFA and that age-induced impairment of flow-induced dilation in SFA was due to a decreased ability to produce and release these vasoactive molecules.
Interestingly, in the presence of L-NNA alone, increases in intraluminal flow induced vasoconstriction in young and old SFA. The mechanism for flow-induced constriction in the presence of L-NNA is not clear. One possible explanation is that increased intraluminal flow elicited corelease of NO and a prostanoid constrictor such that, in the presence of NOS inhibition, the net response to increased intraluminal flow was constriction. Indeed, flow-induced release of constrictor prostaglandins has been reported in arterioles from hypertensive rats (11). In addition, an age-associated increase in the production of a COX-derived vasoconstrictor has been reported in hypertensive human subjects (26). If release of a COX-dependent prostanoid constrictor mediated flow-induced constriction of the SFA, one would expect that combined treatment with L-NNA + Indo would eliminate the constrictor response. Consistent with this prediction, treatment with L-NNA + Indo prevented flow-induced constriction (Fig. 4).
In the present study, ACh was used to assess receptor-mediated, endothelium-dependent, vasodilation. Results indicating that ACh-induced dilation was blunted in SFA from old rats (Fig. 5) confirmed previous studies indicating that ACh-induced dilation is impaired in SFA from senescent rats (30). Because previous work revealed that dilation to ACh is mediated in part by NO (15) and PGI2 (23) in rat skeletal muscle arteries, we sought to determine whether the age-related decrement in ACh-induced dilation involved impairment of the ability of old SFA to release one or both of these vasoactive molecules. The finding that L-NNA (Fig. 6) and Indo (Fig. 7), when administered alone, did not inhibit ACh-induced dilation in SFA from young or old rats suggested the possibility that NO and PGI2 played a minor role in ACh-induced dilation in these arteries. This was surprising given that NO and PGI2 contribute to ACh-induced dilation of first-order arterioles from soleus muscle (21, 23).
Alternatively, the lack of effect of L-NNA or Indo to inhibit ACh-induced dilation may suggest that, in SFA, an interaction existed between NO and PGI2 such that, when either NOS or COX was inhibited, ACh-induced dilation was mediated solely by the unblocked pathway. This speculation is supported by experimental evidence indicating that PGI2 synthesis is enhanced in the presence of NOS blockade (1, 22) and by evidence indicating that PGI2 production is attenuated during infusion of a NO donor (20). Therefore, to further explore the role of NO and PGI2, we assessed ACh-induced dilation in the presence of L-NNA + Indo (double blockade). Importantly, double blockade significantly attenuated ACh-induced dilation in young (not old) SFA such that the response to ACh was no longer greater than old SFA (Fig. 8). These results indicated that an impaired ability to produce and/or release NO and PGI2 contributed to the age-related decrement in ACh-induced dilation in SFA.
It is important to note however, that ACh-induced dilation was not eliminated in the presence of L-NNA + Indo in young or old SFA. The finding that young and old SFA exhibited similar, and significant, ACh-induced dilation in the presence of double blockade indicated that ACh-induced dilation was mediated in part by a pathway independent of NO and PGI2 possibly EDHF. In addition, these data indicate that an age-related decrement in the efficacy of the NOS- and COX-independent vasodilator pathway did not contribute to the impairment of ACh-induced dilation in senescent SFA, as has been reported in mesenteric arteries from senescent rats (8).
The mechanism by which aging impaired NO and PGI2 production in SFA is not known. One possibility is that age-associated reductions in eNOS and COX protein content in SFA decreased the ability to produce NO and PGI2. Importantly, age-related reductions in eNOS protein content have been reported in SFA (30), and reductions in eNOS and COX-1 protein content have been reported in coronary arteries (6) of senescent rats. Alternatively, aging may be associated with increased production of O2-·, resulting in an increased rate of degradation of NO. Indeed, increased generation of O2-· has been reported in aorta and coronary arterioles of senescent rats (6, 9). In addition, aging is associated with decreased SOD-1 protein content in SFA (30), which could attenuate the ability to scavenge O2-·, further reducing the half-life of any NO produced.
It is conceivable that impaired vasodilator responses of old SFA were due in part to an age-related reduction in the ability of vascular smooth muscle to respond to endothelial cell-derived NO. To address this possibility, vasodilator responses to SNP (a NO donor) were assessed in SFA from young and old rats. Importantly, dilator responses to SNP were similar in SFA from young and old rats, indicating that the ability of vascular smooth muscle to respond to NO was not impaired by aging. Thus these results are consistent with the interpretation that age-induced decrements in endothelium-dependent dilation were due to an impaired ability of SFA to produce and/or release endothelial cell-derived NO rather than to a reduced ability of vascular smooth muscle to respond to NO.
In summary, the results of this study indicate that aging induces impairment of endothelium-dependent dilation of SFA, an artery that plays an integral role in the regulation of soleus muscle blood flow during exercise. The age-related decrement in endothelial function was characterized by attenuated vasodilator responses to intraluminal flow and ACh. SFA from aged rats did not exhibit significant dilation in response to flow and exhibited 30-40% less dilation than young SFA in response to ACh. In addition, results indicated that the age-related decrement in endothelium-dependent dilation was due to impairment of NO and PGI2 release by endothelium. Further study will be needed to determine the importance of age-related decrements in endothelium-dependent dilation in the attenuated ability to increase soleus muscle blood flow during exercise in senescent rats.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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