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Cardiovascular Research 2001 50(1):137-144; doi:10.1016/S0008-6363(01)00193-6
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Effect of age on electrical field stimulation (EFS)-induced endothelium-dependent vasodilation in male and female rats

Jennifer C. Sullivan* and Cathy A. Davison

Center for Cardiovascular Sciences, Albany Medical College, Albany, NY, USA

* Corresponding author. Medical College of Georgia, 1459 Laney-Walker Blvd. CB 3213, Augusta, GA 30912, USA. Tel.: +1-706-721-6164; fax: +1-706-721-9799 jsullivan{at}mail.mcg.edu

Received 23 August 2000; accepted 27 December 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods and materials
 3 Results
 4 Discussion
 References
 
Objective: The purpose of this study was to determine whether the effect of age on electrical field stimulation (EFS)-induced endothelium-dependent vasodilation was different in males and females. Methods: Young (3 month) and old (25 month) Fisher 344 rats were studied: young females (YF, n = 34), young males (YM, n = 28), old females (OF, n = 19), and old males (OM, n = 24). Isolated mesenteric resistance arteries (endothelium-intact and denuded) were pressurized, and outer diameter was monitored. EFS-response curves (0.1–8 Hz) were performed in preconstricted arteries in the presence of guanethidine. EFS responses were expressed as percent relaxation from preconstricted diameter. Area under the curve (AUC) was calculated and comparisons were made using ANOVA and t-tests. Results: Males became less responsive to EFS-induced vasodilation with age, while responses among females were unaffected (AUC: YM=344±23, OM=253±25, P = 0.008; YF=397±21, OF=365±25, P = 0.33). Endothelial denudation produced a significant decrease in EFS-induced dilation among all rat groups. The effect of denudation was greater in young animals compared to old. Incubation with the nitric oxide synthase inhibitor N{omega}-nitro-L-arginine (LNA) significantly decreased EFS responsiveness among all of the rat groups. Conclusions: EFS-induced vasodilation declines with age among males. In YF and YM endothelium-dependent EFS-induced vasodilation is mediated by the release of endothelium-derived NO. With age, endothelial function declines, which is likely due to a decrease in the production of endothelium-derived NO. A decline in endothelial-derived NO is likely responsible for the decrease in EFS-induced vasodilation with age among males.

KEYWORDS EFS, electrical field stimulation; YF, young female; OF, old female; YM, young male; OM, old male; LNA, N{omega}-nitro-L-arginine; TTX, tetrodotoxin; NO, nitric oxide


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods and materials
 3 Results
 4 Discussion
 References
 
The vascular endothelium is a dynamic structure that plays a central role in cardiovascular health, in part via the release of vasoactive substances [1,2]. Dysfunction of the endothelium is associated with the development and progression of a number of cardiovascular diseases, including atherosclerosis and thrombosis. Several risk factors for the development of coronary heart disease, such as advancing age and the male gender, are associated with endothelial dysfunction [3–5]. With age there are structural alterations within the vasculature that result in diminished compliance, enhanced vessel stiffness and thickness, and vascular endothelial dysfunction [6,7]. In both coronary and brachial arteries, investigators have reported decreased responses to endothelium-dependent vasodilators without a corresponding decline in response to endothelium-independent vasodilators with advancing age [8–11]. The male gender is also a risk factor for cardiovascular disease. Prior to age 60, men develop cardiovascular disease at twice the rate of women [4,5]. Celermajer et al. [12] reported a gender difference in age-related endothelial dysfunction in the brachial artery, with men experiencing endothelial dysfunction a decade before women. Additionally, other investigators have reported that premenopausal women have greater endothelium-dependent vasodilatory responses [12–14] and nitric oxide (NO) release [15,16] when compared to men. These reports suggest that age-related cardiovascular alterations are different in men and women.

In this study, we examined the effect of age on electrical field stimulation (EFS)-induced vasodilation in male and female rats. EFS induces vasodilation in arterial preparations with elevated tone. EFS-induced dilations have been reported to occur via endothelium-independent and endothelium-dependent mechanisms. Endothelium-independent EFS-induced vasodilation is abolished by incubation with the neurotoxin tetrodotoxin (TTX) or the sensory nerve inhibitor capsaicin [17,18], indicating that dilator responses are mediated by the stimulation of primary sensory nerve fibers and the release of dilatory neurotransmitters [17–20]. Nerve-mediated vasodilator responses are slow in onset and recovery. EFS has also been reported to induce endothelium-dependent vasodilation in the presence of neuronal inhibitors in isolated pulmonary and mesenteric arteries [21–23]. Following removal of the endothelium, these dilator responses are abolished. Endothelium-dependent relaxation in response to EFS has been demonstrated to be mediated by the release of endothelium-derived NO [22,23]. EFS-induced endothelium-dependent vasodilations have a rapid onset and recovery [21–23].

The effects of age and gender on endothelial function have primarily been examined using agonist stimulation of the endothelium. The EFS-stimulation parameters that we used in this study produced an endothelium-dependent vasodilation, allowing us to examine endothelial function in a novel way. The purpose of our study was to determine if the effect of aging on EFS-induced vasodilation is different between males and females.


   2 Methods and materials
Top
 Abstract
 1 Introduction
 2 Methods and materials
3 Results
 4 Discussion
 References
 
2.1 Animals
Male and female Fisher 344 rats of two different ages were used this study: 3-month-old (young rats) and 26-month-old (senescent) rats. The rats were obtained from the colonies at the National Institute on Aging and were barrier-raised. The rats were housed in individual Thoren cages (laminar airflow, autoclaved food, water, and bedding) in temperature and humidity controlled, light-cycled (600–1800 h) quarters with ad libitum access to standard rat chow and water. Rats were fasted overnight prior to sacrifice. All procedures involving the use of animals were approved by the Institutional Animal Care and Use Committee conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Preparation of vascular tissue
The rats were weighed and then euthanized using an overdose of sodium pentobarbital (120 mg/kg, i.p.) followed by thoracotomy. A portion of the small intestine was removed and placed in cold physiological salt solution of the following composition (in mmol/l): NaCl, 130; KCl, 4.7; MgSO4·7H2O, 1.17; KH2PO4, 1.18; NaHCO3, 14.9; dextrose, 5.5; NaCa2EDTA, 0.03; CaCl2·2H2O, 1.6. Using a dissecting microscope, a mesenteric resistance artery was isolated, cleared of fat and connective tissue and placed in a chamber of a dual-chambered arteriograph (Living Systems Instrumentation, Burlington, VT). One end of the vessel was cannulated with a glass microcannula and the vessel lumen was rinsed with physiological salt solution to remove the blood. The distal end of the artery was then secured to another glass microcannula. The artery was secured to the cannulae using nylon ties. The vessel was pressurized to 60 mmHg and the pressure was maintained using an automatic servo device (Living Systems Instrumentation, Burlington, VT).

The artery was bathed by warmed (37°C), gassed (95% O2, 5% CO2) physiological salt solution, at a flow rate of 20 ml/min. The arteriograph was placed on the stage of an inverted microscope and the outer diameter of the vessel was monitored using computerized image analysis consisting of a Framegrabber card (PCVision Plus) and appropriate software (Microsciences Incorporated, Seattle, WA). The vessel was allowed to equilibrate for 45 min. At this point the viability of the vessel was determined by contracting the vessel with 10–6 mol/l phenylephrine followed by 10–5 mol/l acetylcholine to relax the vessel.

2.3 Electrical field stimulation-induced vasodilation
Electrical field stimulation was performed by securing two parallel platinum electrodes (Living Systems Instrumentation, Burlington, VT) on either side of the isolated artery in the arteriograph. The electrical current was driven by a Grass S48 stimulator set to the following parameters: 0.3 ms delay, 1.0 ms duration, 10 V, 60 mA. Between the stimulator and the electrodes was placed a Stimu-Splitter II (Med-Lab Instruments, Loveland CO), a device that serves to modulate waveform distortion and allows for measurement of current at the electrodes.

Vessels were incubated with the adrenergic nerve inhibitor guanethidine (5 µM) [18] and preconstricted to 60% of their resting baseline diameter with phenylephrine. The arteries were stimulated at each frequency (0.1, 0.5, 1, 2, 4 and 8 Hz) until a steady state vasodilatory response was obtained and then allowed to rest between stimulations (approximately 5 min) until the artery returned to the preconstricted diameter. Following a frequency-response curve the artery was allowed to reequilibrate for 30 min and then a second curve was performed in the presence of a pharmacological inhibitor. No more than two curves were performed in each vessel. Preliminary studies confirmed that multiple frequency-response curves could be performed reliably in all of the rat groups.

2.4 Role of sensory nerves
The contribution of sensory nerves to EFS-induced vasodilation was examined using tetrodotoxin and capsaicin. Frequency-response curves were performed in the absence and presence of the neurotoxin tetrodotoxin (TTX, 300 nM) [24] and the sensory nerve inhibitor capsaicin (500 nM) [25]. Arteries were preincubated with TTX for 10-min, and curves were performed in the presence of TTX. Arteries were pretreated with capsaicin for 20 min, the artery was then rinsed and a frequency-response curve was performed. The effects of TTX and capsaicin on EFS-induced vasodilation were examined in different arteries.

2.5 Role of the endothelium
To examine the role of the endothelium in the modulation of EFS-induced vasodilation, frequency-response curves were performed in endothelium-denuded arteries. Endothelium denudation was accomplished by first rubbing the vessel lumen with a human hair and then passing air bubbles through the lumen [26,27]. Denudation was verified by the absence of a vasodilator response to acetylcholine in a vessel preconstricted with phenylephrine. Following the denudation protocol, if an artery failed to have a robust constriction to phenylephrine or had any residual dilation to acetylcholine it was not used in the study. To examine the role of endothelium-derived NO, frequency-response curves were performed in the absence and presence of the nitric oxide synthase inhibitor N{omega}-nitro-L-arginine (LNA; 100 µM) [27].

2.6 Pharmacological analysis of EFS-induced vasodilation
To elucidate the mechanism responsible for EFS-induced vasodilation, pharmacological inhibitors were employed. The role of cholinergic nerves was assessed using atropine (1 µM). The role of CGRP was examined using the CGRP1 receptor antagonist hCGRP8–37 (500 nM). The contribution of histamine was examined using the H1 receptor antagonist pyrilamine (3 µM) and the H2 receptor antagonist tiotidine (1 µM). The role of cyclooxygenase products was determined using the cyclooxygenase inhibitor indomethacin (10 µM).

2.7 Drugs
Acetylcholine, phenylephrine, LNA, tetrodotoxin, guanethidine, capsaicin, atropine, pyrilamine and hCGRP8–37 were all purchased from Sigma Chemical (St. Louis, MO). Buffer reagents were purchased from Fisher Scientific (Pittsburgh, PA). Tiotidine was purchased from Tocris (St. Louis, MO). Stock solutions of acetylcholine (100 mM), phenylephrine (10 mM), LNA (10 mM), tetrodotoxin (1 mM), guanethidine (5 mM), hCGRP8–37 (0.5 mM) were all made in distilled water. Stock solutions for capsaicin (5 mM) were made in 95% ethanol. Stock solutions for pyrilamine (0.3 mM) and atropine (10 mM) were made in buffer. All further dilutions were made in water. The maximum ethanol concentration in the bath did not exceed 0.1%.

2.8 Statistical analysis
All data are expressed as mean±SEM. Vasodilator responses are expressed as a percent of the PE preconstriction. Curves were analyzed by calculating the area under the curve (AUC, % relaxationxmoles/l; GraphPad Prism 2.01). Control responses between the four rat groups were made using a two-way ANOVA (STATISTICA for Windows 4.0; StatSoft, Inc., Tulsa, OK) (factor 1=age; factor 2=gender). Individual comparisons were then performed using a Student Newman–Keuls test. To analyze the effects of pharmacological inhibitors on the AUC, within group comparisons were made using a t-test for dependent samples (STATISTICA for Windows 4.0; StatSoft, Inc., Tulsa, OK). Between group comparisons were made using repeated-measures ANOVA (STATISTICA for Windows 4.0; StatSoft, Inc., Tulsa, OK). Individual comparisons between the rat groups were performed using a t-test for independent samples. To analyze the effect of endothelium denudation on dilator responses, the mean AUC following denudation was compared to the mean AUC for the group control response using a t-test for independent samples.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods and materials
 3 Results
4 Discussion
 References
 
3.1 EFS produces frequency-dependent vasodilation
A typical tracing of an EFS-induced frequency-response curve is shown in Fig. 1. The dilator response is rapid in onset and recovery. Arteries begin to dilate within seconds of turning on the stimulator and the recovery phase of the dilator response begins within seconds of turning the stimulator off.


Figure 1
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  		Fig. 1 EFS produced a frequency-dependent vasodilation. This figure illustrates a typical tracing from a young female. The dilator response is rapid in onset and recovery. The numbers on the graph indicate the frequency of stimulation and the bars indicate the duration of stimulation.

 
3.2 Effect of age on EFS-induced vasodilation in female and male rats
Frequency-response curves for arteries from female and male rats are shown in Figs. 2A and B, respectively. Shown in Fig. 3 are the integrated AUC values for the four rat groups. Baseline diameters (in µm) of the arteries at the beginning of the experiment were: young females (YF): 257±7, old females (OF): 283±5, young males (YM): 272±8 and old males (OM): 294±11. Arteries from YF were slightly but significantly smaller than arteries isolated from OM (YF vs. OM, P = 0.010). Percent constrictions from baseline diameter in response to phenylephrine of the arteries were: YF: 44±1, OF: 46±1, YM: 39±1 and OM: 43±1. Arteries from YM were slightly but significantly less constricted compared to the other rat groups (YM vs. YF, P = 0.015; YM vs. OF, P = 0.005; YM vs. OM, P = 0.047). EFS produced a frequency-dependent vasodilation in all rat groups (Fig. 2). Among the males there was a significant decrease in EFS-induced dilation with age (see Figs. 2B and 3Go). Advancing age had no effect on dilator responses among the females (see Figs. 2A and 3Go). OM were significantly less responsive to EFS-induced dilation compared to the other rat groups, as indicated by a significantly lower AUC (see Fig. 3 for AUC values and Fig. 3 legend for P values).


Figure 2
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  		Fig. 2 Effect of age on EFS-induced vasodilation. EFS-induced dilation was unaltered by age in females (A). Males exhibited a significant decline in EFS-induced dilation with advancing age (B). See Fig. 3 for AUC values. Numbers in parentheses refers to the number of rats in each group. Values represent means±SEM.

 

Figure 3
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  		Fig. 3 Responsiveness to EFS-induced vasodilation. OM were significantly less responsive to EFS compared to the other rat groups, as indicated by the asterisk (*) (OM vs. YF, P<0.001; OM vs. OF, P = 0.004; OM vs. YM, P = 0.008). Number in parentheses refers to the number of rats in each group. Values represent means±SEM.

 
3.3 The role of sensory nerves in EFS-induced vasodilation
The effect of capsaicin treatment and TTX on EFS-induced vasodilation is shown in Table 1. Capsaicin had no effect on EFS-induced dilation in YF, OF or YM. Among OM, following capsaicin treatment there was a significant decrease in EFS-induced vasodilation. TTX had no effect on dilator responses among any of the rat groups (see Table 1 for AUC values, see legend for P values).


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  		Table 1 Effect of capsaicin and TTX on EFS-induced dilationa

 
3.4 The role of the endothelium in EFS-induced vasodilation
Endothelial denudation significantly decreased EFS-induced vasodilation in all rat groups (Fig. 4). Denudation had a greater effect among young animals compared to old animals. Following denudation, YF were significantly less responsive to EFS compared to OF (P = 0.020). Among males, prior to denudation YM were significantly more responsive to EFS compared to OM (P = 0.037). Denudation inhibited vasodilation to a greater extent among YM compared to OM, indicated by the finding that denudation abolished the age effect (P = 0.49).


Figure 4
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  		Fig. 4 Effect of endothelial denudation on EFS-induced vasodilation. Denudation significantly decreased EFS-induced dilation in all rat groups (YF, P<0.001; OF, P = 0.006; YM, P<0.001; OM, P = 0.040). *Indicates significant difference from control. **Indicates significant difference between groups. Control: n = 34 for YF, n = 19 for OF, n = 26 for YM and n = 24 for OM. Denuded: n = 11 for YF and OF, n = 13 for YM and n = 14 for OM. Values represent means±SEM.

 
3.5 The role of nitric oxide in EFS-induced vasodilation
The nitric oxide synthase inhibitor LNA significantly decreased EFS-induced vasodilation in all of the rat groups (Fig. 5).


Figure 5
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  		Fig. 5 Effect of NOS inhibition on EFS-induced vasodilation. The NOS inhibitor LNA (100 µM) significantly decreased EFS-induced dilation in all rat groups (YF, P<0.001; OF, P = 0.006; YM, P<0.001; OM, P = 0.040). *Indicates significant difference from control; n = 6 for YF and OF, n = 9 for YM and n = 8 for OM. Values represent means±SEM.

 
3.6 Pharmacological analysis of EFS-induced vasodilation
Preliminary experiments were performed to characterize the nature of the EFS vasodilatory response. We found that the dilation is resistant to atropine (1 µM), hCGRP8–37 (500 nM), pyrilamine (3 µM) plus tiotidine (1 µM), and indomethacin (10 µM) (data not shown).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods and materials
 3 Results
 4 Discussion
References
 
In this study we found that EFS produces endothelium-dependent vasodilation in isolated mesenteric resistance arteries. Among YF and YM, EFS-induced vasodilation was mediated by the release of endothelium-derived NO. EFS-induced vasodilation declined with age among male rats, but was maintained with age among females. Among both males and females there was a decline in endothelial function with advancing age that was likely due to a decrease in the production of endothelium-derived NO. In OM and OF, there appeared to be extra-endothelial sources of NO. Extra-endothelial sources of NO were more pronounced in OF since EFS-induced dilation was maintained despite the decline in endothelial function.

EFS-induced vasodilation has been reported to occur via endothelium-independent as well as endothelium-dependent mechanisms. All of the studies examining EFS-induced sensory vasodilation in the rat mesentery have been performed using the isolated perfused mesentery. There are numerous reports in the literature that EFS of the perfused mesentery results in endothelium-independent vasodilation. This response has been characterized as TTX and capsaicin sensitive and is mediated by the stimulation of sensory nerves and the release of the dilator neurotransmitter CGRP. Our study is one of few to examine EFS-induced dilation in isolated arteries.

In contrast to the perfused mesentery, EFS of isolated mesenteric resistance arteries produced a frequency-dependent vasodilation that persisted in the presence of TTX and capsaicin, suggesting that the response was not mediated by the stimulation of sensory nerves. Among OM capsaicin significantly inhibited EFS-induced vasodilation, however, since TTX had no effect it is unlikely that this response is due to the stimulation of sensory nerves. Preliminary experiments in YF suggested that CGRP was not involved in EFS-induced vasodilation and a role for cholinergic nerves in the dilation was eliminated, as atropine did not alter EFS-induced dilation. The finding that the dilation is resistant to a number of neuronal inhibitors supports the conclusion that the dilator response is not mediated by sensory nerves. This conclusion is further supported by the finding that following mechanical denudation of the endothelium, EFS-induced vasodilation was significantly inhibited in all of the rat groups.

Our finding supports other reports in the literature that EFS induces endothelium-dependent vasodilation in isolated arteries. Frank and Bevan [21] demonstrated that EFS-induced dilation of isolated pulmonary arteries was endothelium-dependent; vasodilator responses could be restored to an endothelium-denuded artery by an endothelium-intact artery. Using a double vessel preparation, an inverted endothelium-intact artery is placed inside an endothelium-denuded artery, following the addition of the intact artery, dilator responses to EFS are restored to the denuded artery. Further support for endothelial mediation of EFS-induced dilation is derived from a study by Geary et al. [28] in which effluent from electrically stimulated cultured bovine aortic endothelial cells induced relaxation in endothelial denuded rat tail arteries. These studies verified that EFS-induced dilator responses can be mediated by the endothelium. Endothelium-dependent relaxation in response to EFS has been demonstrated to be mediated primarily by the release of NO [22,23].

There are reports in the literature that mast cells are closely associated with sensory nerves and it has been suggested that sensory neurotransmitters released in response to EFS induce degranulation of mast cells and the release of histamine [29,30]. Histamine is known to mediate endothelium-dependent vasodilation via the stimulation of histamine H1 receptors located on the vascular endothelium [31,32]. We report that the histamine receptor antagonists pyrilamine and tiotidine did not alter the response, suggesting that mast cells are not mediating the observed vasodilation. Therefore, under the stimulation parameters we used, EFS appears to directly stimulate endothelium-dependent vasodilation.

In this study we examined the question of whether age differentially affects EFS-induced endothelium-dependent vasodilation in male and female rats. With advancing age, there was a significant decrease in responsiveness to EFS-induced vasodilation among male rats, while dilator responses were maintained with age among females. This finding is consistent with a number of reports in both experimental animals as well as humans in which females have enhanced dilator responses compared to males [13,14,33]. This enhanced dilation observed among females has been postulated to be mediated, at least in part, by estrogen. Our evidence suggests that even at 25 months of age, the females used in this study were still cycling (unpublished data) and therefore had significant amounts of estrogen. One of the mechanisms by which estrogen is thought to enhance dilator response is through the maintenance of the vascular endothelium and stimulation of the production of NO [33–35]. We examined the possibility that there was a gender difference in the effect of aging on vascular endothelial function.

Among both males and females, endothelial denudation suppressed EFS-induced vasodilation to a greater extent among young animals compared to old animals. This suggests that endothelial function, and the ability of the endothelium to respond to EFS, declines with age. Our finding supports experimental studies that have reported a selective decline in agonist-induced endothelium-dependent dilation of isolated mesenteric arteries [36], aorta [37,38] and the perfused mesentery [39] in response to advancing age. Results in animal studies are supported by a number of epidemiological studies. Investigators have reported age-related declines in response to endothelial-dependent vasodilators without a corresponding decrease in response to endothelial-independent vasodilators in both coronary [8,9] and brachial arteries [10,11]. This was actually an unexpected finding, we had expected that with advancing age endothelial function would be preserved among females, since they were still cycling and EFS dilator responses were maintained. It is possible that estrogen is acting to maintain dilator responses via an alternate mechanism.

We examined the role of endothelium-derived vasodilators in mediating EFS-induced vasodilation using pharmacological inhibitors of cyclooxygenase products and NO. We found that cyclooxygenase inhibition using indomethacin had no effect on EFS-induced dilation, suggesting that PGI2 is not mediating the response. Incubation with LNA however, significantly decreased dilator responses in all rat groups. This result supports the findings of other investigators that endothelium-dependent EFS-induced dilation is mediated by the release of NO [22,23].

In young animals, both males and females, LNA abolishes the ability of arteries to dilate in response to EFS. As the response in the presence of LNA was similar to the effect of endothelial denudation, EFS-induced dilation among YM and YF appears to be mediated by the stimulation of endothelium-derived NO.

Similar to the finding in YF, LNA abolishes the EFS dilator response among OF. However, unlike YF, the effect of LNA in OF appears to be greater than the effect of denudation. Since NO production is maintained in OF despite the decline in endothelial function, it is likely that that there are extra-endothelial sources of NO acting to compensate for the loss of endothelium-derived NO. Analogous to observations among OF, the effect of NOS inhibition was greater than the effect of denudation on EFS-induced dilation in the OM, suggesting extra-endothelial sources of NO are present in OM as well. Therefore, with advancing age in females there is a decline in endothelial function, although EFS-induced dilator responses are maintained by the stimulation of NO release from other sources. However, with advancing age among males, extra-endothelial sources of NO are insufficient to restore the depressed EFS response to levels observed in young animals.

Although we did not identify the extra-endothelial source of NO in our study, there are a number of possibilities. NO is known to be localized in sensory nerves [40], sympathetic nerves [41], and in vascular smooth muscle [42,43]. Since incubation with TTX did not alter dilator responses among old animals, it is unlikely that NO is being released from either sympathetic or sensory nerves. Vascular smooth muscle cells have been demonstrated to release NO [44]. Recent studies have identified nNOS as the enzyme mediating NO release in endothelium-denuded rat coronary arteries [42,43]. In addition to nNOS, vascular smooth cells possess iNOS. Studies examining the effect of age on iNOS activity in the rat aorta have reported an increase in the expression and activity of iNOS with advancing age [45,46]. Therefore, it is likely that the extra-endothelial source of NO is the vascular smooth muscle. The effect of gonadal sex hormones on NO production in smooth muscle has not been examined. It is possible however, that estrogen enhances vascular smooth muscle NO release to compensate for the decline in endothelial function.

Our results suggest that while overall NO production is maintained with age, endothelial NO production is decreased. This finding is in accord with reports in the literature. Chauhan et al. [9] reported that depressed acetylcholine-induced dilation among older individuals was antagonized by L-arginine infusion, suggesting that NO production is impaired with advancing age. This postulate has been supported by studies in which direct measurements of nitric oxide release from arteries [47,48] and urine measurements of nitric oxide metabolites [49] have revealed a decrease in nitric oxide with age.

Among males, both young and old, there is a portion of the EFS-induced dilation that is resistant to both denudation and NO inhibition that is not present in females. It is unlikely that nerves mediate the remaining dilation. Preliminary experiments indicated that incubating arteries with buffer containing 25 mM K+ abolished EFS-induced dilation among YM, raising the possibility that a hyperpolarizing factor is being released in response to EFS. Since the endothelium-resistant portion of the response is comparable between young and old males, it is unlikely that this factor is responsible for the observed decline in EFS responsiveness with age.

We conclude that EFS-induced vasodilation declines with age among males. In YF and YM endothelium-dependent EFS-induced vasodilation is mediated by the release of endothelium-derived NO. With age, endothelial function declines, which is likely due to a decrease in the production of endothelium-derived NO. In OM and OF, there appear to be extra-endothelial sources of NO; these sources appear to be more pronounced in OF since EFS-induced dilation is maintained in the face of endothelial dysfunction. Among males there is a portion of EFS-induced vasodilation that is resistant to endothelial denudation, NO inhibition and TTX. Since the endothelium-resistant portion of the response is comparable between young and old males, a decline in endothelial-derived NO is likely responsible for the decrease in EFS-induced vasodilation with age among males.

Time for primary review 22 days.


   Acknowledgements
 
This work was supported by grants (to C.A.D.) from the National Institutes of Health (AG15658), the American Heart Association (9950341N) and (to J.C.S.) Pharmaceutical Research and Manufactures of America Foundation.


   References
Top
 Abstract
 1 Introduction
 2 Methods and materials
 3 Results
 4 Discussion
 References
 

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