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. 2002 Jul 1;542(Pt 1):255–262. doi: 10.1113/jphysiol.2002.019166

Evidence for agonist-specific endothelial vasodilator dysfunction with ageing in healthy humans

Christopher A DeSouza *,†, Christopher M Clevenger *, Jared J Greiner *, Derek T Smith *, Greta L Hoetzer *, Linda F Shapiro *, Brian L Stauffer †
PMCID: PMC2290401  PMID: 12096067

Abstract

Endothelium-dependent vasodilatation declines with advancing age in humans independently of disease. The mechanisms responsible for this decline are not clear. We determined whether the age-related reduction in endothelium-dependent vasodilatation in response to acetylcholine reflects a specific agonist-related defect or rather a more general endothelial cell vasomotor abnormality. Twenty-two young (23-35 years) and 41 older (50-76 years) healthy men were studied. Forearm blood flow (FBF) responses to intra-arterial infusions of acetylcholine, bradykinin, substance P, isoproterenol (isoprenaline) and sodium nitroprusside were measured by strain-gauge plethysmography. There were no differences in resting FBF between the young (3.9 ± 0.2 ml (100 ml tissue)−1 min−1) and older men (4.0 ± 0.2 ml (100 ml tissue)−1 min−1). The increase in FBF at the highest dose of acetylcholine was ∼30 % lower (P < 0.01) in the older (from 4.0 ± 0.2 to 12.3 ± 0.7 ml (100 ml tissue)−1 min−1) compared with young men (from 3.9 ± 0.2 to 17.1 ± 1.5 ml (100 ml tissue)−1 min−1). In contrast to acetylcholine, the FBF responses to the other endothelial agonists were not impaired with age. The maximum increases in FBF in response to bradykinin (19.2 ± 1.0 vs. 20.2 ± 0.9 ml (100 ml tissue)−1 min−1), substance P (15.1 ± 0.8 vs. 16.8 ± 0.7 ml (100 ml tissue)−1 min−1) and isoproterenol (17.5 ± 0.9 vs. 17.5 ± 0.9 ml (100 ml tissue)−1 min−1) were not significantly different between the older and young subjects. There were no age-related differences in the FBF responses to sodium nitroprusside. These results demonstrate that, although acetylcholine-induced vasodilatation is impaired with age, forearm endothelial vasodilatation in reponse to bradykinin, substance P and isoproterenol are well preserved in healthy men. Moreover, these findings suggest that agonist-stimulated endothelium-dependent vasodilatation is not universally impaired with age.

Advancing age is associated with an increased incidence of cardiovascular diseases, in particular atherosclerotic vascular disease (Kannel, 1996; American Heart Association, 2001). Many of the cardiovascular complications associated with ageing (e.g. arterial spasm, thrombosis and myocardial infarction) have been linked to endothelial dysfunction, particularly impaired endothelium-dependent vasodilatation (Taddei et al. 1997b; Shimokawa, 1999). Diminished endothelial vasodilator function occurs early in atherogenesis, before histological and/or angiographic evidence of disease (Yasue et al. 1990). Age-related reductions in endothelium-dependent vasodilatation have been reported in both the brachial (Taddei et al. 1995, 1997b) and coronary (Yasue et al. 1990; Egashiro et al. 1993) arteries of healthy adult humans.

The mechanism(s) responsible for the age-related reduction in endothelium-dependent vasodilatation have not been fully elucidated. It has been suggested that defects in the l-arginine-nitric oxide (NO) pathway involving endothelial cell surface receptors and/or membrane-bound G protein signal transduction pathways may contribute to reduced stimulated NO bioavailability and impaired vasodilator function with age (Shimokawa, 1999). However, previous studies documenting the progressive age-related decline in endothelium-dependent vasodilatation have primarily employed muscarinic receptor agonists, either acetylcholine or methacholine, to assess endothelial vasodilator function (Gerhard et al. 1996; Taddei et al. 1995, 1997b). Thus, based on the available data, it is not clear whether the vasodilator dysfunction associated with ageing is related to an agonist-specific defect or a more general endothelial vasomotor abnormality.

Accordingly, the experimental aim of the present study was to determine whether the age-related reduction in endothelium-dependent vasodilatation in response to acetylcholine reflects an agonist-specific defect or rather global endothelial vasodilator dysfunction. To address this aim, we compared the forearm vascular responses to infusion of acetylcholine to responses to bradykinin, substance P and isoproterenol (isoprenaline) in healthy young and older adult men.

METHODS

Subjects

Twenty-two young (age 23-35 years) and 41 older (age 50-76 years) healthy men participated in the present study. All subjects were normotensive, nonobese and free of overt disease, as assessed from a medical history questionnaire, resting blood pressure measurements and fasting blood biochemical parameters. The older men were further evaluated for clinical evidence of cardiovascular disease with a focused physical examination, and resting and maximal exercise electrocardiograms and blood pressure measurements. None of the subjects was on medication, including vitamins, and all subjects were nonsmokers. Before participation, all the subjects had the research study and its potential risks and benefits explained fully before providing written informed consent according to the guidelines of the University of Colorado at Boulder.

Measurements

Body composition

Body mass was measured to the nearest 0.1 kg using a medical beam balance (Detecto, Webb City, MO, USA). Percentage body fat was determined by dual energy X-ray absorptiometry (DXA, Model DPX-IQ, Lunar Radiation Corporation, Madison, WI, USA). Body mass index was calculated as weight (kilograms) divided by height (metres) squared. Minimal waist circumference was measured according to previously published guidelines (Lohman et al. 1988).

Metabolic measurements

Fasting plasma lipid, lipoprotein, glucose and insulin concentrations were determined by the clinical laboratory affiliated with the General Clinical Research Center, as previously described (DeSouza et al. 1998).

Forearm blood flow

Forearm blood flow (FBF) was measured in both the experimental (nondominant) and contralateral (dominant) forearm using strain-gauge venous occlusion plethysmography (D. E. Hokanson, Bellevue, WA, USA) with both forearms positioned above heart level, as previously described (DeSouza et al. 2000). FBF was recorded four times each minute at rest and throughout each drug infusion protocol. Flows during the last minute of rest and during the last minute of infusion at each drug dose were measured and the mean value reported. All FBF values are presented in millilitres per 100 millilitres forearm volume per minute. Forearm volume was determined using the water displacement method.

Intra-arterial infusion protocol

All studies were performed between 7.00 and 10.00 am after a 12 h overnight fast in a temperature-controlled room. Under strict aseptic conditions a 5 cm, 20 gauge catheter was inserted into the brachial artery of the nondominant arm under local anaesthesia (1 % lignocaine). Heart rate and mean arterial pressure (MAP, assessed as ⅓ pulse pressure plus diastolic pressure) were continuously measured throughout the infusion protocol. Following the measurement of resting blood flow for 5 min, endothelium-dependent vasodilatation was assessed by the FBF responses to incremental doses of acetylcholine (IOLAB Pharmaceuticals, Duluth, GA, USA), bradykinin (Clinalfa AG, Laufelfingen, Switzerland), substance P (Clinalfa AG) and isoproterenol (Isuprel, Abbott Laboratories, Chicago, IL, USA). FBF response to sodium nitroprusside (Nitropress, Abbott Laboratories) was used to assess endothelium-independent vasodilatation. Acetylcholine was infused at rates of 4.0, 8.0 and 16.0 μg (100 ml forearm tissue)−1 min−1, bradykinin at 12.5, 25.0 and 50.0 ng (100 ml forearm tissue)−1 min−1, substance P at 75, 150 and 300 pg (100 ml forearm tissue)−1 min−1, isoproterenol at 5.0, 10.0 and 20.0 ng (100 ml forearm tissue)−1 min−1 and sodium nitroprusside at 1.0, 2.0 and 4.0 μg (100 ml forearm tissue)−1 min−1. The doses of acetylcholine, bradykinin, substance P and isoproterenol selected have been shown to elicit comparable increases in FBF in healthy adults (Gilligan et al. 1994; Newby et al. 1997; Cardillo et al. 1998). Each dose was infused for 5 min and sufficient time (≈20 min) was provided to allow FBF to return to resting levels between drug infusions. To avoid an order effect, the sequence of drug administration was randomized. Some subjects did not receive substance P (7 young and 14 older men) or isoproterenol (7 young and 20 older men) due to drug availability. The length of the infusion protocol was shorter for these subjects. Before the beginning of this study, permission was obtained from the Food and Drug Administration to administer bradykinin and substance P to humans.

Statistical analysis

Differences in baseline subject characteristics between the groups were determined by one-way analysis of variance (ANOVA) and repeated measures ANOVA where appropriate. Because MAP did not change throughout the infusion protocols, forearm vascular conductance (FVC) was calculated as FBF divided by MAP and expressed as arbitrary units (a.u.) × 102. Group differences in the FBF and FVC responses to each vasoactive drug were determined by repeated measures ANOVA. All data are expressed as mean values ± s.e.m. Statistical significance was set at P < 0.05.

RESULTS

Subjects

Selected subject characteristics are presented in Table 1. The mean age difference between the young and older men was 33 years. Body mass, percentage body fat, body mass index and waist circumference were higher (P < 0.05) in the older compared with young men. There were no group differences in baseline FBF or FVC. Although all haemodynamic and metabolic factors were well within clinically normal ranges, the older men generally demonstrated higher (P < 0.05) resting systolic and diastolic blood pressure as well as plasma lipid and lipoprotein concentrations. FBF in the noninfused arm and mean arterial blood pressure remained constant throughout the infusion protocols in both the young and older men (Table 2).

Table 1.

Selected subject characteristics

Variable Young men (n = 22) Older men (n = 41)
Age (years) 28 ± 1 61 ± 1*
Body mass (kg) 78.3 ± 2.8 84.9 ± 1.8
Body fat (%) 13.9 ± 1.6 27.7 ± 1.1*
Waist circumference (cm) 85.9 ± 3.2 99.2 ± 1.8*
Body mass index (kg m−2) 23.1 ± 1.3 27.3 ± 0.7*
SBP (mmHg) 114 ± 3 122 ± 2*
DBP (mmHg) 65 ± 2 76 ± 2*
FBF (ml (100 ml tissue)−1 min−1) 3.9 ± 0.2 4.0 ± 0.2
FVC (a.u.) 4.4 ± 0.3 4.3 ± 0.3
Total cholesterol (mmol l−1) 3.7 ± 0.2 4.5 ± 0.2*
HDL cholesterol (mmol l−1) 1.2 ± 0.1 1.1 ± 0.1
LDL cholesterol (mmol l−1) 2.0 ± 0.2 2.6 ± 0.1*
Triglycerides (mmol l−1) 1.1 ± 0.1 1.4 ± 0.1*
Glucose (mmol l−1) 5.1 ± 0.1 5.3 ± 0.1
Insulin (pmol l−1) 36.0 ± 4.5 40.5 ± 2.6
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SBP indicates systolic blood pressure; DBF diastolic blood pressure; FBF resting forearm blood flow; FVC resting forearm vascular conductance; HDL high-density lipoprotein; LDL low-density lipoprotein. Values are means ± s.e.m.

*

P < 0.05vs. young.

Table 2.

Resting forearm blood flow and mean arterial blood pressure before the administration of each vasoactive agent

Vasoactive agent FBF, noninfused limb (ml (100 ml tissue)−1 min−1) FBF, infused limb (ml (100 ml tissue)−1 min−1) MAP (mmHg)
Young men Older men Young men Older men Young men Older men
Acetylcholine 3.5 ± 0.2 3.4 ± 0.2 3.9 ± 0.2 4.0 ± 0.2 81 ± 1 88 ± 2
Bradykinin 3.2 ± 0.3 3.1 ± 0.3 4.1 ± 0.2 4.2 ± 0.2 81 ± 2 89 ± 2
Substance P 3.3 ± 0.3 3.5 ± 0.2 3.9 ± 0.2 4.0 ± 0.2 83 ± 1 89 ± 2
Isoproterenol 3.4 ± 0.2 3.4 ± 0.2 4.0 ± 0.2 4.3 ± 0.2 81 ± 2 88 ± 2
SNP 3.5 ± 0.3 3.3 ± 0.2 4.0 ± 0.2 4.1 ± 0.2 82 ± 2 88 ± 2
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FBF indicates resting forearm blood flow; MAP mean arterial blood pressure; SNP sodium nitroprusside. Values are means ± s.e.m.

Age and forearm vascular responses to acetylcholine

Figure 1 shows the FBF and FVC responses to acetylcholine in the young and older groups. As expected, there were significant age-group differences in the FBF and FVC responses to acetylcholine. At the highest acetylcholine dose (16.0 μg (100 ml tissue)−1 min−1), the increase in FBF was ∼30 % less (P < 0.01) in the older (from 4.0 ± 0.2 to 12.3 ± 0.7 ml (100 ml tissue)−1 min−1) compared with the young men (from 3.9 ± 0.2 to 17.1 ± 1.1 ml (100 ml tissue)−1 min−1).

Figure 1. FBF and FVC responses to acetylcholine in young (•) and older men (○).

Figure 1

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Values are means ± s.e.m. FBF and FVC responses between groups were significantly different (both P < 0.01).

Age and forearm vascular responses to bradykinin

In contrast to the vascular responses to acetylcholine, there were no significant differences in the FBF or FVC responses to bradykinin between the older and young groups (Fig. 2). At the highest dose of bradykinin (50.0 ng (100 ml tissue)−1 min−1), the magnitude of increase in FBF (≈370 %) was similar in the older (from 4.1 ± 0.2 to 19.2 ± 0.8 ml (100 ml tissue)−1 min−1) and young subjects (from 4.1 ± 0.2 to 20.2 ± 0.7 ml (100 ml tissue)−1 min−1).

Figure 2. FBF and FVC responses to bradykinin in young (•) and older men (○).

Figure 2

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Values are means ± s.e.m. There was no significant difference between the two groups (P = 0.23 for FBF and P = 0.12 for FVC).

Age and forearm vascular responses to substance P

Substance P was administered in 15 of the 22 young and 27 of the 41 older men. As for the vascular responses to bradykinin, there were no significant agegroup differences in the FBF or FVC responses to substance P (Fig. 3). At the highest dose of substance P (300 pg (100 ml tissue)−1 min−1), FBF increased from 4.0 ± 0.2 to 15.1 ± 0.8 ml (100 ml tissue)−1 min−1 in the older men and from 4.0 ± 0.2 to 16.8 ± 0.7 ml (100 ml tissue)−1 min−1) in the young men.

Figure 3. FBF and FVC responses to substance P in young (•) and older men (○).

Figure 3

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Values are means ± s.e.m. There was no significant difference between the two groups (P = 0.27 for FBF and P = 0.18 for FVC).

Age and forearm vascular responses to isoproterenol

Forearm blood flow responses to isoproterenol were determined in 15 of the 22 young and 21 of the 41 older subjects. As for the responses to bradykinin and substance P, there was no significant difference in the FBF or FVC responses to isoproterenol between the older and young groups (Fig. 4). For example, at the highest dose of isoproterenol (100 ng (100 ml tissue)−1 min−1), the magnitude of increase in FBF (≈335 %) was essentially identical in the older (from 4.1 ± 0.3 to 17.5 ± 0.9 ml (100 ml tissue)−1 min−1) and young subjects (from 4.0 ± 0.3 to 17.5 ± 0.9 ml (100 ml tissue)−1 min−1).

Figure 4. FBF and FVC responses to isoproterenol in young (•) and older men (○).

Figure 4

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Values are means ± s.e.m. There was no significant difference between the two groups (P = 0.91 for FBF and P = 0.56 for FVC).

Age and forearm vascular responses to sodium nitroprusside

There were no significant differences between the groups in the forearm vasodilator responses to sodium nitroprusside. The maximum increase in FBF in response to sodium nitroprusside was similar in the older (from 4.1 ± 0.2 to 20.2 ± 0.8 ml (100 ml tissue)−1 min−1) and the young men (from 4.0 ± 0.3 to 20.6 ± 0.9 ml (100 ml tissue)−1 min−1; Fig. 5).

Figure 5. FBF and FVC responses to sodium nitroprusside in young (•) and older men (○).

Figure 5

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Values are means ± s.e.m. There was no significant difference between the two groups (P = 0.85 for FBF and P = 0.32 for FVC).

DISCUSSION

The primary new finding of the present study is that, in marked contrast to the response to acetylcholine, forearm endothelium-dependent vasodilatation in response to bradykinin, substance P or isoproterenol is well preserved with age in healthy men. It is well documented that acetylcholine-mediated endothelium-dependent vasodilatation declines precipitously with advancing age in both men and women independently of disease (Taddei et al. 1995, 1997b; DeSouza et al. 2000). To our knowledge, however, this is the first study to demonstrate that forearm endothelium-dependent vasodilatation in response to bradykinin, substance P or isoproterenol does not decline with age in healthy adult humans. These findings suggest that agonist-stimulated endothelium-dependent vasodilatation is not universally impaired with age.

In the human forearm, endothelial vasodilatation in response to acetylcholine is mediated by M2 and M3 muscarinic receptor subtypes, whose signal is coupled to an intracellular membrane-bound pertussis toxin-sensitive G protein. In turn, the G protein stimulates the phospholipase C- phosphatidylinositol-Ca2+ signalling pathway, leading to increased intracellular calcium and activation of endothelial NO synthase (eNOS) and the production and release of NO (Busse et al. 1993; Shimokawa, 1999). In contrast, bradykinin stimulates the phospholipase C-phosphatidylinositol-Ca2+ intracellular signal transduction pathway through B2-kininergic receptors coupled to a pertussis toxin-insensitive G protein (Brown & Roberts, 2001). Substance P shares the same G protein signalling pathway as acetylcholine but acts via tachykinin receptors on the endothelial cell surface (Saito et al. 1991). Isoproterenol, a β-adrenoceptor agonist, stimulates vasorelaxation, in part by activating the adenylyl cyclase pathway resulting in cAMP activation of eNOS (Cardillo et al. 1998). Like the responses to acetylcholine, the vasodilator responses to bradykinin, substance P and isoproterenol have been shown to be significantly attenuated by the NO synthase inhibitor NG-monomethyl-l-arginine (l-NMMA), confirming their mutual dependence on NO release (Gilligan et al. 1994; Cardillo et al. 1997; Dawes et al. 1997; Newby et al. 1997). Because bradykinin, substance P and isoproterenol exert their vasodilator effects through different endothelial cell surface receptors and signal transduction pathways, the age-related decline in endothelial vasodilatation in response to acetylcholine may reflect a specific defect rather than a more general endothelial vasomotor abnormality.

The mechanisms responsible for the reduction in acetylcholine-mediated vasodilatation with age are poorly understood. Increased production of cyclooxygenase-dependent endothelium-derived constricting factors as well as heightened oxidative stress are both thought to play a role in the age-related decline in endothelial vasodilatation in response to acetylcholine (Taddei et al. 1997a,b). Previous studies have shown that indomethacin (Taddei et al. 1997b), a cyclooxygenase inhibitor, and vitamin C (Taddei et al. 2001), a potent antioxidant, potentiate the forearm vasodilator response to acetylcholine in healthy older adults. However, the favourable affects of both indomethacin and vitamin C on acetylcholine-induced vasodilatation were only observed in adults over the age of 60 years despite the onset of dysfunction decades earlier. Moreover, neither indomethacin nor vitamin C administration completely restored the vasodilator response to acetylcholine to normal levels (Taddei et al. 1997b, 2001). Thus, additional factors are likely to contribute to the age-associated reduction in acetylcholine-mediated endothelium-dependent vasodilatation. For example, age-related changes in muscarinic receptor number and function may play a role in the reduced vasodilator response to acetylcholine (Angus & Lew, 1992). In a recent study, Brodde et al. (1998) reported significant age-related declines in cardiac muscarinic receptor density and affinity in healthy humans. Although they observed this decrease in cardiac tissue, similar age-associated declines in muscarinic receptor density and affinity have been shown to occur in other tissues and vascular beds, including the brain and vasa nervorum (Zaccheo et al. 1991; Yazaw & Honda, 1993; Buyukuysal et al. 1998). Therefore, it is plausible that the selective age-related impairment in acetylcholine-mediated vasodilatation observed in the present study may be due, at least in part, to a reduction in forearm muscarinic receptor number, affinity or G protein coupling (Angus & Lew, 1992).

An interesting finding of the present study was the lack of an age-related decline in the FBF response to isoproterenol. It is well established that ageing is associated with reduced cardiac chronotropic responses to isoproterenol (Bertel et al. 1980; Fitzgerald et al. 1984), but the effects of ageing on β-adrenergic vascular responses are less clear (Van Brummelen et al. 1981; Pan et al. 1986; Klein et al. 1988). For example, Van Brummelen et al. (1981) reported diminished forearm vasodilatation in response to intra-arterial infusion of isoproterenol in older compared with young healthy subjects. In contrast, Klein et al. (1988) observed no significant reduction in vascular β-adrenergic function in healthy older adults. Our data are in apparent conflict with those of Van Brummelen et al. (1981). The primary reason for this discrepancy is probably the different intra-arterial doses of isoproterenol used in each study. They observed age-related differences in FBF in response to very low doses of isoproterenol (0.12-4.0 ng (100 ml tissue)−1 min−1 compared with 25-100 ng (100 ml tissue)−1 min−1 used in the present study), consistent with reduced β-adrenoceptor sensitivity (Fitzgerald et al. 1984). However, at a higher dose of isoproterenol (12 ng (100 ml tissue)−1 min−1), Van Brummelen et al. (1981) reported no difference in the FBF responses between the young and older subjects. The latter finding is in agreement with the results of the present study and, collectively, argues against an age-related decrease in peripheral β-adrenergic vascular responsiveness to isoproterenol.

The varied responses to different endothelial agonists observed in the present study are not unique to ageing. Several conditions characterized by impaired forearm acetylcholine-mediated vasodilatation, such as chronic heart failure, hypertension and hypercholesterolaemia, have been shown to have wide-ranging vasodilator responses to other endothelial agonists (Hirooka et al. 1992; Gilligan et al. 1994; Panza et al. 1994, 1995; Casino et al. 1995). For example, in a series of studies, Panza and coworkers (Panza et al. 1994, 1995; Cardillo et al. 1998) demonstrated that endothelial vasodilator responses to acetylcholine, bradykinin and substance P are impaired, whereas vasodilatation in response to isoproterenol is preserved in patients with hypertension compared with healthy control subjects. In similar studies involving patients with hypercholesterolaemia, the same group of investigators observed significantly reduced vasodilatation in response to acetylcholine and substance P but not bradykinin (Gilligan et al. 1994; Casino et al. 1995). Collectively, the results of these studies and the present investigation underscore the importance of defining the context in which reduced agonist-stimulated endothelium-dependent vasodilatation is observed. Depending on the condition or physiological state, diminished dilatation in response to acetylcholine may not necessarily reflect general endothelial vasodilator dysfunction.

There are several important limitations of the present study. First and foremost, we did not assess forearm vascular responses to each agonist in the absence and presence of l-NMMA to determine whether the contribution of NO to forearm vasodilatation was affected by age. Interestingly, however, Newby et al. (1997) and others (Cockcroft et al. 1994) have reported that, in the forearm of healthy adults, up to 70 % of substance P-induced vasodilatation is NO dependent compared with 30-40 % in the presence of acetylcholine, bradykinin or isoproterenol (Cockcroft et al. 1994; Newby et al. 1997; Quyyumi et al. 1997; Cardillo et al. 1998). Thus, it is reasonable to hypothesize that if the l-arginine-NO system were completely impaired with ageing, substance P-induced vasodilatation would be greatly affected. Yet we observed no significant age-related decrease in the forearm vasodilator responses to substance P in our group of healthy men. Second, the present study design precludes us from ruling out compensatory influences of other endothelium-derived relaxing factors. This is particularly the case with bradykinin, which has been shown to stimulate the release of prostanoids and endothelium-derived hyperpolarizing factor in addition to NO (Cherry et al. 1982; Kemp & Cocks, 1997). Parenthetically, although we did not administer aspirin to inhibit prostanoid release, previous studies have shown that prostacyclin does not play a role in the forearm vascular responses to bradykinin in humans (Bengamin et al. 1989). Third, acetylcholine is vulnerable to destruction in vivo by cholinesterase (Angus & Lew, 1992; Brown & Roberts, 2001). Since we did not measure cholinesterase levels in our study population, we cannot rule out the possibility that increased cholinesterase activity in the older adults reduced the concentration of acetylcholine available to the endothelium, thereby limiting the vasodilator response. However, there is evidence to suggest that this is probably not the case. For example, Chan (1995) reported an age-associated reduction in cholinesterase levels in both plasma and whole blood in healthy adults. In addition, FBF responses to methacholine, a muscarinic receptor agonist resistant to cholinesterase degradation (Brown & Taylor, 2001), have been shown to decline with age progressively in a similar fashion to responses to acetylcholine (Gerhard et al. 1996). Fourth, it is important to recognize that the highest dose of each vasoactive drug used in the present investigation probably did not elicit maximal forearm vasodilatation. Our results, therefore, should be viewed within the context of the dose ranges used. It is currently unknown whether, at a higher dose of acetylcholine (e.g. 64 μg (100 ml tissue)−1 min−1), the age-related difference in FBF would persist or conversely whether, at higher doses of bradykinin or substance P, age-associated impairments would emerge. Nevertheless, the dose range for each vasoactive agent used herein is consistent with those used in previous studies to assess endothelium-dependent vasodilatation in various pathological states (e.g. hypertension, hypercholesterolaemia and chronic heart failure) (Hirooka et al. 1992; Gilligan et al. 1994; Panza et al. 1994, 1995; Casino et al. 1995; Cardillo et al. 1998). Fifth, although all baseline metabolic values were well within clinically normal ranges, the older men did demonstrate higher levels of body fatness, total cholesterol, low-density lipoprotein-cholesterol and triglycerides. As such, we cannot discount the possibility that these factors may have contributed to the age-related impairment in acetylcholine-mediated vasodilatation observed in the present study.

In conclusion, the results of this study demonstrate that, although acetylcholine-induced forearm vasodilatation is impaired with age, the vasodilator responses to bradykinin, substance P and isoproterenol are not compromised in healthy men. These findings suggest that agonist-stimulated receptor-dependent endothelial vasodilator function is not universally impaired with age. Moreover, the age-related decline in acetylcholine-mediated forearm vasodilatation may be due, in part, to an agonist-specific defect, potentially at the level of the muscarinic receptor. Future studies are needed to confirm and identify the nature of this abnormality.

Acknowledgments

We would like to thank all the subjects who participated in the study as well as Yoli Casas and Sharon Blackett for their technical assistance. This study was supported by a National Institutes of Health award HL03840, American Diabetes Association Clinical Research Award, American Heart Association award 0060430Z (Dr DeSouza), and by 2 M01-RR00051 from the General Clinical Research Center Program of the National Center for Research Resources, National Institutes of Health.

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