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. 2006 Dec 7;579(Pt 1):227–236. doi: 10.1113/jphysiol.2006.124313

Ageing reduces nitric-oxide- and prostaglandin-mediated vasodilatation in exercising humans

William G Schrage 2, John H Eisenach 1, Michael J Joyner 1
PMCID: PMC2075375  PMID: 17138603

Abstract

In older humans, infusions of endothelial agonists suggest endothelial dysfunction, due in part to less nitric oxide (NO)- and prostaglandin (PG)-mediated vasodilatation, and a shift toward PG-mediated vasoconstriction. Ageing can also be associated with lower exercise blood flow (exercise hyperaemia), but the vascular mechanisms mediating this remain unknown. Notably, in young adults, inhibition of NO and PGs during exercise decreases exercise hyperaemia by ∼20 and ∼12%, respectively. We tested our first hypothesis that in older humans inhibition of NO would decrease hyperaemia, but that inhibition of PGs would increase hyperaemia by blocking vasoconstrictor PGs. Fifteen older subjects (65 ± 3 years) performed dynamic forearm exercise for 20 min (20 contractions min−1). Forearm blood flow (FBF) was measured beat-to-beat with Doppler ultrasound, while saline or drugs were infused sequentially via brachial artery catheter in the exercising forearm. After achieving steady-state exercise, l-NAME (25 mg) was infused over 5 min to inhibit NO synthase. After a further 2 min of exercise (saline), ketorolac (6 mg) was infused over 5 min to inhibit PGs, followed by a futher 3 min of exercise with saline. Drug order was reversed in seven subjects. l-NAME reduced steady-state exercise hyperaemia by 12 ± 3% in older subjects (P < 0.01), whereas ketorolac had no net effect on blood flow (3 ± 6%, P > 0.4). The effects of l-NAME and ketorolac were independent of drug order. By comparing these results with our previous results in young adults, we tested our second hypothesis that in older humans inhibition of NO or PGs would have less impact on exercise hyperaemia due to less vasodilatation from these signals. Our results suggest that, compared with young adults, in older humans the relative contribution of NO to exercise hyperaemia is reduced ∼45% (22 ± 4 versus 12 ± 3%), but the role of PG in mediating vasodilatation is lost in ageing human skeletal muscle. Lower exercise hyperaemia in older humans may be mediated in part by less NO- and PG-mediated vasodilatation during exercise.

The increase in limb blood flow during exercise (exercise hyperaemia) can be reduced with ageing (Proctor et al. 1998; Beere et al. 1999; Poole et al. 2003; Lawrenson et al. 2004; Musch et al. 2004; Koch et al. 2005), suggesting that under some conditions limitations in oxygen and nutrient delivery (and waste removal) may limit contractile work in ageing humans. The physiological mechanisms for reductions in exercise hyperaemia remain speculative, but could include lower vascular capacity (i.e. structural remodelling) and/or altered vascular signalling.

When peak reactive hyperaemia is used as an index of vascular structural capacity (Proctor et al. 2005; Ridout et al. 2005), there is an age-associated reduction in peak vascular conductance, suggesting the capacity to reach peak blood flows might contribute to lower exercise hyperaemia, but only at peak metabolic demand. Lower peak conductance might also be due to reduced vasodilatation to a given metabolic stimulus, and not necessarily to altered vascular structure.

In this context, it also is reasonable to propose that human ageing is associated with altered vasodilator signalling during exercise. For example, older humans demonstrate blunted forearm vasodilatation to infusions of acetylcholine (Taddei et al. 1995, 2000) or blunted flow-mediated dilatation (Eskurza et al. 2004). This ‘endothelial dysfunction’ is due in part to less nitric oxide (NO)-mediated vasodilatation (Taddei et al. 1995, 2000). Interestingly, the impairment in vasodilatation can be partially reversed by some antioxidant treatments (Eskurza et al. 2004), but not all (Eskurza et al. 2006), supporting the idea that age-related increases in oxidative stress reduce NO-mediated dilatation. Ageing is also associated with a shift away from prostaglandin (PG)-mediated vasodilatation to PG-mediated vasconstriction (Taddei et al. 1995) (via thromboxane, TXA2). For example, infusion of a cyclooxygenase (COX) inhibitor has little effect on acetylcholine-mediated dilatation in young adults, but actually enhances vasodilatation in older adults (Taddei et al. 1995). Taken together, older humans demonstrate less NO- and PG-mediated vasodilatation, both of which may contribute to lower exercise hyperaemia in vivo. One limitation to endothelial agonist infusion studies is that endothelial control is only one component of local vascular control of muscle blood flow; therefore, these results may not directly translate to control of blood flow during exercise.

In young adults, a few studies support an important role for NO (Sheriff et al. 2000; Schrage et al. 2004), while others suggest NO is not obligatory in exercise hyperaemia (Radegran & Saltin, 1999; Frandsen et al. 2001). The case for PGs is less studied. We inhibited NO synthase (NOS) and COX pathways in young healthy adults during exercise, and reported that NOS inhibition diminished exercise blood flow by 20%, and COX inhibition reduced blood flow by 12% (Schrage et al. 2004). Boushel and colleagues, using combined inhibition of both NOS and COX, reported an exercise-intensity-dependent reduction of exercise hyperaemia (Boushel et al. 2002). Thus, during exercise, it appears that NO and vasodilating PGs normally contribute to vasodilatation in young adults; however, it remains unknown whether the age-associated shift away from endothelial NO- and PG-mediated vasodilatation and toward PG-mediated vasoconstriction contributes to blood flow limitations during exercise in older humans.

Therefore, we tested the hypothesis that (1) inhibition of NO would decrease hyperaemia, and inhibition of PGs would increase hyperaemia in healthy ageing adults, and (2) in comparison with younger subjects, in ageing humans inhibition of NO or PGs would have less impact on exercise hyperaemia, due to less ongoing vasodilatation from these signals.

Methods

Subjects

Subjects were recruited from Rochester, MN, USA, and surrounding areas. Fifteen subjects (14 males and 1 female; 55–81 years) were generally healthy and free from cardiovascular problems. Subjects were non-smokers, non-obese (BMI <30 kg m−2). Two subjects were taking Zocor to treat hypercholesterolaemia. All procedures were approved by the Mayo Institutional Review Board and conformed to the standards set by the Declaration of Helsinki. After reviewing the protocol, all subjects provided written informed consent. Subjects reported normal daily activities (walking, household work, gardening) but no regular physical training. Eight subjects reported taking a daily vitamin, but none reported taking antioxidants. Subjects were instructed to refrain from exercise, aspirin, NSAIDS, alcohol and caffeine for 24 h prior to the study day.

Measurements

Heart rate and blood pressure

Heart rate was measured by three-lead electrocardiography. The brachial artery was catheterized with a standard 5 cm 20 gauge Teflon catheter inserted into the non-dominant arm, and flushed with heparinized saline (2 units ml−1, 3 ml h−1). A pressure transducer connected to the arterial catheter measured beat-to-beat blood pressure (Cardio-Cap).

Forearm blood flow

Brachial artery diameter and blood velocity were measured with a Doppler ultrasound probe (12 MHz linear array; Model M12L, Vivid 7, General Electric, USA) with a probe insonation angle calibrated to 60 deg. Except during intermittent diameter measurements, arterial blood velocity was continuously assessed throughout baseline and during the entire 20 min of exercise. Diameter measurements typically resulted in loss of pulse wave signal for 10–15 s. Diameter was taken as a mean of five measurements in late diastole, between muscle contractions. Forearm blood flow (FBF) was calculated as brachial mean blood velocity multiplied by the brachial artery cross-sectional area (Dinenno et al. 2002; Schrage et al. 2004; Tschakovsky et al. 2004).

Experimental procedures

Drugs

Saline and study drugs were administered via the brachial artery catheter using a three-port connector system that permitted simultaneous measurements of arterial pressure during drug infusions. Saline was infused at 2 ml min−1, which did not alter basal blood flow. NG-nitro-l-arginine methyl ester (l-NAME; Aerbio/Clinalfa; 5 mg ml−1, 1 ml min−1) and ketorolac (tradename-Toradol; Abbott; 600 μg ml−1, 2 ml min−1) were diluted in saline immediately before use. Ketorolac was chosen to inhibit COX instead of indomethacin because we have previously shown that ketorolac reduces exercise hyperaemia in young adults by 12 ± 4% (Schrage et al. 2004). All subjects received the same total dose of drugs during exercise, such that the largest forearm (forearm volume was determined by water displacement) would receive a local delivery of drugs equivalent to 2–4 times higher than systemic doses used in previous studies (Lang et al. 1997; Boushel et al. 2002).

Forearm exercise

Dynamic forearm exercise was performed with a hand-grip device by the non-dominant arm, lifting a weight 4–5 cm over a pulley at a duty cycle of 1 s contraction:2 s relaxation (20 contractions min−1). The exercise lasted for 20 min, and the workload corresponded to 10% of maximal voluntary contraction obtained prior to instrumentation.

Exercise protocol with sequential drug infusion

After instrumentation, subjects rested quietly for 30 min and all testing was performed in the supine position (Fig. 1). After 2 min of quiet rest, subjects started 20 min of dynamic exercise. Beat-to-beat brachial artery blood velocity measurements were obtained continuously throughout baseline and exercise. Saline was infused during the first 5 min (control exercise), at which point saline was replaced with l-NAME for 5 min (n = 8). At 10 min exercise, l-NAME was changed to saline for 2 min, followed by a 5 min ketorolac infusion, and saline for the final 3 min of exercise. In the remaining seven subjects, the order of the l-NAME and ketorolac infusions was reversed. Brachial artery diameter was obtained after 2 min of baseline, at steady-state exercise (min 4–5), at the end of l-NAME (min 10), end of saline (min 12, between drugs), end of ketorolac (min 17), and the end of exercise (min 20). Because inhibitors of NOS (such as l-NAME) and COX (such as ketorolac) demonstrate prolonged effects (hours), the protocol was designed such that after ‘single blockade’ with l-NAME or ketorolac, addition of the second drug resulted in ‘double blockade’ condition during exercise.

Figure 1. Experimental timeline.

Figure 1

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After resting measurements, subjects completed 20 min of exercise. After 5 min of control exercise (saline only), l-NAME was infused for 5 min, followed by 2 min saline, 5 min ketorolac, and 3 min saline (n = 8). Drug order was reversed in seven subjects. Brachial artery diameter measurements were obtained several times during the exercise bout (shaded boxes).

Previously published results from young subjects

Data from young subjects has been previously published (Schrage et al. 2004). For the sake of discussion, comparisons between the effects of l-NAME and ketorolac were made only with relative changes in blood flow, since the data from older subjects (present study) and younger subjects were collected with two different ultrasound systems.

Data collection and statistical analysis

All haemodynamic data were digitized and stored on a computer at 200 Hz, and analysed off-line with signal processing software (Powerlab; ADinstruments). Heart rate (HR) was derived from the electrocardiogram and mean arterial pressure (MAP) was derived from the arterial pressure waveform. The mean blood velocity of the Doppler signal was averaged across 30 s intervals during steady-state exercise to reduce contraction-to-contraction-induced variability in blood flow. Forearm vascular conductance (FVC) was calculated as (FBF/MAP) × 100, and expressed in millilitres per·minute per 100 mmHg.

Since all subjects exercised at the same relative workload (10%), a wide range of absolute workload and therefore blood flow responses were obtained. To assess the relative contribution of NO and PGs, we normalized each subject's steady-state exercise blood flow to 100% (no blood flow was defined as 0%).

Student's unpaired t tests were used to compare subject characteristics between groups. The primary analysis was to test whether patients' responses changed in response to l-NAME and ketorolac infusion. Data were analysed using generalized estimating equation (GEE; Zeger et al. 1988) to take into account the clustering of data within subjects. An unstructured working correlation structure was used for the GEE analysis. The GEE approach is easily implemented, is not as sensitive to model assumptions, and has relatively rapid computing time. Since model assumptions are less important using GEE versus ANOVA, we elected to use GEE. A model was run for each dependent variable (FBF, FVC, MAP and HR) and the independent variable was time. We also analysed the data with ANOVA modelling, and those results support the present conclusions equally.

All data are expressed as means ± s.e.m. Significance for all comparisons was P < 0.05.

Results

Subject characteristics

Subjects in the two drug treatment orders displayed similar height, weight, BMI, forearm volume, brachial artery diameter, maximal voluntary contraction, and exercise workloads. Subject characteristics are summarized in Table 1. The main differences between young and ageing subjects were higher blood pressure at rest, and higher LDL and total cholesterol (Table 1).

Table 1.

Subject characteristics

Drug order, aged Drug order, young
Variable LK KL LK KL
Age (years) 67 ± 3 64 ± 3 26 ± 2*  27 ± 3*
Height (cm) 174 ± 2  182 ± 2  167 ± 2    182 ± 2  
Weight (kg) 77 ± 2 87 ± 2 65 ± 2* 87 ± 2
BMI (kg m−2) 26 ± 1 26 ± 1 25 ± 1  25 ± 1
Blood pressure (mmHg) 143 ± 4/79 ± 4 144 ± 7/84 ± 3 112 ± 8/68 ± 4* 123 ± 6/75 ± 3*
Forearm volume (ml) 1189 ± 70  1360 ± 55  1034 ± 93     973 ± 134*
Exercise workload (kg)  4.2 ± 0.2  4.8 ± 0.2  3.5 ± 0.3*   3.4 ± 0.3*
Total cholesterol (mg dl−1) 194 ± 10       159 ± 6*
LDL cholesterol (mg dl−1) 119 ± 8         85 ± 7*
HDL cholesterol (mg dl−1) 55 ± 4       49 ± 5
Triglycerides (mg dl−1) 107 ± 25       128 ± 26
Glucose (mg dl−1) 96 ± 3       85 ± 8
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Data are means ±s.e.m. Within age groups, subjects in each drug treatment order displayed similar age, physical characteristics, and exercise workloads. Compared with ageing subjects, younger subjects exhibited lower blood pressure, lower total and LDL cholesterol, and slightly lower workloads due to higher prevalence of female subjects. BMI, body mass index; LK, drug order where l-name was infused first, followed by ketorolac; KL, ketorolac was infused first, followed by l-name.

*

Significant difference between young (n = 14) and ageing (n = 15) ageing subjects, P < 0.05.

Systemic response to exercise and drug infusions

Arterial pressure and HR during rest and exercise are summarized in Table 2. Arterial pressure in aged subjects did not increase significantly with exercise, or in response to infusion of ketorolac. MAP was higher following infusion of l-NAME (P < 0.01). Similarly, HR was stable from rest to exercise, except during protocol 1, which exhibited a small decrease in HR (5 beats min−1) after infusion of l-NAME (P < 0.02). Systemic responses for young subjects have been reported previously. Comparison of HR between young and ageing subjects revealed that HR was similar at rest, during exercise, and in response to l-NAME infusion in both age groups. MAP was significantly greater at rest in ageing subjects, but the MAP response to exercise and drug infusions were similar between young and ageing subjects (all P > 0.2). The main differences were in the FBF responses to drug infusions discussed below.

Table 2.

Haemodynamic values for forearm exercise and drug infusions

Variable Drug order Baseline Steady-state exercise (min 5) End drug 1 (min 10) Saline (min 12) End drug 2 (min 17) End of exercise (min 20)
HR LK 60 ± 2  62 ± 3 60 ± 2 59 ± 2 55 ± 3 56 ± 2
(beats min−1) KL 55 ± 3  57 ± 4 54 ± 3 55 ± 3 54 ± 3 52 ± 3
MAP LK 102 ± 2   105 ± 3  108 ± 4  106 ± 3  109 ± 4  108 ± 4 
(mmHg) KL 102 ± 5   105 ± 5  106 ± 4  103 ± 3  109 ± 6  113 ± 9 
Artery diameter LK 0.45 ± 0.02  0.45 ± 0.02  0.45 ± 0.02  0.45 ± 0.03  0.45 ± 0.02  0.46 ± 0.02
(cm) KL 0.47 ± 0.01  0.46 ± 0.01  0.47 ± 0.01  0.47 ± 0.01  0.47 ± 0.01  0.47 ± 0.01
FBF LK 86 ± 13 251 ± 37 231 ± 35 217 ± 33 214 ± 37 229 ± 40
(ml min−1) KL 87 ± 14 239 ± 18 246 ± 26 256 ± 28 217 ± 19 213 ± 13
FVC LK 85 ± 14 240 ± 37 220 ± 39 206 ± 35 203 ± 41 216 ± 43
(ml min−1 (100 mmHg−1)) KL 84 ± 11 229 ± 15 234 ± 23 246 ± 23 202 ± 19 189 ± 19
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Data are means ±s.e.m. Subjects displayed similar responses to both exercise and drug infusions for MAP, HR, FBF, and FVC. MAP, mean arterial pressure; HR, heart rate; FBF, forearm blood flow; FVC, forearm vascular conductance; LK, drug order where l-name was infused first, followed by ketorolac; KL, ketorolac was infused first, followed by l-name.

Forearm exercise responses to NOS inhibition followed by COX inhibition

Mean forearm blood flow responses to dynamic exercise are summarized in Fig. 2A. l-NAME reduced FBF (P < 0.0001), which remained reduced during the subsequent saline infusion. Infusion of ketorolac had no effect on the FBF response (P > 0.17; Fig. 2A). When FBF was corrected for blood pressure, the FVC data provided similar results. When FBF and FVC were expressed as a percentage of steady-state exercise, the effects of NOS inhibition were more striking, as were the lack of effects of COX inhibition (Fig. 2B).

Figure 2. Blood flow response to forearm exercise in older adults receiving nitric oxide synthase (NOS) followed by cyclooxygenase (COX) inhibition.

Figure 2

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Data are means ±s.e.m.A, forearm blood flow (FBF) response at rest and during each minute of exercise. Data are group mean responses (±s.e.m.) to l-NAME followed by ketorolac infusion in eight subjects. After reaching steady-state exercise, l-NAME infusion resulted in a reduction in FBF (P < 0.001) that did not change further with subsequent saline or ketorolac infusion (P = 0.82). B, data are FBF responses normalized to steady-state exercise, and show a similar pattern as FBF in A.

Forearm exercise responses to COX inhibition followed by NOS inhibition

Mean FBF responses to dynamic exercise are summarized in Fig. 3A. Ketorolac had no effect on the FBF response to exercise (P > 0.4), which remained stable over the subsequent saline infusion. l-NAME reduced FBF by ∼12% (P < 0.01) which remained reduced through to the end of exercise. When FBF was corrected for blood pressure, the FVC data provided similar results. When FBF and FVC were expressed as a percentage of steady-state exercise, the effects of NOS inhibition were more striking, as were the lack of effects of COX inhibition (Fig. 3B). There were no notable differences in the FBF or drug responses in the only older female, or in the two males taking a statin (Zocor).

Figure 3. Blood flow response to forearm exercise in older adults receiving COX followed by NOS inhibition.

Figure 3

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Data are means ±s.e.m.A, FBF response at rest and during each minute of exercise. Data are group mean responses (±s.e.m.) to ketorolac followed by l-NAME infusion in seven subjects. After reaching steady-state exercise, ketorolac infusion resulted no change in FBF (P = 0.17) that did not change further with subsequent saline infusion (P > 0.05), but decreased significantly with l-NAME infusion (P < 0.01). B, data are FBF responses normalized to steady-state exercise, and show a similar pattern as FBF in A.

Relative contributions of NO and PGs in young versus older adults

To better understand the changes in control mechanisms associated with ageing, we compared the present results of identical protocols (Schrage et al. 2004) used to assess the role of NO and PGs in young adults. All FBF responses were normalized as steady-state exercise FBF = 100%, and zero blood flow = 0%. As seen in Fig. 4, NOS inhibition in older humans reduced FBF by approximately 40% less than young adults (P < 0.02, Fig. 4). Next, COX inhibition reduced FBF in young, but not in older adults (P < 0.001). Lastly, in young subjects, the effects of l-NAME and ketorolac were independent of drug order, suggesting that NO and PGs act independently in regulating exercise hyperaemia in the skeletal muscle of younger humans. However, due to the lack of a significant change in FBF with ketorolac in ageing humans, we could not determine whether or not NO and PGs work independently in ageing muscle.

Figure 4. Comparison of relative changes in steady-state exercise FBF in response to NOS or COX inhibition.

Figure 4

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Data are means ±s.e.m. Summary of results in Figs 2 and 3 and previous work (Schrage et al. 2004) using FBF relative to steady-state exercise (=100%). For instance, if l-NAME reduced FBF to 80% of control exercise, this equalled a 20% decrease in percentage FBF. These data suggest ageing humans mediate exercise hyperaemia with less NO and loss of vasodilator PGs. *Significant difference between young and older adults, P < 0.02.

Discussion

This is the first study to test whether the relative contributions of vasodilator signals responsible for exercise hyperaemia are altered by the ageing process. When compared with our previous work in young adults, the novel findings of this study are that with ageing there is an approximately a 40% loss of the NO-mediated, and complete loss of prostaglandin-mediated contributions to exercise hyperaemia vasodilatation.

Many insights into the effects of ageing on skeletal muscle vasculature have been reported from animal models. For instance, senescent rats display altered exercise hyperaemia (Musch et al. 2004), impaired endothelium-dependent dilatation (Muller-Delp et al. 2002; Woodman et al. 2002, 2003; Spier et al. 2004), and altered eNOS expression (Woodman et al. 2002; Spier et al. 2004). While older humans exhibit lower exercise hyperaemia (Proctor et al. 1998; Beere et al. 1999; Poole et al. 2003; Lawrenson et al. 2004; Musch et al. 2004; Koch et al. 2005), and impaired endothelium-dependent dilatation (Taddei et al. 1995, 1998, 2001), whether similar changes in vascular control occur during exercise, and whether or not the changes are directly responsible for lower exercise hyperaemia, have not been tested in humans.

Numerous studies in young adults have tested the role of NO in exercise-mediated hyperaemia; however, most report that NO is not obligatory (Radegran & Saltin, 1999; Frandsen et al. 2001). Previous studies (Sheriff et al. 2000), in addition to work from our laboratory (Dyke et al. 1995; Schrage et al. 2004), suggest that NO is normally an important contributor to moderate exercise, such that NOS inhibition can reduce exercise hyperaemia by 20–30%. Importantly, no studies in humans have assessed whether the ageing process alters the role of NO or any vasodilating signal during exercise, but the fact that hyperaemia in older humans is also reduced by about 20% (Proctor et al. 1998; Beere et al. 1999; Poole et al. 2003; Lawrenson et al. 2004; Musch et al. 2004; Koch et al. 2005) is an intriguing parallel finding. One caveat to these observations is that our results stem from forearm exercise, and it remains unknown whether the observed age-related loss of NO and PG vasodilatation occurs in the exercising leg.

In contrast to young adults, steady-state FBF in older adults does not decrease to the same extent in response to l-NAME infusion (22 ± 3 versus 12 ± 3%, Fig. 4), suggesting an age-associated loss of NO-mediated vasodilatation during exercise. Less NO-mediated vasodilatation during exercise is consistent with the age-related decreases in acetylcholine responsiveness (Taddei et al. 1995, 2001), and could be the end result of several changes. First, expression of NOS may be reduced in the ageing limb. Next, any given amount of NO produced during exercise may be biologically inactivated by free-radical scavenging. Finally, the vascular smooth muscle of resistance arteries may be less responsive to NO.

Changes in human skeletal muscle NOS expression due to ageing have not been reported. Protein expression of eNOS is reported lower (Woodman et al. 2002) and higher (Spier et al. 2004) in arteries isolated from skeletal muscles of older animals. Since infusion of l-NAME into a resting forearm inhibits the vasodilator response to acetylcholine by at least 70% (F. A. Dinenno, unpublished observations), it is likely that we achieved significant inhibition of eNOS in the working muscle. An important consideration is that NO derived from neuronal NOS (nNOS) in contracting skeletal muscle may be a significant source of NO-mediated dilatation. Interestingly, no data exist on the effects of human ageing on skeletal muscle nNOS expression. Since l-NAME inhibits all forms of NOS, our results cannot differentiate whether ageing reduces the NO signal from nNOS or eNOS, or both. The role if inducible NOS (iNOS) in acute exercise hyperaemia is unknown, but we presume that the contribution of iNOS is negligible in healthy adults.

Many reports indicate ageing subjects are exposed to greater free radical production (Van Der Loo et al. 2000; Hamilton et al. 2001), which in turn diminishes vasodilatation from NO, due to formation of peroxynitrate (Van Der Loo et al. 2000). Even if NO production is normal in older adults, exposure to greater free radical levels may inactivate NO and limit normal NO-mediated vasodilatation. Consistent with this, intravenous infusion of high levels of vitamin C restored endothelial dysfunction (flow-mediated dilatation) in ageing males (Eskurza et al. 2004). In fact, peroxynitrate is a powerful vasoconstrictor signal, and peroxynitrate levels may be greater in older humans (Van Der Loo et al. 2000), since the pathways designed to handle free radicals (i.e. superoxide dismutase, catalase) are also diminished in ageing (Woodman et al. 2002). This idea remains to be tested in humans during exercise.

Vascular smooth muscle responsiveness to vasodilator signals might also be altered by ageing. This idea has primarily been tested with NO donors, such as sodium nitroprusside, and the majority of results suggest that sensitivity to NO is maintained (Muller-Delp et al. 2002; Woodman et al. 2002; Taddei et al. 1995) or decreased modestly (Newcomer et al. 2005). If the present subjects maintained their NO responsiveness, then our results are consistent with less NO production or inactivation by free radicals during exercise in the older humans. On the other hand, if responsiveness to NO was blunted in our older subjects, we may have underestimated the relative amount of NO produced during exercise. In any of these scenarios, future studies in humans will need to determine whether less NO-mediated dilatation is due to less NOS protein, less NO synthesis, and/or greater biological inactivation of NO.

The fact that ketorolac did not change FBF in older adults supports the idea that ageing evokes a shift in PG-mediated control of muscle blood flow (Fig. 4). One study concluded that PGs (vasodilating) do not play an obligatory role in controlling hyperaemia in humans during treadmill walking (Lang et al. 1997). In contrast, using the identical research design in the present study, acute inhibition of PGs with ketorolac during steady-state exercise transiently reduces blood flow ∼12% in young adults (Schrage et al. 2004). Together, work from our laboratory in young and older adults supports the concept of a shift away from PG-mediated vasodilatation. These results from exercising humans do not support our hypothesis that the balance of PGs has shifted toward a predominance of TXA2 during exercise (Taddei et al. 1997), since ketorolac did not increase the FBF responses to contractions. In fact, our results support only a loss of vasodilating PGs in ageing human muscle that is exercising. The effects of ageing on COX expression are not known in humans or animals, but if COX-1 function/expression is unaltered by ageing, then our results would be consistent with reduced PGI2 synthase expression. Alternatively, vascular smooth muscle responsiveness to PGI2 may also be decreased with ageing. Future studies in humans will need to determine whether ageing induces a shift in the biochemical cascade of the COX, PGI2 and TXA2 signalling. It should be noted that ketorolac is a non-specific COX inhibitor, thus we are currently unable to differentiate our results between COX-1 and COX-2 enzymes. The general consensus regarding COX is that COX-2 is inducible in inflammatory states like arthritis, and that COX-1 is the responsible enzyme for production of PGs. We are unaware of studies on the ageing effects on COX-2 expression in skeletal muscle. Regardless of whether ageing human muscles have altered COX-1 or COX-2 expression, the functional result is that ageing humans lose PG vasodilatation to mediate exercise hyperaemia.

Physiological perspectives

Several studies in humans, either using isolated limbs or whole-body exercise, support the concept that hyperaemic (or vascular conductance) responses to exercise are 10–20% lower in older subjects. Lower hyperaemia may be related to the loss of NO (from 22 to 12% relative contribution), and vasoconstriction in active muscles during sympathetic activation which is enhanced in exercising older humans by about 10% (Koch et al. 2003).

Greater vasoconstriction could also limit exercise hyperaemia. Indeed, ageing is also associated with chronic increases in sympathetic nerve activity and circulating norepinephrine (noradrenaline) levels. During exercise, data from several sources indicate (Koch et al. 2003; Dinenno et al. 2005) that ageing is associated with a greater sympathetic restraint of exercise-mediated metabolic vasodilatation. In young adults, the ability of sympathetic activation to restrain blood flow to active muscles is enhanced after combined treatment with l-NAME and ketorolac (Dinenno & Joyner, 2004). In other words, inhibition of NO and PGs in young adults essentially mimics the greater sympathetic vasoconstriction seen in older humans. Taken together with the present results, these findings suggest a loss of NO and (vasodilator) PGs in older adults may interact with augmented sympathetic vasoconstriction and explain reduced exercise hyperaemia with ageing. In the most simplistic sense, if we take a 10% loss of hypaeremia due to reduced NO signalling, and add that to a 10% greater reduction in hyperaemia due to enhanced sympathetic vasoconstriction, we might be able to explain a ∼20% reduction in exercise hyperaemia in observed in older humans.

Experimental considerations and limitations

An important limitation in our comparisons is the difference in baseline MAP. Since MAP was higher in older subjects, it is possible that some of the shift in NO and PGs may be due to mild hypertension in addition to increased age, and that we are unable to differentiate between the two in this design. However since the MAP in our older subjects was ∼11/22 mmHg lower than in hypertensive subjects studied by Taddei et al. 1995 (∼143/80 compared with ∼155/102 mmHg), it seems reasonable to suggest most of the current results are due to ageing. This highlights the important question of whether ageing humans who suffer from hypertension exhibit even greater loss of NO and PGs mediating exercise hyperaemia.

Delivering sufficient pharmacological inhibitors is always a concern in vascular studies. It is possible that the amounts of l-NAME and ketorolac did not completely inhibit NOS and COX, respectively, in the forearm. We think this is unlikely, because our forearm infusions were based on whole body infusions and delivered 2–5 times the whole-body dose, locally (Radegran & Saltin, 1999; Frandsen et al. 2001; Boushel et al. 2002). Further, since we infused these drugs during exercise instead of rest, we feel we vastly increased the likelihood that high levels of inhibitors had access to the resistance arteries engaged in exercise hyperaemia. That said, if the amounts of l-NAME and ketorolac did not completely inhibit the NOS and COX enzymes, then our data underestimate the relative contributions of NO and PGs in controlling exercise hyperaemia. We think this is unlikely, since our findings suggest ageing humans have less NO- and PG-mediated vasodilatation, supporting the idea that the high local delivery of drugs may be even more effective in inhibiting these pathways in older subjects.

The signals contributing to vascular control of muscle blood flow may vary between exercise intensities. We report age-associated reductions in exercise-mediated NO and PG signalling at moderate intensity dynamic forearm exercise. Previous work using combined inhibition of NO and PGs in the leg suggest the importance of these vasodilator signals increases as workload increases. Future studies will need to inhibit NOS and COX during higher relative exercise intensities in older humans to determine whether the age-related loss of NO and PGs contributes to greater differences in blood flow.

Three differences in experimental design between the current study and the study performed in young adults deserve mention. First, the current study used a newer ultrasound that allowed for simultaneous measures of diameter and velocity, improving our ability to track potential drug-induced changes in FBF. However, we are limited in comparing direct FBF between the two systems. We feel this does not limit our results, given that – due to absolute differences in workload, and therefore exercise FBF between males and females – data analysis required the use of normalized FBF to look at the relative roles of NO and PGs. Second, ageing subjects used precisely a 10% workload, whereas young adults used standard workloads that on average corresponded to 9–10% workload. Third, the current study utilized a dose of ketorolac twice that used in the young adults. We used this dose to ensure that the transient reduction in FBF caused by COX inhibition in previous study was not due to lack of complete COX inhibition (or a wash-out effect), versus a compensatory response to loss of PGs. So, the old subjects received a dose of ketorolac that was twice that given to the young. This approach further strengthens our findings of a loss of vasodilator PGs in ageing, as we show a lack of effect of ketorolac in ageing, when inhibition should be even more complete. Otherwise the study designs were identical.

The idea that hyperaemia could be limited by excessive vasoconstriction is not limited to norepinephrine or TXA2. Indeed, endothelin-1 (ET-1) levels are reported to be higher in ageing humans (Eskurza et al. 2004), and endothelin receptor antagonism has been shown to improve endothelial function (Ghiadoni et al. 2000). The reciprocal relationship with NO and ET-1 would support the idea that a less NO in ageing subjects might also lead to greater restraint of blood flow due to higher [ET-1]. We did not test for a difference in the contribution of ET-1 in our study, but given the small muscle mass, moderate exercise intensity and short duration, we suggest that ET-1 did not have a significant impact on our findings.

Observations are also emerging in this field that suggest the forearm circulation and leg circulations differ (Newcomer et al. 2004), and possibly the effects of ageing on these circulations (DeSouza et al. 2000; Proctor et al. 2005; Ridout et al. 2005). Since many of the studies reporting lower hyperaemia in ageing subjects used leg exercise, future studies will need to confirm the shift in NO and PGs in the exercising leg of young and older humans.

Conclusions

In summary, NOS inhibition reduces FBF by ∼12%, and COX inhibition does not alter FBF in older humans performing dynamic forearm exercise. These results are consistent with the concept of age-associated reduction of NO- and PG-mediated vasodilatation during exercise in older adults. These altered mechanisms may also explain why older humans demonstrate lower exercise hyperaemia and greater sympathetic restraint of blood flow to exercising muscles.

Acknowledgments

We are grateful to the superb subjects for their time and enthusiasm. Many thanks to Ruth A. Kraft for subject recruitment, and to Shelly K. Roberts, R.N., Karen P. Krucker, Christopher P. Johnson, Branton G. Walker, Tasha L. Pike and Madhuri Somaraju for technical assistance. Thanks to Sunni A. Barnes, PhD, from the Department of Biostatistics, for statistical consulting. Research support from the following grants is gratefully acknowledged: NIH grants HL-69692 (W.G.S.), HL-46493 (M.J.J.), and General Clinical Research Center RR-00585.

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