Ageing reduces the compensatory vasodilatation during hypoxic exercise: the role of nitric oxide

Non-technical summary

In young healthy humans, the combination of exercise and hypoxia produces a compensatory vasodilatation to ensure the maintenance of oxygen to the active muscles. We have previously demonstrated that nitric oxide (NO) is a major factor responsible for the compensatory vasodilatation. In this paper we show that the compensatory vasodilator response to hypoxic exercise is attenuated in older adults due to less NO signalling. These findings provide important information on the impact of ageing and the role of NO in the regulation of skeletal muscle blood flow during conditions of reduced oxygen availability.

Abstract

Abstract

We tested the hypotheses that (1) the compensatory vasodilatation in skeletal muscle during hypoxic exercise is attenuated in ageing humans and (2) local inhibition of nitric oxide (NO) synthesis in the forearm of ageing humans will have less impact on the compensatory dilatation during rhythmic exercise with hypoxia, due to a smaller compensatory dilator response. Eleven healthy older subjects (61 ± 2 years) performed forearm exercise (10% and 20% of maximum) during saline infusion (control) and NO synthase inhibition (N^G-monomethyl-l-arginine; l-NMMA) under normoxic and normocapnic hypoxic (80% arterial O₂ saturation) conditions. Forearm vascular conductance (FVC; ml min⁻¹ (100 mmHg)⁻¹) was calculated from forearm blood flow (ml min⁻¹) and blood pressure (mmHg). To further examine the effects of ageing on the compensatory vasodilator response to hypoxic exercise we compared the difference in ΔFVC (% change compared to respective normoxic exercise trial) between the older subjects (present study) and previously published data from an identical protocol in young subjects. During the control condition, the compensatory vasodilator response to hypoxia was similar between the old and young groups at 10% exercise (28 ± 6%vs. 40 ± 8%, P = 0.11) but attenuated at 20% exercise (14 ± 4%vs. 31 ± 6%, P < 0.05). l-NMMA during hypoxic exercise only blunted the compensatory vasodilator response in the young group (P < 0.05). Our data suggest that ageing reduces the compensatory vasodilator response to hypoxic exercise via blunted NO signalling.

Introduction

In young healthy humans submaximal exercise during hypoxia produces a compensatory vasodilatation relative to the same level of exercise under normoxic conditions (Rowell et al. 1986; Roach et al. 1999; Calbet et al. 2003; Wilkins et al. 2006, 2008; Casey et al. 2009, 2010). This compensatory vasodilatation during hypoxic exercise occurs to ensure an adequate oxygen delivery to the active muscles. We have previously demonstrated that nitric oxide (NO)-mediated mechanisms contribute to the compensatory vasodilatation at rest and during incremental hypoxic exercise (Casey et al. 2010). However, some evidence suggests that the forearm blood flow response to acute hypoxia at rest is inversely related to age (Kravec et al. 1972). Moreover, ageing has been reported to be associated with a lower exercise hyperaemia under normoxic conditions which may be due in part to reduced endothelial-derived NO and/or prostaglandins (Schrage et al. 2007; Crecelius et al. 2010). To our knowledge, no study has examined the effect of ageing on the vasodilator response during hypoxia exercise. Therefore, the aim of the current study was to investigate the compensatory vasodilator response to hypoxic exercise in healthy ageing humans. We hypothesized that older subjects would demonstrate (1) an attenuated compensatory vasodilator response compared to young healthy subjects and (2) inhibition of NO would have less impact on the compensatory dilatation during hypoxic exercise in ageing humans, due to blunted NO signalling in the older subjects.

Methods

Subjects

A total of 11 older healthy subjects (6 female and 5 male; 55–70 years) volunteered to participate in the study. Subjects completed written informed consent and underwent a standard screening and were healthy, non-obese (BMI < 28 kg m⁻²), non-smokers and were not taking any vasoactive medications. Two subjects were taking bisphosphonates to prevent and/or treat osteoporosis, two subjects were taking aspirin (withheld for 1 week prior to study), and one subject was taking Synthroid to treat hypothyroidism (withheld 3 days prior to study). Subjects reported normal daily activities but no regular physical training. Seven subjects reported taking a daily vitamin. Studies were performed after an overnight fast and refraining from exercise and caffeine for at least 24 h. All female subjects were postmenopausal and were not taking any form of hormone replacement therapy. All study protocols were approved by the Institutional Review Board and were performed according to the Declaration of Helsinki.

Forearm exercise

Subjects performed rhythmic forearm exercise with a handgrip device by the non-dominant arm at 10 and 20% of each subject's maximal voluntary contraction (MVC, mean 34 ± 3 kg, range 21–55 kg), determined at the beginning of each experiment. The weight was lifted 4–5 cm over a pulley at a duty cycle of 1 s contraction and 2 s relaxation (20 contractions per minute) using a metronome to ensure correct timing. The average weight used for forearm exercise was 3.4 ± 0.3 and 6.9 ± 0.6 kg for 10 and 20% MVC, respectively.

Arterial and venous catheterization

A 20 gauge, 5 cm (Model RA-04020, Arrow International, Reading, PA, USA) catheter was placed in the brachial artery of the exercising arm under aseptic conditions after local anaesthesia (2% lidocaine) for administration of study drugs and to obtain arterial blood samples. The catheter was connected to a three-port connector in series, as previously described in detail (Dietz et al. 1994). One port was linked to a pressure transducer positioned at heart level (Model PX600F, Edwards Lifescience, Irvine, CA, USA) to allow measurement of arterial pressure and was continuously flushed (3 ml h⁻¹) with heparinized saline with a stop-cock system to enable arterial blood sampling. The remaining two ports allowed arterial drug administration. Deep venous blood was sampled via an 18 gauge, 3 cm catheter inserted retrogradely in an antecubital vein (Joyner et al. 1992).

Heart rate and systemic blood pressure

Heart rate was recorded via continuous 3-lead ECG. A pressure transducer connected to the arterial catheter measured beat-to-beat blood pressure (Cardiocap/5, Datex-Ohmeda, Louisville, CO, USA)

Forearm blood flow

Brachial artery mean blood velocity and brachial artery diameter were determined with a 12 MHz linear-array Doppler probe (Model M12L, Vivid 7, General Electric, Milwaukee, WI, USA). Brachial artery blood velocity was measured throughout each condition with a probe insonation angle previously calibrated to 60 deg. Brachial artery diameter measurements were obtained at end diastole between contractions during steady-state conditions. Forearm blood flow (FBF) was calculated as the product of mean blood velocity (cm s⁻¹) and brachial artery cross-sectional area (cm²) and expressed as millilitres per minute (ml min⁻¹).

Systemic hypoxia

The hypoxic conditions involved a self-regulating partial rebreathe system that effectively clamps end-tidal CO₂ at baseline levels despite large changes in minute ventilation during hypoxia (Banzett et al. 2000; Weisbrod et al. 2001; Wilkins et al. 2006, 2008). The level of inspired O₂ was titrated to achieve an arterial O₂ saturation (assessed via pulse oximetry) of ∼80%. The amount of O₂ provided in the inspiratory gas was controlled by mixing N₂ with medical air via an anaesthesia gas blender. Carbon dioxide concentrations were monitored (Cardiocap/5) and ventilation was assessed via a turbine (Model VMM-2a, Interface Associates, Laguna Nigel, CA, USA).

Pharmacological infusions

N^G-monomethyl-l-arginine (l-NMMA; NOS inhibitor; Bachem, Switzerland) was infused at a loading dose of 5 mg min⁻¹ for 5 min and then at a maintenance dose of 1 mg min⁻¹ for the remainder of the study. Acetylcholine (ACh; a non-specific muscarinic agonist) was infused intra-arterially at 2.0, 4.0 and 8.0 μg (dl forearm volume)⁻¹ min⁻¹ for 2 min each to determine the endothelium-dependent vasodilator response before and after NOS inhibition with l-NMMA.

Blood gas and catecholamine analysis

Brachial artery and deep venous blood samples were analysed with a clinical blood gas analyser (Bayer 855 Automatic Blood Gas System, Boston, MA, USA) for partial pressures of O₂ and CO₂ ( and ), pH and O₂ saturation (). Arterial and venous O₂ content was calculated using the measured and values. Arterial and venous plasma catecholamine (adrenaline and noradrenaline) levels were determined by HPLC with electrochemical detection.

Experimental protocol

A schematic diagram of the general experimental design is illustrated in Fig. 1. Each subject completed a resting baseline condition followed by rhythmic forearm exercise at 10% which was immediately increased to 20% MVC during normoxia and normocapnic hypoxia. Exposure to normoxia or hypoxia was alternated and randomized. Resting baseline and forearm exercise (normoxia and hypoxia) were performed during a control (saline) infusion, followed by l-NMMA infusion. Due to the long half-life of l-NMMA, study drugs were always administered in the same order. A rest period of at least 20 min was allowed between conditions under each drug infusion. During each infusion and each condition (normoxia and hypoxia) arterial and venous blood was sampled at rest and at steady-state exercise for blood gas analysis and plasma catecholamine determination.

Figure 1. Schematic diagram of experimental protocol.

Measurements were obtained at baseline and incremental exercise (10% and 20% of maximum) under normoxic and hypoxic conditions. Both normoxic and hypoxic trials were performed during control (saline) and l-NMMA infusions. ACh, acetylcholine.

Data analysis and statistics

Data were collected at 200 Hz, stored on a computer and analysed off-line with signal processing software (WinDaq, DATAQ Instruments, Akron, OH, USA). Mean arterial pressure (MAP) was determined from the brachial artery pressure waveform and heart rate was determined from the electrocardiogram. Values for minute ventilation, end-tidal CO₂ and O₂ saturation (pulse oximetry) were determined by averaging minutes 4 and 5 at rest and during each exercise intensity. Forearm blood flow and arterial pressure were determined by averaging values from the 4th minute at rest and each exercise bout. Forearm vascular conductance (FVC) was calculated as (FBF/arterial pressure) × 100 and expressed as ml min⁻¹ (100 mmHg)⁻¹. The change (Δ) in FBF and FVC due to hypoxia at rest and due to hypoxic exercise (10 and 20%) was calculated by subtracting resting FBF and FVC during normoxia at each drug infusion (saline or l-NMMA) from FBF and FVC values obtained during hypoxia (at rest and during exercise) within each drug infusion. Blood gas and catecholamine values were determined from blood samples obtained during normoxia and hypoxia with each drug infusion. Arteriovenous oxygen difference during forearm exercise was calculated by the difference between arterial and venous O₂ content. Venous–arterial noradrenaline difference (v–a noradrenaline difference) was calculated by the difference between venous and arterial noradrenaline concentrations.

All values are expressed as means ± SEM. To determine the effect of hypoxia with each pharmacological treatment, differences in absolute FBF and FVC at rest (normoxia and hypoxia) and differences in ΔFBF and ΔFVC at rest and during each exercise intensity (normoxia and hypoxia) were determined via repeated measures analysis of variance (ANOVA). Haemodynamic, respiratory, blood gases and catecholamine variables were compared via repeated measures ANOVA to detect differences between responses during hypoxia at rest and during exercise across pharmacological infusions. Appropriate post hoc analysis determined where statistical differences occurred. Statistical difference was set a priori at P < 0.05.

To further examine the effects of ageing on the compensatory vasodilator response to hypoxic exercise we compared via ANOVA the difference in ΔFVC (% change compared to the respective normoxic exercise trial) between the older subjects (present study) and previously published data from an identical protocol in young subjects (6 males and 6 females; 26 ± 2 years) (Casey et al. 2010). These comparisons were made during the control saline trials as well as the l-NMMA trials to test our second hypothesis that inhibition of NO would have less impact on the compensatory dilatation during hypoxic exercise in ageing humans, due to blunted NO signalling in the older subjects.

Results

All 11 subjects completed the study. The subjects were 61 ± 2 years of age, 171 ± 3 cm in height and weighed 74 ± 4 kg (BMI: 25 ± 1 kg m⁻²). Their average total, LDL and HDL cholesterol and triglycerides were 191 ± 13, 117 ± 12, 55 ± 4 and 95 ± 9 mg dl⁻¹, respectively.

Systemic haemodynamic and respiratory responses (Table 1)

Table 1.

Systemic haemodynamic and respiratory responses at rest and with incremental exercise during normoxia and hypoxia under control (saline) and NOS inhibition (l-NMMA)

	Normoxia			Hypoxia
	Rest	10%	20%	Rest	10%	20%
Older adults (n = 11)
Control (saline)
Mean arterial pressure (mmHg)	102 ± 4	106 ± 4	109 ± 4†	103 ± 4	106 ± 5	107 ± 4
Heart rate (beats min−1)*	66 ± 3	70 ± 4	72 ± 4†	78 ± 3	79 ± 3	81 ± 5
Minute ventilation (l min−1; BTPS)*	7.2 ± 0.6	7.1 ± 0.5	9.6 ± 1.1	11.7 ± 1.2	14.5 ± 1.4	16.9 ± 2.3†
End-tidal CO2 (mmHg)	36 ± 1	36 ± 1	37 ± 1	36 ± 1	36 ± 1	36 ± 1
l-NMMA
Mean arterial pressure (mmHg)	105 ± 4	108 ± 4	111 ± 5†	106 ± 4	109 ± 5	111 ± 4†
Heart rate (beats min−1)*	66 ± 4	69 ± 4	73 ± 4†	81 ± 4	82 ± 4	84 ± 4
Minute ventilation (l min−1; BTPS)*	8.3 ± 1.0	8.6 ± 0.8	11.0 ± 1.3	14.7 ± 1.5	18.1 ± 1.9†‡	21.1 ± 2.2†‡
End-tidal CO2 (mmHg)	37 ± 1	37 ± 1	37 ± 1	36 ± 1	36 ± 1	36 ± 1
Young adults (n = 12)
Control (saline)
Mean arterial pressure (mmHg)	89 ± 2	90 ± 2	93 ± 2	88 ± 2	90 ± 2	91 ± 3
Heart rate (beats min−1)*	62 ± 3	66 ± 3†	68 ± 3	73 ± 4	77 ± 5†	80 ± 5
Minute ventilation (l min−1; BTPS)*	6.3 ± 0.7	8.7 ± 0.7	9.1 ± 0.8	10.1 ± 1.7	13.7 ± 2.7†	14.7 ± 2.1
End-tidal CO2 (mmHg)	37 ± 1	36 ± 1	37 ± 1	36 ± 1	36 ± 1	37 ± 1
l-NMMA
Mean arterial pressure (mmHg)	91 ± 2	92 ± 2	95 ± 2	92 ± 2	92 ± 2	93 ± 2
Heart rate (beats min−1)*	62 ± 3	65 ± 3	68 ± 3†	76 ± 5	81 ± 5†	82 ± 5
Minute ventilation (l min−1; BTPS)*	8.3 ± 1.0	10.2 ± 1.3	11.9 ± 1.5†	14.4 ± 3.1‡	16.5 ± 2.9	19.0 ± 3.0†‡
End-tidal CO2 (mmHg)	37 ± 1	37 ± 1	37 ± 1	36 ± 1	37 ± 1	37 ± 1

Values are means ± SEM.

^*P < 0.05 vs. normoxia

^†P < 0.05 vs. rest

^‡P < 0.05 vs. Control.

Data for young adults are from previously published work (Casey et al. 2010). BTPS, Body temperature and pressure, saturated.

The group data (mean ± SEM) for haemodynamic and respiratory responses due to combined forearm exercise and hypoxia with each drug infusion are presented in Table 1. As expected, heart rate and minute ventilation increased as a consequence of systemic hypoxia and incremental forearm exercise. Mean arterial pressure was elevated during normoxic and hypoxic exercise at 20% MVC but did not differ between drug conditions (saline vs.l-NMMA). By design, end-tidal CO₂ was maintained throughout rest and incremental exercise under both normoxic and hypoxic conditions for both protocols.

Forearm exercise (Table 2)

Table 2.

Forearm haemodynamics at rest and with incremental exercise during normoxia and hypoxia under control (saline) and NOS inhibition (l-NMMA)

				Δ from normoxia rest
	Rest	10%	20%	10%	20%
Older adults (n = 11)
Forearm blood flow (ml min−1)
Control (saline)
Normoxia	66 ± 7	213 ± 23†	338 ± 44†	147 ± 18	272 ± 40
Hypoxia	79 ± 11	255 ± 30*†	367 ± 49*†	188 ± 26*	300 ± 44
l-NMMA
Normoxia	53 ± 7	191 ± 22†	313 ± 35†	136 ± 20	258 ± 33
Hypoxia	61 ± 10	217 ± 30†‡	340 ± 45†‡	164 ± 24*‡	288 ± 41
Forearm vascular conductance (ml min−1 100 mmHg−1)
Control (saline)
Normoxia	65 ± 7	200 ± 20†	309 ± 38†	135 ± 16	244 ± 34
Hypoxia	76 ± 9	240 ± 27*†	340 ± 46*†	175 ± 23*	275 ± 40
l-NMMA
Normoxia	53 ± 6	178 ± 20†	286 ± 27†	126 ± 19	233 ± 26
Hypoxia	56 ± 8	199 ± 24†‡	312 ± 39*†‡	146 ± 20‡	259 ± 36
Young adults (n = 12)
Forearm blood flow (ml min−1)
Control (saline)
Normoxia	55 ± 5	205 ± 22†	329 ± 35†	150 ± 17	274 ± 29†
Hypoxia	68 ± 8*	245 ± 27*†	388 ± 42*†	190 ± 22*	333 ± 37*†
l-NMMA
Normoxia	41 ± 4‡	174 ± 23†	334 ± 44†	131 ± 18	291 ± 38†
Hypoxia	44 ± 5‡	202 ± 24†‡	341 ± 38†‡	161 ± 21*‡	300 ± 35†‡
Forearm vascular conductance (ml min−1 100 mmHg−1)
Control (saline)
Normoxia	62 ± 6	225 ± 22†	354 ± 34†	163 ± 17	292 ± 28†
Hypoxia	76 ± 9*	273 ± 28*†	429 ± 39*†	211 ± 23*	367 ± 34*†
l-NMMA
Normoxia	44 ± 5‡	187 ± 23†	349 ± 41†	140 ± 17	302 ± 35†
Hypoxia	49 ± 5‡	219 ± 24†‡	362 ± 37†‡	175 ± 20*‡	318 ± 32†‡

Values are means ± SEM.

^*P < 0.05 vs. normoxia

^†P < 0.05 vs. rest

^‡P < 0.05 vs. Control.

Data for young adults are from previously published work (Casey et al. 2010).

Presented in Table 2 are group data (mean ± SEM) forearm haemodynamics at rest and with increasing exercise intensity during control saline and l-NMMA infusion. Although not significant, resting FBF and FVC during systemic hypoxia tended to be higher than normoxia under the control (saline) conditions (P = 0.09 and 0.08, respectively). The absolute FBF and FVC to incremental hypoxic exercise were higher compared to normoxic exercise of the same intensity during the control saline infusion (main effect of hypoxia; P < 0.05; Table 2). However, the change (Δ) in FBF and FVC (relative to normoxic baseline values) to incremental hypoxic exercise was only greater than normoxic exercise during the 10% trials (P < 0.05; Fig. 2A and B), with no difference in ΔFBF and ΔFVC observed during the 20% trials (P = 0.22 and 0.12, respectively; Fig. 2C and D).

Figure 2. Change (Δ) in forearm blood flow (FBF) and forearm vascular conductance (FVC) due to hypoxic exercise during saline (control) and l-NMMA administration.

At 10% or 20% forearm exercise, NO synthase inhibition (l-NMMA) reduced forearm blood flow (A and C) and vascular conductance (B and D) compared to control (saline) during hypoxic exercise. ^†P < 0.05 vs. normoxia. *P < 0.05 vs. control (saline).

Intra-arterial administration of l-NMMA did not significantly decrease normoxic baseline (resting) blood flow below values observed during control saline infusion (P = 0.18). Additionally, l-NMMA infusion did not reduce absolute FBF or FVC (Table 2) or the ΔFBF or ΔFVC during normoxic exercise at 10 and 20% MVC compared to control saline (Fig. 2A–D). Although there was a trend for a reduced FBF and FVC during hypoxia at rest following l-NMMA administration it did not reach significance (P = 0.08 and 0.06, respectively; Table 2). Infusion of l-NMMA decreased absolute FBF and FVC (Table 2) with hypoxic exercise at 10% MVC (P < 0.05) and at 20% MVC (P < 0.05) compared to control saline infusions. However, the ΔFBF and ΔFVC with hypoxic exercise was only decreased during l-NMMA at 10% MVC compared to control saline infusions (Fig. 2A–D).

Compensatory vasodilatation in young versus older adults

To better understand the compensatory vasodilator responses to hypoxic exercise associated with ageing, we compared the present results to our previously published data in young adults (Casey et al. 2010). As seen in Fig. 3 the hypoxic compensatory vasodilatation (%) during control saline conditions tended to be lower in older adults at 10% MVC (P = 0.11) and was substantially lower at 20% MVC (P < 0.05). In young adults l-NMMA reduced the compensatory vasodilator response to hypoxic exercise at 10 and 20% MVC. However, l-NMMA had no effect on the compensatory vasodilatation in older adults (P = 0.96 and P = 0.87 for 10 and 20% MVC, respectively).

Figure 3. Impact of ageing on the compensatory vasodilator response (% change in ΔFVC compared to respective normoxic exercise condition) to hypoxic exercise.

Older adults tended to have an attenuated compensatory vasodilator response at 10% MVC (P = 0. 1) during control saline trials, whereas at 20% MVC the compensatory vasodilatation was substantially lower with ageing. l-NMMA significantly reduced the compensatory vasodilatation in young adults at both exercise intensities. However, there was no difference in the compensatory vasodilatation between control and l-NMMA trails in the older adults. Data for young adults is from previously published work (Casey et al. 2010). *P < 0.05 vs. control (saline); †P < 0.05 vs. young group.

Vasodilator responses to exogenous acetylcholine

The change in FVC increased incrementally with each dose of ACh (2.0, 4.0 and 8.0 μg (dl forearm volume)⁻¹ min⁻¹) in the older adults (P < 0.05; Fig. 4). l-NMMA administration did not reduce the vasodilator response compared to the saline trials in the older adults (P = 0.27–0.38). When compared to our previously published data in young adults (Casey et al. 2010), the vasodilator responses to ACh under saline (control) conditions did not differ between age groups (P = 0.15). However, the vasodilator responses were substantially lower at all three doses of ACh in the presence of l-NMMA in the young adults (P < 0.01; Fig. 4).

Figure 4. Vasodilator response to exogenous acetylcholine (ACh) during saline and l-NMMA administration in young and older adults.

The change (Δ) in forearm vascular conductance (FVC) in response to increasing doses of ACh were not significantly different between young and older adults during the saline (P = 0. 5) or l-NMMA trials (P = 0.38). However, l-NMMA blunted the vasodilator responses (ΔFVC) to ACh at all three doses in the young adults. Data for young adults is from previously published work (Casey et al. 2010). *P < 0.01 vs. control (saline) for young adults.

Blood gases (Table 3)

Table 3.

Arterial and venous blood gas responses at rest and with incremental exercise during normoxia and hypoxia under control (saline) and NOS inhibition (l-NMMA)

	Normoxia			Hypoxia
	Rest	10%	20%	Rest	10%	20%
Older adults (n = 11)
Control (saline)
(%)*	97 ± 0	96 ± 0	96 ± 0	81 ± 1	80 ± 1	80 ± 1
(Torr)*	100 ± 2	97 ± 2	96 ± 2	46 ± 1	45 ± 1	46 ± 1
(Torr)*	37 ± 2	25 ± 1†	27 ± 2	31 ± 1	22 ± 1†	23 ± 1
Arterial O2 content (ml l−1)*	170 ± 5	176 ± 6	177 ± 6	143 ± 5	145 ± 4	149 ± 5
Venous O2 content (ml l−1)*	122 ± 10	76 ± 8†	79 ± 10	104 ± 9	68 ± 7†	64 ± 4
a–v O2 (ml l−1)*	48 ± 7	100 ± 5	99 ± 7	39 ± 5	77 ± 5	85 ± 3
O2 consumption (ml min−1)	3.4 ± 0.4	21.0 ± 2.3†	33.4 ± 5.1†	2.7 ± 0.4	19.2 ± 2.4†	31.1 ± 4.1†
l-NMMA
(%)*	96 ± 0	96 ± 0	97 ± 0	81 ± 1	80 ± 1	81 ± 1
(Torr)*	95 ± 2	95 ± 2	97 ± 1	47 ± 0	46 ± 1	46 ± 1
(Torr)	32 ± 2	22 ± 1†	24 ± 1	26 ± 2*‡	19 ± 1‡	21 ± 1
Arterial O2 content (ml l−1)*	172 ± 6	171 ± 6	174 ± 6	146 ± 5	146 ± 5	150 ± 5
Venous O2 content (ml l−1)*	104 ± 11	66 ± 6†	66 ± 4	87 ± 11‡	55 ± 7†	57 ± 3
a–v O2 (ml l−1)	67 ± 9	105 ± 6†	108 ± 5	60 ± 8‡	91 ± 6†	94 ± 5
O2 consumption (ml min−1)	3.2 ± 0.4	19.9 ± 2.7†	33.4 ± 3.7†	3.1 ± 0.5	18.6 ± 2.6†	31.0 ± 3.8†
Young adults (n = 12)
Control (saline)
(%)*	97 ± 0	97 ± 0	97 ± 0	82 ± 0	82 ± 0	83 ± 0
(Torr)*	107 ± 3	107 ± 4	109 ± 3	48 ± 1	48 ± 1	49 ± 1
(Torr)*	38 ± 2	26 ± 1†	27 ± 1	30 ± 1	22 ± 1†	23 ± 1
Arterial O2 content (ml l−1)*	171 ± 5	173 ± 5	172 ± 5	144 ± 4	147 ± 4	149 ± 4
Venous O2 content (ml l−1)*	118 ± 8	68 ± 4†	72 ± 4	95 ± 7	58 ± 4†	59 ± 3
a–v O2 (ml l−1)*	53 ± 6	105 ± 4†	100 ± 3	49 ± 6	88 ± 4†	90 ± 3
O2 consumption (ml min−1)	3.1 ± 0.6	21.9 ± 2.9†	33.6 ± 4.0†	3.6 ± 0.5	21.6 ± 2.5†	35.2 ± 4.3†
l-NMMA
(%)*	97 ± 0	97 ± 0	97 ± 0	82 ± 0	81 ± 1	82 ± 1
(Torr)*	107 ± 3	106 ± 3	109 ± 3	47 ± 1	46 ± 2	49 ± 1
(Torr)*	34 ± 3	24 ± 1†	28 ± 1†	25 ± 1‡	20 ± 0†‡	23 ± 0†
Arterial O2 content (ml l−1)*	173 ± 5	173 ± 5	176 ± 5	143 ± 4	141 ± 5	148 ± 4
Venous O2 content (ml l−1)*	105 ± 10	68 ± 7†	80 ± 7†	67 ± 5‡	51 ± 2†	61 ± 3†
a–v O2 (ml l−1)	68 ± 9	106 ± 7†	95 ± 6	76 ± 4‡	91 ± 5*†	87 ± 4
O2 consumption (ml min−1)	3.8 ± 0.4	18.1 ± 1.6†	32.3 ± 3.5†	3.4 ± 0.4	18.1 ± 2.7†‡	29.4 ± 4.0†‡

Values are means ± SEM.

^*P < 0.05 vs. normoxia

^†P < 0.05 vs. rest

^‡P < 0.05 vs. control.

Data for young adults are from previously published work (Casey et al. 2010).

By design, systemic hypoxia reduced arterial oxygen saturation, arterial and arterial oxygen content (P < 0.01 for all) at rest and with incremental forearm exercise during both drug trials (saline or l-NMMA). Acute hypoxia also decreased venous oxygen content during each drug infusion (P < 0.05). Under control (saline) conditions the lower arterial and venous oxygen content during hypoxic exercise led to a lower a–v oxygen difference compared to normoxic exercise (P < 0.05). However, the a–v oxygen difference during hypoxic exercise with l-NMMA did not differ to those observed during normoxic exercise. Forearm oxygen consumption increased similarly with normoxic and hypoxic exercise at 10 and 20% MVC during control saline infusion (Table 3). During l-NMMA administration, forearm oxygen consumption also increased with normoxic and hypoxic exercise. Forearm oxygen consumption was not different between drug conditions (control vs.l-NMMA) for either the normoxic or hypoxic trials.

Catecholamines (Table 4)

Table 4.

Adrenaline and noradrenaline at rest and incremental exercise during normoxia and hypoxia under control (saline) and NOS inhibition (l-NMMA)

	Normoxia			Hypoxia
	Rest	10%	20%	Rest	10%	20%
Older adults (n = 11)
Control (saline)
Arterial noradrenaline (pg ml−1)*	220 ± 25	247 ± 30	263 ± 25†	253 ± 24	284 ± 19	314 ± 30†
Venous noradrenaline (pg ml−1)*	359 ± 33	326 ± 36	328 ± 30	437 ± 49	385 ± 27	383 ± 33†
v–a noradrenaline difference (pg ml−1)*	138 ± 17	79 ± 13†	66 ± 9†	184 ± 35	101 ± 12†	79 ± 11†
Arterial adrenaline (pg ml−1)*	63 ± 7	67 ± 9	70 ± 11	96 ± 9	118 ± 16	171 ± 49†
Venous adrenaline (pg ml−1)	45 ± 11	51 ± 8	71 ± 12	60 ± 15	95 ± 16*	149 ± 41*†
l-NMMA
Arterial noradrenaline (pg ml−1)*	207 ± 19	223 ± 18	271 ± 19†	251 ± 20	277 ± 21	318 ± 22†
Venous noradrenaline (pg ml−1)*	331 ± 34	315 ± 30	352 ± 25	428 ± 38	406 ± 39	410 ± 28
v–a noradrenaline difference (pg ml−1)	124 ± 21	92 ± 13	81 ± 11†	178 ± 24*	129 ± 20*†	92 ± 11†
Arterial adrenaline (pg ml−1)*	74 ± 13	82 ± 14‡	88 ± 13‡	99 ± 11	127 ± 17	181 ± 51†
Venous adrenaline (pg ml−1)	57 ± 13	70 ± 12‡	79 ± 14	71 ± 15	88 ± 13	152 ± 43*†
Young adults (n = 12)
Control (saline)
Arterial noradrenaline (pg ml−1)*	108 ± 12	119 ± 13	136 ± 13†	124 ± 11	132 ± 10	141 ± 12†
Venous noradrenaline (pg ml−1)	161 ± 12	164 ± 15	182 ± 20	193 ± 9	176 ± 9	176 ± 11
v–a noradrenaline difference (pg ml−1)	45 ± 8	36 ± 10	36 ± 17	64 ± 10	40 ± 5	30 ± 6
Arterial adrenaline (pg ml−1)*	31 ± 4	35 ± 7	47 ± 7	50 ± 8	64 ± 8	79 ± 12
Venous adrenaline (pg ml−1)	15 ± 2	27 ± 4	33 ± 5	20 ± 5	47 ± 6*†	66 ± 11*†
l-NMMA
Arterial noradrenaline (pg ml−1)*	125 ± 13	128 ± 15	145 ± 17	118 ± 12	136 ± 15	150 ± 18†
Venous noradrenaline (pg ml−1)	174 ± 16	158 ± 17	178 ± 18	182 ± 11	177 ± 12	190 ± 15
v–a noradrenaline difference (pg ml−1)	43 ± 10	24 ± 5†	25 ± 4	54 ± 11	31 ± 6†	28 ± 5
Arterial adrenaline (pg ml−1)*	31 ± 4	40 ± 6	47 ± 7	51 ± 9	76 ± 13	86 ± 12
Venous adrenaline (pg ml−1)	19 ± 2	37 ± 5†	48 ± 7†	23 ± 5	62 ± 9*†	76 ± 10*†

Values are means ± SEM.

^*P < 0.05 vs. normoxia

^†P < 0.05 vs. rest

^‡main effect of drug, P < 0.05 vs. control.

Data for young adults is from previously published work (Casey et al. 2010).

Systemic hypoxia increased venous and arterial adrenaline during control saline and l-NMMA infusions (P < 0.05). Venous and arterial adrenaline increased with higher intensity exercise (20% MVC) under hypoxic conditions during control saline and l-NMMA infusions (P < 0.05). Additionally, systemic hypoxia increased venous and arterial noradrenaline both at rest and during incremental exercise.

Young vs. older adult comparisons

Tables 1–4 contain data from our previous study in young adults (Casey et al. 2010). The inclusion of the data from the young adults in the tables is to make comparisons between age groups across trials more straightforward. Aside from the primary aim of the study (age-related differences in hypoxic FBF and FVC; Fig. 3 and Table 2), we would like to point out that MAP was greater at rest and during exercise under all conditions in the older group (P < 0.01). This is consistent with several of our previous exercise studies (Proctor et al. 1998; Dinenno et al. 2005; Schrage et al. 2007).

Discussion

This is the first study to examine whether ageing alters the compensatory vasodilator response to hypoxic exercise. Our primary findings are (1) compensatory vasodilatation during moderate intensity (20% MVC) hypoxic exercise is attenuated in older adults and (2) this attenuated response is probably due to blunted NO signalling. These conclusions are supported by the fact that older adults did not demonstrate a significant difference in ΔFBF and ΔFVC during hypoxic exercise at 20% MVC compared to respective normoxic exercise conditions. Moreover, when compared to our previous study using an identical protocol in young adults (Casey et al. 2010), the compensatory vasodilator response (% change in ΔFVC compared to respective normoxic exercise trial) was substantially less with ageing (Fig. 3). Additionally, inhibition of NO failed to reduce the compensatory vasodilatation during hypoxic exercise in ageing humans, thus suggesting a blunted NO signalling in older adults.

Ageing is associated with a number of changes in arterial function and structure that are thought to compromise muscle blood flow or alter its regulation during dynamic exercise (Proctor & Parker, 2006). Along these lines, leg blood flow has been shown to be attenuated during normoxic exercise at submaximal and maximal workloads in older adults (Proctor et al. 1998, 2003; Beere et al. 1999; Poole et al. 2003; Lawrenson et al. 2004). However, this finding has not been consistent in the human forearm (Jasperse et al. 1994; Donato et al. 2006; Kirby et al. 2009). In the current study the older subjects demonstrated similar forearm blood flow and conductance responses to submaximal normoxic exercise as the younger comparison group used from our previously published study that utilized an identical protocol (Casey et al. 2010). Since NO has been reported not to be obligatory (Green et al. 2005; Martin et al. 2006; Casey & Joyner, 2009; Casey et al. 2010) or contribute minimally (Schrage et al. 2004) in forearm exercise-mediated hyperaemia under normoxic conditions in young adults we were not surprised by the similar responses between age groups.

Although the increase in blood flow to contracting forearm muscles during normoxic exercise appears to be preserved in older adults, we confirmed our primary hypothesis that there is a shift towards an impaired vasodilator response during exercise under conditions of systemic hypoxia. Additionally, the lack of change in compensatory vasodilatation following administration of l-NMMA suggests that the impaired vasodilator response with ageing is due to blunted NO signalling and is in agreement with our secondary hypothesis. The attenuated NO-mediated vasodilatation during hypoxic exercise could be due to several changes related to the production, activity and/or action of NO. First, there is some evidence that eNOS protein expression is lower in arteries of older compared with young rodents (Csiszar et al. 2002; Woodman et al. 2002), although this is not consistent across studies and appears to be dependent on the type of artery studied (van der Loo et al. 2000; Spier et al. 2004). However, recent evidence suggests that eNOS protein expression is not reduced in vascular endothelial cells of older men (Donato et al. 2009). Second, age-related increases in free radical scavenging can inactivate NO (via formation of peroxynitrite) (van der Loo et al. 2000) and possibly blunt NO-mediated vasodilatation during exercise. In this context, intra-arterial infusion of the anti-oxidant ascorbic acid has been shown to improve blood flow to the contracting forearm in older men and women during normoxic exercise (Kirby et al. 2009). Lastly, it is possible that the older subjects in the present study had altered vascular smooth muscle responsiveness to NO or other vasodilator signals. Unfortunately, we did not directly (i.e. infusion of sodium nitroprusside) test the responsiveness of the vascular smooth muscle in the present study. However, previous studies have demonstrated that sensitivity to NO is maintained in older adults (Taddei et al. 1996, 2000; Kirby et al. 2010). Taken together, if NO responsiveness was maintained in the present study, less NO production and/or a pronounced inactivation via free radicals potentially explain the blunted compensatory vasodilatation during hypoxic exercise in older adults.

Experimental considerations

The attenuated compensatory vasodilator response during hypoxic exercise in the older adults could be a result of a greater sympathetic vasoconstrictor restraint. Both ageing and hypoxia can have profound effects on the sympathetic nervous system. First, ageing is associated with increased basal sympathetic nerve activity and circulating noradrenaline levels (Sundlof & Wallin, 1978; Esler et al. 1995; Narkiewicz et al. 2005). Consistent with these previous reports, the older subjects in the current study demonstrated 2-fold higher venous noradrenaline levels at rest and during exercise than young adults from our previous study (Casey et al. 2010). Ageing is also associated with impaired modulation of postjunctional α-adrenergic vasoconstriction in contracting muscles (Dinenno et al. 2005). Taken together, the potential for an enhanced sympathetic restraint of muscle blood flow during hypoxic exercise in older adults exists. However, if a reduced functional sympatholysis occurred in the older adults there would probably have also been an attenuated blood flow during normoxic exercise compared to the young adults from our previous study (Casey et al. 2010).

Second, acute exposure to hypoxia increases sympathetic vasoconstrictor outflow to skeletal muscle vascular beds (Saito et al. 1988; Rowell et al. 1989) and can mask vasodilatation at rest (Weisbrod et al. 2001) and during forearm exercise (Wilkins et al. 2008). An exaggerated sympathetic response to hypoxic exercise in older adults could have blunted the vasodilator response during hypoxic exercise. However, changes in muscle sympathetic nerve activity in response to acute hypoxia does not appear to be enhanced in older men (Davy et al. 1997). Furthermore, the heart rate and blood pressure responses to hypoxic exercise were similar between the older (current study) and younger adults (Casey et al. 2010) of identical protocols. Therefore, it is unlikely that the blunted compensatory vasodilatation in older adults during hypoxic exercise was a result of an exaggerated sympathetic vasoconstrictor restraint.

As illustrated in Fig. 4, older subjects in the current study demonstrated a 15–30% lower vasodilator response to ACh (across doses) compared to the young adults; however, this difference did not reach statistical significance (P = 0.15). Similar, but significant age-related reductions (∼25–30%) in ACh-induced forearm vasodilatation have been reported by others (Taddei et al. 2001; Donato et al. 2008; Westby et al. 2011). The lack of significance in the current study, despite similar age-related reductions (%), may be related to differences in techniques used to measure the responses (Doppler vs. venous occlusion plethysmography) and/or the ACh doses administered. Of particular interest to the current study, l-NMMA failed to attenuate the vasodilator responses to ACh in the older group, whereas it substantially reduced the response in young adults. These findings are consistent with previous studies demonstrating a lack of change in ACh-induced forearm vasodilatation following NOS inhibition (Taddei et al. 2000, 2001; Al-Shaer et al. 2006) and suggest a decreased NO bioavailability with ageing. Since the same loading and maintenance dose of l-NMMA was used in both groups, we do not feel the lack of change in the older group following l-NMMA administration is due to issues related to inadequate dosing.

Conclusions

This study when integrated with our previous findings in young adults (Casey et al. 2010) demonstrates that the compensatory vasodilator response to hypoxic exercise is attenuated in older adults. Furthermore, the present study suggests that the blunted vasodilator response is due to less NO signalling. We believe these findings provide important information on the impact of ageing and the role of NO in the regulation of skeletal muscle blood flow during conditions of reduced oxygen availability. From a clinical perspective, if this attenuated vasodilator response occurs in other vascular beds (i.e. coronary circulation) with ageing, older individuals may be at risk of reduced perfusion of the myocardium and consequently myocardial injury during exercise with hypoxia or ischaemia.

Glossary

Abbreviations

FBF

forearm blood flow

FVC

forearm vascular conductance

l-NMMA

N^G-monomethyl-l-arginine

MAP

mean arterial pressure

MVC

maximal voluntary contraction

NOS

nitric oxide synthase

oxygen saturation

Author contributions

D.P.C.: conception and design of protocol, and revising the article critically for important intellectual content; T.B.C.: drafting the article; M.J.J.: conception and design of protocol, and revising the article critically for important intellectual content. All authors contributed to drafting the article, analysis and interpretation of data, and all approved the final version to be published. All experiments were performed at the Mayo Clinic, Rochester, MN, USA.