Lifelong physical activity preserves functional sympatholysis and purinergic signalling in the ageing human leg

Authors


S. P. Mortensen: The Copenhagen Muscle Research Centre and Centre of Inflammation and Metabolism, Rigshospitalet, Section 7641, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. Email: stefan.mortensen@rh.regionh.dk

Key points

  • • Ageing is associated with a reduced exercise hyperaemia and impaired ability to override sympathetic vasoconstrictor activity (functional sympatholysis).
  • • We find that sedentary elderly have a lower vasodilator response to ACh and ATP in the leg compared to young, but also that this age-related reduction is partially (ACh) or completely (ATP) offset in lifelong physically active elderly subjects.
  • • An increase in sympathetic vasoconstrictor activity induced by tyramine reduces exercise hyperaemia in sedentary elderly, but not active elderly and young subjects.
  • • Interstitial ATP levels during exercise and P2Y2 receptor content are more related to the physical activity level than age.
  • • Physical activity can prevent the age-related impairment in functional sympatholysis and maintain a sufficient O2 delivery during moderate intensity exercise despite a loss of endothelial function.

Abstract  Ageing is associated with an impaired ability to modulate sympathetic vasoconstrictor activity (functional sympatholysis) and a reduced exercise hyperaemia. The purpose of this study was to investigate whether a physically active lifestyle can offset the impaired functional sympatholysis and exercise hyperaemia in the leg and whether ATP signalling is altered by ageing and physical activity. Leg haemodynamics, interstitial [ATP] and P2Y2 receptor content was determined in eight young (23 ± 1 years), eight lifelong sedentary elderly (66 ± 2 years) and eight lifelong active elderly (62 ± 2 years) men at rest and during one-legged knee extensions (12 W and 45% maximal workload (WLmax)) and arterial infusion of ACh and ATP with and without tyramine. The vasodilatory response to ACh was lowest in the sedentary elderly, higher in active elderly (P < 0.05) and highest in the young men (P < 0.05), whereas ATP-induced vasodilatation was lower in the sedentary elderly (P < 0.05). During exercise (12 W), leg blood flow, vascular conductance and inline image was lower and leg lactate release higher in the sedentary elderly compared to the young (P < 0.05), whereas there was no difference between the active elderly and young. Interstitial [ATP] during exercise and P2Y2 receptor content were higher in the active elderly compared to the sedentary elderly (P < 0.05). Tyramine infusion lowered resting vascular conductance in all groups, but only in the sedentary elderly during exercise (P < 0.05). Tyramine did not alter the vasodilator response to ATP infusion in any of the three groups. Plasma [noradrenaline] increased more during tyramine infusion in both elderly groups compared to young (P < 0.05). A lifelong physically active lifestyle can maintain an intact functional sympatholysis during exercise and vasodilator response to ATP despite a reduction in endothelial nitric oxide function. A physically active lifestyle increases interstitial ATP levels and skeletal muscle P2Y2 receptor content.

Abbreviations 
LVC

leg vascular conductance

MAP

mean arterial blood pressure

MSNA

muscle sympathetic nerve activity

NA

noradrenaline

NO

nitric oxide

WLmax

maximum workload

Introduction

Ageing is associated with a reduced endothelial function (Taddei et al. 1995), limb blood flow and cardiovascular function during exercise (Grimby et al. 1966; Wahren et al. 1974; Proctor et al. 1998). Resting muscle sympathetic nerve activity (MSNA) is increased with ageing (Ziegler et al. 1976; Dinenno et al. 1999) and limb blood flow is reduced (Dinenno et al. 1999). During exercise, the sympathetic nervous activity is increased (Alam & Smirk, 1937; Mitchell et al. 1983; Seals & Victor, 1991) and it is targeted to both resting and contracting skeletal muscle (Hansen et al. 1994; Ray & Mark, 1995; Strange, 1999). In contracting muscles of young, healthy individuals the increased sympathetic vasoconstrictor activity can be attenuated or even abolished (functional sympatholysis) (Remensnyder et al. 1962; Thomas et al. 1994; Hansen et al. 1996; Tschakovsky et al. 2002; Rosenmeier et al. 2004), but in ageing men, functional sympatholysis during exercise is impaired (Koch et al. 2003; Dinenno et al. 2005; Kirby et al. 2011) even though resting postjunctional α-adrenergic responsiveness to exogeneus noradrenaline (NA) release is reduced (Dinenno et al. 2002). While regular exercise has been shown to attenuate or offset the age-related lowering of endothelial function in the human forearm (DeSouza et al. 2000; Taddei et al. 2000), the effect of physical activity on functional sympatholysis remains unknown. In young individuals, functional sympatholysis is impaired after 2 weeks of limb immobilization, suggesting that some level of physical activity is required to maintain functional sympatholysis (Mortensen et al. 2012). Whether a physically active lifestyle can offset the age-related impairment in functional sympatholysis remains unknown.

ATP released from red blood cells and the endothelium is believed to play a role in blood flow regulation (Ellsworth et al. 1995; González-Alonso et al. 2002). Intraluminal ATP induces local vasodilatation (Rosenmeier et al. 2004) and can modulate sympathetic vasoconstrictor activity by acting on endothelial P2Y2 receptors (Kirby et al. 2008; Rosenmeier et al. 2008). The vasodilatory responsiveness and the sympatholytic effect of intraluminal ATP has been reported to be maintained in the ageing forearm (Kirby et al. 2011), but this may not apply to the legs because the endothelial function of the legs appears to be more affected by ageing than the forearm (Thijssen et al. 2011). In contrast to intraluminal ATP, interstitial ATP is generally believed to induce local vasoconstriction by acting on P2X receptors (Burnstock, 2007) and possibly by increasing sympathetic vasoconstrictor activity via group IV afferents (Li & Sinoway, 2002). Whether interstitial [ATP] is altered with ageing and contributes to the lower exercise hyperaemia and increased MSNA remains unknown.

The purpose of the present study was to examine if a lifelong (>30 years) physically active lifestyle can maintain endothelial function, functional sympatholysis and exercise hyperaemia in the leg. To accomplish these aims, we measured leg haemodynamics during exercise and arterial ACh and ATP infusion with and without simultaneous infusion of tyramine to elevate NA release in young men, lifelong sedentary elderly men and lifelong active elderly men. We hypothesized that lifelong physical activity can offset the age-related decline in endothelial function, functional sympatholysis and exercise hyperaemia.

Methods

Volunteers

Eight healthy young men who were not engaged in regular physical activity (less than 2 h of moderate intensity exercise per week during the last 3 years), eight healthy lifelong sedentary elderly men (less than 2 h of moderate intensity exercise per week during the last 30 years), and eight healthy lifelong endurance-trained elderly men (more than 5 h of high-intensity exercise per week during the last 30 years) were studied (Table 1). All men had no signs of ischaemia, had been non-smokers for >30 years, and none of the men had been diagnosed with cardiovascular disease, renal dysfunction, insulin resistance, diabetes, or hypercholesterolaemia. Five of the active elderly men had extra systoles at rest, but not during exercise, whereas the remaining subjects had no arrhythmias at rest or during exercise (ECG).

Table 1.  Characteristics of the male subjects
  Young Sedentary elderly Active elderly
  1. P < 0.05, ††P < 0.001: different from young men; ‡P < 0.05, ‡‡P < 0.001: different from lifelong sedentary elderly men.

Age (years)23 ± 166 ± 2††62 ± 2††
Weight (kg)79 ± 479 ± 276 ± 3
Height (cm)183 ± 2175 ± 3†178 ± 2
Body fat (%)18 ± 326 ± 115 ± 1‡
inline image (l min−1)3.6 ± 0.12.1 ± 0.1†3.7 ± 0.2‡
inline image (ml min−1 kg−1)46 ± 226 ± 1††49 ± 2‡‡
MAP (mmHg)85 ± 298 ± 495 ± 3†
Systolic blood pressure (mmHg)122 ± 2150 ± 5†152 ± 6†
Diastolic blood pressure (mmHg)66 ± 370 ± 369 ± 3
Total cholesterol (mmol l−1)3.8 ± 0.45.3 ± 0.4†4.9 ± 0.2†
LDL cholesterol (mmol l−1)1.9 ± 0.33.3 ± 0.4†2.9 ± 0.2
HDL cholesterol (mmol l−1)1.4 ± 0.11.4 ± 0.21.5 ± 0.2
Triglycerides (mmol l−1)0.8 ± 0.11.5 ± 0.3†1.0 ± 0.1
Leg mass (kg)12.5 ± 0.711.1 ± 0.411.8 ± 0.5
Peak workload during knee-extensor exercise (W)70 ± 547 ± 3†78 ± 6‡

The study was approved by the Ethics Committee of Copenhagen and Frederiksberg communities (H-3-2009-090) and conducted in accordance with the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all subjects before enrolment into the study.

Preliminary testing

Before the experimental day the subjects visited the laboratory to become accustomed to the one-legged knee-extensor model (Andersen & Saltin, 1985) and to perform an incremental bicycle ergometer test (Excalibur Sport, Lode, The Netherlands) in which pulmonary maximal oxygen uptake (inline image) was determined with a metabolic system (Quark CPET system, Cosmed, Rome, Italy). An incremental test was also performed in a one-legged knee-extensor ergometer to determine maximal workload.

Catherization

Subjects refrained from caffeine, alcohol, and exercise for 24 h before the experimental day. On the day of the experiment the men arrived at the laboratory after a light breakfast. After local anaesthesia, catheters were placed in the femoral artery and vein of the experimental leg and in the femoral artery of the non-experimental leg. A muscle biopsy was obtained from m. vastus lateralis of the non-experimental leg. In addition, three microdialysis probes (CMA 63, CMA microdialysis, Solna, Sweden) with a 30 mm membrane (20 kDa cut-off) was inserted into the thigh muscle (m. vastus lateralis) of the experimental leg under local anaesthesia. Thirty minutes after insertion of the probes, the men performed a 10 min exercise bout (12 W) with the purpose of minimizing the tissue response to insertion trauma.

Experimental protocol

Following 30 min of rest, the subjects received a femoral arterial infusion of: (1) ACh (10, 25 and 100 μg min−1 (kg leg mass)−1) and (2) ATP (0.4 ± 0.0, 1.6 ± 0.0 and 4.3 ± 0.0 μmol min−1, i.e. 0.04, 0.15 and 0.4 μmol min−1 (kg leg mass)−1, respectively; A7699 Sigma Aldrich, MO, USA) with and without co-infusion of tyramine (7.9 μmol min−1, i.e. 0.7 μmol min−1 (kg leg mass)−1: Sigma) (see Fig. 1 in online Supplemental material). Each dose of ACh or ATP was infused for 2.5 min and measurements were obtained after 2.0 min. The order of the ACh and ATP trials were randomized and separated by 30 min.

After 30 min of rest, the men completed 10 min of one-leg knee-extensor exercise at 12 W and 45% WLmax (31 ± 2, 21 ± 2 and 35 ± 2 W for the young, sedentary elderly and active elderly men, respectively) (separated by 10 min of rest) under the following conditions: (1) infusion of saline (control) and (2) infusion of tyramine (Supplemental Fig. 2, available online only). Arterial and venous blood samples (1–5 ml) were drawn simultaneously before each trial and during exercise (2.5 and 7.5 min).

Microdialysis

Microdialysate was collected for 10 min during resting conditions and during one-leg knee-extensor exercise. The microdialysis probes were perfused at a rate of 5 μl min−1 with Ringer acetate solution and to determine the relative exchange of ATP a small amount (2.7 nm) of [2-3H]ATP (<0.1 μCi ml−1) was added to the perfusate for calculation of probe recovery (see extended methods in the online Supplemental material for details).

Femoral arterial blood flow

Femoral arterial blood flow was measured with an ultrasound machine (LOGIQ E9, GE Healthcare) equipped with a linear probe operating an imaging frequency of 9 MHz and Doppler frequency of 4.2–5.0 MHz (see extended methods in the online Supplemental material for details).

Measurements

Arterial pressures were monitored with transducers positioned at the level of the heart (Pressure Monitoring Kit, Baxter, IL, USA). Leg mass was calculated from whole-body dual-energy X-ray absorptiometry scanning (Prodigy, GE Medical Systems, WI, USA). Blood gases, haemoglobin, lactate and glucose concentrations were measured using an ABL725 analyser (Radiometer, Copenhagen, Denmark). Plasma [NA] and [adrenaline] were determined with a radioimmunoassay (LDN, Nordhorn, Germany). Interstitial [ATP] in the microdialysis perfusate and dialysate was measured in duplicates with the luciferin–luciferase technique using an internal ATP standard (BioTherma AB, Dalarö, Sweden).

Quantification of purinergic P2Y2 receptor expression

Approximately 5 mg dry wt of the biopsy was homogenized in homogenization buffer and the protein concentration of the lysate samples was determined. Lysate proteins were separated using 10% SDS gels (Bio-Rad Laboratories, CA, USA) and transferred to PVDF membranes (Immobilion Transfer Membrane, Millipore, MA, USA). The membranes were incubated with primary polyclonal antibodies against the purinergic P2Y2 receptor (APR-010, Alomone laboratories, USA). Secondary antibody horseradish peroxidase-conjugated goat anti-rabbit (P-0448, Dako, Glostrup, Denmark) was used for detection. The protein content was expressed in arbitrary units relative to standard samples run on each gel (see extended methods in the online Supplemental material for details).

Statistical analysis

A one- (ACh) and two-way (Exercise/ATP with and without tyramine) repeated measures ANOVA was performed to test significance within and between trials. A two-way ANOVA was used to test significance between the young, elderly sedentary and elderly active men within trials. After a significant F test, pair-wise differences were identified using Tukey's honestly significant difference (HSD) post hoc procedure. The significance level was set at P < 0.05 and data are means ± SEM. No difference in leg blood flow or blood gas variables were observed between the measurement at 2.5 and 7.5 min and the presented data are the mean of the two measurements.

Results

Leg haemodynamics during ACh infusion

Baseline leg blood flow and vascular conductance was lower in both of the elderly groups compared to the young men (P < 0.05). ACh infusion increased leg blood flow and leg vascular conductance (LVC) to 4.0 ± 0.6 l min−1 and 51 ± 8 ml min−1 mmHg−1, respectively, in the young men, but it was lower in both the sedentary (0.8 ± 0.6 l min−1 and 9 ± 2 ml min−1 mmHg−1, respectively) and active elderly (2.8 ± 0.5 l min−1 and 33 ± 6 ml min−1 mmHg−1, respectively) men (Fig. 1). The active elderly men had a higher vasodilatory response to ACh compared to the sedentary elderly men (P < 0.05). Blood variables are presented online in Supplemental Table 1.

Figure 1.

Leg haemodynamics at rest and during femoral arterial ACh infusion in young, sedentary elderly and active elderly men 
*Different from baseline conditions, P < 0.05; †different from young men (same condition), P < 0.05; ‡different from sedentary elderly men (same condition), P < 0.05.

Leg haemodynamics during ATP infusion

Arterial ATP infusion increased leg blood flow and LVC in all three groups, but this increase was lower in the sedentary elderly men compared to the young and active elderly men (P < 0.05), whereas it was similar in the active elderly and young men (Fig. 2). Co-infusion of tyramine with ATP did not alter the vasodilatory response to ATP in any of the groups. Blood variables are presented in online Supplemental Tables 2 and 3.

Figure 2.

Leg haemodynamics at rest and during arterial ATP infusion in young, sedentary elderly and active elderly men with and without tyramine infusion 
Hatched bars indicate tyramine infusion trial. *Different from baseline conditions, P < 0.05; †different from young men (same condition), P < 0.05; ¤different from without tyramine, P < 0.05.

Leg haemodynamics during exercise

Exercise (12 W) increased leg blood flow to 2.1 ± 0.2 l min−1 in the young men and it was not different in the active elderly (1.8 ± 0.1 l min−1; P= 0.103), but lower in the sedentary elderly men (1.7 ± 0.1 l min−1) (Fig. 3; P < 0.05). There was no difference in leg blood flow between the active and sedentary elderly men at 12 W (P= 0.737). Arterial blood pressure was higher at rest and during exercise at 12 W in the sedentary elderly compared to the young men (P < 0.05), whereas there was no difference between the active elderly and young men. LVC was similar between groups during seated rest but lower in the sedentary and active elderly men during exercise (12 W) compared to the young men (P < 0.05), whereas there was no difference between the sedentary and active elderly men (P= 0.412). Leg arteriovenous (a–v)O2 difference was higher in the active elderly at rest compared to the young (P < 0.05) whereas there was no difference in leg a–vO2 difference during exercise between groups. Leg inline image was lower in the sedentary elderly men during exercise (12 W) compared to the young men (P < 0.05) in parallel with an increase in leg lactate release (P < 0.05). Leg inline image was similar in the young and active elderly men whereas the leg lactate release was unaltered in the young men and negative in the active elderly men during exercise (P < 0.05). Blood variables are presented online in Supplemental Table 4.

Figure 3.

Leg haemodynamics and leg lactate release at rest and during exercise at 12 W and 45% of maximal workload with and without tyramine infusion in young, sedentary elderly and active elderly men 
Hatched bars indicate tyramine infusion trial. The workload at 45% WLmax was 31 ± 2, 21 ± 2 and 35 ± 2 W for the young, sedentary elderly and active elderly men, respectively. *Different from baseline conditions, P < 0.05; †different from young men (same condition), P < 0.05; ‡different from sedentary elderly men (same condition), P < 0.05; ¤different from without tyramine, P < 0.05.

When tyramine was infused at rest, leg blood flow and LVC was lowered in all three groups (P < 0.05). During exercise, tyramine lowered leg blood flow and LVC in the sedentary elderly men compared to the control trial (12 W and 45% WLmax, P < 0.05), whereas leg blood flow, mean arterial blood pressure (MAP) and LVC was unaltered in the young and active elderly men compared to the control trial. However, the leg a–vO2 difference increased in the young and sedentary elderly men compared to the control trial (P < 0.05), whereas leg inline image and leg lactate release was unaltered by tyramine. Blood variables during the tyramine trial are presented online in Supplemental Table 5.

Plasma noradrenaline and adrenaline during exercise

At rest plasma [NA] was higher in the active elderly compared to the young and sedentary elderly men (P < 0.05), but similar in all three groups during exercise (Fig. 4). Tyramine infusion increased plasma [NA] in all three groups at rest (P < 0.05), but it was increased more and to similar levels in the sedentary and active elderly men (P < 0.05), whereas it was not different during exercise. There was no difference in plasma [adrenaline] between groups and the [adrenaline] did not change with exercise.

Figure 4.

Plasma noradrenaline and adrenaline concentrations at rest and during exercise at 12 W and 45% of maximal workload with and without tyramine infusion in young, sedentary elderly and active elderly men 
Hatched bars indicate tyramine infusion trial. The workload at 45% WLmax was 31 ± 2, 21 ± 2 and 35 ± 2 W for the young, sedentary elderly and active elderly men, respectively. *Different from baseline conditions, P < 0.05; †different from young men (same condition), P < 0.05; ¤ different from without tyramine, P < 0.05.

Muscle interstitial ATP concentrations during exercise

Resting interstitial [ATP] was similar between groups (Fig. 5). Exercise increased interstitial [ATP] in all three groups (P < 0.05) but it was higher (12 W and 45% WLmax) in the active elderly men compared to the young and sedentary elderly men (P < 0.05).

Figure 5.

Muscle interstitial ATP concentrations at rest and during exercise at 12 W and 45% of maximal workload in young, sedentary elderly and active elderly men 
The workload at 45% WLmax was 31 ± 2, 21 ± 2 and 35 ± 2 W for the young, sedentary elderly and active elderly men, respectively. *Different from baseline conditions, P < 0.05; †different from young men (same condition), P < 0.05; ‡different from sedentary elderly men (same condition), P < 0.05.

Skeletal muscle purinergic P2Y2 receptor content

Skeletal muscle purinergic P2Y2 receptor content was lower in the sedentary elderly compared to the young and active elderly (P < 0.05), whereas P2Y2 receptor content tended (P= 0.072) to be higher in the active elderly compared to the young (Fig. 6). See online Supplemental Fig. 3 for representative blots.

Figure 6.

P2Y2 receptor content in vastus lateralis muscle of young, sedentary elderly and active elderly men 
†Different from young men, P < 0.05; ‡different from sedentary elderly men, P < 0.05.

Discussion

The main findings are: (1) exercise hyperaemia and leg inline image was lower and lactate release higher in the sedentary elderly men compared to the young men, whereas exercise hyperaemia, leg inline image and lactate release were not different in the active elderly and young men; (2) tyramine administration lowered resting blood flow to similar levels in all three groups, but only in the sedentary elderly men during exercise; (3) tyramine increased resting plasma NA concentrations more in the sedentary and active elderly compared to the young men; (4) interstitial ATP concentrations were higher in the active elderly men during exercise compared to the young and sedentary elderly men; and (5) the vasodilatory responsiveness to infused ATP and P2Y2 receptor content was reduced in the sedentary elderly men, but similar in the active elderly men compared to the young men. Together, these findings demonstrate that physical activity can prevent the age-related impairment in functional sympatholysis and maintain a sufficient O2 delivery such that leg inline image is not compromised during moderate intensity exercise despite a lower endothelial nitric oxide (NO) function.

Exercise blood flow and functional sympatholysis

The present study demonstrates that an active lifestyle can maintain an intact functional sympatholysis during exercise in the ageing muscle. The attenuated functional sympatholysis in the sedentary elderly men is in agreement with previous observations that have demonstrated that functional sympatholysis is impaired in the ageing forearm (Dinenno et al. 2005). The effect of tyramine administration on leg blood flow and leg vascular conductance was similar at 12 W and 45% WLmax demonstrating that the attenuated functional sympatholysis in the sedentary elderly was coupled to the effect of ageing and not to a higher relative intensity at 12 W compared to the young and trained elderly. The higher lactate release in the sedentary elderly, suggest that the lower leg inline image was caused by impaired aerobic metabolism and not a lower metabolic demand. Mitochondrial respiration capacity appears to be unaffected by ageing (Rasmussen et al. 2003; Larsen et al. 2012) and the underlying cause of the lower leg inline image in the sedentary elderly therefore appears to be an impaired leg O2 delivery. Although exercise hyperaemia was not different in the active elderly and young men, it was also not different in the active and sedentary elderly at the same workload, which could indicate that there was a general effect of ageing on exercise hyperaemia independent of the physical activity level. Importantly, leg inline image was maintained in the active elderly and the exercising leg was taking up lactate rather than releasing it, suggesting that O2 delivery was sufficient to maintain aerobic metabolism. Leg blood flow is reduced after a period of training (Saltin et al. 1968, 1976; Kalliokoski et al. 2001; Nyberg et al. 2012b; Mortensen et al. 2012) and it therefore seems likely that lifelong training adaptations within the skeletal muscle that result in an optimized blood flow distribution and improved conditions for oxygen diffusion can explain the similar blood flow between the elderly men.

The lower response to infused ACh in the elderly trained compared to young men is in contrast to a previous observation in the leg, indicating that physical activity can completely prevent the age-related reduction in endothelial function (Newcomer et al. 2005). A possible explanation for this difference is the lower fitness level of the young subjects in the previous study (∼34 ml min−1 kg−1) compared to the present study (∼46 ml min−1 kg−1). The observation that an adequate exercise hyperaemia can be maintained despite a lower endothelial NO function is in agreement with the observation that NO is not obligatory for exercise hyperaemia in the leg (Rådegran & Saltin, 1999; Frandsen et al. 2001) and an acute increase in NO bioavailability did not increase exercise hyperaemia in the same elderly subjects in the present study (Nyberg et al. 2012a). Collectively, the present results demonstrate that physical activity can completely offset the age-related impairment in functional sympatholysis and maintain a sufficient exercise hyperaemia and inline image despite a lower endothelial NO function.

Resting blood flow was lower in both elderly groups, but blood flow was reduced to similar levels during tyramine infusion in all three groups, suggesting that postjunctional α-receptor vasoconstrictor activity was reduced in the elderly men. This is similar to observations in the forearm (Dinenno et al. 2002), suggesting that the age-related changes in postjunctional α-receptor vasoconstrictor activity are not limb specific. Moreover, the effect of exogenous NA stimulation on resting LVC was similar in the sedentary and active elderly, indicating that physical activity does not alter these age-related changes. Whether the difference in NA release in response to tyramine and difference in postjunctional α-receptor vasoconstrictor activity at rest may have affected the observed differences in sympatholysis between the young and elderly during exercise is unclear, but because the dose of tyramine used in the present study induces maximal vasoconstrictor activity in young subjects (Mortensen et al. 2012) it is unlikely that the young subjects had a lower vasoconstrictor response than the elderly subjects. Regardless of these potential differences between young and elderly, it is clear that functional sympatholysis during exercise was better in the active elderly compared to the sedentary elderly.

ATP levels, responsiveness and P2Y2 receptor expression

The vasodilator response to arterially infused ATP was maintained in the active elderly, but lower in the sedentary elderly in parallel with a reduced skeletal muscle P2Y2 receptor content.

Despite the differential vasodilator response to infused ATP, the sympatholytic properties of ATP were preserved in both ageing groups. ATP-induced vasodilatation has been reported to be unaltered with ageing in the forearm of sedentary, moderately-trained elderly (Kirby et al. 2010), whereas the sympatholytic properties of ATP are also maintained in the forearm (Kirby et al. 2011). Although the responsiveness to infused ATP appears not to be coupled to the skeletal muscle activity level or P2Y2 receptor content in young men (Mortensen et al. 2012), the present observations suggest that a reduced P2Y2 receptor content plays a role in the reduced vasodilatory response to ATP with ageing. The reduced response to infused ATP in the sedentary elderly may play an important role in the attenuated functional sympatholysis during exercise.

The interstitial levels of ATP were higher in the active elderly compared to the sedentary elderly. The similar ATP levels in the sedentary elderly and young men, but higher ATP levels in the active compared to the sedentary elderly suggest that exercise training increases interstitial ATP whereas ageing does not affect ATP levels. ATP is released from skeletal muscle during exercise (Tu et al. 2010) and the concentration is tightly coupled to the exercise intensity (Hellsten et al. 1998). The functional role of ATP in blood flow regulation remains undisclosed, but ATP could potentially be involved in three of the main regulatory responses to exercise: Firstly, the coupling between interstitial [ATP] and the degree of functional sympatholysis suggests that interstitial ATP could be involved in the local modulation of sympathetic vasoconstrictor activity. Secondly, interstitial ATP could stimulate vasodilatory substances such as it has been shown for interstitial adenosine (Nyberg et al. 2010). Thirdly, ATP has been suggested to stimulate group III/IV afferent fibres and consequently increase sympathetic nerve activity during exercise (Li & Sinoway, 2002). The similar plasma NA levels between groups but higher ATP levels in the trained group indicate that ATP may not be the main stimulant of afferent fibres, because exercise training is associated with a lower and not increased MSNA at the same workload (Ray, 1999). Alternatively, the afferent fibres may become less sensitive to metabolites such as ATP with training, leading to a lower response despite increased levels of ATP (Sterns et al. 1991). The possible triplicate role of interstitial ATP is intriguing, especially given that interstitial ATP can act directly on P2Y2 receptors located on smooth muscle cells (Mortensen et al. 2009), that interstitial ATP levels are severalfold higher than plasma ATP levels (Mortensen et al. 2011), and that there is a tight coupling between interstitial ATP levels and exercise intensity (Hellsten et al. 1998).

Conclusion

These findings demonstrate that lifelong physical activity can maintain an intact functional sympatholysis with ageing and an adequate exercise hyperaemia that allows aerobic metabolism to be maintained despite a lower endothelial NO function. The preserved sympatholytic properties of ATP, but lower responsiveness to infused ATP and reduced P2Y2 receptor content in the sedentary elderly men indicate that impaired ATP signalling may play a role in the attenuated functional sympatholysis during exercise with ageing. The coupling between interstitial ATP concentrations and the degree of functional sympatholysis suggest that interstitial ATP could play an important role in modulating sympathetic vasoconstrictor activity and thereby optimize blood flow distribution within the active muscle.

Appendix

Author contributions

The experiments were conducted at the Copenhagen Muscle Research Centre, Rigshospitalet, Denmark. The contributions of the authors were as follows: conception and design of the study: S.P.M. and B.S.; collection, analysis, and interpretation of data: S.P.M., M.N., K.W. and B.S.; drafting the article or revising it critically for important intellectual content: S.P.M., M.N., K.W. and B.S. All authors approved the final version of the manuscript.

Acknowledgements

This study was supported by a grant from the Lundbeck Foundation. S.P.M. was supported by a grant from the Danish Council for Independent Research – Medical Sciences. M.N. was supported by a grant from the Lundbeck Foundation. The authors have no conflicts of interest to disclose.

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