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The Copenhagen Muscle Research Centre, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
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Abstract |
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20 W), and then compared the responses with the combined infusion of the vasoconstrictor drug tyramine, which evokes endogenous release of noradrenaline from sympathetic nerve endings. In all three hyperaemic conditions, LBF equally increased from 0.5 ± 0.1 l min–1 at rest to 3.6 ± 0.3 l min–1, with no change in MAP. Tyramine caused significant leg vasoconstriction during adenosine infusion (53 ± 5 and 56 ± 5% lower LBF and leg vascular conductance, respectively, P < 0.05), which was completely abolished by both ATP infusion and exercise. In six additional subjects resting in the sitting position, intrafemoral artery infusion of ATP increased LBF and leg vascular conductance 27 ± 3-fold, despite concomitant increases in venous noradrenaline and muscle sympathetic nerve activity of 2.5 ± 0.2- and 2.4 ± 0.1-fold, respectively. Maximal ATP-induced vasodilatation at rest accounted for 78% of the peak LBF during maximal bicycling exercise. Our findings in humans demonstrate that circulating ATP is capable of regulating local skeletal muscle blood flow and O2 delivery by causing substantial vasodilatation and negating the effects of increased sympathetic vasoconstrictor activity.
(Received 20 February 2004;
accepted after revision 12 May 2004;
first published online 21 May 2004)
Corresponding author J. González-Alonso: The Copenhagen Muscle Research Centre, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark. Email: jga{at}cmrc.dk
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Introduction |
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Contraction-induced modulation of sympathetic vasoconstriction has been hypothesized to involve metabolites released from the contracting skeletal muscle, which presumably modulate signal transduction pathways subservient to the activation of postjunctional 
1 and 2-adrenoreceptors located on the vascular smooth muscle (Nishigaki et al. 1991; Ohyanagi et al. 1992; Thomas et al. 1994; Hansen et al. 1996, 1999; Buckwalter et al. 2001; Tschakovsky et al. 2002; Rosenmeier et al. 2003a; Wray et al. 2004). The muscle and interstitial metabolites that have been implicated include H+, Pi, K+, prostaglandins, adenosine and nitric oxide (NO) (McGillivray-Anderson & Faber, 1990, 1991; Nishigaki et al. 1991; Ohyanagi et al. 1992; Thomas et al. 1994; Tateishi & Faber, 1995; Thomas & Victor, 1998; Hansen et al. 2000). Although previous studies have provided strong experimental evidence in rats and humans supporting a role for NO produced by the contracting muscle (Thomas & Victor, 1998; Thomas et al. 1998; Chavoshan et al. 2002), this concept has recently been challenged (Rosenmeier et al. 2003b; Dinneno & Joyner, 2003; Buckwalter et al. 2004). While the precise mechanism(s) by which muscle contraction leads to attenuation of -adrenergic vasoconstriction remains incompletely understood, there are data to suggest that contraction-induced reduction in tissue oxygenation (measured by near infrared spectroscopy) plays a primary role (Hansen et al. 2000). Moreover, decreased O2 delivery relative to utilization (induced by ischaemia, hypoxic hypoxaemia and CO inhalation) can attenuate sympathetic vasoconstriction in resting muscle as well (Hansen et al. 2000; Hanada et al. 2003), suggesting that even in the absence of muscle contraction mechanisms are at work in the skeletal muscle microvasculature to preserve O2 uptake under conditions of reduced arterial O2 content and sympathetic activation.
We have now considered an alternative unifying mechanism for the attenuation of sympathetic vasoconstriction and regulation of muscle blood flow during contraction and during conditions with reductions in blood O2 content. Over the last few years, the erythrocyte has been hypothesized to function as an O2 sensor, which contributes to the control of local blood flow and O2 delivery by releasing ATP into the circulation in proportion to the offloading of O2 from the haemoglobin molecule (Ellsworth et al. 1995; Dietrich et al. 2000; Jagger et al. 2001; González-Alonso et al. 2002; Ellsworth, 2004). The tight coupling between alterations in circulating plasma ATP and changes in the oxygenation state of haemoglobin with normoxia, hypoxia, hyperoxia and CO + normoxia in both exercising and non-exercising human limbs and in vitro vessel preparations perfused with red blood cells strongly supports this hypothesis (González-Alonso et al. 2002; Ellsworth, 2004). ATP is a potent vasodilator when infused in intact humans (Folkow, 1949; Rongen et al. 1994; González-Alonso et al. 2002) or when infused locally in in vitro vessel preparations (Ellsworth et al. 1995; McCullough et al. 1997; Ellsworth, 2004). Mechanistically, ATP can induce vasodilatation by binding to the purinergic P2y receptors located on the vascular endothelial cells to release endothelium-derived hyperpolarization factors (EDHF), NO and/or prostaglandins, which diffuse to the vascular smooth muscle and result in vasodilatation (Ellsworth et al. 1995; Wihlborg et al. 2003). Whether ATP can attenuate sympathetic vasoconstriction in humans has not been studied. Clearly, a prerequisite for demonstrating a pivotal role of circulating ATP in the control of resting and exercising skeletal muscle blood flow and O2 delivery is that ATP should be capable of both causing potent vasodilatation and abolishing -adrenergic vasoconstriction.
Therefore, the primary aim of this investigation was to test the hypothesis that circulating ATP can override -adrenergic vasoconstriction in human skeletal muscle and thus mimic the exercise-induced attenuation. A secondary aim was to test the hypothesis that circulating ATP can evoke large increases in leg blood flow that reach the maximal vasodilatory capacity of the exercising leg despite increasing sympathetic outflow. To test the first hypothesis, we compared the vasoconstrictor effects of tyramine in the leg during three matched hyperaemic conditions produced by low intensity knee-extensor exercise or ipsilateral intrafemoral artery infusion of ATP or adenosine in the resting leg (the latter used as a vasodilator control). In addition, both vasodilators were infused during exercise to further evaluate their vasodilatory capacity in contracting skeletal muscle in the presence and absence of tyramine. To test the second hypothesis, MSNA (peroneal microneurography), femoral venous noradrenaline and leg blood flow were first measured during incremental intrafemoral artery infusion of ATP at rest and then compared to the responses (with the exception of MSNA) during maximal bicycling exercise. Additionally, blood gases were measured in each experimental condition to support the validity of the haemodynamic measures and investigate the effects of altered blood flow and O2 delivery on resting and exercising skeletal muscle O2 uptake.
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Methods |
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In the first study, the eight subjects (7 males and 1 female) underwent three different protocols, separated by 20 min of rest, to determine: (1) the vasoconstrictor effects of the drug tyramine during adenosine-, ATP- and exercise-induced hyperaemia (protocol 1), (2) the vasodilatory effects of ATP and adenosine during exercise and tyramine infusion (protocol 2), and (3) the vasodilatory effects of ATP and adenosine during exercise without the presence of tyramine (protocol 3; Fig. 1). Two interventions were performed at rest and three during knee-extensor exercise at an intensity of 20 W (25% of peak power), a model that allows the exercise to be confined to the quadriceps muscle (Andersen & Saltin, 1985). Before performing the three protocols, leg blood flows were determined during exercise. Resting leg blood flow was then increased in a stepwise manner by infusion of adenosine and ATP (Harvard pumps) until blood flow values matched those obtained during exercise. To do so, adenosine (Item Development AB, Stocksund, Sweden) dissolved in isotonic saline (1.25 mg ml–1) was infused at a rate of 16 µmol min–1 whereas ATP (Sigma A7699) dissolved in isotonic saline (1 mg ml–1) was infused at a rate of 1 µmol min–1. The aim during combined infusion of adenosine and tyramine was to evoke a vasoconstrictor response in the resting leg of 50%, without causing increases in arterial blood pressure, as previously documented in the forearm (Rosenmeier et al. 2003b). Tyramine (Sigma T-2879) dissolved in isotonic saline (0.52 mg ml–1) was infused at a rate of 13.2 µmol min–1 through all the tyramine trials. In separate studies in the resting leg, tyramine infusion at this dose reduced blood flow by 44 ± 3%, without altering mean arterial pressure.
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In the second study (n= 6), ATP was infused in the femoral artery during stepwise 2 min infusion periods to determine the maximal vasodilatory capacity of the resting leg and its effect on MSNA (peroneal microneurography) while seated upright (Fig. 2). The infusion rates given were 1, 2, 4, 8, 16, 32 and 64 µmol min–1. Due to technical difficulties, microneurographic data at the higher infusion rates were only obtained in three subjects. Leg blood flow (LBF) was then measured during incremental upright bicycling exercise to exhaustion (76 ± 4, 152 ± 9, 228 ± 13, 323 ± 20 and 361 ± 21 W; Lode ergometer) to compare the maximal vasodilatory capacities of the resting and maximally exercised leg (Fig. 2). During the ATP infusion and the exercise protocols, blood samples for ATP and catecholamine analyses were obtained following the LBF measurement, commencing after 1 min of infusion or exercise. LBF was measured again after blood withdrawal.
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ATP in plasma was determined with the luciferin-luciferase technique (Lundin, 2000) using a luminometer with two automatic injectors (ORION Microplate Luminometer, Berthold Detection System GmbH, Pforzheim, Germany). Blood samples (2.7 ml) for determination of plasma ATP were obtained using syringes containing EDTA (S-monovette, 2.7 ml KE; Sarstedt, Nümbrecht, Germany) and were centrifuged immediately for 30 s at 14000 r.p.m. (4°C; Sigma 1–15 K, Osterode am Harz, Germany). Plasma was then pipetted into pre-chilled tubes, frozen down in dry ice and stored for later analysis. The duration of the whole procedure from blood withdrawal to plasma separation was 90 s. Plasma ATP was measured at room temperature (20°C) using a commercially available ATP Kit (ATP Kit SL 144–041; BioTherma AB, Dalarö, Sweden) with an internal ATP standard procedure. Samples were measured in duplicates. The coefficient of variation of 10 repeated resting plasma samples was 11%. Plasma haemoglobin was also analysed spectrophotometrically to determine if haemolysis had occurred during the handling of the samples. Samples showing an elevation in plasma haemoglobin were excluded from the analysis. Plasma noradrenaline and adrenaline concentrations were determined with high performance liquid chromatography with electrochemical detection (Hallman et al. 1978). Arterial and femoral haemoglobin concentration and O2 saturation were determined spectrophotometrically (OSM-3 Hemoximeter, Radiometer). PO2 was determined with the Astrup technique (ABL 5, Radiometer, Copenhagen, Denmark). Leg vascular conductance was calculated as the quotient between LBF and mean arterial pressure. Leg O2 delivery was calculated by multiplying LBF by arterial O2 content. Leg O2 uptake (Leg 
) was calculated by multiplying the LBF by the difference in O2 content between the femoral artery and vein (a-v O2 difference).
Statistical analysis
A one-way repeated measures analysis of variance (ANOVA) was performed to test significance between and within treatments. Following a significant F test, pair-wise differences were identified using Tukey's honestly significant difference (HSD) post hoc procedure. When appropriate, significant differences were also identified using Student's paired t tests. The significance level was set at P < 0.05. Data are presented as mean ±S.E.M.
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Results |
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During pharmacologically induced vasodilatation, LBF increased to similar levels as during exercise hyperaemia (3.6 ± 0.3 l min–1) from resting values of 0.5 ± 0.1 l min–1, while mean arterial pressure and arterial O2 content remained unchanged. Hence, the elevations in leg vascular conductance and O2 delivery during hyperaemia were proportional to the increases in LBF (Fig. 3). Tyramine infusion reduced LBF during adenosine infusion from 3.8 ± 0.3 to 1.7 ± 0.2 l min–1 and leg vascular conductance from 29 ± 4 to 12 ± 4 ml min–1 mmHg–1 (both P < 0.05). In contrast during exercise and ATP infusion, tyramine did not alter either LBF (P= 0.53) or leg vascular conductance (Fig. 3). Reflecting the decline in LBF with combined infusion of tyramine and adenosine, leg a-v O2 difference increased from 6.4 ± 0.5 ml l–1 with adenosine infusion to 20.1 ± 2.2 ml l–1 with combined adenosine and tyramine (P < 0.05; Fig. 3). Conversely, no change in leg a-v O2 difference was observed with the addition of tyramine during ATP infusion (7.9 ± 1.7 and 9.5 ± 1.6 ml l–1, respectively; P= n.s.). However, both in the presence and absence of tyramine, leg 
was maintained at resting levels with adenosine and ATP infusions (0.03 l min–1) but increased 10-fold during exercise (0.33 ± 0.02 l min–1) in association with almost a doubling in leg a-v O2 difference from resting values (i.e. 109 ± 3 (exercise) versus 64 ± 3 ml l–1 (resting), respectively; Fig. 3). Femoral venous adrenaline was elevated from 1.7 nmol l–1 during hyperaemia to 3.0 nmol l–1 (P < 0.05) throughout all the tyramine infusions (Fig. 4), while noradrenaline (epinephrine) concentrations remained unchanged (0.3 nmol l–1). No significant changes in femoral venous and arterial ATP were observed during combined adenosine and tyramine infusion or during combined light exercise and tyramine infusion (0.7–1.0 µmol l–1). However, a trend for an elevation in plasma ATP was observed during combined ATP and tyramine infusion (from 1.5 to 3.1 µmol l–1). During hyperaemia, heart rate was identical with adenosine, ATP and exercise (91 ± 4 beats min–1) (Fig. 4).
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During the three exercise interventions, LBF increased to 3.4 ± 0.3 l min–1, whereas mean arterial pressure and arterial O2 content remained unchanged compared to resting values. Therefore, leg vascular conductance and O2 delivery increased in proportion to the elevation in LBF (Fig. 5). The superimposition of tyramine during exercise did not alter LBF or leg vascular conductance. However, the addition of ATP or adenosine during exercise and tyramine further increased LBF to 5.3 ± 0.3 l min–1 or 6.3 ± 0.4 l min–1, respectively (P < 0.05), whereas the a-v O2 difference declined from 125 to 129 (± 4) ml l–1 during exercise + tyramine to 47 ± 3 ml l–1 with addition of ATP and 60 ± 3 ml l–1 with addition of adenosine. Thus, despite large differences in O2 delivery among conditions, leg 
was maintained at 0.3 l min–1 during exercise by reciprocal changes in leg blood flow and a-v O2 difference (Fig. 5). With combined tyramine and ATP or adenosine infusions, femoral venous noradrenaline was significantly elevated (2.9–4.7 nmol l–1; P < 0.05), being higher than values with ATP and adenosine infusion in the absence of tyramine (1.7–2.4 nmol l–1; Fig. 6). Heart rate increased significantly when ATP and adenosine were infused during exercise but remained unchanged during tyramine infusion (Fig. 6).
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With a fixed-dose ATP infusion of 1 µmol min–1, total MSNA increased to 140 ± 12%(n= 6, P < 0.05). Incremental intrafemoral artery infusion of ATP in resting subjects resulted in dose-dependent increases in LBF, leg vascular conductance, circulating noradrenaline and total MSNA, all reaching a plateau at an infusion rate of 32 µmol min–1 (Figs 7 and 8). ATP infusion also resulted in progressive increases in heart rate with maintained mean arterial pressure. Femoral venous plasma ATP in the infused leg tended to decrease (P= 0.067) with increasing ATP infusion rate, while arterial plasma ATP remained unchanged. During incremental cycling exercise, LBF and leg vascular conductance increased progressively and reached a peak value at 90% of peak power. In contrast, circulating noradrenaline increased exponentially after 60% of peak power was reached (Fig. 7). Maximal LBF during ATP infusion accounted for 78 ± 2% of the peak LBF during maximal exercise (7.2 ± 0.3 versus 9.3 ± 0.7 l min–1, respectively; n= 4). By comparison, femoral venous noradrenaline was 9-fold lower at peak vasodilatation during ATP infusion than during maximal bicycle exercise (6.1 ± 0.5 versus 59.9 ± 8.7 nmol l–1, respectively).
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Discussion |
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Circulatory ATP attenuates sympathetic vasoconstriction in a manner similar to exercise while circulating adenosine does not
Despite augmented -adrenoreceptor stimulation evoked by tyramine infusion, no reduction in blood flow or vascular conductance was observed during ATP infusion or exercise, yet a substantial vasoconstrictor response was seen during adenosine infusion. Leg a-v O2 difference was also unchanged with a combined ATP and tyramine infusion but was elevated with a combined adenosine and tyramine infusion such that leg 
was maintained at resting levels in both hyperaemic conditions. While modulation of -adrenergic vasoconstriction has generally been associated with muscle contraction (McGillivray-Anderson & Faber, 1990; Nishigaki et al. 1991; Ohyanagi et al. 1992; Hansen et al. 1996, 2000; Thomas & Victor, 1998; Thomas et al. 1998; Ruble et al. 2002; Tschakovsky et al. 2002; Chavoshan et al. 2002; Rosenmeier et al. 2003a,b; Dinneno & Joyner, 2003; Wray et al. 2004; Fadel et al. 2004), the present investigation demonstrates a similar modulation in association with ATP infusion. This finding is compatible with a more universal role of ATP as a circulating metabolite, which might be involved in the maintenance or elevation of limb blood flow under conditions of elevated MSNA produced by exercise alone or exercise in combination with stressors such as hypoxia, CO-hypoxia, anaemia, heat stress and dehydration. For example, recent evidence demonstrates that blood flow to the resting leg is maintained despite a 2- to 4-fold elevation in MSNA with handgrip exercise and/or hypoxic hypoxia and CO-hypoxia (Hanada et al. 2003). Previous (González-Alonso et al. 2002) and present findings also indicate that blood flow in the control leg is maintained during ATP infusion despite a 2.5-fold elevation in sympathetic activity. Moreover, blood flow to the exercising limbs is elevated with hypoxia despite the concurrent enhanced sympathoexcitation (González-Alonso et al. 2001, 2002; Dinneno & Joyner, 2003). As hypothesized in the introduction, a prerequisite for demonstrating a pivotal role of a metabolite in exercise hyperaemia and resting limb blood flow control is its dual ability to cause marked vasodilatation and inhibit -adrenergic vasoconstriction. Clearly, ATP fulfils such requirements as the haemodynamic responses to ATP infusion mimicked exercise responses, while also allowing the maintenance of resting limb blood flow in the face of a sympathetic vasoconstrictor challenge. Therefore, the present results support a central role of circulating ATP in the attenuation of -adrenergic vasoconstriction observed in exercising and resting skeletal muscle. Nevertheless, experiments reducing ATP levels in plasma and inhibiting the ATP receptor sites in the vascular endothelium are warranted to conclusively prove that endogenous ATP is the putative signal leading to inhibition of -adrenergic vasoconstriction.
ATP infusion mimics exercise-induced increases in skeletal muscle blood flow despite increasing MSNA
Another major finding of this study is that ATP infusion augments leg MSNA and circulating noradrenaline, while evoking profound leg vasodilatation and elevating O2 delivery to levels normally observed during intense dynamic leg exercise in humans. The potency of ATP as a vasodilator in vitro and in vivo conditions is well documented (Folkow, 1949; Rongen et al. 1994; Ellsworth et al. 1995; González-Alonso et al. 2002) and is further supported by the present finding in healthy young subjects that peak LBF during ATP infusion is only 22% lower than the 9.3 l min–1 peak LBF during maximal bicycling exercise, being similar to the maximal LBF values reported during one-legged cycling and one-legged knee-extensor exercise (Klausen et al. 1982; Andersen & Saltin, 1985). While its vasodilatory potency is well established, the capacity of circulating ATP to increase MSNA and circulating noradrenaline has not been reported to our knowledge. We have considered several possible underlying mechanisms for this increase. First, it is possible that ATP could directly activate the chemosensitive group III and IV afferent afferent nerve endings in the muscle interstitium also involved in the activation of the muscle metaboreflex (Mitchell et al. 1983; Li & Sinoway, 2002; Hanna et al. 2002; Hanna & Kaufman, 2003). However, femoral venous plasma ATP in the infused leg tended to decline during ATP infusion in association with the increase in O2 saturation from 70% at baseline to 97% at peak ATP infusion, while arterial ATP remained unchanged with maintained O2 saturation (98%). Thus, it is unlikely that ATP infusion exerted its sympathoexcitatory effect via direct increases in leg muscle interstitial ATP concentrations, because an elevation in extracellular and thus femoral venous [ATP] is required for this to happen (Mo & Ballard, 2001).
In the absence of an increase in femoral venous [ATP] it is also unlikely that the sympathoexcitatory actions of ATP involved direct effects on receptors located in the central nervous system or in the central circulation. Likewise, a dominant role of arterial baroreceptors seems improbable since ATP infusion did not reduce mean arterial pressure. More likely, indirect effects of ATP infusion causing unloading of cardiopulmonary baroreceptors might play a role, as the 7 l min–1 increase in LBF was quite probably mirrored by a parallel increase in cardiac output, as suggested by the simultaneous rise in heart rate, and possibly a reduction in central venous pressure. Results from experiments selectively depressing central venous pressure using low levels of lower body negative pressure strongly support the ability of cardiopulmonary baroreceptors to increase MSNA in the absence of changes in arterial blood pressure (Jacobsen et al. 1993). Although the precise mechanism underlying the ATP infusion-mediated sympathoexcitation requires further experimentation, the present findings clearly demonstrate the ability of circulating ATP to directly or indirectly augment MSNA (Fig. 9).
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In the presence or absence of tyramine during exercise, ATP infusion evoked a remarkably similar elevation in blood flow and O2 delivery to the exercising leg, even though circulating noradrenaline was different by a factor of 2. Similarly, adenosine infusion during exercise and tyramine infusion almost doubled blood flow and O2 delivery, indicating that exercise completely nullified the effect of tyramine and concomitant elevated circulating noradrenaline seen with adenosine alone at rest. Intraarterial infusion of adenosine evokes a rapid and potent dose-dependent vasodilatation in the leg to a magnitude comparable with that found here with ATP infusion (Rådegran & Calbet, 2001). Of note is that both adenine nucleotides cause a more potent vasodilatation when infused in the resting human leg than the infusion of the NO donor sodium nitroprusside or acetylcholine (2–5 l min–1), and they do so without the profound hypotension produced by the latter vasodilators (Rådegran & Saltin, 1999). This makes adenosine an ideal enhancer of vasodilatation, together with ATP, as demonstrated in our study by the further increase in LBF with the combined infusion of ATP and adenosine as compared to infusion of ATP alone. However, it is important to note that the rate of adenosine infusion needed to achieve this effect was very high (16 000 nmol min–1) in comparison with the plasma adenosine concentrations during exercise (10–20 nmol l–1) (Saito et al. 1999; Tune et al. 2000) and the 1000 nmol min–1 ATP infusion needed to increase LBF to the same level. This supports a contributory rather than essential role of adenosine in exercise hyperaemia, because ATP does not seem to be largely degraded to adenosine in the circulation as previously demonstrated using P1 receptor blockade with theophylline during ATP infusion (Rongen et al. 1994). Adenosine blockade did not prevent ATP-induced vasodilatation, suggesting that ATP exerts its vasodilatory effect primarily via P2y stimulation rather than P1 stimulation via adenosine (Rongen et al. 1994). Together, these data show the capacity of circulating ATP to enhance vasodilatation and O2 delivery in contracting skeletal muscle and the potential of circulating adenosine to augment this response.
Modulation of sympathetic vasoconstriction by circulating ATP: potential mechanisms
Although the precise mechanism by which ATP overrides the increases in vasoconstrictor drive is not readily evident, the present difference in vasoconstrictor responses between ATP and adenosine at rest does not involve a differential reduction in presynaptic release of noradrenaline, since the venous noradrenaline increased to the same level during tyramine infusion in both conditions. Because neither ATP nor adenosine can readily cross the endothelium (Mo & Ballard, 2001), it is unlikely that ATP acts via direct modulation of -adrenoreceptors located on vascular smooth muscle cells. Another argument against this direct pathway is that extraluminar ATP would cause vasoconstriction by binding to abundant vasoconstrictor P2X purinergic receptors in vascular smooth muscle (Buckwalter et al. 2003), an unlikely possibility here given that blood flow and vascular conductance were unchanged with infusion of ATP and tyramine. Rather, the sympatho-inhibitory difference between these two related metabolites is probably due to activation of different signal transduction pathways and receptor types (Burnstock & Kennedy, 1986). On one hand, adenosine binds avidly to P1 purinergic receptors inducing vasodilatation by releasing endothelial prostaglandin and/or NO (Ray et al. 2002). On the other hand, ATP binds strongly to P2 purinergic receptors located on vascular endothelial cells inducing vasodilatation by triggering the release of endothelium-derived hyperpolarization factors (EDHF), NO and/or prostaglandins (Ellsworth et al. 1995; Wihlborg et al. 2003). Because prostaglandin has not been demonstrated to participate in functional sympatholysis in humans (Hansen et al. 2000; Frandsen et al. 2000), and since there still is conflicting evidence with regard to NO's role in this phenomenon (Rosenmeier et al. 2003b; Dinneno & Joyner, 2003; Buckwalter et al. 2004), the possibility exists that ATP primarily acts via EDHF and that this signalling pathway explains the distinctly different actions of ATP and adenosine. In support of this, the ATP-induced vasodilatation has been shown to be unaltered despite concomitant infusion of the NO synthase (NOS) inhibitorL-NMMA in the forearm (Rongen et al. 1994). Furthermore, EDHF has been shown to activate KATP channels located both on endothelial cells and the vascular smooth muscle cells (Brayden, 1990). These KATP channels have previously been implicated in exercise-induced attenuation of -adrenergic vasoconstriction during exercise (Thomas et al. 1997). Of note, the KATP channel opener diazoxide is the only other compound to our knowledge with the ability to abolish sympathetic vasoconstriction in quiescent skeletal muscle, as demonstrated in anaesthetized rats (Thomas et al. 1997). Future studies should elucidate if ATP exerts its dual vasodilatory and sympatholytic actions via EDHF or other unknown signal-transduction mechanism.
Integration of the vasoconstrictor and vasodilatory activities in the control of O2 delivery to resting and contracting skeletal muscle: role of the erythrocyte
Compelling evidence in humans indicates that blood flow to contracting and resting skeletal muscle is exquisitely regulated to maintain O2 delivery in a variety of conditions that drastically alter blood O2 content and MSNA such as hypoxia, CO-hypoxia, hyperoxia, heat stress and dehydration (González-Alonso et al. 1998, 2001, 2002, 2004; González-Alonso & Calbet, 2003). Human studies independently manipulating the amount of O2 circulating in arterial plasma (PaO2) and the amount of O2 bound to haemoglobin (O2Hb) with normoxia, hypoxia, hyperoxia, CO + normoxia and CO + hyperoxia also show that changes in blood flow, MSNA and circulating ATP are closely linked to alteration in O2Hb but unrelated to large changes in PaO2 (i.e. 40–600 mmHg) (González-Alonso et al. 2001, 2002; Hanada et al. 2003). This suggests that the main vascular O2-sensing locus is located in the erythrocyte itself and that the red blood cell senses and signals its O2 availability, thereby matching O2 delivery to tissue O2 demand (Ellsworth et al. 1995; Dietrich et al. 2000; Jagger et al. 2001; González-Alonso et al. 2002; Hanada et al. 2003; Ellsworth, 2004). The present observation that increasing O2 delivery to resting or exercising skeletal muscle does not enhance muscle aerobic metabolism implies that excessive O2 delivery has no beneficial effects in muscle, but rather could be counterproductive as it unnecessarily taxes the heart. In this context, the O2-sensing and -signalling erythrocyte provides an optimal mechanism to regulate local skeletal muscle blood flow and O2 delivery by releasing ATP into the circulation in direct proportion to the number of unoccupied O2 binding sites in the haemoglobin molecule (Jagger et al. 2001; González-Alonso et al. 2002). Although human experiments blocking ATP release from the erythrocyte and inhibiting the ATP receptor sites in the vascular endothelium need to be performed to conclusively prove a role for circulating ATP and the erythrocyte in skeletal muscle hyperaemia, available in vitro data strongly support this theory by demonstrating that: (1) ATP is released from red blood cells with exposure to hypoxia in the presence of hypercapnia (Bergfeld & Forrester, 1992), hypoxia alone (Ellsworth et al. 1995) and mechanical deformation (Sprague et al. 1996), and (2) ATP infused locally in first- and second-order arterioles produces a potent vasodilatory response which is conducted upstream (Ellsworth et al. 1995; McCullough et al. 1997; Ellsworth, 2004). Furthermore, the finding presented here that in healthy humans circulating ATP is capable of modulating sympathetic vasoconstriction supports a novel role of ATP released from the erythrocyte as a modulator of sympathetic vasoconstriction in exercising or resting skeletal muscle. The present observations in normal humans may have broader implications for our understanding of disordered neurocirculatory control in disease states accompanied by excessive muscle hypoxia and intense sympathetic activation at rest or during exercise, such as severe pulmonary disease, congestive heart failure or in cardiogenic shock.
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References |
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. Data are means ±S.E.M. for 8 subjects. *Significantly different from hyperaemia (P < 0.05). 
Significantly different from combined ATP and tyramine and exercise and tyramine (P < 0.05).
. Data are means ±S.E.M. for 8 subjects. *Significantly different from exercise (P < 0.05). 
