J Physiol 586.20 (2008) pp 4993–5002 4993

Activation of ATP/UTP-selective receptors increases bloodflow and blunts sympathetic vasoconstriction in humanskeletal muscle

Jaya B. Rosenmeier1, Gennady G. Yegutkin2 and Jose Gonzalez-Alonso1,3

1The Copenhagen Muscle Research Centre, Rigshospitalet, Denmark2MediCity Research Laboratory, Turku University and National Public Health Institute, Turku, Finland3Centre for Sports Medicine and Human Performance, Brunel University, Uxbridge, UK

Sympathetic vasoconstriction is blunted in the vascular beds of contracting skeletal muscle inhumans, presumably due to the action of vasoactive metabolites (functional sympatholysis).Recently, we demonstrated that infusion of ATP into the arterial circulation of the restinghuman leg increases blood flow and concomitantly blunts α-adrenergic vasoconstriction in asimilar manner to that during moderate exercise. Here we tested the hypothesis that ATP, ratherthan its dephosphorylated metabolites, induces vasodilatation and sympatholysis in restingskeletal muscle via activation of ATP/UTP-selective receptors. To this aim, we first measuredleg blood flow (LBF), mean arterial pressure (MAP), cardiac output (Q ), leg arterial–venous(a–v) O2 difference, plasma ATP and soluble nucleotidase activities during intrafemoral arteryinfusion of adenosine, AMP, ADP, ATP or UTP in nine healthy males. Comparison of thedoses of nucleotides and adenosine required for a similar increase in LBF from ∼0.5 l min−1

at baseline to ∼3.5 l min−1 (without altering MAP but increasing Q significantly) revealed thefollowing rank order of vasoactive potency: ATP (100) = UTP (100) >> adenosine (5.8) > ADP(2.7) > AMP (1.7). The infusions did not cause any shifts in plasma ATP level or soluble serumnucleotidase activities. Combined infusion of the vasodilatory compounds and the sympatheticvasoconstrictor drug tyramine increased plasma noradrenaline in all hyperaemic conditions, butonly caused leg and systemic vasoconstriction and augmented O2 extraction during adenosine,AMP and ADP infusion (LBF from 3.2 ± 0.3 to 1.8 ± 0.2 l min−1; 3.7 ± 0.4 to 1.7 ± 0.2 l min−1

and 3.3 ± 0.4 to 2.4 ± 0.3 l min−1, respectively, P < 0.05). These findings in humans suggest thatthe vasodilatory and sympatholytic effects of exogenous ATP in the skeletal muscle vasculatureare largely mediated via ATP itself rather than its dephosphorylated metabolites, most likely viabinding to endothelial ATP/UTP-selective P2Y2 receptors. These data are consistent with a roleof ATP in skeletal muscle hyperaemia in conditions of increased sympathetic nerve drive suchas exercise or hypoxia.

(Received 18 April 2008; accepted after revision 14 August 2008; first published online 14 August 2008)Corresponding author J. B. Rosenmeier: Copenhagen Muscle Research Centre, Rigshospitalet, Section 7652,Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. Email: jaya@dadlnet.dk

The potent and widespread vascular actions of purinenucleotides and nucleosides have long been recognized(Drury & Szent-Gyorgyi, 1929), and particularly ATP hasbeen implicated in a number of physiological functionssuch as the hyperaemia seen during exercise and hypoxia(Gonzalez-Alonso et al. 2002). Extracellular nucleotidesmediate their effects via a series of cell surface P2receptors composed of ligand-gated P2X receptors andG-protein-coupled P2Y receptors where the principalphysiological agonists of the human P2Y receptors areADP (P2Y1, P2Y12, P2Y13), UTP/ATP (P2Y2), UTP

(P2Y4), UDP (P2Y6) and ATP (P2Y11) whereas adenosinebinds to nucleoside-selective P1 receptors (Ralevic &Burnstock, 1998; Abbracchio et al. 2006). Activationof purinergic receptors in blood vessels induces vaso-dilatation or vasoconstriction depending upon receptorlocalization and subtype and therefore are thought toplay important roles in the regulation of vascular toneand blood flow (Ralevic & Burnstock, 2003). To terminatesignalling, ectonucleotidases are present in the circulationand on cell surfaces, rapidly degrading extracellularATP into ADP, AMP and adenosine (Gordon, 1986;

C© 2008 The Authors. Journal compilation C© 2008 The Physiological Society DOI: 10.1113/jphysiol.2008.155432

4994 J. B. Rosenmeier and others J Physiol 586.20

Zimmermann, 2006; Yegutkin, 2008). Thus, activation ofthe receptors represents a net balance between nucleotiderelease, inactivation and receptor affinity.

We have recently demonstrated that ATP, when infusedintra-arterially into resting limb skeletal muscle at thelevels released during moderate exercise (Rosenmeier et al.2004), not only increases leg blood flow via purinergicreceptor stimulation but also directly or indirectlyoverrides increases in muscle sympathetic vasoconstrictoractivity, thereby simulating what has been demonstratednumerous times to occur during exercise (a phenomenontermed functional sympatholysis) (Remensnyder et al.1962; Thomas & Victor, 1998; Buckwalter et al. 2001;Tschakovsky et al. 2002; Dinenno & Joyner, 2003;Rosenmeier et al. 2003a,b). Because extracellular ATP canbe rapidly degraded to ADP, AMP and adenosine (Gordon,1986; Zimmermann, 2006; Yegutkin, 2008), it remainsunknown whether the vasodilatory and sympatholyticeffects of ATP are partially or totally mediated via ATP itselfor its dephosphorylated compounds and which particularreceptor subtypes are involved.

We therefore used pharmacological dissection in thisstudy to characterize the purinergic receptors responsiblefor endothelium-derived vasodilatation in human vesselsby using adenosine and various purine and pyrimidineagonists and further comparing their vasoactive potency.To test which receptors are capable of producingsympatholysis, the different endothelial vasodilators werealso investigated under conditions of tyramine-mediatedsympathetic vasoconstriction. We hypothesize that ATP,rather than its dephosphorylated metabolites, inducesvasodilatation and sympatholysis in resting skeletal musclevia activation of ATP-selective receptors. Lastly, sincestrenuous exercise and hypoxia are characterized bysignificantly increased blood flow and activation ofintravascular nucleotide turnover (Gonzalez-Alonso et al.2002; Yegutkin et al. 2007), we also determined whetherthese vasoactive compounds when infused into thecirculation of resting subjects would affect circulating ATPand soluble nucleotidase activities to the same extent asseen during exercise.

Methods

Subjects

This study was performed in nine healthy male subjectswith a mean ± S.D. age of 27 ± 2 years, body weight of75 ± 3 kg, and height of 178 ± 5 cm. The subjects werefully informed of any risks and discomforts associatedwith the experiments before giving their informed writtenconsent to participate. The studies conformed to the codeof Ethics of the World Medical Association (Declaration ofHelsinki) and were approved by the Ethics Committee forCopenhagen and Frederiksberg communities.

Experimental protocols

Subjects reported to the laboratory at 8 am or 1 pm,following the ingestion of a light breakfast or lunch.Upon arrival, they rested in a supine position whilethree catheters were placed under local anaesthesia (1%lidocaine (lignocaine)) into the femoral artery and veinof the right leg and in the femoral artery of the left legusing the Seldinger technique. The femoral artery and veincatheters were positioned 1–2 cm distal from the inguinalligament. A thermistor (Edslab probe 94–0.3–2.5F) tomeasure venous blood temperature was inserted throughthe femoral venous catheter orientated in the anterogradedirection for the determination of femoral venous bloodflow. Adenosine (1.25 mg ml−1; Item Development AB,Stocksund, Sweden) and nucleotides AMP, ADP, ATPand UTP (all from Sigma) were dissolved in isotonicsaline and infused at a rate sufficient to increase LBF to∼3.5 l min−1. Next, the combined infusion of vasodilatorynucleotides and adenosine with tyramine (Sigma T-2879;infused at a rate of ∼13.2 μmol min−1) was performedto evoke a vasoconstrictor response in the resting legof ∼50% with the adenosine infusion, without causingsignificant increases in arterial blood pressure. Following30 min of rest, subjects first received separate infusions ofadenosine, AMP, ADP, ATP or UTP for 4 min followedby combined infusions of vasodilator and tyramine for anadditional 4 min period while they were resting seated inthe upright position. Trials were separated by 30 min ofrest and were counterbalanced across the subjects, exceptfor the adenosine trial, which was always performed firstand repeated at the end of the study. This comparisondemonstrated a high reproducibility of the effect ofadenosine + tyramine infusion on leg vasoconstriction.Separate tyramine infusion was performed once in eachsubject. The whole protocol lasted ∼4.5 h.

Blood flow and vascular conductance

Femoral venous blood flow (an index of leg bloodflow; LBF) was determined by the constant infusionthermodilution technique as previously described(Gonzalez-Alonso et al. 2000). Cardiac output wascalculated by multiplying stroke volume by heart rate,using the Modelflow method to determine stroke volumefrom directly measured arterial blood pressure (BeatScope 1.1a; Finapress Medical Systems BV, Amsterdam,the Netherlands) (Bogert & van Lieshout, 2005;Gonzalez-Alonso et al. 2006). LBF represents the average oftwo measurements made after 1 min of the start of separateinfusion of AMP, ADP, UTP, ATP or adenosine and after1 min of subsequent combined infusion of these purinesor pyrimidines and the vasoconstrictor drug tyramine.Arterial blood pressure was continuously monitored in allconditions by a pressure transducer (Pressure Monitoring

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Kit, Baxter) positioned at the level of the inguinal regionand connected to a haemodynamic monitor (Dialogue,Danica Elektronic, Copenhagen, Denmark). Mean arterialpressure was calculated by integration of the pressurecurve whereas heart rate was determined from an electro-cardiogram. All data were continuously recorded usinga Powerlab system (ADInstruments, Sydney, Australia).Leg vascular conductance was calculated as the quotientbetween LBF and mean arterial pressure.

Blood analysis and measurementof soluble nucleotidase activities

Blood samples (1.5 ml for blood gases, 2.7 ml for plasmaATP, 2 ml for noradrenaline and adrenaline analysesand 5 ml for serum nucleotidase assays) were drawnfrom the femoral vein of the right leg and femoralartery of the left leg at baseline and after 2 min ofpurine and pyrimidine infusion, as previously described(Rosenmeier et al. 2004). Plasma noradrenaline andadrenaline concentrations were determined with highperformance liquid chromatography with electrochemicaldetection (Hallman et al. 1978). Arterial and femoralhaemoglobin concentration, O2 saturation and PO2 wereanalysed with an automatic blood gas analyser (ABL720,Radiometer, Copenhagen, Denmark). ATP concentrationin EDTA-containing plasma samples was determined bythe luciferin–luciferase technique (Lundin, 2000) usingan ATP kit (BioTherma AB, Sweden) and an ORIONMicroplate Luminometer with two automatic injectors(Berthold Detection System, Germany).

Soluble nucleotidase activities were measured byincubating serum at 37◦C for 60 min in a final volumeof 60 μl RPMI-1640 medium containing 5 mmol l−1

β-glycerophosphate in the following ways: (1) forevaluation of total ATPase activity, 5 μl of serum wasincubated with 100 μmol l−1 ATP with tracer [8-14C]ATP(specific activity 57 mCi mmol−1; Amersham), and(2) for ADPase/NTPDase activity serum (10 μl) wasincubated with 50 μmol l−1 ADP and tracer [2,8-3H]ADP(27.5 Ci mmol−1; Perkin Elmer) in the presence ofadenylate kinase inhibitor Ap5A (80 μmol l−1). Radio-labelled nucleotides and their dephosphorylated productswere separated by thin layer chromatography (TLC)using Alugram SIL G/UV254 sheets (Macherey-Nagel,Germany), visualized in UV light and quantified byscintillationβ-counting, as described previously (Yegutkinet al. 2003).

Statistical analysis

A one-way repeated measures analysis of variance(ANOVA) was performed to test significance between andwithin treatments. Following a significant F test, pair-wisedifferences were identified using Tukey’s honestly

significant difference (HSD) post hoc procedure. Whenappropriate, significant differences were also identifiedusing Student’s paired t tests. The significance level wasset at P < 0.05. Data are presented as mean ± S.E.M.

Results

Haemodynamic responses, vasodilator potencyand intravascular nucleotide turnover duringseparate infusions of purines and pyrimidines

During pharmacologically induced vasodilatation, LBFincreased from a baseline level of∼0.5 l min−1 to 3.2 ± 0.3,3.7 ± 0.4, 3.4 ± 0.4, 3.3 ± 0.3 and 3.6 ± 0.3 l min−1 foradenosine, AMP, ADP, ATP and UTP, respectively, whilemean arterial pressure remained unchanged (Fig. 1).

Figure 1. Leg haemodynamics during purine or pyrimidineinfusion alone and in combination with tyramine infusionLeg blood flow, mean arterial pressure and leg vascular conductanceduring separate intrafemoral artery infusion of adenosine, AMP, ADP,ATP or UTP or combined with tyramine infusion. Data aremean ± S.E.M. for 9 subjects. ∗Significantly different from control,P < 0.05.

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Consequently, leg vascular conductance increased from∼4.2 ml min−1 mmHg−1 at baseline to 28.5 ± 2.7,30.0 ± 3.3, 28.5 ± 3.1, 29.3 ± 2.5 and 30.4 ± 3.3 ml min−1

mmHg−1 for adenosine, AMP, ADP, ATP and UTP,respectively. During hyperaemia, heart rate increased to∼68 beats min−1 in all conditions from similar restinglevels of ∼59 beats min−1 accompanying an increase instroke volume from ∼87 to ∼103 ml beat−1 (Fig. 2).Thus, cardiac output increased similarly in all conditions(Fig. 2).

Figure 2. Plasma noradrenaline and systemic haemodynamicsduring purine or pyrimidine infusion alone and in combinationwith tyramine infusionFemoral venous noradrenaline concentration, heart rate, strokevolume and cardiac output during separate intrafemoral arteryinfusion of adenosine, AMP, ADP, ATP or UTP or combined withtyramine infusion. Data are mean ± S.E.M. for 9 subjects. ∗Significantlydifferent from control, P < 0.05.

Comparable infusion rates of ∼1 μmol min−1 wererequired for both ATP and UTP to achieve increasesin LBF from ∼0.5 to 3.6 l min−1. Compared toATP/UTP, other infused compounds, adenosine, AMPand ADP, displayed much lower vasodilatory potencyand caused similar increases in LBF only at ratesof 20.9 ± 2.8, 71.3 ± 4.6 and 44.6 ± 2.6 μmol min−1,respectively. Thus, comparison of the relative vasoactivepotencies of exogenous nucleotides and adenosinerevealed the following rank order: ATP (100) = UTP(100) >> adenosine (5.8) > ADP (2.7) > AMP (1.7).

Blood samples were collected from the femoral veinof the right leg and femoral artery of the left leg atbaseline and after intrafemoral artery infusion of ATP,UTP and ADP, at concentrations inducing an increase inblood flow above baseline of ∼3 l min−1. Use of the highlyATP-specific bioluminescent assay revealed that humanplasma normally contains nanomolar concentrationsof ATP and these circulating nucleotide levels remainunchanged in the arterial and femoral venous plasmaunder conditions of pharmacological nucleotide-inducedvasodilatation (∼500 nmol l−1; Fig. 3A). Moreover,radio-TLC enzyme assay demonstrated that infusion ofvasodilatory nucleotides also does not affect the rates of[14C]ATP (Fig. 3B) and [3H]ADP (Fig. 3C) hydrolyses bysoluble nucleotidases circulating in human serum. Lastly,separate purine/pyrimidine infusion did not alter femoralvenous or arterial noradrenaline concentration (Fig. 2).

Haemodynamic responses during combinedpurine/pyrimidine and tyramine infusion

During separate tyramine infusion, LBF decreased from0.5 ± 0.1 to 0.3 ± 0.1 l min−1 (44 ± 3%), without alteringmean arterial pressure. During pharmacologically inducedvasodilatation and combined tyramine infusion, LBFdeclined to 1.7 ± 0.2 l min−1 (56%), 1.7 ± 0.2 l min−1

(53%) and 2.3 ± 0.3 l min−1 (30%) during adenosine,AMP and ADP, respectively, but remained unchangedduring ATP and tyramine infusion (3.3 ± 0.3 to3.3 ± 0.3 l min−1; P = 0.97), and UTP and tyramineinfusion (3.6 ± 0.4 to 3.7 ± 0.4 l min−1 P = 0.79). Thesame pattern of response was observed in leg vascularconductance (Fig. 2). With superimposition of tyramine,heart rate and cardiac output declined during adenosine,AMP and ADP, but not during ATP and UTPconditions (Fig. 2). Femoral venous noradrenalineconcentration increased during all the combined infusionsof purine/pyrimidine and tyramine (from ∼2 to3–4 nmol l−1) (Fig. 2).

Reflecting the LBF responses, leg a–v O2 differenceincreased from 5 to 8 ml l−1 during separate infusionsof adenosine, AMP and ADP to 26.0 ± 2.2 ml l−1 withcombined adenosine and tyramine infusion (P < 0.05),

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15.3 ± 3.1 ml l−1 with combined AMP and tyramineinfusion and 8.5 ± 0.8 ml l−1 (P = 0.07) with combinedADP and tyramine infusion. However, leg a–v O2

difference remained unchanged during combined ATPand tyramine infusion compared to separate infusionof ATP (7.9 ± 1.7 and 5.7 ± 1.6 ml l−1, respectively;P = 0.75) and combined UTP and tyramine infusioncompared to separate UTP infusion (7.1 ± 0.9 and7.1 ± 1.5 ml l−1, respectively; P = 0.98; Table 1).

Discussion

There were three important findings in this study. First,intra-arterial infusion of ATP and UTP, but not ofADP, AMP or adenosine, fully blunts the effects ofthe tyramine-mediated elevation in sympathetic vaso-constrictor activity on leg muscle and systemic perfusion.Second, by comparing the relative vasoactive potenciesof ATP, ADP, AMP and adenosine, when infusedintra-arterially, we demonstrated that ATP exerts physio-logically relevant vasodilatation at much lower doses thanits dephosphorylated metabolites. Further, the infusiondoses of ATP and UTP which have the P2Y2 receptoras the only common receptor were similar. Third,nucleotide-induced vasodilatation does not affect plasmaATP and serum nucleotidase activities. Collectively, thesefindings suggest that the vasodilatory and sympatholyticeffects of exogenous ATP in the human skeletal musclevasculature are largely mediated via ATP itself ratherthan its dephosphorylated metabolites, most likely viabinding to endothelial ATP/UTP-selective P2Y2 receptors.

ATP/UTP-selective receptor stimulation opposespharmacologically induced vasoconstriction

A major finding of this study was that intra-arterialinfusion of ATP and UTP, but not of ADP, AMP oradenosine, inhibited the effects of tyramine-mediatedincreases in sympathetic vasoconstrictor activity onreducing muscle and systemic perfusion in restinghumans. To characterize the purinergic receptorsresponsible for endothelium-derived vasodilatation andpharmacologically induced sympatholysis, we used herea pharmacological dissection model that extended ourprevious approach using exercise and infusion of ATPand adenosine in combination with tyramine infusion(Rosenmeier et al. 2004). Because the adenine nucleosidesand nucleotides infused in this study are known tostimulate different receptors in humans (i.e. adenosine(P1), AMP (P1 via degradation to adenosine), ADP(P2Y1, P2Y12, P2Y13), UTP (P2Y2, P2Y4), and ATP (P2Y2,P2Y4, P2Y11)) (Ralevic & Burnstock, 1998; Abbracchioet al. 2006), we can establish that the only known

receptors in common amongst all these compounds areP2Y2 and P2Y4. Yet, ATP is known to be a competitiveantagonist to P2Y4 receptors in humans (Kennedy et al.2000), making it unlikely that this receptor plays amajor role in the strikingly similar vasodilatation andsympatholysis found with ATP and UTP. In favour of apreferential action of the P2Y2 receptor are also the factsthat: (i) human skeletal muscle expresses mainly P2Y2

purinergic receptors located in the vascular endothelium(S. P. Mortensen, J. Gonzalez-Alonso, L. Bune, B. Saltin,H. Pilegaard & Y. Hellsten, unpublished observations),(ii) the expression of P2Y4 receptors on endothelial cellsfrom the human umbilical vein is very scarce (Wanget al. 2002) and cannot be detected at the mRNAlevel in human skeletal muscle (S. P. Mortensen et al.,unpublished observations), (iii) unlike adenosine, thebreakdown product of UTP; i.e. uridine, is an inactive

Figure 3. Plasma ATP and soluble serum nucleotidase activitiesduring infusion of exogenous nucleotides and adenosine inhumans‘Basel’ stands for baseline (no infusion condition). ATP, ADP and UTPwere infused into the femoral artery of the right leg at respective ratesof ∼1, 40 and 1 μmol l−1. Blood was collected from the right femoralvein and left femoral artery before and after nucleotide infusion.Circulating ATP was measured in EDTA-plasma using aluciferin–luciferase assay (A), while soluble nucleotidases were assayedin human serum by TLC using 100 μmol l−1 [14C]ATP (B) and50 μmol l−1 [3H]ADP (C) as substrates. Data are mean ± S.E.M. for 6subjects.

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Table 1. Blood parameters during purine and pyrimidine infusion alone and in combination with tyramine infusion

Baseline Purine or pyrimidine infusion Purine or pyrimidine + tyramine infusion

Ado AMP ADP ATP UTP Ado AMP ADP ATP UTP Ado AMP ADP ATP UTP

Haemoglobin (g l−1)a 137 ± 4 134 ± 5 132 ± 5 132 ± 5 130 ± 5 131 ± 4 135 ± 5 134 ± 4 135 ± 5 131 ± 5 136 ± 5 134 ± 4 133 ± 5 131 ± 5 129 ± 4v 137 ± 5 132 ± 4 130 ± 5 130 ± 5 128 ± 6 134 ± 5 134 ± 4 132 ± 5 132 ± 5 128 ± 4 131 ± 5 133 ± 5 131 ± 4 131 ± 5 128 ± 5

O2 saturation (%)a 98.2 ± 0.2 98.7 ± 0.2 98.1 ± 0.1 98.4 ± 0.2 98.5 ± 0.2 98.8 ± 0.1 98.3 ± 0.2 98.4 ± 0.2 98.0 ± 0.1 98.0 ± 0.2 99.0 ± 0.2 98.3 ± 0.1 98.3 ± 0.2 98.6 ± 0.2 99.0 ± 0.2v 63.1 ± 2.7 61.1 ± 2.9 64.8 ± 1.9 62.7 ± 2.8 66.3 ± 3.0 96.7 ± 0.4 95.4 ± 1.1 95.3 ± 0.7 95.9 ± 0.7 96.0 ± 0.6 85.9 ± 0.4∗† 83.4 ± 0.2∗† 88.2 ± 0.5∗† 94.6 ± 0.6 98.2 ± 0.5

PO2 (mmHg)a 100 ± 4 101 ± 3 100 ± 4 101 ± 2 100 ± 2 105 ± 2 106 ± 2 104 ± 3 106 ± 3 108 ± 2 112 ± 5 110 ± 6 108 ± 2 109 ± 2 110 ± 2v 35 ± 4 36 ± 3 35 ± 3 31 ± 3 32 ± 4 79 ± 1 76 ± 2 74 ± 2 76 ± 2 77 ± 3 54 ± 4∗† 56 ± 3∗† 60 ± 3∗† 70 ± 3 79 ± 3

Data are mean ± S.E.M. for 9 subjects. ∗Significantly different from separate purine or pyrimidine infusion, P < 0.05. †Significantlydifferent from ATP and UTP, P < 0.05.

metabolite with limited additional pharmacological effects(Vassort, 2001), and (iv) UTP is only known be releasedfrom endothelial cells during pathological conditions suchas cardiac ischaemia in humans (Wihlborg et al. 2006).Taken together, these observations in humans suggestthat the P2Y2 receptor might be physiologically relevantfor the regulation of vascular tone and blood flow inconditions of transient ATP or UTP release into the blood-stream combined with increases in noradrenaline-inducedvasoconstriction.

LBF responses to separate purine/pyrimidineinfusion and combined purine/pyrimidine + tyramineadministration were met by parallel cardiac outputresponses. This raises the question of whether the leginfusions were having systemic effects that alter LBFindirectly. Arguing against this possibility, only ATP andUTP infusion prevented the decline of both LBF andcardiac output. It is reasoned that if the metabolitesand tyramine acted via systemic effects, cardiac outputwould be the same across all the interventions given theequal initial hyperaemia and subsequent vasoconstrictorchallenge. Further, blood flow to non-infused tissues (i.e.contralateral leg and upper body tissues) did not increasein any of the conditions, in agreement with the previousobservations during intrafemoral artery ATP infusionthat contralateral limb blood flow remains unchangedand that LBF and cardiac output increase proportionally(Gonzalez-Alonso et al. 2002, 2008). Lastly, cardiac outputand LBF do not increase when adding ATP into the venousblood returning to the heart during administration ofATP in the femoral vein (Gonzalez-Alonso et al. 2008).Therefore, these findings jointly indicate that the infusedmetabolites and tyramine are having primarily a localeffect.

Although the precise mechanism by which ATP or UTPoverrides the vasoconstrictor influence of noradrenalineis not readily evident, the present difference in vaso-constrictor responses in the leg and systemic circulationsbetween ATP/UTP and the other purines and pyrimidinesdoes not appear to involve a blunting in presynaptic

release of noradrenaline since tyramine infusion increasedfemoral venous noradrenaline concentration to the samelevel in all conditions (Fig. 2). Because neither ATP,ADP, AMP, nor adenosine can readily cross the end-othelium (Mo & Ballard, 2001), it is unlikely thatthese compounds act via direct modulation of post-junctional α-adrenoreceptors located on vascular smoothmuscle cells. Rather, the sympatho-inhibitory differencebetween these related compounds are probably dueto either activation of different receptor subtypes,signal transduction pathways, and/or second metaboliterelease involved in the communication betweenthe endothelial and smooth muscle cells in theskeletal muscle microvasculature (Burnstock & Kennedy,1986).

Binding of ATP to endothelial P2Y receptors isknown to stimulate the release of NO, adenosine,prostaglandins and endothelial-derived hyperpolarizationfactors (EDHF) leading to vascular smooth musclerelaxation (Collins et al. 1998; Olearczyk et al. 2004;van Ginneken et al. 2004; Winter & Dora, 2007). Inthe context of the present findings, these downstreamsignals could also lead to the modulation of directnoradrenaline-induced α-adrenoceptor stimulation. Thefindings by Kirby et al. (2008) demonstrating thatexogenous ATP (but not adenosine) can abolish directpostjunctional α1- and α2-adrenergic vasoconstrictionindicate that the postjunctional α-adrenoceptors areindeed involved in ATP/UTP-induced sympatholysis.However, evidence for the exact intracellular signalsinvolved in this phenomenon is lacking. NO, adenosineor prostaglandins seem unlikely signals since they arenot obligatory for functional sympatholysis (Thomas &Segal, 2004). EDHF is also an unlikely signal because itis not exclusively triggered by ATP or UTP (Wihlborget al. 2003). EDHF can be released by acetylcholine,which does not have sympatholytic properties (Busse et al.2002). Thus, elucidation of the precise mechanisms forthe ATP/UTP-induced sympatholysis in human skeletalmuscle is warranted.

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Potency differences between adenineand uridine compounds

We have recently shown that intrafemoral artery ATPinfusion, but not intrafemoral vein infusion, canelevate blood flow up to ∼80% of the maximalleg exercise hyperaemia established during incrementalcycling to exhaustion (∼9 l min−1) (Rosenmeier et al.2004; Gonzalez-Alonso et al. 2008). Although ATP andUTP in this study were found to be equipotent withregard to leg blood flow at infusion rates of ∼1 μmolmin−1, progressive infusion of UTP in three subjectsdemonstrated a plateau response in leg blood flowat ∼6 l min−1, a peak value which is lower than the∼8 l min−1 peak leg blood flow observed with ATPinfusion (Rosenmeier et al. 2004; Gonzalez-Alonso et al.2008). This poses the question of whether the degradationof ATP to its other derivatives such as ADP contributesto the higher peak ATP-induced vasodilatation. Thisis an attractive possibility in the light of the recentobservation that strenuous exercise-induced hyperaemiais accompanied by concurrent up-regulation of solublenucleotide-inactivating enzymes (Yegutkin et al. 2007).Yet the present observation that the rates of adenosine(21 μmol min−1), AMP (71 μmol min−1) and ADP(44 μmol min−1) infusion needed to achieve the sameleg blood flow levels of about 3.5 l min−1 are very highin comparison with that of ATP and UTP (1 μmolmin−1) suggests that ATP-dephosphorylated metabolitesdo not contribute importantly to ATP-induced vaso-dilatation and sympatholysis. Although a rapid uptake andmetabolic breakdown mechanisms of adenosine mightpartially explain its lower vasodilator potency (Murrant& Sarelius, 2002), adenosine infusion is not capable ofopposing direct sympathetic vasoconstriction in humans(Tschakovsky et al. 2002; Rosenmeier et al. 2003b; Kirbyet al. 2008), rendering it an unlikely candidate forfunctional sympatholysis. Therefore, the present findingsupports a smaller contribution of adenosine, AMPand ADP to limb muscle hyperaemia, because ATP, forinstance, does not seem to be largely degraded to adenosinederivates in the circulation as previously demonstratedusing P1 receptor blockade with theophylline during ATPinfusion (Rongen et al. 1994) and the soluble purinergicenzyme concentration did not increase during infusion asdiscussed below.

Nucleotide-induced leg muscle vasodilatation doesnot affect plasma ATP and serum nucleotidaseactivities

As previously mentioned, exercise hyperaemia isaccompanied by transient increases in circulatingnucleotide levels (i.e. plasma ATP, ADP and AMP)as well as by up-regulation of soluble nucleotide

pyrophosphatase/phosphodiesterase (NPP) and NTPDaseactivities (Yegutkin et al. 2007). With the currentinvestigation we also wanted to determine whetherregulation of leg haemodynamics by infusion ofvasoactive nucleotides to the resting subjects wouldaffect intravascular nucleotide turnover to the sameextent as strenuous exercise. Infusion of vasodilatorynucleotides ATP, ADP and UTP was not accompaniedby significant shifts in plasma ATP (Fig. 3A) as wellas in other circulating nucleotides (data not shown),thus suggesting that, subsequent to signal trans-duction, nucleotides are immediately eliminated from thebloodstream.

In keeping with this rationale of rapid nucleotidebreakdown, we believe that previously reported findingson exercise-mediated increases in circulating ATP, ADPand AMP levels (Gonzalez-Alonso et al. 2002; Mortensenet al. 2007; Yegutkin et al. 2007) reasonably indicate that anet balance between nucleotide release and inactivationis markedly disturbed under exercising hyperaemicconditions and therefore, actual amounts of nucleotidesliberated in the bloodstream of exercising humansshould exceed their measured plasma concentrationsof ∼1 μmol l−1. Interestingly, cultured endothelialcells have been shown to release E-NTPDases andecto-5′-nucleotidase along with ATP in response to theapplication of shear force (Yegutkin et al. 2000). Byanalogy, it would be anticipated that the increased shearrate might also activate the release of membrane-boundnucleotidases in vivo, thus underlying the recentlydescribed phenomena of transient up-regulation ofserum NTPDase and NPP under exercising hyperaemicconditions (Yegutkin et al. 2007). However, directionalmanipulation of LBF by injection of ATP and othervasoactive nucleotides into the femoral artery of theresting subjects did not affect the rates of [14C]ATPand [3H]ADP hydrolyses by human serum (Fig. 3), thusruling out the possibility that the increased shear ratedirectly stimulates the release of soluble purine-convertingactivities.

Experimental limitations

There are limitations in manipulating vascular adeninenucleotides and adenosine with intravascular injectionsto investigate the role ATP and its dephosphorylatedmetabolites on the regulation of skeletal muscle bloodflow during conditions of enhanced sympatheticvasoconstrictor drive such as moderate and intenseexercise and/or hypoxia. Because nucleotides andadenosine in the bloodstream do not pass easily throughthe interstitium (Mo & Ballard, 2001), the intravascularinjections employed here are likely to mimic the effectsof purines when they are released from red blood cells,

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endothelial cells or generated extracellularly from othersources (Gonzalez-Alonso et al. 2002; Rosenmeier et al.2004; Erlinge & Burnstock, 2008). Their effects probablyare largely mediated via stimulation of P1 and P2Yendothelial receptors. During exercise or hypoxia,however, nucleotides and/or adenosine mightalso increase in concentration in the interstitium(Hellsten et al. 1998; MacLean et al. 1998). Inter-stitial purines probably act mainly on the vascularsmooth muscle receptors, possibly on abundant vaso-constrictor P2X receptors, and possibly on sympatheticprejunctional receptors (Erlinge & Burnstock,2008).

Tyramine is a commonly used pharmacological tool toinduce limb muscle vasoconstriction via stimulation ofendogenous release of noradrenaline from sympatheticnerve endings (Rongen et al. 1994; Dinenno &Joyner, 2003). It must be emphasized that venousnoradrenaline concentration does not always accuratelyreflect noradrenaline release from sympathetic neurons(Esler et al. 1990). Hence, an exact extrapolation cannotbe made between the effects of tyramine and those ofnaturally evoked increases in sympathetic activity. Herewe assumed that the equal increase in femoral venousnoradrenaline with superimposition of tyramine infusionreflected an equal release of noradrenaline from thesympathetic nerve terminals and equivalent increase invasoconstrictor drive in all conditions. However, thenaturally evoked sympathetic activity occurs in burstsand groups of impulses and three different transmittersare released, namely noradrenaline, ATP and neuro-peptide Y, where the relative importance of each ofthese compounds in contributing to vasoconstriction isdifferent depending on the exact patterning of activityand the prevailing conditions. In future studies it willbe important to test the ability of infused ATP/UTPto inhibit the vasoconstrictor influence of a naturallyevoked increase in muscle sympathetic nerve activity(MSNA).

In conclusion, the present findings demonstrate thatexogenous ATP and UTP possess the capacity to producepotent limb muscle vasodilatation and override vaso-constriction evoked by tyramine-induced release ofnoradrenaline at rest, possibly via their common P2Y2

receptor. These data are consistent with the additionalobservations in this study showing a greater vasodilatorypotency of ATP than its dephosphorylated metabolitesin the leg and the unchanged plasma nucleotide andsoluble nucleotide concentrations in the blood. BecauseATP and UTP are discharged in normal physiologicalor pathophysiological conditions where the sympatheticnervous drive or MSNA is also elevated, such as exercise(Yegutkin et al. 2007), hypoxia (Gonzalez-Alonso et al.2002; Hanada et al. 2003), anaemia (Gonzalez-Alonsoet al. 2006) and cardiac ischaemia (Erlinge et al. 2005),it seems possible that the present findings might have

implications for the understanding of circulatory controlduring exercise and certain disease states.

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Acknowledgements

Special thanks are given to Stefan Mortensen for hisassistance with the study as well as to Paula Croft fromEurope ADInstruments Ltd for her technical support. J. B.

Rosenmeier was supported by the Danish Heart Foundationand Novo Nordisk Foundation. G. G. Yegutkin was supportedby the Finnish Academy, Sigrid Juselius Foundation. J.Gonzalez-Alonso was supported by grants from the DanishNational Research Foundation.

C© 2008 The Authors. Journal compilation C© 2008 The Physiological Society