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Eur J Appl Physiol
DOI 10.1007/s00421-014-2932-8
ORIGINAL ARTICLE
The effect of purinergic P2 receptor blockade on skeletal muscle
exercise hyperemia in miniature swine
S. P. Mortensen · R. M. McAllister · H. T. Yang ·
Y. Hellsten · M. H. Laughlin
Received: 21 April 2014 / Accepted: 3 June 2014
© Springer-Verlag Berlin Heidelberg 2014
230 ± 9 beats/min, whereas blood pressure was unaltered.
Arterial ATP infusion caused a ~twofold increase in blood
flow in 15 of the 16 sampled muscle tissues and this effect
was abolished after RB2 infusion.
Conclusions These results indicate that P2 receptors play
a role in regulating skeletal muscle blood flow during exer-
cise in miniature swine.
Keywords ATP · Blood flow · P2Y receptors ·
Vasodilation · Skeletal muscle
Abbreviations
Adr Adrenal
Bic Br Biceps brachii
Bic Fem Biceps femoris
Brach Brachialis
Dia Diaphragm
EDHF Endothelium-derived hyperpolarizing factor
Int Intestines
Kid Kidney
LV Left ventricle
Pan Pancreas
RB2 Reactive blue 2
RF Rectus femoris
RV Right ventricle
Semit Semitendinosus
Stom Stomach
Sup Superficial portion
TAH Triceps brachii accessory head
TLH Triceps brachii long head
TltH Triceps brachii lateral head
TMH Triceps brachii medial head
VI Vastus intermedius
VL Vastus lateralis
VM Vastus medialis
Abstract
Purpose ATP could play an important role in skeletal
muscle blood flow regulation by inducing vasodilation via
purinergic P2 receptors. This study investigated the role of
P2 receptors in exercise hyperemia in miniature swine.
Methods We measured regional blood flow with radiola-
beled-microsphere technique and systemic hemodynamics
before and after arterial infusion of the P2 receptor antago-
nist reactive blue 2 during treadmill exercise (5.2 km/h,
~60 % VO2max) and arterial ATP infusion in female
Yucatan miniature swine (~29 kg).
Results Mean blood flow during exercise from the
16 sampled skeletal muscle tissues was 138 ± 18 mL/
min/100 g (mean ± SEM), and it was reduced in 11
(~25 %) of the 16 sampled skeletal muscles after RB2
was infused. RB2 also lowered diaphragm blood flow and
kidney blood flow, whereas lung tissue blood flow was
increased (all P < 0.05). Infusion of RB2 increased arte-
rial lactate concentration during exercise from 1.6 ± 0.5
to 3.4 ± 0.6 mmol/L and heart rate from 216 ± 12 to
Communicated by Carsten Lundby.
S. P. Mortensen (*)
Department of Cardiovascular and Renal Research, Institute
of Molecular Medicine, University of Southern Denmark,
J.B.Winsløwsvej 21-3, 5000 Odense C, Denmark
e-mail: smortensen@health.sdu.dk
R. M. McAllister · H. T. Yang · M. H. Laughlin
Department of Biomedical Sciences, University of Missouri,
Columbia, MO, USA
Y. Hellsten
Department of Nutrition, Exercise and Sports, University
of Copenhagen, Copenhagen, Denmark
Eur J Appl Physiol
1 3
Introduction
The complex regulation of precisely matching O2 delivery
to the metabolic demand of contracting muscle is thought
to be brought about by an interaction between locally
formed vasoactive substances and sympathetic vasocon-
striction (Laughlin et al. 2011; Hellsten et al. 2012b). Apart
from functioning as an intracellular energy source, nucleo-
tides function as a signaling molecule (Erlinge and Burn-
stock 2008; Hellsten et al. 2012b; Ellsworth and Sprague
2012). In skeletal muscle, ATP has been proposed to con-
tribute to the local regulation of skeletal muscle blood
flow by inducing local vasodilation (Ellsworth et al. 1995;
González-Alonso et al. 2002; Mortensen et al. 2011) and
overriding sympathetic vasoconstrictor activity (Remen-
snyder et al. 1962; Rosenmeier et al. 2004; Kirby et al.
2008). In vitro data indicate that ATP is released from
erythrocytes when exposed to hypoxia (Bergfeld and For-
rester 1992; Ellsworth et al. 1995) and upon deformation
(Sprague et al. 1996). ATP and UTP are also released from
endothelial cells in response to shear stress and hypoxia
(Burnstock 1999; Saiag et al. 1995; Mortensen et al. 2011).
Nucleotides exert their cardiovascular effect via activa-
tion of a group of Purinergic P2 receptors, composed of
ligand-gated P2X receptors and G-protein-coupled P2Y
receptors. Initially, P2Y receptors located in the endothe-
lium were thought to induce vasodilation, whereas smooth
muscle P2X receptors were thought to induce vasocon-
striction (Gordon 1986; Burnstock 2007; Burnstock and
Ralevic, 2014). However, subsequent studies showed a more
complex pattern of P2 receptor distribution and function
(Mortensen et al. 2009a; Burnstock 2010). The principal
physiological agonists of the P2Y receptors are ATP (P2Y2,
P2Y11), ADP (P2Y1, P2Y12, P2Y13), UTP (P2Y2, P2Y4)
and UDP (P2Y6) (Ralevic and Burnstock 1998; Abbrac-
chio et al. 2006). In the human leg, ATP-sensitive P2Y2 and
P2X1 receptors are located in the endothelium of microves-
sels and capillaries (Mortensen et al. 2009a; Thaning et al.
2010). Although indirect evidence suggests that ATP could
play a role in blood flow regulation, the physiological role
of extracellular ATP and P2 receptors remains unknown.
To investigate the role of P2 receptors in skeletal mus-
cle blood flow regulation, we measured regional blood flow
in Yucatan miniature swine during treadmill running and
arterial ATP infusion before and after administration of the
non-selective P2 receptor antagonist Reactive blue 2 (RB2)
(Ralevic and Burnstock 1998; Brunschweiger and Müller
2006). We used this porcine model because its tissue-spe-
cific blood flow responses to exercise are well character-
ized (McAllister et al. 2008). Furthermore, use of the radi-
olabelled microsphere technique permitted muscle-specific
blood flow determination for multiple muscles of varying
function (flexor/extensor) and fiber type composition. We
hypothesized that ATP plays an important role in skeletal
muscle exercise hyperemia and that infusion of RB2 there-
fore would lower exercising muscle blood flow.
Methods
Eleven female Yucatan miniature swine (28.6 ± 1.5 kg) were
included in the experiment. The study was carried out in
accordance with the recommendations in the Guide for the
Care and Use of Laboratory Animals of the National Institutes
of Health. The treatments and other procedures were approved
by the Animal Care and Use Committee of the University of
Missouri and all efforts were made to minimize suffering.
Surgery to implant a catheter in the left atrium and
catheters in both internal thoracic arteries was conducted
8–10 days before exercise tests. To implant the left atrial
catheter, after cutting the sternabrae on the midline and open-
ing the pericardium, the tip of the left atrial appendage was
incised while its base was clamped. The catheter (Micro-
Renathane; outside diameter, 0.095 in.; Braintree Scientific)
was advanced ~5 cm into the atrium, and then it was secured
to the atrial wall using a purse-string suture. A short loop of
catheter was left in the pericardium as the latter was closed.
To implant the internal thoracic arterial catheters, the arteries
were isolated in the cranial thorax. After incising the arteries,
two catheters (Micro-Renathane; outside diameter, 0.080 in.;
Braintree Scientific) were advanced cranially to the junction
of the internal thoracic artery and aorta and secured. After
closing the sternabrae using stainless steel sutures, the cath-
eters were tunneled subcutaneously using a trocar from the
thoracic inlet to a position between the scapulae. At the exit
site, the catheters were wrapped, secured to the dorsal sur-
face, and covered with a jacket until the day of exercise test-
ing with blood flow determinations. Routine postoperative
care was provided, and each animal was familiarized with
treadmill running for 4–5 days before its first exercise test.
Experimental protocol
The miniature swine (n = 6) ran on a motorized treadmill
for 13 min [5.2 km/h corresponding to an estimated ~60 %
of VO2max (McAllister et al. 2008)]. After 5 min of run-
ning, the P2 receptor antagonist RB2 (Alexis Biochemicals,
San Diego, CA) was infused at a rate of 5.6 ± 0.3 mg/min
for 5 min. Arterial blood sampling and blood flow determi-
nation (see below) were performed before (3–5 min) and
after (11–13 min) RB2 administration (Fig. 1).
The selected dose of RB2 was found to induce the best
inhibition to femoral arterial ATP-induced vasodilation
in resting Danish farm pigs without altering resting car-
diac output (4.7 ± 0.7 and 4.6 ± 0.7 L/min during control
Eur J Appl Physiol
1 3
and RB2, respectively; thermodilution), MAP (85 ± 1 and
87 ± 1 mmHg, respectively) and hindlimb blood flow
(0.68 ± 0.07 and 0.63 ± 0.05 L/min, respectively; ultrasound
Doppler) (n = 3) (Mortensen SP & Hellsten Y, Unpublished
observations). To test the efficacy of this dose of RB2 under
similar experimental conditions, ATP infusion into an inter-
nal thoracic artery (30 µmol/min; Sigma) was performed at
rest in five miniature swine. The selected dose of ATP was
the highest that could be infused systemically while maintain-
ing the pigs in a resting, standing position. Blood sampling
and blood flow determination were performed during base-
line conditions and during ATP infusion before (3–5 min)
and after (11–13 min) RB2 administration. Arterial pressures
were monitored with transducers positioned at the level of the
heart (Pressure Monitoring Kit, Baxter, Deerfield, IL). Heart
rate was calculated from the arterial blood pressure curve.
Regional blood flow determination
The radiolabeled-microsphere technique was used to deter-
mine tissue blood flows with the animal standing on the
treadmill before exercise, as well as during submaximal
running as described previously (McAllister et al. 2008).
In brief, for each blood flow determination, microspheres
labeled with 46Sc, 103Ru, or 141Ce (15 µm diameter; Perkin
Elmer) in 1.0 mL of saline with Tween 80 were infused via
the left atrial catheter. Microspheres were sonicated and
vortexed before infusion; their infusion was followed by a
10-mL saline flush. A reference blood sample was obtained
simultaneously via the internal thoracic arterial catheter
using a syringe pump (Harvard Apparatus). The rate of
withdrawal of this reference blood sample was 4.12 mL/
min. After completion of the exercise test, the animal was
anesthetized (Thiopental, 10 mg/kg) and then euthanized
by removal of the heart. Tissue samples were then dis-
sected, weighed, and counted in a gamma counter (model
CompuGamma CS; LKB Wallac), and tissue blood flows
were calculated using standard procedures. For each tis-
sue, conductance was calculated as the quotient of blood
flow and mean arterial pressure. Viscera sampled included
the liver, spleen, pancreas, stomach, jejunum, colon, kid-
neys, and adrenals. Forelimb skeletal muscles sampled
included the triceps brachii lateral head [TltH; both deep
(D) and superficial (S) portions], triceps brachii accessory
head (TAH), triceps brachii medial head (TMH), triceps
brachii long head (TLH; both D and S portions), biceps
brachii (Bic Br), and brachialis (Brach). Hindlimb muscles
sampled included the vastus lateralis (VL; both D and S
portions), vastus intermedius (VI), vastus medialis (VM),
rectus femoris (RF; both D and S portions), biceps femoris
(Bic Fem), and semitendinosus (Semit). In addition, the left
and right ventricles, diaphragm, and lung were sampled.
Lactate and glucose concentrations were measured using
an EML 105 analyzer (Radiometer, Copenhagen, Denmark).
Plasma catecholamines concentrations were determined
with a radioimmunoassay (LDN, Nordhorn, Germany).
Statistical analysis
A one-way repeated measures ANOVA was performed to
test significance within trials. Following 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
mean ± SEM unless otherwise indicated.
Results
Effect of RB2 infusion on systemic and regional
hemodynamics during exercise
Skeletal muscle blood flow during exercise before and
after RB2 infusion
Mean blood flow during exercise from the 16 sampled skel-
etal muscle tissues was 138 ± 18 mL/min/100 g (Fig. 2).
Administration of RB2 lowered skeletal muscle hyper-
emia in 11 of the 16 sampled muscle tissues by ~25 %
(P < 0.05), whereas there was no change in skeletal muscle
blood flow with RB2 infusion in the remaining five muscle
tissues. Infusion of RB2 increased heart rate from 216 ± 12
to 230 ± 9 beats/min, whereas arterial blood pressure
remained unchanged (132 ± 12 and 126 ± 14 mmHg
Fig. 1 Experimental protocol.
The pigs ran on the treadmill
(5.2 km/h) or received arterial
ATP infusion (30 µmol/min) for
13 min. After 5 min of running/
infusion, reactive blue 2 was
infused for 5 min. Blood flow
was determined at rest (ATP
only) and after 3 and 11 min of
exercise/infusion
Eur J Appl Physiol
1 3
during control and RB2, respectively). Accordingly, skel-
etal muscle vascular conductance was reduced by ~21 % in
the 11 muscle sections that exhibited reduced blood flow
after RB2 administration (P < 0.05).
Cardiothoracic and visceral hemodynamics during exercise
before and after RB2 infusion
Reactive Blue 2 infusion lowered blood flows to the dia-
phragm muscle and kidneys (left and right) (P < 0.05),
whereas blood flow was increased in lung tissue (P < 0.05)
and remained unchanged in the remaining tissues (Fig. 3).
Vascular conductance was lower in the diaphragm and tended
to be lower in the right (P = 0.060) and left (P = 0.057) kid-
ney, but was increased in lung tissue (P < 0.05).
Blood variables during exercise before and after RB2
infusion
Reactive Blue 2 infusion increased arterial lactate
concentration from 1.6 ± 0.5 to 3.4 ± 0.6 mmol/L
(P < 0.05), whereas glucose remained unchanged. Plasma
noradrenaline concentrations increased from 3.0 ± 0.9 to
4.7 ± 0.4 nmol/L after RB2 infusion (P < 0.05).
Effect of ATP and RB2 infusion on systemic and skeletal
muscle hemodynamics
Skeletal muscle blood flow during baseline conditions
and during ATP infusion before and after RB2 infusion
Arterial infusion of ATP increased skeletal muscle perfusion
and vascular conductance values in 15 of the 16 sampled
muscle tissues (P < 0.05; Fig. 4), whereas arterial blood pres-
sure remained unchanged compared to baseline conditions
(117 ± 5 versus 123 ± 7 mmHg). After infusion of RB2,
skeletal muscle perfusion and vascular conductance values
were reduced compared with ATP infusion only (P < 0.05),
whereas blood pressure (130 ± 9 mmHg) was similar to ATP
alone. Compared to baseline conditions, skeletal muscle per-
fusion and vascular conductance values during ATP + RB2
were similar despite an increase in blood pressure (P < 0.05).
Cardiothoracic and visceral hemodynamics
during baseline conditions and during ATP infusion
before and after RB2 infusion
Arterial infusion of ATP increased blood flow in the ven-
tricles (left and right), spleen, pancreas, kidneys (left
Fig. 2 Skeletal muscle blood
flow and vascular conduct-
ance during treadmill running
(5.2 km/h) with and without P2
receptor blockade (reactive blue
2). TltH triceps brachii lateral
head, Sup superficial portion,
TAH triceps brachii accessory
head, TMH triceps brachii
medial head, TLH triceps
brachii long head, Bic Br biceps
brachii, Brach brachialis. VL
vastus lateralis, Sup superficial
portion, VI vastus intermedius,
VM vastus medialis, RF rectus
femoris, Bic Fem biceps femo-
ris, Semit semitendinosus. Data
are mean ± SEM for 6 pigs,
*P < 0.05
Eur J Appl Physiol
1 3
and right) and adrenals (left and right) (P < 0.05; Fig. 5),
whereas there was no change in blood flow to the lung, dia-
phragm, liver, stomach, small and large intestines. When
ATP was infused after RB2 infusion, blood flow to the ven-
tricles and kidney (left) was reduced (P < 0.05), whereas
there was no change in the remaining visceral tissues.
ATP infusion increased heart rate (106 ± 8 versus
127 ± 10 beats/min; P < 0.05), whereas there was no
change in heart rate (122 ± 11 beats/min) after RB2
infusion.
Discussion
This study investigated the role of P2 receptors in skel-
etal muscle blood flow regulation in exercising miniature
swine. The results show that blockade of P2 receptors low-
ers exercise hyperemia and abolishes the ATP-induced
increases in muscle blood flow. These observations indicate
that nucleotides play an important role in skeletal muscle
blood flow regulation and that P2 receptors are essential for
ATP-induced vasodilation in miniature swine.
This study is the first to examine the role of nucleotide
sensitive purinergic P2 receptors in exercise hyperemia.
The measured blood flows to the muscles during control
exercise in the current study are similar to those reported
previously in miniature swine running at similar speeds
(Laughlin et al. 1989; McAllister et al. 2008). When P2
receptors were inhibited, it was found that skeletal mus-
cle exercise hyperemia was lower in 11 of the 16 sampled
muscle tissues without altering arterial pressure. Three of
the five muscle samples not showing a significant decrease
in BF were the slow-twitch postural muscles of the triceps
brachii (medial head, THM and accessory head, TAH) and
quadriceps muscle. This may suggest that the P2 inhibi-
tion had less effect on the postural muscles with the great-
est slow-twitch fiber type composition. ATP and RB2
had similar vascular effects in all of the sampled muscle
tissues, suggesting that all tissues express P2 receptors.
However, the relative importance of ATP in the control
of blood flow could differ with fiber type composition in
skeletal muscle vascular beds due to differences in redun-
dancy and contribution of other vasodilator systems. For
example, it has been reported that treatment of pigs with
Fig. 3 Cardiac and visceral
blood flows and vascular
conductance during treadmill
running (5.2 km/h) with and
without P2 receptor block-
ade (reactive blue 2). LV left
ventricle, RV right ventricle,
Dia diaphragm, Pan pancreas,
Stom stomach, Int intestines,
Kid kidney, Adr adrenal. Data
are mean ± SEM for 6 pigs,
*P < 0.05
Eur J Appl Physiol
1 3
dipyridamole (which blocks cellular uptake of adenosine)
produced increases in blood flow during exercise at 70 %
maximal only in the heart, diaphragm, medial head of the
triceps muscle (Laughlin et al. 1989). In the current study,
inhibition of P2 receptors did not significantly decrease
blood flow in the deep long head of triceps, the medial
head of the triceps or in the heart, thus, it is possible that
these muscles have a greater contribution of adenosine for
vasodilation during exercise than the other muscles stud-
ied and this allows them to maintain blood flow during P2
receptor inhibition. Also, the muscle recruitment pattern
may change when blood flow is blunted by inhibition of
P2 receptors and/or other compensatory mechanisms may
maintain blood flow when P2 receptors are inhibited.
Indirect evidence from in vitro (Ellsworth et al. 1995;
Dietrich et al. 2000; McCullough et al. 1997) and in vivo
(González-Alonso et al. 2002; Rosenmeier et al. 2004;
Kirby et al. 2008; Mortensen et al. 2009a, b) studies indi-
cate that intraluminal ATP could play an important role in
skeletal muscle hyperemia, but the effect of blocking ATP-
sensitive receptors on skeletal muscle exercise hyperemia
has not previously been examined. ATP-sensitive P2Y2 and
P2X1 receptors are located in the endothelium of human
skeletal muscle (Mortensen et al. 2009a). The present data
confirm that P2 receptors play an important role in mediat-
ing ATP-induced vasodilation as RB2 abolished the vaso-
dilatory response to ATP (Hammer et al. 2003) and also
suggest that the signaling pathway of ATP was inhibited
during exercise. Antagonists that are specific to ATP-sen-
sitive receptors are currently not available and we therefore
used RB2. Reactive blue 2 has been reported to selectively
inhibit P2Y receptors within a certain plasma concentra-
tion range (Burnstock and Warland 1987), but it could also
inhibit P2X receptors (Brunschweiger and Müller 2006)
(Ralevic and Burnstock 1998; Abbracchio et al. 2006). In
the present study, we chose the RB2 dose that produced the
largest inhibition of ATP-induced vasodilation and plasma
RB2 levels could not be determined. Thus, not only P2Y
receptors but also P2X receptors may have been inhib-
ited in the present experimental conditions. Furthermore,
RB2 may also inhibit ecto-ATPase (Chen et al. 1996) and
thereby alter plasma ATP levels. Apart from ATP, the other
nucleotides (ADP, UTP and UDP) also act via P2 receptors
and may therefore also have contributed to P2 receptor acti-
vation during exercise. However, although a previous study
has shown that plasma ATP and ADP increase to similar
levels during exercise (Yegutkin et al. 2007), ATP is a >30
times more potent vasodilator than ADP (Rosenmeier et al.
Fig. 4 Skeletal muscle blood
flow and vascular conductance
during basal conditions and
arterial ATP infusion (30 µmol/
min) with and without P2 recep-
tor inhibition induced by RB2
infusion. TltH triceps brachii
lateral head, Sup superficial por-
tion, TAH triceps brachii acces-
sory head, TMH triceps brachii
medial head, TLH triceps
brachii long head, Bic Br biceps
brachii, Brach brachialis. VL
vastus lateralis, D deep portion,
S superficial portion, VI vastus
intermedius, VM vastus media-
lis, RF rectus femoris, Bic Fem
biceps femoris, Semit semiten-
dinosus. Data are mean ± SEM
for 5 pigs, asterisk signifies dif-
ference from basal conditions,
P < 0.05. Section sign different
from ATP alone. P < 0.05
Eur J Appl Physiol
1 3
2008). In addition, resting plasma UTP levels are ~tenfold
lower than ATP, and UTP levels have only been shown to
increase during pathological conditions (Wihlborg et al.
2006). Collectively, these observations therefore sug-
gest that ATP is the main agonist of P2 receptors during
exercise.
The vascular effects of RB2 were not confined to skel-
etal muscle, but also lowered renal and diaphragm blood
flow and increased lung tissue perfusion. Purinergic recep-
tors are widely distributed across organs but systematic
comparisons of P2 receptor localization and quantity in
different tissues have not been performed. The involvement
of purinergic receptors in renal function is well known, but
nucleotides have been reported to induce both vasodilation
and constriction depending on species and experimental
setup (Inscho 2009). In the present experimental condi-
tions, P2 receptors were found to play a role in mediating
vasodilation in the renal microcirculation and it was also
observed that ATP induces vasodilation in kidney tissue.
The latter is consistent with observations in dogs where
ATP appears to stimulate NO formation (Majid et al. 1999).
In contrast to muscle and kidney tissue, blood flow in the
lung was increased after RB2 infusion. Nucleotides have
been suggested to play an important role in regulating the
pulmonary vascular tone by both vasodilation (Sprague
et al. 1996) and vasoconstriction (Rubino and Burnstock
1996; Mitchell et al. 2012). The present results support a
primary role of nucleotides in inducing pulmonary vaso-
constriction during exercise. The increased lung perfusion
values appear not to be a direct effect of RB2, as it did not
occur during the ATP infusion trial, but it is possible that
increased peripheral arterio-venous shunting during exer-
cise in the RB2 trial could have resulted in an overestima-
tion of the perfusion values (Duncker et al. 2000).
In vivo and in vitro evidence suggest that blood flow to
skeletal muscle involves a complex interaction among sev-
eral vasodilator systems including adenosine (Rådegran
and Calbet 2001), NO and prostacyclin (Saunders et al.
2005; Schrage et al. 2006; Mortensen et al. 2007; Hellsten
et al. 2012a). The present data suggest that ATP also con-
tributes to this regulation. Both ATP and adenosine induce
vasodilation by increasing NO and prostacyclin formation
(Mortensen et al. 2009a, c; Smits et al. 1995), although
adenosine appears to be more NO and prostacyclin depend-
ent (Mortensen et al. 2009a, c). However, when NO and
prostacyclin formation is inhibited simultaneously, exer-
cise hyperemia is reduced by only ~30–35 % (Mortensen
et al. 2007; Saunders et al. 2005), suggesting that other
Fig. 5 Cardiac and visceral
blood flows and vascular con-
ductance during basal condi-
tions and arterial ATP infusion
(30 µmol/min) with and without
P2 receptor inhibition induced
by RB2 infusion. LV left
ventricle, RV right ventricle,
Dia diaphragm, Pan pancreas,
Stom stomach, Int intestines,
Kid kidney, Adr adrenal. Data
are mean ± SEM for 5 pigs,
asterisk signifies difference
from basal conditions, P < 0.05.
Section sign different from ATP
alone. P < 0.05
Eur J Appl Physiol
1 3
mechanisms also are involved. ATP could also contribute
to blood flow regulation by inhibiting sympathetic vaso-
constriction [functional sympatholysis (Remensnyder et al.
1962)] (Rosenmeier et al. 2004; Kirby et al. 2008), but it
remains unknown if this mechanism is P2 receptor depend-
ent. Furthermore, the role of endothelium-derived hyper-
polarizing factors (EDHFs) in ATP and exercise hyperemia
remains unclear (Hillig et al. 2003; Mortensen et al. 2007;
van Ginneken et al. 2004). Finally, compensatory mecha-
nisms may exist that maintain blood flow when a system
is inhibited. Inhibition of P2 receptors did not alter cardiac
tissue blood flow, despite the proposed effects on nucleo-
tides on cardiac function (Erlinge and Burnstock 2008).
However, heart rate and circulating norepinephrine concen-
trations were increased after RB2 infusion during exercise,
which is likely to have increased myocardial demand for
O2 and thus blood flow.
We used RB2, a non-selective P2 receptor antagonist
and therefore we cannot discriminate between the relative
importance of the nucleotides (ATP, ADP, UTP and UDP)
(Burnstock and Ralevic 2014). In humans, plasma ATP
and ADP increase to similar levels during exercise (Yegut-
kin et al. 2007) but ATP is a >30 times more potent vaso-
dilator than ADP (Rosenmeier et al. 2008). Also, resting
plasma UTP levels are ~tenfold lower than ATP, and UTP
levels have only been shown to increase during pathologi-
cal conditions (Wihlborg et al. 2006). Therefore, it appears
likely that ATP is the main agonist of P2 receptors during
exercise.
In conclusion, the present data obtained in exercising
miniature swine show that P2 receptors play an important
role in skeletal muscle blood flow regulation and indicate
that ATP is the main activator of P2 receptors during exer-
cise. When more selective antagonists become available,
the specific role of ATP, P2Y and P2X receptors in this reg-
ulation remains to be determined.
Acknowledgments This work was supported by a grant from the
Novo Nordisk foundation. The technical assistance of Cory Weimer,
Dave Harah and Denise Holiman is gratefully acknowledged.
Conflict of interest None.
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