PHYSIOLOGICAL REVIEWS
Vol. 81, No. 2, April 2001
Printed in U.S.A.
Adenosine 5⬘-Triphosphate: a P2-Purinergic
Agonist in the Myocardium
GUY VASSORT
Institut National de la Santé et de la Recherche Médicale U. 390, Centre Hospitalier Universitaire
Arnaud de Villeneuve, Montpellier, France
I. Introduction
II. Extracellular Adenosine 5⬘-Triphosphate: Sources, Metabolism, and Receptors
A. Sources of extracellular ATP
B. Mechanisms of ATP release
C. ATP degradation by ecto-ATPases
D. Sources and metabolism of UTP and diadenosine polyphosphates
E. ATP binding sites
F. Ionotropic P2X receptors
G. Metabotropic P2Y receptors
H. Adenine dinucleotide receptors
I. Pharmacological concerns
III. Physiological and Physiopathological Effects of Extracelluar Adenosine 5⬘-Triphosphate
A. Contractile effects
B. Chronotropic effects
C. Pro- and anti-arrhythmic effects of ATP
D. Hypertrophy
E. Other general effects of ATP in cardiac cells
F. Effects of diadenosine polyphosphates on heart
IV. Molecular and Cellular Mechanisms
A. Modulation of cardiac transmembrane ionic currents
B. Signal transduction pathways
V. Concluding Remarks
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Vassort, Guy. Adenosine 5⬘-Triphosphate: a P2-Purinergic Agonist in the Myocardium. Physiol Rev 81: 767– 806,
2001.—ATP, besides an intracellular energy source, is an agonist when applied to a variety of different cells including
cardiomyocytes. Sources of ATP in the extracellular milieu are multiple. Extracellular ATP is rapidly degraded by
ectonucleotidases. Today ionotropic P2X1–7 receptors and metabotropic P2Y1,2,4,6,11 receptors have been cloned and their
mRNA found in cardiomyocytes. On a single cardiomyocyte, micromolar ATP induces nonspecific cationic and Cl⫺
currents that depolarize the cells. ATP both increases directly via a Gs protein and decreases Ca2⫹ current. ATP activates
the inward-rectifying currents (ACh- and ATP-activated K⫹ currents) and outward K⫹ currents. P2-purinergic stimulation
increases cAMP by activating adenylyl cyclase isoform V. It also involves tyrosine kinases to activate phospholipase C-␥
to produce inositol 1,4,5-trisphosphate and Cl⫺/HCO⫺
3 exchange to induce a large transient acidosis. No clear correlation
is presently possible between an effect and the activation of a given P2-receptor subtype in cardiomyocytes. ATP itself
is generally a positive inotropic agent. Upon rapid application to cells, ATP induces various forms of arrhythmia. At the
tissue level, arrhythmia could be due to slowing of electrical spread after both Na⫹ current decrease and cell-to-cell
uncoupling as well as cell depolarization and Ca2⫹ current increase. In as much as the information is available, this review
also reports analog effects of UTP and diadenosine polyphosphates.
I. INTRODUCTION
Interest in ATP as a molecule was for many years
devoted to the concept of the “high-energy phosphate
bond” introduced by Lippman (292); this despite the fact
http://physrev.physiology.org
that roles for extracellular purines had been described by
Drury and Szent-Györgyi in 1929 (137) soon after the
discovery of ATP (144, 295). In fact, the crude tissue
extracts used by Drury and Szent-Györgyi contained
mostly AMP and induced a negative chronotropic effect
0031-9333/01 $15.00 Copyright © 2001 the American Physiological Society
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GUY VASSORT
and a decrease in blood pressure. ATP was later shown to
produce transient tachycardia at low doses or to slow
heart and to induce atrioventricular block at higher doses
(207, 454). These effects were associated with vasodilatation of coronary vessels and led Berne (36) to postulate
that adenosine was the mediator. Adenosine then became
the topic of most studies concerning purines and the
cardiovascular system.
ATP is an amphiphilic compound showing both hydrophilic and strong hydrophobic interaction. Adenine, its
6-aminopurine base, has pKa ⬍1, 4.1, and 9.8. Like free
fatty acids, ATP binds to BSA, which is generally used to
mimic protein solutions and establish an oncotic pressure. Up to 94%, but not all, ATP could be liberated from
BSA by the addition of palmitate. Interaction of BSA with
AMP is considerably weaker (21). ATP strongly binds
divalent cations such as to form ⬃70% MgATP2⫺, 22%
CaATP2⫺, and 2.4% free ATP4⫺ in a physiological solution
(441). The diffusion coefficient of Na2ATP in water at
25°C is 7.01 ⫻ 10⫺6 cm2/s not far from 7.54 ⫻ 10⫺6 cm2/s
for CaCl2 (196).
Autonomic nonadrenergic and noncholinergic nerves
(71) were suggested to contain ATP (72), and a tentative
model of storage and release was proposed (68). Nerves
utilizing ATP as their principal transmitter were named
“purinergic” (67). Based on the actions of purine nucleotides and nucleosides in a wide variety of tissues, Burnstock (69) proposed the P1- and P2-receptor classification, which was then extended to the heart (74). P1
purinoceptors are much more sensitive to adenosine and
AMP than to ADP and ATP. The reverse is true for P2
purinoceptors. P2 receptors are unaffected by methylxanthines such as caffeine and theophylline that selectively
and completely inhibited the P1 purinoceptors. Subclasses of P1 adenosine receptors were introduced when
their stimulation was shown to inhibit (A1 subtype) or
activate (A2 subtype) adenylyl cyclase activity. A similar
lack of homogeneity of P2 receptors was indicated by the
use of phosphate-modified ATP and ADP (166, 302); this
was reinforced by the observation that apamin, a K⫹channel blocker, antagonizes the inhibitory action of ATP
in guinea pig cecum and stomach but not its excitatory
action in guinea pig bladder and uterus (439). The first
clear subdivision of P2 receptors proposed P2X purinoceptors that mediate vasoconstriction with ␣,␤-methylene
adenosine 5⬘-triphosphate (␣,␤-met-ATP) as a potent agonist, and P2Y purinoceptors that mediate vasodilatation
with 2-methylthioadenosine 5⬘-triphosphate (2-MeSATP)
as a potent agonist (73).
P2 purinoceptors were initially classified according
to pharmacological studies. This pharmacological nomenclature has now been replaced by molecular-based nomenclature. The first defined subtypes according to pharmacological criteria include the following: P2X for
excitatory and P2Y for inhibitory, then P2U for those also
Volume 81
activated by UTP and P2T at which ADP induces platelet
aggregation, finally P2Z for a large molecular pore activated by ATP4⫺ and P2D which is activated by dinucleotides. The second system is based on the cloning of P2
receptors where, the IUPHAR, Committee on Receptor
Nomenclature (164, 165), has defined as P2X1-n the ligandgated ion channel and as P2Y1-n the G protein-coupled
receptors. Very slow progress has been made to establish
the correspondence of the cloned to the pharmacologically defined P2 subtypes. This has proven difficult to
realize in part because some P2 purinoceptors are functional heteroligomers and in part because the molecule
binding to a given receptor is often a hydrolysis product
of that which was added to the bath. Moreover, this field
is characterized by the paucity of receptor subtype-selective agonists and antagonists that have their own propensity to undergo rapid hydrolysis and interconversion just
like the natural agonists. It is also a problem that commercially available compounds might already contain
breakdown products that act as effective agonists (42,
198, 342, 513), a point already considered in 1934 by
Gillepsie (180).
After a characterization of the sources and extracellular metabolism pathways of ATP and the various purinergic receptors known today, the present review
concentrates on the mechanical, chronotropic, and
arrhythmogenic effects of ATP at the cellular level and
under the whole heart as well as in pathological conditions. These pieces of information are then interpreted on
the basis of our present knowledge concerning the electrophysiological consequences and signal transduction
pathways activated by applying ATP to isolated cardiomyocytes. Inasmuch as it is possible, similar pieces of
information are given for UTP and diadenosine polyphosphastes. Much more is known about the effects of ATP in
wider aspects of cardiovascular system, and this has been
extensively reviewed elsewhere (59, 143, 191, 272, 355,
402, 488).
II. EXTRACELLULAR ADENOSINE
5ⴕ-TRIPHOSPHATE: SOURCES,
METABOLISM, AND RECEPTORS
A. Sources of Extracellular ATP
Adenine nucleotides are continually present in quite
variable amounts in the extracellular space of the heart.
More precise knowledge of ATP exocytosis could be expected with the development of new bioluminescence
technologies (464) involving cell surface-attached firefly
luciferase (28) or atomic force microscopy in combination with myosin functional cantilevers (431). Basal ATP
found in the coronary effluent from isolated, saline-perfused hearts had been shown to range below 1 nM (54,
April 2001
ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
491). This rather low value reflects the rapid degradation
of ATP, which is over 95% during a single coronary passage. With the use of microdialysis, ATP in the interstitial
space has been estimated to be 40 nM (276). These levels
increase markedly during electrical stimulation as already
noted in 1962 (1): hypoxia or ischemia (36, 163, 276, 359,
491, 519), challenge by cardiotonic agents (54, 117, 244,
491), increased blood flow (117, 492), mechanical stretch
(483), and even work load in frog heart (136).
There is strong evidence supporting the notion that
ATP is a cotransmitter in perivascular sympathetic nerves
(68). Other likely sources of the nucleotide include the
following: 1) ischemic myocytes (36, 163, 359, 519), 2)
activated platelets (118, 209, 328), 3) nerve terminals (70,
210, 408), 4) inflammatory cells (133), 5) erythrocytes (33,
141, 449), 6) endothelial cells (46, 401, 526), 7) smooth
muscle cells (45, 245, 369), and 8) exercising muscle cells
(162, 364).
B. Mechanisms of ATP Release
The mechanism by which ATP is transported across
the cell membrane or released from muscle cells is not
fully understood. In living cells, the electrochemical gradient favoring ATP efflux is near nine orders of magnitude
(versus 6 for Ca2⫹) considering intra- and extracellular
ATP concentrations to be 10 mM and 10 nM, respectively,
a cell resting potential of ⫺90 mV and MgATP2⫺ as the
preferred flowing anion. Thus membrane permeability to
ATP should be very low, which is in agreement with the
molecular size of ATP.
ATP can be released by exocytosis from platelets
and nerves like other neurotransmitters. It can also
leak out during cell lysis (139, 182). In recent years, the
cystic fibrosis transmembrane conductance regulator
(CFTR) has been suggested to act as an ATP channel
and enables intracellular ATP to cross the cell membrane (2, 3, 6, 366, 432, 509). This is strongly disputed
(see Ref. 123 for review), and it has been shown that
mechanical stimulation of the cell surface, rather than
CFTR activation, is sufficient to release ATP (510). A
recent work (457) suggests that the permeation pathways for Cl⫺ and ATP are distinct, in which case CFTR
would be a regulator of the ATP channel. Identification
of the molecular basis of the CFTR-associated ATP
channel is of critical interest.
Supporting the idea that an anion channel can allow
for ATP to cross through membranes, it has been recently
reported that the mitochondrial voltage-dependent anion
channel (412) as well as the 116-pS Cl⫺ channel in cardiac
sarcoplasmic reticulum (SR) conduct ATP and adenine
nucleotides (247).
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C. ATP Degradation by Ecto-ATPases
The breakdown of ATP and ADP to form adenosine
was first reported nearly 50 years ago (235), and it is long
recognized that this susceptibility to degradation does
limit the potency of ATP and its degradable analogs (515).
In considering the biological responses to extracellular
ATP in multicellular tissues and even isolated cells, it
should be noted that such effects are not only complicated by the presence of multiple P2-purinergic receptors
but also by the rapid catabolism of ATP to produce adenosine. This cascade of surface-located enzymes converts
P2 into P1 signaling. The extracellularly formed adenosine can itself modulate cardiac functions and also serve
purine salvage after reuptake via plasma membrane-located adenosine transporters (471). The majority of the
ATP perfused into the heart is dephosphorylated during a
single passage through the coronary vasculature (29, 158,
359). The half-life of ATP is 0.2 s when perfused in blood
through the lung vasculature (413) instead of ⬃10 min in
whole blood ex vivo (479). It is likely that this catabolism
is mostly due to endothelial cell ectonucleotidases. This
hydrolysis of ATP, ADP, and AMP was first demonstrated
in isolated rat myocytes by Bowditch et al. (56), but the
nature and characteristics of the enzymes were not studied. Kinetic properties of the extracellular reaction sequences ATP 3 ADP 3 AMP 3 adenosine catalyzed by
ectonucleotidases has been investigated at the surface of
adult rat cardiomyocytes by following the catabolism of
3
H-labeled nucleotides (158, 324) and by NMR (35) or in
the ventricular muscle by microdialysis (276). It is worth
noting that a significant ATP catabolism occurs even on
isolated cardiomyocytes. Thus ATP is rapidly hydrolyzed
to 65 and 33% after 3 and 15 min, respectively, by plasma
membrane at 20°C (42).
A variety of extracellular enzymes utilize ATP to
induce biological responses (256, 324, 325, 368, 373, 541,
542) (Table 1). In many tissues including heart, extracellular ATP as well as other nucleotide tri- and diphosphates appear to be hydrolyzed by E-type enzymes. They
include ecto-ATPases and ecto-ATP diphosphohydrolases
or ecto-ATPDases. All (but the ␣-sarcoglycan or Adhalin)
lack the Walker consensus ATP binding motif; instead,
they have several apyrase conserved domains, a putative
ATP ␤-phosphate 1 binding motif, found in proteins as
diverse as actins, 70-kDa heat shock protein, and sugar
kinases. Ecto-ATPases are insensitive to known inhibitors
of various intracellular ATPases such as ouabain and
vanadate (P-type) or N-ethylmaleimide (V type) and F⫺
for the alkaline phosphatase. The turnover number of the
ecto-ATPases is estimated to be 500,000 molecules/min, a
value 50 times higher than the value of the Na⫹-K⫹ATPase and two orders of magnitude higher than the one
of the SR Ca2⫹-ATPase (380). These enzymes are activated by either Ca2⫹ or Mg2⫹ and inhibited however at
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TABLE
GUY VASSORT
Volume 81
1. Ecto-nucleotidases and enzymes involved in the extracellular hydrolysis of nucleotides
Names
Ecto-ATPDase
ATP diphosphohydrolase
Ecto-apyrase, CD39
Ecto-ATPase
Ca2⫹-Mg2⫹-ATPase (myoglein)
t-Tubule Mg2⫹-ATPase
Adhalin, ␣-sarcoglycan
Ecto-nucleotide pyrophosphate/phosphodiesterase
(autotaxin)
Ecto-alkaline phosphatase
Ecto-nucleotide-diphosphate kinase (NDPK)
Ecto-protein kinase, CK1CK2
Catalytic Properties*
Inhibitors
Reference Nos.
ATP 3 AMP ⫹ PPi
Azide
Evans blue
25, 146, 326
238, 249
ATP 3 ADP ⫹ Pi
Suramin
RB2 PPADS
ATP analogs
BzATP, ADP
PPADS
257
128, 324, 480
26, 119, 249, 415
38
93, 181
ATP 3 ADP ⫹ Pi
ATP 3 AMP ⫹ PPi
cAMP 3 AMP
ATP 3 ADP ⫹ Pi
ADP 3 AMP ⫹ Pi
AMP 3 adenosine ⫹ Pi
ATP ⫹ NDP 7 ADP ⫹ NTP
ATP 3 ADP ⫹ PO⫺
4
518
279
85, 271
* All enzymes hydrolyze not only adenine nucleotides but all purines and pyrimidine nucleotides. An ecto-adenylate kinase that catalyzes ATP
⫹ AMP 3 ADP ⫹ ADP has not yet been reported. Bold references relate to cardiac tissues. RB2, reactive blue 2; PPADS, pyridoxal-phosphate-6azophenyl-2⬘,4⬘-disulfonic acid; BzATP, 3⬘-O-(4-benzoyl)benzoyl ATP.
near millimolar concentration by Cu2⫹, Zn2⫹, or La3⫹
(539). Aluminium is present at 1–10 ␮M in the extracellular fluid and is bound by ATP with a significantly higher
affinity than either Ca2⫹ or Mg2⫹; it has, however, no
effect on the hydrolysis of ATP by the enzymes (266).
Ecto-nucleotidases have been proposed to be coreleased
with ATP from nerve terminals (472).
Ecto-ATPDase is a highly glycosylated protein with
six potential N-linked glycosylation, five apyrase-conserved regions, and two potential transmembrane domains. Both NH2 and COOH terminals are short with
COOH terminals demonstrating phosphorylation consensus sites (92, 304). This 82-kDa protein is identical to a
lymphoid cell activation antigen CD39, an ecto-apyrase
(227, 238, 507). It hydrolyzes nearly equally ATP and ADP
to produce PPi and Pi, respectively. It is inhibited by azide
(265). Ecto-ATPDase has been found in heart tissues by
Northern hybridization (238, 249). It has also been purified from the bovine heart and immunocytochemically
localized in Purkinje and ventricular cells (25). Using an
ATPDase purified from the bovine aorta, the same group
demonstrated that the ATP analogs modified on the phosphate chain were not hydrolyzed at variance with those
modified on the purine base. Thus 2-chloro-ATP, 2-MeSATP, and 8-bromo-ATP demonstrated similar Michaelis
constant (Km) and maximal hydrolysis rate (16 –20
␮mol 䡠 min⫺1 䡠 mg protein⫺1) (377).
Ecto-ATPases are millimolar cation-dependent, lowspecificity enzymes that hydrolyze all nucleotide triphosphates (NTPases). A microsomal Mg2⫹-ATPase has originally been found in low-density vesicles that contained
plasma membrane of various rat tissues with the following properties: 1) Km for ATP of 0.2 mM; 2) ATP-induced
inactivation of the enzyme prevented by concanavalin A;
3) hydrolysis of all NTP tested but no nonnucleotide
phosphocompounds; and 4) Ca2⫹, Mn2⫹, Zn2⫹, or Co2⫹
could substitute for Mg2⫹, but Ca2⫹, Sr2⫹, Ba2⫹, or Be2⫹
could not fulfill the bivalent cation requirement (26). They
can be distinguished from ecto-ATPDases by their inability to hydrolyze ADP and other nucleotide diphosphates
at a rate ⬎1–2% that of their ATP hydrolysis rate. Also,
they are insensitive to azide. However, 8-azido-ATP is a
good substrate of ecto-ATPase activity while it is an irreversible partial inhibitor after photoactivation (410). Another difference with ecto-ATPDases is fast inactivation
of their ATP hydrolysis activity, an effect prevented by
concanavalin A, a known inhibitor of 5⬘-nucleotidase
(368). The first vertebrate ecto-ATPase was cloned and
sequenced from the chicken muscle (257). It has a molecular mass of 54.4 kDa. Ecto-ATPases share some homologies with CD39 ecto-ATPDases in that they have two
transmembrane domains, four external putative glycosylation sites, and four apyrase-conserved regions. Like ectoATPDases, they do not show Walker consensus sequence.
The COOH terminal contains a single putative cAMP/
cGMP-dependent protein kinase phosphorylation site as
well as a single putative tyrosine kinase phosphorylation
site (257).
A Ca2⫹/Mg2⫹ ecto-ATPase activity has long been reported in the plasma membrane of cardiac myocytes (128,
414, 480, 531). Rat cardiac sarcolemmal Ca2⫹/Mg2⫹ ectoATPase (Myoglein) requires millimolar concentrations of
Ca2⫹ or Mg2⫹ for maximal ATP hydrolysis. It has been
purified to apparent homogeneity, and a partial cDNA
clone has been produced that revealed 100% homology to
human platelet CD36 (241). In its native form, this enzyme
has a molecular mass of 180 kDa and two subunits of ⬃90
kDa each (240). The limited sequence information indicates that there is no sequence similarity with any of the
previously reported ecto-ATPases, chicken gizzard
smooth muscle ecto-ATPase (257), or CD39 (242, 507),
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ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
but partial homology with SR and sarcolemmal pump
Ca2⫹-ATPases.
␣-Sarcoglycan (Adhalin) has recently been shown to
have ecto-ATPase activity in skeletal muscle (38). It is
different from the t-tubule ecto-ATPase of 56 kDa (119).
Its deduced amino acid sequence for ATP binding reveals
two Walter consensus sequences present in several intracellular ATPases. It binds ATP in a Mg2⫹-dependent,
Ca2⫹-independent manner. ATP binding is inhibited by
3⬘-O-(4-benzoyl)benzoyl ATP (BzATP). The authors thus
suggest that ␣-sarcoglycan also modulates the activity of
P2X (P2X7) receptor by buffering the extracellular ATP
concentration (38).
An ecto-nucleoside diphosphokinase (NDPK) has
also been described but is, as yet, not reported in cardiac
muscle. It controls the interconversion of P2-receptor
agonists with a rate of extracellular transphophorylation
up to 20-fold the rate of nucleotide hydrolysis (279). The
latter can just couple ATP- and UTP-dependent stimulations. Other ecto-enzymes that also catalyze the hydrolysis of ATP such as alkaline phosphatase (518) or autotaxin, a nucleotide phosphodiesterase/ATP pyrophosphatase
(93, 181), have not yet been studied in cardiac tissues.
Furthermore, the ATP degradation product AMP will
be hydrolyzed to adenosine by 5⬘-nucleotidase, a phosphatase active mostly at alkaline pH (25, 146, 326, 377,
380, 433).
The kinetic properties of the ATP hydrolysis catalyzed by ectonucleotidases at the surface of cardiomyocytes were characterized (324). It is worthy of noting that
the activity of these ecto-nucleotidases is modulated by
Mg2⫹ (305), protein kinase C (PKC), and ␣1-adrenergic
stimulation (260, 262, 347, 420) as well as during preconditioning or postmyocardial infarction (263, 276, 329) and
also differs in isolated perfused versus in situ hearts
(276). The activity of the ecto-ATPase is not affected by
treadmill exercise training or by exhaustive exercise
(120). In isolated cardiomyocytes, exogenous adenosine,
via A1-receptor activation and a Gi protein, decreases both
ectosolic and cytosolic 5⬘-nucleotidase activity (261).
Intracellular protein kinases are important in the regulation of cellular functions. Protein kinases that use
extracellular ATP to phosphorylate proteins localized at
the external surface of the plasma membrane (ecto-protein kinases) have now been demonstrated in a variety of
tissues. Several surface proteins are reported to be phosphorylated in nerve cells and in aortic endothelial cells
(140, 142; see Ref. 139 for review). In addition to an
ecto-protein kinase with catalytic properties of atypical
PKC-␨ implicated in hippocampal long-term potentiation
(85), a cAMP-dependent kinase has been shown to phosphorylate the atrial natriuretic peptides (270, 271). In view
of the fact that ATP and cAMP are released in the extracellular space, one should expect a role for these ectokinases in the cardiac tissues.
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D. Sources and Metabolism of UTP
and Diadenosine Polyphosphates
Most P2Y receptors (P2Y2, 4, 6) and, maybe, some P2X
receptors are also activated by uracil nucleotides. This
suggests the presence of extracellular UTP and derivatives. Recently, it has been demonstrated that mechanical
stimulation induces a 15-fold increase in UTP release
from 1321 N1 human astrocytoma (278). A recent review
has appeared (10). Presently, little is known in heart. UTP
stimulates cardiomyocytes. Most studies deal with the
mechanisms of nucleoside plasma salvage leading to uracil nucleotide synthesis (175, 297). Total uracil nucleotides is 1.7 ␮mol/g protein in myocardium and in freshly
isolated cardiomyocytes much less than the ATP content
(27 ␮mol/g protein) in myocardium (176, 354).
Adenosine polyphosphates are a group of adenosine
dinucleotides that consist of two adenosine molecules
bridged by a variable number of phosphates thus abbreviated ApnA (n ⫽ 2– 6). They are ubiquitous molecules
found in prokaryotes and eukaryotes. Intracellular ApnAs
appear to alert the cell under stress conditions, thus their
name “alarmones.” The potency and efficacy of ApnAs to
inhibit ATP-activated K⫹ channel activity were described
in cardiac cells and appeared similar to those of intracellular ATP (236). Also, ApnAs stimulate a Ca2⫹-induced
Ca2⫹ release channel from skeletal muscle SR as well as
increasing the binding of ryanodine to the calcium release
channel by, respectively, nine- and threefold in skeletal
and cardiac muscles (206). Another alarmone receptor is
hemoglobin. ApnAs bind preferentially with high affinity
to the deoxy conformation of hemoglobin in a ratio of one
per tetramer. This binding markedly enhances the Bohr
effect (52). Recently, it has been reported that the mammalian myocardium contains abundant amounts of Ap5A
(237) or of Ap4A in humans (485). In mammalian tissues,
ApnA are produced and released from platelets or chromaffin cells where it is costored with ATP and catecholamines. Ischemia induces a 10-fold decrease in the
ApnA myocardial concentration (237, 264, 338). ApnAs are
degraded in rat plasma to ATP (from Ap5A) and AMP, and
these nucleotides are further degraded to adenosine.
However, compared with ATP, Ap3A and Ap4A have a
relatively long half-life (300). The rate of degradation is
dose dependent: the biological half-life was ⬃3 s at 1
mg/kg after intravenous infusion (253). The endothelial
ecto-diadenosine polyphosphate hydrolase of cultured adrenomedullary vascular endothelial cells is activated by
Mg2⫹ and Mn2⫹ and inhibited by Ca2⫹, adenosine 5⬘-O-(3thiotriphosphate) (ATP␥S), and suramin (308). These
compounds are considered to act as extra- and intracellular signaling molecules, and many of their properties are
close to those of ATP (22, 259, 353).
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E. ATP Binding Sites
Purified heart sarcolemma membranes were found to
bind a slowly hydrolyzable analog of ATP, [35S]ATP␥S, in a
specific manner and exhibited two apparent affinity sites
(532). The high-affinity site, which may represent the ATP
receptor according to the authors, had a dissociation constant (Kd) of 4.7– 8.3 nM and a maximal binding (Bmax) of
9.5–18.4 pmol/mg protein, whereas the low-affinity site had a
Kd of 655–1.227 nM and a Bmax of 812–2.955 pmol/mg protein. [35S]ATP␥S binding was displaced by GTP, UTP, CTP,
and ITP. The number of high-affinity binding sites decreases
during oxidative stress (334). Similar high- and low-affinity
binding sites for ␣,␤-[3H]met-ATP, then considered to be a
P2X agonist, were also reported in rat heart membranes
(327). In another study, 8-azido-ATP (8-Az-ATP) was used
for labeling intact rat ventricular myocytes. After minimizing
background labeling by large concentrations of UTP, two
bands, one near 48 kDa and a less pronounced at 90 kDa, are
labeled by radioactive 8-Az-ATP (177). 2-MeSATP and
ATP␥S both partially and specifically inhibited labeling of
the 48-kDa band. This was related to activation of the fast
Ca2⫹ transient by 2-MeSATP but not by ATP␥S and of the
Na-Pi cotransport by ATP␥S only. However, much caution
should be taken during these binding studies, not only because it would be difficult to distinguish sites with relatively
similar binding affinities but mostly because secured experimental conditions are difficult to settle. Optimal binding
conditions to obtain reliable equilibrium parameters require
a 15-min incubation, a time during which ⬎75% of the radioligand must remain intact, a situation not easily reached in
view of the high ecto-ATPase activies (42).
F. Ionotropic P2X Receptors
Several reviews directly aimed at P2X receptors have
appeared (17, 59, 64, 112, 149, 178, 349 –351, 403, 448, 459).
TABLE
P2X1
P2X2*
rP2X3
hP2X3
P2X4*
P2X5
P2X6
P2X7
P2X2/3
P2X1/5
P2X2/6
Volume 81
In this section I outline the characteristics of the P2X
receptors in as far as they help understanding the following focus of attention on cardiac cells and tissue. Particular attention is paid to recent publications not covered
by previous reviews. It is a general feature of all P2
purinoceptors to bind ATP4⫺, uncomplexed to cations,
whereas ATPMg2⫺ is the substrate of ATPases and kinases.
1. Characteristics
P2X receptors are ion channels opened by micromolar extracellular ATP. The seven subunits currently
known (Table 2) are encoded by different genes. Each
subunit has a topology that is fundamentally different
from that of other known ligand-operated channels. P2X
receptors are made of proteins, with 379 – 472 amino acids, inserted in the membrane to form a pore comprising
two hydrophobic transmembrane domains with a large
extracellular hydrophyllic loop. The putative extracellular
loop of cloned P2X1–7 has 10 conserved cysteine and 14
conserved glycine residues as well as two to six potential
N-linked glycosylation sites. Glycosylation of at least two
of these asparagine residues that sit on the extracellular
loop is needed for P2X2 receptor expression at the cell
surface and its function (339, 474, 475).
A) P2X1 RECEPTOR. In 1994, two joined papers in Nature
described a new family of ligand-gated ion channels defined by the P2X receptor (60, 487). Valera et al. (487) first
generated a unidirectional cDNA library from the
poly(A)⫹ RNA of vas deferens that was screened in oocytes after injection with RNA transcribed in vitro. The
cation-selective channel generated by expression of this
P2X1 receptor activates and desensitizes rapidly and
shows a relative high permeability for Ca2⫹. The order of
agonist potency is 2-MeSATP ⱖ ATP ⬎ ␣,␤-met-ATP ⬎
ADP, UTP, GTP; ACh and 5-hydroxytryptamine were ineffective. A recent report on P2X1 receptors in acutely
2. Ionotropic P2X purinoceptor: characteristics and presence in the heart
Amino
Acids
[ATP],
␮M
399
472
397
397
388
455
379
595
1
10
1
1
20
15
10
200
6
[␣,␤-Met-ATP],
␮M
2
⬎⬎100
1
1
100
⬎⬎100
⬎⬎100
⬎⬎100
1
5
12
TNT-ATP,
nM
1
⬎⬎100
1
⬎⬎100
⬎⬎100
1
PPADS,
␮M
2
2
1
⬎100⫹
2
⬎100
45
1
⬎100
Cation
Na⫹/K⫹/Ca2⫹
Na⫹/K⫹
Na⫹/K⫹
Na⫹/K⫹/Ca2⫹
Na⫹/K⫹
Na⫹/K⫹/Ca2⫹weak
Na⫹/K⫹/Ca2⫹
Pore
Na⫹/K⫹/Ca2⫹
Desensitization
Fast
Slow
Fast
Fast
Slow
Slow
Slow
Slow
Fast/slow
Heart
⫹
⫹
0
⫹
⫹(⫹)
⫹
⫺
⫺
⫺
⫺
⫺
Reference
Nos.
47, 348
348
47, 173
171, 348, 447
172
99
406, 460
84, 285
477
280
* Splice variants P2X2-2 (265 amino acids) (61) and P2X4a (361 amino acids) (478) have been found. ⫹ The agonist response of mouse ortholog
mP2X4 is potentiated by P2X antagonists (478). (⫹), Found in fetal but not adult. ␣,␤-Met-ATP, ␣,␤-methylene adenosine 5⬘-triphosphate; TNT-ATP,
2⬘,3⬘-O-(2,4,6-trinitrophenyl)adenosine 5⬘-triphosphate.
April 2001
ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
dissociated smooth muscle cells of the rat tail artery
proposes that UTP is an effective agonist but 100-fold less
potent than ATP (318). Both suramin and pyridoxal-phosphate-6-azophenyl-2⬘,4⬘-disulfonic acid (PPADS), but not
amiloride, block this current. The conductance, 19 pS at
negative potential, shows anomalous inward rectification.
These electrophysiological properties are close to those
observed in smooth muscle (24). The cloned 399-amino
acid-long protein is mostly extracellular and contains two
transmembrane domains plus a pore-forming motif that
bears a striking similarity with the P domain of the inward
rectifying K⫹ channel, Kv 2.1 (205). A similar molecular
architecture was reported for the mechanosensitive channels of Caenorhabditis elegans (211) and the related
amiloride-sensitive epithelial sodium channel (77). However, there is no primary sequence homology between
these channels and the P2X purinoceptor.
B) P2X2 RECEPTOR. Essentially the same structure and
properties were reported by Brake et al. (60) for a P2X2
channel cloned from a complementary DNA library constructed from PC12 mRNA screened in oocytes. The homomeric P2X2 channel is most readily characterized by its
insensitivity to ␣,␤-met-ATP and its lack of desensitization during ATP applications of up to 10 s. Its conductance is 21 pS at ⫺100 mV in 150 mM NaCl. The behavior
of this cloned P2X2 receptor resembles native P2X receptors on PC12 cells but differs from those on vascular
smooth muscle and vas deferens where ␣,␤-met-ATP acts
as a potent agonist. ATP, ATP␥S, and 2-MeSATP were
roughly equipotent agonists. CTP and 2⬘-deoxy-ATP
(dATP) elicited small currents, whereas UTP, GTP, ADP,
and AMP were inactive. As in neurons (96, 286), extracellular Zn2⫹ potentiates the ATP effect on the cloned receptor by shifting the EC50 for ATP to 15 ␮M. The intracellular COOH terminus of P2X2 receptor contains several
consensus sequences for phosphorylation by protein kinase A (PKA) and by PKC. After its reexpression in HEK293 cells, dialyzing with phorbol 12-myristate 13-acetate
(PMA) fails to alter the ATP-induced cationic current.
However, 8-bromo-cAMP or the purified catalytic subunit
of PKA causes a reduction in the magnitude of the ATPactivated current without altering its kinetics of inactivation or its reversal potential (90).
Both groups (60, 487) noticed the similarity of the
P2X1 and P2X2 receptors with a partial cDNA called RP-2
that was isolated from cells induced to die (358). This led
them to suggest that extracellular ATP might initiate programmed cell death as is known from the application of
ATP to various cells.
C) P2X3 RECEPTOR. A single cDNA (P2X3 receptor) was
obtained from a rat dorsal root ganglion library (84, 285).
Both ATP and ␣,␤-met-ATP evoked fast-activating and
rapidly desensitizing current. However, only the coexpression of P2X3 and P2X2 yielded ATP-activated currents
that closely resembled the ␣,␤-met-ATP-sensitive, nonde-
773
sensitizing current in sensory neurons. Direct evidence
for heteromeric assembly of P2X2 and P2X3 receptors was
provided by Baculovirus expression (400). That led to the
new idea that these channels are formed by subunit heteropolymerization at variance with the homomeric channels formed by the cloned P2X1 and P2X2 receptors (60,
487). It is notable that after expression of these cDNA, the
irreversible block by PPADS requires lysine at position
249 of P2X1. P2X3 lacks this lysine, and the inhibition by
PPADS is fast and shows rapid recovery (285).
D) P2X4 RECEPTORS. P2X4 was initially cloned from rat
brain (43, 447). It is distantly related to P2X1, P2X2, and
P2X3. Its expression gives an ATP-activated cation-selective channel that is highly sensitive to Ca2⫹ and whose
agonist sensitivity is increased by Zn2⫹ without altering
maximal response (447). Its conductance is 9 pS. This
ligand-gated channel is activated by ATP ⬎ ATP␥S ⬎ 2MeSATP ⬎ ADP ⬃ ␣,␤-metATP. However, a distinct characteristic is its insensitivity to the currently used P2Xpurinoceptor antagonists, suramin, PPADS, and reactive
blue 2 (RB2). Surprinsingly, however, the agonist response
of the mouse ortholog of the P2X4 receptor, mP2X4, is
potentiated by suramin, RB2, and in part by PPADS (478).
E) P2X5 AND P2X6 RECEPTORS. The ATP-induced current
after expression of the P2X5 receptor in HEK-293 cells
showed rapid activation with minimal desensitization and
a lack of effect of ␣,␤-met-ATP (99). Like with P2X1 and
P2X2, this current was inhibited by suramin and PPADS.
Together with P2X1, P2X5 forms an heteromeric receptor
(477). The same group (99) reported the cloning of the
P2X6 purinoceptor that, like P2X4, was not sensitive to the
antagonists suramin and PPADS.
F) P2X7 RECEPTOR. The P2X7 presently cloned from rat
and human macrophages and brain (406, 460) shows a
unique feature with its long intracellular COOH terminal,
240 amino acids of which at least 177 are involved in the
induction of the nonselective cation channel and do not
contain any known signaling motifs. P2X7 receptors share
many similarities with previously named P2Z receptors
through which ATP permeabilizes macrophages and other
cells and leads to cell death (132). Thus P2X7 and P2Z
show both high sensitivity to BzATP relative to ATP and
marked potentiation of the responses with reduced external divalent cation concentration so that ATP4⫺ could be
considered as the agonist (98). Indeed, the binding of
ATP4⫺ to P2X7 receptors induces within milliseconds the
opening of a channel selective for small cations and
within seconds a larger pore whose single-channel conductance is 409 pS in macrophages and which is permeable to molecules up to 900 Da (108, 460). These effects of
ATP are antagonized by PPADS not by suramin (87).
Several experimental situations suggest a role for
connexin43 (Cx43) in the ATP-induced cell leakage of
cations and small molecules. First, mouse macrophage
J774 cells that do not express Cx43 are ATP resistant. It
774
GUY VASSORT
was, thus, suggested that Cx43 forms “half-gap junctions”
in response to extracellular ATP (40). Second, both ATP
and low-Ca solutions similarly evoked dye leakage from
Novikoff hepatoma cells (293). The latter solution is
known to induce hemigap-junction channels with singlechannel conductance of 145 pS in catfish retina (124).
Third, in HEK-293 cells overexpressing Cx43, as well as in
ventricular cardiomyocytes, low-Ca solutions and metabolic inhibition open hemichannels (234). The latter case
represents a physiopathological situation during which,
not only cations, but also ATP could leak out of the
cardiac cells and might induce autocrine stimulation.
P2X7 receptors are insensitive to UTP and inhibited
by isoquinolene derivatives 1-(N,O-bis[5-isoquinolinesulphonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN62), oxidized ATP (460), and calmidazolium (88, 224, 495). P2X7
receptors in macrophages and lymphocytes cause activation of phospholipase D (143) and modulate lipopolysaccharide signaling and inducible nitric oxide synthase expression in macrophages (220).
2. Regulation
As for K⫹ channels, several cloned P2X-receptor subunits are required to form an ion channel that enables the
formation of homomeric and heteromemic receptors that
might provide distinct or novel characteristics. To what
extent such in vitro constructs might reflect physiological
conditions is illustrated below.
A) HOMO- AND HETEROMERIC RECEPTORS. Like P2X2 and
P2X3 that combine to form a unique heteromeric channel
(285, 400), with specific properties (496, 497), others assemble. In cells expressing the heteromeric P2X1/5, ␣,␤met-ATP evoked biphasic currents with a pronounced
nondesensitizing plateau phase (477). The heteromeric
assembly was suggested by in situ hybridization studies
(280) and is confirmed by coimmunoprecipitation (477).
Similarly, P2X4 and P2X6 are major subunits with highly
overlapping mRNA distribution in the mammalian central
nervous system (99).
A systematic study of subunit P2X coassembly has
been tested by protein-protein interactions using a coimmunoprecipitation assay (476). P2X4 and P2X7 do not
form heteromers, whereas P2X6 needs to be associated in
heteromers with P2X1,2,4,5 to form an active channel. It
has been proposed that a novel structural motif of quaternary structure of P2X receptors together (as with voltage-dependent Na⫹, Ca2⫹, or K⫹ channels) with a bundle
of ␣-helices contributed by the putative transmembrane
segment M2 near the COOH terminal (405) might lead to
a tetrameric (as with the nicotinic channel) or pentameric
organization that forms a pore. In another elegant study,
it is suggested that P2X1, P2X4, or P2X3 receptors form
stable trimers (344). The dose-response curves for ATP
activation of the 30-pS P2X2 channel showing a Hill coef-
Volume 81
ficient of 2.3 also suggests the channel to be a trimer
(131).
B) PERMEABILITY. The substituted cysteine accessibility
method was used to identify parts of the molecule that
form the ionic pore of the P2X2 receptor (405). L338 and
D349 are on either side of the channel gate with D349
located near the middle of P2X2 channel. It is a negatively
charged amino acid conserved among all the seven P2X
receptors. It is thus possible that D349 is the site of
permeant cation binding and be responsible for ionic
selectivity (131). The asparagine residue Asn-333 was
found to regulate the conductance such that the unitary
conductance of 80 pS in 100 Na⫹ was roughly halved
when it was replaced by isoleucine (N333I) (336).
The mechanism of inward rectification has been extensively studied in several types of channels. Small peptides that formed the channel might aggregate to lead to
an intrinsic inward rectification (252). More often inward
rectification results from open channel block by Mg2⫹
(310) and polyamines (296, 343). The ATP-receptor subunit P2X2 expressed in Xenopus oocytes and in HEK-293
cells shows profound inward rectification in all patchclamp recording conditions. That suggests single-channel
conductance may decrease when membrane potential becomes more positive, and furthermore, there is a substantial contribution of voltage-dependent gating to inward
rectification of the steady-state current-voltage relation
(537). P2X activation by external ATP (see sect. IVA1) was
suggested.
Externally applied ATP activates a voltage-independent conductance on single smooth muscle cells dispersed from rabbit ear artery (31). This is also true in
amphibian atrial cells (167). However, weak inward rectifying properties are shown for the ATP-induced current
in rat ventricular, guinea pig atrial (204, 311, 424), and
rabbit sinoatrial node (SAN) cells (436).
With the use of a combination of whole cell patchclamp and fura 2 fluorescence measurements in HEK-293
cells transfected with hP2X4, it was determined that the
recombinant channel allows a substantial amount of Ca2⫹
to permeate. The 8% current carried by Ca2⫹ is very close
to the value previously reported for native ATP-induced
currents in sympathetic neurons (411). Such a high Ca2⫹
permeation through hP2X4 receptors suggests that activation of these proteins may directly carry Ca2⫹ that will
contribute to synaptic transmission (94).
The Ca2⫹ permeability of P2X1 receptors is greater
than that of P2X2 receptors (PCa/PNa 3.9 and 2.2, respectively) despite no difference between the two receptors
with respect to their permeability to a series of monovalent organic cations (148). In later work, advantage was
taken of the clearly different properties of P2X2 and P2X3
(␣,␤-met-ATP sensitivity and desensitization) to compare
Ca2⫹ permeability in homomeric and heteromeric expressed receptors (496). P2X3 has a relatively low Ca2⫹
April 2001
ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
permeability (PCa/PNa 1.2–1.5) that dominates the heteromer P2X2/3 Ca2⫹ permeability while the heteromeric receptor is nearly as sensitive to external Ca2⫹ inhibition as
the P2X2 receptor. These properties reinforced the view
that the native ␣,␤-met-ATP-sensitive receptor in nodose
ganglion neurons is a P2X2/3 heteromultimer (496). P2X2/3
desensitization is much slower than that of P2X3 subtype
alone; thus the heterogeneous expressed P2X2/3 acquires
more effective Ca2⫹ dynamics than P2X2 or P2X3 receptor
alone (481).
C) DESENSITIZATION. P2X receptors can be divided into
two broad groups according to whether they show fast
desensitization within 100 –300 ms or slowly if at all (Table 1). Desensitizing currents and the mechanisms of
P2X-receptor desensitization have been recently reviewed
(403). Briefly, rapidly desensitizing P2X receptors are activated by ATP, ␣,␤-met-ATP, and 2-MeSATP. They include recombinant P2X1 and P2X3. The nondesensitizing
␣,␤-met-ATP-insensitive P2X receptors are the expressed
cloned P2X2, P2X4, P2X5, P2X6, and P2X7 receptors. Several mechanisms have been proposed to account for desensitization. Desensitization implies the first or the second hydrophobic domain since substitution of these
domains in chimeric P2X1 or P2X2 receptors confer the
characteristic (516).
The rate of desensitization of the heterologously expressed P2X1 receptor channel in HEK-293 cells has been
reported to be 50 –150 ms, while it is 5–10 s when recorded from Xenopus oocytes (147, 516). A slower rate of
desensitization of the P2X1 receptor from rat vas deferens
stably expressed into HEK-293 cells was observed by the
second day of culture. This effect was reversed by cytochalasins B and D (362). Mutations, M332I and T333S,
identified in the porelike domain near or within the second putative transmembrane domain also prevented
changes in kinetics.
Desensitization of P2X2 receptors expressed in either
Xenopus oocytes or HEK cells is markedly accelerated by
increasing ATP concentrations (538). However, this concentration-dependent effect could in part be attributable
to the concomitant acidification that occurs when high
concentrations of ATP are dissolved in weakly buffered
solutions (456). Desensitization of P2X2 receptor was proposed to be controlled by alternative splicing (61). The
splice isoform P2X2b or P2X2–2R, which lacks a stretch of
COOH-terminal amino acids (Val370-Gln438), exhibits
rapid and complete desensitization, whereas the wild-type
channel desensitizes slowly (268, 442). The Pro373Pro376 sequence of P2X2R represents a functional motif
that is critical for the development of the slow desensitization profile (267).
The truncated P2X3 clone lacking the NH2-terminal
intracellular region expresses functional channels that do
not desensitize in oocytes (254). Desensitization of the
ATP-gated cation channel P2X3 is abolished by removal of
775
external Ca2⫹ or by cyclosporin pretreatment (254). The
rate of desensitization is also decreased by injection of
the autoinhibitory peptide CaNA457– 481 in the oocyte.
Thus it is thought that P2X3 desensitizes through a Ca2⫹dependent calcineurin-mediated phosphorylation on NH2terminal residues (254).
3. P2X receptors in the heart
Most efforts in cloning of P2X receptors and their
tissue distribution has been devoted to the brain. Much
less is known for the heart and cardiomyocytes in particular. Immunoreactivity has been first reported with antisera against P2X1 receptors on rat cardiac tissues that
showed positive staining at the intercalated disk (501).
One of the most remarkable characteristics of the rat
P2X3 receptor was its apparent tissue expression pattern
with transcripts restricted to nociceptive neurons (84,
285). However, major species difference seems to exist,
since P2X3 was found in human heart (173) despite the
fact that this gene is highly conserved from rat to human,
coding for channel proteins that share 93% identity. The
majority of sequence variations are located within the
putative extracellular loop. P2X3 is also found in human
fetal heart where it appears to be the most abundant
P2X-receptor subtype (47). Electrophysiological characterization showed however minor differences with an
inhibitory potency of suramin five times lower in human
than in rat (IC50 15 vs. 3 ␮M, respectively) (173). It is also
remarkable that hP2X3 had a high affinity for CTP (EC50
⬃20 ␮M). The authors could not rule out the possibility
that the detection of human P2X3 RNA in heart and spinal
cord reflects neuronal transcripts located in primary sensory afferents densely innervating these structures.
In situ hybridization histochemistry was recently performed together with RT-PCR on the rat cardiovascular
system (348). In heart sections, the authors noticed that
mRNA transcripts for P2X1, P2X2, and P2X4 all colocalized in smooth muscle cells of coronary vessels with no
specific apparent positivity in myocardium. However, RTPCR from microdissected tissues from various areas of
the heart confirm the presence of P2X1, P2X2, and P2X4
receptor mRNAs, with strong signals in the atria and only
the typical band for P2X4 in ventricles (348). Furthermore,
two splice variants of the P2X2 were identified. The discrepancy between the PCR and in situ hybridization data
might result from a too low level of mRNA expression for
detection or, as well, tiny blood vessels and nerve endings
may also contribute.
Northern blot and RT-PCR analysis demonstrate
P2X4 transcripts in many tissues of the rat including blood
vessels and heart (447). In a subsequent study, GarciaGuzman et al. (171), characterization of recombinant human P2X4 receptor reveals pharmacological differences in
the two species. The homology to the rat P2X4 receptor
776
GUY VASSORT
shows 87% identity. Zn2⫹ increases the apparent gating
efficiency at low concentration (5–10 ␮M) but inhibits the
ATP-evoked current at 100 ␮M and higher. hP2X4 and
rP2X4 receptors display similar agonist potency profiles,
but the human receptor has a notably higher one.
The P2X5 purinoceptor was cloned in rat heart (172)
simultaneously to its cloning in brain (99). Like in brain,
functional expression of the recombinant rat P2X5 receptor shows a current that resembles mostly the P2X2 phenotype: slow desensitization, inhibition by PPADS and
suramin, and no activation by ␣,␤-met-ATP. The EC50 for
ATP is 8 ␮M. The rank order of potency is ATP ⱖ 2MeSATP ⬎ AMP ⬃ ADP ⬎ dATP ⬃ ␤,␥-met-ATP. No
current is recorded after application of ␣,␤-met-ATP,
CTP, GTP, UTP, or adenosine, each at 500 ␮M (172).
P2X1 receptor mRNA level is increased 2.7-fold in
rats under congestive heart failure (216).
G. Metabotropic P2Y Receptors
To date a total of 13 P2Y receptor-like DNA sequences have been cloned (P2Y1–11, tp2Y, and fb1). However, the P2Y7 receptor is definitely a leukotriene B4 receptor, and there is no functional evidence that P2Y5,
P2Y9, and P2Y10 receptors are nucleotide receptors.
Therefore, there are currently five genuine human P2Y
receptors (P2Y1,2,4,6,11). It is most likely that the chicken
P2Y3 receptor is the avian ortholog of the P2Y6 receptor,
whereas Xenopus P2Y8 and tp2y are probably orthologs of
the P2Y4 receptor. Members of the P2Y-receptor family
couple to heteromeric G proteins which, in turn, activate
intracellular second messenger systems to modulate the
physiological function of the cells. All have a wide tissue
distribution (Table 3).
TABLE
Volume 81
A number of specific reviews have appeared (18, 44,
59, 100, 191, 255, 403).
1. Characteristics
A) P2Y1 RECEPTOR. The first cloning of a P2Y receptor
from chick brain was reported in 1993 (514). Subsequently, P2Y1 was isolated from a range of species including rat, mouse, bovine, and human with a wide tissue
distribution including heart (12, 47, 199, 473, 511). Pharmacological characterization of the P2Y1 receptor indicates that only purines are active. UTP and derivatives are
not active at this receptor subtype (443). More precisely,
the P2Y1 receptor had been initially characterized by an
agonist potency order of 2-MeSADP ⬎ 2-MeSATP ⬎ ADP
⬎ ATP (57, 58, 427, 514). However, this has been questioned (198, 283). Whereas ADP and 2-MeSADP are potent
full agonists, purified ATP and 2-MeSATP rather act as
weak antagonists on the heterologously expressed hP2Y1.
This observed adenine nucleoside diphosphate specificity
of the P2Y1 receptor is very similar to the pharmacological selectivity of the P2T receptor in platelets (217). It is
now well established that part of the ADP effect on platelets (calcium release, shape change) is mediated by the
P2Y1 receptor. Other effects (cyclase inhibition) are mediated by a still uncharacterized distinct receptor,
whereas aggregation requires both receptors (283). The
apparent discrepancy with ATP being a weak but full
agonist on P2Y1 expressed in HEK and an antagonist on
the same receptor expressed in Jurkat T cells was analyzed in a recent study and attributed to differences in the
degree of P2Y1-receptor reserve (361). In another recent
work, adenosine 2⬘-phosphate-5⬘-phosphate (A2P5P) and
adenosine 3⬘-phosphate-5⬘-phosphate (A3P5P) inhibited
ADP-induced platelet shape change and aggregation and
3. Mammalian metabotropic P2Y purinoceptortor: characteristics and presence in the heart
Antagonists†
P2Y
Ortholog
hP2Y1
Amino Acids
rP2Y4
hP2Y6
RB2
Heart
⫹⫹
⫹⫹
⫹⫹
12
473
511
373
UTP ⫽ ATP ⬎ Ap4A
(2-MeSATP, ADP, UDP inactive)
⫹⫹
0
⫺
47
365
365
UTP, ATP␥ S ⬎⬎ ATP, ITP
(2-MeSATP, UDP inactive)
ATP ⬃ UTP ⬎ Ap4A ⬎ ATP␥S
UDP ⬎⬎ UTP ⬎ ATP ⬎ ADP
⫹
0
0
⫹⫹/0
⫺
0
81, 407, 513
48
0
0
⫹⫹⫹
⫺
⫺
⫺
⫺
⫺
⫺
48, 513
47
80, 511
216
361
379
rP2Y6
hP2Y11
PPADS
2-MeSADP ⬎ ADP ⬎ ATP*
mP2Y2
rP2Y2
hP2Y4
Suramin
362
mP2Y1
rP2Y1
bP2Y1
hP2Y2
Agonists
371
ATP ⬎ 2-MeSATP ⬎⬎⬎ ADP
(UTP, UDP inactive)
* ATP was suggested to be an antagonist (197, 198, 283). † A3P5P (57), A2P5P, and A3P5P (197, 198) are specific antagonists at hP2Y1. ⫹, 0,
Degree of antagonistic effects; (⫺), not determined. 2-MeSATP, 2-methylthio-adenosine 5⬘-triphosphate; ATP␥S, adenosine 5⬘-O-(3-thiotriphosphate).
April 2001
ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
competitively antagonized Ca2⫹ movements in response
to ADP in fura 2-loaded platelets, a B10 clone of brain
capillary endothelial cells, and Jurkat cells expressing the
human P2Y1. In contrast, these compounds had no effects
on ADP-induced inhibition of adenylyl cyclase in platelets
or B10 cells (197).
B) P2Y2 RECEPTOR. Concurrently in 1993, a receptor was
isolated from mouse neuroblastoma cells and found to be
activated by UTP and by ATP with equal potency and
efficacy. ATP␥S and ITP are less potent while 2-MeSATP
and ␣,␤-met-ATP are weak partial agonists (299). Orthologs of P2U, now named P2Y2, have subsequently been
isolated from human and rat (365, 511). P2Y2 is expressed
in a wide variety of tissue including heart (81, 407, 511).
After expression in Xenopus oocytes, P2Y2 coupled to
two classes of G proteins to increase an endogenous
Ca2⫹-dependent Cl⫺ channels and mediate a pertussis
toxin-sensitive increase in the inward-rectifier K⫹ channels of the Kir3.0 subfamily (330).
C) P2Y3 RECEPTOR. The P2Y3 was cloned from chick
brain and exhibits the following rank order of agonist
potency: UDP ⬎ UTP ⬎ ADP ⬎ ATP (512). It is most
likely the avian ortholog of the mammalian P2Y6 receptor.
D) P2Y4 RECEPTOR. Another clone originally thought to
be the pyrimidinoceptor, P2Y4, was isolated from human
genomic DNA libraries (104, 340). The hP2Y4 receptor is
highly selective for UTP, whereas ATP, ADP, and ITP
appear to be weak partial agonists (104, 340, 342). This
rules out P2Y4 as a true pyrimidinoceptor (440). It is not
antagonized by suramin, nor by PPADS (but see for
rP2Y4), and is blocked by RB2. Pyrimidinoceptors were
mainly found in rat sympathetic neurons. Recently, a
rP2Y4 has been cloned (48, 513). It shares 83% overall
identity with the hP2Y4 and is less related to the rP2Y2
with 51% identity. Its agonist profile is close to the native
P2U receptor: ATP ⬃ UTP ⬎ Ap4A ⬎ ATP␥S ⬎ 2-MeSATP.
It is also strongly activated by ITP and UDP, although
the latter could be a partial agonist. These observations, including suramin efficiency, help differentiating
the rP2Y4 from the orthologs of P2Y2 (48, 154). ATP and
UTP act on the same receptor, since they show crossdesensitization. Besides, the hP2Y4 is among the few G
protein-coupled receptors to show no N-glycosylation
consensus sequence (104), but the rat ortholog does
(513).
E) P2Y6 RECEPTOR. The P2Y6 was cloned from a rat
aortic smooth muscle library and from a human placenta
cDNA library; the two orthologs show 88% amino acid
identity (80, 103). As for P2Y4 receptors, the selectivity of
P2Y6 receptors for UDP had to be reexamined because of
the use of UDP preparations contaminated by UTP and
the degradation of UTP into UDP during incubation of the
cells (342). With these precautions the P2Y6 receptor is
activated most potently by UDP and weakly by UTP, ATP,
and ADP.
777
F) P2Y11 RECEPTOR. The newly cloned human P2Y receptor provisionally called hP2Y11 is characterized by
considerably larger second and third extracellular loops
(101). It exhibits only 33% homology with hP2Y1, its closest homolog, and 28% homology with hP2Y2. It shows one
potential site for N-linked glycosylation and two potential
sites for phosphorylation by PKC or calmodulin-dependent protein kinases. hP2Y11 couples positively to both
phosphoinositide and adenylyl cyclase, a unique feature
among the P2Y family. Following stable expression in
1321 N1 astrocytoma and CHO-K1 cells, the rank order of
agonist potency for the two pathways is ATP␥S ⬃ BzATP
⬎ dATP ⬎ ATP ⬎ adenosine 5⬘-O-(2-thiodiphosphate)
(ADP␤S) ⬎ 2-MeSATP ⬎ ADP with UTP and UDP being
inactive, thus showing hP2Y11 is presently the most adenine nucleotide selective P2Y receptor (106).
G) DETERMINANTS OF ATP BINDING ON P2Y PURINOCEPTORS.
Site-directed mutagenesis of P2Y2 led to the suggestion
that the charged amino acids His-262, Arg-265 in TM6 and
Arg-292 in TM7 interact with the phosphate groups of the
nucleotides (145). The amino acid sequence of hP2Y4
shows some similarities with the P2Y2 receptor at sites
directly involved in the binding of negatively charged
phosphate groups. Three residues are conserved: His-262,
Arg-265, and Arg-292, whereas Arg-265 is replaced by a
Lys-259 in P2Y1 as in the P2Y6 (105, 340). Moreover,
Lys-289 residue also plays a major role, since its substitution by Arg shifts the preference of the P2Y2 receptor
for triphosphate nucleotides to diphosphate nucleosides
(145). Note that this Lys residue is conserved in both P2Y4
and in P2Y6; the latter which, however, has a clear preference for UDP.
2. Coupling to G proteins and second messengers
Members of the P2Y family are quite diverse in sequence, more so than other known G protein-coupled
receptor families. The third intracellular loop and the
COOH-terminal tail, the two regions implicated in G protein specificity in the other G protein-coupled receptors,
vary greatly among the P2Y receptors. Nevertheless, P2Y
receptors have all been shown to be coupled through
a Gq/11 protein to the inositol 1,4,5-trisphosphate (IP3)
pathway.
Activation of the recombinant P2Y1 receptor mediates IP3 formation and increases intracellular Ca2⫹ concentration ([Ca2⫹]i), but it does not change cAMP (443).
However, in a recent study BzATP, an antagonist of rat
and human P2Y1 expressed in Jurkat cells, prevents the
ADP-induced inhibition of the cAMP pathway (493). IP3
and [Ca2⫹]i increases can stimulate a variety of pathways
including PKC, phospholipase A2 (PLA2), and nitric oxide
synthase which subsequently can activate other pathways
such as phospholipase D (PLD) and mitogen-activated
protein kinase (MAPK).
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GUY VASSORT
Activation of P2Y4 and P2Y6 receptors also leads to
the formation of inositol phosphates (103, 104) and a rise
in [Ca2⫹]i (80, 340). However, the two receptors seem to
involve distinct G proteins. The P2Y4 but not the P2Y6
response was inhibited by pertussis toxin (80, 102, 409).
hP2Y11 has the unique property of simultaneously activating the adenylyl cyclase with the same rank order of
potency of agonists (101, 106). Surprisingly, the ATP derivative AR-C67085, a potent inhibitor of ADP-induced
platelet aggregation, was the most potent agonist at the
recombinant hP2Y11 receptor (106).
3. P2Y receptors in the heart
The expression of P2Y1,2,4,6 receptor transcripts was
reported by RT-PCR in rat heart (511). All receptor sequences could be amplified from neonatal whole heart,
with P2Y6 appearing the most abundant transcript of the
four. However, in neonatal cardiomyocytes, P2Y1 is expressed at higher levels than the others. In adult myocytes, P2Y1, P2Y2, and P2Y6 could be amplified while P2Y4
could not be detected (511). Two major conclusions can
be made from this work: 1) the need to specifically work
on isolated cardiomyocytes, and 2) P2Y expression varies
during development with the arrest of P2Y4 expression in
adult cardiomyocytes. P2Y2, P2Y4, and P2Y6 receptors
were recently cloned in fetal human heart using degenerated oligonucleotides (47). Other works reporting the
presence of one or the other of the P2Y receptors are
quoted in Table 2. P2Y11 was not found in human heart
using Northern blot analysis (101). In a recent study however, using a new quantifying RT-PCR protocol, P2-receptor mRNA expression was compared in control and rats
under congestive heart failure. In the sham-operated rats,
P2Y receptors are expressed at a higher level than P2X1
receptors, with P2Y6 being the most abundant. In failing
hearts, a prominent change was seen: P2X1 and P2Y2
receptor mRNA levels were increased 2.7 and 4.7, respectively (216). Extending their study to adult human myocardium, the authors could detect P2X1, P2Y1, P2Y2, P2Y6,
and P2Y11 receptors in both right and left atria and ventricles, while no P2Y4 receptor was seen.
H. Adenine Dinucleotide Receptors
The selectivity and activity of adenine dinucleotides
for neuronally derived recombinant P2 purinoceptors
were studied using P2X2 and P2Y1 subtypes expressed in
Xenopus oocytes (378). Ap4A is as active as ATP but less
potent (EC50 ⬃15 and 4 ␮M, respectively) on P2X2 receptors. Other adenine dinucleotides are inactive. In a previous work it had been shown that Ap5A is a partial agonist
at the human ortholog hP2X1 (147). However, Ap5A potentiates the ATP responses but not the Ap4A response at
Volume 81
the P2X2 subtype with an EC50 of ⬃3 nM, and Ap5A also
enhances the efficacy of suramin (378). In a recent work,
Ap5A also potentiated the ATP response in rat cerebellar
astrocytes (233). Such an effect appears to be mediated
by the metabotropic P2Y receptors. At the P2Y1 subtype,
Ap3A is equipotent and active as ATP. Ap4A is a weak
partial agonist and other dinucleotides are inactive. In
another study, Ap4A is reported to be a potent agonist at
P2Y2 but not P2Y1, P2Y4, and P2Y6 expressed in 1321 NI
human astrocytoma cells (341). Thus some dinucleotides
have the capacity to potentiate ATP responses at both
P2X and P2Y receptors.
To date, in cardiac tissues, only the presence of Ap4A
receptor has been reported (202, 203, 504), with at least
77% of the active receptors on the plasma membranes
(502). Recent work demonstrates photocross-linking of
an Ap4A derivative with a 50-kDa polypeptide, suggesting
a homogeneous population of receptors (42). At the highaffinity binding site, the apparent Kd values for Ap4A and
ATP␥S are 0.08 and 0.04 ␮M, respectively; there is also a
low-affinity site for ATP␥S with a Kd at 1.0 ␮M. The P2X
agonist ␣,␤-met-ATP did not compete effectively with
these two agonists and indicates that the Ap4A receptor is
a P2Y receptor in cardiac plasma membranes. The same
group had previously suggested Ap4A binds to a specific
30-kDa polypeptide dinucleotide receptor. This observation resulted from receptor proteolysis. Ap4A receptor
was shown to undergo at least two proteolytic processing
steps, one of which is carried out by a serine protease,
and this serine protease is required for receptor activation
(202, 503, 504). Specific dinucleotide receptors activated
by ApnAs, but not ATP or UTP, have been suggested on
synaptic terminals in guinea pig diencephalon and cerebellum (379). This follows the identification of binding
sites that are highly selective, or even specific, for ApnA
as opposed to mononucleotides. This possibly represents
P2D receptors, P2YApnA receptors, or also P4 receptor
that are distinct from those activated by ATP to induce an
elevation in intrasynaptosomal Ca2⫹.
I. Pharmacological Concerns
Despite much effort there is a still strong need of
potent and subtype-specific P2 receptor agonists and antagonists. Several substances that display selectivity for
P2X or P2Y subtypes have been recently carefully reviewed (403) and are summarized in Tables 1 and 2. In the
following I would just like to draw attention to side
effects of most of the presently available compounds that
make their use difficult even in isolated cells.
It had been already recognized that the ecto-nucleotidase activity accounts, in most part, for the difference
in the concentration curves of P2X agonists in vascular
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ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
smooth muscle contraction and electrophysiological response, since ATP degradation products might act besides ATP itself (250). Inhibitors of the ecto-ATPases
include NaN3 or AP5A as well as many of the so-called
purinergic antagonists, suramin, PPADS, RB2, FLP 66301,
and FLP 67156 (39, 66, 82, 110, 111, 223, 524, 540). All the
nonhydrolyzable ATP analogs also do (83, 377). Consequently, such an inhibition of ecto-ATPases might, in part,
account for the observed facilitation effects of ATP in
tissues or in cell incubation (82). Suramin is also an active
inhibitor of protein-tyrosine phosphatases (530), a side
effect of potential importance knowing the involvement
of tyrosine kinase pathways after ATP stimulation (394).
PPADS also appears to inhibit UTP- and ATP-induced
Ca2⫹ mobilization by a nonspecific mechanism independent of P2Y-receptor recognition that might involve the
inhibition of intracellular IP3 channels (494). Moreover,
there might be even species selectivity. Thus rat recombinant P2X4 and P2X6 receptors are not blocked by
PPADS, but the human homolog of the P2X4 receptor is
(171).
Recently, 2⬘,3⬘-O-(2,4,6-trinitrophenyl)adenosine 5⬘triphosphate (TNT-ATP) has been reported to inhibit specifically P2X1 and P2X3 and heteromeric P2X2/3 with an
IC50 of 1 nM. However, its use in tissue might be of limited
interest due to its fast breakdown (497).
BzATP, an antagonist of P2X7 receptor, is also a
photoaffinity probe that binds covalently to the nucleoside sites of ATPases (20, 360); it prevents ATP binding to
␣-sarcoglycan, a skeletal muscle ecto-ATPase (38), and to
P2Y1 receptors (493).
Quinidine was first used by Burnstock (69) as a P2receptor inhibitor. It is interesting to note that quinidine
antagonizes the ATP-induced nonselective cationic current in rat ventricular cells (91) and the late phase of
positive inotropy in guinea pig atria (135) in line with the
view that a part of contractile activity might result from
P2X-mediated Ca entry (169).
DIDS is a commonly used anion transport inhibitor. It
has also been reported to block a number of presumably
purinoceptor-mediated responses. Indeed, DIDS causes a
long-lasting blockade of P2X receptor activity in rat vas
deferens (65, 112, 319). This effect is in line with the fact
that DIDS decreases the binding of [32P]ATP in rat parotid
acinar cells (319). DIDS is now known to be a P2-receptor
antagonist (65, 147, 445).
Ion selectivity of the P2X-receptor channels resembles that of nicotinic ACh, glutamate-activated, or serotonin-activated channels. It might be of interest to
test blockers of these channels (local anesthetics
QX314, QX222, D-tubocurarine, dizocilpine, and phencyclidine). In fact, D-tubocurarine is a blocker of P2X2
receptors (60).
779
III. PHYSIOLOGICAL AND
PHYSIOPATHOLOGICAL EFFECTS
OF EXTRACELLULAR ADENOSINE
5ⴕ-TRIPHOSPHATE
A. Contractile Effects
Most initial reports describe negative inotropic effects after applying ATP in mammalian hearts, particularly in the atrium (75, 137, 159, 207, 212). These effects
should be attributable to A1-adenosine receptor activation
after ATP breakdown in the tissue. Later studies on
guinea pig and rat atria revealed positive inotropic effects
of ATP that developed after a transient rapid decrease
(135, 168, 169, 306). In these studies, ATP, ADP, AMP,
adenosine, ␣,␤-met-ATP, ␤,␥-met-ATP, as well as UTP
were shown to have this dual effect. Desensibilization by
long exposure to ␣,␤-met-ATP (135), suramin, but not
RB2 could antagonize the positive inotropic effect (168),
whereas 8-phenyltheophylline (8-PT) or 8-cyclopentyl-1,3dipropylxanthine (DPCPX) prevented the negative effects
(168, 306). It is noteworthy that 2-MeSATP induces only a
negative inotropic effect (168). A similar dual inotropic
effect of ATP is observed in human atrial strips (J. Alvarez
and G. Vassort, unpublished results). Pyridoxal 5⬘-phosphate, an active form of vitamin B6, shows antagonism
toward ATP-induced positive inotropic effect in perfused
rat heart, with both pyridoxal and phosphate moities
being essential for the action (508).
Fifty years ago, Green and Stoner (185) reported that
after a brief period of cardiac arrest, adenine nucleotides
had a strong positive inotropic effect in isolated perfused
mammalian hearts. They reported that increasing states
of phosphorylation of the adenosine molecule correlated
well with the increasing inotropic effects. They suggested
that the adenine nucleotides exerted directly their effect
on the myocardium rather than through vasodilatation.
Several reports established ATP as a full positive inotropic agent in frog ventricles (11, 161, 183, 489 and see
references therein). ATP also induces positive inotropic
effects in the ventricle of axolotl (321) but has no effect in
turtle heart, dogfish, and trout ventricle (320, 322, 323).
Positive inotropy in frog heart is also induced by UTP,
ITP, CTP, GTP, and their derivatives (489, 490, 527). The
fast initial positive phase is followed, after a transient
decrease, by a secondary increase of variable amplitude
according to the agonists. ATP and its derivatives exert
clear positive inotropy in rat papillary muscle (282, 421)
and enhance cell shortening in various mammalian ventricular cardiomyocytes (Fig. 1) (91, 116, 381, 421).
A beneficial effect of adenine nucleotides on contractility of postischemic papillary muscles is long known; a
situation where ATP effect was tentatively attributed to
its high-energy supplier properties such as it would con-
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FIG. 1. Inotropic effects of ATP. A: in the frog heart, the application of ATP (20 mg%) is able to compensate for the
partial removal of extracellular Ca2⫹. [From Antoni et al. (11). Copyright 1960 Springer-Verlag.] B: ATP at 1 ␮M in the
presence of 1 mM Ca2⫹ induces parallel changes in transient Ca2⫹ and cell shortening in an indo 1-loaded rat ventricular
myocyte. [From Danziger et al. (116).] C: negative and positive inotropic effects of ATP in a guinea pig left atria,
respectively, in absence and presence of 0.1 ␮M 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). [From Mantelli et al.
(306).] D: positive inotropic effects of 100 ␮M ATP on an auricular strip isolated from a pertussis toxin-treated rat before
and after 0.5 ␮M isoproterenol (ISO) stimulation. [From Scamps et al. (421).]
tribute to high-energy phosphate replenishment (434).
However, with the present knowledge, positive inotropy
in control and diseased cells could be attributable to an
increase in L-type long-lasting Ca2⫹ current (ICaL ) (204,
421– 423) that mediates an increase in Ca2⫹ transient.
Moreover, ATP could enhance SR Ca2⫹ release, although
this mechanism is unclear (41, 91, 204, 389). Other mechanisms have been suggested as well. They include an
interaction of ATP and UTP with P2X receptors that
mediate Ca2⫹ entry (169). A similar mechanism in cultured chick embryo ventricular myocytes could account
for the observation that ATP roughly doubles cell shortening (381). ATP analogs mimick this effect with an efficacy and potency order of ATP ⬎ ADP ⬎ AMP ⬎ adenosine. 2-MeSATP but not ␣,␤-met-ATP has a strong
positive effect while UTP is weak. In contrast, UTP, like
ATP, triggers a strong phosphoinositide-stimulating hydrolysis while 2-MeSATP is a weak agonist, thus excluding a PLC-dependent effect (381). Positive inotropy can, in
part, be attributed to the alkalinizing effect of ATP (391,
468) which sensitizes the contractile machinery to Ca2⫹
(150). No direct effect on the contractile proteins was
observed contrary to such observation after ␣1-adrenergic-mediated inotropy (388, 467).
In isolated ferret ventricular myocytes, extracellular
ATP (10⫺7 to 10⫺3 M) inhibits ICaL and decreases Ca2⫹
transient and contraction. These effects were sustained
(399). In this study, performed in a Na⫹-free, K⫹-free
solution, there was also no evidence for ATP activating a
nonselective cation current, I(ATP). In this case the Ca2⫹
conductance via I(ATP) might be too small to measure.
B. Chronotropic Effects
In their seminal work, Drury and Szent-Györgyi (137)
reported a negative chronotropic effect of purines. Following studies suggested that ATP has dose-dependent
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ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
effects; small doses producing tachycardia while relatively larger doses of ATP slow the heart and induce
atrioventricular nodal conduction block (207, 455). ATP
decreases the slow diastolic depolarization in Purkinje
fibers but increases it in frog heart (269). However, these
negative chronotropic effects might be the result of ATP
degradation to adenosine (36) and the action of the latter
on sinoatrial and atrioventricular nodes (372). This has
been shown to be the case even during administration of
ATP into the sinus node blood supply (i.e., intracoronary)
in dogs (232), in which model the negative chronotropic
action of ATP was attenuated by theophylline, a known
antagonist of adenosine at the P1-receptor site (19, 371).
The negative chronotropic action of ATP is independent
of prostaglandins (375). These observations indicate that
negative chronotropy is in part due to P1-purinergic activation by ATP or rather by its degradation product, adenosine (523). Thus ATP (Striadyne) was used to treat paroxysmal supraventricular tachycardia (331, 419). On the
other hand, ATP caused cardiac acceleration in 40% of the
rabbit hearts using the Langendorff-perfusion method
(463). An excitatory effect of ATP in isolated cat atria was
revealed by the previous addition of acetylcholine (4).
These excitatory effects of ATP were not antagonized by
either theophylline or pertussis toxin pretreatment, but
were almost completely blocked by neomycin and indomethacin (463). On the basis of these data, it was concluded that the positive chronotropic effect of ATP was
mediated by P2-purinoceptors coupled to prostaglandin
synthesis via a pertussis toxin-insensitive pathway involving the stimulation of PLC (463).
It is difficult to anticipate the direct effect of ATP on
sinus node rhythm. Extracellular ATP activates a timeindependent, weakly inwardly rectifying current that is
nonselective for monovalent cations (436). This current is
not activated either by ADP, AMP, or adenosine, suggesting that the action of ATP is mediated by a P2 purinoceptor. In view of the critical role of ICa in the genesis of the
action potential in SAN cells, it was proposed that extracellular ATP may play an important role in the regulation
of heart rate (396). It is even more difficult to extrapolate
data obtained in vitro (396, 463) to the human heart in
vivo. Numerous studies in cats, dogs, and humans have
indicated that extracellular ATP exerts a negative chronotropic action on cardiac pacemakers that is mediated in
part by the vagus nerve in addition to the action of adenosine (30, 371, 374), an effect already reported in 1950
(185). The vagal effect is due to a cardio-cardiac depressor reflex elicited by the action of ATP on vagal afferent
nerve terminals in the left ventricle (243), similar to its
action on pulmonary vagal afferent terminals (374). In
both organs (i.e., heart and lungs), the triggering of
the vagal reflex by ATP is mediated by P2X receptors
(243, 374).
In cultured adult guinea pig myocytes, chronotropic
781
effects of ATP could be greatly enhanced in the presence
of cardiac neurons that also possess P2 purinoreceptors
(213). Thus ATP increases contractile rate in intrinsic
cardiac neuron-myocyte cocultures by 40% under control
conditions and much more (100%) after blockade of ␤-adrenergic receptors. In contrast, ATP induces much
smaller effects (26%) in noninnervated myocyte cultures.
C. Pro- and Anti-Arrhythmic Effects of ATP
ATP was shown to produce transient tachycardia at
low doses or to slow heart and to induce atrioventricular
and His bundle blocks at higher doses (156, 207, 454). In
fact, transient ectopic beats of atrioventricular junctional
origin were consistently observed at the onset of the
atrioventricular block produced by ATP. These premature
beats were attributed to reentry phenomena, since positive chronotropic effects of ATP were never observed
(484). Although these effects can be attributed to the fast
formation of adenosine from ATP, in isolated rat ventricular myocytes, the sudden application of ATP at micromolar concentration induces cell depolarization and triggers automaticity (Fig. 2) (91, 424). A similar ATPtriggered automaticity in rat papillary muscles requires
the uncaging of ATP by ultraviolet-light flash (Vassort,
unpublished results) while the application of ATP by
changing the bath solution was without significant effect.
These effects could be attributable to activation of the
fast transient nonselective cationic current [I(ATP)] and
cell depolarization.
In isolated ventricular myocytes of the guinea pig,
ATP alone does not exert significant electrophysiological
effect; however, when it is applied with drugs known to
increase [Ca2⫹]i, ATP facilitates the induction of afterdepolarizations and triggered activity in ⬃60% of the cells
(446). In the presence of isoproterenol, ATP increases the
amplitude of the transient inward current (Iti), delayed
afterdepolarizations (DADs), and ICaL. In the presence of
either BAY K 8644 or quinidine, ATP further prolongs the
action potential duration and also increases the amplitude
of early afterdepolarizations (EADs) (446). These findings
extend earlier observations regarding the interaction between the effects of catecholamines and ATP on ICaL (125,
535) and support the hypothesis that the release of ATP
into the extracellular space under pathophysiological
conditions could be arrhythmogenic (276, 424).
In addition to these various effects at the cellular
level, several other mechanisms triggered by extracellular
ATP suggest that P2-purinergic stimulation can induce
arrhythmia in the cardiac tissue. Both the transitory acidosis and the increase in intracellular Ca2⫹ decrease intercellular coupling (317). Tyrosine phosphorylation of
protein Cx43 might reinforce this situation, since it closes
the gap junction (277, 385). Complementary aspects are
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Volume 81
FIG. 2. Arrhythmogenic effects of ATP. A: the extracellular application of 100 ␮M ATP on a rat ventricular cell
induces membrane depolarization together with cell automaticity. Vm, membrane potential. [From Christie et al. (91).]
B: ATP-induced delayed afterdepolarization and transient inward current in the presence of isoproterenol (10 nM ISO
⫹ 100 ␮M ATP) recorded on a guinea pig single ventricular cell. The action potential recorded at a basal stimulation
frequency of 0.5 Hz (left) is followed by membrane potential recorded after 15-s overdrive stimulation applied at a
frequency of 4 Hz (middle) and membrane current after a 10-s train of 300-ms depolarizing pulses from ⫺80 to ⫹40 mV
at a frequency of 2 Hz (right). [From Song and Belardinelli (446).] C: the addition of 10 ␮M ATP to the bathing solution
induces oscillatory Ca2⫹ transients in a rat ventricular cell loaded with indo 1; however, this effect of ATP requires the
presence of Mg2⫹. [From Pucéat et al. (389).] D: ATP at 20 ␮M stops the spontaneous activity of cultured neonatal rat
ventricular myocytes loaded with fluo 3 that are beating at a high plating density. [From Jaconi et al. (231).]
the slowing of activation spread due to a decreased Na⫹
current (425) as well as the increase in tissue heterogeneity following the facilitation of IK(ATP) and action potential shortening (13).
Is there a role for IP3 in cardiomyocyte arrhythmia?
The signaling role of IP3 has been clearly established in
many cell types in which it induces Ca2⫹ release from the
endoplasmic reticulum (ER) (37, 94). IP3 is generated
following the action of many neurohormones that activates a Gq-dependent PLC-␤ or, in the case of ATP or
angiotensin II, the tyrosine kinase-dependent PLC-␥ (184,
395). In the heart, IP3 has been involved in cardiac arrhythmias (138, 230, 520), heart failure (307), and graft
rejection (152). In a recent work on neonatal rat cells, it is
shown that uncaging IP3 by ultraviolet light or by applying
ATP (or PGF2␣ that activates PLC-␤) induces Ca2⫹ release
around the nucleus from a ryanodine- and caffeine-insensitive compartment, probably the ER (231). Surprisingly
in view of the reports in adult cardiomyocytes, uncaged
IP3 or ATP slowed or abolished the spontaneous Ca2⫹
spiking of cells cultured at high plating density. Such an
antiarrhythmic effect was prevented by neomycin,
genistein, or microinjected blocking anti-PLC␥ antibody,
all treatments that demonstrate the requirement of IP3
formation in this purinergic effect. However, uncaging
Ca2⫹ from EGTA increases beating rate or triggers arrhythmias. Further works demonstrated that mitochondria that lie close to ER pump Ca2⫹ as indicated by the
transient dissipation of their membrane potential. Thus
IP3 generated by various neurohormones and mitochondria are key components in the neurohormonal regulation
of rhythmic activity in cultured neonatal rat cardiomyocytes that have a developed ER but limited SR (231).
D. Hypertrophy
Hormones and mechanical stretch can induce cardiac growth. Extracellularly applied ATP constitutes a
stimulus sufficient to induce changes in the pattern of
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ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
expression of immediate-early genes such as c-fos and
jun-B that is mediated by a Ca2⫹-dependent pathway in
neonatal rat ventricular myocytes (392, 534).
However, ATP does not induce cell hypertrophy.
Studies on the intracellular signaling mechanisms responsible for myocardial cell growth have focused on MAPK,
p42 and p44MAPK (50, 382, 417, 470), or p38MAPK (95,
529). These kinases are activated by both Ser-Thr kinases
and tyrosine kinase and are involved during various neurohormonal stimulations and mechanical stress. Although
ATP activates p42 and p44MAPK (382) and p38MAPK (5), it
does not, like carbachol, transactivate cardiac-specific
promoter/luciferase reporter genes, nor increases atrial
natriuretic factor expression (382). These studies furthermore suggest that activation of c-fos, jun-B, PKC, and
MAPK are not sufficient by themselves to stimulate hypertrophy. ATP might also activate an inhibitory pathway,
as suggested by the fact that it prevents phenylephrineinduced hypertrophy (533). These effects are probably
mediated by P2Y receptors (533). However, it has been
reported that the autocrine release of ATP mediated by
stress induces hypertrophy of cultured rat neonatal myocytes. This effect involves P2Y receptors and the activation of stretch-induced ERK (483).
E. Other General Effects of ATP in Cardiac Cells
1. Glucose transport inhibition
Extracellular ATP markedly inhibits glucose transport (157) by decreasing the amount of glucose transporters in the plasma membrane with a concomitant increase
in intracellular microsomal membranes. P2X purinoceptors are not involved since a drastic reduction in extracellular Na⫹ or Ca2⫹ does not alter this effect of ATP. The
rank order potency of P2 receptor agonists (ATP ⱖ
ATP␥S ⱖ 2-MeSATP ⬎ ADP ⬎ ␣,␤-met-ATP) does not
match that of any known P2Y purinoceptor subtype. Inhibition of transmembrane glucose transport was specific,
since ATP did not inhibit the rate of glycolysis or the rate
of pyruvate decarboxylation. The physiological significance of this inhibitory effect of ATP on glucose transport
is unclear. It is noteworthy that glucose transport is reduced and is rate limiting in hypertrophied failing heart
(462), a condition during which ATP release and extracellular content are expected to be enhanced.
2. Agonist-induced internalization of purinoceptors
In the face of persistent stimulation, the response of
most receptors fades away. The process of receptor desensitization, one of the many forms of the G proteincoupled receptor regulation, has received much attention.
Desensitization occurs generally via feedback regulation
by the second messenger-stimulated kinases PKA or PKC
783
that the Gs and Gq protein-coupled receptors activate,
respectively. This was first reported for the ␤2-adrenergic
receptor (55). Phosphorylation is followed by ␤-arrestin
binding, a crucial step in internalization of most heptahelical receptors (153, 281).
Our knowledge of the turnover sequence activation/
desensitization of the purinoceptor is rather limited. The
agonist-induced desensitization and sequestration of the
P2Y2 receptor is attributable to its COOH terminus. Thus
truncation of 18 or more amino acids does not alter
UTP-stimulated increases in [Ca2⫹]i, while the concentration of UTP necessary to desensitize the receptor is increased 30-fold (174). The tagging of P2Y2 receptor at its
NH2 terminus with a hemagglutinin-epitope sequence reveals that the receptor undergoes agonist-promoted
movement to an intracellular compartment. However, this
internalization does not establish any functional consequence and is not required for agonist-induced desensitization (450).
3. ATP and preconditioning
The mechanism of preconditioning, a brief ischemic
treatment known to protect cardiac tissue from damage
to subsequent ischemic episodes, is still a matter of debate. The possibility that p38MAPK activation after ATP
release intervenes in the process has been foreseen.
p38MAPK is activated by adenosine soon after ischemia
(49, 316), a period during which ATP is released (276) and
could be degraded. ATP itself activates p38MAPK (5). ATP
is also expected to be released during stretch by many cell
types including cardiac cells (483), an experimental condition sharing conditioning mechanisms of protection
with ischemic conditioning (189). Infarct size was reduced by various antagonists that share in common the
prevention of an increase in K⫹ currents. They include
blockers of adenosine receptors and PKC as well as gadolinium, Gd3⫹, a blocker of stretch-activated channels
including the ATP-activated K⫹ channel (5), and by glibenclamide, a blocker of the IK(ATP) known to be enhanced
by local intracellular ATP depletion following the activation of adenylyl cyclase by isoproterenol and ATP
(13, 428).
4. Prostaglandin release
Biosynthesis and release of prostaglandins are increased by applying ATP to the perfused rabbit heart, an
effect blocked by indomethacin (337). This effect was
mimicked by ischemia, a condition which among other
things increases extracellular ATP; ATP as well as UTP
can induce prostaglandin release from cultured porcine
endothelial cells and bovine aortic smooth muscle cells
(121, 370). This P2Y-receptor effect involves mostly an
increase in [Ca2⫹]i (79, 298).
The large increase in prostaglandin synthesis corre-
784
GUY VASSORT
lates with the acceleration of sinus pacemaker activity
following the addition of ATP in the cardiac perfusate
(463). However, ATP-induced prostaglandin release has
not been characterized at the cardiomyocyte level. The
involvement of prostaglandins to mediate the effects of
ATP has been denied for both the increase in ICaL current
(423) and cAMP synthesis (387).
F. Effects of Diadenosine Polyphosphates on Heart
In spontaneously beating guinea pig atria, diadenosine polyphosphates, ApnAs, exert a negative chronotropic effect. They also reduce maximal contractile force
and slow its development (218, 486). In guinea pig papillary or human ventricular muscles, Ap6A alone was ineffective, but it attenuated isoprenaline-stimulated force
(486). This is at odds with the primary observations that
diadenosine penta- and hexaphosphate, Ap5A and Ap6A,
have been shown to elicit phasic contraction in vas deferens and urinary bladder (453) and constrict rat aorta
via an increase in [Ca2⫹]i (429). These negative chronotropic and inotropic effects are mediated by A1-adenosine
receptors. This has been confirmed in recent studies of
Ap4A, whose effects are abolished by DPCPX (338, 485).
However, in human atrial and ventricular preparations,
Ap4A alone increases contractile force, an effect which is
abolished by suramin (485). The positive inotropic effect
was accompanied by prolonged contractile time parameters similarly to those observed during ␣1-adrenoceptor
TABLE
I(ATP)
I(ATP)SaS
Cl⫺
K⫹
stimulation (469), although it was insensitive to prazozin.
The mechanism is unknown; the authors tentatively suggest activation of a PLC.
The origin of bradycardia induced by ApnAs in perfused heart and multicellular preparations was attributed
to A1-adenosine receptor activation (62). However, a detailed analysis at first using dipyridamole, an inhibitor of
cellular adenosine uptake, indicates that in the tissue
ApnAs had no direct effect on A1-adenosine receptor.
Rather, the authors demonstrated that it is its degradation
product adenosine that activates IK(ACh). In the same
work, an activation of IK(ATP) is also observed. The latter
is attributed to intracellular AMP increase following the
hydrolysis of ApnAs after its cellular influx (62) rather
than to a direct effect of ApnAs (237).
IV. MOLECULAR AND CELLULAR MECHANISMS
A. Modulation of Cardiac Transmembrane
Ionic Currents
1. Purinergic-induced nonspecific cationic currents
The fast application to cardiac cells of ATP in the
micromolar range elicits a rapid activating, fast desensitizing inward current I(ATP) (Table 4). Recovery occurs
within a few minutes. Initially reported by Friel and Bean
(167) in frog atrial cells, this observation was extended to
mammalian atrial and ventricular cells (41, 91, 204, 363,
4. ATP-induced effects on cardiac ionic currents
Current
Na⫹
Ca2⫹
Volume 81
Species
Frog atria
Mammalian
Rabbit sinoatrial node
Guinea pig atria
Rat, mouse ventricle
Guinea pig ventricle
Rat ventricle
Frog atria
Frog ventricle
Rat ventricle
Rat ventricle
Guinea pig, rabbit atria
Ferret ventricle
Hamster ventricle
Frog atria
Guinea pig atria
Rat atria
Guinea pig atria
Guinea pig ventricle
Rat ventricle
Rat ventricle
Pathway
Effects
Reference Nos.
P2X
/
/
/
/
P-Tyr on CFTR
/
Voltage shift
/
/
/
direct Gs
/
/
/
IK(ACh)
IK(ACh)
IK(ACh)
IKs
P-Tyr on IK
IK(AA)
IK(ATP)
cAMP synthesis
⫹
⫹
⫹
⫹
⫹
⫹
0
⫺
⫹
⫹/⫺
⫹
⫹
⫺
⫺
⫺
⫹
⫹
167
41, 91, 363, 424, 536
436
311
239
89, 438
363
425
183, 527
7
91, 116, 125, 126, 389, 435
423
204
398, 399
500
167
170, 190, 312, 315
521
313, 315
309
5
13, 14
⫹
⫹
⫹
⫹
⫹
(/), Nondetermined pathway; (⫹), (⫺), increase or decrease in peak current amplitude; (0), no effect. CFTR, cystic fibrosis transmembrane
conductance regulator. See text for definitions of currents.
April 2001
ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
424, 536). It has also been seen in human cells (Alvarez
and Vassort, unpublished results). The current has a reversal potential just below 0 mV. It could not be attributed
to a Cl⫺ channel since neither internal nor external Cl⫺
ionic substitution altered the current. Rather, I(ATP) is
attributable to a nonselective cation channel allowing
Na⫹, K⫹, and Ca2⫹, but not N-methyl-D-glucamine
(NMDG) to flow through. I(ATP) has linear (91, 167) or
inwardly rectifying current-voltage relationships (204,
363, 424). Inward rectification of K⫹ currents has been
attributed to intracellular Mg2⫹ and/or polyamines. Variations in the rectifying properties of the I(ATP)-voltage
relationships are of unknown origin; at first sight, they
may be attributed to differences in the experimental conditions. Fluctuation analysis after ATP application yields
a very low unitary conductance (363). Like ATP, 2-MeSATP is a very efficient agonist with EC50 0.7–3 ␮M (363,
426) while ATP␥S and ␣,␤-met-ATP as well as GTP, ITP,
UTP, ADP, AMP, and adenosine are not active (167, 424).
However, ␣,␤-met-ATP at 100 ␮M, and ATP␥S at 250 ␮M,
were reported to induce this current in atrial mammalian
cells (91, 204). Ca2⫹ channel antagonists such as Ni2⫹,
Co2⫹, or verapamil do not alter I(ATP). However, increasing external Ca2⫹ reduces I(ATP) (167). Opposite effects
are seen when removing external Ca2⫹ (424), although
I(ATP) was unaffected by ryanodine treatment (204). Decreasing intracellular Ca2⫹ by BAPTA decreases and prolongs I(ATP) (91, 421, 536). Mg2⫹ are required in the bathing solution for ATP to induce I(ATP) (91, 424, 536). It is
worth noting that generally divalent cations including
Mg2⫹ reduce the affinity for ATP at P2X receptors (288).
I(ATP) is blocked by quinidine (91). It is also inhibited
by PPADS and cibacron blue (14, 426). However, these
P2-receptor antagonists are known to interact with different ATP-binding sites apart from the specific purinoceptors.
The first thought, supported by the rapid activation
and deactivation and by the nonspecificity of cation transfer, would be to relate I(ATP) to ligand-operated channels.
Such was the initial proposal in smooth muscle cells (31,
32) and sensory neurons (23). After eliminating “permeabilization” of the cells the way ATP does in mast cells
(97), Friel and Bean (167) strengthened the comparison
with nicotinic ACh receptor channels, although they
could not rule out the involvement of a second messenger
mechanism (probably Ca2⫹).
The fast kinetic characteristics suggest to relate this
current to either P2X1 or P2X3 receptors. However, on
both receptor subtypes, ␣,␤-met-ATP is an active agonist
while in cardiac cells this poorly hydrolyzable analog
prevents the effects of ATP (167) without causing desensitization (426, 536). Also, in cardiac cells, fluctuation
analysis of the ATP-induced current with Na⫹ as the main
charge-carrying ion yields an apparent very small unitary
conductance of 0.08 pS at a holding potential of ⫺120 mV
785
while 2-MeSATP evokes large whole cell currents, although the unitary conductance could not be detected
using the same type of analysis (363). This situation is far
from the single-channel unitary conductance (10 – 60 pS)
reported for the expressed clones of P2X1 and P2X2 receptors (60, 487, 537).
With these observations, Scamps and Vassort (424)
initially proposed a four-step cascade to account for the
effects of rapid ATP application that implied the activation of the anion exchanger Cl⫺/HCO⫺
3 . Both I(ATP) and
acidosis requested Mg2⫹ and were not activated by UTP,
CTP, ITP, GTP, and ADP (390). However, probenecid, an
inhibitor of Cl⫺/HCO⫺
3 exchanger, does not prevent I(ATP)
(M. Ugur and G. Vassort, unpublished results), and this
hypothesis must be presently discarded. Led by the Mg2⫹
dependency and the observation that extracellular ATP
induces phosphorylation of several membrane proteins,
Christie et al. (91) proposed that the polyphosphate chain
serves as a phosphate donor through action of a protein
kinase intimately associated with the receptor. Furthermore, they suggested that either or both 32- and 47-kDa
proteins could be involved in the formation of an aqueous
pore that allows cation influx. Their view was supported
by the weaker effect of ATP␥S and of ␣,␤-met-ATP in
their hands. In conclusion, the present site of action of
Mg2⫹ is unclear. Among several possibilities, one can
suggest the following: Mg2⫹ interaction with P2X channels is compulsory for their opening; ATPMg2⫺ is the
ligand of a given cardiac P2X subtype; or ATPMg2⫺ is the
substrate of an ecto-kinase that modulates P2X channels.
Presently, the protein support of this fast-activating, transient, nonselective cationic current, or its mechanism of
induction by ATP on cardiac cells is unsettled.
More recently, another ATP-activated cationic current was reported in rabbit sinoatrial cells (436). This
current increases in a dose-dependent manner over the
concentration range of 0.01–1 mM ATP, which indeed was
the only active agonist. This current could be carried by
various cations including Tris and NMDG, whose apparent permeabilities are only five times less than K⫹, Na⫹,
or Ca2⫹. It shows a reversal potential near 0 mV and is
weakly inward rectifying. This current does not appreciably desensitize within 3 min of ATP application. A similar
long-lasting ATP-induced current was also elicited in rat
ventricular cells over a very broad range of ATP concentrations (Ugur and Vassort, unpublished results). The ATP
effect does not require the presence of Mg2⫹. BzATP is a
weaker agonist than ATP, whereas neither PPADS nor
oxidized ATP prevents the sustained ATP-induced currents (Ugur and Vassort, unpublished results). These
pharmacological characteristics make it difficult to
clearly attribute this current to the activation of a P2X7
receptor.
Several mammalian sequences homologous to Drosophila Trps have been identified that represent a new
786
GUY VASSORT
family of Ca2⫹-permeable cation channels with at least
TRPC1 present in heart (517). Their mechanisms of activation are still poorly known but appear quite variable.
They include intracellular Ca2⫹ increase as follows from
angiotensin II activation of expressed TRPC3 (543). Similar effects of ATP have to be investigated.
These two ATP-induced nonspecific cationic currents have two major cellular consequences. First they
depolarize the cell membrane and so might initiate spontaneous automaticity that will lead to arrhythmia. Arrhythmia might also occur as a consequence of Ca2⫹ entry
and Ca2⫹ overload through these channels. However, it
has been reported that extracellular EGTA and Ca2⫹
channel blockers (nifedipine, verapamil) reduce the ATPinduced Ca2⫹ transient while BAY K 8644 increases it;
that suggests a more direct relation with the L-type Ca2⫹
channel (126). How much of the ATP-induced effects on
intracellular Ca2⫹ can be attributed to the nonspecific
cationic current remains to be determined. Although such
Ca2⫹ entry should be expected in cardiac cells, the Ca2⫹
transient elicited by ATP has been generally attributed to
other mechanisms (see below).
2. Cl⫺ current
Extracellular ATP (5–50 ␮M) activates an outwardly
rectifying, time-dependent Cl⫺ current (ICl) in single
guinea pig atrial myocytes. ADP, AMP, and adenosine also
activate ICl (311). This is also true in single rat and mouse
ventricular myocytes (222, 239, 284); however, there was
no evidence for such a current in guinea pig ventricular
cells (363). In mouse ventricular cells, this current is
blocked by DIDS (which is now known to be a P2-receptor antagonist) and is not activated by either AMP or
adenosine. ATP␥S also elicits the Cl⫺ current (284). The
differential action of adenosine on ICl, activated by extracellular ATP in atrial and ventricular myocytes, could
reflect different Cl⫺ channels in these cells or species
variability.
Neither the purinoceptor subtype nor the signal
transduction pathway mediating the action of ATP on ICl
is clearly known. Levesque and Hume (284) reported that
activation of ICl is not likely to be dependent on increased
[Ca2⫹]i, since ATP activates the current in cells dialyzed
with 10 mM EGTA or 20 mM BAPTA, a much faster Ca2⫹
chelator. The ATP-induced Cl⫺ current exhibits a weak
outward rectifying conductance like the Cl⫺ current activated by cAMP (357). However, a Cl⫺ current was not
activated by isoproterenol or forskolin application in
mouse ventricular cells, although such occurs in other
species (15, 193). Furthermore, adenosine, which is
known to reverse the ␤-adrenergic-induced increase in
cAMP in mouse ventricular cells (525) and ICl(cAMP) in
rabbit ventricle (194), does not prevent the effects of ATP.
Volume 81
This supports the contention that cAMP is not involved in
the activation of the ATP-induced Cl⫺ conductance.
Attenuation of isoprenaline-induced ICl in guinea pig
ventricular myocytes by ATP has also been reported
(404). In addition, it has been shown that genistein at 100
␮M, but not daidzein, activates the cardiac chloride conductance (89, 438). This effect is antagonized by Na3VO4,
an inhibitor of phosphotyrosine phosphatase. Comparison of ICl activated by genistein and ICl activated by
forskolin led the authors to suggest that genistein activates the cAMP-dependent CFTR channel. Similarities in
potency and efficacy of ATP for intracellular acidification
(390) and in Cl⫺ current activation were noted (239). It
has thus been proposed that activation of a Cl⫺ current
near resting potential could lead to intracellular Cl⫺ depletion and thus to the activation of the Cl⫺/HCO⫺
3 exchanger and subsequent intracellular acidification (239).
The physiological importance of ATP enhancement of Cl⫺
currents is not known. However, with a Cl⫺ reversal
potential around ⫺45 mV in cardiac cells (122), these
currents are potentially arrhythmogenic because they depolarize the membrane and shorten action potential plateau. The Cl⫺ channels should be considered potential
target sites for the development of antiarrhythmic agents.
3. Na⫹ current
Extracellular ATP decreases the inward Na⫹ current
(INa) (425). Thus, in rat single cardiac ventricular myocytes, similar to its effect on ICa (423), extracellular ATP
in the micromolar range causes a leftward shift (5– 8 mV)
in both activation and availability characteristics of INa so
that the availability is reduced at resting potential (91,
425). At hyperpolarized potentials, INa could be slightly
increased due to the voltage shift in its activation kinetics
(425). ATP␥S and ␣,␤-met-ATP exert similar effects but
not UTP, ␤,␥-met-ATP, ADP, and adenosine. The voltage
shift induced upon application of extracellular ATP is not
affected by cholera toxin treatment, suggesting that a Gs
protein and cAMP are not involved (425). The reduction in
Na⫹ current will slow the spread of activation and might
contribute to the arrhythmogenic effects of ATP.
4. Ca2⫹ current
More than two decades ago Goto et al. (183) using
the double sucrose-gap technique reported that extracellular ATP and ADP enhance the calcium inward current
(ICa) and the ICa-dependent phasic tension in muscle bundles isolated from the right atrium of the bullfrog. In a
subsequent study, the same group determined that the
action of ATP on ICa and tension does not require the
hydrolysis of ATP and is probably mediated by a receptor
located at the outer surface of the affected cell membrane
(527). This followed the primary observation that ATP is,
like isoprenaline, able to restore electrical and mechani-
April 2001
ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
cal activities in Ca2⫹-deficient solutions (11) or to induce slow action potentials in K⫹-depolarized guinea pig
hearts (430).
A) ACTIVATION. In single frog ventricle cells under
whole cell patch clamp, ATP (1 ␮M) increases ICaL by up
to twofold; at higher ATP concentrations, the increase in
ICa is smaller, and at 100 ␮M, ATP reduces this current
(7). The ATP-induced increase in Ca2⫹ current is prevented by perturbations that block either signal transduction pathways involving the activation of PLC or its activity (7). These data were interpreted to suggest that the
ATP-induced increase in Ca2⫹ current in frog ventricular
myocytes is mediated by P2-purinoceptor and phosphoinositide turnover on the basis that neomycin specifically
inhibits PLC, a probably misleading assumption (7).
Later detailed studies demonstrated that the extracellular application of micromolar ATP increases the Ltype Ca2⫹ current, ICaL, in mammalian cells isolated from
rat ventricular myocardium (91, 421, 423, 426, 536) and
from guinea pig and rabbit atrial myocytes (204). ATP␥S
exerts a similar effect, but adenosine is much less effective, and GTP, UTP, CTP, and ITP are without effect (421).
The rank order of efficacy and potency of ATP analogs in
increasing ICaL amplitude is 2-MeSATP ⬃ ATP ⬃ ATP␥S,
while ␣,␤-met-ATP, ␤,␥-met-ATP, and ␤,␥-imido-ATP have
no effect (426). The single-channel conductance (⬃17 pS)
is not changed by ATP␥S application on rat ventricular
cells. However, both the probability of channel opening
and availability of functional channels are enhanced after
P2-purinergic stimulation (422). In contrast to the induction of the nonspecific cationic current by ATP, the enhancement of ICaL does not require the presence of Mg2⫹
(424, 536). The activation of P2 purinoceptor leads to an
increase of ICaL via the activation of cholera toxin-sensitive Gs protein but is independent of cAMP or phosphoinositide turnover (423, 536) (Fig. 3). The ATP-induced
increase in ICaL does not involve a phosphorylation by
PKA, since it occurs in the presence of 500 ␮M cAMP in
the patch pipette (423) and is not prevented by the intracellular application of PKI (Alvarez and Vassort, unpublished results). ATP activates the tyrosine kinase pathway; however, the role of tyrosine phosphorylation on
ICaL in cardiomyocytes is, as yet, unknown. Src and FAK
but not MAPK are involved in enhancing the Ca2⫹ current
in smooth muscle cells, an effect that should be related
to the association of Src to the ␣1-subunit of the Ca2⫹
channel (219). Some other effects could also be involved
in relation with the known interaction of tyrosine kinase
with G proteins. Similarly, ATP increases the transient,
low-threshold Ca2⫹ current (ICaT) in frog atrial cells via
a pathway that probably does not involve phosphorylation (9).
B) INHIBITION. In isolated ferret ventricular myocytes,
extracellular ATP in the micromolar range inhibits ICaL in
a time- and concentration-dependent manner (398, 399).
787
2⫹
FIG. 3. Schematic regulation of the L-type Ca
current (ICaL) following P2-purinergic stimulation. A Gs protein positively couples to the
channel activity while the mechanisms of inhibition are unknown. PKG,
protein kinase G.
Stable ATP analogs, 2-MeSATP, ATP␥S, and ␣,␤-metATP,
also cause inhibition, whereas suramin prevents it (398).
The inhibitory effect of ATP cannot be blocked by either
the muscarinic cholinergic antagonist atropine or the
adenosine A1-receptor antagonist DPCPX (399). The ATPinduced inhibitory effect is not dependent on Ca2⫹ influx
or Ca2⫹ load, since it is observed with Ba2⫹ as chargecarrying ion or in the presence of BATPA (398), thus
eliminating a mechanism related to the known Ca2⫹-dependent Ca2⫹ inactivation of ICaL. ATP also inhibits ICa in
guinea pig single SAN cells in a concentration-dependent
manner (396, 446). The rank order of potency of ATP and
related compounds inhibiting ICa in SAN cells is as follows: ATP ⫽ ␣,␤-met-ATP ⬎ 2-MeSATP ⱖ ATP␥S ⬎
UTP ⫽ ADP ⬎ AMP ⱖ adenosine (396). This potency
order has not been reported with regard to previously
identified P2-purinoceptor subtypes, suggesting the mediation of a novel purinergic receptor. Inhibition of ICaL by
ATP is also observed in the presence of guanosine 5⬘-O(3-thiotriphosphate) (GTP␥S) (398, 399, 423) and was
then poorly reversible (500). In guinea pig SAN cells,
DPCPX and 8-PT as well as PPADS and suramin do not
prevent the inhibitory effect of ATP of ICa which is, however, abolished by the PKC inhibitors staurosporine and
calphostin. The authors proposed that in these cells the
ATP-induced inhibition involves neither the P1 nor the P2
purinoceptors (396, 397).
Activation of the heterologously expressed P2Y2 receptor induces inhibition of the N-type Ca2⫹ current in rat
sympathetic neurons (154). ATP and UTP are equally
active with an EC50 (⬍0.5 ␮M) similar to their EC50 for
phosphoinositide signaling and their ability to stimulate
Ca2⫹ release and IP3 production. Despite this, the authors
suggested a direct action of a Gi protein inhibiting N-type
channel gating (134). A Gi protein-mediated inhibition has
been reported for the T-type current (8) and might account in part from the ATP-induced inhibition observed in
frog heart cells at high ATP concentration.
788
GUY VASSORT
Inhibition of ICaL is known to be mediated by activation of the cGMP-dependent protein kinase particularly
when the cell has been previously stimulated by a ␤-adrenergic agonist (192, 201). An inhibition of basal ICaL by
PKG was also reported (226, 466). Because purinergic
stimulation increases cGMP in cardiac cells (423), such a
pathway might well be involved in ICaL inhibition.
External ATP reduces binding of isradipine by 90%
on cardiomyocytes in 30 mM K⫹ buffer in the presence or
not of Ca2⫹ and Mg2⫹ (452). The reduction in binding sites
might correspond in some ways to the inactivation of
Ca2⫹ channels leading to ICaL inhibition.
In rat ventricular cells, a transient inhibition of ICaL
was observed when applying suddenly ATP that preceded
the ATP-induced increase in ICaL (421). This inhibitory
effect was attributed to the transient acidosis of the cells
(389, 390), which is known to reduce ICaL (274). How
much of the inhibitory effects described in other cells are
attributable to ATP-induced acidosis has to be checked by
the use of an inhibitor of the Cl⫺/HCO⫺
3 exchanger such
as probenecid.
5. K⫹ currents
A number of K⫹ channels are present in cardiac
myocytes that determine the shape of the action potential
and frequency of beating. Several observations have indicated that most of these channels are regulated by extracellular ATP.
⫹
A) INWARD RECTIFYING K
CHANNELS, IK(ACh) AND IK(ATP).
Friel and Bean (167) first reported in bullfrog and bovine
atrial cells that extracellular ATP activates an inwardly
rectifying K⫹ channel whose current-voltage relationship
was similar to that activated by ACh. Further works on
mammalian cells (170, 315, 521) demonstrated that P2
receptors, like the muscarinic cholinergic receptors and
the A1-adenosine receptors, are directly coupled to a K⫹
channel via a pertussis toxin-sensitive Gk protein. UTP
also activates IK(ACh), an effect inhibited by pertussis
toxin (521). These observations suggest the involvement
of P2Y2 or P2Y4 receptors.
Activation of IK(ACh) by P2-purinergic agonists decreases monotonically in the continuous presence of the
agonist in the bath solution. As well, ATP application only
transiently increases IK(ACh) activated by ACh before depressing the ACh effect (190, 313). This depressing effect
of ATP is not mediated by a pertussis toxin-sensitive G
protein and is not affected by PKC inhibitor. The potency
order of ATP analogs in reducing IK(ACh) ATP ⱖ 2-MeSATP ⱖ ␣,␤-met-ATP indicates involvement of a P2Y
receptor (312).
Recent studies have shown that extracellular ATP
enhances the current flow through the intracellular ATPsensitive K⫹ channel IK(ATP) once it has been partially
activated under conditions of metabolic stress (i.e., 100
Volume 81
␮M of intracellular ATP) (13). The K(ATP) channel consists
of a weak inward rectifier subunit Kir6.2 plus a member of
the adenine nucleotide binding cassette (ABC) superfamily, SUR2 (228). K(ATP) channel activation during acute
ischemia/hypoxia has been shown to exert a protective
effect on the heart (186, 187). Studies in cardiac myocytes
in vitro have suggested that the activation of A1-adenosine
receptors could result in the activation of K(ATP) channels
(229, 258). However, at least in the hypoxic guinea pig
heart in vivo, endogenous adenosine failed to activate
K(ATP) channels (523). Several analogs of ATP, i.e., ␣,␤met-ATP, 2-MeSATP, and ATP␥S, exert a similar effect to
that of ATP in activating IK(ATP) in rat ventricular myocytes, whereas UTP and ADP have a relatively small effect
and AMP and adenosine have no effect (14). The enhancement of IK(ATP) by extracellular ATP is inhibited by cholera toxin as well as by inhibition of adenylyl cyclase (Fig.
4) (13). Thus it has been suggested that the mechanism of
this effect is the Gs-dependent activation of adenylyl cyclase which causes an increase in cAMP production and
thereby reduces intracellular ATP level (13).
⫹
B) DELAYED OUTWARD RECTIFYING K
CHANNELS, IK. ATP
increases the delayed outward rectifier K⫹ current (IK) in
guinea pig atrial cells (312, 314, 315). ADP also enhances
IK while adenosine is without effect. PTX and theophylline as well as buffering intracellular Ca2⫹ or PKC inhibition do not affect the activation by ATP (312, 314, 315).
ATP was shown to selectively enhance the slow component of the delayed rectifier K⫹ current (IKs), leaving the
fast component IKr unaffected since ATP increases similarly the current and its tail and since ATP effect is
unaffected by E-4031, a known blocker of IKr (313).
In various tissues, several K⫹ currents can be modulated by tyrosine kinase, which is known to be activated
by ATP. Thus the m1-muscarinic ACh receptor potently
inhibits a cloned delayed-rectifier K⫹ channel, Kv1–2,
through its direct phosphorylation via PYK2 (151, 221).
The direct association of Src and tyrosine phosphoryla-
FIG. 4. Proposed scheme accounting for the enhancement of the
ATP-dependent K⫹ current [IK(ATP)] by P2-purinergic stimulation. The Gs
protein specifically couples to a given isoform of adenylyl cyclase, ACV,
whose activation induces a localized ATP depletion.
April 2001
ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
tion of hKv1.5 suppresses channel current (208). In guinea
pig ventricular cells, the delayed rectifier K⫹ current IK is
concentration dependently increased by ATP with an
EC50 of 1.86 ␮M. The ATP effect develops slowly; it is not
affected by PKA and PKC inhibitors while genistein suppresses the response. That leads the authors to hypothesize that tyrosine phosphorylation is involved in P2Yreceptor stimulation of IK (309).
In rat ventricular cells, ATP in the micromolar range
also activates a delayed outward K⫹ current. The latter
effect of ATP is mimicked by the application of arachidonic acid and blocked by AACOCF3, a PLA2 inhibitor as
well as by inhibition of the cAMP pathway (5). These
effects could be attributable to the activation of a twopore domain K⫹ channel of the TWIK-1 family such as
TRAAK (155).
C) HYPERPOLARIZATION-ACTIVATED CURRENT, If . The hyperpolarization-activated current If is sensitive to neuromodulation. ␤-Adrenergic stimulation facilitates If via a
direct cAMP-induced leftward shift of its activation curve.
These effects are antagonized by ACh (129) and would
account for the modulation of SAN activity. As well, adenosine inhibits automaticity probably through the same
mechanism (528). There is yet no report on the effects of
purinergic agonist on If. However, one might anticipate
that ATP by increasing cAMP activates If and has a positive chronotropic effect.
B. Signal Transduction Pathways
789
creases the cAMP level by twofold; at the lower basal
cAMP levels, a fourfold stimulation was observed (387).
The effect of ATP on cAMP production is poorly potentiated by forskolin and is additive to that of submaximal
concentrations of isoproterenol. The ATP-induced activation of the adenylyl cyclase is mediated by a 45-kDa Gs
protein, similar to that observed with isoproterenol stimulation. Both ATP and isoproterenol increase cAMP in
HEK-293 cells expressing type V adenylyl cyclase,
whereas cAMP was only increased by ␤-adrenergic stimulation of HEK-293 expressing type IV and type VI adenylyl cyclases. Thus, in rat cardiomyocytes, purinergic and
␤-adrenergic stimulations differentially activate cyclase
isoforms; adenylyl cyclase V appears the specific target of
the purinergic stimulation (387). A paracrine effect involving PLA2 activation and the formation of prostaglandins
could be excluded, since this work was conducted on
isolated cardiac cells. However, in intact tissues, an increase in cAMP following activation of prostaglandin synthesis (PGI2) should be acknowledged, since ATP activates this pathway in endothelial cells (370).
The increase in cAMP has not yet been related to any
particular purinoceptor subtype in cardiac cells (Fig. 5). A
candidate would be the recently cloned P2Y11 (101). In
other cells such as Madin-Darby canine kidney cells, ATP
increases cAMP preferentially through P2Y2 relative to
P2Y1 and P2Y11 (383, 384). Considering that ATP is rapidly
degraded to adenosine, it should also be noted that ventricular cardiac cells possess both cAMP-decreasing A1
The effects of extracellular ATP on second messengers such as cAMP, cGMP, and IP3 have been investigated
not only in whole heart tissues but also, in the most recent
studies, in isolated cardiomyocytes.
1. cAMP and cGMP
A) CAMP. Whether ATP modulates intracellular cAMP
in cardiac cells has long been controversial. An ATPinduced twofold increase in cAMP had long been reported
in frog ventricle associated with the increase in contractile force (161). However, the authors noted that most of
the increase in force was antagonized by indomethacin,
implying an indirect effect via prostaglandin. Indomethacin was not used during cAMP assays. Zheng et al. (535)
and Scamps et al. (423) observed that ATP does not
significantly affect basal cAMP level in rat ventricular
cardiomyocytes, but it facilitates the isoproterenol-induced increase in cAMP (535). In cardiomyocytes isolated
from fetal mice, basal cAMP level is not changed by ATP,
which, however, partially antagonizes the effect of isoproterenol (525). Reinvestigating the effects of purinergic
agonists on rat neonatal and adult ventricular cardiomyocytes, we found that stimulation with ATP␥S in the
presence of the phosphodiesterase inhibitor IBMX in-
FIG. 5. Models for the control of cAMP level by P2Y receptors. In
most cases P2Y receptors are reported to inhibit adenylyl cyclase (AC).
However, activation of the adenylyl cyclase can occur 1) directly via a Gs
protein coupled to a type V AC (387) and might follow stimulation of
receptor (101); 2) via Ca2⫹ increase and calmodulin by which AC types
I, III, and VIII are positively modulated (107, 458); Ca2⫹ also inhibits
phosphodiesterase; and 3) via prostaglandin (PGE2) release that activates an external Gs protein-coupled receptor R (383, 384). PDE, phosphodiesterase; PLC-␤, phospholipase C-␤; PLA2, phospholipase A2; IP3,
inositol 1,4,5-trisphosphate.
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GUY VASSORT
and cAMP-increasing A2 adenosine receptors (27, 51,
451), although this has been disputed (200, 437).
B) CGMP. ATP, in the presence of IBMX, increases the
cGMP content of isolated rat cardiomyocytes (423). A
slowly developing increase in cGMP has also been reported in frog ventricles associated with the secondary
decrease in contractile force (161).
C) UTP. Flitney and Singh (160) also reported that in
the frog heart UTP, similarly to ATP, increases both cAMP
and cGMP. Note, however, that in whole heart both effects could be secondary to activation of the prostaglandin synthesis or to transphorylation by the NDPK. Indirect
effect of UTP on cAMP content was precisely reported in
mouse macrophages (290). In these cells UTP and UDP,
but not ATP and its analogs, cause cAMP accumulation by
activating several PKC isoforms.
2. IP3
Most P2Y receptors (i.e., P2Y1, P2Y2, P2Y4, and P2Y6)
are coupled to PLC-␤. In a recent study, ATP and UTP
were both shown to activate Gq/11 and Gi-3 but not Go,
Gi1–2, or Gs protein in membranes from gastric and aortic
smooth muscle as well as from heart (not isolated cardiomyocytes) (333). IP3 formation induced by ATP and UTP
was mediated concurrently by Gq/11-dependent activation
of PLC-␤1 and G13␤␥-dependent activation of PLC-␤3.
Phosphoinositide hydrolysis was thus partially inhibited
by pertussis toxin. More specifically, in rat ventricles
(282) and isolated fetal mouse cardiomyocytes (525), ATP
accelerates phosphatidylinositol turnover and induces
IP3 formation. The ATP-induced IP3 formation in rat ventricular myocytes should be attributed, in most part, to
the activation of the PLC-␥ by a tyrosine kinase pathway (395).
The role of IP3 in cardiac cells is still unclear. In adult
mammalian cardiomyocytes, IP3 releases Ca2⫹ from the
SR in mechanically skinned or chemically permeabilized
cells (251, 352, 498, 499) and in voltage-clamped guinea
pig cardiomyocytes (179). Despite that SR-Ca2⫹ release
evoked by IP3 is slower than that evoked by Ca2⫹, IP3
enhances contractions (418, 499). In neonatal rat cells,
caged IP3 as well as purinergic stimulation triggers a
spatially restricted intracellular Ca2⫹ release from a ryanodine- and caffeine-insensitive Ca2⫹ store enriched in IP3
receptors. PLC-␥ activation is required for the ATP effects. ATP, also in an IP3-dependent manner, induces a
depolarization of the mitochondrial membrane that is
associated with a transient Ca2⫹ influx concomitant to the
arrest in Ca2⫹ spiking of cardiomyocytes (231). These
observations led the authors to propose that an integrated
control of the filling state of Ca2⫹ pools accounts for the
IP3-dependent purinergic regulation of cardiac arrhythmic
activity.
Volume 81
3. PKC
Direct evidence in favor of an ATP-induced increase
in PKC activity had been obtained. ATP triggers redistribution from cytosol to the membrane of both ⑀- and
␦-PKC, two Ca2⫹-insensitive PKC isoforms expressed in
neonatal and adult cardiac cells. PKC also induces the
phosphorylation of myristoylated alanine-rich C-kinase
substrate (MARCKS) and the expression of c-fos and
jun-B in neonatal cells, two events known to be mediated
by the kinase (534). These observations are of physiological relevance with regard to the likely specific role of
PKC isoforms in cardiac function (392).
4. Tyrosine kinase activation
Phosphorylation is a major modulating factor of protein activity. Specific kinases phosphorylate proteins on
serine and threonine or on tyrosine residues. The first
type of residue requires activation of PKA, PKC, and PKG
as well as Ca2⫹-calmodulin kinase; the second implies
either tyrosine kinases that display an intrinsic tyrosine
kinase activity in their intracellular domain or the nonreceptor tyrosine kinases activated by G protein-coupled
receptors (225). Various effects of tyrosine kinase have
been reported on Ca2⫹ and K⫹ channels and on the gap
junctions involved in cell-to-cell conduction (see Ref. 386
for review). A useful comparison could be made with the
effects of angiotensin II whose role of tyrosine kinase in
signal transduction in vascular smooth muscle has been
recently reviewed (34). However, today, very little is
known in the heart. Some comparison and arguments are
taken from observations in other tissues.
Several nonreceptor tyrosine kinases are expressed
in isolated cardiomyocytes: two members of the Src family pp60c-src and p59Fyn, Src and Fyn (394, 416), and FAK
(394). Several other nonreceptor tyrosine kinases are
found in whole heart. PYK2 from the FAK family was
shown in whole rat heart (130) but not in isolated neonatal or adult rat cells (394). Fes (78), Arg BP2 mostly
located at the Z disks (506), and Tec (248) were also
reported. Presently, there is no report about the presence
of receptor tyrosine kinases in cardiomyocytes and heart.
ATP, like carbachol, phenylephrine, and endothelin,
activates p42 and p44MAPK isoforms in rat neonatal ventricular myocytes (382). It also activates them as well as
p38MAPK in rat adult cardiomyocytes (5). Activation of
the G protein-coupled P2Y2 receptors by ATP and UTP
stimulates p42 and p44MAPK in endothelial cells (367) and
in PC12 cells (444). In PC12 cells also, the activation of
the Ca2⫹-dependent tyrosine kinase PYK2 after ATP stimulation of P2X2 receptors leads to MAPK activation (461).
The first evidence that ATP activates the tyrosine
kinase pathways (Fig. 6) in adult rat cardiomyocytes was
the labeling by an antiphosphotyrosine antibody of several proteins on Western blot, among them PLC-␥ (395).
April 2001
ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
791
FIG. 6. Tyrosine kinase pathways involved during P2purinergic stimulation. The activation of Fyn, a tyrosine
kinase of the src family, leads to the phosphorylation of
the Cl⫺/HCO⫺
3 exchanger and of the phospholipase C
(PLC)-␥ to induce, respectively, acidosis and phosphatidylinositol bisphosphate hydrolysis to produce diacylglycerol
(DAG) and inositol trisphosphate (IP3). IP3 might trigger
Ca2⫹ release from intracellular stores, particularly the endoplasmic reticulum (ER). MgATP, rather than ATP, is
required for the activation of the Cl⫺/HCO⫺
3 exchanger.
PIP2, phosphatidylinositol 4,5-bisphosphate.
Both phosphorylation and translocation of PLC-␥ depend
on the presence of extracellular Ca2⫹. These events, as
well as ATP-induced IP3 formation, are prevented by
genistein and herbimycin A, two potent tyrosine kinase
inhibitors (395). The anionic exchanger AE1 is also
strongly phosphorylated on Tyr residues in ATP-stimulated cells (394). ATP stimulation of cardiomyocytes
strongly increases both Src and Fyn activities. Fyn also
associates to FAK, which is then phosphorylated on a Tyr
residue, although its activity is unchanged (394). In addition to PLC-␥ and the Cl⫺/HCO⫺
3 exchanger, many other
proteins are phosphorylated on Tyr residues (395).
In earlier works it has been shown that several
growth factors and lymphocyte cell-surface antigens,
whose action is mediated by members of the Src family,
modulate G protein functioning (53, 465) and adenylyl
cyclase activity (16, 335). Overexpression of avian
pp60c-src in fibroblasts results in an enhanced intracellular
cAMP in response to ␤-adrenergic hormones that act
through the stimulatory Gs protein (76, 301). More precisely, the Gs␣ subunit can be phosphorylated by Src
tyrosine kinase on a NH2-terminal and a COOH-terminal
Tyr37 and Tyr377 residues in vitro (332), and the rate of
binding of GTP␥S to Gs protein is increased by pp60c-src
(195). In this regard, it is interesting to note that caveolin,
a major structural component of caveolae membranes,
associates with H-Ras, c-Src, and other tyrosine kinases of
Src family as well as with the ␣-subunit of G proteins
including Gs; it negatively regulates both c-Src and GsATPase activity (287, 289).
Notwithstanding that ATP activates tyrosine kinase,
ATP (100 ␮M) and isoproterenol (1 ␮M) were just additive
in increasing cAMP in neonatal cells (387). The use of
supramaximal concentrations might have obscured synergistic effects; however, genistein did not alter ATPinduced cAMP increase (387). Such a modulating/controlling effect of tyrosine kinase after ATP stimulation might
occur on IP3 formation. Gq␣ and G11␣ can be phosphor-
ylated on Tyr-356. IP3 formation is reduced after activating the m1-ACh receptor by carbachol in cells expressing
a mutated G11␣ (Y356F) protein. That suggests tyrosine
phosphorylation events occur before activation of Gq/11␣
and regulate its interaction with G protein-coupled receptors, thus accounting for the more active stimulation of
PLC-␤ (294, 482).
The purinoreceptor subtype activating the tyrosine
kinase cascade is unknown. However, it was previously
shown (91, 390, 424) that the ATP-induced nonspecific
cationic current and the ATP-induced acidosis requires
the presence of Mg2⫹, a characteristic that is not generally
recognized with the presently available cloned purinoceptors.
5. PLA2 and PLD
Besides the activation of PLC and the production of
IP3, second messenger signaling of ATP cascades also
include activation of PLA2 (86, 291, 522). In the latter
report in MDCK-D1 cells (522), the ATP and UTP increased PLA2 activity and arachidonic acid release involving either PKC-␣ or other PKC isoforms acting through
MAPK activation. Previous reports indicate that ATP activates arachidonic acid metabolism in both whole heart
and isolated cardiac myocytes (113, 463). Arachidonic
acid is thought to mediate sinus rhythm acceleration via a
cyclooxygenase metabolite, i.e., prostaglandins (463), or
to enhance contractile force and intracellular Ca2⫹ transient after Ito inhibition and action potential prolongation
(113). Arachidonic acid increase might also mediate the
ATP-induced activation of a two-pore domain K⫹ current
in rat cardiac cells (5). Another consequence could be an
ATP-induced increase in cGMP content, since arachidonic
acid has been reported to activate soluble guanylyl
cyclase.
ATP effects also include activation of PLD mediated
by PKC-␨ (376) or both a G protein, probably Rho, and
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GUY VASSORT
Volume 81
tyrosine kinases (139, 275). Thus it was observed ATP
markedly enhances PLD stimulation by GTP␥S; this effect
is mimicked by vanadate, an inhibitor of protein tyrosine
phosphatases and inhibited by tyrosine kinase inhibitors.
The rank order of efficacy for stimulation of PLD activity
in the presence of GTP␥S is ␤,␥-met-ATP ⬎ ATP␥S ⫽
ATP ⫽ ADP ⬎ 2-MeSATP ⬎ ␣,␤-met-ATP ⫽ UTP and is
independent from PLC activation in rat liver plasma membranes (303).
6. Intracellular Ca2⫹
In quiescent and stimulated cells, the extracellular
application of ATP in the micromolar range causes an
about threefold increase in [Ca2⫹]i (91, 116, 125, 126, 204,
389, 435). This increase results, in most part, from a large
Ca2⫹ release by the SR, since caffeine and ryanodine
markedly reduce it, although they do not prevent it all (41,
125, 204) even in the absence of external Ca2⫹ (389). In
the frog heart, despite the limited SR, a role of this Ca2⫹
store in the facilitating effect of ATP had also been reported (345, 346). However, in most of these studies, a
requirement for external Ca2⫹ was reported. This led
some groups to suggest a relation with the increase in ICaL
associated with extracellular ATP (116, 125, 126, 204).
Furthermore, ATP directly gates a nonselective cationic
channel, I(ATP) (41, 167, 536), through which a significant
Ca2⫹ influx could also occur (see sect. VIA). This is in line
with the observation that both I(ATP) and the ATP-induced
increase in Ca2⫹ are Mg2⫹ dependent (91, 389, 424) and
with the ATP-mediated influx of Mn2⫹ that quenched the
indo 1 fluorescence signal (389). Other suggested mechanisms include phosphorylation of an extracellular protein
leading to activation of a novel ion channel (91), as well as
ATP-induced acidosis (389), leading to both I(ATP) and the
increase in [Ca2⫹]i (424).
Several ATP analogs, ATP␥S, ␣,␤-met-ATP, ATP␥S,
and 2-MeSATP, also induce an increase in [Ca2⫹]i while
␤,␥-met-ATP, ␤,␥-imido-ATP, AMP, and ADP are inactive
(91, 116, 204, 389). In their study Christie et al. (91) noted
that the ATP effect is biphasic with a large transient (1
min) peak followed by a weaker sustained Ca2⫹ increase.
This was not reproduced by the other agonists, which
furthermore could desensitize the ATP effect as they do
for ATP (426). Thus, to summarize, ATP first activates
I(ATP) carried by various cations including Ca2⫹; I(ATP)
depolarizes the cell and triggers INa followed by ICaL so
that tetrodotoxin could suppress the effect of ATP on
[Ca2⫹]i (41).
Increase in Ca2⫹ transient and contractile force
might be also indirect, secondary to action potential prolongation following K⫹ current inhibition, or secondary to
activation of the reverse mode of the Na⫹/Ca2⫹ exchanger
following Na⫹ load. Thus arachidonic acid that is produced after ATP application inhibits Ito; during the sub-
FIG. 7. Regulation of intracellular pH (pHi) by ATP. A: the sudden
application of 10 ␮M ATP on a Snarf-1-loaded ventricular myocyte
triggers first cell acidosis followed by alkalosis. These effects are attributable in most part to the successive activation of the Cl⫺/HCO⫺
3 exchanger and of the Na⫹/H⫹ antiport. [From Pucéat et al. (390).] B: after
⫹
⫹
blockade of the Na /H antiport with ethylisopropylamiloride (EIPA),
10 ␮M ATP still accelerates the recovery from acidosis on a rat ventricular cell submitted to a NH4 pulse (20 mM for 1.5 min). This effect is
attributed to the activation of the Na⫹/HCO⫺
3 cotransport (467, 468).
[From Terzic et al. (468). Copyright 1992 Springer-Verlag.]
sequently prolonged action potential, the Ca2⫹ transient
and force increases (113–115). Also, extracellular ATP as
well as ATP␥S stimulates the Na⫹-Pi cotransport (127). As
a consequence to the Na⫹ load in the presence of high
extracellular Pi, there is activation of the reverse form of
the Na⫹/Ca2⫹ exchanger that may trigger a hypercontracture. However, a direct stimulating effect of P2-receptor
activation on the Na⫹/Ca2⫹ exchanger has also been proposed (214, 215). In the latter case, high ATP concentrations have to be used (estimated EC50 ⬃70 ␮M).
7. Intracellular pH
Intracellular pH is a main determinant of several
cellular activities: ionic channels, contractile proteins,
and metabolism. Purinergic stimulation activates the
three major pH-regulating systems: the Na⫹/H⫹ anti⫺
⫺
porter, the Na⫹/HCO⫺
3 symporter, and the Cl /HCO3 exchanger (Fig. 7). Intracellular alkalinization is prevented
by amiloride derivatives and is attributable in most part to
activation of the Na⫹/H⫹ antiport (391, 505). Under experimental acid load, ATP also activates an amilorideinsensitive HCO⫺
3 -dependent alkalinizing mechanism
(468). The signal transduction pathways have not been
clearly established in both cases but do not seem to
directly involve PKC.
April 2001
ATP: A P2-PURINERGIC AGONIST IN THE MYOCARDIUM
The most remarkable effect on intracellular pH of a
sudden application of micromolar extracellular ATP is a
large (0.4 pH unit) and transient (1 min) acidosis that
requires Cl⫺ in the extracellular milieu that has been
attributed to the activation of the anionic Cl⫺/HCO⫺
3 exchanger (390). The activation of the exchanger is associated with a band 3-like protein phosphorylation on a
tyrosine site (393, 394). More recent work has shown that
isolated cardiomyocytes express both AE1 and AE3 isoforms. ATP induces tyrosine phosphorylation of AE1 (Fig.
6) while acidosis still occurs in cell in which AE3 expression was blocked. More precisely, ATP activates the tyrosine kinase Fyn and the association of both Fyn and
FAK with AE1. Tyrosine kinase inhibitors and microinjection of either anti-Cst.1 antibody or recombinant COOHterminal Src kinase (CSK), both of which prevent activation of Src kinases, significantly depress the ATP-induced
activation of the anion exchanger. As well microinjection
of an anti-FAK antibody directed against the domain of
Fyn binding or overexpression of a dominant negative
FAK (FAKF397) significantly blocks ATP-induced activation of the exchanger (394).
In conclusion, it is proposed that ATP by activating
not-yet-identified receptors implicates both the alkalinizing ion exchangers, Na⫹-H⫹ antiport and Na⫹-HCO⫺
3 cotransport, and the acidifying AE1 and thus provides the
cell with a complementary functional buffering system
bringing a beneficial protective way to face pH disturbances.
V. CONCLUDING REMARKS
The cloning of 7 P2X and 13 P2Y receptors during the
last 5 years has considerably advanced our basic knowledge. However, the precise functions of those of these
proteins which are expressed in cardiac myocytes are not
clear. Not only is this due to the probable simultaneous
expression of multiple receptors in each cardiomyocyte
but also to the fact that P2X receptors can form complex
homo- or heterotrimeric receptors whose activity differs
with each oligomer (99, 131, 344). A similar complexity
may also apply to P2Y receptors following the recent
demonstration that G protein-coupled GABAB receptors
form heterodimers with specific and different properties
compared with the individual homomeric receptors (246,
273). Analysis should also take into account that P2Y
receptors might connect with only specific parts of an
otherwise general pathway such as occurs with the coupling to isoform V of adenylyl cyclase (387). Furthermore,
as for other proteins, receptor phosphorylation can modulate channel activity that is shown by P2X2 receptors
(90). In addition, some peculiar effects of ATP such as the
activation of the tyrosine kinase pathways (387, 394) have
yet to be associated with any particular purinergic recep-
793
tor subtype. Therefore, there is still a long way to go to
understand the behavior of integrated cellular P2 receptors from the expression of single cloned proteins. However, cloning and expression of these proteins are primary
and necessary steps to develop efficient pharmacological
tools, an urgent need in the face of the present paucity.
Several P2X mRNAs have been found in heart tissues;
in as far as they are translated, the role of these proteins
remains to be determined. As mentioned earlier, there is
no clear P2X-related effect of ATP on cardiomyocytes
despite I(ATP) showing some broad similarities (rapid inactivation, nonselectivity, and cation permeability) with
P2X-purinoceptor activation after reexpression. However,
the three studies (91, 424, 536) in which the effects of
Mg2⫹ were investigated report their need to elicit I(ATP).
That led these authors to suggest the involvement of
ectoenzymes. This is at odds with the general belief that
ATP4⫺ is the preferred agonist of P2 purinoceptors. Mg2⫹
might also control receptor affinity; however, divalent and
trivalent cations reduce ATP-activated currents in native
and endogenous P2X receptors (91, 403, 424, 536), and
Mg2⫹, in particular, have been shown to reduce P2X2
affinity to ATP (288). Mg2⫹ are also requested for the activation of the tyrosine kinase pathway and the Cl⫺/HCO⫺
3
exchanger. Moreover, in cardiomyocytes, it is noteworthy
that ATP and its analogs modified on the purine base that
are known good substrates for ecto-ATPases induce I(ATP).
The same compounds were also reported good agonist on
P2Y1 purinoceptors. These effects were suppressed under
conditions in which care was taken to purify the agonist
solutions from contaminating ADP by treatment with a creatine phosphate/creatine phosphate kinase regenerative system. Similar control experiments need to be performed to
determine the exact origin of I(ATP).
ATP is released into the interstitial milieu from most
cell types. UTP, by transphosphorylation and diadenosine
polyphosphates, after degradation is another potential
source of extracellular ATP. At the same time ATP is
degraded to adenine nucleotides and nucleosides so that
the response of the cardiomyocyte should be the integral
of the various effects of these multiple compounds generated by the different surrounding tissues. Furthermore,
under pathological conditions, this process could be accelerated by the macrophages and lymphocytes that have
high ecto-ATPase activities and which invade the interstitial space. Also, upregulation of the enzymatic chain hydrolyzing extracellular ATP after ischemia could be expected in cardiac tissues as was observed in the forebrain
after transient occlusion of brain arteries (63). Cardiomyocytes also exhibit both autocrine and paracrine purinergic activities. In this context, it has been reported that
ATP might mediate the cell-to-cell spread of the Ca2⫹
signal in the absence of gap-junctional coupling such as
occurs in mast cells or astrocytes (188, 356). Cardiac
muscle is considered to be a syncytium. However, the
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GUY VASSORT
spread of electrical activation should be reduced in the
presence of ATP both by INa depression and cell uncoupling after intracellular Ca2⫹ release and acidosis. In
these circumstances, anomalous activity might occur and
propagate thanks to ATP diffusion and P2X-receptor activation that leads to cell depolarization and contraction,
stretching of the neighboring cells, and further ATP
release.
At this point it should be clarified that ATP is an
agonist with both physiological and pathological effects.
The first include positive or negative inotropy and chronotropy. These effects seem to be mostly mediated by
P2Y receptors. However, when applied abruptly, ATP induces acidosis, cell depolarization, and arrhythmia mostly
by acting through P2X receptors. It is noteworthy that
most extracellular effects of ATP occur in the micromolar
range, at a concentration at least 1,000-fold less than for
its intracellular metabolic effects. Such a variation in
stimulation profiles is not generally recognized for agonists acting within the same concentration range on a
tissue with the same physiological status. Other possible
roles of ATP concern various physiopathological aspects
such as hypertrophy, preconditioning, and apoptosis. The
latter was early mentioned by Brake et al. (60) who reported the extensive sequence similarity between P2X2
and the partial cDNA called RP-2 and isolated in immature
thymocytes undergoing programmed cell death. More recently, apoptosis of dendritic cells was shown to depend
on P2X7/P2X receptor by a pathway, in most part, independent of known caspases (109). A common feature to
these receptors is the increase in [Ca2⫹]i following their
activation. Apoptosis is a process well established in cardiac cells under stress but for which an involvement of
ATP remains to be elucidated.
Few studies have investigated the presence of P2X
receptors on isolated cardiomyocytes rather than on
membranes obtained from the whole heart, but it is precisely this which is required to extend our knowledge of
the change in P2-receptor subtype expression with age
(47, 48, 511) or pathological conditions (216). These studies used PCR protocols. Very few antibodies are currently
available. Further research will benefit from the recent
developments in genome-based methods: expressed sequence tags, DNA microassays, mRNA differential displays, or the serial analysis of gene expression. The acquired information about developmental and compensatory mechanisms are important to design possible pharmacological treatments.
Finally, ApnAs are a new class of signaling molecules
that act both intra- and extracellularly to alter both cardiac chronotropy and inotropy. The specificity of diadenosine phosphate receptors and their signal transduction
pathways are still poorly understood. Furthermore,
ApnAs slowly formed nucleotides and nucleosides that
further multiply the difficulties of analysis. Cloning of
Volume 81
these receptors and detailed studies after reexpression
are prerequisite to better understand the roles of ApnAs in
cardiac tissues.
I express my thanks to Drs. F. Aimond, J. Alvarez, A.
Babenko, I. Korichneva, M. Pucéat, and F. Scamps for their
experimental collaboration and many fruitful discussions over
the past years. I am very grateful to Drs I. Findlay , J. Alvarez,
and C. Frelin for critical comments on the manuscript and to
Michèle Benoit for skillful secretarial help. Thanks are also due
to Drs. J. M. Boeynaems, D. Erlinge, C. Frelin, and V. Ralevic for
providing data in press.
Address for reprint requests and other correspondence: G.
Vassort, INSERM U. 390, CHU Arnaud de Villeneuve, 34295
Montpellier Cedex 5, France (E-mail: vassort@montp.inserm.fr).
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