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Department of Medicine, Cardiovascular Division, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Although ATP,
acting through a P2 purinoceptor, can
stimulate a pronounced positive inotropic effect in cardiac ventricular myocytes, the receptor-effector mechanism that underlies this stimulatory cardiac action is not well understood. The objectives of
the present study were to develop the cultured chick embryo ventricular
myocytes as a novel model for the cardiac P2
purinoceptor and to determine the mechanism underlying its positive
inotropic effect. ATP caused an 89 ± 8.9%
(n = 14 cells) increase in the myocyte
contractility, with an efficacy and potency order of ATP > ADP > AMP 
adenosine. 2-Methylthio-ATP (2-MeS-ATP) but not 
,
-methylene-ATP was able to stimulate myocyte contractility, with
a maximal increase of 54 ± 2.6%
(n = 11 cells). Although UTP potently
stimulates phosphoinositide hydrolysis, it had an only modest positive
inotropic effect (27 ± 7% maximal increase; n = 8 cells). In contrast to previous
suggestions, the 2-MeS-ATP-stimulated positive inotropic response does
not require the action of phospholipase C (PLC), such as that of the
inositol phosphates; the UTP effect on contractility appears to be
mediated via the 2-MeS-ATP-sensitive P2
receptor. The PLC inhibitor U-73122 had no effect on the
2-MeS-ATP-stimulated increase in contractility, providing further
evidence against a role for PLC in the inotropic effect of 2-MeS-ATP.
An adenosine 3',5'-cyclic monophosphate-independent
Ca2+ entry-stimulating mechanism
appears to underlie a direct coupling of the receptor to stimulation of
the myocyte contractility. This new PLC- and adenosine
3',5'-cyclic monophosphate-independent positive inotropic
mechanism represents a target for developing novel positive inotropic
therapeutics.
heart; receptor; purinergic; contractility; purines
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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ATP EXERTS A NUMBER of pronounced effects in the
cardiovascular system (for reviews, see Refs. 21, 24). In the cardiac myocyte, ATP causes a pronounced positive inotropic effect (12, 18), which appears to be mediated by a cell surface ATP
receptor or P2 purinoceptor. Activation of the
P2 purinoceptor by ATP, which can be released
from platelets, endothelial cells, or hypoxic cardiac tissues (5, 10,
14, 15, 22, 33) as a paracrine and autocrine regulatory agent, may
provide important inotropic support in both healthy and diseased
hearts. ATP can also act in synergy with a -adrenergic agonist to
augment myocyte contractility (37) as a cotransmitter released with
norepinephrine from the sympathetic nerve endings. How ATP causes the
increase in myocyte contractility is not well understood. A number of
P2-purinoceptor cDNAs have been cloned,
including at least four subtypes for the P2Y
receptor and seven for the P2X receptor as
well as that encoding the P2 receptor found on
macrophages and platelets (7).
Activation of the P2Y receptor has been shown to cause either an inhibition of or no effect on adenylyl cyclase activity and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation (26, 36). Coupling of the P2Y receptor to the inhibition of adenylyl cyclase and cAMP accumulation is mediated by the inhibitory G (Gi) protein. The P2Y receptor is also linked to stimulation of phosphatidylinositol 4,5-bisphosphate (PIP2)-phospholipase C (PLC), leading to an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (for a review, see Ref. 17). Ins(1,4,5)P3, by mobilizing intracellular Ca2+, can stimulate myocyte contractility. Diacylglycerol, by activating protein kinase C (PKC), can increase myofilament sensitivity to Ca2+ and thus enhance cardiac contractility. The P2X receptor subfamily represents a ligand-gated ion channel that permits entry of Na+ and Ca2+ into the cell (6, 8, 32). Although a P2 purinoceptor appears to mediate stimulation of Ca2+ entry and an increase in the cytosolic Ca2+ level in isolated cardiac myocytes, the subtype of the P2 purinoceptor that mediates the increase in cardiac myocyte contractility and the mechanism underlying this stimulatory effect remain unknown. A myocyte model system for the cardiac P2 purinoceptor is lacking. The role of PIP2-PLC in mediating the P2 agonist-stimulated myocyte contractility is not clear.
The objectives of the present study were to develop a myocyte model using cultured chick embryo ventricular myocytes to characterize the cardiac P2 purinoceptor and to investigate the cellular mechanism underlying the P2 receptor-mediated positive inotropic effect.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Preparation of cultured cardiac cells. Ventricular cells were cultured from chick embryos 14 days in ovo according to previously described procedures (2, 19). After trypsin was neutralized with a medium containing horse serum, the cells were centrifuged and resuspended in culture medium containing 6% fetal bovine serum, 40% medium 199 (GIBCO), 0.1% penicillin-streptomycin, and a salt solution. The cells were plated at a density of 400,000 cells/ml and cultivated in a humidified 5% CO2-95% air mixture at 37°C. The cells grew to confluence on day 3 in culture and exhibited rhythmic spontaneous contraction.
Measurement of 45Ca uptake into myocardial cells and cAMP level. Determination of 45Ca uptake was made as previously described (20). Cultures were incubated with L-[3,4,5-3H(N)]-leucine (152.2 Ci/mmol) for 24 h before 45Ca uptake. [3H]leucine incorporated into the cellular protein allowed normalization of 45Ca content to milligrams of cell protein. After exposure to 45Ca, the cells were washed free of 45Ca with four rinses of ice-cold buffer containing (in mM) 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1.0 CaCl2, 4 KCl, 0.5 MgCl2, 142 NaCl, and 1 lanthanum, pH 7.35. Such a washing procedure removed >99% of the extracellular marker 51Cr-EDTA and ensured complete removal of the extracellular 45Ca. Uptake of 45Ca was quantitated for 90 s. The cells were solubilized for 2 h in a solution containing 1% sodium dodecyl sulfate and 10 mM sodium borate. Aliquots of solution containing dissolved cells were assayed for radioactivity and protein content. cAMP was then extracted with the addition of a 0.1 volume of 1 N HCl to the medium, followed by boiling for 10 min. The extracted cAMP was assayed according to a previously described radioimmunoassay method (Ref. 19; Amersham, Arlington Heights, IL). The effect of the agonist on cAMP accumulation was linear for 10 min, at which time cAMP was extracted for assay.
Determination of contractile amplitude. Measurement of contractile amplitude in cultured cardiac cells was carried out via an opticovideo motion-detection system as previously described (2, 20). Cells were paced at 2 Hz to focus on the change in contractile amplitude. Myocytes were exposed to a perfusion medium that contained the various nucleotide analogs indicated as well as the following components (in mM): 4 HEPES (pH 7.4), 137 NaCl, 3.6 KCl, 0.5 MgCl2, 1.1 CaCl2, and 5.5 glucose. Measurement of contractile amplitude was carried out on only one cell per coverslip. Both the basal contraction amplitude and the amplitude measured during adenine nucleotide exposure were determined. Measurement of the phosphoinositide response. Inositol phosphates were determined according to the basic method of Berridge et al. (3), and further modified as described by Barnett et al. (1). Cells were preincubated with 5 µCi/ml of myo-[3H]inositol for 24 h and washed with Dulbecco's modified Eagle's medium containing 15 mM LiCl and incubated in this LiCl buffer for 10 min at 37°C before being exposed to ATP or other nucleotide analogs. After extraction with 1 ml of chloroform-methanol-HCl (at 1:2:0.05, vol/vol), the various inositol phosphates were separated on a 1.0-ml anion-exchange column (AGx8 resin, formate form), and inositol 1-phosphate (InsP1), inositol 1,4-bisphosphate [Ins(1,4)P2], and Ins(1,4,5)P3 were eluted sequentially with 100 mM formic acid-200 mM ammonium formate, 100 mM formic acid-600 mM ammonium formate, and 100 mM formic acid-1 M ammonium formate, respectively. The columns were calibrated with each inositol phosphate standard to confirm complete separation of InsP1, Ins(1,4)P2, and Ins(1,4,5)P3. Recovery of each inositol phosphate was >95%.Ins(1,4,5)P3-radioreceptor assay. To complement results on the Ins(1,4,5)P3 level determined by anion-exchange chromatography, the effect of ATP-receptor agonists on the Ins(1,4,5)P3 level was quantitated via an Ins(1,4,5)P3-radioreceptor assay. The growth medium in which the ventricular cells were grown was replaced with a HEPES-buffered solution (pH 7.35) containing (in mM) 1.0 CaCl2, 4 KCl, and 0.5 MgCl2, and then exposed to ATP. The reaction was terminated by a 0.2 volume of ice-cold trichloroacetic acid, which was removed by extraction with a solution of 1,1,2-trichloro-1,2,2-trifluoroethane-trioctylamine. The Ins(1,4,5)P3 in the aqueous phase was determined by competing with [3H]Ins(1,4,5)P3 for binding to the Ins(1,4,5)P3 receptor supplied (DuPont, Boston, MA) (20).
Materials. Embryonic chick eggs were from Spafas (Storrs, CT). The cAMP radioimmunoassay kit was obtained from Amersham. [3H]leucine, myo-[3H]inositol, the Ins(1,4,5)P3-radioreceptor assay kit, and 45Ca were obtained from Dupont (Boston, MA). Adenosine, ADP, AMP,,-methylene-ATP,
,
-methylene-ATP, and UTP were obtained from Sigma Chemical (St.
Louis, MO); 2-methylthio-ATP (2-MeS-ATP) was from RBI (Natick, MA).
1(6-((17-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122) and
1(6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidine-dione (U-73343) were from BIOMOL (Plymouth Meeting, PA).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Characterization of the positive inotropic response to
ATP and adenine nucleotides. ATP stimulated a marked
increase in the myocyte contractile amplitude, with a half-maximal
effective concentration (EC50)
of 0.16 ± 0.1 (SE) µM and a maximal increase of 89 ± 8.9% occurring at 3 µM (n = 14 cells;
Fig. 1). ADP also caused a significant increase in myocyte contractility, with a similar
EC50 (0.40 ± 0.3 µM;
n = 8 cells) although it was less
efficacious (maximal increase of 47 ± 10.5%;
n = 8). AMP and adenosine were less
effective in stimulating myocyte contractility (maximal percent
increase in contractile amplitude was 10 ± 4.3%,
n = 7 cells and 16 ± 3.7%, n = 24 cells, respectively),
indicating that the inotropic effect of ATP is mediated by the
P2 rather than by the
P1 purinoceptor (Fig. 1). To characterize the
subtype of P2 purinoceptor that mediates the positive inotropic
response to ATP, a number of P2 receptor
subtype-selective agonists were tested. The
P2-receptor agonist 2-MeS-ATP caused
a large increase in the contractile amplitude (EC50 of 0.06 ± 0.05 µM;
n = 11 cells), whereas ,-methylene-ATP or
,-methylene-ATP, which are agonists at some of the
P2X receptors, was ineffective at
stimulating the myocyte contractility (Fig. 2). UTP, capable of activating the
UTP-sensitive P2Y receptor, had a modest
stimulatory effect on the myocyte contractility, with an
EC50 of 0.3 ± 0.1 µM and a
maximal percent increase in contractile amplitude of 27 ± 7%
(n = 8 cells; Fig. 2),
consistent with a role of an UTP-sensitive P2Y
receptor in mediating the positive inotropic response to ATP. Because
2-MeS-ATP is a potent agonist at some of the
P2X receptors such as the
P2X2,
P2X4,
P2X5, and
P2X6 subtypes, it is possible that
a P2X receptor can also mediate the
ATP-induced positive inotropic effect in the cardiac myocyte.
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Subtype of cardiac P2 purinoceptor coupled to stimulation of phosphatidylinositol hydrolysis. Because P2Y receptors can be coupled to activation of PIP2-PLC, with consequent stimulation of phosphatidylinositol (PI) hydrolysis (16), we determined whether a UTP-sensitive cardiac P2Y receptor can also couple to stimulation of PIP2-PLC and whether the increase in PIP2-PLC activity mediates the positive inotropic response. ATP caused a significant increase in the levels of InsP1, Ins(1,4)P2, and Ins(1,4,5)P3 (Fig. 3A), and after 30 min of stimulation by ATP, there was a nearly sixfold (570 ± 110%; n = 12 cells) increase in total inositol phosphates, with an EC50 of 15 ± 10 µM (n = 12 cells; Fig. 3B). The increase in the Ins(1,4,5)P3 isomer was confirmed by an Ins(1,4,5)P3-radioreceptor assay (basal level, 42 ± 6 pmol/mg; in the presence of ATP, 96 ± 4 pmol/mg; n = 3 experiments). The Ins(1,4,5)P3 increase was transient, peaking at 45 s. UTP is also coupled to a pronounced stimulation of inositol phosphate production, with an increase in total inositol phosphates of 500 ± 90% at its maximal concentration (300 µM) and an EC50 of 11 ± 10 µM (n = 12 cells; Fig. 3B). Neither the ATP- nor the UTP-stimulated PI response was attenuated by prior treatment of the myocytes with 5 ng pertussis toxin/ml over 24 h (data not shown), a treatment protocol that caused complete ADP ribosylation of Gi by the endogenous NAD+ in these cultures (19, 20).
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,-methylene-ATP and ,-methylene-ATP were ineffective in
stimulating PI hydrolysis (data not shown). The data are consistent
with the notions that a UTP-sensitive cardiac
P2Y receptor is closely coupled to the
activation of PIP2-PLC, whereas a
separate 2-MeS-ATP-sensitive P2
receptor is potently coupled to stimulation of myocyte contractility but is inefficiently coupled to
PIP2-PLC. Alternatively, a
P2X or
P2Y receptor, activated by 2-MeS-ATP,
may be selectively coupled to the stimulation of myocyte contractility,
whereas the UTP-sensitive P2Y receptor
is coupled only to PIP2-PLC, the
activation of which has no effect on the myocyte contractility. If this
notion is correct, the stimulatory effect of 2-MeS-ATP on PLC activity
is due to its agonist activity at the PLC-coupled
P2Y receptor, whereas the positive
inotropic effect of UTP is due to its agonist activity at a
2-MeS-ATP-sensitive P2 purinoceptor. To
provide further evidence for this notion, a number of
cross-desensitization experiments were carried out.
Role of PIP2-PLC in mediating the
P2 agonist-induced positive inotropic response.
The UTP- and 2-MeS-ATP-mediated PI hydrolysis was partially
desensitized by a 90-min prior incubation of the myocytes with 100 µM
UTP (Fig. 4). However, prior treatment of
the myocytes with 100 µM 2-MeS-ATP for 80 min had no effect on the
basal level of inositol phosphates [control cells: 9,883 ± 320 counts/min (cpm), n = 3 experiments; 2-MeS-ATP-treated cells: 9,214 ± 410 cpm,
n = 3 experiments] or on the
increase in PI hydrolysis that was subsequently stimulated by either
UTP (control cells: 45,313 ± 1,820 cpm,
n = 3 experiments; 2-MeS-ATP-treated
cells: 46,576 ± 1,694 cpm, n = 3 experiments) or 2-MeS-ATP (control cells: 15,842 ± 2,010 cpm,
n = 3 experiments; 2-MeS-ATP-treated
cells: 14,265 ± 1,902 cpm, n = 3 experiments). The role of the PLC-coupled
P2Y receptor in mediating the positive
inotropic response was examined next. An 80-min exposure to 100 µM
2-MeS-ATP caused a significant desensitization in the ATP-induced
positive inotropic response (73 ± 4% decrease in the extent of
stimulation in myocyte contractility; n = 8 cells). The 2-MeS-ATP-treated
myocytes also showed a diminished positive inotropic response to
2-MeS-ATP and virtually no increase in myocyte contractile amplitude in
response to UTP (Fig.
5A). However, a 90-min prior exposure to 100 µM UTP had no
effect on the 2-MeS-ATP- or UTP-stimulated increase in myocyte
contractility (Fig. 5B). These data
are consistent with the notion that a
P2Y receptor, activated with a potency
order of UTP 2-MeS-ATP, is coupled to stimulation of
PIP2-PLC, whereas a
P2X or
P2Y receptor, activated with
a potency order of 2-MeS-ATP UTP, is coupled only to
stimulation of the myocyte contractility. The
aminosteroid U-73122, a known inhibitor of the
receptor-mediated activation of PLC, was used to further determine
whether PLC plays a role in the 2-MeS-ATP-stimulated increase in
myocyte contractility. U-73122 at 1 (data not shown) or 10 µM (Fig.
6) had no effect on the
2-MeS-ATP-stimulated increase in myocyte contractility. Ten micromolar
U-73122, a concentration known to completely inhibit the PLC-mediated
PI hydrolysis (data not shown; Refs. 29, 31), caused a slight
depression in myocyte contractility but did not affect
2-MeS-ATP-induced stimulation of contractility (increase in contractile
amplitude in response to 2-MeS-ATP plus U-73122, 48.4 ± 8%;
n = 12 cells from 3 cultures). The
inactive structural analog of U-73122, U-73343, also had no effect on
the 2-MeS-ATP-stimulated increase in myocyte contractility (data not
shown).
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A cAMP-independent 45Ca entry underlies
the P2 receptor-mediated positive inotropic
response.
Previous studies indicated that ATP can induce an increase in myocyte
contractile amplitude (3) as well as an increase in
Ca2+ entry and cytosolic
Ca2+ (9, 13, 26). Both 2-MeS-ATP
and ATP caused a pronounced increase in the transsarcolemmal uptake of
45Ca, whereas
,-methylene-ATP (Fig. 7) or
,-methylene-ATP had virtually no stimulatory effect on the
45Ca uptake (data not shown).
Neither 2-MeS-ATP nor ATP was able to stimulate cAMP accumulation
(basal cAMP level: 12.2 ± 2 pmol/mg, n = 9 cells; cAMP levels in the
presence of 2-MeS-ATP and ATP: 13.1 ± 1.6 pmol/mg,
n = 5 cells and 13.2 ± 1.1 pmol/mg, n = 5 cells, respectively).
None of the other P2-receptor agonists
such as UTP, ,-methylene-ATP, or ,-methylene-ATP was able
to induce cAMP accumulation (data not shown). As a positive control,
isoproterenol caused a 6.7 ± 1.4-fold increase in the level of cAMP
(n = 5 cells). Such data indicate that
a cAMP-independent Ca2+
entry-stimulating mechanism mediates the
P2-receptor agonist-induced stimulation
of myocyte contractility.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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ATP exerts a number of stimulatory effects in the heart, including
vasodilatation of the coronary vasculature (21, 24), stimulation of
transsarcolemmal Ca2+ entry (25,
26, 28), acidification (23), depolarization, (27), cytosolic
Ca2+ transients (12-14), and
contractility of the cardiac myocyte (12, 18). These studies have
largely investigated the effects of ATP on cardiac
Ca2+ current and cytosolic
Ca2+ transients that appear to be
mediated by a purinoceptor of the P2
subtype, whereas others studied ATP-induced
Na+ current,
Cl
/
exchange, acidification, and depolarization, which appear to require
high concentrations of ATP and the presence of
Mg2+ and are not mediated by the
P2 purinoceptor (23, 27). A few studies
(12, 18, 25) investigated the effects of ATP and ATP analogs on the
contractility of the rat cardiac ventricular myocytes and intact
papillary muscle, which clearly demonstrated an ATP-induced positive
inotropic response. Although a P2Y
receptor was suggested to mediate the increase in cytosolic
Ca2+ level (4), further
characterization of the P2 receptor
involved is lacking. The cellular mechanism underlying this inotropic
effect remains poorly understood. This lack of understanding may be
due, in part, to the absence of a myocyte model for the cardiac
P2 purinoceptor. Cultured chick embryo
ventricular myocytes have served as a useful experimental model for a
number of receptor-effector systems including the
P1 (adenosine) receptor. Because these
myocytes remain stable in culture for at least 3 days, they allow
various interventions such as desensitization studies and equilibration of
myo-[3H]inositol
within the myocyte pool for the study of PI hydrolysis. The feasibility
of preparing a relatively large number of myocytes also facilitates the
biochemical determination of phosphoinositide levels. The stability of
the cultured myocytes enables reliable and reproducible determination
of changes in contractility in response to various agonists. The
objective of the present study was to develop these cultured
ventricular myocytes as a model system in which a stable and
reproducible ATP-induced positive inotropic response would facilitate a
full characterization of the receptor(s) involved as well as a study of
its underlying mechanism.
ATP caused a pronounced stimulation of the myocyte contractility, with
an order of efficacy of ATP > ADP > AMP adenosine, consistent
with the notion that this positive inotropic response is mediated by a
P2 purinoceptor. That the order of
potency and efficacy is ATP > 2-MeS-ATP > UTP > ,-methylene-ATP or ,-methylene-ATP is consistent with
the notion that the subtype of P2
receptor mediating the positive inotropic effect is a
P2Y receptor or a 2-MeS-ATP-sensitive
P2X receptor such as
P2X2,
P2X4,
P2X5, or P2X6 receptor (7).
Because UTP has a significant positive inotropic effect, it is possible that a UTP-sensitive P2 receptor, either one of the known P2Y receptors or a novel receptor, also mediates some of the ATP-stimulated positive inotropic effect (7, 17). Although the EC50 values for ATP- and UTP-stimulated PI hydrolysis were higher than those for the increase in contractile amplitude, 1 µM ATP or UTP was able to cause a significant increase in the inositol phosphate level (increase was 85 ± 10 and 90 ± 15% for ATP and UTP, respectively; n = 3 experiments). Because UTP causes a pronounced increase in the level of inositol phosphates and Ins(1,4,5)P3 is known to stimulate the release of Ca2+ from the sarcoplasmic reticulum (34, 35), it is possible that the receptor-mediated increase in Ins(1,4,5)P3 causes the positive inotropic effect. On the other hand, because 2-MeS-ATP causes only a modest stimulation of inositol phosphate production, the 2-MeS-ATP-sensitive P2 receptor may be coupled directly to the stimulation of myocyte contractility independent of the increase in inositol phosphate production. According to this hypothesis, ATP causes its positive inotropic effect by activating both a PLC-coupled P2Y receptor and a 2-MeS-ATP-sensitive P2 receptor. Alternatively, the positive inotropic effect of UTP is due to its agonist activity, although modest, at the 2-MeS-ATP-sensitive P2 purinoceptor, whereas the stimulatory effect of 2-MeS-ATP on the inositol phosphate level is from its cross-activity at the UTP-sensitive P2Y receptor. Two lines of evidence support the latter notion. First, prior treatment of the myocyte with UTP desensitized both the UTP- and 2-MeS-ATP-induced stimulation of inositol phosphate production. On the other hand, prior treatment of the myocyte with 2-MeS-ATP had no effect on the increase in inositol phosphates caused by the subsequent stimulation with UTP or 2-MeS-ATP, suggesting that only the UTP-sensitive P2Y receptor is coupled to inositol phosphate production. Second, prior treatment of the myocyte with 2-MeS-ATP caused a significant desensitization of the positive inotropic response to either UTP or 2-MeS-ATP. On the other hand, prior exposure to UTP had no effect on the UTP- or 2-MeS-ATP-stimulated myocyte contractility, suggesting that only the 2-MeS-ATP-sensitive P2 purinoceptor is coupled to the stimulation of myocyte contractility.
That ATP can stimulate PI hydrolysis in cultured chick ventricular myocytes is similar to the findings obtained in mouse (36) and rat (18) ventricular myocytes. The present study showed that the stimulatory effect of ATP is mediated via a UTP-sensitive P2Y receptor. In contrast to previous suggestions (18), the present data suggest that the positive inotropic response to ATP, such as that mediated via the 2-MeS-ATP-sensitive P2 receptor, can occur independent of PLC activation. The formation of Ins(1,4,5)P3 is not necessary for the 2-MeS-ATP-stimulated positive inotropic response. This notion was further supported by the finding that although 1 µM 2-MeS-ATP caused a maximal positive inotropic effect, it had no effect on the inositiol phosphate level. U-73122, a known PLC inhibitor, had no effect on the 2-MeS-ATP-stimulated increase in myocyte contractility even at 10 µM, providing further evidence against a role of PLC in mediating the inotropic response to 2-MeS-ATP.
Further investigation of the underlying cellular mechanism showed that
the ATP agonists were able to cause a significant stimulation in
transsarcolemmal Ca2+ entry.
However, none of the ATP agonists caused an increase in the cell cAMP
content, similar to the finding obtained in rat ventricular myocytes
(26). The order of efficacy of ATP > 2-MeS-ATP > UTP > ,-methylene-ATP in stimulating
Ca2+ entry is similar to that of
the efficacy of the same analogs in stimulating myocyte contractility.
These data suggest that a cAMP-independent,
Ca2+ entry-stimulating mechanism
underlies the 2-MeS-ATP-sensitive P2
purinoceptor-mediated increase in myocyte contractile amplitude. The
present data are compatible with prior findings that the classic P2Y agonist 2-MeS-ATP can stimulate
Ca2+ entry via both a nonselective
cation channel and an L-type Ca2+
channel (25, 28) and an increase in
Ca2+ transients (4) in rat
ventricular myocytes. It is unlikely that an ATP-induced acidification
with a consequent stimulation of cytosolic
Ca2+ contributes to
the positive inotropic effect of ATP because ATP-induced acidification
required a Mg-ATP complex at a concentration 100-fold higher than that
causing the increase in myocyte contractility. The exact identity of
the P2 purinoceptor that mediates the
increase in myocyte contractility remains less clear. That some of the recently cloned P2X receptors can be
potently activated by 2-MeS-ATP and that mRNAs encoding these receptors
are expressed in abundant amount in the heart (16, 30) raise the
possibility that a P2X receptor
mediates the positive inotropic effect of ATP. This P2X receptor is unlikely to be a
P2X1 or
P2X3 receptor because the current
mediated by a P2X1 or
P2X3 receptor desensitizes
completely within a few seconds (11) and because these two
P2X receptors can be activated by
,-methylene-ATP. Identification of the exact P2 receptor subtype mediating the
positive inotropic response awaits the development of agonists and
antagonists capable of distinguishing the various
P2X receptor subtypes and of distinguishing these P2X from
P2Y receptors. Overall, the present
study demonstrates that a novel cAMP- and PLC-independent
Ca2+ entry pathway, likely
mediating the direct coupling of a P2
purinoceptor to stimulation of myocyte contractility, exists in the
intact cardiac cell. This high-affinity stimulatory
P2 purinoceptor pathway represents a
potentially novel target for the development of new positive inotropic
therapeutics.
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ACKNOWLEDGEMENTS |
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This work was supported by an established investigatorship of the American Heart Association and National Heart, Lung, and Blood Institute Grants HL-48225 and HL-44188.
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FOOTNOTES |
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Address for reprint requests: B. T. Liang, 504 Johnson Pavilion, 3610 Hamilton Walk, Univ. of Pennsylvania Medical Center, Philadelphia, PA 19104.
Received 14 March 1997; accepted in final form 15 July 1997.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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), ADP (
), and AMP (
). A:
effects of these agonists on basal level of contractility. Data are
means ± SE; n = 14 cells for ATP,
8 cells for ADP, and 7 cells for AMP from 3-4 cultures.
B: representative contractility
tracing for ATP-induced stimulation of myocyte contractility.
) for 30 s, and levels of inositol 1-phosphate
(InsP1), inositol
1,4-bisphosphate
[Ins(1,4)P2], and
inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3] were
measured as described in METHODS. 
