A new cyclic AMP-independent, Gs-mediated stimulatory mechanism via the adenosine A2a receptor in the intact cardiac cell.

The objectives of this study were to investigate the mechanism underlying the adenosine A2a receptor (A2aR)-mediated positive inotropic response and to define its contractile function using chick embryo ventricular cells as a model. Activation of the A2aR caused a marked stimulation of calcium entry and cell contractility, which were blocked by verapamil or nifedipine. The effects elicited by maximal concentrations of the A2aR agonist 2-[4-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine and the β-adrenergic agonist isoproterenol were additive, indicating that the two receptors do not share a common stimulatory mechanism. The cAMP antagonist (Rp)-adenosine cyclic 3′:5′-monophosphorothioate was ineffective in inhibiting the A2aR-mediated stimulation of contractility or the L-type calcium channel, while it completely abolished the isoproterenol effects. Activation of the A2aR had no effect on Na+/Ca2+ exchange or inositol 1,4,5-trisphosphate accumulation. Blocking of the A2aR resulted in unopposed A1 receptor-mediated inhibitory effects and led to an inhibition of basal contractility and an enhanced anti-adrenergic effect by A1 agonist. The adenosine A2a receptor mediates a new cyclic AMP-independent mechanism and a new contractile function in the cardiac cell.

Multiple pathways mediate stimulation of cardiac cell contractility. The classical and best characterized pathway is that mediated by the ␤-adrenergic receptor, giving rise to an increase in cAMP with subsequent calcium entry and increase in myocyte contractility (1)(2)(3)(4). The ␣-adrenergic receptor-mediated augmentation of cardiac contractility involves stimulation by inositol phosphates and protein kinase C that is cyclic AMPindependent (5)(6)(7)(8). Adenosine exerts pronounced contractile effects in the heart. In the cardiac ventricular cell, adenosine attenuates the increase in the force of contraction elicited by ␤-adrenergic agonist, known as the anti-adrenergic effect, while adenosine by itself has no direct effect on the basal level of contractility. The anti-adrenergic effect is mediated by the inhibitory A 1 subtype of the adenosine receptor (for review, see Refs. 9 -13). Using fetal chick embryo ventricular cells as a model, prior studies demonstrated the presence of a high affinity adenosine A 2a receptor capable of mediating positive ino-tropic response when the A 1 receptor is blocked (14,15). The marked positive inotropic response mediated by the A 2a receptor suggests a significant physiological or pathophysiological role of this receptor in the cardiac cell. Since the inhibitory A 1 receptor coexists with the stimulatory A 2a receptor and since the A 1 receptor mediates the anti-adrenergic effect of adenosine, the question arises regarding specifically the function of the A 2a receptor in modulating the basal as well as the ␤-adrenergic stimulated contractile states. Furthermore, the mechanism underlying the A 2a receptor-mediated positive inotropic response is not known. Although activation of the A 2a receptor can be coupled to stimulation of cAMP accumulation, it is not known whether cAMP mediates the increase in myocyte contractility.
The objectives of this study, using cultured fetal ventricular cells as a model system, were to investigate the mechanism underlying the adenosine A 2a receptor-mediated positive inotropic response and to study the contractile function subserved by the A 2a receptor. Cardiac ventricular cells cultured from chick embryos retain many of the biological properties of the intact heart and represent a useful model for study of cardiac function and contractility (16 -23). Activation of adenosine receptors in these cultured heart cells produced physiological effects similar to those elicited by adenosine in the mammalian heart (14,15,23). The present data demonstrate that the A 2a receptor and the ␤-adrenergic receptor do not share a common pathway leading to stimulation of myocyte contractility or calcium entry; that a cyclic AMP-independent, G s -mediated mechanism is largely responsible for the A 2a receptor-mediated stimulatory response; and that a physiological action of the activated A 2a receptor is to oppose the inhibitory effect of A 1 receptor agonist on basal and ␤-adrenergic stimulated contractile states.

Methods
Preparation of Cultured Cardiac Cells-Atrial and ventricular cells were cultured from chick embryos 14 days in ovo according to previously described procedures (15,23). Cells grew to confluence on day 3 of culture and exhibited rhythmic spontaneous contraction. Unless otherwise indicated, all experiments were carried out on day 3 of culture. Cultures were treated with adenosine deaminase (2 units/ml) for 24 h to keep the endogenous adenosine at a minimal level; they were also treated with pertussis toxin (5 ng/ml for 24 h) to uncouple the inhibitory A 1 receptor from its effector(s) as described previously (14,15). Blocking of the A 1 receptor facilitated quantitation of the A 2 receptor-mediated functional responses. In experiments in which cholera toxin was used to activate the stimulatory G protein (G s ), the cultures were treated with 2 g/ml cholera toxin for 3 h. This dose and duration gave consistent stimulation of cyclic AMP and 45 Ca 2ϩ influx.
Determination of Contractile Amplitude-Measurement of contractile amplitude in cultured cardiac cells was carried out according to previously described methods (15,18). The contractile amplitude of the cultured cell was determined by an optico-video motion detection sys-tem with a video motion analyzer (Colorado Video Inc., Boulder, CO) as described previously. The perfusion medium contained the various adenosine analogs indicated as well as the following components: 4 mM HEPES (pH 7.4), 137 mM NaCl, 3.6 mM KCl, 0.5 mM MgCl 2 , 0.6 mM CaCl 2 , 5.5 mM glucose, and 6% horse serum. Measurement of contractile amplitude was carried out on only one cell/coverslip, and each culture dish contained five coverslips. After achieving a steady state of beating in medium without adenosine analogs, the medium was switched to that containing the indicated adenosine drugs. Both the basal contractile amplitude and the amplitude measured during adenosine analog exposure were determined. The stimulatory effect of the various adenosine analogs on the contractile state was predominantly on the amplitude of contraction (14,15). There was no significant consistent effect of any of the analogs on the spontaneous rate of contraction.
Measurement of Cyclic AMP Level-Cultured ventricular cells were treated with pertussis toxin and adenosine deaminase as described above. On day 3 of culture, the media were replaced with culture medium lacking fetal bovine serum, and cells were incubated with the indicated agonist(s) and antagonist(s). Cyclic AMP was extracted and assayed according to a previously described radioimmunoassay method (Ref. 15;Amersham Corp.). The effect of agonist on cyclic AMP accumulation was linear for 10 min, at which time, cyclic AMP was extracted for assay.
Measurement of 45 Ca Uptake into Myocardial Cells-Determination of 45 Ca uptake was made according to a modification of a previously described method (18). Cultures were incubated with L- [3,4, 45 Ca uptake, growth media in which ventricular cells were grown and maintained on 12-well culture plates were replaced with HEPES-buffered solution (pH 7.35) containing 1.0 mM CaCl 2 , 4 mM KCl, and 0.5 mM MgCl 2 (buffer A) at 37°C for 10 min and then incubated in the same medium except that calcium was omitted and 1 mM EGTA was added (buffer B) and subsequently incubated in buffer A containing 45 Ca (5-10 Ci/ml) and adenosine analogs for the times indicated. The step of exposure of myocytes to calcium-free medium helped minimize mixing of 45 Ca with unlabeled Ca 2ϩ near or at the cell surface, optimized 45 Ca at the cell surface for subsequent uptake, and resulted in reproducible quantitation of 45 Ca uptake. Cells were then washed free of the 45 Ca media by four rinses with ice-cold buffer A containing 1 mM lanthanum. This washing procedure removed Ͼ99% of the extracellular marker 51 Cr-labeled EDTA and thus ensured complete removal of the extracellular 45 Ca. The presence of lanthanum in the wash buffer allowed displacement of any 45 Ca that may have attached to the extracellular surface of the cells. Influx of 45 Ca was quantitated for 90 s to allow determination of its uptake into the rapidly exchanging pool, which has been shown to be due either to calcium entry through the L-type calcium channel or to Na ϩ /Ca 2ϩ exchange (18). Determination of 45 Ca influx during this initial phase of its uptake in the presence of various agents permitted assessment of their effects on the activity of the calcium channel or Na ϩ /Ca 2ϩ exchange (24,25). For all data comparing the effect of different agents on 45 Ca influx, one-way ANOVA 1 followed by group comparison with the t test was carried out at each time point of 45 Ca influx. Cells were then exposed to CGS21680, carbamylcholine, or ATP at the concentrations indicated. The reaction was terminated by the addition of 0.2 volume of ice-cold trichloroacetic acid. The trichloroacetic acid was removed by extraction with a solution of 1,1,2-trichloro-1,2,2-trifluoroethane/trioctylamine. The IP 3 in the aqueous phase was determined by competing with

Effects of Adenosine A 2a and ␤-Adrenergic
Receptor Activation on Cardiac Cell Contractility and Calcium Influx-The A 1 receptor coexists with the A 2a receptor and mediates inhibition of the stimulatory responses caused by A 2a agonist in cultured fetal chick embryo ventricular cells (14,15). In cells where the A 1 receptor was first inactivated by prior treatment with pertussis toxin, the A 2a receptor-selective agonist CGS21680 caused a marked increase in myocyte contractile amplitude that was additive to the increase elicited by maximally effective concentration of the ␤-adrenergic agonist isoproterenol ( Fig.  1A), indicating that the two receptor pathways do not share a common mechanism of positive inotropic response. The positive inotropic response to isoproterenol is mediated by an increase in the trans-sarcolemmal influx of extracellular calcium. CGS21680 was also able to stimulate a significant increase in 45 Ca influx (Fig. 1B), which was again additive to the calcium influx effect elicited by isoproterenol (Fig. 1C), providing further evidence that the two receptors do not share a common pathway. In comparing the ability of CGS21680 and isoproterenol to stimulate myocyte contractile amplitude, the CGS21680 (10 M)-induced increase in contractile amplitude (20 Ϯ 2%, mean Ϯ S.E., n ϭ 14) was less than that caused by isoproterenol (31 Ϯ 2.5%, n ϭ 15; p Ͻ 0.01). The extent of the CGS21680 (1 M)-stimulated increase in 45 Ca influx, measured at the 60-s time point (45 Ϯ 6%, n ϭ 5), was also less than the isoproterenol-induced stimulation (79 Ϯ 8%, n ϭ 6; p Ͻ 0.01).

Role of Cyclic AMP in Mediating Stimulatory Effect of Adenosine A 2a Agonist on Contractility and Calcium Influx-Activa-
tion of the ␤-adrenergic receptor in the cardiac cell causes a large increase in cAMP accumulation, which, in turn, serves as a primary mediator of its positive inotropic effect (1)(2)(3)(4). In contrast to isoproterenol, CGS21680 (1 M) was able to induce only a modest increase in cAMP, but elicited a marked increase in 45 Ca influx. The percent increases above basal levels in the presence of CGS21680 and isoproterenol were 40 Ϯ 5% (n ϭ 5) and 317 Ϯ 30% (n ϭ 16), respectively (the basal cAMP level was 13.2 Ϯ 1.6 pmol/mg (n ϭ 12)). Isoproterenol (3 nM) stimulated an increase in cAMP similar to that elicited by 1 M CGS21680, but was not able to cause any increase in either 45 Ca influx or myocyte contractility (Fig. 2). (R p )-cAMP-S, which can antagonize the action of intracellular cAMP (27)(28)(29)(30)(31), blocked all of the isoproterenol-stimulated increase in calcium influx and myocyte contractility (Fig. 2B), but had only a modest inhibitory effect on the CGS21680-induced calcium response (Fig. 2C) ((R p )-cAMP-S caused a 24.8 Ϯ 2.4% inhibition of the CGS21680-stimulated calcium response at the 90-s time point (n ϭ 5).) (R p )-cAMP-S alone had no effect on the basal contractility or the basal uptake of 45 Ca (data not shown). Similar percent inhibition by (R p )-cAMP-S was also evident at the 15-, 30-, and 60-s time points of 45 Ca influx (Fig. 2C). Increasing (R p )-cAMP-S to 100 M did not result in further inhibition of the CGS21680-induced increase in 45 Ca influx (inhibition ϭ 26.4 Ϯ 1% at the 90-s time point (n ϭ 5)). The ability of CGS21680 to stimulate myocyte contractility appeared to be 1 The abbreviations used are: ANOVA, analysis of variance; IP 3 minimally affected by (R p )-cAMP-S (Fig. 2C). However, a small inhibition of contractile amplitude by (R p )-cAMP-S may be difficult to quantitate and cannot be ruled out completely. Although the data are consistent with a role of cyclic AMP in mediating the calcium influx response to the A 2a agonist, they demonstrate the existence of a cAMP-independent mechanism in causing a stimulation of calcium influx and provide further evidence that the A 2a and ␤-adrenergic receptors use different pathways to cause stimulatory responses.
Cellular Mechanisms Underlying A 2a Receptor-mediated Stimulation of Calcium Influx-Activation of the A 2a receptor could cause an increase in the trans-sarcolemmal calcium influx by stimulating the voltage-sensitive L-type calcium channel or by augmenting Na ϩ /Ca 2ϩ exchange. Verapamil (1 M) blocked the spontaneous contraction of cultured ventricular cells and completely attenuated the CGS21680-elicited increase in 45 Ca influx in the absence (Fig. 3A) or presence (data not shown) of (R p )-cAMP-S, indicating that the effect on calcium influx is due to stimulation of the L-type calcium channel. Similar results were obtained using nifedipine (1 M) as the calcium channel antagonist (data not shown). To examine whether Na ϩ /Ca 2ϩ exchange is involved in this calcium re-sponse, the effect of CGS21680 was determined in media in which choline chloride substituted for NaCl in the presence of verapamil. Decreasing the extracellular Na ϩ induced a marked stimulation of 45 Ca influx in the presence of verapamil (Fig.  3B), indicating a Na ϩ /Ca 2ϩ exchange-mediated increase in calcium influx. The addition of CGS21680 did not cause any further stimulation of 45 Ca influx. In the absence of verapamil, CGS21680 increased the extent of 45 Ca influx further, above that caused by lowering the extracellular sodium concentration (Fig. 3C).
Stimulation of cAMP accumulation by adenosine A 2a agonist is likely mediated by the G s -induced activation of adenylyl cyclase (9 -13). Although the present data do not indicate a major role for the A 2a receptor-G s -cAMP pathway in the calcium influx response, activation of G s mediated by the A 2a receptor may be involved in causing a cAMP-independent stimulation of calcium influx. Direct activation of G s by cholera toxin, which stimulated cAMP accumulation (67 Ϯ 4%, mean Ϯ S.E., n ϭ 5), induced a large increase in 45 Ca influx that was only partially blocked by 100 M (R p )-cAMP-S (Fig. 3D). The percent inhibition of cholera toxin-stimulated 45 Ca influx by (R p )-cAMP-S was 40 Ϯ 8, 54 Ϯ 15, 43 Ϯ 8, and 48 Ϯ 6% at the  (Fig. 3D), which was also able to block all of the 45 Ca influx caused by isoproterenol or by cholera toxin (data not shown). Thus, direct activation of G s by cholera toxin can stimulate, through the L-type calcium channel, both cAMP-dependent and -independent increases in 45 Ca influx. CGS21680 (at 1 or 10 M) had no effect on the level of inositol 1,4,5-trisphosphate (41 Ϯ 5 pmol/mg (control) versus 45 Ϯ 4 pmol/mg (1 M CGS21680) and 38 Ϯ 6.5 pmol/g (10 M CGS21680)) (data were means Ϯ S.E. of quadruplicates and were representative of three other experiments). As a positive control, both the muscarinic cholinergic agonist carbamylcho-line (300 M) and the purinergic agonist ATP (300 M) caused a large increase in the level of inositol trisphosphate (carbamylcholine, 70 Ϯ 5 pmol/mg; and ATP, 92.5 Ϯ 6 pmol/mg) (data were typical of three other experiments). Thus, at the concentrations of CGS21680 that caused a marked increase in calcium influx or myocyte contractility, there was no stimulation of inositol 1,4,5-trisphosphate accumulation. These data indicate that the CGS21680-mediated stimulatory contractile or 45 Ca influx response is not mediated by inositol 1,4,5-trisphosphate.

Adenosine A 2a Receptor Activation Attenuates A 1 Receptormediated Anti-adrenergic Contractile and Calcium Influx Effects-
The present data predict that concomitant activation of both the A 2a and A 1 receptors by adenosine agonist should alter the basal as well as the ␤-adrenergic stimulated levels of con-

FIG. 2. Role of cyclic AMP in mediating adenosine A 2a and ␤-adrenergic agonist effects on cardiac contractility and calcium influx.
The effects of isoproterenol (ISO; 3 nM) on the level of myocyte contractile amplitude (A) were determined. In experiments in which the effect of the cAMP antagonist (R p )-cAMP-S was examined, cells were preincubated with 10 M (R p )-cAMP-S for 1 h and were then continuously exposed to (R p )-cAMP-S during the actual measurement of the contractile and 45 Ca influx responses to isoproterenol (B) and to CGS21680 (C). The level of 45 Ca influx determined in the presence of isoproterenol (0.3 M) plus (R p )-cAMP-S was less than that obtained with isoproterenol alone (p Ͻ 0.01), but was similar to that in the control (CON; p Ͼ 0.1). In contrast, the level of 45 Ca influx in the presence of CGS21680 (1 M) plus (R p )-cAMP-S was less than that determined in the presence of CGS21680 alone (p Ͻ 0.05), but was greater than that obtained in the control (p Ͻ 0.01) (one-way ANOVA followed by t test was carried out at each time point of 45 Ca influx). In (R p )-cAMP-S-treated cells, the level of basal contractile amplitude was similar to that obtained with isoproterenol (0.3 M) (p Ͼ 0.1), whereas the level of basal contractile amplitude was significantly less than that determined in the presence of CGS21680 (10 M) (p Ͻ 0.01).
tractility. (R)-PIA is an A 1 receptor-selective agonist that can also activate the A 2a receptor at the higher concentrations (14,26). Prior studies indicated that (R)-PIA was an A 1 receptor agonist capable of inhibiting the isoproterenol-stimulated increase in myocyte contractility in chick embryo cardiac cells (14,15,23). The present data demonstrate that (R)-PIA (10 M) can stimulate both 45 Ca influx and contractility in cells in which the A 1 receptor pathway is blocked (Fig. 4A). The A 2a receptor-selective antagonist CSC blocked the (R)-PIA-induced stimulation of calcium influx and contractility, indicating that the stimulatory effects of (R)-PIA are mediated by the A 2a receptor. (R)-PIA further increased the level of 45 Ca influx and myocyte contractility stimulated by isoproterenol (Fig. 4, B and  C), an additive effect that was consistent with agonist activity of (R)-PIA at the A 2a receptor. Thus, (R)-PIA can activate both the A 1 and A 2a receptors in these ventricular cells. Whether activation of the A 2a receptor can modulate the basal and anti-adrenergic contractile responses elicited by A 1 receptor agonist was examined next.
Blocking of the A 2a receptor caused not only a depression of the basal contractility (Fig. 5A), but also a further inhibition of the isoproterenol-stimulated positive inotropic response by (R)-PIA (Fig. 5B). A previous study demonstrated that atrial myocytes cultured from the same chick embryos exhibited no positive inotropic response to CGS21680 or other adenosine receptor agonists (14), indicating the absence of a functional A 2a receptor in the atrial myocyte. If atrial myocytes do not express A 2a receptors, the presence of CSC should not influence the ability of (R)-PIA to inhibit the basal or isoproterenolstimulated increase in contractility. In fact, Fig. 5C demonstrates that CSC had no effect on the ability of (R)-PIA to inhibit basal contractile amplitude (basal contractile amplitude ϭ 77 Ϯ 2%, mean Ϯ S.E., n ϭ 10 ((R)-PIA) versus 78 Ϯ 1.6%, n ϭ 10 ((R)-PIA plus CSC); p Ͼ 0.1, paired t test). Furthermore, CSC did not affect the (R)-PIA-mediated inhibition of the isoproterenol-stimulated increase in contractility (maximal isoproterenol stimulation by (R)-PIA alone of 76 Ϯ 3% versus percent maximum of 75 Ϯ 4% by (R)-PIA plus CSC; p Ͼ 0.1, paired t test). These data serve as a control for the experiments carried out in the ventricular cells and indicate that CSC has no intrinsic nonspecific contractile effect. In the absence of verapamil, the extent of 45 Ca uptake stimulated by CGS21680 plus choline chloride was significantly greater than that determined in the presence of choline chloride alone (p Ͻ 0.01), which was, in turn, greater than the extent of 45 Ca influx in the control (CON; p Ͻ 0.01) (C). The effects of cholera toxin (CTX) on 45 Ca influx were determined in the presence or absence of (R p )-cAMP-S (100 M) and/or verapamil (1 M) (D). Cholera toxin (2 g/ml) stimulated a marked increase in 45 Ca influx (p Ͻ 0.01). (R p )-cAMP-S abolished part of the cholera toxin-stimulated 45 Ca influx (p Ͻ 0.05); verapamil reduced the remaining portion of the increase in 45 Ca influx to the control level (p Ͻ 0.05).

DISCUSSION
Adenosine plays an important role in modulating the contractile state of the cardiac cell (for review, see Refs. 9 -13). Previous studies carried out in this laboratory have demonstrated the existence of a high affinity adenosine A 2a receptor capable of mediating a marked positive inotropic response in cultured fetal chick embryo ventricular cells (14,15). However, the mechanism underlying the A 2a receptor-mediated positive inotropic effect is not known. Although the A 2a receptor is coupled to stimulation of cyclic AMP accumulation, it is not clear whether cyclic AMP mediates all of the positive inotropic effect of A 2a receptor agonist. It is not known whether the A 2a receptor and the ␤-adrenergic receptor share a similar mechanism of positive inotropic effect. Furthermore, the role of the A 2a receptor in regulating the contractile state of the cardiac cell is not known. Since the inhibitory A 1 receptor coexists with the stimulatory A 2a receptor in these cultured ventricular cells and since the A 1 receptor mediates the anti-adrenergic effect of adenosine, the question arises regarding the specific role of the A 2a receptor in modulating the basal as well as the ␤-adrenergic stimulated contractile states. The main findings of this study are as follows. The mechanism underlying the A 2a receptor-mediated positive inotropic effect involves a cyclic AMPindependent, A 2a receptor/G s -mediated stimulation of the Ltype calcium channel and differs from the mechanism of the ␤-adrenergic stimulated inotropic response. A normal function of the activated A 2a receptor is to attenuate the A 1 receptormediated anti-adrenergic response.
Four lines of evidence support the existence of a new mechanism responsible for the A 2a receptor-mediated positive inotropic response. First, the positive inotropic effects elicited by maximally effective concentrations of isoproterenol and CGS21680 were additive, indicating that the ␤-adrenergic and adenosine A 2a receptors do not share a common positive inotropic mechanism. Similarly, the stimulation of calcium influx caused by maximally effective concentrations of isoproterenol and CGS21680 was also additive, providing further evidence for the distinct pathways used by the two receptors. Second, while the ␤-adrenergic receptor is coupled to pronounced stimulation of cyclic AMP accumulation, A 2a receptor activation caused only a modest increase in cyclic AMP, with nearly 10-fold less stimulation of the cyclic AMP level. Third, at a concentration of isoproterenol that caused an increase in the cyclic AMP level similar to the increase elicited by the maximally effective concentration of CGS21680, there was no increase in either myocyte contractile amplitude or calcium influx. Fourth, while (R p )-cAMP-S was able to block all of the positive inotropic and calcium influx responses elicited by isoproterenol, (R p )-cAMP-S could inhibit only a small portion of the calcium influx response stimulated by CGS21680. These data indicate that a distinct mechanism, different from that used by the ␤-adrenergic receptor, mediates the stimulatory contractile and calcium influx responses to adenosine A 2a receptor agonist.
Since verapamil or nifedipine (1 M) was able to block all of the CGS21680-stimulated increase in calcium influx, an A 2a receptor-mediated stimulation of the L-type calcium channel appeared to be responsible for the increase in calcium influx. Lowering the extracellular sodium in the presence of verapamil caused a marked stimulation of calcium influx, secondary to a Na ϩ /Ca 2ϩ exchange-mediated increase in calcium entry. The inability of CGS21680 to cause a further increase in calcium influx in this type of medium suggests that CGS21680 does not stimulate Na ϩ /Ca 2ϩ exchange. In the absence of verapamil, CGS21680 was able to cause a further increase in the level of calcium influx that was stimulated by lowering the extracellu- The level of 45 Ca influx obtained in the presence of (R)-PIA plus CSC was less than that determined in the presence of (R)-PIA alone (p Ͻ 0.01), but was similar to that in the control (p Ͼ 0.1). The level of contractile amplitude obtained in the presence of (R)-PIA plus CSC was less than that obtained with (R)-PIA alone (p Ͻ 0.01), but was similar to that in the control (p Ͼ 0.1) (by one-way ANOVA and t test). The effect of (R)-PIA (10 M) on the isoproterenol (ISO)-stimulated level of 45 Ca influx (B) and of myocyte contractility (C) was next determined. The level of 45 Ca influx determined in the presence of isoproterenol plus (R)-PIA was greater than that obtained with isoproterenol alone (p Ͻ 0.01), which was, in turn, similar to that determined in the presence of isoproterenol plus (R)-PIA and CSC (p Ͼ 0.1). Similarly, the contractile amplitude determined in the presence of isoproterenol plus (R)-PIA was greater than that obtained with isoproterenol alone (p Ͻ 0.01). lar sodium. These data provide further evidence for the notion that the CGS21680-induced calcium effect is mediated by the L-type calcium channel and does not involve Na ϩ /Ca 2ϩ ex-change. Since CGS21680 could not stimulate inositol 1,4,5trisphosphate accumulation, it is unlikely that the inositol trisphosphate is involved in mediating the CGS21680-induced stimulatory responses.
Although (R p )-cAMP-S inhibited partially the A 2a receptormediated calcium influx response, the percent inhibition was modest (25%). These data indicate that a cAMP-independent pathway is capable of, and likely plays a primary role in, mediating the A 2a agonist-induced increase in calcium influx. Activation of G s by A 2a receptor agonist likely mediates the cAMP-independent stimulation of the L-type calcium channel, based on the following evidence. First, direct activation of G s by cholera toxin resulted in stimulation of both cAMP and calcium influx; cholera toxin-stimulated calcium influx exhibited both (R p )-cAMP-S-sensitive and -insensitive components. Both components can be blocked by the L-type channel antagonists verapamil and nifedipine. Since activation of G s can stimulate the L-type calcium channel independent of cAMP (32,33), these data are consistent with the notion that cholera toxinactivated G s can cause a cAMP-independent stimulation of the L-type calcium channel. Second, A 2a agonist-stimulated calcium influx also exhibited (R p )-cAMP-S-sensitive and -insensitive components, which were blocked by verapamil or nifedipine. These data indicate that the A 2a receptor is also capable of mediating a cAMP-independent activation of the L-type channel. Similarities in the action and effect of CGS21680 compared with those of cholera toxin are consistent with the notion that the A 2a agonist-induced augmentation of nifedipine-sensitive calcium influx involves a cAMP-independent, A 2a receptor/G smediated stimulation of the L-type calcium channel. Preliminary data demonstrate that transfection of the myocytes with rat G ␣s causes an increased A 2a agonist-mediated, nifedipinesensitive 45 Ca influx in the presence of (R p )-cAMP-S, providing evidence that exogenous G ␣s can couple the A 2a receptor to a cAMP-independent stimulation of the calcium channel. Although ␤-adrenergic receptor activation, via G s , can stimulate the L-type calcium channel directly independent of cAMP in the cardiac cell membrane (32,33), the present data suggest that a cyclic AMP-independent, G s -mediated pathway, which functionally coupled the cell-surface A 2a receptor to stimulation of the L-type calcium channel, exists in the intact cardiac cell.
These results indicate that cAMP mediates most if not all of the ␤-adrenergic stimulated increases in myocyte contractility and calcium influx in the cultured chick embryo ventricular cell, similar to findings in other cardiac cells (1)(2)(3)(4). The reason for the ability of the G s -linked adenosine A 2a receptor, but not the G s -linked ␤-adrenergic receptor, to couple to stimulation of the calcium channel independent of cAMP is not clear. Whether this differential ability of the two G s -linked receptors to couple to activation of the calcium channel can be explained by a predetermined coupling of the A 2a receptor to a pool of G s capable of mediating a cAMP-independent stimulation of the calcium channel, which are separate from those coupled to the ␤-adrenergic receptor, remains unknown.
The existence of an A 2a receptor-mediated signaling mechanism distinct from that of the ␤-adrenergic receptor raised the possibility that activation of the A 2a receptor can modulate not only the basal but also the anti-adrenergic contractile responses elicited by adenosine A 1 receptor agonist. Prior studies indicated that (R)-PIA is an A 1 receptor agonist capable of inhibiting basal (atrial cells) and isoproterenol-stimulated (both atrial and ventricular cells) increases in contractile amplitude (9 -13, 14, 15, 23). The present data demonstrate that (R)-PIA at the higher concentration can also activate the A 2a receptor in the cultured ventricular cell. Blocking the A 2a re-FIG. 5. Adenosine A 2a receptor effects on A 1 receptor-mediated anti-adrenergic contractile and calcium responses. Atrial and ventricular cells were prepared and pretreated with adenosine deaminase only. The effects of CSC (1 M) on the basal and anti-adrenergic responses to (R)-PIA (10 M) were examined. After achieving steady state, ventricular cells were exposed to (R)-PIA and then to (R)-PIA plus CSC (A). The level of contractile amplitude determined in the presence of (R)-PIA plus CSC was less than that in the control or the amplitude obtained with (R)-PIA alone (one-way ANOVA followed by t test; p Ͻ 0.01). In another experiment, ventricular cells were exposed to isoproterenol (ISO; 0.3 M), then to isoproterenol plus (R)-PIA, and finally to isoproterenol plus (R)-PIA and CSC (B). The level of contractile amplitude determined in the presence of isoproterenol plus (R)-PIA and CSC was less than that obtained with isoproterenol plus (R)-PIA (p Ͻ 0.01), which was, in turn, less than that obtained in the presence of isoproterenol alone (p Ͻ 0.01). Similar experiments were carried out to examine the effect of CSC (1 M) on the (R)-PIA (10 M)-mediated direct negative inotropic or anti-adrenergic response in atrial cells (C). In atrial cells, the levels of contractile amplitude obtained in the presence of CSC were not different from those determined in its absence (p Ͼ 0.1). ceptor with the selective antagonist CSC caused not only a decrease in basal contractile amplitude, but also a further inhibition of isoproterenol-stimulated contractility in response to (R)-PIA. These data indicate that activation of the A 1 receptor, if unopposed by the A 2a subtype, could cause a direct negative inotropic effect as well as an enhanced anti-adrenergic response in the ventricular cell. The lack of effect of CSC on the A 1 receptor-mediated anti-adrenergic or direct negative inotropic effect in atrial cells, which do not express an A 2 receptormediated functional response (14), provides an important control and suggests that the effect of CSC in ventricular cells is not due to a nonspecific contractile response to CSC. These data are consistent with a prior study indicating that CSC acts as a selective antagonist at the adenosine A 2a receptor in the cultured chick embryo ventricular cell (15). The data also indicate that potential agonist effect on the A 2a receptor should be considered when studying the cardiac action of adenosine agonist.
Overall, this study demonstrates a new stimulatory signaling mechanism mediated by the adenosine A 2a receptor and elucidates a novel contractile function of the A 2a receptor in the intact cardiac ventricular cell. Whether other cell-surface cardiac receptor(s) are also coupled to such G s -mediated stimulatory pathway remains to be determined. Demonstration of this new cAMP-independent, G s -mediated stimulatory mechanism in the cardiac cell, however, should have significant general implications for the regulation of basic cardiac function.