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Regulation of Ca2+ current in frog ventricular cardiomyocytes by guanosine 5'-triphosphate analogues and isoproterenol.

Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. To gain insight into the role of G proteins in the regulation of ICa in intact cells, the effect of internal perfusion with hydrolysis-resistant GTP analogues, guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-thiotriphosphate (GTP gamma S), on ICa stimulated by isoproterenol (Iso) or forskolin (Forsk) was examined. Significant differences were observed between the effects of the two GTP analogues. Internal perfusion of GppNHp resulted in a near-complete (approximately 80%) and irreversible inhibition of Iso-stimulated ICa. In contrast, internal perfusion with GTP gamma S resulted in only a partial (approximately 40%) inhibition of Iso- or Forsk-stimulated ICa. The fraction of the current not inhibited by GTP gamma S remained persistently elevated after the washout of Iso but declined to basal levels upon washout of Forsk. Excess internal GTP or GppNHp did not reduce the persistent ICa. Internal adenosine 5'-thiotriphosphate (ATP gamma S) mimicked the GTP gamma S-induced, persistent ICa. GppNHp sometimes induced a persistent ICa, but only if GppNHp was present at high concentration before Iso exposure. Inhibitors of protein kinase A inhibited both the GTP gamma S- and ATP gamma S-induced, persistent ICa. We conclude that: (a) GTP gamma S is less effective than GppNHp in inhibiting adenylyl cyclase (AC) via the inhibitory G protein, Gi; and (b) the persistent ICa results from a long-lived Gs-GTP gamma S complex that can activate AC in the absence of Iso. These results suggest that different hydrolysis-resistant nucleotide analogues may behave differently in activating G proteins and imply that the efficacy of G protein-effector molecule interactions can depend on the GTP analogue with which the G protein is activated.


INTRODUCTION
Many ion channels are regulated both directly and indirectly by G proteins (Levitan, 1988). One common approach for studying G protein involvement in ion channel regulation has been to perfuse cells internally with hydrolysis-resistant GTP analogues to stably activate G proteins. Relatively little quantitative information exists, however, on the effects of these analogues on ionic currents, differences between analogues, or complications resulting from activation of multiple types of G proteins in cells. Because the cardiac Ca current is a prime model for ion channel modulation by neurotransmitters, we have used internal perfusion of cardiac myocytes with nucleotide analogues to provide quantitative information about how G proteins function in the intact ceils.
Studies in cell-free and reconstituted systems have demonstrated that the regulation of AC involves two species of G proteins, a stimulatory G protein (Gs) linked to the 13-adrenergic receptor and an inhibitory G protein (Gi) linked to the muscarinic cholinergic receptor. [3-Adrenergic stimulation of AC is thought to involve hormonestimulated exchange of GTP for bound GDP on the Gs protein and subsequent dissociation of 0qGTP) and 133' subunits (Gilman, 1987). The GTP-liganded et subunit then binds AC and stimulates its catalytic activity. Hydrolysis of GTP and reassociation of a~GDp) and 133' subunits terminates the G protein's catalytic activity. The mechanism of Gi-mediated inhibition of AC is complex. It is not clear whether inhibition involves merely the binding and neutralization of a~ by 133' subunits released from Gi (reviewed by Gilman, 1987;Ross, 1989), or whether ai or 133' might directly inhibit AC (see discussion in Pfeuffer and Helmreich, 1988). More recent experiments have provided evidence for direct actions of both ai (Wong, Federman, Pace, Zachary, Evans, Pouyssegur, and Bourne, 1991) and 133' (Tang and Gilman, 1991) on AC.
Since reconstituted systems differ in important ways from intact cells (including protein stoichiometries and tissue sources, as well as disruption of subcellular compartmentalization, ion gradients, and transmembrane potentials), we have studied the G protein regulation of AC in intact cells utilizing internal perfusion of hydrolysis-resistant analogues of GTP and voltage clamp measurements of ionic currents in frog ventricular myocytes (Parsons, Lagrutta, White, and Hartzell, 1991). A similar approach was used in the pioneering work of Breitwieser and Szabo (1988) to study G protein turnover in intact atrial cells. They measured the muscarinic K channel, which is thought to be directly gated by a G protein, and thus were able to infer rates of activation and deactivation of the G protein. The cardiac Ca channel, which is phosphorylated by a cAMP-dependent kinase, provides a rapid assay for AC activity in intact cells. However, cAMP-dependent phosphorylation of the Ca channel involves a cascade of intracellular reactions, of which there may be several ratelimiting steps (Frace, M6ry, Fischmeister, and Hartzell, 1993) that make difficult a similar detailed quantitative kinetic analysis of the G proteins that regulate AC. Nevertheless, the frog cardiomyocyte is an excellent model for studying G protein regulation of AC in intact cells in the steady state because: (a) the Ca current is a defined function of intracellular cAMP White and HartzeU, 1988); (b) cAMP-dependent phosphorylation is the only stimulatory mechanism for [3-adrenergic stimulation of Ica (Hartzell, M6ry, Fischmeister, and Szabo, 1991;; and (c) the cAMP-dependent stimulation of Ica is amazingly stable during intracellular dialysis .
In our previously published work , we observed that guanylyl 5'-imidodiphosphate (GppNHp) inhibited/Ca stimulated by either isoproterenol (Iso) or low concentrations of forskolin (Forsk). We suggested that this inhibition resulted from the inhibition of AC by activation of a Gi-like protein via basal nucleotide exchange in the absence of muscarinic agonist. In this article, we compare the effects of guanosine 5'-thiotriphosphate (GTPTS) and GppNHp to further characterize the activation of Gs. We report that although GppNHp completely inhibited Isostimulated Ica, GTP~/S only partially inhibited /Ca and also induced a persistent /Ca resulting from a long-lived Gs-GTP3,S complex that activated AC in the absence of Iso. Unlike Gi, which is activated by GppNHp in the absence of a muscarinic agonist , the activation of G~ depends on [3-adrenergic stimulation.

Preparation
Bullfrogs (Rana catesbeiana) were killed by decapitation and double pithing. Ventricular myocytes from freshly dissected hearts were isolated by enzymatic and mechanical dissociation and kept in maintenance media as previously described (Hartzell and Simmons, 1987).

Solutions and Cell Perfusion
During electrophysiological recordings of/Ca, cells were bathed in and superfused with 115 mM NaC1, 20 mM CsCI, 1.8 mM MgCI2, 1.8 mM CaCI2, 5 mM Na pyruvate, 5 mM glucose, 0.3 p.M tetrodotoxin, and 10 mM HEPES at pH 7.4. During the course of an experiment, the cell under analysis was exposed to various test compounds (ACh and Iso from Sigma Chemical Co., St. Louis, MO; Forsk from Calbiochem-Novabiochem Corp., LaJolla, CA; and Sotalol provided by Ken Minneman, Emory School of Medicine, Atlanta, GA) by positioning it in front of flowpipes containing superfusion solution supplemented with these chemicals. Cells were usually perfused internally with 118 mM CsCI, 5 mM K2EGTA, 2.8 mM Na~K2ATP, 4.04 mM MgCI~, 5 mM Na2 creatine phosphate, and 10 mM K-PIPES at pH 7.15. The internal solution was changed by a system permitting continuous perfusion of the patch pipette   (White and Hartzell, 1988), assuming the same stability constants for GppNHp and GTP,/S as for GTP.  In most experiments, GTP~/S was used without repurification. However, commercially available GTP~/S was often significantly contaminated with 260-nm absorbing material (presumably nudeotides). For some experiments, GTP~S or GppNHp was repurified to >95% by HPLC as previously described .

Recording Protocols
Electrophysiological recordings were conducted using the whole-cell configuration of the patch clamp technique as previously described (Fischmeister and Hartzeil, 1987)./ca was defined as the net inward current elicited by voltage pulses from -80 to 0 mV for 400 ms. Changes in series resistance during experiments were monitored by continued observation for abrupt changes in the amplitude or kinetics of the/ca transient. If either was observed, then series resistance was usually recalculated from the capacitive transients associated with small voltage steps away from the holding potential or the experiment was terminated.

Data Analysis and Statistics
Calcium current densities were calculated by dividing/Ca by cell capacitance and assuming 1 ~F = 1 cm 2. Data are expressed as mean -standard error with n equal to the number of cells studied. The time course of stimulation of/Ca by Iso was described by a parameter, t,, which was defined as the time after initial stimulation with Iso when the magnitude of/ca was equal to 63.2% of the difference between the basal/Ca and maximal Iso-stimulated Ic~.

Inhibition of Ca Current by IntraceUular GTP,/S
GTPyS-mediated inhibition oflso-stimulated Ica. We previously reported  that GppNHp inhibited Iso-stimulated /Ca nearly completely. To determine whether similar results were obtained with other hydrolysis-resistant analogues of GTP, cells were internally perfused with GTP',/S. The cell in Fig. I, A and B was first perfused with a solution lacking any added guanine nucleotides and then exposed to 0.3 IxM Iso. After Ica stabilized, internal 50 IxM GTP~/S produced a partial decrease in the current (Fig. 1, A and B). On average, 0.3 IxM Iso resulted in an 11.7 + 1.1-fold (n = 39) stimulation of Ica and inhibition by 50 I~M GTP~/S resulted in a internal perfusion with GTP-/S from the experiment depicted in A. Current traces were not leak subtracted. Capacitive transient was blanked for 1.5 ms. (C) Effect of 500 IxM GTP'yS on Ica elevated by 0.3 I~M Iso. The cell was first exposed to 0.3 IxM Iso and then internally perfused with 500 I~M GTP~/S after lca stabilized. After the partial inhibition of the Iso-stimulated lca, 500 v,M GppNHp was added internally. Neither internal GppNHp nor washout of Iso significantly changed the partial inhibition of the Iso-stimulated Ic~ by GTP~/S. (D) Persistent/ca after GTP~/S. Cell was exposed to 0.3 I~M Iso and then perfused internally with 50 ~M GTP~/S. The current was only partially inhibited. After the inhibition reached an apparent steady state, Iso was washed out (at 20 min). The current remained unchanged for 5 min of washing out Iso. In control cells not exposed to GTP~/S, the Iso response washed out completely in the same time period (open squares). (E) Summary of effects of internal perfusion with GTP~/S on Iso-stimulated Ica. lca density (lzA/cm 2) is plotted for several experimental conditions using 0.1-10 I~M Iso and 50 I~M GTP~/S. Each bar represents the mean /Ca density value for the number of cells tested under each condition. Error bars are SEM. 42.2 + 3.8% (n = 15) decrease in Iso-stimulated Ic~ ( Table I A , 1 and 2). Note that "percentage decrease" is the mean of the decreases observed in each individual cell. These values differ slightly from the decrease calculated from Ic~ densities in Table I. Exposure to 100-fold greater concentrations of Iso did not reverse the partial inhibition of Iso-stimulated/Ca (n = 6) (data not shown). Similar incomplete inhibition was obtained with 500 I~M internal GTP'yS ( Fig. 1 C) (Table I A, 3). GTP~/S inhibited Iso-stimulated /ca in most experiments (see Figs. 1 and 2). However, in four cells 50 ~M GTP~/S produced a transient inhibition followed by a stimulation of 2.0 -0.3-fold (Table I A , 4). The Iso-stimulated/Ca density before internal GTP~/S in these four cells was less than half that of the group inhibited by internal GTP~/S (Table I A, 2 and 4), suggesting that the ability to respond to Iso was not the same in these cells.
GTP~,S blocks the effects of GppNHp. The 42% inhibition of Iso-stimulated/Ca by GTP~/S contrasts with the ~ 80% decrease in /Ca caused by internal perfusion of GppNHp during Iso stimulation of/Ca . However, replacing GTP~/S with internal GppNHp for > 25 min resulted in no additional decrease in/Ca ( Fig. 1 C). This finding suggests that the partial inhibition of Iso-stimulated/Ca by GTP~/S was not due to the incomplete activation of Gi by GTP~/S. Persistent stimulation of Ica. After /Ca was partially inhibited by GTP'yS, the current that was not inhibited remained elevated even when Iso was washed out ( Fig.  1 D). This is also observed in Fig. 1 C after internal perfusion with both GTP~/S and GppNHp. We will term this current that is stimulated above basal levels in the absence of 13-adrenergic agonist "persistent/ca," although it should be noted that this current exhibits the same voltage-dependent properties and inactivation as the basal current. In other words, the amplitude of the current is persistently stimulated above basal levels, but the current still requires depolarization to be activated. The effects of Iso and GTP~/S on/ca are summarized in the bar graph in Fig. 1 To define the mechanism of GTP~S-mediated inhibition of stimulated /Ca, the effect of GTP~/S on/Ca was studied under three experimental conditions: (a) basal or unstimulated/Ca; (b) Ica elevated by direct activation of AC via Forsk (HartzeU and ; and (c) /Ca stimulated by activation of protein kinase A with internal perfusion of cAMP.
Effect of GTP3,S on basal Ica. We previously reported that GppNHp had no effect on basal Ica . Similar results were observed with GTP~/S. Cells were internally perfused for at least 10 min in the absence of Iso or Forsk with either 50 I~M GTP~/S or 50 I~M GTP or a control internal solution containing no added guanine nucleotides. Under all three conditions a similar approximately twofold increase in basal Ica was observed. This runup is probably due to equilibration of the pipette solutions (Cs +, Mg 2+, etc.) with the cell. These results support the suggestion that GTP',/S has no effect on/Ca in the absence of the activation of AC. Fig. 2 A was first perfused with a solution lacking any added guanine nucleotides and then exposed to 3 ~.M Forsk. When 50 txM GTP~/S was added internally, Ica steadily decreased to an intermediate level (Fig. 2 A). On average, 3 IxM Forsk resulted in a 9.4 -0.9-fold stimulation of lca (n = 30) and 50 ~M GTP~/S resulted in a 46.6 +-4.7% decrease (n = 17) in 3 I~M Forsk-stimulated/Ca (Fig. 2 A; Table I B, 1 and 2). Because Forsk stimulates the activity of AC in the absence of activation of Gs (Seamon and Daly, 1986), these results suggest that even in the absence of Gs activation, GTP~/S can inhibit/Ca. .,, f The cell was exposed to 3 IzM Forsk and internally perfused with 50 IxM GTP~tS. Ic, steadily decreased, but reached the steady-state level, which was elevated above basal Ic~. (B) Partial inhibition of Forsk-stimulated lc, by 500 p,M GTP~S. The cell was exposed to 3 p,M Forsk and internally perfused with 500 I~M GTP3,S. Ic, steadily decreased, but reached the steady-state level, which was elevated above basal/Ca. Subsequent washout of Forsk resulted in a rapid decline in the Ica. (C) Summary of effects of internal perfusion with GTP3,S on Forsk-stimulated Ic~. lc~ density (l~A/cm 2) is plotted for several experimental conditions using 3 p,M Forsk and 50 p,M GTP~/S. Each bar represents the mean /ca density value for the number of cells tested under each condition. Error bars are SEM.

GTP),S-mediated inhibition of Forsk-stimulated lca. The cell in
A similar incomplete inhibition of 3 p.M Forsk-stimulated lca was also observed with 500 p~M GTP~/S (Fig. 2 B ). On average, 500 ~M GTP~/S resulted in a 31.6 _+ 4.6% decrease (n = 4) in 3 ~M Forsk-stimulated Ica (Table I B, 3). However, upon washout of Forsk the elevated/Ca rapidly returned to basal levels. On average, 90.8 -2.2% of the initial Forsk-stimulated Ica reversed upon washout of Forsk after internal perfusion with GTP3,S (Fig. 2 B). This is in contrast to the finding with Iso and GTP~/S (Fig. 1, C or D) in which > 60% of the initial Iso-stimulated /Ca remained persistently elevated after the washout of Iso. Results are summarized in Fig. 2 Some variation in the effect of GTP',/S on Forsk-stimulated/Ca was observed. The cells depicted in Fig. 2, A and B, and 3 A exhibit the typical response. However, in some cells transient inhibition and sometimes stimulation of the Forsk-stimulated/Ca was observed. 4 of 21 cells (19%) that had been stimulated with Forsk (0.3-3 I~M) and then internally perfused with GTP~S exhibited an increase in /Ca" The average percentage increase in the Forsk-stimulated /Ca was 78.8 -+ 37.2% (n = 4). As we observed with the atypical responses of Iso-stimulated/ca to GTP~S (Table I, A, 4), the mean amplitude of the Forsk-stimulated Ica obtained before internal perfusion with GTP~/S was less for cells in which GTP~/S was stimulatory than for cells in which GTP,/S was inhibitory (Table I B, 4).

GTPyS blocks the effects of ACh. We examined the ability of ACh to further inhibit
Forsk-stimulated Ica that had been incompletely inhibited by GTP,/S. In Fig. 3 A, 3 ~M Forsk stimulated/Ca ~ 15-fold./Ca was then briefly inhibited by a 3-rain exposure to 20 ~M ACh. After washout of ACh, internal perfusion of GTP~/S (50 ~M) partially inhibited the Forsk-stimulated/Ca. After ~ 15 min of internal GTP~/S, a second 3-min exposure to 20 ~M ACh had only very small inhibitory effects on Ica. On average, ACh had no effect on Forsk-stimulated Ica that was not inhibited by GTP~/S (average 3.4 + 7.8% decrease, n = 5) (Fig. 3 B, Table I C). Like the Forsk-stimulated Ica that was partially inhibited by GTP~/S in Fig. 2, the ACh-resistant current was not persistently activated either./Ca rapidly returned to control levels after the removal of Forsk. On average, 90.2 + 5.2% (n = 5) of the initial Forsk-stimulated Ica reversed upon washout of Forsk after internal perfusion with 50 ~M GTP,/S and exposure to ACh (Fig. 3 B). These experiments demonstrate that after internal GTP',/S, the ACh-resistant Forsk-stimulated/Ca does not remain elevated in the absence of Forsk.
Effects of GTPyS on cAMP-stimulated Ic~. We examined the possibility that GTP~/S might be inhibiting Ica at a site beyond AC. For example, activation of a phosphodiesterase (PDE) would increase the degradation of cAMP and result in a decrease in Ica. However, in none of the cells studied did GTP~/S result in inhibition of cAMP-stimulated Ica (n = 7). We observed either no change or a small stimulation of cAMP-elevated/Ca after internal GTP~/S. These results do not support the hypothesis that GTP~/S-mediated inhibition of Iso or Forsk-stimulated/Ca is via the stimulation of a PDE.
Taken together these results suggest that GTP~,S activates an inhibitory G protein whose primary site of action is AC, because the inhibitory effects on/Ca were observed only under conditions that activate AC (e.g., Iso or Forsk, but not basal or cAMP). Similar results and conclusions were reported for GppNHp . Persistent Ca Current Induced by GTPyS As described above, the fraction of Ic~ that was not inhibited by GTP~/S remained persistently stimulated after washout of Iso (Fig. 1). This is in contrast to the results after internal GTP~/S with Forsk-stimulated Ic~. A persistent Ic~ was not observed with internal GTP~S after the washout of Forsk (Figs. 2 and 3). Regardless of the degree of inhibition of Iso-stimulated/Ca by GTP~/S, the noninhibited /Ca always remained ~ ACh20 ACh20  elevated after washout of the [3-agonist (Fig. 1). On average, only a 7.3 + 2.5% (n = 15) decrease in/Ca followed the washout of Iso, leaving a persistent Ica that was 9.0 + 1.4-fold greater than basal Ic~ (Table I A, 2). Sotalol (5 p.M), a [3-adrenergic antagonist, did not significantly reduce the persistent /Ca (4.2 +--4.0% decrease, n = 5) (see Fig. 6 A). Similar results were obtained with 500 p.M GTP-/S (Table I A, 3). We hypothesize that because the a subunits of G proteins do not hydrolyze GTP~/S at a significant rate (reviewed by Ross, 1989), the inhibition of Iso-stimulated/Ca was due to the irreversible activation of an inhibitory G protein, and the persistent/Ca was due to irreversible activation of a stimulatory G protein. Consistent with this hypothesis, persistent/ca was unchanged by prolonged (> 20 min) washing out of GTP~/S with excess GTP (Fig. 4A). In Fig. 4A  ~M GTP failed to reduce significantly the persistent lc~. Fig. 4 B depicts a similar experiment except that GTP-fS was washed out with a 10-fold excess of GppNHp. GTP~/S partially inhibited the Iso-stimulated /Ca; however, /Ca remained elevated upon washout of Iso. Subsequent internal perfusion with 500 lzM GppNHp for ~ 20 min failed to reduce the persistent/ca. On average, GppNHp after GTP'yS produced an insignificant decrease in Ica of only 3.0 -+ 1.4% (n = 7). This is in contrast to the ~ 80% decrease in/Ca caused by internal perfusion of GppNHp during Iso stimulation of/c, . These findings are consistent with the idea that the GTP~/S-induced persistent/Ca is due to the irreversible activation of G~.
GTPyS-mediated persistent Ica not inhibited by ACh. The persistently stimulated/Ca that remained in the absence of Iso was unaffected by application of 10 ~M ACh. On average, after prolonged internal perfusion with GTP'yS, ACh produced an insignificant decrease (3.1 + 3.3%, n = 5) of the persistent/Ca. These effects are summarized in Fig. 4   cAMP-dependent protein kinase Hartzell and Fischmeister, 1992). Thus, we predicted that AC was the site of action of the irreversibly activated G protein. To test this hypothesis, we examined the effect of inhibitors of cAMP-dependent protein kinase on the persistent Ic, (Fig. 5). The cell in Fig. 5 was stimulated with 0.3 I~M Iso and internally perfused with 500 llM GTP3,S, and then Iso was washed out. Subsequent internal perfusion with 16 IxM Wiptide inhibited the persistent Ic,. Similar results were obtained with another protein kinase A inhibitor, (Rp)cAMPS, which also blocked persistent Ic, (Table I A, 2). Similar results were obtained with 0.1 or 10 I~M Iso (n = 4). These experiments support the hypothesis that persistent /Ca results from the formation of a long-lived 0q(GTPTS) that continues to activate AC after the removal of Iso.
Persistent Ca current is enhanced by GTPyS before Iso. In the preceding experiments, GTPTS was perfused during Iso exposure. Quantitatively different results were obtained when GTP~S was perfused before Iso exposure. After internal perfusion with 50 I~M GTP~/S for > 10 min, subsequent exposure to 0.3 ~tM Iso resulted in a 7.8 -+ 1.4-fold (n = 13) increase in/ca (Table I D). After removal of Iso, a large persistent/Ca remained (Fig. 6 A). In the presence of GTP~/S,/Ca decreased 18.7 __ 24.0% (n = 12) upon washout of Iso and was 7.5 --+ 1.4-fold larger than the unsfimulated /ca (Table I D , Fig. 6 B). ACh (10 I~M) or sotalol (5 p,M) did not significantly inhibit the persistent /ca that remained (Fig. 6A). On average, the persistent Ic~ was only negligibly reduced by ACh (8.2 +-2.3%, n = 9) ( Table I D  Summary of effects of prior internal perfusion with GTP~S on /ca. /Ca density is plotted for several experimental conditions using 0.3 ~M Iso, 50 I~M GTP~S, and l 0 i.tM ACh. by sotalol (3.3 -0.4%, n = 3). In contrast, under control conditions (in the absence of GTP~S), near-complete inhibition of Iso-stimulated/Ca by ACh  or sotalol (open squares, Fig. 6 A) was observed./ca stimulated by Iso was 1.8 times larger when the cell was perfused with GTP~/S before Iso exposure than when the cell was perfused with GTP',/S during Iso exposure (cf. Table I, A and D).

Persistent Ca Current Induced by ATPyS
Because GppNHp almost completely inhibits Iso-stimulated /Ca, we have suggested that the predominant effect of internal GppNHp in frog ventricular myocytes is to activate the inhibitory G protein, Gi . In contrast, it appears that the effects of GTP~/S involve both Gi and Gs because GTP~/S produces only incomplete inhibition and persistence of the noninhibited current. The difference between the effects of GppNHp and GTP~/S could be explained by the fact that GTP,tS can serve as a substrate for nucleoside diphosphate kinase (NDPK), whereas GppNHp cannot (Heidb/ichel, Callewaert, Vereecke, and Carmeliet, 1990). To explore whether the observed differences between the internal perfusion with GTP~/S and GppNHp were due to the thiophosphate donor property of GTP~yS, another thiophosphate nucleotide, ATP~/S, was perfused internally.
Internal perfusion with ATP'¢S resulted in a persistent/Ca after Iso stimulation. The cell in Fig. 7 A was internally perfused with 2.5 mM ATP~/S for > 10 min before stimulation with 0.3 I~M Iso. Upon exposure to Iso, Ica increased > 10-fold over ~ 25 min (te = 10.1 rain). The average rate of increase in Iso-stimulated Ica with internal 2.5 mM ATP~S was te = 8.4 -1.6 min (n = 6). This was similar to the rate of Iso stimulation in the presence of internal 50 I~M GTP'yS (re = 5.2 -0.7 min, n = 13), but was much slower than the increase with 50 p~M GTP (te = 1.3 _ 0.1 min, n = 3). Upon stabilization of the current and washout of Iso in the presence of ATPTS,/ca remained elevated. On average the Iso-stimulated persistent /Ca in the presence of ATP,/S was 4.8 + 1.2-fold (n = 6) greater than the unstimulated /Ca. Washout of Iso reduced/Ca only 4.3 --_ 3.0% (Table I E).
Effects of ATPyS on basal Ca current. In the presence of Iso, both GTPTS and ATPTS, but not GppNHp , regularly induced an /Ca that remained elevated above basal levels after the washout of the agonist. These findings suggested that transfer of the thiophosphate fi'om the nucleoside thiophosphate (transphosphorylation) contributed to the development of the persistent /Ca. These data raise the question of whether the putative transphosphorylation required the presence of Iso. Cells were internally perfused for > 10 min in the absence of Iso with either 2.5 mM ATP~¢S (no added ATP), 50 I~M GTP, or a control solution containing no added guanine nucleotides. Under all three conditions a similar approximately twofold increase in basal Ica was observed (Fig. 7 B). This runup of Ic~ is probably due to equilibration of the pipette solution (Cs ÷, Mg 2+, etc.) with the cell. We have shown that internal perfusion of either GppNHp (Parsons et ai., 1991) or GTP-~S (Table I D GppNHp  in which internal perfusion of the GTP analogue was initiated after exposure to Iso and during the steady-state stimulation of /Ca to [3-agonist.) When GppNHp was perfused before Iso exposure, there was considerable variability in the subsequent Iso response. In some cells, stimulation by Iso was essentially abolished (Fig. 8 A). Most often Iso stimulation resulted in a persistent Ic~ that was reduced in magnitude compared with control Iso responses (Fig. 8 B). However, in other cells Iso stimulated a large persistent/Ca (Fig. 8 C). The stimulatory response of Ica to Iso was highly variable with this experimental paradigm as compared with internal perfusion of GppNHp during stimulation with Iso (Fig. 8 D; cf. ISO before GppNHp versus GppNHp before ISO). Despite the variation, on average/Ca was approximately two times larger with GppNHp before exposure to Iso than Iso before GppNHp (Table I F ). In contrast to these results just described with 500 p.M GppNHp, persistent Ic~ was never observed with 50 ~,M GppNHp.

DISCUSSION
Internal perfusion of frog ventricular myocytes with hydrolysis-resistant analogues of GTP was used to examine the role of G proteins in the autonomic modulation of cardiac Ca channels. In our previous studies we showed that GppNHp activated an inhibitory G protein via basal nucleotide exchange in the absence of muscarinic agonists and virtually completely inhibited Iso-or Forsk-stimulated/Ca . In this article we showed that GTP~/S only incompletely inhibited Iso stimulation of/Ca and that the portion of the current that was not inhibited was persistently stimulated even in the absence of Iso. These results suggest that the two analogues of GTP differ in their ability to mediate inhibition of/Ca and to induce persistent/ca after the washout of Iso.
Although the molecular identity of the G proteins involved in the effects that we Internal perfusion with GppNHp before Iso exposure reduces Iso response. The cell was exposed to 0.3 I~M Iso, which resulted in an increase in Ica which was reversed upon washout of Iso. The cell was then internally perfused with 500 ~M GppNHp and exposed again to 0.3 I~M Iso. The second response to Iso was only 50% as large as the first. However, Ic~ remained elevated after the washout of Iso. (C) Large persistent lca after internal perfusion of GTP~/S before stimulation with Iso. After internal perfusion with 500 IxM GppNHp, Iso (1 I~M) produced a large increase in Ic~. This have observed is not known, for simplicity of discussion we will designate the G protein involved in Iso stimulation of Ica as Gs and the G protein involved in inhibition of lca as Gi. We feel this latter designation is justified because pertussis toxin blocks the inhibitory effects of ACh on /Ca in cardiomyocytes from chick (Pfaffinger, Martin, Hunter, Nathanson, and Hille, 1985), guinea pig (Hescheler et al., 1986), and frog (Nakajima, Wu, Irisawa, and Giles, 1990). Furthermore, antibodies against the COOH terminus of mammalian Gs and Gi o~ subunits recognize bands of appropriate molecular weights on Western blots of frog heart membrane homogenates (A. M. Spiegel, T. D. Parsons, and H. C. Hartzell, unpublished observations).

Hydrolysis-resistent Analogues of GTP Activate Gi
The similarity of the inhibitory effects of ACh, internal GppNHp, and GTP~/S on Isoand Forsk-stimulated /Ca suggests that all three agents act through the same mechanisms, although there are quantitative differences. Neither ACh , GppNHp , nor GTP~S has effects on basal /Ca. Furthermore, ACh (Fischmeister and Shrier, 1989), GppNHp , and GTP~S inhibit Iso-stimulated Ic~ in what appears to be a noncompetitive manner; e.g., up to a 100-fold excess of Iso does not reverse the inhibition. Both ACh and GppNHp are thought to inhibit Iso-stimulated Ic~ by inhibition of AC via a mechanism involving Gi Hartzell and Fischmeister, 1987;Hartzell, 1988;. Thus, we assume that the inhibitory effects of GTP~/S are mediated via the irreversible activation of Gi. The ability of internal GTP~/S to antagonize the Iso stimulation of/Ca in frog cardiac cells has been observed by other investigators (Fischmeister and Shrier, 1989;Nakajima et al., 1990). Nakajima et al. (1990) demonstrated complete inhibition of /Ca stimulated by 1 IxM Iso when the cell was previously perfused with 500 I~M GTP~S or when GTP~,S was perfused after Iso. Fischmeister and Shrier (1989) reported that exposure of cells to Iso in the presence of GTP,/S induced responses that varied significantly from cell to cell, which we have also explanation for the differences in the amount of Nakajima et al. (1990).
The lack of effect of hydrolysis-resistant nucleotide somewhat different from what has been reported observed. We do not have an inhibition observed by us and analogues on basal Ica in frog is for mammalian cardiac cells. GppNHp (Heschler et al., 1986) and GTP~S (Shuba, Hesslinger, Trautwein, McDonald, and Pelzer, 1990) in guinea pig ventricular myocytes cause an increase in basal Ica. The inhibition of/ca by ACh is also different in guinea pig (Heschler et al., 1986) than it is in frog cardiomyocytes (Fischmeister and Shrier, 1989): in guinea pig the inhibition appears to be competitive with Iso, whereas in frog it appears to be noncompetitive.

Mechanisms of Partial Inhibition of Stimulated Ica
Internal perfusion with GTP~/S resulted in only partial inhibition of Iso-or Forskstimulated /ca as compared with near-complete inhibition by ACh  or GppNHp . Furthermore, exposure to ACh after a steady-state partial inhibition had been produced by GTP3,S resulted in no additional inhibition. This incomplete inhibition by GTP~/S and the apparent functional uncoupling of the muscarinic receptor from inhibition of AC could, in principal, be due either to incomplete activation of Gi or to the relative inability of GTP~/S-liganded Gi to inhibit AC.
It seems unlikely that these effects can be explained by incomplete activation of Gi by GTP'yS. After Iso-or Forsk-stimulated Ica has been partially inhibited by GTP~/S, no additional inhibition was produced by (a) 10-fold higher concentrations of GTP~/S, (b) GppNHp, or (c) ACh. However, both GppNHp and ACh can completely inhibit Iso-stimulated /ca in the absence of GTP~tS. This suggests that the incomplete inhibition of stimulated/Ca produced by GTP~/S is not due to incomplete activation of Gi by GTP~/S. We propose that the incomplete inhibition of Iso-and Forsk-stimulated Ica by GTP~/S involves a reduced ability of ai~GTPvS) tO inhibit AC, as compared with eti~GTe) or 0q(GppNHp ). 0q(GTPvS) could have a relatively lower affinity for AC or be less efficacious at inhibiting AC than ¢Xi(GppNHp ), This would result in partial inhibition of Iso-or Forsk-stimulated Ic~ and a reduced responsiveness to ACh due to less inhibition per activated Gi. Thus, although GTP~S has a higher affinity than other GTP analogues for Gs (Northup, Smigel, and Gilman, 1982), Gi (Bokoch, Katada, Northup, Ui, and Gilman, 1984), transducin (Kelleher, Dudycz, Wright, and Johnson, 1986), and GK , the activity of the G protein-nucleotide complex may depend on the nucleotide bound and may be unrelated to the affinity of the nucleotide for the G protein.
The decreased ability of eti(GTP~S) to inhibit Ic~ could theoretically be due to a greater activation of Gs by GTP~/S than by GppNHp and competition between the effects of Gi and Gs on AC. If the potency or efficacy of GTP~/S is greater than GppNHp at activating Gs, a higher ratio of activated Gs to Gi would occur and Gi may not be able to inhibit AC completely. We discount this hypothesis because the stimulatory effect of Forsk is incompletely inhibited by GTP2tS to the same extent as the effect of Iso. Because Forsk stimulates AC via a mechanism that apparently does not require Gs (Seamon and Daly, 1986), it is unlikely that the incomplete inhibition of Forsk-stimulated Ic~ by GTP~/S occurs because GTP~/S activates more Gs than GppNHp does. The observation that Forsk stimulation of/ca reversed immediately upon washout of Forsk (even in the presence of GTP~/S) verifies the assumption that '~s~G~S) had not formed. Similarly, no difference in the ability of ACh to inhibit either Iso-or Forsk-stimulated/ca after internal GTP~/S was observed. This line of reasoning suggests that different ratios of Gs/Gi activated by GTP~/S or GppNHp cannot explain the differences in inhibition mediated by these analogues. The idea that GppNHp does not significantly activate Gs because of its relatively lower affinity for Gs is excluded by the observation that increasing the GppNHp concentration does not decrease the amount of inhibition produced by GppNHp, as would be expected if increasing GppN Hp were able to activate Gs . Thus, we conclude that a role for Gs in the incomplete inhibition of stimulated /Ca or in the GTP~Smediated uncoupling of the muscarinic ACh receptor from inhibition-stimulated/Ca is unlikely.
In conclusion, internal perfusion of frog cardiomyocytes with GTP3,S or GppNHp results primarily in the irreversible activation of Gi via basal nucleotide exchange.
However, the ability of ai(GTevS) to inhibit AC appears to be reduced compared with Oq(GppNHp)-

Incomplete Inhibition and Persistence Do Not Necessarily Share a Common Mechanism
The fraction of/Ca that was not inhibited by GTP~/S remained persistently stimulated after the washout of ~-adrenergic agonists. A similar large persistent/ca was only very rarely observed with internal GppNHp. An obvious question that arises is: Are incomplete inhibition and persistence related? We will argue that persistence and poor inhibition are due to different mechanisms. Above, we have proposed that the ability of 0q(dppNHp ) tO inhibit AC was greater than that of oti(GVP~S) and that the degree of inhibition depends on the nucleotide bound to the G protein. Below, we will argue that GTP~/S induces a persistent/Ca, whereas GppNHp rarely does, because GTP~/S can activate Gs by a pathway that is not utilized by GppNHp. Thus, whereas partial inhibition is caused by different efficacies of analogue-liganded ai subunits, persistence is due to the ability of GTP~/S to preferentially activate Gs.
This conclusion is both surprising and seemingly contradictory. However, it is supported by the fact that partial inhibition and persistence can be separated. For example, internal GTP-/S only incompletely inhibits Forsk-stimulated/ca, but after washout of Forsk, Ic~ is not persistent. Conversely, exposure to Iso after internal GTP3,S usually produces a large persistent Ic: in the apparent absence of partial inhibition. Subsequent internal perfusion with GppNHp after development of persistent /Ca has no effect. Since all our data as well as those of Szabo (1985, 1988) show a rapid basal nucleotide exchange on Gi, it is very likely that Gi is fully liganded with GTP analogue under these conditions. Thus, persistence is not simply due to incomplete activation of ai (as discussed above). Obviously, however, persistence would not be observed unless GTP~/S also was relatively ineffective at producing inhibition.

Nature of Persistent Ic:
Possible mechanisms. The persistent/Ca is probably due to persistent stimulation of AC by Gs and not to some other effect of GTP~/S. The strongest evidence was that blockers of protein kinase A largely inhibited the persistent /Ca. This demonstrates that the persistent/Ca is dependent on continuing protein kinase activity, suggesting that persistent activation of Ic~ is due to persistent stimulation of AC. Breitwieser and Szabo (1985) observed persistent stimulation of/Ca by Iso in frog atrial myocytes perfused with GppNHp. We rarely observed a large persistent/Ca with GppNHp. However, when high concentrations of GppNHp were present for a long time before Iso application, a persistent Ica was sometimes observed. The GppNHpinduced persistent/Ca was also variable in magnitude and often small in comparison to the larger, more consistently observed persistent Ic~ with GTP'yS.
The observation that the persistent /Ca is larger after internal perfusion with GTPTS than with GppNHp suggests that the activation of Gs by GTP3,S must be somehow different from the activation by GppNHp. The additional observation that ATP3,S mimics GTP~/S in the development of the persistent/Ca leads us to favor the idea that GTP'yS acts via an additional pathway to activate Gs. GTP~/S and ATP~/S can be used as substrates for the synthesis of GTP~/S from cellular GDP by NDPK, whereas GppNHp cannot.
The ability of ATP~/S to produce a persistent/Ca, as did GTP'yS, was consistent with the idea that GTP~/S was activating Gs to produce a persistent Ica, but the fact that ATP~/S might substitute for ATP and result in thiophosphorylation of the Ca channel complicated the interpretation of the ability of ATP~/S to induce a persistent/Ca-This presumably would result in the generation of a population of Ca channels that are resistant to dephosphorylation by phosphatases and are persistently activated (see Trautwein and Hescheler, 1990). (Rp)cAMPS, an inhibitor of protein kinase A, blocked the majority of the persistent Ica in our experiments. This finding implied that: (a) thiophosphorylation of the Ca channel was not the major source of persistent /ca; (b) despite replacement of ATP with ATP~S in the pipette, sufficient levels of intracellular ATP were maintained by cellular metabolism (creatine phosphate was in ii:!:i:i:!:iG~P~:i:i:i:i:i:i:~i:ii:!:!:!:!:!:!:!:!:!:!:i GTP~ orGppNHp : clase. NDPK in the presence of agonist provides a pathway for the irreversible activation of o~ that is available to ATP~/S and GTP~/S, but not GppNHp. Conversely, GTP~/S or GppNHp, but not ATP-yS, can irreversibly activate 0q, even in the absence of agonist. The small dashed arrow shows that the proposed subcellular compartmentalization G, is not a rigid barrier and that GppNHp can sometimes support the irreversible activation of cq. Omitted from the diagram for simplicity is the rest of the well-established cAMP-dependent phosphorylation cascade that regulates/ca in cardiac cells. the internal solution and glucose and pyruvate were in the bathing solution); and (c) ATP rather than ATP-/S acted as a preferential substrate for protein kinase A in the phosphorylation of the Ca channel or the thiophosphorylated channel can be rapidly dephosphorylated.
Proposed model. We propose that persistent/Ca is produced by GTP,/S but not by GppNHp because guanine nucleotide triphosphates have restricted access to Gs and because guanine nucleotides are provided preferentially to Gs by the enzyme NDPK (Fig. 9). We suggest that NDPK transfers the ~-phosphate of a nucleoside-5'triphosphate outside the Gs compartment to a nucleoside-5'-diphosphate within the compartment to activate Gs. This enzyme would serve to maintain a high local concentration of guanine nucleotide for Gs activation when GTP or GTP',/S was present, but would not do so with GppNHp because GppNHp is not a substrate for NDPK. GppNHp can enter this compartment, as evidenced by the fact that prolonged perfusion of high concentrations of GppHNp does produce persistent/Ca (Fig. 8). We do not necessarily envision this compartment as a defined physical entity, but rather as a virtual compartment created by the enzymatic funneling of selected substrates to the site of nucleotide exchange. In contrast, either GTP analogue can irreversibly activate Gi even in the absence of agonist; thus no such compartmentalization appears to exist for Gi. Under certain restricted conditions, however, adenine nucleotides can activate Gi via conversion to guanine nucleotides via NDPK (Otero, Breitwieser, and Szabo, 1988;Heidb~ichel et al., 1990;Szabo and Otero, 1990).
Other studies have proposed a regulatory function for enzymes located in close proximity to cardiac ion channels that act to control the local cytoplasmic environment of these channels. Weiss and Lamp (1989) demonstrated that glycolytic enzymes in close association with the ATP-sensitive K channel can act as a preferential source of ATP. A similar type of subcellular compartmentalization has been proposed for membrane-bound NDPK in the activation of the muscarinic K channel. In the absence of GTP, muscarinic K channel activity can be restored in excised patches of atrial myocytes by cytosolic exposure to several nucleosides including ATP and ATP~/S, but not AMP-PNP, an ATP analogue with an imido-linked terminal phosphate. Heidbiichel and colleagues have proposed that under physiological conditions NDPK acts a local intracellular buffer of GTP (Heidbiichel et al., 1990). Further experiments will be needed to determine whether or not the transphosphorylation mechanism reported here plays a role in the physiological activation Gs by Iso and GTP.
Alternative interpretations. We believe that the ability of GTP~S to induce a persistent Ica is best explained by a functional subcellular compartmentalization of Gs created by NDPK described above. However, another mechanism that could explain the ability of GTP~/S to induce a large persistent/Ca is that asiGTP~S) is more effective at stimulating AC than (~s(GppNHp) • This hypothesis has proved difficult to address because of our inability to define experimental conditions in which GppNHp reproducibly produces a large persistent /Ca. However, there are some data that support this suggestion that 0~s(GppNHp ) has low efficacy in stimulating AC . After inhibition of Forsk-stimulated/Ca by GppNHp, exposure to Iso had no stimulatory effect on /ca. However, after Iso, reexposure to Forsk produced a dramatic increase in/ca. This increase was due to an ~ 100-fold increase in the EC50 for Forsk. We interpret the rescue of Forsk-stimulated /ca by Iso to be due to Iso stimulating the loading of Gs with GppNHp. This activation of Gs increases the affinity of AC for Forsk, but I:ls(GppNHp ) alone is incapable of stimulating the catalytic activity of AC.
Several other potential sites for the regulation of/ca by G proteins may exist in cardiomyocytes. Internal perfusion of hydrolysis-resistant GTP analogues could in theory activate a multitude of different G proteins that might result in a persistent /Ca. We will consider two possible alternatives to AC as sites of action for irreversibly activated G proteins. Several authors have suggested that Gs may have a direct effect on the channel (Yatani, Codina, Imoto, Reeves, Birnbaumer, and Brown, 1987;Imoto, Yatani, Reeves, Codina, Birnbaumer, and Brown, 1988;Yatani, Imoto, Codina, Hamilton, Brown, and Birnbaumer, 1988;Yatani and Brown, 1989). However, we believe that cAMP-dependent phosphorylation is the major regulatory pathway of /Ca in these cells. This is supported by the observation that PKI completely inhibits the Iso-stimulated/Ca Parsons et al.i 1991). Furthermore, PKI also inhibited the majority of the persistent/ca. Thus, we do not believe that the direct action of Gs on the Ca channel has a significant role in the development of the persistent/Ca. G protein-mediated inhibition of PDE has been suggested to underlie the stimulatory effects of glucagon on frog cardiomyocytes (M6ry, Brechler, Pavoine, Pecker, and Fischmeister, 1990). Our observation that the internal GTP~/S can sometimes further increase /ca previously activated by internal cAMP is consistent with this claim. However, we do not believe that the irreversible inhibition of a PDE mediates the persistent lca described here because the persistent/Ca is only observed with Iso-stimulated/Ca and not with Forsk-stimulated/Ca-If the effects of GTP~S were mediated by a PDE, the persistent/Ca would be expected after any transient increase in intracellular cAMP in the presence of GTP',/S. However, a persistent Ic~ has not been observed after internal GTP,/S and subsequent washout of either Forsk or cAMP (Parsons, T.D., R.E. White and H.C. Hartzell, unpublished observations).