Regulation of the inositol 1,4,5-trisphosphate-activated Ca2+ channel by activation of G proteins.

Streptolysin O-permeable pancreatic acini were used to study the regulation of the inositol 1,4,5-trisphosphate (IP)-activated Ca channel (IPACC) by agonists and antagonists. Measurements of the apparent affinity for IP (KIP) showed that the IPACC is dynamically controlled during cell stimulation and inhibition, i.e. agonists decreased and antagonists increased KIP. KIP was also independently regulated by thimerosal, Ca content of the stores, the incubation temperature, activation of protein kinases, and inhibition of protein phosphatases, but none of these mechanisms contributed to the regulation by agonists and antagonists. Incubating the cells with low concentration of GTPS or AlF reproduced the effect of the agonist on KIP. Moreover, low [GTPS] allowed activation of the IPACC by agonists at basal levels of IP and markedly impaired channel inactivation by antagonists. Channel sensitization by GTPS also restored the ability of thimerosal to mobilize Ca from internal stores with no change in cellular IP levels. The combination of low [GTPS] and thimerosal locked the channel in an open, antagonist-insensitive state. All modulatory effects of GTPS are independent of phospholipase C activation and IP production. We propose that the dynamic regulation of the IPACC by a G protein-dependent mechanism can play a major role in triggering and maintaining Ca oscillations at low agonist concentrations when minimal or no changes in IP level take place.

Ca 2ϩ mobilizing agonists stimulate the production of inositol 1,4,5-triphosphate (IP 3 ), 1 which releases Ca 2ϩ from intracellular stores located in the endoplasmic reticulum (ER) (1). Stimulation with high agonist concentration leads to a persistent activation of the IP 3 -dependent Ca 2ϩ channel, which results in a single transient change in free cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) (2). On the other hand, at low agonist concentrations usually oscillations in [Ca 2ϩ ] i are observed (1,2). Only partial correlation exists between the type of the [Ca 2ϩ ] i signal generated and IP 3 production. In most cases, high agonist concentration increases IP 3 to a supermaximal concentration, while at low agonist concentration it is difficult to demonstrate stimulated production of IP 3 (1,2). In addition, several agonists such as parathyroid hormone acting on osteoblasts (3,4) or bradykinin acting on 3T6 fibroblasts (5) cause substantial Ca 2ϩ release from intracellular stores with no apparent increase in IP 3 . However, basal levels of IP 3 , although varying widely among different cell types, are almost always higher than that needed for maximal Ca 2ϩ release (1, 6 -9).
A plausible explanation for Ca 2ϩ release in the absence of IP 3 production and in the face of high basal levels of IP 3 is that low [agonists] trigger Ca 2ϩ release by regulating the affinity of the IP 3 -activated Ca 2ϩ channel (IPACC) for IP 3 . Studies in several cellular systems and the isolated and reconstituted IPACC showed the presence of multiple mechanism of channel regulation. Besides activation by IP 3 , the channel can be regulated by Ca 2ϩ in a biphasic manner (10 -13). ATP can regulate the channel directly (10,11,14) or by an indirect mechanism (13,15), which may involve phosphorylation/dephosphorylation reactions (13). Recent elegant work by Cameron et al. (16) showed the intimate association of the cerebellar IPACC with calcineurin. The channel's affinity to IP 3 was regulated by the combined action of protein kinase C and calcineurin (16). The same system was used to show that protein kinase A reduces the apparent affinity of the IPACC to IP 3 (14,16). In hepatocytes, protein kinase A increased the affinity for IP 3 in inducing Ca 2ϩ release (17,18), which was further augmented by inhibition of protein phosphatases with okadaic acid (18). The affinity of the IPACC for IP 3 was also shown to be regulated by thiol-reactive agents such as thimerosal (TMS) (19 -24) and by temperature (25). The relationship between all the different modes of regulating IP 3 affinity and Ca 2ϩ release are not known.
The above regulatory modes offer many mechanisms for the agonists to effect Ca 2ϩ release and initiate Ca 2ϩ oscillations without the need to increase IP 3 levels. Indeed, in rat liver, prestimulation with agonists was shown to modify the behavior and/or affinity for IP 3 (26). More recently we showed that agonists can activate and antagonists inactivate the IPACC independent of IP 3 metabolism (27,28). How such a regulation is achieved, what mechanism the agonists use to modulate Ca 2ϩ release and the antagonists to terminate the release is not known.
In the present studies we used agonist/antagonist responsive, streptolysin O (SLO)-permeabilized pancreatic acini to demonstrate that in the same cells the IPACC can be regulated by multiple and independent mechanisms. More importantly, a G protein-dependent mechanism is used by agonists to activate the channel at basal levels of IP 3 . Such activation dramatically impairs the ability of the antagonist in inactivating the IPACC. Once activated by the G protein-dependent mechanism, the channel can be locked in the open state by TMS. These findings point to the possible mechanism by which agonists can initiate Ca 2ϩ release and Ca 2ϩ oscillations with minimal changes in IP 3 concentrations.

EXPERIMENTAL PROCEDURES
Preparation of Pancreatic Acini-Acini were prepared from a rat pancreas using standard collagenase digestion (27). In brief, the pancreas was removed, minced, and incubated for 5-6 min at 37°C in a solution composed of (in mM) 140 NaCl, 5 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 Hepes (pH 7.4 with NaOH), 10 glucose, 10 pyruvate, 0.1% bovine serum albumin, 0.02% soybean trypsin inhibitor (solution A) and supplemented with 165 g/ml collagenase P. After digestion, the acini were collected and washed by centrifugation and kept in solution A on ice until use.
Measurement of Ca 2ϩ Uptake and Release in Permeabilized Cells-This procedure was identical with that described before (27,28). About 100 mg acini were washed twice with a solution containing 145 mM KCl and 10 mM Hepes (pH 7.4 with NaOH) and once with the same solution that was treated with Chelex 100. The acini were transferred to a fluorimeter cuvette containing a warm (37°C) incubation medium composed of the Chelex-treated solution, 3 mM ATP, 5 mM MgCl 2 , 10 mM creatine phosphate, 5 units/ml creatine kinase, 10 M antimycin A, 10 M oligomycin, 0.02% soybean trypsin inhibitor, 1 M Fluo 3, and 0.4 unit/ml SLO. Fluo 3 fluorescence was recorded at an excitation wavelength of 488 nm and emission wavelength of 530 nm. Calibration of Fluo 3 signals was as described before (27,28) (29).
Measurement of IP 3 -Acini were washed and incubated in permeabilization medium as for measurement of Ca 2ϩ uptake and release. After a 1.5-min incubation at 37°C, if required, TMS or U73122 was added, and, after a 2.5-min incubation, the cells were stimulated with agonists. At the indicated times, 100-l samples were transferred to 100 l of solution containing 15% perchloric acid, mixed and kept on ice for about 20 min to allow complete protein precipitation. After a 2-min centrifugation at 10,000 ϫ g, the supernatants were transferred to clean tubes. Standards of IP 3 were prepared in permeabilization medium and processed in a similar manner. Perchloric acid was precipitated, and IP 3 was extracted by the addition of 0.2 ml of tri-n-octylamine and 0.2 ml of freon. The samples were mixed vigorously, centrifuged for 30 s at 10,000 ϫ g to separate the phases, and 15-25 l of the upper layer was used for mass measurement of IP 3 by a standard radioligand binding assay (13). 3 -The maintained intact signaling system in the SLO-permeabilized cells allowed examination of the acute effect of agonists and antagonists on the IP 3 -activated Ca 2ϩ channel (IPACC). Fig. 1 shows the properties and demonstrates the advantages of this experimental system. Pancreatic acinar cells are fully permeabilized to small molecules within 10 -15 s of exposure to the concentration of SLO used and reduced [Ca 2ϩ ] of the incubation medium to the 50 -80 nM range within 2 min of incubation at 37°C. Addition of increasing concentrations of IP 3 resulted in discrete events of [Ca 2ϩ ] increase, typical of the quantal behavior of Ca 2ϩ release (30). Plotting the increments in medium [Ca 2ϩ ] as a function of IP 3 concentration showed a saturation kinetic with an apparent affinity for IP 3 (K app IP 3 ) of 0.43 Ϯ 0.02 M (Fig. 1a, Table I) and a Hill coefficient of 1.42 Ϯ 0.026 (n ϭ 33). Stimulating the cells with low concentrations of the muscarinic agonist, carbachol, caused small Ca 2ϩ release. Titration of Ca 2ϩ release in these cells showed that carbachol stimulation decreases the K app IP 3 by about 2.53-fold to 0.17 M (Fig. 1b, Table I), without changing the Hill coefficient. On the other hand, stimulation by maximal [carbachol] and inactivation by the antagonist atropine was followed by a 1.42-fold increase in the K app IP 3 to about 0.61 Ϯ 0.02 M (Fig. 1c, Table I), again without an apparent change in the Hill coefficient. Thus, agonist stimulation increases whereas antagonist inhibition decreases the apparent affinity of the IPACC to its ligand IP 3 . These changes in K app IP 3 are independent of the total amount of Ca 2ϩ release since the release was measured at the same Ca 2ϩ buffering conditions.

Regulation of K app for IP
To study the possible mechanism by which the agonist and  (21) and increase the affinity for IP 3 in several cell types (19 -24). Fig. 2c shows that unlike the case in intact cells, TMS (up to 500 M) alone was unable to cause Ca 2ϩ release in permeable cells. However, as little as 100 M TMS decreased K app IP 3 by about 4-fold (Table I). Fig. 2c and Table I show the results with 100 M TMS since this concentration was sufficient to maximally modify the effects of the agonist and GTP␥S on IPACC (see below). The effect of Ca 2ϩ content in the IP 3 -mobilizable Ca 2ϩ pool on IPACC has been studied extensively in several cell types (31)(32)(33)(34)(35)(36)(37)(38). Fig. 2d shows that overloading the IP 3 -sensitive pool with Ca 2ϩ caused small, but significant increase in the affinity to IP 3 (see also Table I). Similar K app IP 3 was measured after one (not shown), two (Fig. 2d), and four pulses of 5 M Ca 2ϩ (not shown). Finally, the temperature of the incubation medium had a profound effect on K app IP 3 but without changing the quantal nature of Ca 2ϩ release. Thus, Fig. 2e shows that after 2.5 min of cell permeabilization and Ca 2ϩ loading at 37°C and a subsequent 3-min incubation at 0°C, increasing concentrations of IP 3 induce discrete and finite Ca 2ϩ release events. The release was much slower than that at 37°C, which allowed a clear resolution of each Ca 2ϩ release event. In separate experiments, Ca 2ϩ release at submaximal [IP 3 ] was followed up to 15  Table I. min with no sign of deviation from quantal behavior (not shown). Table I shows that at 0°C K app IP 3 was reduced by about 8-fold.
An important question concerning the various modes of regulation of K app IP 3 was the relationship between them to determine whether they regulate the IPACC by the same or by independent mechanisms. Fig. 3 and Table I show the additive and independent effect of the different modes. Thus, overloading the Ca 2ϩ pool did not prevent or reduce the effect of GTP␥S (Fig. 3a), TMS (Fig. 3b), or low concentration of carbachol (Table I) on the K app IP 3 . Similarly, the effect of low temperature on K app IP 3 was additive with the effect of GTP␥S (Fig. 3c), TMS (Fig. 3d), and carbachol (Table I). Interestingly, addition of all agents after cooling to 0°C had no effect on K app IP 3 . The cells had to be treated with carbachol, GTP␥S, or TMS at 37°C before cooling to observe their effect on K app IP 3 , indicating activation of biochemical pathways by all agents, including  3

in activating Ca 2ϩ release
The apparent affinity was calculated from experiments similar to those in Fig. 1-3 and 8. Numbers in parentheses indicate number of independent determinations. Incubation with agonist and GRP␥S was usually for 30 s and incubation with TMS was for 1 min. In experiment e, the acini were first incubated for 2.5 min at 37°C, then transferred to a thermostated cuvette and incubated for 3 min at 0°C before the first addition of IP 3 . The trace shows the last part of the incubation at 0°C under control conditions. After each treatment, the K app IP 3 was measured by incremental additions of IP 3 . In experiments a-d, additions of IP 3 were as those shown in trace a. The numbers in trace e indicate the concentrations of IP 3 used for this titration. The results of all similar experiments are summarized in Table I.  Table I. TMS, to regulate K app IP 3 .
Several previous studies reported regulation of K app IP 3 by activation of protein kinases (13, 14, 16 -18). To evaluate the role of protein kinases in the SLO-permeabilized system, we tested the effect of several protein kinase activators (12-Otetradecanoylphorbol 13-acetate, cAMP, cGMP), protein kinase inhibitors (genistein, H7), and phosphatase inhibitors (KT62, okadaic acid, cyclosporine) on K app IP 3 . None of these agents affected K app IP 3 in unstimulated SLO-permeabilized cells. Further, these agents did not prevent or augment the effect of TMS, Ca 2ϩ load, or low temperature (not shown). Surprisingly, all kinase activators and phosphatase inhibitors tested reduced or prevented the effect of GTP␥S and carbachol, whereas the kinase inhibitors augmented the effect of the agonists. Table I lists the effect of the most effective compounds, okadaic acid and H7. Treatment with 0.12 M okadaic acid largely prevented the effect of 30 s of treatment with 10 M GTP␥S. H7 at 0.5 mM, a concentration sufficient to inhibit most protein kinases (38), had no effect in unstimulated cells but it increased the effect of submaximal [GTP␥S]. The lack of selectivity in the effect of kinase and phosphatase activators/inhibitors raises questions as to the physiological significance of these findings. However, their value for the present studies is in showing the similarity between their effects on GTP␥S and carbachol, which were different from those of TMS, Ca 2ϩ load, and low temperature.

Regulation of K app IP 3 Is Independent of PLC Activation-At
this stage of the study, the possibility arose that the effects of carbachol and GTP␥S on K app IP 3 were simply due to partial stimulation of PLC and a persistent, small global or local increase in [IP 3 ]. Three types of experiments were performed to exclude this possibility. The first is shown in Fig. 4, a-c, in which U73122 was used to inhibit PLC. U73122 did not reduce the effect of either carbachol (Fig. 4b), GTP␥S (Fig. 4c), or TMS (not shown) on K app IP 3 , although it inhibited the small Ca 2ϩ release evoked by low concentrations of carbachol. Table II shows that stimulation with 10 M carbachol had a small effect on IP 3 , whereas U73122 slightly reduced the basal level of IP 3 .
In the presence of U73122, IP 3 level was not changed by carbachol or GTP␥S. The second evidence is provided by the finding that prestimulation with low [GTP␥S] had a marked effect on K app IP 3 with minimal or no effect on IP 3 levels, Ca 2ϩ release, or the size of the IP 3 -mobilizable Ca 2ϩ pool. The third and most convincing evidence against stimulation of PLC by carbachol or GTP␥S as the cause of the change in K app IP 3 is shown in Fig. 4, d and e. In these experiments, 25 g/ml IP 3 competitive inhibitor heparin (39,40) were added to the incubation medium to increase the K app IP 3 under control conditions from 0.43 M (Table I) to 1.67 M (Fig. 4d). This should dilute the effect of any IP 3 generated by GTP␥S or carbachol stimulation by about 4-fold (1.67/0.43) to virtually eliminate the effect of the agents on K app IP 3 . Fig. 4e shows that this is not the case. Pretreatment with GTP␥S in the presence of 25 g/ml heparin had the same effect on K app IP 3 as in control cells. Similar results were obtained with heparin concentrations between 5 and 50 g/ml and with cells stimulated with 10 M carbachol (not shown). Hence, together the three protocols indicate that carbachol and GTP␥S modified K app IP 3 independent of PLC stimulations.
G Protein-dependent Regulation of IPACC-The finding that GTP␥S can modulate K app IP 3 independent of PLC activation suggested that activation of a G protein(s) by GTP␥S was sufficient to modulate interaction of IPACC with its ligand. Further support for this notion was obtained by testing the effect of AlF 3 , which activates mainly heterotrimeric but not small G proteins (41). All the effects of GTP␥S presented above and in subsequent figures could be initiated by 1 mM NaF ϩ 0.2 M AlCl 3 (not shown). 10 mM NaF ϩ 2 M AlCl 3 induced strong Ca 2ϩ release comparable with that observed with 100 M GTP␥S (see below) or 2 mM carbachol.
More dramatic evidence for an effect of G protein(s) activation on the activity of the IPACC was obtained when the effect of a low concentration of GTP␥S on the response to carbachol, atropine, and TMS was studied. Fig. 5a shows that exposure of control or stimulated cells to 100 M TMS in the absence of GTP␥S augmented the effect of low concentrations of carbachol on Ca 2ϩ release (Fig. 5, b and c), without affecting IP 3 levels during the first 20 s of cell stimulation (Table II). Performing complete concentration dependence of both IP 3 production and Ca 2ϩ release showed that pretreatment with 100 M TMS had no effect on IP 3 production while increasing the apparent affinity for carbachol-mediated Ca 2ϩ release by about 6.3-fold.
On the other hand, GTP␥S profoundly modified the effect of carbachol. Treating the cells with as little as 2 M GTP␥S for 30 s was sufficient to cause maximal Ca 2ϩ release by 10 M carbachol (Fig. 5d). That the effect of GTP␥S was independent of PLC activation became even more evident when the effect of 2 M GTP␥S on the dose response to carbachol and atropine was measured. Fig.  6 shows that under control conditions the concentration dependence curves for carbachol stimulation of IP 3 production and Ca 2ϩ release were identical. Half-maximal stimulation (EC 50 ) of Ca 2ϩ release was at 210 Ϯ 13 M (n ϭ 8), and the EC 50 for IP 3 production was 236 Ϯ 27 M (n ϭ 4). In the presence of 2 M GTP␥S, the EC 50 for IP 3 production was reduced by about 2.9-fold to 82 Ϯ 11 M (n ϭ 4), whereas the EC 50 for Ca 2ϩ release was reduced by 90-fold to 2.35 Ϯ 0.13 M (n ϭ 8).
The effect of low [GTP␥S] on signal termination by atropine is shown in Fig. 7. In the absence of GTP␥S, atropine inhibited the IPACC of cells stimulated with 2 mM carbachol (measured from the rate of [Ca 2ϩ ] reduction in carbachol-stimulated cells, see Fig. 1  The fact that atropine inhibited PLC activation in the presence of GTP␥S clearly shows that the effect of GTP␥S was independent of PLC activation, since binding of nonhydrolyzable GTP analogues to the ␣ subunit of G proteins, including G ␣s (41,42) and G ␣q (43), irreversibly stimulates the ␣ subunits and prevents inhibition by antagonists.
Stabilization of IPACC in an Active State-In previous studies, we reported that antagonists inactivate the IPACC independent of IP 3 metabolism (27, 28). In Fig. 1 and Table I, we showed that termination of carbachol stimulation with atropine significantly increased K app IP 3 . To better understand the mechanism of channel inactivation, we first tested the effect of atropine on the various mechanisms shown to affect K app IP 3 . Fig. 8 illustrates the effect of carbachol stimulation and atropine inhibition of the ability of GTP␥S and TMS to modify K app IP 3 . Similar experiments were performed to test the effect of Ca 2ϩ load and low temperature. Carbachol stimulation followed by atropine inhibition did not prevent the reduction in K app IP 3 caused by TMS (Fig. 8c), Ca 2ϩ load, or low temperature (not shown), while completely preventing the effect of GTP␥S (Fig. 8b). Incubating the cells with atropine alone without carbachol stimulation had no effect on the ability of GTP␥S to reduce K app IP 3 (not shown). Table I shows that after carbachol and atropine treatment the K app IP 3 (Table I). These experiments indicate that, in permeabilized cells, once the IPACC was inactivated by atropine, the inactivation could not be relieved by activation of G proteins.
Because the effect of carbachol was modified by both GTP␥S and TMS, to understand how they may modify channel activity we tested the effect of TMS on the modulation of IPACC by GTP␥S. Interestingly, GTP␥S markedly sensitized the effect of TMS to cause maximal discharge of the Ca 2ϩ stores. This is illustrated in Fig. 9. Fig. 9a shows that addition of 100 M TMS to cells treated with 5 M GTP␥S induced rapid and maximal Ca 2ϩ release. Similar results were obtained when GTP␥S was added to TMS-treated cells, but the time course of Ca 2ϩ release was significantly slower. Accordingly, as all other effects of GTP␥S, the effect shown in Fig. 9 was time-(not shown) and concentration-dependent (Fig. 9c). In the absence of TMS, high concentrations of GTP␥S could release Ca 2ϩ , which was probably due to stimulation of PLC to generate IP 3 . TMS actually partially inhibited the production of IP 3 generated by all concentrations of GTP␥S while sensitizing activation of Ca 2ϩ release by GTP␥S. It is important to note that despite the absence of an increase in IP 3 the effect of TMS and GTP␥S on Ca 2ϩ release was still inhibited by heparin (Fig. 9c). This would suggest that TMS ϩ GTP␥S sensitized the IPACC to a level that maximal and rapid Ca 2ϩ release was observed at the level of IP 3 present in unstimulated cells.
In the next stage, we tested the effect of channel sensitization by GTP␥S and TMS on the inactivation induced by atropine. Fig. 10a shows that 1 mM atropine completely inactivated channels activated by 10 M carbachol and 2 M GTP␥S. However, treating the cells with 100 M TMS prior to stimulation with carbachol and GTP␥S completely prevented channel inactivation by atropine. The small reduction in medium [Ca 2ϩ ] due to atropine probably reflects the activity of channels that were not accessed by GTP␥S. The channels were permanently stabilized in an active state since medium [Ca 2ϩ ] remained elevated for at least 15 min with no sign of decline. To show that TMS and GTP␥S did not inhibit the SERCA pumps and that the maintained high level of medium [Ca 2ϩ ] was due to stabilizing the IPACC in an active state, the channel was inhibited by heparin. Addition of heparin resulted in channel inhibition and, consequently, rapid Ca 2ϩ uptake into the IP 3 -sensitive pool at a rate comparable to that measured in Fig. 10a after addition of atropine.
As indicated above, TMS alone (not shown) or GTP␥S alone ( Fig. 10a and Fig. 7), although reducing the affinity for atropine, never prevented channel inactivation by atropine. That activation of G proteins by GTP␥S and channel sensitization by TMS was required to prevent channel inactivation is further emphasized in the experiments shown in Fig. 10, c and d. As we showed before (28), stimulation of Ca 2ϩ release with 100 M GTP␥S did not prevent channel inactivation by atropine (Fig.  10c), even though IP 3 levels under these conditions were very high (28). On the other hand, treating the cells with 2 M (not shown) or 100 M GTP␥S (Fig. 10d) and 100 M TMS in the absence of agonist stimulation was sufficient to prevent channel inactivation by atropine.
An important control for the experiments in Fig. 10 is to show that TMS did not prevent the hydrolysis of IP 3 initiated by atropine. The results of such experiments are shown in Fig.  11. Even in the presence of 100 M carbachol and 5 M GTP␥S, TMS accelerated, rather than inhibited, the hydrolysis of IP 3 . After 2 min of exposure to atropine, IP 3 was reduced to basal levels, while the channel was fully activated (Fig. 10b).  Table I. FIG. 9. GTP␥S together with TMS induces maximal Ca 2؉ release. Experiment a in the upper panel shows that 100 M TMS causes rapid and maximal Ca 2ϩ release from acini treated with 5 M GTP␥S for 30 s. This Ca 2ϩ release can be inhibited largely by 50 g/ml heparin (b). Experiment c in the lower panel shows the dependence of Ca 2ϩ release (E, q) and IP 3 production (Ç, å) on GTP␥S concentration in the absence (E, Ç) or presence (q, å) of 100 M TMS. For Ca 2ϩ release, the protocol in experiment a was used except that the cells were incubated with or without 100 M TMS for 1 min before addition of GTP␥S. IP 3 production was measured as described in the legend to Fig. 6 except that the cells were treated with or without 100 M TMS for 1 min before being stimulated with the indicated concentration of GTP␥S for 1 min.  experiments (b, d) after atropine inhibition, the cells were exposed to 100 g/ml heparin. Results similar to those in experiment d were obtained with 2 and 5 M GTP␥S. The experiment shown and the one using 5 M GTP␥S were repeated at least seven times with different cell preparations.
[IP 3 ] (1). Modulation of the IP 3 -activated Ca 2ϩ channel (IPACC) can be potentially important in view of the high basal IP 3 levels in most cells (1, 2, 6 -9). Although several regulatory mechanisms of IPACC have been reported (10 -26), the relationship between them and their role in agonist-dependent regulation of Ca 2ϩ release is not known. The present study suggests the existence of multiple and independent mechanisms for regulation of IPACC, prominent among which is regulation by G protein activation. The latter is used by agonists and antagonists to modulate the K app IP 3 in a reciprocal manner and thus facilitate Ca 2ϩ release during agonist stimulation and impair the release during antagonist inhibition. Such a mechanism can contribute to the cyclical activation and inactivation of the IPACC during Ca 2ϩ oscillations (44). Below we discuss the evidence in support of these findings.
Comparing the effect of many agents and conditions in the same cell type and experimental system showed that K app IP 3 can be modulated by several independent mechanisms (Table  I). Thus, additive effects were found between TMS, Ca 2ϩ load, low temperature, and the agonists. The important implication of these findings is that although the IPACC can be regulated by various mechanisms, none of them appear to contribute to the regulation by agonists. Of course, regulation by Ca 2ϩ load can have important physiological significance in that when the stores are loaded, they are primed for release by small additional change in K app IP 3 . It is likely that in empty stores the IPACC has the lowest affinity for IP 3 , which will facilitate channel inactivation at the termination of cell stimulation. Nonetheless, the regulation of K app IP 3 by Ca 2ϩ content in the ER is relatively modest and occurs by a mechanism different from that used by agonists.
Interestingly, despite the fact that variations in K app IP 3 between compartmentalized Ca 2ϩ pools account in large part for quantal Ca 2ϩ release (28), none of the modulators of K app IP 3 changed the quantal properties of Ca 2ϩ release. This includes low temperature (Figs. 2 and 3). We particularly studied the effect of low temperature in detail since a previous report suggested that quantal Ca 2ϩ release becomes continuous at low temperature, and this process is reversible (25). However, we failed to convert the quantal to a continuous Ca 2ϩ release by short or long incubation at 25 or 0°C or by any other modulator of K app IP 3 . In the earlier studies by Kindman and Meyer (25), one concentration of IP 3 was used to demonstrate submaximal Ca 2ϩ release at 37°C and maximal release at 0°C, without considering the effect of the temperature on K app IP 3 . Such an effect as demonstrated in the present study (Table I) can account well for the differences between the two studies. Our results of maintained quantal behavior under all conditions suggest that all modulators of K app IP 3 , including agonists, affect all IPACC equally rather than equalize K app IP 3 of channels of different compartments.
The present studies show that K app IP 3 is dynamically controlled. Low concentrations of agonists, which minimally activate PLC, markedly reduced K app IP 3 of the IPACC. Moreover, termination of cell stimulation with antagonists increased K ap -pIP 3 to a level above that measured in control cells. The antagonist was effective only if the cells were first stimulated with carbachol. The antagonist had no effect in control cells, and, when added before GTP␥S, it did not prevent the GTP␥S-dependent reduction in K app IP 3 . The combined effects of the agonist and antagonist indicate that the K app IP 3 of the IPACC is dynamically controlled during cell stimulation/inhibition cycles. One advantage of such a dynamic control is that small changes in IP 3 levels can lead to maximal opening or closing of the channel. This will be translated into a high cooperativity for interaction of IP 3 with the IPACC and in channel activation/inactivation.
Probably the most interesting finding of the present studies is that agonists and antagonists appear to modulate K app IP 3 by a mechanism dependent on activation of G proteins. The first indication of this was the similarity between the effect of the agonist and preincubation with GTP␥S on K app IP 3 . Both affected K app IP 3 in cells treated with TMS, high Ca 2ϩ load or incubated at low temperature. Both effects were inhibited similarly by activators of protein kinases, inhibitors of protein phosphatases, and augmented by inhibitors of protein kinases. Inhibition of agonist-activated cells by atropine to increase K app IP 3 inhibited the effect of GTP␥S, but not of any other agent or treatment. Together, these observations strongly suggest that agonist stimulation and GTP␥S reduced the K app IP 3 by the same mechanism. It is possible that the agonists and GTP␥S activated heterotrimeric, rather than small G proteins, since all effects of GTP␥S could be reproduced with low concentrations of AlF 3 . The effect of GTP␥S described in the present study is different from the previously described modification of the size of the IP 3 -sensitive Ca 2ϩ pool by GTP (45)(46)(47). GTP␥S inhibited the GTP-induced expansion of the Ca 2ϩ pool, whereas GTP␥S increased the K app IP 3 and millimolar concentrations of GTP were required to mimic the effect of low concentrations of GTP␥S and AlF 3 .
That G proteins are involved in the effect of the agonist/ antagonist and their full impact becomes more evident when the effect of their activation on agonist/antagonist-dependent changes in Ca 2ϩ release and IP 3 levels are considered. Even in the absence of preincubation, low concentrations of GTP␥S (0.2-2 M) markedly increased the potency of the agonist and decreased the potency of the antagonist in affecting Ca 2ϩ release. Thus, when irreversible activation of G proteins was allowed by the presence of GTP␥S, the agonists exceedingly sensitized the IPACC to trigger Ca 2ϩ release at basal [IP 3 ]. Measurement of IP 3 levels showed that GTP␥S had a small effect on IP 3 production during agonist stimulation and did not FIG. 11. TMS accelerates atropine-induced IP 3 hydrolysis. Acini incubated in permeabilization medium for 2.5 min at 37°C (å) were stimulated with 100 M carbachol and 2 M GTP␥S (q, E) and in the presence of 100 M TMS (E). After 20 s of stimulation, a portion of cells stimulated in the absence (f) or the presence of TMS (Ⅺ) were transferred to vials containing atropine to give a final concentration of 1 mM. At the indicated times, samples were removed to assay the levels of cellular IP 3 . This experiment represents 2 others with similar results. The same behavior was found in two experiments where 10 M carbachol was used. In the absence of GTP␥S, TMS augmented IP 3 hydrolysis even more than that shown in the figure (three independent experiments). prevent initiation of IP 3 hydrolysis by the antagonist. The latter excludes the possibility that the effects of GTP␥S, or for that matter the agonist and the antagonist, on Ca 2ϩ release was dependent on PLC activity (41)(42)(43). In fact, in cells stimulated with carbachol and GTP␥S, 10 -100 M atropine completely inactivated PLC without having any inactivation effect on the IPACC (Fig. 7). These experiments, therefore, indicate that agonists use G protein activation to reduce K app IP 3 and stabilize the channel in an open state. Once the channel is activated, complete inactivation of G proteins by the antagonist beyond (or different from?) that required to modulate PLC activity, is needed for channel inactivation. Indeed, preliminary experiments showed that muscarinic, bombesin, and cholecystokinin antagonists were between 100-and 1000-fold more effective in preventing cell stimulation when added before the respective agonist than in reversing agonist effects when added to stimulated cells. 2 Further insight into the regulation of IPACC by G proteins was obtained when the effect of TMS on GTP␥S-, agonist-, and antagonist-dependent Ca 2ϩ release was studied. TMS and low GTP␥S caused maximal Ca 2ϩ release at basal IP 3 concentration. It is interesting that in intact cells 100 M TMS caused significant Ca 2ϩ release (21 and data not shown), whereas in permeabilized cells up to 500 M TMS did not release Ca 2ϩ but only decreased K app IP 3 . Incubating the cells with as little as 0.2 M GTP␥S was sufficient to restore the ability of TMS to cause Ca 2ϩ release with no change in IP 3 levels. Together, these observations indicate that: (a) TMS and GTP␥S modulate the IPACC by different mechanisms, (b) the IPACC is regulated by G proteins in permeabilized and probably in intact cells, and (c) TMS appears to modulate the interaction of the G protein-dependent mechanism with the IPACC to stabilize the channel in an open state. Indeed, treating the cells with TMS and GTP␥S, with or without agonist, completely prevents channel inactivation by the antagonist. This effect was absolutely dependent on the combined action of TMS and GTP␥S to the extent that incubating the cells with very high GTP␥S (100 M) did not prevent the effect of atropine, while stimulation with 2 M GTP␥S and 100 M TMS locked the channel in an antagonistinsensitive state. It is important to note that TMS increased, rather than decreased, the rate of IP 3 hydrolysis under all conditions, including incubation with GTP␥S.
The implication of the studies with TMS, GTP␥S, agonist, and antagonist is that all point to the involvement of G proteins in the regulation of K app IP 3 by agonist stimulation and antagonist inhibition. They also point to the broad extent of such regulation. Such a regulatory mechanism can be very attractive in explaining how agonists can stimulate Ca 2ϩ oscillations in the absence of stimulated IP 3 production. Our results suggest that minimal activation of G proteins (low GTP␥S, AlF 3 , or agonist concentrations), which is not sufficient to appreciably activate PLC, is sufficient to markedly decrease the K app IP 3 and trigger large Ca 2ϩ release at basal [IP 3 ]. This regulation is a dynamic process as revealed by the antagonist-induced increase in K app IP 3 beyond that found in resting cells. It is well established in many cell systems that Ca 2ϩ oscillations occur only at low agonist concentration, when usually no change in IP 3 is observed. It is easy to see how the dynamic regulation of K app IP 3 described here can contribute or even dominate the mechanism by which agonists signal Ca 2ϩ oscillations. Understanding how activation of G proteins modulate K app IP 3 is essential to evaluate its role in Ca 2ϩ oscillation in particular and Ca 2ϩ signaling in general. This should be the challenge for future studies.