Alternate Coupling of Receptors to Gs and Gi in Pancreatic and Submandibular Gland Cells*

Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gqclass heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cβ either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and β-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gsand Gi. This inference was based on the inhibitory action of antibodies specific for Gαs, Gαi, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of β-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Gα15 −/− knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors.

A family of heterotrimeric guanine nucleotide-binding proteins (G proteins) transduces a variety of signals across the plasma membrane by sequential interactions with receptor and effector proteins (e.g. second messenger-generating enzymes and ion channels). These interactions result from guanine nucleotide-driven conformational changes in G protein ␣ subunits (1). Agonist-bound receptors catalyze the exchange of GDP for GTP on the ␣ subunits of their cognate G proteins to promote dissociation of ␣ from a high affinity complex of ␤␥ subunits. Dissociated subunits are competent to modulate the activity of effectors. GTP hydrolysis ultimately returns G␣ to the GDPbound state, thus allowing reformation of inactive heterotri-mer. Sixteen distinct genes encode G protein ␣ subunits in mammals. The family is commonly divided into four classes based on amino acid sequence identity and function: G s , G i , G q , and G 12 . Members of a newly identified family of regulators of G protein signaling (RGS proteins) 1 have been shown to stimulate the GTPase activity of G i and G q class ␣ subunits, thus attenuating signaling (2).
One of the more thoroughly characterized examples of G protein-mediated signal transduction is carried out by the hormone-sensitive adenylyl cyclase system. Relevant receptors communicate with homologous G proteins, one of which (G s ) activates adenylyl cyclase while others (G i ) inhibit the enzyme (1). The second messenger (cAMP) mediates diverse cellular responses, primarily by activating cAMP-dependent protein kinase (PKA). In the case of Ca 2ϩ -mobilizing agonists, G protein activation is followed by stimulation of phospholipase C␤ (PLC␤) to generate IP 3 in the cytosol, which initiates the [Ca 2ϩ ] i signal by release of Ca 2ϩ from internal stores (1,3). PLC␤ can be activated by each of the four G q class ␣ subunits or by G␤␥ subunits released from G i class proteins (4). Only G i -mediated PLC␤ activation is inhibited by pertussis toxin (4). In this study we sought to learn the mechanism by which G s -coupled receptors evoke Ca 2ϩ signaling.
Several G s -coupled receptors can activate dual signaling cascades. For example, increases in both cAMP and [Ca 2ϩ ] i have been observed by histamine acting on H 2 receptors in parietal cells (5), parathyroid hormone acting on osteoblasts (6), and isoprenaline acting on cardiac myocytes (7) or salivary gland cells (8,9). In contrast to the simple paradigm that each receptor molecule can activate a single class of G protein (10), activation of more than one signaling cascade could be due to coupling of one receptor type to two classes of G proteins. This model is supported by experiments in heterologous expression systems. Overexpression of histaminergic H 2 (11), parathyroid hormone (12), luteinizing hormone (13), P2Y11 (14), vasopressin V2, dopamine D1A, and adenosine A2A (15) receptors resulted in stimulation of adenylyl cyclase and PLC␤. The ␤-adrenergic receptor (which is considered to be a classical G scoupled receptor) and the vasopressin V2, dopamine D1A, and adenosine A2A can functionally interact with the G q family member, G 15 , when both proteins are overexpressed in COS cells (15,16).
An alternate mechanism for stimulation of Ca 2ϩ signaling by G s -coupled receptors is activation of PLC␤ by PKA. In several cell types, increasing cellular cAMP with forskolin (5,8,9) or membrane permeable cAMP analogues (5) increased [Ca 2ϩ ] i similar to stimulation of G s -coupled receptors. In a recent study we showed that stimulation of submandibular gland (SMG) duct cells with forskolin results in PLC␤-mediated and IP 3 -dependent Ca 2ϩ release from internal stores (9). These findings suggest that, at least in some cell types, stimulation of PKA can activate PLC␤ to generate a Ca 2ϩ signal. Phosphorylation-dependent switching of receptor specificity for G proteins is another mechanism by which a single receptor could activate more than one G protein (17). As outlined recently by Lefkowitz (18), receptor-dependent activation of G s stimulates adenylyl cyclase, generates cAMP, and activates PKA. Phosphorylation of the receptor by PKA is proposed to switch its coupling specificity from G s to G i . Receptor-dependent activation of G i could thus release sufficient G␤␥ to activate PLC␤. Activation of PLC␤ generates IP 3 (which releases Ca 2ϩ from internal stores) and diacylglycerol to activate protein kinase C. Hence, PKA-dependent switching of receptor coupling to different classes of G proteins (the G s /G i switching model) is a potential mechanism for activation of multiple signal transduction cascades by the same receptor.
In the work presented here we sought to determine if any of the above models applied to classical G s -coupled receptors that evoke Ca 2ϩ signals in cells freshly isolated from native tissues. We used vasoactive intestinal peptide (VIP) stimulation of pancreatic acinar cells and isoprenaline (Iso) stimulation of SMG duct cells to show that switching or augmentation of receptor coupling to G i could account for activation of cAMP and Ca 2ϩ signaling systems in vivo.
Cell Preparation-Production of G␣ 15 (Ϫ/Ϫ)-mutant mice was described (22,23). Single pancreatic acinar and submandibular gland (SMG) duct cells from wild type (WT) and G␣ 15 (Ϫ/Ϫ)-mice were prepared by standard collagenase and trypsin digestion procedures (24,25). In brief, mice were sacrificed by exposure to a methoxyfluranesaturated atmosphere. The pancreas and SMG were removed and cleaned by injection of PSA. Minced tissues were incubated in a PSA solution containing 0.1 mg/ml collagenase (type CLSP, Worthington) before a short treatment with a trypsin/EDTA solution to release single cells. The cells were washed with PSA and kept on ice until use.
Current Recording-The Ca 2ϩ -activated Cl Ϫ current of pancreatic acinar and SMG duct cells was recorded as detailed (21), using the whole cell configuration of the patch clamp technique (26). The cells were dialyzed with the pipette solution for 8 -10 min before the first stimulation to allow equilibration of proteins and antibodies when included in the pipette solution. Membrane potential was held at Ϫ40 mV to record the inward current. The output signal recorded with a pClamp 6 and DigiData 1200 interface was filtered at 20 Hz. Due to significant variations in the current magnitude between preparations, results are given primarily as the number of responding cells. For each protocol similar results were obtained with cells from at least three mice. Fig. 1 summarizes the signaling pathways by which G scoupled receptors (R s ) may trigger a Ca 2ϩ signal. Stimulation of a G q -coupled cholinergic receptor with carbachol (R q ) was FIG. 1. Signaling pathways tested in this study. Double slashes through arrows in the pathway signify inhibition by the indicated agents. A positive control for Ca 2ϩ release was tested by carbachol stimulation of G q -coupled muscarinic m3 receptors (R q ), which stimulate PLC␤ via G q class ␣ subunits. Ca 2ϩ signaling in this work is followed by measuring the activity of Ca 2ϩ -activated Cl Ϫ current. a, G s -coupled receptors (R s ) such as the VIP or ␤-adrenergic receptor might activate the G q class heterotrimeric G protein, G␣ 15 . A wide variety of G s -coupled receptors can couple to G 15 , activate PLC␤ to produce IP 3 , and release Ca 2ϩ from intracellular stores (15,16). This potential pathway would be absent from G␣ 15 knockout mice. b, R s activation of G s and stimulation of adenylyl cyclase (AC) to increase production of cAMP, activates PKA which could activate PLC␤. c, agonist stimulation of R s typically activates G s and stimulates AC to increase production of cAMP (inhibited by carboxyl-terminal G␣ s antibodies). PKA activity is required (directly or indirectly) for generation of *R s , thereby eliciting a switch (17) or an augmentation to account for activation of G i by G s -coupled receptor agonists. G␤␥ released by activation of G i could stimulate PLC␤ activity, produce IP 3 , and release Ca 2ϩ from intracellular stores. PTX and antibodies (Abs) to the carboxyl terminus of G␣ i inhibit receptor activation of G i . PIP 2 antibodies prevent PLC␤ hydrolysis of PIP 2 and production of IP 3 . The Ca 2ϩ ionophore, A23187, bypasses the need for IP 3 production needed for Ca 2ϩ release from intracellular stores. used as a positive control. Three mechanisms were tested: (a) activation of G␣ 15 by R s , (b) direct activation of PLC␤ by PKA, and (c) switching or augmentation of coupling specificity of R s from G s to G i . We tested these mechanisms using two G scoupled receptors which evoke different types of Ca 2ϩ signals: pancreatic acinar cells stimulated with VIP and SMG duct cells stimulated with Iso. Ca 2ϩ signaling was followed by measuring the activity of the Ca 2ϩ -activated Cl Ϫ current in each cell type. Previous work showed that pancreatic acinar and SMG cells express the Ca 2ϩ -activated Cl Ϫ channel (21,25,27) and this current faithfully reflects changes in [Ca 2ϩ ] i (21,27). Fig. 2a shows that stimulation of pancreatic acinar cells with a saturating concentration of VIP-induced [Ca 2ϩ ] i oscillations which lasted for the duration of cell stimulation, as previously reported (28). Maximal stimulation of the G q -coupled muscarinic m3 receptor with 1 mM carbachol in the same cells resulted in a typical biphasic response of a spike and a plateau. This response was highly reproducible in mouse pancreatic acinar cells; similar responses were observed in 15/15 cells from 13 mice. Fig. 2b shows that stimulation of SMG duct cells with the ␤-adrenergic agonist Iso caused a sustained increase in the Ca 2ϩ -activated Cl Ϫ current with no apparent oscillations. Following removal of Iso, stimulation with carbachol caused a large biphasic response. The Cl Ϫ current responses are similar in shape and time course to the previously reported changes in [Ca 2ϩ ] i caused by these agonists in SMG cells (8,9). Among cells which responded to carbachol, prior stimulation with Iso elicited a response similar to that in Fig. 2b in 19/25 SMG duct cells from 17 mice.

RESULTS AND DISCUSSION
␤-Adrenergic, vasopressin V2, dopamine D1A, and adenosine A2A receptors overexpressed in COS cells can couple to G␣ 15 , but not other members of the G q class, and stimulate PLC␤ activity (15,16). This would suggest that G␣ 15 has the unique ability to couple to receptors which are usually coupled to G s . Currently, there are no good biochemical tools to specifically evaluate G␣ 15 function in native cells. Genetics provide an alternative approach. We measured the effect of VIP and Iso on Ca 2ϩ signaling in cells prepared from mutant G␣ 15 (Ϫ/Ϫ)-mice to rule out the possibility that G␣ 15 contributes to Ca 2ϩ signaling by G s -coupled receptors in SMG and pancreatic acinar cells. Fig. 2c shows that VIP-and carbachol-induced Ca 2ϩ signaling was completely normal in pancreatic acini for G␣ 15 The same results were obtained in six out of six experiments with acini from six mice. Fig. 2d shows that Iso-and carbacholinduced Ca 2ϩ signaling was normal in SMG duct cells from G␣ 15 (Ϫ/Ϫ)-mice. Similar results were obtained in four out of six experiments with SMG ducts prepared from the six mice that were used to study the response of pancreatic acinar cells. These findings exclude coupling to G␣ 15 as obligatory for activation of Ca 2ϩ signaling by the G s -coupled receptors. Coupling of R s to other members of the G q class is also excluded by experiments with antibodies described below.
Experiments with RGS4 supplied our first evidence that activation of Ca 2ϩ signaling by VIP and Iso involves more than activation of G s . RGS4 accelerates GTP hydrolysis by G q and G i class ␣ subunits but not G␣ s (29,30). In Fig. 3, infusion of 100 pM RGS4 through a patch pipette into pancreatic acinar (Fig.  3a) or SMG duct (Fig. 3b) cells completely inhibited the Ca 2ϩ response to VIP and Iso, respectively. The control shows that the response to subsequent stimulation with carbachol was markedly reduced, as we reported recently (23). Measurement of cAMP production in streptolysin O-permeabilized cells showed that inhibition of Ca 2ϩ signaling by RGS4 was not due to inhibition of cAMP production by the G s -coupled receptors (not shown). The results with RGS4 exclude model b of Fig. 1 as the mechanism by which R s evokes a Ca 2ϩ signal.
In the next set of experiments we systematically tested the model for PKA-dependent G s /G i switching (or augmentation) of receptor specificity shown in Fig. 1c (17, 18). We first tested if stimulation of G s is obligatory for launching a Ca 2ϩ signal by the VIP and Iso receptors. This was achieved by introducing antibodies specific for G␣ s into the cells through a patch pipette. Antibodies to the carboxyl terminus of G␣ s were used because they have been reported to block receptor-mediated activation of adenylyl cyclase (31). Fig. 4 shows that the antibodies specific for G␣ s inhibited Ca 2ϩ oscillations induced by VIP stimulation of pancreatic acinar cells and the Ca 2ϩ signal stimulated by Iso acting on SMG duct cells without affecting the oscillations or the biphasic response evoked by stimulation of the G q -coupled m3 receptor with carbachol. Similar findings were observed in 4 additional acinar and 3 additional duct cells. As discussed below, infusion of G␣ q specific antibodies did not effect VIP-or Iso-evoked Ca 2ϩ signaling. Therefore, G s stimulation was essential for launching a Ca 2ϩ signal by the two classical G s -coupled receptors.
If PKA-dependent phosphorylation were involved, then inhibition of PKA activity should block G s -but not G q -dependent signaling (Fig. 1c). The R s in both cell types met this criterion as shown in Fig. 5. In control experiments, Ca 2ϩ oscillations were initiated by stimulation of pancreatic acinar cells with VIP. After termination of VIP stimulation by removing the agonist, very similar oscillations were initiated by stimulating the same cells with low concentrations of carbachol, which acts through the G q -coupled muscarinic receptor. Finally, the cell was stimulated with a supermaximal concentration of carbachol (Fig. 5). Similar results were obtained in 14 cells. In four separate experiments, the VIP response was completely abolished when pancreatic acinar cells were treated with 10 M H89, a selective and potent inhibitor of PKA (32), whereas the ability of a low concentration of carbachol to induce oscillations or of a supermaximal concentration to induce a biphasic response was unaltered (Fig. 5b). Similarly, treatment of SMG duct cells with 10 M H89 abolished Iso-dependent [Ca 2ϩ ] i increase, without affecting the carbachol-dependent response (Fig. 5d). Inhibition of the response to Iso was observed in all 6 SMG duct cells treated with H89. The requirement for PKA stimulation was further verified by testing the effect of the potent and selective inhibitor of PKA, Rp-8-CPT-cAMP-S. Infusing the cells with 10 M Rp-8-CPT-cAMP-S through the pipette abolished the response to VIP (n ϭ 7) and Iso (n ϭ 5) in all cells tested (Fig. 5, c and e). Again, control experiments in the same cells showed that all forms of G q -dependent responses were unaffected by inhibition of PKA with Rp-8-CPT-cAMP-S. These inhibitory effects of the two PKA inhibitors argue against the possibilities that unregulated VIP or ␤-adrenergic receptors are coupled directly to G i (33) or that G␣ s directly modulates Ca 2ϩ channels (34) in these systems.
To directly address a role for G i in Ca 2ϩ signaling by VIP and Iso we measured the effect of infusing the cells with PTX or antibodies specific for certain members of the G i subclass of ␣ subunits. Preliminary studies showed that concentrations of PTX below 20 ng/ml in the pipette solution did not consistently inhibit VIP-induced signaling. At concentrations above 50 ng/ ml, PTX rapidly caused a large, time-dependent, nonselective increase in membrane conductance, as if PTX caused cell permeabilization. We therefore limited our testing to the effect of 20 ng/ml PTX on Ca 2ϩ signaling in pancreatic acinar cells. Fig.  6 shows that treatment with PTX inhibited VIP but not carbacholdependent Ca 2ϩ signaling. Similar results were obtained in four experiments. In 13 additional experiments, PTX-treated acinar cells lysed before the experimental protocol could be completed. We were unable to find a concentration of PTX that inhibited the Iso response in SMG duct cell without causing cell lysis.
Antibodies generated against peptides representing the carboxyl termini of G␣ i and G␣ q subunits inhibit receptor-initiated activation of these G proteins (20,21,35). The results obtained by infusing antibodies into pancreatic acinar cells are illustrated in Fig. 7. Two types of polyclonal antibodies against G i were used, one recognizing G␣ i3 and G␣ o or one specific for G␣ i1 and G␣ i2 (19). Fig. 7a shows that infusing 17.5 g/ml antibodies specific for G␣ i3 and G␣ o had no effect on Ca 2ϩ signaling induced by G s -or G q -coupled receptors. Similar results were obtained in four cells. However, these antibodies were not without effect, as seen for SMG cells (described below). Fig. 7b shows that infusing pancreatic acinar cells with 9 g/ml G␣ i1,i2 -  H89 (b and d). After about 10 min incubation with H89, pancreatic acinar cells were stimulated with 10 nM VIP, then with the submaximal concentration of 0.5 M carbachol (Car) to induce G q -dependent oscillations, and finally with the supermaximal concentration of 1 mM carbachol to induce a biphasic response. SMG duct cells were stimulated with 10 M Iso and then 1 mM carbachol. The number of observations under each condition is given in the text. specific antibodies completely inhibited the response to VIP without affecting the response to carbachol. Similar results were observed in six cells. An important control is shown in Fig.  7c. In contrast to the effect of G i -specific antibodies, infusing the cells with G␣ q,11 antibodies (at sufficient concentration to abolish the oscillation and largely inhibit the sustained response to carbachol) had no effect on the ability of VIP to induce oscillations. In seven experiments with cells infused with 80 g/ml anti-G␣ q IgG the response to VIP remained normal, while the response to the low concentration of carbachol was abolished and the response to supermaximal concentration of carbachol was inhibited by 83 Ϯ 7%. Fig. 8. In six cells infused with G␣ q,11 reactive IgG, the response to supermaximal concentrations of carbachol was reduced by 91 Ϯ 6% while the response to Iso was not affected (Fig. 8b). Unlike the findings in pancreatic acinar cells stimulated with VIP, both G i antibody preparations effectively inhibited the response to Iso in SMG duct cells. G␣ i1,i2 -specific antibodies, at a concentration of 9 g/ml, completely inhibited the Ca 2ϩ response to Iso (Fig. 8c). Infusion of only 3.5 g/ml G␣ i3,o antibodies completely inhibited the response to Iso in two cells and partially (63 Ϯ 14%) in three cells (Fig. 8d). At a concentration of 7 g/ml the anti-G␣ i3,o completely inhibited the response to Iso in five cells (Fig. 8e).

Activation of G i by Iso is further suggested by the results for SMG duct cells shown in
The findings in Figs. 7 and 8 provide strong evidence that activation of Ca 2ϩ signaling by G s -coupled receptors is independent of members of the G q class. The inhibitory G q antibodies used in the present work recognizes the predominant G q class ␣ subunits expressed in these cells, G␣ q , G␣ 11 , and G␣ 14 (22). Furthermore, these antibodies were shown to inhibit Ca 2ϩ signaling evoked by several G q -coupled receptors in pancreatic (21) and other cell types (36,37). At a concentration inhibiting the oscillatory and the biphasic response to cholinergic stimulation, the antibodies had no apparent effect on the response to either VIP or Iso. This data supports the conclusion that inhibition of VIP-and Iso-induced Ca 2ϩ signaling by RGS4 was due to acceleration of GTPase activity of a G i class ␣ subunit(s).
The use of PTX and G␣ i antibodies indicates that receptormediated activation of G i was required for activation of Ca 2ϩ signaling by VIP or Iso. It is notable that both G i antibody preparations inhibited Iso-stimulated Ca 2ϩ signaling in SMG duct cells whereas only the G␣ i1,i2 -specific preparation was effective for inhibiting VIP-stimulated signaling in the pancreatic acinar cells. This minor difference between the two systems may be attributed to cell type-specific expression patterns of G␣ i isoforms or the degree of G␣ i selectivity exhibited by putative PKA-phosphorylated VIP and ␤-adrenergic receptors. It is puzzling that the ␤-adrenergic Ca 2ϩ response is inhibited completely by either G i antibody preparation. If the ␤-adrenergic receptor couples to all members of the G i class, then each antibody preparation would be expected to only partially inhibit and a mixture of the antibodies to completely inhibit signaling by these receptors. The complete inhibition of signaling by either antibody preparation suggests that partial inhibition of IP 3 production by stimulation of the ␤-adrenergic receptor had reduced IP 3 below a threshold level needed to trigger Ca 2ϩ release. This interpretation is supported by previous work showing that Iso released Ca 2ϩ from the IP 3 mobilizable Ca 2ϩ pool (9) without causing a detectable increase in global IP 3 concentration (8).
In the G s /G i switching model, activated receptor, phosphorylated by PKA, couples to G i (18). This predicts that G␤␥ released from G i could activate PLC␤. Thus, inhibition of G␤␥ or PLC␤ activity is expected to inhibit the effect of the G scoupled receptors on [Ca 2ϩ ] i . To test these predictions, we measured the effect of the G␤␥ scavenging protein ␤ARK1 (21,38) and of the inhibitory PIP 2 antibody (39, 40) on VIP-dependent Ca 2ϩ signaling. Fig. 9a shows that infusing 5 M ␤ARK1 into pancreatic acinar cells completely inhibited the response to VIP. As we (21) and others (37) reported earlier, ␤ARK1 also inhibited the response to stimulation of the G q -coupled muscarinic receptor. Inhibition by ␤ARK1 was upstream of the Ca 2ϩ increase because elevation of [Ca 2ϩ ] i with A23187 strongly activated the Cl Ϫ current. Results similar to those in Fig. 9a, including the positive control with A23187, were obtained in five experiments. Fig. 9b shows that cytoplasmic PIP 2 antibodies completely inhibited the response to VIP and reduced the response to carbachol by 88 Ϯ 11% (n ϭ 7). These experiments indicate that both VIP and carbachol stimulate PLC␤ to cause the hydrolysis of PIP 2 .
In summary, our examination of the [Ca 2ϩ ] i increase triggered by G s -coupled receptors supports a model for switching or augmentation of receptor coupling to extend to G i in native cells freshly isolated from tissue. We conclude that the pathway involves activation of G s and PKA, receptor stimulation of G i , and activation of PLC␤ by G␤␥ (derived from G i ). We acknowledge that the PKA substrate(s) responsible for activation of G i are not known but, as suggested by the switching model (17,18), they could be the same receptors that were initially coupled only to G s . We use caution, however, in referring to the Ca 2ϩ pathway (Fig. 1c), as a receptor switching model. PKAdependent phosphorylation of the VIP or ␤-adrenergic receptors could allow G i to replace G s but the data are also consistent with broadening of receptor coupling to G s plus G i . One mode for augmentation of receptor coupling can be envisioned if it is assumed that most ␤-adrenergic or VIP receptors are productively coupled to G s but a smaller subpopulation are poised to couple to G i . Effective G i coupling would occur only when the receptors are phosphorylated by PKA. Because expression of a mutant (phosphorylation negative) ␤-adrenergic receptor prevented PKA-dependent activation of G i in HEK 293 cells (17), it is unlikely that phosphorylation of proteins downstream of the VIP or ␤-adrenergic receptors are responsible for activation of G i in pancreatic acinar or submandibular gland cells. An alternative to the assumption that mutant receptor is unable to couple to G i (17) is that the mutant cannot regulate its interaction with an RGS protein that may ordinarily suppress G i activation stimulated by the ␤-adrenergic receptor. A role for regulation of RGS protein function by receptor phosphorylation is attractive, not only because RGS proteins exhibit selectivity among receptor signaling complexes (23,41,42), but also because phosphorylation is not necessary for purified ␤-adrenergic receptors to activate G i in vitro (33). Additional experimental tools are needed to distinguish between these and other potential mechanisms. Independent of the mode of coupling it is clear that in pancreatic acinar and submandibular cells G s -coupled receptors activate Ca 2ϩ signaling by coupling to G i and this coupling requires activation of G s .
An equally important conclusion is that VIP and ␤-adrenergic receptor regulation of Ca 2ϩ release is completely independent of G q class proteins. The observation that PKA-dependent switching/augmentation in receptor/G protein coupling occurs in two different native cell types via two different receptors (that generate different types of Ca 2ϩ signals) suggests a generalization of the mechanism by which G s -coupled receptors generate a second signal to activate a distinct signaling cascade.