INTRODUCTION

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GLUCAGON, A PANCREATIC HORMONE that plays an important role in maintaining glucose homeostasis, is secreted from the -cells in response to low blood glucose levels and stimulates hepatic glucose output by increasing glycogenolysis and gluconeogenesis while at the same time inhibiting glycolysis (5). In addition to its metabolic actions in the liver, glucagon is also involved in the regulation of adipose, cardiac, renal, gastrointestinal, and pancreatic functions, including the potentiation of glucose-induced insulin secretion (5). These effects are mediated by specific glucagon receptors, which have been shown to have a wide tissue distribution corresponding to the observed multiple functions of glucagon (12).

The glucagon receptor belongs to the superfamily of seven transmembrane-spanning receptors that couple to heterotrimeric guanine nucleotide-binding proteins (G proteins). Furthermore, on the basis of structural homology, glucagon receptors, together with those for glucagon-like peptide 1 (GLP-1), gastric inhibitory polypeptide (GIP), secretin, vasoactive intestinal polypeptide (VIP), growth hormone-releasing factor, corticotropin-releasing factor, pituitary adenylate cyclase-activating polypeptide, parathyroid hormone, and calcitonin (5, 18), form a subfamily of closely related receptors that is now emerging as a group of G protein-coupled receptors able to activate multiple signaling pathways. All these receptors are able to stimulate adenylate cyclase, but, in addition, many have been found to activate alternative intracellular second messengers. For example, stimulation of the parathyroid hormone receptor leads to intracellular accumulation of cAMP, inositol trisphosphates (IP₃), and Ca²⁺ (1), and splice variants of the pituitary adenylate cyclase-activating polypeptide receptor are able to differentially couple to adenylate cyclase and phospholipase C (23). The cloned receptors for calcitonin (7), GLP-1 (9, 28), GIP (29), and glucagon (16) have been shown to stimulate cAMP production and a rise in intracellular free Ca²⁺ concentration ([Ca²⁺]_i).

It had long been postulated that the cellular effects of glucagon are mediated not only by cAMP but also by [Ca²⁺]_i, since it has been reported that, in perfused rat liver or cultured hepatocytes, glucagon is able to increase the [Ca²⁺]_i by inducing a Ca²⁺ influx as well as by stimulating the release of intracellular Ca²⁺ stores (3, 6, 21). However, the mediator(s) of this rise in [Ca²⁺]_i remains to be determined, particularly since there appears to be some controversy as to whether this Ca²⁺ response to glucagon is a cAMP-mediated effect or a result of a separate signaling pathway. Experiments demonstrating that the effects of glucagon on Ca²⁺ mobilization in rat liver cells can be reproduced by cAMP analogs have led some investigators to conclude that the glucagon-mediated rise in [Ca²⁺]_i is mediated by cAMP (8, 24, 25). In contrast, others have reported cAMP-independent effects of glucagon on intracellular Ca²⁺ mobilization (22, 27). On the basis of their studies demonstrating that TH-glucagon, a biologically active glucagon analog that is unable to activate adenylate cyclase, could stimulate inositol phosphate production in rat hepatocytes, Wakelam et al. (27) had proposed the possible existence of two distinct hepatic glucagon receptors: one that couples to adenylate cyclase and the other to the breakdown of inositol phospholipids. However, we have since demonstrated that the cloned rat hepatic glucagon receptor is capable of mediating both a cAMP and a Ca²⁺ response (16).

In the current study we have used baby hamster kidney (BHK) cells expressing the cloned human glucagon receptor (19) to further characterize our original observation that the glucagon receptor is able to stimulate an increase in [Ca²⁺]_i. By video imaging of fura 2-loaded cells, we have monitored the Ca²⁺ response to glucagon and have addressed the questions as to the source of the glucagon-mediated rise in [Ca²⁺]_i and the possible molecular mechanisms involved. In view of glucagon's central role in regulating glucose metabolism and our previous findings that a mutated glucagon receptor may in some manner contribute to the development of non-insulin-dependent diabetes (10, 11), the characterization of the molecular signaling pathways activated by glucagon is not only important for understanding the normal physiological mechanisms involved in glucagon action in target tissues such as the liver, fat, or pancreas, but it may also lead to the identification of defects in glucagon signal transduction that may occur in diabetes.

	MATERIALS AND METHODS

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Cell culture and transfections. BHK cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 10 µg/ml streptomycin, and 2 mM L-glutamine at 37°C in 5% CO₂-95% air. The transfection of BHK cells with the human glucagon receptor cDNA pLJ6 (19) was carried out using the Liptofectamine reagent, as previously outlined in detail (11). The clones analyzed in the present study have been characterized previously for the level of glucagon receptor expression (~3 × 10⁶ receptors/cell) and for their ability to bind ¹²⁵I-labeled glucagon (dissociation constant ~10 nM) as well as stimulate cAMP production in response to glucagon (EC₅₀ ~2 pM) (11). For the BHK cells coexpressing the glucagon and muscarinic (M₁) receptors, the glucagon receptor-expressing BHK cells were transfected with the M₁ cholinergic receptor plasmid pM1-R by use of the calcium-phosphate method (20).

[Ca²⁺]_i measurements. BHK cells (~3 × 10⁴), seeded onto thin, circular coverslips (22 mm) that had been precoated with poly-D-lysine (10 µg/ml), were cultured for 3 days. Before Ca²⁺ measurements, the cells were loaded with 7 µM fura 2-AM in the presence of the nonionic detergent Pluronic F-127 (25%) at 37°C for 30 min, and then an equal volume of HEPES-buffered RPMI culture medium was added. The cells were then incubated for a further 30 min to allow complete deesterification of fura 2. Subsequently, the cells were washed three times in NaCl-HEPES buffer (in mM: 145 NaCl, 5 KCl, 1 Na₂HPO₄, 0.5 Mg₂SO₄, 20 HEPES, 5 glucose, 1 CaCl₂, pH 7.5). Finally, the coverslips were mounted in a recording chamber and placed on an inverted microscope (model 135 TU, Zeis axioscope, Oberkochen, Germany). The recording chamber was continuously perfused with NaCl-HEPES buffer supplemented with 1% BSA at 1.5-2 ml/min. All test compounds were applied through the perfusate to give the concentrations indicated. For experiments that required Ca²⁺-free medium, the CaCl₂ in the NaCl-HEPES buffer was replaced with 1 mM EGTA.

View larger version (79K): [in this window] [in a new window]   Fig. 1.   Glucagon-induced mobilization of Ca2+ in baby hamster kidney (BHK) cells expressing cloned human glucagon receptors. Each panel represents pseudocolor ratio images of fura 2-labeled cells obtained at time of peak response to concentration of glucagon (in nM) indicated at bottom left. Colors represent range of free Ca2+ concentrations according to scale. Original magnification, ×300.

IP₃ determinations. BHK cells transfected with the human glucagon receptor or the human M₁ acetylcholine receptor were seeded (5 × 10⁴ cells) into 60-mm tissue culture dishes and allowed to grow for 3-4 days to ~90% confluency. For an estimate of cell number, the cells from two dishes were counted for each cell line. The cells were then incubated for 1 h in DMEM culture medium containing 10 mM myo-inositol and for an additional 30 min in the presence of 10 mM myo-inositol and 10 mM LiCl₂ in DMEM. Cells were subsequently stimulated for 30 s with various concentrations of glucagon or carbachol prepared in DMEM containing 1% BSA. The reactions were terminated by the addition of 2 ml of ice-cold 15% TCA. The cells were then scraped off and transferred onto ice for 20 min, and then they were centrifuged for 15 min at 250 g at 4°C. The supernatants were then transferred to polypropylene tubes containing 10 µl of 5 mM EDTA. To the remaining pellets 0.5 ml of 15% TCA was added, and the pellets were incubated for an additional 20 min on ice. The resulting supernatant was combined with the first and extracted four times with one volume of diethyl ether (H₂O saturated). The aqueous phase was freeze-dried, resuspended in 2 ml of H₂O, and adjusted to pH 7.5 with 1 M NaHCO₃.

cAMP measurements. Cells were seeded out in six-well plates (3 × 10⁵ cells/well) and cultured overnight. Before stimulation the cells were washed once in Hanks' balanced salt solution and again in RPMI 1640 medium, and finally in 1 ml of RPMI medium supplemented with 0.5% fetal bovine serum and 0.45 mM IBMX was added to each well. The cells were stimulated with glucagon and carbachol, and after 20 min of incubation at 37°C, 1 ml of 65% ethanol was added to each well. The cells were scraped off and transferred to Eppendorf tubes, which were then centrifuged at 300 g for 15 min. The resulting supernatants were dried down overnight in a Speed-Vac and stored at 20°C until they were assayed. The cAMP concentrations were determined using the cAMP ¹²⁵I scintillation proximity assay (Amersham). The dried cell extracts were resuspended in 1 ml of assay buffer and diluted 1:150. For the assay the acetylation protocol described by the manufacturer was followed, and the cAMP determinations were normalized to cell number.

Reagents. All tissue culture flasks and dishes were from Nunc (Roskilde, Denmark). Thin (0.2-mm), circular (22-mm-diameter) glass coverslips were purchased from Struers Kebo Lab (Albertslund, Denmark). Cell culture medium and components, FCS, and Lipofectamine reagent were from GIBCO BRL (Paisley, Scotland). Poly-D-lysine, forskolin, pertussis toxin, cholera toxin, thimerosal, EGTA, LaCl₃, carbachol, and myo-inositol were purchased from Sigma Chemical (St. Louis, MO). Glucagon was obtained from Novo Nordisk (Bagsvaerd, Denmark); GLP-1-(736) amide and VIP were from Peninsula Laboratories (Belmont, CA). The cAMP antagonist, an Rp diastereomer of 8-bromoadenosine-3',5'-cyclic monophosphothioate (Rp-8-BrcAMPS), and the cAMP agonistic Sp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Sp-8-BrcAMPS) were obtained from BIOLOG Life Science Institute (Bremen, Germany). Ryanodine was from Alomone Labs (Jerusalem, Israel). The phospholipase C inhibitor U-73122 and its inactive analog U-73343 were obtained from Calbiochem (Bad Soden, Germany). Fura 2-AM and fura 2 pentapotassium salt were from Molecular Probes (Eugene, OR). The IP₃ ³H assay system and cAMP ¹²⁵I scintillation proximity assay system (dual range) from Amersham (Little Chalfont, England) were used for IP₃ and cAMP measurements.

	RESULTS

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Specific, dose-dependent rise in [Ca²⁺]_i mediated by glucagon. In BHK cells stably expressing the cloned human glucagon receptor, we found that stimulation by glucagon results in a transient, synchronized, and dose-dependent rise in [Ca²⁺]_i (Figs. 1 and 2). The response is rapid and recovers to prestimulus levels within 2-3 min. As can be noted in Fig. 1, it is not only the amplitude of the response to glucagon that increases in a dose-dependent manner but also the number of responding cells rises with increasing concentrations of glucagon, such that at high levels (25 nM) all cells respond. Although the amplitude of the Ca²⁺ response to glucagon varies from cell to cell, it appears that all responding cells do so in a synchronous manner. There is a delay in the response that becomes progressively less when higher concentrations of glucagon are used; there is a 75 ± 3 s lapse from the time 5 nM glucagon is introduced to the time the rise in [Ca²⁺]_i peaks, whereas the delay is only 53 ± 1, 38 ± 1, and 31 ± 2 s for 10, 25, and 50 nM glucagon, respectively (n = 6). The observed effects of glucagon on the increase in [Ca²⁺]_i appear to be specific, mediated by the binding and activation of specific glucagon receptors, since the related hormones, GLP-1-(736) amide and VIP, were not able to elicit a rise in [Ca²⁺]_i in these cells (Table 1). Finally, in nontransfected BHK cells, no Ca²⁺ response to glucagon could be observed.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent rise in intracellular free Ca2+ concentration ([Ca2+]i) in response to glucagon in BHK cells expressing human glucagon receptors. Increase in [Ca2+]i in response to glucagon was determined as difference between peak and baseline Ca2+ values and is represented as means ± SD from data obtained from 24 cells from at least 3 different experiments.

Toxin sensitivity of the glucagon-mediated rise in [Ca²⁺]_i. The glucagon-induced rise in [Ca²⁺]_i was found to be mediated by the activation of a pertussis toxin-sensitive G protein, since pretreatment of the human glucagon receptor expressing BHK cells with pertussis toxin resulted in the near ablation of the Ca²⁺ response to glucagon (Table 1). Under identical conditions, pertussis toxin had no effect on the carbachol-stimulated Ca²⁺ response in BHK cells expressing the M₁ cholinergic receptors. In contrast, a glucagon-mediated rise in [Ca²⁺]_i was still observed in cells pretreated with cholera toxin, although the amplitude of the response was decreased in comparison to that observed in nontreated cells (Table 1).

Source of the glucagon-mediated rise in [Ca²⁺]_i. To determine the source of the observed increase in [Ca²⁺]_i, we examined the Ca²⁺ response to glucagon in the absence of extracellular free Ca²⁺, which was achieved by using Ca²⁺-free medium to which was added a further 1 mM EGTA. Under these conditions, glucagon was still able to elicit a robust rise in [Ca²⁺]_i of the same magnitude as obtained in the presence of extracellular Ca²⁺ (Fig. 3, A and B), suggesting that this transient rise in [Ca²⁺]_i is a result of the release of intracellular Ca²⁺ stores. This is also confirmed by our finding that LaCl₃, a general Ca²⁺ channel blocker, did not affect the Ca²⁺ response to glucagon (Fig. 3C).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Characterization of glucagon-induced rise in [Ca2+]i in BHK cells expressing human glucagon receptors. A: stimulation with 200 nM glucagon over time period covered by horizontal line (control). B: stimulation with 200 nM glucagon in Ca2+-free medium supplemented with 1 mM EGTA. C: stimulation with 200 nM glucagon in presence of 0.6 mM LaCl3. Each trace represents response in a single cell and is representative of at least 30 single-cell recordings from 3 different experiments.

Mechanisms involved in the glucagon-mediated rise in [Ca²⁺]_i. Binding of glucagon to its receptors results in activation of heterotrimeric G proteins (G_s subtype), stimulating the production of cAMP, which ultimately functions as the second messenger mediating many of glucagon's cellular effects. However, because it has also been reported that stimulation of hepatocytes with glucagon leads to activation of phospholipase C and production of IP₃, we have examined the putative role of this second messenger in glucagon receptor signaling. As evident from Fig. 4A, incubation of the cells with the phospholipase C inhibitor U-73122 completely abolished the ability of glucagon to mobilize intracellular Ca²⁺. The inactive isoform of the inhibitor U-73343 was without effect (Fig. 4B), indicating that activation of phospholipase C is indeed involved in the glucagon-induced Ca²⁺ response. Furthermore, incubation of the cells with 50 µM ryanodine had no effect on the Ca²⁺ response to subsequent stimulation with glucagon (Fig. 4C). These findings are consistent with our observation that glucagon can stimulate a twofold increase in IP₃ production (Fig. 5A). This response, however, appears to be relatively modest compared with the 100-fold increase obtained when carbachol is used to stimulate BHK cells expressing M₁ cholinergic receptors, which are known to activate phospholipase C, resulting in the formation of IP₃ and release of IP₃-sensitive intracellular Ca²⁺ stores. Figure 5B also shows that stimulation with carbachol does not lead to any concomitant increase in cAMP production, indicating that signaling by the M₁ receptors, when transfected into BHK cells, is essentially normal. The effectiveness of the phospholipase C inhibitor U-73122 is illustrated in Fig. 5C, where treatment of the cells with this agent (15 µM) decreases the carbachol-mediated IP₃ response by 67.5 ± 7.8% (n = 4) compared with untreated cells. In addition, the specificity of U-73122 can be seen in Fig. 5D, where this inhibitor was found to have no effect on adenylate cyclase activity as determined by the accumulation of cAMP in response to glucagon.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Mechanisms involved in glucagon-stimulated increase in [Ca2+]i. Fura 2-loaded BHK cells expressing human glucagon receptors were stimulated with 200 nM glucagon after incubation for 10 min with 15 µM U-73122 (A), 15 µM U-73343 (B), or 50 µM ryanodine (C) for time periods indicated by horizontal lines. Each trace represents change in [Ca2+]i within a single cell and is representative of at least 20 single-cell recordings from 3 experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Accumulation of D-myo-inositol 1,4,5-trisphosphate (IP3) and cAMP in BHK cells expressing human glucagon receptors (BHK; Glu) or muscarinic (M1) receptors (BHK; M1). A: glucagon- and carbachol-stimulated IP3 production. B: glucagon- and carbachol-stimulated cAMP production. Effect of 10 min of pretreatment with 15 µM U-73122 on carbachol-stimulated accumulation of IP3 (C) or glucagon-stimulated cAMP production (D) is shown. [IP3]i and [cAMP]i, intracellular free IP3 and cAMP concentration, respectively. Values are means ± SD of 4 determinations.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Desensitization of glucagon- and carbachol-mediated rise in [Ca2+]i in BHK cells coexpressing human glucagon receptors and M1 acetylcholine receptors. Cells were stimulated for time intervals indicated by horizontal lines. Each trace represents response in a single cell and is representative of at least 30 single-cell recordings from 3 different experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Role of protein kinase A in glucagon-mediated Ca2+ response. BHK cells expressing wild-type human glucagon receptor were stimulated with 200 nM glucagon after a previous stimulation with 10 µM forskolin (A), 100 µM Rp diastereomer of 8-bromoadenosine 3',5'-cyclic monophosphothioate (Rp-8-BrcAMPS, B), or 100 µM Sp diastereomer of 8-bromoadenosine 3',5'-cyclic monophosphothioate (Sp-8-BrcAMPS, C) for time period indicated by horizontal bar. D: BHK cells expressing mutant (Gly40Ser) glucagon receptors were stimulated with 200 nM glucagon. Each trace represents response in a single cell and is representative of at least 20 single-cell recordings from 3 different experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Modulation of glucagon- and carbachol-evoked Ca2+ responses. A: BHK cells expressing human glucagon receptors were stimulated with 200 nM glucagon after pretreatment with 10 µM forskolin and 100 µM thimerosal. BHK cells expressing human M1 receptors were stimulated with 1 nM and 100 nM carbachol (B) or 1 nM carbachol after previous stimulation with 10 µM forskolin (C). Each trace represents change in [Ca2+]i within a single cell and is representative of at least 20 single-cell recordings from 3 experiments.

	DISCUSSION

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We previously demonstrated that glucagon stimulates a concentration-dependent rise in intracellular cAMP production in BHK cells expressing human glucagon receptors (11, 19). Here we provide evidence to indicate that activation of the cloned human glucagon receptor expressed in BHK cells leads to a rise in [Ca²⁺]_i that does not appear to be due to cAMP-mediated activation of Ca²⁺ influx. This suggests that the glucagon receptor, like many others of the secretin receptor family, can stimulate two independent signaling pathways: one mediated by cAMP and the other by Ca²⁺.

From studies aimed at characterizing the rise in [Ca²⁺]_i stimulated by glucagon, we found it to be independent of extracellular Ca²⁺, since it was not affected by blocking of membrane Ca²⁺ channels or by removal of extracellular Ca²⁺. These findings indicate that the source of the glucagon-stimulated rise in [Ca²⁺]_i is intracellular Ca²⁺ stores. Thus the general features of the glucagon-mediated rise in [Ca²⁺]_i appear to differ from those described in response to activation of GLP-1 and GIP receptors, the two most closely related to the glucagon receptor. In contrast to the Ca²⁺ response to glucagon, the Ca²⁺-mobilizing effects of GLP-1 in pancreatic -cells have been demonstrated to be dependent on extracellular Ca²⁺ and mimicked by stimulation with cAMP. This response has been proposed to involve influx of extracellular Ca²⁺ through voltage-dependent Ca²⁺ channels as well as a release of Ca²⁺ from intracellular stores (15). From further studies on -cells, it has been proposed that the mobilization of Ca²⁺ from intracellular stores in response to GLP-1 reflects Ca²⁺-induced Ca²⁺ release, since it was blocked by ryanodine (9). On the other hand, the rise in [Ca²⁺]_i elicited by GIP through the cloned receptor expressed in COS-7 cells has been shown to be biphasic, where the first transient rise in [Ca²⁺]_i has been attributed to the mobilization of intracellular thapsigargin-sensitive Ca²⁺ stores and the second sustained rise in Ca²⁺ has been found to be a result of Ca²⁺ influx through a cation channel (29). Despite these differences among the Ca²⁺ signaling mediated by the various related receptor types, the characteristics of the Ca²⁺ response that we observe after activation of the cloned human glucagon receptor nonetheless are supported by previous studies in which endogenous hepatic glucagon receptors were found to have Ca²⁺-mobilizing effects that could not be reproduced by cAMP (22, 27). We do recognize, however, that by overexpressing the glucagon receptor in BHK cells we run the risk of "forcing" the coupling to effectors that may not necessarily occur in cells expressing the endogenous receptors. Nevertheless, previous studies using cultured primary hepatocytes have demonstrated that glucagon is able to activate cAMP- and Ca²⁺-mediated signaling pathways, which supports our findings with use of the BHK cells transfected with the cloned glucagon receptor. In addition, signaling by the cloned M₁ receptors in BHK cells overexpressed to the same extent as the glucagon receptors seems to have the same characteristics as signaling by endogenous M₁ receptors, that is, activation of phospholipase C and release of intracellular Ca²⁺ without a concomitant increase in cAMP production (Fig. 5, A and B).

To address the question as to the mechanism of glucagon-induced Ca²⁺ signaling, we have shown that phospholipase C and IP₃ are indeed involved, resulting in the release of intracellular Ca²⁺ stores. This is demonstrated by our observation that the phospholipase C inhibitor U-73122 completely abolishes the rise in [Ca²⁺]_i stimulated by glucagon (Fig. 4A). In addition, we observed a twofold increase in IP₃ in response to glucagon stimulation, although this was small compared with the response observed with carbachol (Fig. 5A). This difference in the magnitude of IP₃ formation provides an explanation for our heterologous desensitization experiments, from which it is apparent that, even at a maximal concentration, glucagon only partially empties the Ca²⁺ stores mobilized by IP₃ after activation of M₁ cholinergic receptors. The fact that stimulation with carbachol could desensitize the Ca²⁺ response to a subsequent stimulation with glucagon whereas the opposite is not the case suggests that glucagon and carbachol mobilize Ca²⁺ from the same IP₃-sensitive intracellular stores but to varying degrees. This is further exemplified by the finding that thapsigargin is still able to release Ca²⁺ stores after stimulation by glucagon but not by carbachol (Fig. 6, E and F). Finally, Hofer et al. (14) found that the intraorganellar sequestration of Ca²⁺ in BHK cells is not affected by ryanodine, which supports our finding that this agent has no effect on glucagon's Ca²⁺ response (Fig. 4C). Although it is thus apparent that phospholipase C-catalyzed IP₃ production is involved in the Ca²⁺ response to M₁ and glucagon receptor activation, the upstream signaling mechanisms leading to this phospholipase C activation are clearly different, since we find that pertussis toxin blocks the glucagon-mediated rise in [Ca²⁺]_i (Table 1). Because M₁ cholinergic receptors couple to phospholipase C by a pertussis toxin-resistant G protein (G_q) (2), the glucagon-induced Ca²⁺ signal must involve a G protein distinct from that used by the M₁ receptors. It has been shown that the -subunit of the pertussis toxin-sensitive G_i is able to activate the ₂-isoform of phospholipase C (17), and since it has been recently shown that the luteinizing hormone receptor is able to couple to G_s and G_i to activate adenylate cyclase and phospholipase C, with the -subunits released from either G protein contributing to the stimulation of phospholipase C -isoforms (13), it is possible that this mechanism may also be involved in the glucagon-mediated Ca²⁺ response.

Our results also indicate that G_s- and G_i-activated pathways are involved in the Ca²⁺ response to glucagon, since Rp-8-BrcAMPS, which acts as an inhibitor of PKA, was found to reduce the glucagon-stimulated increase in [Ca²⁺]_i (Fig. 7B), whereas neither forskolin nor the cAMP agonist Sp-8-BrcAMPS was able to elicit a Ca²⁺ response on its own. Furthermore, the IP₃-sensitizing agent thimerosal, although unable to elicit a Ca²⁺ response itself, leads to an increase in IP₃ if the cells have previously been exposed to forskolin (Fig. 8A). Further substantiation of the possible intereaction between cAMP and IP₃ is given by our finding that a subthreshold concentration of carbachol (1 nM) is also able to evoke a Ca²⁺ response after prior treatment with forskolin (Fig. 8, B and C). This suggests that although cAMP is not responsible for the rise in [Ca²⁺]_i stimulated by glucagon, cAMP can potentiate the Ca²⁺ response. It is interesting to note that the induction of Ca²⁺ oscillations that occurred after thimerosal (Fig. 8A) or Rp-8-BrcAMPS (Fig. 7B) treatment was similar to our observation in BHK cells expressing the Gly40Ser mutant receptor (Fig. 7D), which we have reported to be present in a subset of diabetic patients (10). We previously demonstrated that this mutation results in a decreased sensitivity of the receptor to glucagon in terms of binding and cAMP production (11). However, the mechanisms underlying the induction of this oscillatory response to glucagon under certain conditions and the significance of these oscillations remain to be determined.

Thus, taking into consideration all our findings, we suggest the following model for glucagon-mediated Ca²⁺ signaling in which two distinct signaling pathways are involved. On the basis of sensitivity to pertussis and cholera toxins, it appears that the glucagon receptor can couple to multiple G proteins: G_s and possibly G_i. In addition, the glucagon-stimulated cAMP production appears to play a permissive role in the rise in [Ca²⁺]_i, such that cAMP, although itself unable to elicit a response, in concert with the small incremental release of IP₃ is able to mediate the observed rise in [Ca²⁺]_i in response to glucagon. Previously, we proposed that a similar mechanism underlies the GLP-1-stimulated release of Ca²⁺ from intracellular stores in pancreatic -cells, such that a cAMP-induced phosphorylation of the IP₃ receptor (mediated by PKA) may enhance the Ca²⁺ mobilization in response to the low levels of IP₃ arising from activation of phospholipase C by localized Ca²⁺ influx (9). Therefore, although we did not see an increase in Ca²⁺ in response to cAMP stimulation alone (via forskolin), the cAMP produced in response to activation of glucagon receptors in these cells may potentiate the effects of the rather small increase in IP₃ we observed after glucagon stimulation. This model is supported by our observation that thimerosal or a subthreshold concentration of carbachol is able to induce a Ca²⁺ response in the presence of forskolin. In summary, we have analyzed the general characteristics of the glucagon-mediated rise in [Ca²⁺]_i and have provided evidence to suggest that, contrary to earlier proposals of two glucagon receptor subtypes in the liver (27), the cloned human glucagon receptor is able to couple to multiple G proteins, thereby activating two distinct signaling pathways. It remains a challenge, however, to elucidate the physiological role of these cAMP-Ca²⁺ signaling pathways stimulated by glucagon in its target tissues.