Glucagon-mediated Ca2+ signaling in BHK cells expressing cloned human glucagon receptors

Lars H. Hansen1, Jesper Gromada2, Pierre Bouchelouche3, Ted Whitmore4, Laura Jelinek4, Wayne Kindsvogel4, and Erica Nishimura1

1 Department of Molecular Signaling, Hagedorn Research Institute, DK-2820 Gentofte; 2 Islet Cell Physiology, Novo Nordisk A/S, DK-2100 Copenhagen; 3 Department of Clinical Biochemistry, Roskilde County Hospital, DK-4600 Køge, Denmark; and 4 ZymoGenetics Inc., Seattle, Washington 98102

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

From video imaging of fura 2-loaded baby hamster kidney (BHK) cells stably expressing the cloned human glucagon receptor, we found the Ca2+ response to glucagon to be specific, dose dependent, synchronous, sensitive to pertussis toxin, and independent of Ca2+ influx. Forskolin did not elicit a Ca2+ response, but treatment with a protein kinase A inhibitor, the Rp diastereomer of 8-bromoadenosine-3',5'-cyclic monophosphothioate, resulted in a reduced glucagon-mediated Ca2+ response as well as Ca2+ oscillations. The specific phospholipase C inhibitor U-73122 abolished the Ca2+ response to glucagon, and a modest twofold increase in inositol trisphosphate (IP3) production could be observed after stimulation with glucagon. In BHK cells coexpressing glucagon and muscarinic (M1) acetylcholine receptors, carbachol blocked the rise in intracellular free Ca2+ concentrations in response to glucagon, whereas glucagon did not affect the carbachol-induced increase in Ca2+. Furthermore, carbachol, but not glucagon, could block thapsigargin-activated increases in intracellular free Ca2+ concentration. These results indicate that, in BHK cells, glucagon receptors can activate not only adenylate cyclase but also a second independent G protein-coupled pathway that leads to the stimulation of phospholipase C and the release of Ca2+ from IP3-sensitive intracellular Ca2+ stores. Finally, we provide evidence to suggest that cAMP potentiates the IP3-mediated effects on intracellular Ca2+ handling.

phospholipase C; adenosine 3',5'-cyclic monophosphate; G proteins; inositol trisphosphate; intracellular calcium stores; baby hamster kidney cells

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

GLUCAGON, A PANCREATIC HORMONE that plays an important role in maintaining glucose homeostasis, is secreted from the alpha -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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

The [Ca2+]i measurements were performed essentially as outlined previously (4, 9, 26). Briefly, the cells were illuminated with alternating wavelengths (340 and 380 nm) by using interference filters mounted on a filter wheel and observed through a 510-nm emission filter. The images were captured in real time with a low-light-level intensified charge-coupled devise videocamera system. The system was calibrated for free Ca2+ concentrations by use of the formula
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB> ⋅ &bgr; ⋅ (R − R<SUB>340/380 min</SUB>)/(R<SUB>340/380 max</SUB> − R)
where Kd is the dissociation constant of the dye (225 nM for fura 2), beta  is the proportionality coefficient (4), and R is the wavelength ratio. Calibrations of the fura 2 fluorescence signal were performed by dialyzing single cells with 100 µM fura 2 pentapotassium salt and Ca2+-EGTA buffers with free Ca2+ concentrations ranging from 0 to 39.8 µM. Thus R340/380 max was determined under saturating Ca2+ concentrations, whereas R340/380 min was measured for the same field under Ca2+-free conditions. The ratio images (R340/R380) were obtained by dividing the 340-nm images by the 380-nm image on a pixel-by-pixel basis, whereas the numeric R340/R380 values were calculated after the pixel gray values obtained for each wavelength were averaged. Ratio values were then converted to Ca2+ concentrations by use of the calibration curve, and the background (field without cells) was subtracted.

For the experiments shown in Table 1 and Fig. 1, the recordings were made using the Tardis imaging software (Applied Imaging, Tyne & Wear, UK). For the toxin sensitivity studies, the cells were pretreated with 10 µg/ml pertussis or cholera toxin for 24 h at 37°C in the culture medium. The recordings in Figs. 3, 4, and 6-8 were made using a fluorescence imaging system (IonOptix, Milton, MA).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Toxin sensitivity and specificity of glucagon-induced increase in [Ca2+]i in BHK cells stably transfected with human glucagon receptor cDNA


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.

Amersham's D-myo-inositol 1,4,5-trisphosphate (IP3) 3H assay system was used to measure IP3 in the BHK cell extracts according to the manufacturer's instructions. For each cell extract, duplicate determinations of a 1:10 dilution were assayed. The samples were counted for 4 min in a beta scintillation counter, and the amount of IP3 in each sample was determined by interpolation from the standard curve.

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-(7---36) 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-(7---36) 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 (Gs 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.

Further characterization of the Ca2+ response to glucagon was carried out in BHK cells coexpressing glucagon and M1 receptors. As expected, repeated stimulation of these cells with the same agonist, glucagon (Fig. 6A) or carbachol (Fig. 6B), results in desensitization of the Ca2+ response. Interestingly, we found that although prior stimulation with carbachol could prevent the Ca2+ response to a subsequent glucagon treatment (Fig. 6D), glucagon pretreatment was not able to prevent the rise in [Ca2+]i elicited by a subsequent carbachol stimulation (Fig. 6C). These results indicate that glucagon releases Ca2+ from the IP3-sensitive intracellular stores but that, unlike carbachol, glucagon is not capable of emptying these stores completely. This is further substantiated by our finding that, after depletion of thapsigargin-sensitive intracellular Ca2+ stores, glucagon is unable to induce a rise in [Ca2+]i (data not shown). In addition, unlike carbachol, which can deplete thapsigargin-sensitive intracellular Ca2+ stores (Fig. 6F), glucagon only partially empties the thapsigargin-releasable intracellular Ca2+ stores, since treatment with thapsigargin immediately after glucagon stimulation can still elicit a rise in [Ca2+]i (Fig. 6E).


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.

To investigate whether cAMP may be involved in the glucagon-induced rise in [Ca2+]i, we examined the effects of forskolin, which stimulates the accumulation of intracellular cAMP by directly activating adenylate cyclase. As can be seen in Fig. 7A, forskolin was not in itself able to elicit a Ca2+ response in these cells, whereas the subsequent addition of glucagon was still able to stimulate a rise in [Ca2+]i. Consistent with these results, the activator of protein kinase A (PKA), Sp-8-BrcAMPS, was also unable to stimulate an increase in [Ca2+]i (Fig. 7C). In addition, we examined the effects of Rp-8-BrcAMPS, a cAMP analog that specifically inhibits PKA (8), the effectiveness of which has been demonstrated in BHK cells by its ability to inhibit the translocation of the catalytic subunit of PKA, as determined by video-imaging techniques (O. Thastrup, personal communication). Here we found that Rp-8-BrcAMPS inhibits the Ca2+ response to glucagon, as can be seen by the diminished [Ca2+]i transients (Fig. 7B). Furthermore, in some of the Rp-8-BrcAMPS-treated cells, glucagon-evoked Ca2+ oscillations could be observed; the amplitude of the repetitive spikes appeared to be somewhat lower than that of the initial response (Fig. 7B). These data suggest that the rise in [Ca2+]i after glucagon stimulation is most likely not mediated by cAMP itself but that it may in some way modulate or potentiate the Ca2+ response. Further support of this suggestion is the finding that, together with forskolin, the IP3 receptor-sensitizing agent thimerosal is able to elicit a Ca2+ response (Fig. 8A), whereas on its own, thimerosal does not affect the [Ca2+]i (data not shown). A Ca2+ response of normal magnitude is observed after stimulation with glucagon, which is then followed by a number of Ca2+ oscillations (Fig. 8A). To further test this hypothesis that cAMP potentiates the Ca2+ response to IP3, we examined the effect of cAMP on the carbachol-stimulated rise in [Ca2+]i in BHK cells expressing M1 receptors. We found that subthreshold concentrations of carbachol could induce a Ca2+ response in the presence of forskolin (Fig. 8, B and C). Finally, in BHK cells transfected with a mutant (Gly40Ser) glucagon receptor that had previously been identified in some diabetic patients (10), we observed a reduction in the Ca2+ response to glucagon (Fig. 7D), and, interestingly in some of these cells, glucagon-induced Ca2+ oscillations occurred that were similar to those seen in cells treated with Rp-8-BrcAMPS or thimerosal (Figs. 7B and 8A, respectively).


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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -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 beta -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 (Gq) (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 beta gamma -subunit of the pertussis toxin-sensitive Gi is able to activate the beta 2-isoform of phospholipase C (17), and since it has been recently shown that the luteinizing hormone receptor is able to couple to Galpha s and Galpha i to activate adenylate cyclase and phospholipase C, with the beta gamma -subunits released from either G protein contributing to the stimulation of phospholipase C beta -isoforms (13), it is possible that this mechanism may also be involved in the glucagon-mediated Ca2+ response.

Our results also indicate that Gs- and Gi-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: Gs and possibly Gi. 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 beta -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.

    ACKNOWLEDGEMENTS

The technical assistance of Ane Bøjet Baunsgaard is gratefully acknowledged. We thank Dr. Nils Billestrup for insightful comments regarding the manuscript.

    FOOTNOTES

This work was supported in part by the Danish Research Academy. The Hagedorn Research Institute is an independent research unit of Novo Nordisk.

Address for reprint requests: E. Nishimura, Dept. of Molecular Signaling, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark.

Received 19 March 1997; accepted in final form 9 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Abou-Samra, A.-B., H. Jyppner, T. Force, M. W. Freeman, X.-F. Kong, E. Schipani, P. Urena, J. Richards, J. V. Bonventre, J. T. J. Potts, H. M. Kronenberg, and G. V. Segre. Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free calcium. Proc. Natl. Acad. Sci. USA 89: 2732-2736, 1992[Abstract].

2.   Ashkenazi, A., E. Peralta, J. Winslow, J. Ramachandran, and D. Capon. Functionally distinct G proteins selectively couple different receptors to PI hydrolysis in the same cell. Cell 56: 487-493, 1989[Medline].

3.   Benedetti, A., P. Graf, R. Fulceri, A. Romani, and H. Sies. Ca2+ mobilization by vasopressin and glucagon in perfused livers. Biochem. Pharmacol. 38: 1799-1805, 1989[Medline].

4.   Bouchelouche, P. N. Dynamic, real time imaging of ion activities in single living cells using fluorescence video microscopy and image analysis. Scand. J. Clin. Lab. Invest. 53: 27-39, 1993.

5.   Burcelin, R., E. B. Katz, and M. J. Charron. Molecular and cellular aspects of the glucagon receptor: role in diabetes and metabolism. Diabetes Metab. 22: 373-396, 1996[Medline].

6.   Bygrave, F. L., A. Gamberucci, R. Fulceri, and A. Benedetti. Evidence that stimulation of plasma-membrane Ca2+ inflow is an early action of glucagon and dibutyryl cyclic AMP in rat hepatocytes. Biochem. J. 292: 19-22, 1993[Medline].

7.   Chabre, O., B. Conklin, H. Lin, H. Lodish, E. Wilson, H. Ives, L. Catanzariti, B. Hemmings, and H. Bourne. A recombinant calcitonin receptor independently stimulates adenosine 3',5'-cyclic monophosphate and Ca2+/inositol phosphate signaling pathways. Mol. Endocrinol. 6: 551-556, 1992[Abstract].

8.   Connelly, P. A., L. H. Botelho, R. B. Sisk, and J. C. Garrison. A study of the mechanism of glucagon-induced protein phosphorylation in isolated rat hepatocytes using (Sp)-cAMPS and (Rp)-cAMPS, the stimulatory and inhibitory diastereomers of adenosine cyclic 3',5'-phosphorothioate. J. Biol. Chem. 262: 4324-4332, 1987[Abstract/Free Full Text].

9.   Gromada, J., S. Dissing, K. Bokvist, E. Renström, J. Frøkjaer-Jensen, B. S. Wulf, and P. Rorsman. Glucagon-like peptide I increases cytoplasmic calcium in insulin-secreting beta TC3 cells by enhancement of intracellular calcium mobilization. Diabetes 44: 767-774, 1995[Abstract].

10.   Hager, J., L. Hansen, C. Vaisse, N. Vionnet, A. Philippi, W. Poller, G. Velho, C. Carcassi, L. Contu, C. Julier, F. Cambien, P. Passa, M. Lathrop, W. Kindsvogel, F. Demenais, E. Nishimura, and P. Froguel. A missense mutation in the glucagon receptor gene is associated with non-insulin-dependent diabetes mellitus. Nat. Genet. 9: 299-304, 1995[Medline].

11.   Hansen, L. H., N. Abrahamsen, J. Hager, L. Jelinek, W. Kindsvogel, P. Froguel, and E. Nishimura. The Gly40Ser mutation in the human glucagon receptor gene associated with NIDDM results in a receptor with reduced sensitivity to glucagon. Diabetes 45: 725-730, 1996[Abstract].

12.   Hansen, L. H., N. Abrahamsen, and E. Nishimura. Glucagon receptor mRNA distribution in rat tissues. Peptides 16: 1163-1166, 1995[Medline].

13.   Herrlich, A., B. Kyhn, R. Grosse, A. Schmid, G. Schultz, and T. Gudermann. Involvement of Gs and Gi proteins in dual coupling of the luteinizing hormone receptor to adenylyl cyclase and phospholipase C. J. Biol. Chem. 271: 16764-16772, 1996[Abstract/Free Full Text].

14.   Hofer, A. M., W.-R. Schlue, S. Curci, and T. E. Machen. Spatial distribution and quantitation of free luminal [Ca] within the InsP3-sensitive internal store of individual BHK-21 cells: ion dependence of InsP3-induced Ca release and reloading. FASEB J. 9: 788-798, 1995[Abstract/Free Full Text].

15.   Holz, G. G., IV, C. A. Leech, and J. F. Habener. Activation of a cAMP-regulated Ca2+-signaling pathway in pancreatic beta -cells by the insulinotropic hormone glucagon-like peptide-1. J. Biol. Chem. 270: 17749-17757, 1995[Abstract/Free Full Text].

16.   Jelinek, L. J., S. Lok, G. B. Rosenberg, R. A. Smith, F. J. Grant, S. Biggs, P. A. Bensch, J. L. Kuijper, P. O. Sheppard, C. A. Sprecher, P. J. O'Hara, D. Foster, K. M. Walker, L. H. J. Chen, P. A. McKernan, and W. Kindsvogel. Expression cloning and signaling properties of the rat glucagon receptor. Science 259: 1614-1616, 1993[Medline].

17.   Katz, A., D. Wu, and M. I. Simon. Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C. Nature 360: 686-689, 1992[Medline].

18.   Laburthe, M., A. Couvineau, P. Gaudin, J.-J. Maoret, C. Rouyer-Fessard, and P. Nicole. Receptors for VIP, PACAP, secretin, GRF, glucagon, GLP-1, and other members of their new family of G protein-linked receptors: structure-function relationship with special reference to the human VIP-1 receptor. In: VIP, PACAP, and Related Peptides: Second International Symposium, edited by A. Arimura, and S. I. Said. New York: NY Acad. Sci., 1996, p. 94-109 vol. 805)

19.   Lok, S., J. L. Kuijper, L. J. Jelinek, J. M. Kramer, T. E. Whitmore, C. A. Sprecher, S. Mathewes, F. J. Grant, S. H. Biggs, G. B. Rosenberg, P. O. Sheppard, P. J. O'Hara, D. C. Foster, and W. Kindsvogel. The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization. Gene 140: 203-209, 1994[Medline].

20.   Maniatis, T., E. Fritsch, and J. Sambrook. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

21.   Mine, T., I. Kojima, and E. Ogata. Sources of calcium mobilized by glucagon in isolated rat hepatocytes. Acta Endocrinol. 119: 301-306, 1988[Medline].

22.   Mine, T., I. Kojima, and E. Ogata. Evidence of cyclic AMP-independent action of glucagon on calcium mobilization in rat hepatocytes. Biochim. Biophys. Acta 970: 166-171, 1988[Medline].

23.   Spengler, D., C. Waeber, C. Pantaloni, F. Holsboer, J. Bockaert, P. H. Seeburg, and L. Journot. Differential signal transduction of five splice variants of the PACAP receptor. Nature 365: 170-175, 1993[Medline].

24.   Staddon, J. M., and R. G. Hansford. 4beta -Phorbol 12-myristate 13-acetate attenuates the glucagon-induced increase in cytoplasmic free Ca2+ concentration in isolated rat hepatocytes. Biochem. J. 238: 737-743, 1986[Medline].

25.   Staddon, J. M., and R. G. Hansford. Evidence indicating that the glucagon-induced increase in cytoplasmic free Ca2+ concentration in hepatocytes is mediated by an increase in cyclic AMP concentration. Eur. J. Biochem. 179: 47-52, 1989[Abstract].

26.   Stroop, S., D. Thompson, R. Kuestner, and E. Moore. A recombinant human calcitonin receptor functions as an extracellular calcium sensor. J. Biol. Chem. 268: 19927-19930, 1993[Abstract/Free Full Text].

27.   Wakelam, M. J. O., G. J. Murphy, V. J. Hruby, and M. D. Houslay. Activation of two signal-transduction systems in hepatocytes by glucagon. Nature 323: 68-71, 1986[Medline].

28.   Wheeler, M., M. Lu, J. Dillon, X.-H. Leng, C. Chen, and A. Boyd III. Functional expression of the rat glucagon-like peptide-1 receptor, evidence for coupling to both adenylyl cyclase and phospholipase-C. Endocrinology 133: 57-61, 1993[Abstract].

29.   Wheeler, M. B., R. W. Gelling, C. H. S. McIntosh, J. Georgiou, J. C. Brown, and R. A. Pederson. Functional expression of the rat pancreatic islet glucose-dependent insulinotropic polypeptide receptor: ligand binding and intracellular signaling properties. Endocrinology 136: 4629-4639, 1995[Abstract].


Am J Physiol Cell Physiol 274(6):C1552-C1562
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society