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ENDOCRINE AND DIABETES

Differential Modulation of Ca_v1.2 and Ca_v1.3-Mediated Glucose-Stimulated Insulin Secretion by cAMP in INS-1 Cells: Distinct Roles for Exchange Protein Directly Activated by cAMP 2 (Epac2) and Protein Kinase A

Department of Medicinal Chemistry and Molecular Pharmacology (G.L., N.H., G.H.H.) and Biochemistry and Molecular Biology Graduate Program (S.M.P.J.), Purdue University, West Lafayette, Indiana

Received October 20, 2005; accepted March 23, 2006.

	Abstract

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Using insulin-secreting cell line (INS)-1 cells stably expressing dihydropyridine-insensitive mutants of either Ca_v1.2 or Ca_v1.3, we previously demonstrated that Ca_v1.3 is preferentially coupled to insulin secretion and [Ca²⁺]_i oscillations stimulated by 11.2 mM glucose. Using the same system, we found that insulin secretion in 7.5 mM glucose plus 1 mM 8-bromo-cAMP (8-Br-cAMP) is mediated by both Ca_v1.2 and Ca_v1.3. Treatment of INS-1 cells or INS-1 cells stably expressing Ca_v1.2/dihydropyridine-insensitive (DHPi) channels in the presence of 10 µM nifedipine, with effector-specific cAMP analogs 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP [8-pCPT-2'-O-Me-cAMP; 100 µM; Exchange Protein directly Activated by cAMP 2 (Epac2)-selective] or N⁶-benzoyl-cAMP [50 µM; Protein Kinase A (PKA)-selective] partially increased insulin secretion. Secretion stimulated by a combination of the two cAMP analogs was additive and comparable with that stimulated by 1 mM 8-Br-cAMP. In INS-1 cells stably expressing Ca_v1.3/DHPi in the presence of 10 µM nifedipine, N⁶-benzoyl-cAMP, but not 8-pCPT-2'-O-Me-cAMP, significantly increased glucose-stimulated insulin secretion. However, the combination of N⁶-benzoyl-cAMP and 8-pCPT-2'-O-Me-cAMP significantly increased glucose-stimulated secretion compared with N⁶-benzoyl-cAMP alone. In INS-1 cells, 8-Br-cAMP potentiation of insulin secretion in 7.5 mM glucose is blocked by thapsigargin (1 µM) and ryanodine (0.5 µM). In contrast, ryanodine has no effect on insulin secretion or [Ca²⁺]_i oscillations stimulated by 11.2 mM glucose in INS-1 cells. Our data suggest that both Ca_v1.2 and Ca_v1.3 mediate insulin secretion stimulated by 7.5 mM glucose and cAMP via a mechanism that requires internal stores of Ca²⁺. Furthermore, cAMP modulation of secretion mediated by Ca_v1.2 seems to involve both Epac2 and PKA independently. In contrast, cAMP modulation of Ca_v1.3-mediated secretion depends upon PKA activation, whereas the contribution of Epac2 is dependent upon PKA activation.

The glucose dependence of the insulinotropic action of GLP-1 implies that there is cross-talk between the L-type VDCC-mediated secretion and the GLP-1 signaling pathways (Holz et al., 1992). After post-translational modification, the active form of GLP-1 [GLP-1 (7-36)] binds to the G-protein-coupled GLP-1 receptor in the pancreatic -cell, which activates adenylate cyclase and results in elevated cAMP production (Holz et al., 1995). Although the prosecretory properties of GLP-1 in -cells are believed to be mediated by PKA (Thorens and Waeber, 1993), recent studies have revealed alternative cAMP signal transduction pathways that seem to be PKA-independent. One such pathway involves a newly characterized family of cAMP-binding proteins designated as the cAMP-guanine nucleotide exchange factors (GEFs or Epac) (Kawasaki et al., 1998).

In -cells, the increase in [Ca²⁺]_i in response to glucose and GLP-1 has recently been reported to be mediated by cAMP-GEFII (referred to here as Epac2). This PKA-independent action of GLP-1 is linked to the release of ryanodine-sensitive intracellular Ca²⁺ stores and is facilitated by the interaction of Epac2 with the monomeric G-protein Rab3 (Ozaki et al., 2000; Kashima et al., 2001). Glucose alone can induce an oscillatory or sustained [Ca²⁺]_i increase, and L-type calcium channels expressed in the plasma membrane of -cells play an essential role in these [Ca²⁺]_i changes (Liu et al., 2004). GLP-1-induced cAMP elevation can further enhance [Ca²⁺]_i responses in -cells compared with glucose alone. This alteration in [Ca²⁺]_i response is critical for the potentiation of insulin secretion induced by cAMP (Lu et al., 1993). Single-cell [Ca²⁺]_i imaging studies in rat and human pancreatic -cells indicate that cAMP induces [Ca²⁺]_i oscillations via Ca²⁺-induced Ca²⁺ release (CICR) from intracellular Ca²⁺ stores (Holz et al., 1999). Although the specific receptor(s) mediating intracellular Ca²⁺ release remains uncertain, Ca²⁺ influx through L-VDCC is required for the stimulatory action of both GLP-1 (Yada et al., 1993) and cAMP (Holz et al., 1999) on [Ca²⁺]_i in pancreatic -cells.

Pancreatic -cells of rats and humans express two distinct L-VDCC subtypes, Ca_v1.2 and Ca_v1.3 (Seino et al., 1992; Horvath et al., 1998). Both channel subtypes contain four highly homologous transmembrane domains, with a higher degree of divergence in the intracellular loops linking each domain. The gating properties of these two channels are slightly different, and they are both sensitive to L-VDCC blockers (Bell et al., 2001; Scholze et al., 2001) such as the dihydropyridine nifedipine and the benzothiazepine diltiazem (Hockerman et al., 1997). Because Ca_v1.3 is the dominant L-VDCC isoform expressed in human and rat pancreatic -cells (Seino et al., 1992), it was proposed that Ca_v1.2 and Ca_v1.3 play different roles in insulin secretion. We previously reported that Ca_v1.3 is preferentially coupled to glucose-stimulated insulin secretion (Liu et al., 2003) and glucose-stimulated [Ca²⁺]_i oscillations (Liu et al., 2004). However, the specific role of these two L-VDCC subtypes in the insulinotropic effect of cAMP has not been investigated. Identifying Ca²⁺ influx channel(s) responsible for cAMP-potentiated insulin secretion would further our understanding of the molecular mechanisms involved in the action of cAMP-elevating agents and may provide new insights into the therapeutic usage of GLP-1 to treat type II diabetes.

The present study explored the roles of the L-VDCC subtypes Ca_v1.2 and Ca_v1.3 in cAMP-potentiated insulin secretion at a submaximal glucose concentration in the rat pancreatic -cell line INS-1. We have established a pharmacological knockdown system with stable INS-1 cell lines expressing either of two drug-insensitive mutant channels, Ca_v1.2/DHPi or Ca_v1.3/DHPi. We further probed this system using two cAMP analogs, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-cAMP and N⁶-benzoyl-cAMP to examine the contribution of Epac2 and PKA, respectively (Christensen et al., 2003). In the presence of 7.5 mM glucose, both Ca_v1.2 and Ca_v1.3 were able to mediate cAMP-potentiated insulin secretion in INS-1 cells. This seems to require intracellular Ca²⁺ stores and the combined action of both Epac2 and PKA.

	Materials and Methods

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Materials. 8-(4-Chloro-phenylthio)-2'-O-methyladenosine-cAMP and N⁶-benzoyl-cAMP were from BIOLOG Life Science Institute (Bremen, Germany). 8-Br-cAMP and H89 were from Sigma (St. Louis, MO). Adenosine 3',5'-cyclic monophosphorothioate and Rpisomer (Rp-cAMPS) were from Calbiochem (La Jolla, CA).

Cell Culture. INS-1 cells were cultured in RPMI 1640 medium supplemented with 10 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol as previously reported (Asfari et al., 1992). Cultures were incubated at 37°C in a humidified 95% air/5% CO₂ atmosphere. Characterization of the stable INS-1 cell lines expressing either Ca_v1.2/DHPi or Ca_v1.3/DHPi channels fused to GFP is described elsewhere (Liu et al., 2003). In brief, the presence of the mutant channels in these stable cell lines was confirmed by reverse transcription-polymerase chain reaction with two primer sets and Western blotting with an anti-GFP antibody. Current displaying the characteristic pharmacology of the DHPi channels (i.e., insensitivity to nifedipine and sensitivity to diltiazem; Hockerman et al., 2000) was detected in each cell line using the whole-cell patch-clamp technique. The whole-cell Ba²⁺ current density was not significantly different in the DHPi cell lines and untransfected INS-1 cells.

Insulin Secretion and Cellular Insulin Content. INS-1 cells were cultured in 12-well dishes and allowed to grow until 80 to 90% confluent. Cells were rinsed twice with modified Krebs-Ringer-bicarbonate HEPES (KRBH) buffer (115 mM NaCl, 24 mM NaHCO₃, 5 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, 25 mM HEPES, and 0.5% bovine serum albumin, pH 7.4), and preincubated in 1 ml of KRBH buffer for 30 min at 37°C, 5% CO₂. After aspiration of the buffer, the cells were stimulated in KRBH buffer supplemented with different stimuli (+ or - indicated drugs) for 30 min at 37°C. The buffer containing secreted insulin was collected and centrifuged at 700g for 3 min to remove any detached cells. The cellular insulin was extracted by incubation in 1 ml of acid-ethanol (ethanol/H₂O/HCl, 14:57:3) overnight at 4°C and centrifuged at 700g for 3 min. Supernatants were diluted 10-fold with glycine-NaOH buffer (0.2 M glycine and 0.5% bovine serum albumin, pH 8.8) before insulin secretion assay. Assays were performed using the High Range Rat Insulin ELISA kit (ALPCO, Windham, NH) according to the manufacturer's instructions.

Measurement of [Ca²⁺]_i. INS-1 cells were split into four-well, glass-bottomed chambers (Nalge Nunc International, Naperville, IL) and cultured in complete medium for 48 h before experiments. Cells were washed with KRBH buffer and incubated with 5 µM of the calcium indicator indo-1 AM (Molecular Probes, Eugene, OR) in KRBH buffer for 30 min in the dark. After washing with KRBH buffer, cells were incubated for an additional 30 min in KRBH buffer. Ca²⁺ imaging experiments were carried out at the Purdue University Cytometry Laboratories. Indo-1 AM-loaded cells in glass-bottomed chambers were observed via confocal laser scanning microscopy with a Bio-Rad MRC 1024 (Bio-Rad, Hercules, CA) system based on an inverted Nikon Diaphot 300 microscope (Nikon, Tokyo, Japan). The stage was thermostatically controlled to maintain a temperature of 37°C at the bottom of the chamber. The confocal system was equipped with a 60x PlanApo 1.4 NA oil immersion objective lens and 100-mW argon ion water-cooled laser (Coherent Inc., Santa Clara, CA). Single cells or small clusters of cells, isolated optically by means of a diaphragm, were studied by indo-1 fluorescence. Indo 1 AM-loaded cells were excited at 363 nM, and the emissions at wavelengths of 405 nm (F₄₀₅) and 460 nm (F₄₆₀) were used to calculate the fluorescence ratio (F₄₀₅/F₄₆₀). Cells were excited at a frequency of 1 Hz, and the fluorescence images were collected simultaneously. [Ca²⁺]_i was calculated from F₄₀₅/F₄₆₀ using a standard curve generated with a Ca²⁺ concentration buffer kit with Mg²⁺ (Molecular Probes).

View larger version (14K): [in this window] [in a new window]   Fig. 1. L-type VDCC activity is required for 8-bromo-cAMP-potentiated glucose-stimulated insulin secretion. A, effect of different glucose concentrations on insulin secretion in INS-1 cells. Glucose (11.2 or 18 mM) stimulated significantly higher level of insulin secretion compared with 0 mM glucose (*, p < 0.05, one-way ANOVA). B, 8-Br-cAMP-potentiated insulin secretion in INS-1 cells. Either 7.5 mM glucose (Glu) or 1 mM 8-Br-cAMP (8-Br) did not significantly induce insulin secretion compared with basal level (2 mM glucose). However, in the presence of 7.5 mM glucose, 1 mM 8-Br-cAMP (Glu + 8-Br) significantly potentiated insulin secretion. Basal, 2.1 ± 0.45% (n = 3); Glu, 2.83 ± 0.48% (n = 3); 8-Br, 2.63 ± 0.68% (n = 3); and Glu + 8-Br, 4.87 ± 0.39% (n = 3) (*, p < 0.05 compared with basal level; one-way ANOVA with Tukey post hoc test). C, nifedipine, L-type VDCC blocker, or thapsigargin, ER Ca2+-ATPase inhibitor, completely inhibited 8-Br-cAMP-potentiated insulin secretion. INS-1 cells were preincubated with KRBH buffer or 1 µM thapsigargin for 30 min. Cells were then incubated with KRBH buffer containing indicated reagents for another 30 min before insulin assay. 8-Br-cAMP (1 mM) together with 7.5 mM glucose (glu + 8-Br) significantly potentiated glucose-induced insulin secretion. Nifedipine (10 µM) completely inhibited this effect (glu + 8-Br + Nif). glu, 2.67 ± 0.05% (n = 3); glu + 8-Br, 5.56 ± 0.44% (n = 3); glu + 8-Br + Nif, 2.87 ± 0.71% (n = 3). After thapsigargin pretreatment, 1 mM 8-Br-cAMP can no longer potentiate insulin secretion in INS-1 cells (glu, 3.1 ± 0.02%, n = 3; glu + 8-Br, 2.71 ± 0.14%, n = 3). **, p < 0.01 compared with Glu, one-way ANOVA with Tukey post hoc test.

	Results

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Ca²⁺-Induced ER Ca²⁺ Release Is Required for cAMP-Potentiated Insulin Secretion in Response to a Submaximal Glucose Concentration. Untransfected INS-1 cells secrete insulin in response to glucose in a dose-dependent fashion. The dose-response curve for glucose stimulation of insulin secretion from these cells is shown in Fig. 1A (EC₅₀, 9.4 mM). Note that although concentrations of 11.2 and 18 mM glucose clearly stimulate secretion, 7.5 mM glucose is not sufficient to significantly stimulate insulin secretion. Likewise, 1 mM 8-Br-cAMP alone also did not significantly increase insulin secretion above the basal level (Fig. 1B). In contrast, 7.5 mM glucose together with 1 mM 8-Br-cAMP significantly enhanced insulin secretion (Fig. 1B). Previously, single-cell [Ca²⁺]_i imaging studies suggested that the insulinotropic effect of cAMP is mediated by L-type Ca²⁺ channel-dependent CICR (Holz et al., 1999). Consistent with this result, pretreatment of cells with the ER Ca²⁺-ATPase inhibitor thapsigargin (1 µM) abolished cAMP-potentiated insulin secretion (Fig. 1C), suggesting the involvement of ER Ca²⁺ release in this process. The 8-Br-cAMP-potentiated insulin secretion in response to 7.5 mM glucose in INS-1 cells was completely inhibited by the L-VDCC blocker nifedipine (Fig. 1C).

To address the possibility that Ca²⁺-induced Ca²⁺ release is involved in the insulinotropic action of cAMP, we performed Ca²⁺ imaging experiments in INS-1 cells pretreated with ryanodine. Ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP₃R) localized in the ER can mediate intracellular Ca²⁺ release and have both been identified in pancreatic -cells (Blondel et al., 1993; Gamberucci et al., 1999). Because RyR is sensitive to elevated [Ca²⁺]_i, it has been suggested that RyR may mediate cAMP-induced CICR in -cells (Holz et al., 1999). In skeletal muscle, the Ca²⁺ permeability of RyR is modulated by ryanodine in a biphasic manner. Although low ryanodine concentrations (0.01-10 µM) potentiate RyR-mediated Ca²⁺ release, saturating ryanodine concentrations (10-300 µM) are inhibitory (Lattanzio et al., 1987).

We first tested whether ER Ca²⁺ release is required for high glucose-stimulated [Ca²⁺]_i oscillation in INS-1 cells. After pretreatment with either 0.5 or 20 µM ryanodine, the glucose-stimulated [Ca²⁺]_i oscillations were still observed in untransfected INS-1 cells (Fig. 2A). We next examined the role of ER Ca²⁺ stores in insulin secretion in response to two different stimuli, high glucose alone (11.2 mM) or mildly elevated glucose (7.5 mM) together with 8-Br-cAMP. INS-1 cells maintained insulin secretion in response to high glucose after pretreatment with low (0.5 µM) or high (20 µM) ryanodine concentration (Fig. 2B). In the presence of mildly elevated glucose, 1 mM 8-Br-cAMP was still able to enhance insulin secretion in INS-1 cells pretreated with 20 µM ryanodine (Fig. 2C). However, this insulinotropic effect of cAMP was completely abolished in cells pretreated with 0.5 µM ryanodine (Fig. 2C), similar to the effect observed with thapsigargin pretreatment (Fig. 1C). Both the Ca²⁺ imaging and insulin secretion experiments indicate that, unlike the response to high glucose concentrations alone, ryanodine-sensitive intracellular Ca²⁺ stores seem to be required for the insulinotropic action of cAMP in the presence of mildly elevated glucose.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Ca2+ release from internal stores is required for cAMP-potentiated insulin secretion but not high glucose-stimulated [Ca2+]i oscillation and insulin secretion in INS-1 cells. A, [Ca2+]i responses to glucose in untransfected INS-1 cells. Glucose (glu; 18 mM) triggered [Ca2+]i oscillations, which are not inhibited by either 0.5 or 20 µM ryanodine. Traces shown are representative of 16 cells observed in each condition. B, insulin secretion stimulated by 11.2 mM glucose is not inhibited by either 0.5 or 20 µM ryanodine. INS-1 cells were preincubated with either 0.5 or 20 µM ryanodine in KRBH buffer for 30 min before stimulation with 11.2 mM glucose. Ryanodine (0.5 µM), basal, 1.25 ± 0.12% (n = 3); glu, 3.55 ± 0.44% (n = 3). Ryanodine (20 µM), basal, 0.74 ± 0.14% (n = 3); glu, 2.79 ± 0.11% (n = 3) (**, p < 0.01; ***, p < 0.001, compared with basal, one-way ANOVA with Tukey post hoc test). C, insulin secretion stimulated by 7.5 mM glucose plus 1 mM 8-Br-cAMP is inhibited by 0.5, but not 20, µM, ryanodine. Cells were preincubated with either 0.5 µM ryanodine (RY) or 20 µM ryanodine in KRBH buffer. Cells were then incubated with either 7.5 mM glucose (glu) or 7.5 mM glucose together with 1 mM 8-Br-cAMP (glu + 8-Br) for 30 min. Ryanodine (0.5 µM) preincubation inhibited the response to cAMP, whereas 20 µM ryanodine did not have any effect on cAMP-potentiated insulin secretion. RY (0.5 µM), glu, 2.42 ± 0.6% (n = 3); glu + 8-Br, 2.51 ± 0.15% (n = 3). RY (20 µM), glu, 1.88 ± 0.33% (n = 3); glu + 8-Br, 4.8 ± 0.78% (n = 3) (*, p < 0.05 compared with 7.5 glu in 20 µM ryanodine, one-way ANOVA with Tukey post hoc test).

View larger version (22K): [in this window] [in a new window]   Fig. 3. cAMP-potentiated insulin secretion in response to 7.5 mM glucose involves both Epac2 and PKA. A, INS-1 cells were incubated with either 7.5 mM glucose (Glu) or 7.5 mM glucose together with 100 µM 8'-pCPT-2'-O-Me-cAMP (Glu + Epac) or 50 µM N6-benzoyl-cAMP (Glu + PKA) or a combination of the three (Glu + Epac + PKA) in KRBH buffer for 30 min. Treatment with Epac-cAMP or PKA-cAMP alone resulted in a partial increase in insulin secretion relative to 7.5 mM glucose alone, but a combination of both analogs resulted in significantly greater response compared with either analog alone. In every case, stimulated secretion is completely blocked by 10 µM nifedipine (Nif). Inclusion of 10 µM of the PKA inhibitor H89 significantly reduced secretion stimulated by Glu + Epac + PKA. Glu, 4.00 ± 0.84 (n = 9); Glu + Epac, 7.40 ± 0.61% (n = 6); Glu + Epac + Nif, 3.69 ± 0.61% (n = 6); Glu + PKA, 9.31 ± 2.19% (n = 4); Glu + PKA + Nif, 3.73 ± 0.49% (n = 4); Glu + Epac + PKA, 16.4 ± 1.20% (n = 3); Glu + Epac + PKA + Nif, 2.04 ± 0.18 (n = 3); and Glu + Epac + PKA + H89, 6.9 ± 1.08% (n = 3). B, Rp-cAMPS (100 µM) inhibits the stimulation of insulin secretion by 50 µM N6-benzoyl-cAMP in the presence of 7.5 mM glucose in INS-1 cells. Glu, 1.29 ± 0.06% (n = 21); Glu + PKA, 3.34 ± 0.73% (n = 6); and Glu + PKA + Rp-cAMPS, 1.37 ± 0.13% (n = 15). C, Rp-cAMPS (100 µM) does not inhibit the stimulation of insulin secretion by 100 µM 8'-pCPT-2'-O-Me-cAMP in the presence of 7.5 mM glucose in INS-1 cells. Glu, 1.29 ± 0.06% (n = 21); Glu + Epac, 1.73 ± 0.10% (n = 14); Glu + Epac + Rp-cAMPS, 1.77 ± 0.203% (n = 3). *, p < 0.05; ***, p < 0.001 compared with Glu, one-way ANOVA with Tukey post hoc test; $, p < 0.05 compared with Glu, one-way ANOVA with Dunnett's post hoc test; ##, p < 0.01, ###, p < 0.001 compared with Glu + Epac + PKA, one-way ANOVA with Tukey post hoc test.

Ca_v1.2 and Ca_v1.3 Can Mediate cAMP-Potentiated Insulin Secretion at Submaximal Glucose Concentrations. We established INS-1 cell lines stably expressing either Ca_v1.2/DHPi or Ca_v1.3/DHPi fused to GFP in an effort to examine the distinct contributions of Ca_v1.2 and Ca_v1.3 to cAMP-potentiated glucose-stimulated insulin secretion (Liu et al., 2003, 2004). Because both of these L-VDCC subtypes present in cells are sensitive to L-type channel blockers, the endogenous L-type channels in these cell lines can be silenced with nifedipine, and the independent contribution of each subtype can be assessed. Mutation of two amino acids in the IIIS5 region of Ca_v1.2 or Ca_v1.3 results in a channel that is insensitive neutral DHPs such as isradipine and nifedipine but remains sensitive to the benzothiazepine diltiazem (Hockerman et al., 2000) (Fig. 4A). Using this system, we have shown previously that similar to untransfected INS-1 cells, insulin secretion in response to 11.2 mM glucose in the Ca_v1.2/DHPi cells was completely inhibited by nifedipine, whereas Ca_v1.3/DHPi cells demonstrated 40% DHP-insensitive insulin secretion. These results indicated that Ca_v1.3, but not Ca_v1.2, is preferentially coupled to 11.2 mM glucose-stimulated insulin secretion (Liu et al., 2003).

View larger version (27K): [in this window] [in a new window]   Fig. 4. Cav1.3-mediated [Ca2+]i oscillations in INS-1 cells are not sensitive to ryanodine. A, Cav1.2/DHPi and Cav1.3/DHPi channels. Two amino acids in the transmembrane segment IIIS5 of Cav1.2 or Cav1.3 were mutated to generate channels insensitive to dihydropyridine block. The C termini of both mutant constructs were fused to GFP. B, effect of ryanodine pretreatment on glucose-stimulated [Ca2+]i oscillation in Cav1.2/DHPi cells. Glucose (18 mM)-stimulated [Ca2+]i oscillation was maintained after Cav1.2/DHPi cells were pretreated with 0.5 µM ryanodine. However, this [Ca2+]i oscillation was completely inhibited by 10 µM nifedipine. C, effect of ryanodine pretreatment on glucose-stimulated [Ca2+]i oscillation in Cav1.3/DHPi cells. After 0.5 µM ryanodine pretreatment, glucose-stimulated [Ca2+]i oscillation maintained in Cav1.3/DHPi cells, and this oscillation is not sensitive to nifedipine. Traces shown in B and C are representative of 16 cells observed under each condition.

To address the possibility that a particular L-VDCC subtype serves as the Ca²⁺ influx channel involved in glucose-stimulated Ca²⁺-induced Ca²⁺ release in INS-1 cells, we pretreated Ca_v1.2/DHPi or Ca_v1.3/DHPi cells with an inhibitory concentration of ryanodine (0.5 µM) and examined [Ca²⁺]_i oscillations in response to high glucose. Similar patterns of glucose-stimulated [Ca²⁺]_i oscillations have been observed in untransfected INS-1 cells and Ca_v1.2/DHPi cells in our previous study (Liu et al., 2004). Ca_v1.2/DHPi cells responded to high glucose with nifedipine-sensitive [Ca²⁺]_i oscillations after 0.5 µM ryanodine pretreatment (Fig. 4B), consistent with the responses of these cells to L-VDCC antagonists without ryanodine pretreatment (Liu et al., 2004). However, in Ca_v1.3/DHPi cells, 11.2 mM glucose was still able to induce nifedipine-resistant [Ca²⁺]_i oscillations in the presence of ryanodine (Fig. 4C), indicating that Ca_v1.3 is preferentially coupled to glucose-stimulated [Ca²⁺]_i oscillation, as reported previously (Liu et al., 2004). These data are also consistent with our observation that ryanodine-sensitive intracellular Ca²⁺ stores are not involved in high glucose-stimulated [Ca²⁺]_i oscillations in untransfected INS-1 cells (Fig. 2A).

To further explore the specific roles of Ca_v1.2 and Ca_v1.3 in stimulus-secretion coupling, the cAMP-potentiated insulin secretion in response to mildly elevated glucose was measured in Ca_v1.2/DHPi and Ca_v1.3/DHPi cells (Fig. 5A). Treatment with 1 mM 8-Br-cAMP increased insulin secretion in response to 7.5 mM glucose in both stable cell lines. Moreover, both channels showed augmentation of insulin secretion that is insensitive to 10 µM nifedipine but blocked by 500 µM diltiazem. The detection of DHP-insensitive responses to 7.5 mM glucose plus 1 mM 8-Br-cAMP in these cell lines suggests that both Ca_v1.2 and Ca_v1.3 are able to mediate cAMP-potentiated insulin secretion under mildly elevated glucose conditions. It is interesting to note that unlike the submaximal (40%) DHPi response of Ca_v1.3/DHPi to high glucose that we have previously reported (Liu et al., 2003), both Ca_v1.2/DHPi and Ca_v1.3/DHPi cells exhibited full responses to the synergistic effect of 7.5 mM glucose and 8-Br-cAMP in the presence of nifedipine.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Both Cav1.2 and Cav1.3 are able to mediate cAMP-potentiated insulin secretion in INS-1 cells. A, in the presence of 7.5 mM glucose (Glu), 1 mM 8-Br-cAMP-potentiated insulin secretion (+8-Br) in Cav1.2/DHPi cells or Cav1.3/DHPi cells was not inhibited by 10 µM nifedipine (+8-Br + Nif) but blocked by coapplication of 10 µM nifedipine + 500 µM diltiazem (+8-Br + Nif + Dil). In Cav1.2/DHPi cells, Glu, 3.75 ± 0.68%; +8-Br, 8.63 ± 0.55%; +8-Br + Nif, 7.76 ± 0.41%; +8-Br + Nif + Dil, 4.42 ± 0.24% (n = 4). In Cav1.3/DHPi cells, Glu, 3.79 ± 0.66%; +8-Br, 8.68 ± 1.14%; +8-Br + Nif, 8.46 ± 1.33%; +8-Br + Nif + Dil, 3.41 ± 0.52% (n = 6) (*, p < 0.05; ***, p < 0.001 compared with Glu, one-way ANOVA with Tukey post hoc test). B, both Epac and PKA contribute to cAMP-potentiated coupling of Cav1.2 and Cav1.3 to insulin secretion. DHPi cells were incubated with either 7.5 mM glucose (Glu) or 7.5 mM glucose together with 100 µM 8'-pCPT-2'-O-Me-cAMP (+Epac) or 50 µM N6-benzoyl-cAMP (+PKA) or a combination of the three (+Epac + PKA) in the presence of 10 µM nifedipine in KRBH buffer for 30 min. Diltiazem (500 µM) was added to inhibit the DHPi channels (+Epac + PKA + Dilt). In Cav1.2/DHPi cells, Glu, 3.15 ± 0.41%; +Epac, 5.40 ± 0.30%; +PKA, 5.66 ± 0.35%; +Epac + PKA, 9.49 ± 0.42%; +Epac + PKA + Dilt, 3.60 ± 0.74% (n = 5). In Cav1.3/DHPi cells, Glu, 2.10 ± 0.06%; +Epac, 3.67 ± 0.60%; +PKA, 4.51 ± 0.47%; +Epac + PKA, 7.21 ± 0.55%; and +Epac + PKA + Dilt, 2.22 ± 0.52 (n = 15). **, p < 0.01; ***, p < 0.001 compared with Glu; ###, p < 0.001 compared with Glu + PKA (one-way ANOVA with Tukey post hoc test).

	Discussion

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Ca²⁺ Influx via Either Ca_v1.2 or Ca_v1.3 Can Mediate Insulin Secretion in Response to 7.5 mM Glucose and cAMP. Previously, we found that the VDCC Ca_v1.3, but not Ca_v1.2, is preferentially coupled to insulin secretion (Liu et al., 2003) and [Ca²⁺]_i oscillations (Liu et al., 2004) in response to high glucose concentrations in INS-1 cells. Here, we investigated the role of L-VDCCs in stimulus-secretion coupling in INS-1 cells in response to mildly elevated glucose concentrations in combination with cAMP. In contrast to our studies using high glucose concentrations alone, we found that both Ca_v1.2 and Ca_v1.3 are able to mediate insulin secretion in response to 7.5 mM glucose and cAMP. What can account for this difference in channel selectivity in the face of these two distinct stimuli? Our data in Figs. 2 and 4 (and our previous studies) suggest that the Ca²⁺ influx across the plasma membrane mediated by Ca_v1.3 drives both insulin secretion and [Ca²⁺]_i oscillations in response to 11.2 mM glucose in INS-1 cells because neither are inhibited by unloading of intracellular Ca²⁺ stores by thapsigargin or 0.5 µM ryanodine. In contrast, cAMP potentiation of mildly elevated glucose-stimulated insulin secretion seems to involve release of Ca²⁺ from internal stores because both thapsigargin and 0.5 µM ryanodine, which empties ER Ca²⁺ stores via activation of RyR, inhibit secretion under these conditions. Furthermore, the release of Ca²⁺ from internal stores can be triggered by Ca²⁺ influx across the plasma membrane via either Ca_v1.2 or Ca_v1.3. The DHP-insensitive insulin secretion observed in Ca_v1.3/DHPi cells in response to 11.2 mM glucose was 40% of that observed in the absence of nifedipine (Liu et al., 2003). In contrast, both Ca_v1.2/DHPi and Ca_v1.3/DHPi cells exhibit insulin secretion in response to 7.5 mM glucose and 1 mM 8-Br-cAMP in the presence of nifedipine that is not significantly different from that observed in the absence of nifedipine (Fig. 5A). This observation further supports the notion that, in the presence of mildly elevated glucose and high cAMP concentrations, Ca²⁺ influx via Ca_v1.2 or Ca_v1.3 serves as a trigger for Ca²⁺ release from internal stores and does not directly drive insulin secretion.

Previous studies have also implicated CICR in cAMP-potentiated-insulin secretion. Measurements of [Ca²⁺]_i changes in response to endogenous cAMP elevation in -cells is reported to require CICR (Holz et al., 1999; Kang et al., 2001). The intracellular Ca²⁺ release channel responsible for cAMP-induced CICR has not been clearly identified. However, some studies have indicated the involvement of RyR in this event (Holz et al., 1999). RyR2 is expressed in INS-1 cells (Gamberucci et al., 1999) and is able to mediate Ca²⁺ release from ER (Gamberucci et al., 1999; Bruton et al., 2003). Similar to thapsigargin, we found that 0.5 µM ryanodine pretreatment emptied ER Ca²⁺ stores and prevented the cAMP-dependent CICR and, therefore, abrogated insulin secretion in response to mildly elevated glucose (Fig. 2C). However, 20 µM ryanodine, which decreases the Ca²⁺ permeability of RyR (Lattanzio et al., 1987), did not influence the action of 8-Br-cAMP (Fig. 2C). Our results suggest that the pool of Ca²⁺ required for insulin secretion stimulated by 7.5 mM glucose and cAMP is sensitive to ryanodine but is not released via RYR2 in INS-1 cells. In neurons, it is reported that ER acts as a single functional Ca²⁺ store shared by both RyR and IP₃R (Solovyova and Verkhratsky, 2003). Recently, the IP₃ receptor was implicated in CICR in mouse -cells (Berggren et al., 2004); thus, it is an attractive candidate as the mediator of CICR in INS-1 cells.

cAMP Potentiation of 7.5 mM Glucose-Stimulated Insulin Secretion Involves the Combined Action of Epac2 and PKA. Epac2 has been shown previously to modulate the cAMP-dependent insulin secretion (Kashima et al., 2001) and CICR (Kang et al., 2003) in -cells independently of PKA. In our present study, we found that Epac2 triggered cAMP-dependent insulin secretion in response to 7.5 mM glucose independently of PKA (Figs. 3 and 5B). Although this potentiation is significantly different from basal, the maximal response under these conditions is seen only when PKA is also activated. The observation that, in untransfected INS-1 cells, inhibition of PKA with H89 reduces secretion stimulated by 7.5 mM glucose plus both selective cAMP analogs to a level that is not significantly different from that of 7.5 mM glucose alone (Fig. 3A) may reflect the ratio of LVDCC subtypes present in INS-1 cells. Ca_v1.3 is prevalent in this cell line, whereas Ca_v1.2 is present in a much smaller amount (Horvath et al., 1998). Thus, the small population of Ca_v1.2 channels present in untransfected INS-1 cells may mediate a small amount of Epac2-dependent, PKA-independent secretion that becomes statistically insignificant in the presence of H89.

Epac2 and PKA Differentially Modulate Ca_v1.2- and Ca_v1.3-Coupled Insulin Secretion in Response to 7.5 mM Glucose and cAMP. Because we found that both Ca_v1.2 and Ca_v1.3 are able to mediate insulin secretion in response to 7.5 mM glucose and cAMP (Fig. 5A), we examined the contribution of Epac2 and PKA in the potentiation of secretion in both of the DHPi cell lines. We reasoned that, although both Ca_v1.2 and Ca_v1.3 seem to mediate CICR in the presence of elevated cAMP concentrations, they could use distinct cAMP effector pathways. We found that, similar to untransfected INS-1 cells, Ca_v1.2-coupled insulin secretion is partially enhanced by Epac2 and PKA independently of each other (Fig. 5B). However, Ca_v1.3-coupled secretion seems not to be enhanced by Epac2 in the absence of PKA activation (Fig. 5B). In all cases examined, the combined action of Epac2 and PKA seems sufficient to account for the insulinotropic action of 8-Br-cAMP (Fig. 5B). It is interesting that Epac2 exerts PKA-independent activity only in Ca_v1.2/DHPi cells because it has been previously reported that RIM 2, a downstream target of Epac2 (Ozaki et al., 2000), binds to the intracellular II-III loop of Ca_v1.2, but not Ca_v1.3 (Shibasaki et al., 2004). Phosphorylation of Ca_v1.3 may be necessary for Epac2 to modulate secretion mediated by that channel. Indeed, inspection of the II-III loop region of Ca_v1.3 reveals two putative PKA phosphorylation sites that are not present in Ca_v1.2. On the other hand, Epac2 modulation of Ca_v1.3-mediated secretion is consistent with the presence of a putative PKA phosphorylation site on Epac2 (Kawasaki et al., 1998). Several studies have suggested possible Epac2- or PKA-mediated regulatory mechanisms at the level of channel gating and protein-protein interactions. Previous studies have suggested that in human embryonic kidney 293 cells, the phosphorylation of Ca_v1.2 by PKA (Gromada et al., 1998) causes a leftward shift in the V_1/2 of activation, requiring a smaller depolarization to initiate channel gating. Likewise, HIT-T15 insulinoma cells transfected with the G_s-coupled GLP-1 receptor demonstrate an increase in the VDCC currents, presumably due to a leftward shift in the V_1/2 of activation (MacDonald et al., 2002). A rightward shift in the voltage dependence of steady-state inactivation VDCC in mouse pancreatic -cells is observed upon endogenous cAMP elevation by treatment with GLP-1 (Britsch et al., 1995). In addition to modulation of L-type channel activity, PKA activation has been shown recently to amplify CICR from internal stores via IP₃ receptors in rat pancreatic cells (Dyachok and Gylfe, 2004). Finally, cAMP also plays a role in replenishing ER stores of Ca²⁺, by stimulating the thapsigargin-sensitive reuptake of Ca²⁺, via a PKA-dependent mechanism in rat pancreatic -cells (Yaekura and Yada, 1998). Thus, priming of CICR and ER Ca²⁺ refilling, in contrast to increasing Ca²⁺ influx via L-type channels, are potential mechanisms to explain why insulin secretion in response to mildly elevated glucose concentrations plus cAMP involves CICR, but secretion in response to elevated glucose concentrations alone does not.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Proposed models for the involvement of Cav1.2 and Cav1.3 in insulin secretion in response to either 11.2 mM glucose alone or 7.5 mM glucose together with cAMP. Left, Cav1.3 is preferentially coupled to glucose-stimulated insulin secretion in INS-1 cells. Upon 11.2 mM glucose stimulation, Ca2+ influx through Cav1.2 and Cav1.3 form a local high [Ca2+]i domain beneath each channel. Cav1.3 is preferentially coupled to the insulin-containing granules, probably via Cav1.3 II-III loop, which may position insulin-containing granules within the Cav1.3-mediated high [Ca2+]i domain and induce insulin secretion. Alternatively, the lower activation voltage of Cav1.3 versus Cav1.2 may result in preferential activation of Cav1.3 by glucose. Under these conditions, Ca2+ release from internal stores is not involved. Right, both Cav1.2 and Cav1.3 are able to mediate cAMP-potentiated insulin secretion. In the presence of cAMP, 7.5 mM glucose-induced membrane depolarization activates Ca2+ entry via Cav1.2 and Cav1.3. The Ca2+ influx through both channels is able to trigger Ca2+-induced Ca2+ release from internal stores. This release of Ca2+ is not mediated by RYR, but the required pool of Ca2+ is sensitive to both thapsigargin and 0.5 µM ryanodine. The IP3R, or some other ryanodine-insensitive mechanism, probably mediates the Ca2+ release. cAMP-induced PKA activity is required for this action and is proposed to affect the Ca2+ sensitivity of the Ca2+ release channels on the ER membrane and/or the Ca2+ influx channels (Cav1.2 and/or Cav1.3) on the plasma membrane. Epac2 or PKA activation can independently potentiate Cav1.2-mediated insulin secretion. PKA activation can independently potentiate Cav1.3-mediated insulin secretion, whereas PKA activity is required for Epac2 potentiation of Cav1.3-mediated insulin secretion.

	Footnotes

 
This work was supported by a Purdue Research Foundation Grant (to G.L. and G.H.H.), by American Diabetes Association Research Award 1119990378 (to G.H.H.), and by National Institutes of Health Research Grant R01 DK064736 (to G.H.H.).

In memory of Claire E. Hockerman and Nathan G. Hockerman.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.097477.

ABBREVIATIONS: VDCC, voltage-dependent calcium channel; GLP, glucagon-like peptide; PKA, protein kinase A; GEF, guanine nucleotide exchange factor; INS-1, insulin-secreting cell line-1; Epac2, exchange protein directly activated by cAMP 2; CICR, Ca²⁺-induced Ca²⁺ release; 8-Br-cAMP, 8-bromo-cAMP; H89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl; Ca_v1.2/DHPi, Ca_v1.2 channel insensitive to dihydropyridines; Ca_v1.3/DHPi, Ca_v1.3 channel insensitive to dihydropyridines; Rp-cAMPS, adenosine 3',5'-cyclic monophosphorothioate (Rp-isomer); GFP, green fluorescent protein; DHPi, dihydropyridine-insensitive; KRBH, Krebs-Ringer-bicarbonate HEPES; ANOVA, analysis of variance; ER, endoplasmic reticulum; RyR, ryanodine receptor; IP₃R, inositol 1,4,5-triphosphate receptors.

Address correspondence to: Gregory H. Hockerman, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907-2091. E-mail: gregh{at}pharmacy.purdue.edu

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