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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

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.

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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