Received for publication October 21, 2005.
Revised March 21, 2006.
Accepted for publication March 23, 2006.

Differential modulation of Ca_v1.2 and Ca_v1.3-mediated glucose-stimulated insulin secretion by cAMP in INS-1 cells: distinct roles for Epac2 and PKA

Abstract

Using 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-Br-cAMP is mediated by both Ca_v1.2 and Ca_v1.3. Treatment of INS-1 cells, or Ca_v1.2/DHPi cells in the presence of 10 µM nifedipine, with either effector-specific cAMP analogs 8-pCPT-2'-O-Me-cAMP (100 µM; Epac2-selective) or N6-Benzoyl-cAMP (50 µM; PKA-selective) partially increased insulin secretion. Secretion stimulated by a combination of the two cAMP analogs was additive and comparable to that stimulated by 1 mM 8-Br-cAMP. In Ca_v1.3/DHPi cells 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 to 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+. Further, cAMP modulation of secretion mediated by Ca_v1.2 appears to involve both Epac2 and PKA independently. In contrast, cAMP modulation of Ca_v1.3 mediated secretion depends upon PKA activation, while the contribution of Epac2 is dependent upon PKA activation.

Key words: Epac2, L-type calcium channels, PKA, cAMP, insulin secretion, voltage-gated calcium channels

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