Differential modulation of Cav1.2 and Cav1.3-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.
Using insulin-secreting cell line (INS)-1 cells stably expressing dihydropyridine-insensitive mutants of either Cav1.2 or Cav1.3, we previously demonstrated that Cav1.3 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 Cav1.2 and Cav1.3. Treatment of INS-1 cells or INS-1 cells stably expressing Cav1.2/dihydropyridine-insensitive (DHPi) channels in the presence of 10 microM nifedipine, with effector-specific cAMP analogs 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP [8-pCPT-2'-O-Me-cAMP; 100 microM; Exchange Protein directly Activated by cAMP 2 (Epac2)-selective] or N6-benzoyl-cAMP [50 microM; 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 Cav1.3/DHPi in the presence of 10 microM 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 microM) and ryanodine (0.5 microM). 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 Cav1.2 and Cav1.3 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 Cav1.2 seems to involve both Epac2 and PKA independently. In contrast, cAMP modulation of Cav1.3-mediated secretion depends upon PKA activation, whereas the contribution of Epac2 is dependent upon PKA activation.
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907-2091, USA.
Guohong
G
Liu
Sarah Melissa P
SM
Jacobo
Nathan
N
Hilliard
Gregory H
GH
Hockerman
D000818:Animals; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002352:Carrier Proteins / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000502:physiology*; D051379:Mice
C485995:Cacna1d protein, mouse; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; C419385:L-type calcium channel alpha(1C); C486707:Rapgef4 protein, mouse; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose