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.
Liu
Guohong
G
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907-2091, USA.
Jacobo
Sarah Melissa P
SM
Hilliard
Nathan
N
Hockerman
Gregory H
GH
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