Mol081943 191..205

Tolbutamide and gliclazide block the KATP channel Kir6.2/Sur1, causing membrane depolarization and stimulating insulin secretion in pancreatic beta cells. We examined the ability of the EPAC-selective cAMP analog 8-pCPT-29-O-Me-cAMP-AM to potentiate the action of these drugs and the mechanism that might account for it. Insulin secretion stimulated by both 200 mM tolbutamide and 20 mM gliclazide, concentrations that had equivalent effects on membrane potential, was inhibited by thapsigargin (1 mM) or the L-type Ca channel blocker nicardipine (2 mM) and was potentiated by 8-pCPT-29-O-MecAMP-AM at concentrations $2 mM in INS-1 cells. Ca transients stimulated by either tolbutamide or gliclazide were inhibited by thapsigargin or nicardipine and were significantly potentiated by 8-pCPT-29-O-Me-cAMP-AM at 5 mM but not 1 mM. Both tolbutamide and gliclazide stimulated phospholipase C activity; however, only gliclazide did so independently of its activity at KATP channels, and this activity was partially inhibited by pertussis toxin. 8-pCPT-29-O-Me-cAMP-AM alone (5 mM) did not stimulate insulin secretion, but did increase intracellular Ca concentration significantly, and this activity was inhibited by 25 mM 2-aminoethoxydiphenylborate (2-APB) or the removal of extracellular Ca. 8-pCPT-29-OMe-cAMP-AM potentiation of insulin secretion stimulated by tolbutamide was markedly inhibited by 2-APB (25 mM) and enhanced by the PKC inhibitor bisindolylmaleimide I (1 mM). Our data demonstrate that the actions of both tolbutamide and gliclazide are strongly potentiated by 8-pCPT-29-O-MecAMP-AM, that gliclazide can stimulate phospholipase C activity via a partially pertussis toxin-sensitive mechanism, and that 8-pCPT-29-O-Me-cAMP-AM potentiation of tolbutamide action may involve activation of a 2-APB-sensitive Ca influx.