Cyclic AMP/cAMP‐GEF pathway amplifies insulin exocytosis induced by Ca2+ and ATP in rat islet β‐cells

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Abstract
Background Cyclic AMP (cAMP) plays a pivotal role in insulin secretion induced by incretins. The effects of the second messenger extend to many sites and there has been much controversy on the mechanisms. The aim of this study was to examine how cAMP amplified insulin exocytosis. Methods Rat islets were permeabilized with α-toxin to measure insulin exocytosis in the fixed conditions of Ca2+ and ATP. The effects of several agents on insulin exocytosis were observed in perifusion experiments. Results Cyclic AMP enhanced the Ca2+-induced insulin release by around 30%, independent of Ca2+ concentrations between 10 and 3000 nmol/L. A cAMP-GEF selective cAMP analogue, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate, also amplified insulin release. The effect disappeared in the absence of ATP. Conversely, cAMP-independent gradual increase in insulin release was observed with ATP. These results suggested that the site of action of cAMP-GEF existed proximal to that of ATP. An analogue selective to PKA, N6-Benzoyladenosine-3′,5′-cyclic monophosphate, had little effect. Also, a PKA-selective inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, reduced insulin releases induced by 1000 nmol/L Ca2+, but did not influence the relative increase produced by Ca2+ and cAMP. Conclusion Cyclic AMP potentiated Ca2+ and ATP-induced exocytosis to a similar relative extent independent of Ca2+ concentrations. The process appeared to be mainly mediated by cAMP-GEF. In addition, the cAMP/cAMP-GEF pathway may enhance insulin release by replenishing the readily releasable pool. Copyright © 2005 John Wiley & Sons, Ltd.