Intracellular cAMP and Ca2+ are important second messengers that regulate insulin secretion in pancreatic β-cells; however, the molecular mechanism underlying their mutual interaction for exocytosis is not fully understood. In the present study, we investigated the interplay between intracellular cAMP and Ca2+ concentrations ([cAMP]i and [Ca2+]i respectively) in the pancreatic β-cell line MIN6 using total internal reflection fluorescence microscopy. For measuring [cAMP]i, we developed a genetically encoded yellow fluorescent biosensor for cAMP [Flamindo (fluorescent cAMP indicator)], which changes fluorescence intensity with cAMP binding. Application of high-KCl or glucose to MIN6 cells induced the elevation of [cAMP]i and exocytosis. Furthermore, application of an L-type Ca2+ channel agonist or ionomycin to induce extracellular Ca2+ influx evoked the elevation of [cAMP]i, whereas application of carbachol or thapsigargin, which mobilize Ca2+ from internal stores, did not evoke the elevation of [cAMP]i. We performed RT (reverse transcription)–PCR analysis and found that Ca2+-sensitive Adcy1 (adenylate cyclase 1) was expressed in MIN6 cells. Knockdown of endogenous ADCY1 by small interference RNA significantly suppressed glucose-induced exocytosis and the elevation of both [cAMP]i and [Ca2+]i. Taken together, the findings of the present study demonstrate that ADCY1 plays an important role in the control of pancreatic β-cell cAMP homoeostasis and insulin secretion.

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