title: Extracellular calcium influx activates adenylate cyclase 1 and potentiates insulin secretion in MIN6 cells.
abstract: Intracellular cAMP and Ca(2+) 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 Ca(2+) concentrations ([cAMP](i) and [Ca(2+)](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 Ca(2+) channel agonist or ionomycin to induce extracellular Ca(2+) influx evoked the elevation of [cAMP](i), whereas application of carbachol or thapsigargin, which mobilize Ca(2+) from internal stores, did not evoke the elevation of [cAMP](i). We performed RT (reverse transcription)-PCR analysis and found that Ca(2+)-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 [Ca(2+)](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.
authors.affiliation: Life Function and Dynamics, ERATO, JST (Japan Science and Technology Agency), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. kitaguct@aoni.waseda.jp
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kitaguchi
authors.forename: Manami
authors.initials: M
authors.lastname: Oya
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Wada
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsuboi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D051379:Mice; D014162:Transfection
chemical_list: D007328:Insulin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; C106670:adenylyl cyclase 1; D005947:Glucose; D002118:Calcium