Extracellular calcium influx activates adenylate cyclase 1 and potentiates insulin secretion in MIN6 cells
Extracellular calcium influx activates adenylate cyclase 1 and potentiates insulin secretion in MIN6 cells
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DOI：
10.1042/bj20121022
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发表时间：
2013-03-01
影响因子：
4.1
通讯作者：
Miyawaki, Atsushi
中科院分区：
文献类型：
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作者：
Kitaguchi, Tetsuya;Oya, Manami;Miyawaki, Atsushi
胞内cAMP和Ca 2+是调节胰腺β细胞中胰岛素分泌的重要第二信使;然而，它们相互作用的分子机制尚未完全了解。在本研究中，我们使用全内反射荧光显微镜研究了胰腺β细胞系MIN 6中细胞内cAMP和Ca 2+浓度（分别为[cAMP]（i）和[Ca 2 +]）之间的相互作用。为了测量[cAMP]（i），我们开发了一种用于cAMP的遗传编码黄色荧光生物传感器[Flamindo（荧光cAMP指示剂）]，其随着cAMP结合而改变荧光强度。高浓度KCl或葡萄糖作用于MIN 6细胞可引起[cAMP]（i）和胞吐作用的升高。此外，应用L型Ca 2+通道激动剂或离子霉素诱导细胞外Ca 2+内流引起[cAMP]升高，而应用卡巴胆碱或毒胡萝卜素从内部储存中动员Ca 2+，并没有引起[cAMP]升高（i）。我们进行了RT（逆转录）-PCR分析，发现Ca 2+敏感的Adcy 1（腺苷酸环化酶1）在MIN 6细胞中表达。通过小干扰RNA敲低内源性ADCY 1显著抑制葡萄糖诱导的胞吐作用以及[cAMP]（i）和[Ca 2 +]（i）的升高。综上所述，本研究的结果表明，ADCY 1在控制胰腺β细胞cAMP稳态和胰岛素分泌中起重要作用。
Intracellular cAMP and Ca2+ are important second messengers that regulate insulin secretion in pancreatic beta-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+], respectively) in the pancreatic beta-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] 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 beta-cell cAMP homoeostasis and insulin secretion.