Yamamoto Hideyuki, Matsumoto Kazuya, Araki Eiichi, Miyamoto Eishichi
Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto, Japan.
J Pharmacol Sci. 2003 Sep;93(1):30-4. doi: 10.1254/jphs.93.30.
The exocytosis of insulin from pancreatic beta-cells is closely related to intracellular elevation of Ca(2+). The effects of Ca(2+) may be mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Four subunits of CaMKII, termed alpha, beta, gamma, and delta, are encoded by distinct genes, and various isoforms of these subunits exist as different splicing variants. In the brain, phosphorylation of synapsin I by the alpha isoform induces neurotransmitter release. In order to clarify whether phosphorylation of synapsin I by CaMKII was involved in insulin exocytosis, we cloned the isoforms of CaMKII and synapsin I from mouse insulinoma MIN6 cells. We found that beta'e and delta2 are the major isoforms of CaMKII and that synapsin Ib is a major isoform of synapsin I in MIN6 cells. It was interesting that delta2 and synapsin Ib were co-localized with insulin secretory granules in the cells. Treatment of MIN6 cells with glucose and tolbutamide rapidly activated CaMKII. Immunoblot analysis with two antibodies against synapsin I phosphorylated by CaMKII demonstrated the increase in phosphorylation of synapsin I by the secretagogues. Furthermore, the secretagogue-induced phosphorylation of synapsin I and insulin secretion were potentiated by transient overexpression of the beta'e or delta2 isoform. These results suggest that activation of CaMKII and the concomitant phosphorylation of synapsin I induce insulin exocytosis from pancreatic beta-cells.
胰岛素从胰腺β细胞的胞吐作用与细胞内Ca(2+)浓度升高密切相关。Ca(2+)的作用可能由Ca(2+)/钙调蛋白依赖性蛋白激酶II（CaMKII）介导。CaMKII的四个亚基，即α、β、γ和δ，由不同的基因编码，这些亚基的各种同工型以不同的剪接变体形式存在。在大脑中，α同工型对突触素I的磷酸化诱导神经递质释放。为了阐明CaMKII对突触素I的磷酸化是否参与胰岛素胞吐作用，我们从小鼠胰岛素瘤MIN6细胞中克隆了CaMKII和突触素I的同工型。我们发现β'e和δ2是CaMKII的主要同工型，而突触素Ib是MIN6细胞中突触素I的主要同工型。有趣的是，δ2和突触素Ib在细胞内与胰岛素分泌颗粒共定位。用葡萄糖和甲苯磺丁脲处理MIN6细胞可迅速激活CaMKII。用两种针对CaMKII磷酸化的突触素I的抗体进行免疫印迹分析表明，促分泌剂可增加突触素I的磷酸化。此外，β'e或δ2同工型的瞬时过表达增强了促分泌剂诱导的突触素I磷酸化和胰岛素分泌。这些结果表明，CaMKII的激活以及突触素I的伴随磷酸化诱导了胰腺β细胞的胰岛素胞吐作用。
