Roles of G-proteins and cytoskeletal porteins in transmitter release.

本课题旨在阐明gtp结合蛋白和细胞骨架蛋白在递质释放机制中的作用。在此之前，我们已经提出Go，一种异三聚体gtp结合蛋白，负调控钙<2+>-依赖性儿茶酚胺在通透性染色质细胞中的释放。我们还提出肌球蛋白轻链激酶可能参与Ca^<2+>依赖性胞外分泌的机制。在该项目中，我们试图阐明钙离子（Go）和肌球蛋白轻链激酶（myosin light chain kinase）在通透化的染色质细胞中调控Ca^<2+>依赖性释放的机制。我们得到了以下结果：通过使用已知可直接激活gtp结合蛋白的肽mastoparan和使用抗氧化石墨烯抗体，我们发现氧化石墨烯对atp依赖的胞吐启动过程具有负调控作用。这种抑制性氧化石墨烯被GAP43激活，蛋白激酶C的激活似乎通过消除GAP43对氧化石墨烯的作用来刺激Ca^<2+>依赖性释放。我们发现肌球蛋白轻链激酶对atp依赖的胞吐启动至关重要，通过使用特定的肌球蛋白轻链激酶抑制剂，wortmannin和SM-1。组织化学研究表明，对肌球蛋白轻链激酶的抑制抑制了皮层f -肌动蛋白的重组，从而表明皮层f -肌动蛋白在染色质囊泡向胞外部位的启动中起重要作用。

The aim of this research project is to clarify the roles of GTP-binding proteins and cytoskeletal proteins in the mechanism of transmitter release.Previously we have suggested that Go, one of heterotrimeric GTP-binding proteins, negatively regulates Ca^<2+>-dependent release of catecholamine in permeabilized chromaffin cells.We also have suggested a possible involvement of myosin light chain kinase in the mechanism of Ca^<2+>-dependent exocytosis.In the project, we attempted to elucidate the mechanism whereby Ca^<2+>-dependent release is regulated by Go, and by myosin light chain kinase in permeabilized chromaffin cells.We have obtained the following results.1.By use of mastoparan, a peptide known to activate directly GTP-binding proteins, and by use of anti-Go antibodies, we found that the negative regulation by Go is on the process of ATP-dependent priming of exocytosis.This inhibitory Go was suggested to be activated by GAP43, and activation of protein kinase C seemed to stimulate Ca^<2+>-dependent release by abolishing the action of GAP43 on Go.2.We found that myosin light chain kinase is essential for ATP-dependent priming of exocytosis, by use of specific inhibitors of myosin light chain kinase, wortmannin and SM-1.Histochemical studies suggested that the inhibition of myosin light chain kinase inhibits reorganization of cortical F-actin, and thus suggested an important role of cortical F-actin for priming of chromaffin vesicles to the exocytotic site.