Regulation of Exocytosis

1)我们先前已经证明肌球蛋白轻链激酶在肾上腺嗜铬细胞胞吐作用的ATP依赖的启动过程中是必不可少的。通过使用Mychalolide B，一种选择性地抑制肌动蛋白-肌球蛋白相互作用的毒素，我们证明了肌动蛋白-肌球蛋白相互作用在ATP依赖的启动机制中是重要的。为了了解胞吐的动力学过程，我们结合使用碳纤维直流安培法和视频增强对比DIC显微镜分析了胞吐的释放过程。当细胞受到乙酰胆碱或电极的刺激时，出现高达350pA的电流尖峰，并同时出现颗粒图像的快速闪烁。大约80%的电流尖峰与视频显微反应相关，20%的尖峰被分配给可能发生在焦点外的隐形反应。我们得出的结论是：(I)在牛嗜铬细胞中，用直流电流法记录的电流瞬变可以安全地被认为代表单个囊泡融合事件；以及(Ii)幅度分布主要由儿茶酚胺的扩散曲线决定。3)尿苷5‘-三磷酸(UTP)诱导的细胞内钙离子浓度([Ca]i)增加和多巴胺的释放被用大鼠嗜铬细胞瘤PC12细胞来研究。结果表明，UTP通过钙离子动员和钙离子内流两种途径刺激PC12细胞的P2U-嘌呤受体，引起细胞内[Ca]i升高。UTP-诱发的多巴胺释放需要外在的Ca~(2+)~(2+)通过对锌敏感而对镉不敏感或尼卡地平不敏感的途径进入细胞。这些结果表明，ATP通过不同类型的嘌呤受体调节递质的释放。

We have obtained the following results from this research project on the regulation of exocytosis.1) We have previously demonstrated that myosin lightchain kinase is essential for ATP-dependent priming of exocytosis in adrenal chromaffin cells. By use of mychalolide B,a toxin that selectively inhibit actin-myosin interaction, we demonstrated that actin-myosin interaction is important in ATP-dependent priming mechanism. It was also suggested that ATP-dependent priming is regulated by a trimeric G-protein, Go, and GAP43.2) In order to understand the dynamics aspects of exocytosis, we analyzed the releasing process by combined use of the carbon fiber DC-amperometry and the video-enhanced contrast DIC microscopy. When a cell was stimulated by acetylcholine or by electrode attachment, current spikes as large as 350 pA appeared and rapid flickers of granule image also appeared simultaneously. About 80% of current spikes were correlated with video microscopic responses, and 20% of spikes were assigned to invisible responses presumably taking place off focus. We conclude that (i) in bovine chromaffin cells, current transients recorded with DC amperometry can be securely considered to represent single vesicular fusion events ; and (ii) that the amplitute distribution is determined primarily by the diffusional profile of the catecholamine.3) Uridine 5'-triphosphate (UTP)-evoked increase in intracellular Ca^<2+> concentration ([Ca]i) and release of dopamine were investigated using rat pheochromocytoma PC12 cells. The data demonstrated that UTP stimulates P2U-purinoceptors and induces a rise in [Ca]i both by Ca^<2+>-mobilization and Ca^<2+>-influx in PC12 cells. The UTP-evokes dopamine release requires external Ca^<2+> which may enter the cells through pathway sensitive to Zn^<2+>, but insensitive to Cd^<2+> or nicardipine. These results suggested that ATP modulates transmitter release through different types of purinoceptors.