Regulation of Exocytosis

我们从这个关于胞吐调节的研究项目中获得了以下结果。1)我们之前已经证明肌球蛋白轻链激酶对于肾上腺染色质细胞atp依赖的胞外分泌的启动是必不可少的。通过使用mychalolide B（一种选择性抑制肌动蛋白-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。研究还表明，atp依赖性的启动是由三聚体g蛋白、Go和GAP43.2调控的。)为了了解胞外分泌的动力学方面，我们结合使用碳纤维直流电流法和视频增强对比DIC显微镜分析了释放过程。当细胞受到乙酰胆碱或电极连接刺激时，会出现高达350 pA的电流峰值，同时也会出现颗粒图像的快速闪烁。大约80%的电流峰值与视频微观响应相关，20%的峰值被分配给可能发生在焦点外的不可见响应。我们得出的结论是：(i)在牛染色质细胞中，直流安培法记录的电流瞬变可以被安全地认为代表单个囊泡融合事件；(2)振幅分布主要由儿茶酚胺的扩散曲线决定。3)用大鼠嗜铬细胞瘤PC12细胞研究尿苷5′-三磷酸（UTP）诱导细胞内Ca^<2+>浓度（[Ca]i）升高和多巴胺释放。数据表明，UTP刺激p2u -嘌呤受体，并通过Ca^<2+>-动员和Ca^<2+>-内流在PC12细胞中诱导[Ca]i升高。utp引起的多巴胺释放需要外部Ca^<2+>, Ca^<2+>可能通过对Zn^<2+>敏感的途径进入细胞，但对Cd^<2+>或尼卡地平不敏感。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.