Regulation of Exocytosis

胞吐作用的调节

• 批准号: 06044199
• 负责人: KUMAKURA Konosuke
• 金额: $ 7.3万
• 依托单位: Sophia University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06044199/
• 关键词: Regulation of exocytosis; Chromaffin cells; GTP-binding protein; Phospholyration; Video-Microscope; Vesicular movement; Cytoskeleton; Release from Single cells; Amperometric detection

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.

我们从这个关于胞吐调控的研究项目中获得了以下结果：1）我们以前已经证明，肌球蛋白轻链激酶是肾上腺嗜铬细胞胞吐的ATP依赖性启动所必需的。利用选择性抑制肌动蛋白-肌球蛋白相互作用的毒素mychalpine B，我们证明肌动蛋白-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依赖性启动受三聚体G蛋白、Go和GAP 43的调节。2）为了了解胞吐的动力学方面，我们通过结合使用碳纤维DC-安培法和视频增强对比DIC显微镜来分析释放过程。当细胞被乙酰胆碱或电极刺激时，出现高达350 pA的电流尖峰，同时也出现颗粒图像的快速闪烁。大约80%的电流尖峰与视频显微镜反应相关，20%的尖峰被分配到无形的反应，大概发生在离焦。我们的结论是：（1）在牛嗜铬细胞中，用直流电流分析法记录的电流瞬变可以可靠地被认为代表单个囊泡融合事件;和（ii）振幅分布主要由儿茶酚胺的扩散曲线决定。3）尿苷5 '-三磷酸（UTP）诱发的细胞内Ca^2+浓度（[Ca]i）增加用大鼠嗜铬细胞瘤PC 12细胞研究多巴胺的释放。结果表明，UTP通过Ca^2+动员和Ca^2+内流两种途径刺激P2 U-嘌呤受体并诱导PC 12细胞内[Ca]i升高。UTP诱发多巴胺释放需要外源性Ca^<2+>，Ca^<2+>可能通过对Zn^<2+>敏感而对Cd^<2+>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释放。

项目成果

H.Misonou: "Inhibition of ATP-dependent priming of exocytosis by mastoparan." Neurochem.Res.20(3). 317 (1995)

H.Misonou：“mastoparan 抑制 ATP 依赖性胞吐作用启动。”

K.Kumakura: "The role of myosin light chain Kinase and F-actin for ATP-dependent priming of exocytosis in adrenal Chromaffin cells." Society for Neuroscience Abstract. Part 1. 329 (1995)

K.Kumakura：“肌球蛋白轻链激酶和 F-肌动蛋白在肾上腺嗜铬细胞中 ATP 依赖性胞吐作用启动中的作用。”

Konosuke Kumakura: "Research mote on : Regulation of Exocytosis" Sophia Life Science Bulletin. 14. 69-73 (1995)

Konosuke Kumakura：“研究要点：胞吐作用的调节”索菲亚生命科学通报。

Konosuke Kumakura: "The role of myosin light chain Kinase and F-actin for ATP-dependent priming of expcytosis in adrenal Chromaffin cells." Society for Neuroscience Abstract. Part 1. 329 (1995)

Konosuke Kumakura：“肌球蛋白轻链激酶和 F-肌动蛋白在肾上腺嗜铬细胞中 ATP 依赖性胞吐作用启动中的作用。”

今泉 美佳: "開口放出と3量体Gタンパク質" 細胞工学. 13. 373-380 (1994)

Mika Imaizumi：“胞吐作用和三聚体 G 蛋白”《细胞工程》13. 373-380 (1994)。