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Metabolism and biological activities of lnsP5 and InsP6 in neuronal cells

lnsP5和InsP6在神经元细胞中的代谢和生物活性

基本信息

批准号：
09670104
负责人：
SASAKAWA Nobuyuki
金额：
$ 2.43万
依托单位：
Sophia University (1998)Keio University (1997)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670104/
关键词：
Inositol polyphosphates exocytosis adrenal chromaffin cell シナプトタグミン

项目摘要

Several kinds of stimulants are known to induce inositol1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6) accumulations in intact adrenal chromaffin cells, N1E-115 cells and rat cerebellar granule cells. Recently, we suggested that the binding of inositol polyphosphates to the C2B domain of synaptotagmin may clamp spontaneous fusion of the docked or primed vesicles in adrenal chromaffin cells. In this study, we investigated the CaィイD12+ィエD1-evoked release of InsP5 and InsP6, and the effects of the antibodies against synaptotagmin C2A and C2B domains in digitonin-permeabilized adrenal chromaffin cells. When the cells were stimulated with CaィイD12+ィエD1 (100μM), transient increase of InsP5 and InsP6 in the cytosolic medium were observed. Upon a CaィイD12+ィエD1-challenge, amount of InsP5 and InsP6 in the cytosolic medium reached the maximum at 15 sec. Anti-C2A antibody (C2A Ab) completely inhibited CaィイD12+ィエD1-induced InsP5 and InsP6 accumulation in cytosolic medium. Anti-C2B antibody (C2B Ab) also inhibited CaィイD12+ィエD1-induced InsP5 and InsP6 accumulation in the cytosolic medium. Since pretreatment with C2B Ab during permeabilization, but not with C2A Ab, induced increase of InsP5 and InsP6 in the permeabilizing buffer, the mechanisms of the inhibitory effects of the C2B Ab is different from those of C2A Ab. These results strongly suggest that the binding of increased intracellular CaィイD12+ィエD1 to the C2A domain liberate InsP5 and InsP6 from the C2B domain of synaptotagmin, supporting our idea of the clamp of fusion by synaptotagmin and inositol polyphosphates.

已知几种兴奋剂可诱导完整肾上腺嗜铬细胞、N1 E-115细胞和大鼠小脑颗粒细胞中肌醇1，3，4，5，6-五磷酸（InsP 5）和肌醇六磷酸（InsP 6）的积累。最近，我们提出，肌醇多磷酸的结合突触结合蛋白的C2B结构域可能钳停靠或引发的小泡在肾上腺嗜铬细胞的自发融合。在这项研究中，我们研究了Ca ~（2+）D_12 + Ca ~（2+）D_1诱导的InsP 5和InsP 6的释放，以及抗突触结合蛋白C2 A和C2B结构域的抗体在毛地黄皂苷透化的肾上腺嗜铬细胞中的作用。当用Ca ~（2+）D_1 + Ca ~（2+）D_1（100μM）刺激细胞时，观察到胞浆中InsP_5和InsP_6的瞬时增加。在Ca ~（2+）D_12 + Ca ~（2+）D_1刺激下，细胞内InsP_5和InsP_6的含量在15秒时达到最大值。抗C2 A抗体（C2 A Ab）完全抑制Ca ~（2+）D_12 + Ca ~（2+）D_1诱导的InsP_5和InsP_6在胞液中的积累。抗C2B抗体（C2B Ab）也抑制Ca ~（2+）D_1诱导的InsP_5和InsP_6在胞液中的积累。由于在透化过程中用C2B Ab预处理而不是用C2 A Ab预处理诱导透化缓冲液中InsP 5和InsP 6的增加，因此C2B Ab的抑制作用机制不同于C2 A Ab。这些结果强烈表明，增加的细胞内Ca ~（2+）D_12 + Ca ~（2+）D_1与C2 A结构域的结合将InsP 5和InsP 6从突触结合蛋白的C2B结构域中释放出来，支持我们关于突触结合蛋白和多磷酸肌醇的融合钳的想法。