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Regulation of the intracellular sorting of gastric proton pump in the parietal cell - Analysis using permeabilized cells.

壁细胞胃质子泵细胞内分选的调节 - 使用透化细胞进行分析。

基本信息

批准号：
11672160
负责人：
URUSHIDANI Tetsuro
金额：
$ 2.43万
依托单位：
Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The aim of the present study was to analyze the signal transduction in the gastric parietal cell using two permeabilized cell models.(1) Using β-escin permeabilized gastric gland model, we examined the effects of inhibitory peptides for various kinases and functional domains of several small GTP-binding proteins in order to identify the proteins downstream of cAMP.We found a possible involvement of a myosin light chain kinase-like enzyme was involved in the downstream of A-kinase, whereas the involvement of other kinases was minimum. Among various small GTP-binding proteins, only Arf showed significant effects. When acid secretion by β-escin permeabilized glands was inhibited by GTPγS, Arf was accumulated in the membrane fraction suggesting the possible involvement of Arf in the vesicular transport. These results were published in Am.J.Physiol.(2) We tried to purify the essential components in the cytosol using digitonin-permeabilized glands where cytosolic factors were lost. Combination of various chromatographic technique, we identified two activating factors. Of these, the higher one was identified as phosphatidylinositol transfer protein. We also purified a inhibitory factor, whereas we have not identified it as it appeared to be a complex of several multiple components. We could suggest a novel mechanism of activation of acid secretion that stimulation elicits the release of inhibitory factor from the membrane. These results are featured in two papers one of which is now accepted in Am.J.Physiol.

本研究的目的是利用两种透化细胞模型分析胃壁细胞的信号转导。(1)利用β-七叶皂苷透化的胃腺模型，我们研究了抑制肽对各种激酶和几种小的GTP结合蛋白的功能结构域的影响，以确定cAMP下游的蛋白质，我们发现在A-激酶的下游可能涉及肌球蛋白轻链激酶样酶，而其他激酶的参与是最小的。在各种小GTP结合蛋白中，只有Arf表现出显著的作用。当β-七叶皂苷透化腺体的酸分泌被GTPγS抑制时，Arf在膜组分中积累，表明Arf可能参与囊泡转运。这些结果发表在《美国生理学杂志》上。(2)我们试图用毛地黄皂苷透化的腺体纯化胞质中的必需成分，因为胞质因子在腺体中丢失。结合多种色谱技术，鉴定出两种活化因子。其中，较高的一个被确定为磷脂酰肌醇转移蛋白。我们还纯化了一种抑制因子，但我们尚未鉴定它，因为它似乎是几种多组分的复合物。我们可以提出一种新的机制，激活酸分泌的刺激elaborates释放抑制因子从膜。这些结果在两篇论文中有特色，其中一篇现在被《美国生理学杂志》接受。