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

本研究的目的是利用两种通透性细胞模型分析胃壁细胞的信号转导：(1)利用β-ESCIN通透性胃腺模型，我们研究了抑制肽对几种小的GTP结合蛋白的各种激酶和功能结构域的影响，以确定cAMP下游的蛋白质，发现A-激酶下游可能参与了肌球蛋白轻链激酶样酶的作用，而其他激酶的参与很少。在各种小的GTP结合蛋白中，只有Arf有显著的作用。当β-ESCIN通透性腺体的分泌酸被γS抑制时，膜组分中有Arf积聚，提示Arf可能参与了囊泡的转运。这些结果发表在《美国生理杂志》上。(2)我们试图使用洋地黄渗透的腺体提纯胞浆中的基本成分，在胞浆因子丢失的情况下。结合不同的层析技术，我们确定了两个激活因子。其中，较高的一个被鉴定为磷脂酰肌醇转移蛋白。我们还纯化了一个抑制因子，但我们没有对其进行鉴定，因为它似乎是由几个多组分组成的复合体。我们可以提出一种新的激活酸分泌的机制，即刺激引起膜上抑制因子的释放。这些结果发表在两篇论文中，其中一篇现已被《物理学杂志》接受。

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.