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

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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.