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The Role of Protein Phosphorylation in Amylase Exocytosis from the Parotid Gland.

蛋白质磷酸化在腮腺淀粉酶胞吐作用中的作用。

基本信息

批准号：
01571024
负责人：
TAKUMA Taishin
金额：
$ 1.15万
依托单位：
Higashi Nippon Gakuen University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

Amylase release from parotid acinar cells has been extensively studied as a useful model of cAMP-dependent exocytosis. A crucial role of cAMP-dependent protein kinase in the exocytosis has long been postulated, since the activation of the protein kinase and the phosphorylation of some proteins were observed concurrently with amylase release evoked by beta-adrenergic agonists. However, studies using protein kinase inhibitors (H-8 or heat-stable protein kinase inhibitor) suggested that cAMP-dependent protein phosphorylation is not directly involved in the exocytosis, since the inhibitors mark edly reduced protein phosphorylation without decreasing amylase release. To determine the role of cAMP-dependent protein kinase in the exocytosis, I studied the effects of various cAMP analogues on amylase release and protein phosphorylation in saponin-permeabilized parotid acini. The dose-dependent responses of amylase release and protein phosphorylation evoked by various analogues were closely correlated. The combination of site-selective cAMP analogues synergistically increased amylase release. The cAMP antagonist Rp-cAMPS (Rp-diastereomer of adenosine 3 ; , 5 ; -phosphorothioate) did not stimulate amylase release and inhibited amylase release stimulated by Sp-cAMPS, the Sp-diastereoisomer. These results strongly support the involvement of cAMP-dependent protein kinase in the process of exocytosis. Next I studied the effects of okadaic acid, a potent selective inhibitor of protein phosphatase types 1 and 2A, on amylase release and protein phosphorylation. Okadaic acid by itself weakly stimulated amylase release, but markedly inhibited rather than potentiated amylase release evoked by cAMP. Okadaic acid increased both cAMP-dependent and -independent protein phosphorylation. These results suggest that nonspecific increase in protein phosphorylation does not necessarily enhance amylase release, and rather suppresses full activation of the exocytosis.

腮腺腺泡细胞淀粉酶释放作为cAMP依赖性胞吐作用的一个有用模型已被广泛研究。cAMP依赖性蛋白激酶在胞吐中的关键作用一直被假定，因为蛋白激酶的激活和一些蛋白的磷酸化与β-肾上腺素能激动剂引起的淀粉酶释放同时观察到。然而，使用蛋白激酶抑制剂（H-8或热稳定蛋白激酶抑制剂）的研究表明cAMP依赖性蛋白磷酸化不直接参与胞吐，因为抑制剂显著降低蛋白磷酸化而不降低淀粉酶释放。为了确定cAMP依赖性蛋白激酶在胞吐中的作用，我研究了各种cAMP类似物对皂苷透化腮腺腺泡中淀粉酶释放和蛋白磷酸化的影响。各种类似物引起的淀粉酶释放和蛋白磷酸化的剂量依赖性反应密切相关。位点选择性cAMP类似物的组合协同增加淀粉酶释放。cAMP拮抗剂Rp-cAMPS（腺苷3 ;，5 ; -硫代磷酸酯的Rp-非对映体）不刺激淀粉酶释放，并抑制由Sp-cAMPS（Sp-非对映体）刺激的淀粉酶释放。这些结果强烈支持cAMP依赖性蛋白激酶参与胞吐过程。接下来，我研究了冈田酸（一种1型和2A型蛋白磷酸酶的有效选择性抑制剂）对淀粉酶释放和蛋白磷酸化的影响。冈田酸本身弱刺激淀粉酶的释放，但显着抑制，而不是增强cAMP诱发的淀粉酶释放。冈田酸增加cAMP依赖性和非依赖性蛋白磷酸化。这些结果表明，蛋白磷酸化的非特异性增加不一定增强淀粉酶的释放，而是抑制胞吐的完全激活。