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The Cytophamacological Study on the Responses of the Receptors in the Cell System

细胞系统受体反应的细胞病理学研究

基本信息

批准号：
02454139
负责人：
MIYAMOTO Eishichi
金额：
$ 3.97万
依托单位：
KUMAMOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

When the extracellular signals such as hormones, neurotransmitters, growth factors and so on reach the plasma membranes of the cells, a variety of active substances are produced in the cells. Calcium ion (Ca^<2+>) is now considered to be one of so called second messengers and involved in many physiological and pathophysiological processes of the living systems. The effects of Ca^<2+> in the cells may at least partly be mediated by Ca^<2_>/calmodulin-dependent protein kinase II (CaM kinase II). CaM kinase II is present at the highest concentration in the brain and undergoes autophosphorylation in the presence of Ca^<2+>/CaM. The autophosphorylation renders the enzyme Ca^<2+>-independent and is therefore considered to be the activation of the enzyme. In the present study, the activation of the enzyme was examined using the cultured cells such as the primary culture of neurons and the established cell lines such as PC12 cells, neuro-2A, 3Y1 cell, C6 gliona cell and NG108-15 cell. The isoe … More nzyme of CaM kinase II in NG108-15 cells were extensively studied. The Ca^<2+>/CaM-independent activity (autonomous activity) of the enzyme increased twice within 10 sec by stimulation with 1 muM bradykinin in the cells. The increase in the autonomous activity of the enzyme had two phases; the transient early-peak phase and the long late-plateau phase. The former was abolished by the pretreatment of the cells with 10 mM caffeine or 20 muM BAPTA-AM, and the latter was abolished by the removal of the extracellular Ca^<2+> with 1 mM EGTA or by the pretreatment with 1 muM nifedipine. Stimulation of ^<32>P-labeled NG108-15 cells with 1 muM bradykinin increased the autophosphorylation of CaM kinase II and this increase was abolished by pretreatment with caffeine or BAPTA-AM. These results suggest that CaM kinase II is activated via the inositol phospholipid signaling pathway induced with bradykinin in NG108-15 cells. Thus we were able to show the change in the enzyme activity in response to the drugs in intact cells, which was coupled to the Ca^<2+> mobilization. Less

当激素、神经递质、生长因子等细胞外信号到达细胞的质膜时，细胞内产生多种活性物质。钙离子（Ca^<2+>）目前被认为是第二信使之一，参与生命系统的许多生理和病理生理过程。Ca^<2+>在细胞中的作用可能至少部分由Ca^<2_>/钙调素依赖性蛋白激酶II （calmodulin-dependent protein kinase II, CaM kinase II）介导。CaM激酶II在大脑中浓度最高，在Ca^<2+>/CaM存在时发生自磷酸化。自磷酸化使酶Ca^<2+>-独立，因此被认为是酶的激活。本研究以神经元原代培养细胞和PC12细胞、神经2a细胞、3Y1细胞、C6胶质细胞、NG108-15细胞等已建立的细胞系为研究对象，考察了该酶的活化情况。本文对NG108-15细胞中CaM激酶II酶活性进行了广泛的研究。在1 muM缓激肽的刺激下，该酶的Ca^<2+>/ cam独立活性（自主活性）在10秒内增加了2倍。酶自主活性的提高有两个阶段；短暂的峰前阶段和漫长的高原后阶段。前者可通过10 mM咖啡因或20 muM BAPTA-AM预处理细胞而消除，后者可通过1 mM EGTA或1 muM硝苯地平预处理细胞外Ca^<2+>而消除。用1mum缓激肽刺激^<32> p标记的NG108-15细胞可增加CaM激酶II的自磷酸化，而用咖啡因或BAPTA-AM预处理可消除这种增加。这些结果表明，在NG108-15细胞中，CaM激酶II通过缓激肽诱导的肌醇磷脂信号通路被激活。因此，我们能够在完整细胞中显示响应药物的酶活性的变化，这与Ca^<2+>的动员有关。少