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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质膜时，细胞内产生多种活性物质。钙离子（Ca^<2+>）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<2_>/钙调蛋白依赖性蛋白激酶II（CaM激酶II）介导。CaM激酶II在脑中的浓度最高，在Ca^<2+>/CaM存在时发生自磷酸化。自磷酸化使酶不依赖于Ca^2+，因此被认为是酶的活化。在本研究中，使用培养的细胞如神经元的原代培养物和建立的细胞系如PC 12细胞、neuro-2A、3 Y1细胞、C6胶质瘤细胞和NG 108 -15细胞检测酶的活化。isoe ...更多信息对NG 108 -15细胞中CaM激酶Ⅱ的酶活性进行了研究。用1 μ M缓激肽刺激细胞，10秒内酶的Ca^<2+>/CaM非依赖性活性（自主活性）增加两倍。该酶的自主活性的增加有两个阶段;短暂的早期峰值阶段和长期的后期平台阶段。用10 mM咖啡因或20 μ M BAPTA-AM预处理细胞可消除前者，用1 mM EGTA去除细胞外Ca^<2+>或用1 μ M硝苯地平预处理细胞可消除后者。用<32>1 μ M缓激肽刺激13 P标记的NG 108 -15细胞，可增加CaM激酶II的自磷酸化，这种增加可被咖啡因或BAPTA-AM预处理所消除。这些结果表明，钙调蛋白激酶II激活通过肌醇磷脂信号通路诱导缓激肽在NG 108 -15细胞。因此，我们能够显示完整细胞中酶活性对药物的反应变化，这与Ca^2+动员有关。少