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

当激素、神经递质、生长因子等细胞外信号到达细胞质膜时，细胞内就会产生各种活性物质。钙离子现在被认为是所谓的第二信使之一，参与了生命系统的许多生理和病理生理过程。细胞内钙离子的作用可能至少部分是通过钙调素依赖的蛋白激酶II(CaM Kinase II)介导的。CaM激酶II在大脑中的浓度最高，并在钙离子/CaM存在的情况下进行自动磷酸化。自磷酸化使该酶不依赖于钙，因此被认为是该酶的激活。在本研究中，利用原代培养的神经元和已建立的细胞系，如PC12细胞、Neo-2A、3Y1细胞、C6胶质瘤细胞和NG108-15细胞，检测了该酶的活性。The ISOE…对NG108-15细胞中更多的CaM激酶II酶进行了广泛的研究。在10s内，用1um的缓激肽刺激，该酶的钙非依赖性活性(自主活性)增加了一倍。酶的自主活性的提高有两个阶段：短暂的早期高峰期和较长的后期平台期。前者可被10 mM咖啡因或20 mM BAPTA-AM预处理所消除，后者可被1 mM EGTA或1 mM硝苯地平预先去除细胞外钙离子而取消。用1MU缓激肽刺激~&lt；32&gt；P标记的NG108-15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预先阻断。这些结果表明，在NG108-15细胞中，CaM激酶II是通过缓激肽诱导的肌醇磷脂信号通路被激活的。因此，我们能够在完整的细胞中显示酶活性对药物的响应变化，这与钙动员有关。较少

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