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

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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