Molecular cytobiological study on CaィイD12+ィエD1 signaling in the cells with cultured cells

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Cell stimulants such as nerve impulses, neurotransmitters, active peptides, hormones, autacoids, cytokines, growth factors etc stimulate receptors and ion channels in plasma membranes, and mediate extracellular information into cells. Consequently, second messengers are produced in the cells. Intracellular calcium ion (CaィイD12+ィエD1) increases in the cells by stimulation. CaィイD12+ィエD1 is now established to be one of second messengers and involved in many cellular functions such as various metabolic reactions, biosynthesis of neurotransmitters, stimulus-secretion coupling, stimulus-contraction coupling, neuronal plasticity, cellular morphology, axonal flow, membrane ion conductance, down-regulation of receptors and gene expression. A low concentration of intracellular CaィイD12+ィエD1 is mainly regulated by CaィイD12+ィエD1 channels and pumps. Homeostasis of intracellular CaィイD12+ィエD1 is strictly maintained.In the present study, we attempted to elucidate cellular functions through CaィイD12+ィエD1 signaling, using primary cultures such as hippocampal neurons, cerebral neurons, cerebellar granule cells and astrocytes and established cell lines such as PC12 cells, NG108-15 cells and MIN6 cells. We found that CaM kinases II and IV are activated through elevation of intracellular CaィイD12+ィエD1 in response to the extracellular stimuli such as glutamate. CaM kinase II is localized in both the cytosol and nucleus, while CaM kinase IV is exclusively localized in the nucleus. Addition of cyclosporin A increased glutamate-induced phosphorylation of CaM kinase IV, but did not increase autophosphorylation of CaM kinase II. GST-CaM kinase IV phosphorylated by CaM kinase kinase was dephosphorylated by protein phosphatases 1, 2A and 2C and calcineurin. We characterized the isoforms of CaM kinase II and synapsin I in MIN6 cells. We continuously have a plan to study on in situ dynamic activation of the enzymes in response to receptor stimulation with concomitant cellular functions.