Molecular pharmacological approarches of intracellular calcium regulatory mechanisms

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The purpose of this research is investigate the role of myosin light chain (MLC) phosphorylation in vascular contraction and platelet function with direct pharmacological manipulation of MLC-kinase, since we have developed a series of novel inhibitors of intracellular Ca^<2+> messenger system.1. We found that a newly synthesized compound, 1-(5-chrolonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) is a direct and selective inhibitor of MLC-kinase,with Ki value 3.8 M, and its inhibition was of the competitive type with respect to ATP Superprecipitation and Mg-ATPase activity of actomyosin from bovine aorta was inhibited by the addition of ML-9 in a dose-dependent manner. In chemically skinned smooth muscle cells of rabbit mesenteric artery, ML-9 inhibited the both Ca^<2+>- and Ca^<2+>-independent MLC-kinase-induced contraction. In the intact vascular strips, ML-9 suppressed the KCl-induced contraction concomitant with the inhibition 20-kDa MLC phosphorylation.2. Monophosphorylated and diphosphorylated 20-kDa MLC were demonstrated in thrombin-stimulated human platelets by two different gel electrophoretic methods. The more rapid monophosphorylation was catalyzed by MLC-kinase while the slower and additional phosphorylation was catalyzed mainly by protein kinase C.3. The rate of phosphorylation and daphosphorylation of myosin was closely related to the change of myosin conformation.4. These results suggest the significance of Ca^<2+>, calmodulin-depenpent MLC phosphorylation in the regulation of vascular contractile activity and platelet function.