Molecular pharmacological approarches of intracellular calcium regulatory mechanisms

本研究的目的是利用药物直接调控肌球蛋白轻链（myosin light chain，MLC）激酶，研究MLC磷酸化在血管收缩和血小板功能中的作用，因为我们已经开发了一系列新型的细胞内Ca^<2+>信使系统抑制剂.发现一种新合成的化合物1-（5-chloronaphthalene-1-sulfonyl）-1H-hexahydro-1，4-diazepine（ML-9）是一种直接的、选择性的MLC-激酶抑制剂，Ki值为3.8M，其抑制作用与ATP超沉淀和牛主动脉肌动球蛋白Mg-ATP酶活性有关，并呈剂量依赖性。在化学去皮的兔肠系膜动脉平滑肌细胞中，ML-9抑制Ca^<2+>和Ca^<2+>非依赖性MLC激酶诱导的收缩。在完整的血管条中，ML-9抑制KCl诱导的收缩，同时抑制20-kDa MLC磷酸化.单磷酸化和二磷酸化的20-kDa MLC证明在凝血酶刺激的人血小板通过两种不同的凝胶电泳方法。更快速的单磷酸化是由MLC-激酶催化的，而较慢的和额外的磷酸化主要是由蛋白激酶C催化的。肌球蛋白的磷酸化和二磷酸化速率与肌球蛋白构象的变化密切相关.这些结果表明，Ca^<2+>、钙调素依赖的MLC磷酸化在调节血管收缩活动和血小板功能中具有重要意义。

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.