Molecular pharmacological approarches of intracellular calcium regulatory mechanisms

本研究的目的是研究肌球蛋白轻链（MLC）磷酸化在血管收缩和血小板功能中的作用，通过直接药理操作MLC激酶，因为我们已经开发了一系列新的细胞内Ca^<2+>信使系统抑制剂。我们发现新合成的化合物1-（5-氯代萘-1-磺酰基）- 1h -六氢-1,4-二氮平（ML-9）是一种直接选择性的mlc激酶抑制剂，Ki值为3.8 M，对牛主动脉肌动球蛋白的ATP超沉淀具有竞争性抑制作用，且ML-9的加入对肌动球蛋白mg -ATP酶活性呈剂量依赖性抑制。在兔肠系膜动脉化学皮肤平滑肌细胞中，ML-9抑制Ca^<2+>-和Ca^<2+>-独立的mlc激酶诱导的收缩。在完整的血管条中，ML-9抑制了kcl诱导的收缩，同时抑制了20-kDa MLC磷酸化。用两种不同的凝胶电泳方法在凝血酶刺激的人血小板中证实了单磷酸化和二磷酸化的20 kda MLC。较快速的单磷酸化是由mlc激酶催化的，而较慢的磷酸化主要是由蛋白激酶C.3催化的。肌球蛋白的磷酸化和磷酸化速率与肌球蛋白构象的变化密切相关。这些结果提示Ca^<2+>，钙调素依赖性MLC磷酸化在调节血管收缩活性和血小板功能中的重要意义。

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.