Role of Myosin Light-Chain Kinase in the Regulation of Endothelial Functions and Calcium Signaling

肌球蛋白轻链激酶在内皮功能和钙信号传导调节中的作用

• 批准号: 10670642
• 负责人: WATANABE Hiroshi
• 金额: $ 2.11万
• 依托单位: Hamamatsu University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670642/
• 关键词: Myosin light chain kinase; Endothelial cell; Calcium; Nitric Oxide; Endothelium-derived hyperpolarizing factor; 一酸化窒素; 内皮依存性過分極反応; 内皮依存性血管拡張因子

Activation of smooth muscle myosin light-chain kinase (MLCK) causes contraction. Here we have proven that MLCK controls Ca^<2+> entry (CE) in endothelial cells (ECs) : MLCK antisense oligonucleotides strongly prevented bradykinin (BK)- and thapsigargin (TG)-induced endothelial Ca^<2+> response while MLCK sense did not. We also show that the relevant mechanism is not phosphorylation of myosin light-chain (MLC) : MLC phosphorylation by BK required CE, but MLC phosphorylation caused by the phosphatase inhibitor calyculin A did not trigger Ca^<2+> response. Most importantly, we provide for the first time strong evidence that, in contrast to its role in smooth muscle cells, activation of MLCK in ECs stimulates the production of important endothelium-derived vascular relaxing factors : MLCK antisense and MLCK inhibitors abolished BK- and TG-induced nitric oxide production, and MLCK inhibitors substantially inhibited acetylcholine-stimulated hyperpolarization of smooth muscle cell membrane in rat mesenteric artery. These results indicate that MLCK controls endothelial CE, but not through MLC phosphorylation, and unveil a hitherto unknown physiological function of the enzyme : vasodilation through its action in endothelial cells. The study discovers a counter-balancing role of MLCK in the regulation of vascular tone.

平滑肌肌球蛋白轻链激酶 (MLCK) 的激活会导致收缩。在这里，我们已经证明MLCK控制内皮细胞(EC)中的Ca^2+进入(CE)：MLCK反义寡核苷酸强烈阻止缓激肽(BK)和毒胡萝卜素(TG)诱导的内皮Ca^2+反应，而MLCK有义则不然。我们还表明，相关机制不是肌球蛋白轻链(MLC)的磷酸化：BK引起的MLC磷酸化需要CE，但磷酸酶抑制剂calyculin A引起的MLC磷酸化不会触发Ca ^ 2+ 反应。最重要的是，我们首次提供强有力的证据表明，与其在平滑肌细胞中的作用相反，EC 中 MLCK 的激活刺激重要的内皮源性血管舒张因子的产生：MLCK 反义和 MLCK 抑制剂消除了 BK 和 TG 诱导的一氧化氮产生，MLCK 抑制剂显着抑制乙酰胆碱刺激的一氧化氮产生。 大鼠肠系膜动脉平滑肌细胞膜超极化。这些结果表明MLCK控制内皮细胞CE，但不是通过MLC磷酸化，并揭示了该酶迄今为止未知的生理功能：通过其在内皮细胞中的作用来舒张血管。该研究发现 MLCK 在血管张力调节中具有平衡作用。

项目成果

Kimura M.,Watanabe H.,Takahashi R.,Kosuge K.,Umemura K.,Hayashi H.,Ohashi K.,Ohno R.: "Inhibitory effect of insulin on bradykinin-induced venodilation."J.Hypertens.. 18. 287-292 (2000)

Kimura M.、Watanabe H.、Takahashi R.、Kosuge K.、Umemura K.、Hayashi H.、Ohashi K.、Ohno R.：“胰岛素对缓激肽诱导的静脉扩张的抑制作用。”J.Hypertens.. 18

Watanabe H.,Takahashi R.,Tran QK.,Takeuchi K.,Kosuge K.,Satoh H.,Uehara A.,Terada H.,Hayashi H.,Ohno R.,Ohashi K.: "Increased cytosolic Ca2+ concentration in endothelial cells by calmodulin antagonists."Biochem.Biophys.Res.Commun.. 265. 697-702 (1999)

Watanabe H.、Takahashi R.、Tran QK.、Takeuchi K.、Kosuge K.、Satoh H.、Uehara A.、Terada H.、Hayashi H.、Ohno R.、Ohashi K.：“细胞溶质 Ca2 浓度增加

Tran QK.,Ohashi K.,Watanabe H.: "Calcium signalling in endothelial cells.(Review)"Cardiovasc.Res.. 48. 13-22 (2000)

Tran QK.、Ohashi K.、Watanabe H.：“内皮细胞中的钙信号传导。（综述）”Cardiovasc.Res.. 48. 13-22 (2000)

Watanabe H.,Tran QK.,Takeuchi K.,Fukao M.,Liu MY.,Kanno M.,Hayashi T.,Iguchi A.,Seto M.,Ohashi K: "Myosin light-chain kinase regulates endothelial calcium entry and endothelium-dependent vasodilation."FASEB J.. 15. 282-284 (2001)

Watanabe H.，Tran QK.，Takeuchi K.，Fukao M.，Liu MY.，Kanno M.，Hayashi T.，Iguchi A.，Seto M.，Ohashi K：“肌球蛋白轻链激酶调节内皮钙进入和

Tran QK., Ohashi K., Watanabe H.: "Calcium signalling in endothelial cells."Cardiovasc.Res.. 48. 13-22 (2000)

Tran QK.、Ohashi K.、Watanabe H.：“内皮细胞中的钙信号传导”。Cardiovasc.Res.. 48. 13-22 (2000)