Development of the method(s) to monitor cardiovascular function and its intracellular signal transduction

以下结果已获得：1。第一年：我们开发了监测心血管功能及其内部信号转换的方法。我们引入了一种快速重组蛋白，将细胞醇引入血管光滑麝香条，并使用受体偶联膜渗透。如果使用这个新系统，我们将Rho-kinase识别为Ca-y D12+ y D1-独立于血管光滑麝香的新型信使。This conclusion was supported by the following results.1）Recombinant catManagement tic domain of Rho-kinase is constitutively active and induced the CaイイD12+イエD1-independent contraction of permeManagement ized vascular smooth muscle in the absence of cytosolic GTE。（2）Rho-kanase诱导的接触与肌红蛋白光链磷酸化的提升有关。（3）Rho-kinase-诱导的收缩和增加在myosin光链磷酸化对myosin光链激酶阻断器具有抵抗力。第二年：我们投资信号转换基金 ... More M细胞膜是细胞化信使（Rho-kinase）。We identified several sphingolipids as a novel mediator for the CaイD12+イエD1-independent contraction of vascluar smooth muscle。在添加中，我们发现了鞘脂化合物诱导的Cai-D12 + Yi-D1-独立契约在渗透性血管光滑麝香条中的缺失细胞质GTP，其中被Rho-kinase抑制剂阻断，建议鞘脂诱导的契约是由Rho-kinase介导的。Fluorometry of fura-2 revealed that sphingolipids induced large contraction without elevation of cytosolic Ca D12+ Cytosolic Ca D1 concentration （[Ca D12+ Cytosolic D1]i） in Oracle ebral arteries， supporting the role of sphingolipid as a mediator for the Ca D12+ Cytosolic D1-independent contraction.3。THIRD TEAR ： We started to investigate the CaイイD12+イエD1-independent contraction of human vas？smManagement muscle strips。Sphingolipid诱导了非常小的接触，从患者的胆固醇水平是正常的，但从患者的胆固醇水平较高的条带诱导了较大的接触。这些发现的建议是，一个鞘脂/Rho-kinase路径在非正常血管收缩的发展中发挥了一个重要的作用，其中一个经常与高脂蛋白有关。Less（低）

The following results were obtained :1. FIRST YEAR : We developed the method to monitor cardiovascular function and its intracellular signal transduction. We introduced rapidly recombinant proteins into the cytosol of vascular smooth muscle strips, using the receptor-coupled membrane permeabilization. Using this new system, we identified Rho-kinase as a novel messenger for the CaィイD12+ィエD1-independent contraction of vascular smooth muscle. This conclusion was supported by the following results.1)Recombinant catalytic domain of Rho-kinase is constitutively active and induced the CaィイD12+ィエD1-independent contraction of permeabilized vascular smooth muscle in the absence of cytosolic GTE. 2) Rho-kanase-induced contraction was associated with the elevation of myosin light chain phosphorylation. 3) Rho-kinase-induced contraction and increase in the myosin light chain phoshorylation were resistant to a myosin light chain kinase blocker.2. SECOND YEAR : We investigated signal transduction fro … More m the cell membrane to cytosolic messenger (Rho-kinase). We identified several sphingolipids as a novel mediator for the CaィイD12+ィエD1-independent contraction of vascluar smooth muscle. In addition, we found that the sphingolipids induces CaィイD12+ィエD1-independent contraction in the absence of cytosolic GTP in permeabilized vascular smooth muscle strips, which was blocked by a Rho-kinase inhibitor, suggesting that sphingolipid-induced contraction is mediated by Rho-kinase. Fluorometry of fura-2 revealed that sphingolipids induced large contraction without elevation of cytosolic CaィイD12+ィエD1 concentration ([CaィイD12+ィエD1]i) in cerebral arteries, supporting the role of sphingolipid as a mediator for the CaィイD12+ィエD1-independent contraction.3. THIRD TEAR : We started to investigate the CaィイD12+ィエD1-independent contraction of human vascular smooth muscle strips. Sphingolipid induced very small contraction obtained from patients whose cholesterol levels are normal, but induced large contraction of the strips obtained from patients whose cholesterol levels are high. These finding suggest that a sphingolipid/Rho-kinase pathway plays an important role in the development of abnormal vascular contraction which was frequently associated with hyperlipidemia. Less