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

following results were obtained:1.第一年：我们开发了monitor cardiovascular function and its intracellular signal的方法transduction. We introduced rapidly recombinant proteins into the cytosol of vascular smooth musclestrips, using the receptor-coupled membrane permeabilization. using this new system，we identified Rho-kinase as a novel messenger for the Ca D12+ dj -independent contraction ofvascular smooth muscle. This conclusion was supported by the following results.1)混合组合catalytic domain of Rho-kinase is constitutively active and induced the Ca D12+ D1-independentcontraction of permeabilized vascular smooth muscle in the absence of cytosolic GTE. 2)Rho-kanase-induced contraction was associated with the elevation of myosin light chainphosphorylation. 3) Rho-kinase-induced contraction and increase in the myosin light chainphoshorylation were resistant to a myosin light chain kinase blocker.2. SECOND YEAR：我们投资信号transduction fro细胞membrane to cytosolic messenger (Rho-kinase). We identified several More m the cell membrane to cytosolic messengersphingolipids as a novel mediator for the Ca D12+ D1-independent contraction of vascluar smoothmuscle. In addition，we found that the sphingolipids induces Ca我们发现了D12+ D1-independent contraction in the absence ofcytosolic GTP in permeabilized vascular smooth muscle stripswhich was blocked by a Rho-kinase inhibitorsuggesting sphingolipid-induced contraction is mediated by Rho-kinase. Fluorometry of fura-2revealed that sphingolipids induced large contraction without elevation of cytosolic Ca D12+ D12concentration ([Ca D12+ d D1]i) in cerebral arteries，supporting the role of sphingolipid as a mediator for the Ca D12+ dependent contraction.3.THIRD TEAR：我们开始投资Ca D12+ D1-independent contraction of human vascular smooth muscleSphingolipid induced very small contraction obtained from patients whose cholesterol levelsare normal,but induced large contraction of the strips obtained from patients whose cholesterol level arehigh. These finding suggest that a sphingolipid/Rho-kinase pathway plays an important role in thedevelopment of abnormal vascular contraction was常quently associated with hyperlipidemia.less

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