Molecular and cellular physiological studies on the intracellular signal transduction causing abnormal vascular contraction

获得了以下结果：1.第一年：我们对可能介导信号从细胞膜到胞质信使的信号分子Rho激酶进行了筛选。我们确定了一种鞘脂，鞘氨醇磷酸胆碱（SPC）作为一种新的信使的钙离子非依赖性收缩的血管平滑肌（VSM）介导的Ryo激酶。此外，我们还发现SPC在不存在胞浆GTP的情况下，可诱导透化的VSM条的非Ca ~（2+）依赖性收缩，这一作用可被Rho激酶抑制剂阻断，表明鞘脂诱导的收缩是由Rho激酶介导的。Fura-2荧光测定显示鞘脂诱导的收缩不增加胞浆Ca ~（2+）浓度（[Ca 2 +]i），支持鞘脂作为Ca 2+非依赖性收缩介质的作用.第二年：为了证实Rho激酶参与SPC诱导的Ca 2+非依赖性逆转录酶， 关于我们 在细胞质中引入显性负性Rho激酶，可完全阻断SPC引起的非Ca ~（2+）依赖性收缩。此外，常规和新型蛋白激酶C（PKC）的假底物肽对SPC诱导的收缩没有影响，而它消除了PKC激活剂佛波酯诱导的Ca ~（2+）非依赖性收缩。这些结果表明SPC诱导的VSM非钙依赖性收缩是由Rho激酶介导的，而不是由PKC介导的.第三年：我们开始研究人类和哺乳动物VSM条的Ca 2+非依赖性收缩。从胆固醇水平正常的患者或兔获得的鞘脂诱导非常小的收缩，但从胆固醇水平高的患者或兔获得的条诱导大的收缩。此外，SPC引起的收缩依赖于VSM中胆固醇的组织含量。这些发现表明，鞘脂/Rho激酶途径在VSM异常收缩的发展中起着重要作用，这通常与高脂血症有关。少

The following results were obtained : 1. FIRST YEAR : We performed the screening for on the signal molecule(s) which may mediate signal transduction from the cell membrane to cytosolic messenger, Rho-kinase. We identified a sphingolipid, sphingosylphosphorylcholine (SPC) as a novel messenger for the Ca2 + -independent contraction of vascular smooth muscle (VSM) mediated by Ryo-kinase. In addition, we found that SPC induces Ca2 + -independent contraction in the absence of cytosolic GTP in permeabilized VSM 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 Ca2 + concentration ([Ca2 +]i) in cerebral arteries, supporting the role of sphingolipid as a mediator for the Ca2 + -independent contraction.2. SECOND YEAR : In order to confirm the involvement of Rho-kinase in the SPC-induced Ca2 + -independent contra … More ction of VSM, dominant negative Rho-kinase was introduced into the cytosol of the peremeabilized VSM strips, and it completely abolished the Ca2 + -independent contraction induced by SPC. In addition, pseudosubstrate peptide of conventional and novel protein kinase C (PKC) had no effect on the SPC-induced contraction , whereas it abolished the Ca2 + -independent contraction induced by a PKC activator, phorbol ester. These results indicate that SPC induces Ca2 + -independent contraction of VSM which is mediated by Rho-kinase, but not by PKC.3. THIRD YEAR : We started to investigate the Ca2 + -independent contraction of human and mammalian VSM strips. Sphingolipid induced very small contraction obtained from patients or rabbit whose cholesterol levels are normal, but induced large contraction of the strips obtained from patients or rabbit whose cholesterol levels are high. In addition, the SPC-induced contraction depended on the tissue content of cholesterol in VSM. These finding suggest that a sphingoliopid/Rho-kinase pathway plays an important role in the development of abnormal VSM contraction which was frequently associated with hyperlipidemia. Less