Regulation of Vascular Smooth Muscle Contracting

描述(由申请人提供)：本研究的总体目标是 阐明介导激动剂刺激的血管的细胞机制 平滑肌肉的收缩和松弛。为了实现这些目标，我们必须 识别特定的信号分子并破译它们在信号中的作用 导致平滑肌肉收缩和松弛的转导通路。 当前提案的主要目标是阐明分子 花生四烯酸(AA)诱导的机制及生理意义 钙敏化在血管平滑肌收缩调节中的作用。 钙敏化描述了一种兴奋性激动剂 在恒定的游离钙离子作用下，可引起显著的平滑肌收缩。大有可为 有证据表明，由各种钙敏化激动剂释放的AA可能 有助于这种G-蛋白介导的钙敏化。然而， 磷脂酶A2(PLA2)负责AA的释放，G蛋白 调节AA的释放，以及AA诱导的分子机制 钙离子敏化作用仍不清楚。我们建议检验这样的假设：AA， 由ras介导的钙非依赖性iPLA2的激活释放，有助于 激动剂诱导的钙敏化作用于多个位点：Rho-Kinase (韩国)。蛋白激酶C(PKC)和肌球蛋白磷酸酶(MLCP)。具体目标是 (1)检验iPLA2介导GTP-GammaS诱导的假设， 血管平滑肌钙非依赖性AA释放和钙敏化。 (2)检验ras介导激动剂诱导的血管紧张素转换酶激活的假说。 IPLA2和钙非依赖性AA释放在钙敏感性调节中的作用 平滑的肌肉收缩。(3)检验假设在血管顺畅 肌肉组织，AA通过作用于多个部位抑制肌球蛋白磷酸酶：韩国， PKC和MLCP。A-毒素或β-七叶皂苷钠穿透兔股动脉或 门静脉将被用来确定选择性激活和 AA通路对激动剂和GTP-GammaS诱导的ras激活的抑制作用 ROK，PKC，肌球蛋白磷酸酶抑制和钙敏化的力和 MLC20磷酸化。添加游离将选择性地激活AA途径 AA或过表达iPLA2，并通过使用选择性的 IPLA2抑制剂(BEL)或反义寡核苷酸。 了解血管平滑过程中的细胞信号转导途径 肌肉将有助于确定心血管疾病的病因(S)。这 信息可能导致战略性药物设计和/或分子生物学 针对信使分子的方法提供新的治疗方法 心血管疾病。

DESCRIPTION (provided by the applicant): The overall aim of this research is to elucidate the cellular mechanisms that mediate agonist-stimulated vascular smooth muscle contraction and relaxation. To achieve these goals, we must identify the specific signaling molecules and decipher their role in signal transduction pathways that lead to smooth muscle contraction and relaxation. Major objectives of the current proposal are to elucidate the molecular mechanisms and physiological significance of arachidonic acid (AA)-induced Ca2+-sensitization in regulation of vascular smooth muscle contraction. Ca2+-sensitization describes a mechanism through which an excitatory agonist induces significant smooth muscle contraction under constant free Ca2+. Much evidence indicates that AA, released by various Ca2+-sensitizing agonists, may contribute to this G-protein mediated Ca2+-sensitization. However, the phospholipase A2 (PLA2) responsible for AA release, the G protein that regulates AA release, and the molecular mechanism through which AA induces Ca2+-sensitization remain unclear. We propose to test the hypothesis that AA, released by ras-mediated activation of Ca2+-independent iPLA2, contributes to the agonist-induced Ca2+-sensitization by acting at multiple sites: rho-kinase (ROK). protein kinase C (PKC) and myosin phosphatase (MLCP). Specific aims are (1) To test the hypothesis that iPLA2 mediates the GTPgammaS-induced, Ca2+-independent AA release and Ca2+-sensitization in vascular smooth muscle. (2) To test the hypothesis that ras mediates agonist-induced activation of iPLA2 and Ca2+-independent AA release in regulation of Ca2+-sensitization of smooth muscle contraction. (3) To test the hypothesis that in vascular smooth muscle tissue, AA inhibits myosin phosphatase by acting at multiple sites: ROK, PKC and MLCP. a-toxin or beta-escin-permeabilized rabbit femoral arteries or portal veins will be used to determine the effects of selective activation and inhibition of AA pathway on agonist- and GTPgammaS-induced activation of ras, ROK, PKC, inhibition of myosin phosphatase and Ca2+-sensitization of force and MLC20 phosphorylation. AA pathway will be selectively activated by adding free AA or overexpressing iPLA2, and selectively inhibited by using a selective iPLA2 inhibitor (BEL) or antisense oligonucleotide. Understanding the cellular signal transduction pathways in vascular smooth muscle will help to identify the cause(s) of cardiovascular diseases. This information may lead to strategic drug design and/or molecular biological approaches targeted at messenger molecules that provide new treatments for cardiovascular diseases.