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生成的细胞机制 平滑肌收缩和舒张。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传导中的作用 导致平滑肌收缩和舒张的转导途径。 本提案的主要目标是阐明 花生四烯酸（AA）诱导的 Ca ~（2+）增敏在血管平滑肌收缩调节中的作用。 Ca 2+敏化描述了一种兴奋性激动剂 在恒定游离Ca 2+下诱导显著的平滑肌收缩。多 有证据表明，由各种Ca 2+致敏激动剂释放的AA可能 有助于这种G-蛋白介导的Ca 2 +-敏化。但 磷脂酶A2（PLA 2）负责AA的释放，G蛋白， 调节AA的释放，以及AA诱导 Ca 2+敏感性尚不清楚。我们建议测试假设AA， 通过ras介导的Ca 2+非依赖性iPLA 2激活释放，有助于 激动剂通过多位点作用诱导的Ca 2+增敏：rho激酶 （大韩民国）。蛋白激酶C（PKC）和肌球蛋白磷酸酶（MLCP）。具体的目标是 (1)为了检验iPLA 2介导GTP γ S诱导的， 血管平滑肌中钙非依赖性AA释放和钙敏感化。 (2)为了验证ras介导激动剂诱导的激活的假设， iPLA 2和Ca 2+非依赖性AA释放在调节Ca 2+敏感性中的作用 平滑肌收缩(3)为了验证血管平滑肌中 肌肉组织，AA通过作用于多个位点抑制肌球蛋白磷酸酶：ROK， PKC和MLCP。a-毒素或β-七叶皂苷透化的兔股动脉，或 门静脉将用于确定选择性激活的效果， AA途径对激动剂和GTP γ S诱导ras活化的抑制， ROK、PKC、肌球蛋白磷酸酶抑制和力及肌浆细胞Ca ~（2+）敏感化 MLC 20磷酸化。AA途径将被选择性地激活， AA或过表达iPLA 2，并通过使用选择性抑制剂选择性抑制。 iPLA 2抑制剂（BEL）或反义寡核苷酸。 了解血管平滑肌细胞信号转导通路 肌肉将有助于确定心血管疾病的原因。这 信息可以导致战略药物设计和/或分子生物学 针对信使分子的方法， 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.