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Calcium entry and vascular smooth muscle excitation

钙进入和血管平滑肌兴奋

基本信息

批准号：
7058719
负责人：
KENNETH L BYRON
金额：
$ 32.52万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-06-01 至 2008-05-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Vasoconstrictor hormones cause contraction of vascular smooth muscle (VSM) cells by increasing cytosolic free Ca2+ concentration ([Ca2+]i). The best characterized signal transduction pathway leading to elevation of [Ca2+]i involves inositol trisphosphate-mediated release of Ca2+ from the sarcoplasmic reticulum (SR) and Ca2+ entry via non-selective calcium-permeable cation channels. Voltage-sensitive L-type Ca2+ channels are also known to be important in vasoconstrictor action, though the signaling pathways leading to activation of L-type channels are not well characterized. We have previously demonstrated that physiological concentrations of [Arg8]-vasopressin (AVP, 10-100 pM) induce Ca2+ spiking in A7r5 VSM cells by a novel signaling pathway that leads to activation of L-type Ca2+ channels. Membrane depolarization is presumably required for L-type Ca2+ channel activation. We hypothesize, that AVP exerts a depolarizing effect on the smooth muscle cells. Among the most likely candidates to elicit this effect are non-selective cation channels. We have previously provided evidence for two distinct non-L-type divalent cation entry pathways activated by AVP. The molecular natures of the channels that mediate these effects are not known, but members of the transient receptor potential (TRP) family of non-selective cation channels have been implicated. The present proposal seeks to examine the hypothesis that these divalent cation entry pathways play an important role in the Ca2+ spiking response to physiological AVP concentrations. The specific aims are to: 1) identify which TRP channel homologues are expressed in A7r5 cells and rat mesenteric artery smooth muscle cells (RMASMC) using RT-PCR and Western blotting; 2) characterize the non-selective cation currents activated by 100 pM AVP in A7r5 cells and RMASMC and evaluate their contribution to membrane potential changes using patch clamp techniques; 3) dissociate "capacitative" store-operated Ca2+ entry from non-capacitative AVP-stimulated Ca2+ entry pathways and determine which is/are important in triggering Ca2+ spiking; 4) knock out or overexpress specific TRP channel homologues to determine their role in AVP-stimulated Ca2+ signaling and their relationship to AVP-stimulated nonselective cation currents and Ca2+ entry pathways.

描述(由申请人提供)：血管收缩激素通过增加细胞内游离钙离子浓度([钙]i)引起血管平滑肌(VSM)细胞收缩。最具特征性的导致[Ca~(2+)]i升高的信号转导途径涉及肌醇三磷酸介导的肌浆网(SR)释放Ca~(2+)和通过非选择性钙通透性阳离子通道进入Ca~(2+)。电压敏感的L型钙离子通道在血管收缩作用中也很重要，尽管导致L型钙通道激活的信号通路尚未得到很好的描述。我们先前已经证明，生理浓度的[Arg8]-加压素(AVP，10-100 pm)通过一种新的信号通路诱导A7r5 VSM细胞内钙尖峰，从而激活L型钙通道。膜去极化可能是L型钙通道激活所必需的。我们推测，AVP对平滑肌细胞有去极化作用。最有可能引起这种效应的候选者是非选择性阳离子通道。我们先前已经提供了两个不同的非L类型的二价阳离子进入途径被AVP激活的证据。介导这些效应的通道的分子性质尚不清楚，但已涉及非选择性阳离子通道的瞬时受体电位(Trp)家族成员。目前的建议试图验证这一假说，即这些二价阳离子进入途径在生理AVP浓度下的钙尖峰反应中发挥重要作用。其具体目的是：1)用RT-PCR和Western blotting鉴定A7r5细胞和大鼠肠系膜动脉平滑肌细胞(RMASMC)表达哪些Trp通道同源物；2)表征100 PM AVP激活的A7r5细胞和RMASMC上的非选择性阳离子电流，并利用膜片钳技术评价它们对膜电位变化的贡献；3)从AVP刺激的非容量性钙内流途径中分离“容积性”的储存操作的钙内流，并确定哪些是触发钙尖峰的重要因素；4)敲除或过度表达特定的Trp通道同源物，以确定它们在AVP刺激的钙信号转导中的作用及其与AVP刺激的非选择性阳离子电流和钙离子进入途径的关系。