Calcium entry and vascular smooth muscle excitation

描述（由申请人提供）：血管收缩激素通过增加胞质游离Ca2+浓度（[Ca2+]i）引起血管平滑肌（VSM）细胞的收缩。导致[Ca2+]i升高的最具特征的信号转导途径涉及肌醇三磷酸介导的Ca2+从肌浆网（SR）释放和Ca2+通过非选择性钙透性阳离子通道进入。电压敏感的l型Ca2+通道在血管收缩作用中也很重要，尽管导致l型通道激活的信号通路尚未得到很好的表征。我们之前已经证明，生理浓度的[Arg8]-抗利尿激素（AVP, 10-100 pM）通过一种新的信号通路，导致l型Ca2+通道的激活，诱导A7r5 VSM细胞中的Ca2+峰值。膜去极化可能是l型Ca2+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。我们之前已经提供了AVP激活的两种不同的非l型二价阳离子进入途径的证据。介导这些作用的通道的分子性质尚不清楚，但非选择性阳离子通道的瞬时受体电位（TRP）家族成员已被牵连。目前的建议旨在检查这些二价阳离子进入途径在生理AVP浓度的Ca2+尖峰反应中起重要作用的假设。具体目的是：1)利用RT-PCR和Western blotting鉴定在A7r5细胞和大鼠肠系膜动脉平滑肌细胞（RMASMC）中表达哪些TRP通道同源物；2)利用膜片钳技术表征100 pM AVP在A7r5细胞和RMASMC中激活的非选择性阳离子电流，并评估其对膜电位变化的贡献；3)从非容性avp刺激的Ca2+进入途径中分离出“容性”存储操作的Ca2+进入途径，并确定哪些是触发Ca2+尖峰的重要途径；4)敲除或过表达特异性TRP通道同源物，以确定它们在avp刺激的Ca2+信号传导中的作用，以及它们与avp刺激的非选择性阳离子电流和Ca2+进入途径的关系。

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.