Calcium entry and vascular smooth muscle excitation

性状（由申请方提供）：血管收缩激素通过增加胞质游离Ca 2+浓度（[Ca 2 +]i）引起血管平滑肌（VSM）细胞收缩。导致[Ca 2 +]i升高的最佳特征信号转导途径涉及三磷酸肌醇介导的肌浆网（SR）中Ca 2+的释放和经由非选择性钙渗透阳离子通道的Ca 2+进入。电压敏感性L-型Ca 2+通道在血管收缩作用中也是重要的，尽管导致L-型通道激活的信号传导途径还没有很好地表征。我们以前已经证明，生理浓度的[Arg 8]-加压素（AVP，10-100 pM）诱导Ca 2+尖峰A7 r5 VSM细胞通过一种新的信号通路，导致激活L型Ca 2+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。引起这种效应的最可能的候选者是非选择性阳离子通道。我们以前提供的证据表明，AVP激活两种不同的非L型二价阳离子进入途径。介导这些效应的通道的分子性质尚不清楚，但涉及非选择性阳离子通道的瞬时受体电位（TRP）家族的成员。本提案旨在研究的假设，这些二价阳离子进入途径发挥了重要作用，在Ca 2+尖峰响应生理AVP浓度。具体目标是：1）用RT-PCR和Western blotting鉴定TRP通道同源物在A7 r5细胞和大鼠肠系膜动脉平滑肌细胞（RMASMC）中的表达; 2）用膜片钳技术表征100 pM AVP在A7 r5细胞和RMASMC中激活的非选择性阳离子电流，并评估它们对膜电位变化的贡献; 3）将“容量性”钙库操作的Ca 2+内流与非容量性AVP刺激的Ca 2+内流途径分离，并确定哪些在触发Ca 2+尖峰中是重要的; 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Ca 2+信号传导中的作用以及它们与AVP的关系。刺激非选择性阳离子电流和Ca 2+进入途径。

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.