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）细胞收缩。导致 [Ca2]i 升高的最佳特征信号转导途径涉及肌醇三磷酸介导的 Ca2 从肌浆网 (SR) 释放以及 Ca2 通过非选择性钙渗透性阳离子通道进入。已知电压敏感的 L 型 Ca2 通道在血管收缩作用中也很重要，但导致 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]-加压素（AVP，10-100 pM）通过一种新的信号传导途径诱导 A7r5 VSM 细胞中的 Ca2 尖峰，从而导致 L 型 Ca2 通道的激活。 L 型 Ca2 通道激活可能需要膜去极化。我们假设 AVP 对平滑肌细胞发挥去极化作用。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。介导这些效应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) 家族的成员与此有关。本提案旨在检验以下假设：这些二价阳离子进入途径在对生理 AVP 浓度的 Ca2 尖峰反应中发挥重要作用。具体目标是： 1) 使用 RT-PCR 和 Western blotting 鉴定哪些 TRP 通道同源物在 A7r5 细胞和大鼠肠系膜动脉平滑肌细胞 (RMASMC) 中表达； 2) 表征 A7r5 细胞和 RMASMC 中 100 pM AVP 激活的非选择性阳离子电流，并使用膜片钳技术评估它们对膜电位变化的贡献； 3) 将“电容性”存储操作的 Ca2 进入与非电容性 AVP 刺激的 Ca2 进入途径分离，并确定哪一个对于触发 Ca2 尖峰是重要的； 4) 敲除或过表达特定的 TRP 通道同源物，以确定它们在 AVP 刺激的 Ca2 信号传导中的作用及其与 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.