Calcium entry and vascular smooth muscle excitation

钙进入和血管平滑肌兴奋

• 批准号: 6749575
• 负责人: KENNETH L BYRON
• 金额: $ 33.3万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6749575
• 关键词: antisense nucleic acid; arginine vasopressin; calcium channel; calcium flux; calcium indicator; cell line; immunocytochemistry; inositol phosphates; laboratory rat; membrane channels; membrane potentials; polymerase chain reaction; protein structure function; sarcoplasmic reticulum; transfection; vascular smooth muscle; vasoconstriction; voltage /patch clamp; voltage gated channel; western blottings

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胞质游离Ca2+浓度（[Ca2+] I）而导致血管平滑肌（VSM）细胞的收缩。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）（SR）和Ca2+进入的释放，并通过非选择性钙的阳离子阳离子通道进入。众所周知，对电压敏感的L型Ca2+通道在血管收缩的作用中也很重要，尽管导致L型通道激活的信号传导途径没有很好地表征。我们先前已经证明，[ARG8] - 瓦索素（AVP，10-100 pm）的生理浓度通过一种新的信号传导途径在A7R5 VSM细胞中诱导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对平滑肌细胞产生去极化作用。引起这种效果的最有可能的候选者包括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本质尚不清楚，但是涉及非选择性阳离子通道的瞬时受体电位（TRP）家族的成员。本提案旨在检查以下假设：这些二价阳离子进入途径在CA2+尖峰对生理AVP浓度的反应中起着重要作用。具体目的是：1）使用RT-PCR和Western blotting确定哪些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细胞中100 pm AVP激活的非选择性阳离子电流和RMASMC，并使用斑块夹技术评估了它们对膜电位变化的贡献； 3）从非电容性AVP刺激的Ca2+进入途径中分离“电容”存储的Ca2+进入，并确定哪个对于触发Ca2+​​ Spiking很重要； 4）敲除或过表达特定的TRP通道同源物，以确定其在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+入口途径的关系。