Ion channels and Ca regulation in vascular SMC function

描述(申请人提供)：这项修订的竞争性更新将阐明存储操作通道(SOC)的生理作用以及iPLA2和PKC在血管收缩、转录因子激活和SMC增殖中的复杂调控。 这一建议的中心假设是，SOC复杂的分子调控及其与其他通道和信号级联的相互作用决定了SMC的主要功能。1)依赖iPLA2的SOC通道的激活不仅为钙内流提供了一条途径，而且也是激活钙离子L通道的重要触发因素。2)依赖于PKCE的复杂的iPLA2调节可能为SMC增殖过程中的SOC和容量性钙内流提供了微调机制；3)转录因子的激活和SMC的增殖依赖于iPLA2和SOC通道。 一种综合的方法，包括先进的电生理、分子、生化和成像技术，将被用来检验iPLA2、PKCepsilon、P-CREB和NFAT4的水平，以及它们在新鲜分离和培养的SMC以及在完整的血管(新鲜或器官培养)中的移位和串扰。所有这些方法都是在Pi的实验室中建立和使用的，初步数据和最近的出版物有力地支持了拟议研究的可行性。具体目标是： 目的1.建立iPLA2和SOC通道作为脑动脉钙离子内流和收缩的主要决定因素的新作用。我们将评估SOC及其与钙L通道在激动剂诱导的收缩和肌源性张力中的复杂作用； 目的2.明确PKCE在SMC增殖过程中iPLA2依赖的SOC通道和CCE调控中的复杂作用； 目的3.探讨SOC及其iPLA2、PKC和溶血磷脂依赖的调节在CREB和NFAT激活、SMC增殖和分化中的作用。。

DESCRIPTION (provided by applicant): This revised competitive renewal will elucidate the physiological role of the store-operated channel (SOC) and its complex regulation by iPLA2 and PKC in vascular constriction, activation of transcriptional factors and SMC proliferation. The central hypothesis of this proposal is that complex molecular regulation of SOC and their interplay with other channels and signaling cascades determines major SMC functions. Three specific aims are designed to test the following specific hypotheses: 1) iPLA2-dependent activation of SOC channels provide not only a path for Ca2+ entry, but also a crucial trigger for activation of Ca2+ L channels, Ca2+ influx and constriction in cerebral arteries, 2) complex PKCe-dependent regulation of iPLA2 may provide a fine-tuning mechanism for SOC and capacitative Ca2+ entry during SMC proliferation, 3) activation of transcription factors and SMC proliferation depends on iPLA2 and SOC channels. An integrative approach that involves advanced electrophysiological, molecular, biochemical and imaging techniques will be used to test these hypotheses on the level of iPLA2, PKCepsilon, P-CREB and NFAT4, their translocation and cross-talk in freshly isolated and cultured SMC, and in intact blood vessels (fresh or organ culture). All these methods are established and used in PI's lab, and the feasibility of the proposed studies is strongly supported by preliminary data and recent publications. Specific aims are: Aim 1. To establish a novel role for iPLA2 and SOC channels as major determinants of Ca2+ influx and constriction in cerebral arteries. We will assess a complex role of SOC and its cross-talk with Ca2+ L channels during agonist-induced constriction and myogenic tone; Aim 2. To define a complex role of PKCe in iPLA2-dependent regulation of SOC channels and CCE during SMC proliferation; Aim 3. To establish the role of SOC and its iPLA2, PKC and lysophospholipid-dependent regulation in activation of CREB and NFAT, SMC proliferation and differentiation. .