Store-operated Ca2+ influx & iPLA2 in vascular SMC

商店操作的 Ca2 流入

• 批准号: 6759324
• 负责人: Victoria M Bolotina
• 金额: $ 40.25万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-13 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6759324
• 关键词: Xenopus; angiotensin II; calcium flux; calmodulin; clinical research; confocal scanning microscopy; enzyme activity; human tissue; immunocytochemistry; isozymes; laboratory mouse; laboratory rabbit; membrane channels; muscle cells; phenylephrine; phospholipase A2; polymerase chain reaction; protein localization; vascular smooth muscle; western blottings

DESCRIPTION (provided by applicant): My long-term goal is to define the key elements in capacitative Ca2+ influx, and to establish the molecular mechanism of regulation of native store-operated cation (SOC) channels in vascular smooth muscle cells (SMC). Recently, after establishing the physiological importance of store-operated (capacitative) Ca2+ influx in agonist-induced contraction and nitric oxide-induced relaxation of SMC, we successfully characterized a novel small conductance (3 pS) SOC channel that is responsible for capacitahve Ca2+ influx in SMC. We also established that calcium influx factor (CIF) activates this native SOC channel. In spite of the tremendous importance of this store-operated pathway, the molecular mechanism of CIF-induced activation of SOC channels remains unknown. Our recent studies resulted in totally novel and very intriguing results which strongly suggest that Ca2+-independent phospholipase A2 (iPLA2), which has never been linked to this process before, can play a crucial role in activation of SOC channels and vascular contraction. We found that inhibition of expression and/or functional activity of iPLA2 prevents activation of Ca2+ influx, and impairs agonist-induced contraction. Our preliminary data showed that physiological activation/inhibition of SOC channels could be mimicked by displacement/association of inhibitory CaM from/to iPLA2 in membrane-delimited fashion, and that CIF could displace inhibitory CaM from iPLA2 resulting in its activation. We also have evidence that lysophospholipid products of iPLA2 activity can activate SOC channels. The overall goal of my proposal is to establish the novel role of iPLA2 in capacitative Ca2+ influx pathway and vascular contraction, and to determine the molecular mechanism of CIF-induced iPLA2- dependent activation of SOC channels. The hypothesis of this proposal is that iPLA2 is a novel molecular determinant of capacitative Ca2+ influx and vascular contraction, and that SOC channel activation is a result of membrane delimited CIF-induced displacement of inhibitory CaM from iPLA2, which is located in plasma membrane close to SOC channel. This will be tested using electrophysiological, molecular, biochemical, imaging and physiological approaches on the level of single SOC channels, whole-cell currents, intracellular Ca2+, iPLA2 expression and activity in vascular SMC, as well as contractility of intact blood vessels. All these methods are established and successfully used in Pl's lab. The feasibility of the model and proposed studies are fully supported by extensive preliminary data. Specific aims of this proposal are : Aim 1. To establish iPLA2 as a novel determinant in regulation of store-operated channels and vascular contraction. We will: Establish that iPLA2 is absolutely required for store-dependent activation of SOC channels and capacitative Ca 2+ influx, establish the novel role of iPLA2 in vascular contraction, determine which specific isoforms of iPLA2 are involved in store-operated pathway, and determine the location of iPLA2 in SMC. Aim 2. To define the molecular mechanism of iPLA2-dependent activation of store-operated channels in SMC. We will test all the steps in our novel model of CIF-induced iPLA2- and CaM-mediated activation of SOC channels, and will determine if iPLA2 can be activated by CIF, and by depletion of Ca 2+ stores in SMC, establish direct correlation between CaM-dependent regulation of iPLA2 and CaM-dependent regulation of SOC channels in SMC, test the ability of CIF to displace CaM from iPLA2, and its correlation with CIF-induced activation of store- operated channels, and determine which specific product(s) of iPLA2 activate native store-operated channels.

描述(申请人提供)：我的长期目标是明确电容性钙离子内流的关键因素，并建立调节血管平滑肌细胞(SMC)天然存储操作阳离子(SOC)通道的分子机制。最近，在确定了钙离子内流在激动剂引起的SMC收缩和一氧化氮引起的SMC松弛中的生理意义后，我们成功地描述了一种新的小电导(3pS)SOC通道，该通道与SMC的电容性钙内流有关。我们还证实了钙内流因子(CIF)激活了这一天然的SOC通道。尽管这种储存操作的途径非常重要，但CIF诱导SOC通道激活的分子机制仍不清楚。我们最近的研究得出了全新而有趣的结果，这些结果有力地表明，以前从未与此过程相关的钙非依赖性磷脂酶A2(IPLA2)在SOC通道的激活和血管收缩中发挥关键作用。我们发现，抑制iPLA2的表达和/或功能活性可以阻止钙离子内流的激活，并削弱激动剂诱导的收缩。我们的初步数据表明，SOC通道的生理激活/抑制可以通过以膜分隔的方式将抑制性CaM从iPLA2置换/结合到iPLA2来模拟，而CIF可以取代抑制性CaM而导致其激活。我们也有证据表明，iPLA2活性的溶血磷脂产物可以激活SOC通道。本研究的总体目标是确定iPLA2在容量性钙内流途径和血管收缩中的新作用，并确定CIF诱导的依赖于iPLA2的SOC通道激活的分子机制。 提出的假设是，iPLA2是一种新的钙离子容量性内流和血管收缩的分子决定因素，而SOC通道的激活是由膜限定的CIF诱导的抑制性CaM从位于质膜上靠近SOC通道的iPLA2移位的结果。这将利用电生理、分子、生化、成像和生理学方法，从单个SOC通道水平、全细胞电流、细胞内钙离子、血管SMC中iPLA2的表达和活性以及完整血管的收缩能力等方面进行测试。所有这些方法都是在Pl的实验室中建立并成功应用的。大量的初步数据充分支持了该模型和拟议研究的可行性。这项建议的具体目的是：目的1.建立iPLA2作为调节商店操作的通道和血管收缩的新的决定因素。我们将：确定iPLA2对于SOC通道的钙化激活和容量性钙内流是必不可少的，建立iPLA2在血管收缩中的新作用，确定哪些特定的iPLA2异构体参与商店操作的通路，并确定iPLA2在SMC中的位置。目的2.明确iPLA2依赖的SMC储存操作通道激活的分子机制。我们将测试CIF诱导的iPLA2和CaM介导的SOC通道激活新模型中的所有步骤，并确定iPLA2是否可以被CIF激活，以及通过耗尽SMC中的钙库来激活iPLA2，建立依赖CaM的iPLA2调控和SMC中CaM依赖的SOC通道调控之间的直接联系，测试CIF取代CaM的能力，以及它与CIF诱导的商店操作的通道激活的相关性，并确定iPLA2的哪一特定产物(S)激活天然商店操作的通道。