Store-operated Ca2+ influx & iPLA2 in vascular SMC

商店操作的 Ca2 流入

• 批准号: 6893652
• 负责人: Victoria M Bolotina
• 金额: $ 60.38万
• 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/6893652
• 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（由申请人提供）：我的长期目标是定义电容性Ca2+涌入中的关键要素，并确定血管平滑肌细胞中天然存储 - 储存阳离子（SOC）通道调节的分子机制（SMC）。最近，在建立了储存（电容性）Ca2+流入的生理重要性之后，在激动剂诱导的收缩和一氧化氮诱导的SMC弛豫中，我们成功地表征了一种新型的小电导（3 ps）SOC通道，该通道负责SMC中的capacitahve CA2+ Implux，该通道负责。我们还确定，钙涌入因子（CIF）激活了这种天然SOC通道。尽管该商店经营的途径非常重要，但CIF诱导的SOC通道激活的分子机制仍然未知。我们最近的研究产生了完全新颖且非常有趣的结果，这强烈表明Ca2+独立的磷脂酶A2（IPLA2）以前从未与此过程相关，可以在激活SOC通道和血管收缩的激活中起着至关重要的作用。我们发现，IPLA2的表达和/或功能活性的抑制可阻止Ca2+流入的激活，并损害激动剂引起的收缩。我们的初步数据表明，可以通过膜降低的方式从/到IPLA2的抑制性CAM的位移/关联来模仿SOC通道的生理激活/抑制作用，并且CIF可以从IPLA2中取代IPLA2，从而导致其激活。我们还有证据表明，IPLA2活性的溶物磷脂产物可以激活SOC通道。我的建议的总体目标是确定IPLA2在电容性Ca2+涌入途径和血管收缩中的新作用，并确定CIF诱导的IPLA2依赖性SOC通道的分子机制。 该提议的假设是IPLA2是电容Ca2+流入和血管收缩的一种新型分子决定因素，SOC通道激活是位于位于质膜接近SOCMARMBRANE的膜膜上的膜抑制性CAM的抑制性CAM的结果。这将在单个SOC通道，全细胞电流，细胞内CA2+，IPLA2表达和血管SMC中的活性以及完整血管的收缩力以及完整血管收缩的水平上使用电生理学，分子，生化和生理方法进行测试。所有这些方法均在PL的实验室中建立并成功使用。广泛的初步数据充分支持了模型和拟议研究的可行性。该提案的具体目的是：目标1。建立IPLA2作为储存通道和血管收缩调节的新决定因素。我们将：确定IPLA2绝对需要商店依赖性SOC通道激活和电容性Ca 2+涌入，确定IPLA2在血管收缩中的新作用，确定IPLA2的哪些特异性同工型与Store-Operated途径有关，并确定IPLA2在SMC中的位置。目的2。定义SMC中存储操作通道的IPLA2依赖性激活的分子机制。 我们将测试我们新颖的CIF诱导的IPLA2和CAM介导的SOC通道激活模型中的所有步骤，并将确定IPLA2是否可以通过CIF激活IPLA2，以及通过SMC中Ca 2+存储的耗竭，建立CAM依赖性调节IPLA和CAM依赖于SMC的COMC，COF依赖于IPL的COF，COF依赖于IPL的COF，CIF依赖于IPL的COF，COF依赖于IPL的COF，CIF依赖于CIF的COF，CIF依赖于CIF的COF，CIF依赖于CIF，CIF依赖于CIF的COF，CIF依赖于COF，并建立了COIF依赖性。通过CIF诱导的储存通道激活，并确定IPLA2的哪些特定产物激活了天然存储的通道。