Store-operated Ca2+ influx & iPLA2 in vascular SMC

商店操作的 Ca2 流入

• 批准号: 7067126
• 负责人: Victoria M Bolotina
• 金额: $ 58.96万
• 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 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7067126
• 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）：我的长期目标是确定电容性 Ca2+ 内流的关键要素，并建立血管平滑肌细胞 (SMC) 中天然钙池操纵的阳离子 (SOC) 通道调节的分子机制。最近，在确定了存储操作（电容性）Ca2+流入在激动剂诱导的收缩和一氧化氮诱导的SMC舒张中的生理重要性后，我们成功地表征了一种新型小电导（3 pS）SOC通道，该通道负责SMC中的电容性Ca2+流入。我们还确定钙内流因子 (CIF) 激活这种天然 SOC 通道。尽管这种存储操纵途径非常重要，但 CIF 诱导 SOC 通道激活的分子机制仍然未知。我们最近的研究得出了全新且非常有趣的结果，强烈表明以前从未与该过程相关的 Ca2+ 依赖性磷脂酶 A2 (iPLA2) 可以在 SOC 通道的激活和血管收缩中发挥至关重要的作用。我们发现抑制 iPLA2 的表达和/或功能活性可以防止 Ca2+ 内流的激活，并损害激动剂诱导的收缩。我们的初步数据表明，SOC 通道的生理激活/抑制可以通过抑制性 CaM 以膜分隔方式从 iPLA2 置换/关联到 iPLA2 来模拟，并且 CIF 可以从 iPLA2 置换抑制性 CaM，从而导致其激活。我们还有证据表明 iPLA2 活性的溶血磷脂产物可以激活 SOC 通道。我的提案的总体目标是确定 iPLA2 在电容性 Ca2+ 内流途径和血管收缩中的新作用，并确定 CIF 诱导的 iPLA2 依赖性 SOC 通道激活的分子机制。 该提议的假设是，iPLA2 是电容性 Ca2+ 流入和血管收缩的新型分子决定因素，并且 SOC 通道激活是膜界定的 CIF 诱导的抑制性 CaM 从 iPLA2 中移位的结果，iPLA2 位于靠近 SOC 通道的质膜中。将使用电生理学、分子、生物化学、成像和生理学方法对单个 SOC 通道、全细胞电流、细胞内 Ca2+、血管 SMC 中 iPLA2 表达和活性以及完整血管的收缩性水平进行测试。所有这些方法都是在Pl实验室建立并成功使用的。该模型和拟议研究的可行性得到了广泛的初步数据的充分支持。该提案的具体目标是： 目标 1. 将 iPLA2 确立为调节储存操纵通道和血管收缩的新决定因素。我们将：确定 iPLA2 对于 SOC 通道的储存依赖性激活和电容性 Ca 2+ 内流是绝对必需的，确定 iPLA2 在血管收缩中的新作用，确定 iPLA2 的哪些特定异构体参与储存操作途径，并确定 iPLA2 在 SMC 中的位置。目标 2. 明确 SMC 中 iPLA2 依赖的储存操纵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 通道激活的新模型中的所有步骤，并将确定 iPLA2 是否可以被 CIF 激活，并通过消耗 SMC 中的 Ca 2+ 存储，建立 iPLA2 的 CaM 依赖性调节和 SMC 中 SOC 通道的 CaM 依赖性调节之间的直接相关性，测试 CIF 从 iPLA2 中取代 CaM 的能力及其相关性 通过 CIF 诱导的商店运营通道激活，并确定 iPLA2 的哪些特定产品激活本地商店运营通道。