Novel Signaling for Ca2+ and Release in Airway Myocytes

描述(由申请人提供)： 胆碱能神经通过M受体对呼吸道平滑肌细胞(ASMC)起主要神经控制作用。模拟这些受体导致磷脂酶C(PLC)的激活和1，4，5-三磷酸肌醇(IP3)的产生，从而通过IP3受体(IP3Rs)诱导钙释放。这一已知的信号通路对ASMCs的M胆碱钙释放和收缩具有重要作用。然而，我们的初步研究结合以前的发现表明，毒扁豆碱刺激可能激活ADP-核糖环化酶，然后产生环状ADP-核糖(CADPR)，通过直接和/或通过将FK506结合蛋白12.6(FKBP12.6)从这些钙释放通道上解离RyRs来诱导SR释放钙，从而导致ASMCs毒扁豆碱钙释放和相关收缩的放大。这个新的信号通路，ADP-核糖环化酶-cADPR-FKBP12.6-RyR，可能在哮喘中具有高功能，参与了呼吸道肌肉对M受体激动剂和其他多种痉挛诱因的收缩高反应。为了验证这些假说，我们将解决以下问题(具体目的)：(1)cADPR是否介导正常和哮喘ASMC的M胆碱钙释放？(2)正常和哮喘ASMC中是否需要FKBP12.6来调节M胆碱钙释放和cADPR的靶点？(3)RyRs的哪个亚型参与了M胆碱能钙释放，以及cADPR和FKBP12.6在正常和哮喘ASMC中的作用？这些目标将通过同时测量新鲜分离的呼吸道肌细胞中的钙火花或全细胞[钙]i和膜电流来实现。遗传操作(基因过表达和基因敲除)、分子生物学和生化方法也将被用于这项提议。这项研究将扩大我们对正常和哮喘ASMCs毒扁豆碱钙释放的细胞和分子机制的理解，并可能确定哮喘发作的新治疗靶点。由于cADPR、FKBP12.6和/或RyRs在各种类型的细胞中普遍表达，本研究的机制发现将对钙离子介导的各种细胞效应具有普遍的生物学和病理学意义。

DESCRIPTION (provided by applicant): Cholinergic nerves provide a predominant neural control of airway smooth muscle cells (ASMCs) through muscarinic receptors. Simulation of these receptors results in the activation of phospholipase C (PLC) and generation of inositol 1,4,5-triphophate (IP3), which induces Ca2+ release through IP3 receptors (IP3Rs). This well-known signaling pathway is important for muscarinic Ca2+ release and contraction in ASMCs. However, our preliminary study, together with previous findings, suggests that muscarinic stimulation may activate ADP-ribosyl cyclase and then produce cyclic ADP-ribose (cADPR), which induces Ca2+ release from the SR by opening RyRs directly and/or indirectly by disassociating FK506 binding protein 12.6 (FKBP12.6) from these Ca2+ release channels, leading to the amplification of muscarinic Ca2+ release and associated contraction in ASMCs. This novel signaling pathway, ADP-ribosyl cyclase-cADPR-FKBP12.6-RyR, may be hyper-functioned in asthma, contributing to the airway muscle Ca2+ and contractile hyperresponsiveness to muscarinic agonists and other numerous spasomogens. To test these hypotheses, we will address the following questions (specific aims): (1) Does cADPR mediate muscarinic Ca2+ release in normal and asthmatic ASMCs? (2) Is FKBP12.6 required for muscarinic Ca2+ release and the target for cADPR in normal and asthmatic ASMCs? and (3) which subtype of RyRs involves muscarinic Ca2+ release and the roles of cADPR and FKBP12.6 in normal and asthmatic ASMCs? These aims will be implemented using simultaneous measurements of Ca2+ sparks or whole-cell [Ca2+]i and membrane currents in freshly disassociated airway myocytes. Genetic manipulations (gene overexpression and knockout), molecular biological and biochemical methods will be also used in this proposal. This study will extend our understanding of the cellular and molecular mechanisms underlying muscarinic Ca2+ release in normal and asthmatic ASMCs, and may also identify novel therapeutic targets for asthma attacks. Since cADPR, FKBP12.6, and/or RyRs are ubiquitously expressed in a variety of cell types, the mechanistic findings from the proposed study will have general biological and pathological significance for various Ca2+-mediated cellular effects.