Novel Signaling for Ca2+ and Release in Airway Myocytes

描述（由申请人提供）： 胆碱能神经通过毒蕈碱受体对气道平滑肌细胞（ASMCs）提供主要的神经控制。这些受体的模拟导致磷脂酶C（PLC）的活化和肌醇1，4，5-三磷酸（IP 3）的产生，其通过IP 3受体（IP 3Rs）诱导Ca 2+释放。 这一众所周知的信号通路对于ASMCs中的毒蕈碱Ca 2+释放和收缩是重要的。 然而，我们的初步研究，以及以前的研究结果表明，毒蕈碱刺激可能会激活ADP-核糖环化酶，然后产生环ADP-核糖（cADPR），它通过直接和/或间接打开RyR，通过解离FK 506结合蛋白12.6，诱导SR中的Ca 2+释放。（FKBP12.6）从这些Ca 2+释放通道中释放，导致毒蕈碱Ca 2+释放的放大和ASMCs中的相关收缩。这种新的信号通路，ADP-核糖基环化酶-cADPR-FKBP 12.6-RyR，可能在哮喘中功能亢进，导致气道肌肉Ca 2+和对毒蕈碱激动剂和其他许多痉挛剂的收缩高反应性。为了验证这些假设，我们将解决以下问题（具体目标）：（1）cADPR是否介导正常和哮喘ASMCs的毒蕈碱型Ca 2+释放？(2)FKBP12.6是正常和哮喘ASMCs中毒蕈碱Ca 2+释放和cADPR靶点所必需的吗？（3）正常和哮喘ASMCs中哪种RyR亚型参与毒蕈碱型Ca ~（2+）释放以及cADPR和FKBP 12.6的作用？这些目标将通过同时测量新鲜分离的气道肌细胞中的Ca 2+火花或全细胞[Ca 2 +]i和膜电流来实现。基因操作（基因过表达和敲除）、分子生物学和生物化学方法也将用于本提案。这项研究将扩展我们对正常和哮喘ASMCs中毒蕈碱Ca 2+释放的细胞和分子机制的理解，也可能确定哮喘发作的新治疗靶点。由于cADPR、FKBP12.6和/或RyR在各种细胞类型中普遍表达，因此拟议研究的机制发现将对各种Ca 2+介导的细胞效应具有一般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.