Dynamic Calcium Regulation in Airway Smooth Muscle

描述（由申请人提供）：在气道平滑肌（ASM）细胞中，乙酰胆碱（Ach）刺激时，肌浆网（SR） Ca2+通过ryanodine受体通道释放是由环adp核糖（cADPR）介导的，cADPR是β - nad +的代谢物。通过adp -核糖基环化酶和cADPR水解酶合成和降解cADPR的酶分别与单个双功能蛋白CD38相关。在ASM中，RT-PCR和Western blot分析显示CD38的表达。在ASM中，乙酰胆碱引起CD38活化，表现为adp -核糖基环化酶活性和cADPR生成的增加。ASM与cADPR拮抗剂8-Br-cADPR预孵育导致Ca2+反应减弱和对激动剂的收缩。在转染了反义CD38和CD38敲除小鼠细胞的ASM细胞中，激动剂引起Ca2+反应降低。这些研究证实了CD38/cADPR信号在ASM Ca2+调控中的作用。猪ASM细胞中cadpr介导的SR Ca2+释放是激动剂特异性的。8-Br-cADPR抑制Ach-和ET-1-，但不抑制组胺-诱导的Ca2+释放。CD38/cADPR信号与M2毒蕈碱受体偶联，提示受体亚型特异性激活。[Ca2+]i对激动剂的反应升高是气道炎性疾病的特征。发病机制涉及基因表达的改变和参与Ca2+调节的第二信使的激活。炎症因子、il -1 β、tnf - α和ifn - γ上调人ASM细胞中CD38的表达和adp -核糖素环化酶的活性，其中[Ca2+]i对BK、Ach、组胺和凝血酶的反应增强，8-Br-cADPR减弱了这种反应。在暴露于tnf - α的反义CD38转染细胞中，[Ca2+]i对BK的反应低于对照组。这些发现为CD38-cADPR-SR Ca2+释放在ASM高反应性中的作用提供了新的见解。拟议研究的目标是获得多种收缩激动剂激活CD38的证据及其在炎症性疾病（如哮喘）中改变[Ca2+]i调节和ASM收缩性中的作用。本研究的总体假设是，收缩激动剂激活ASM细胞中的CD38/cADPR信号以升高[Ca2+]i，细胞因子上调CD38表达，cADPR介导的钙动员和收缩导致气道高反应性。我们的发现将导致针对这一信号通路的潜在药物的开发。

DESCRIPTION (provided by applicant): In airway smooth muscle (ASM) cells, sarcoplasmic reticulum (SR) Ca2+ release via ryanodine receptor channels during acetylcholine (Ach) stimulation is mediated by cyclic ADP-ribose (cADPR), a metabolite of beta-NAD+. The enzymes for the synthesis and degradation of cADPR through ADP-ribosyl cyclase and cADPR hydrolase respectively are associated with a single bifunctional protein, CD38. In ASM, RT-PCR and Western blot analyses reveal CD38 expression. In ASM, Ach causes CD38 activation, reflected as increased ADP-ribosyl cyclase activity and cADPR production. Pre-incubation of ASM with 8-Br-cADPR, a cADPR antagonist results in attenuated Ca2+ responses and contraction to agonists. Agonists elicit decreased Ca2+ responses in ASM cells transfected with anti-sense CD38 and cells from CD38 knockout mice. These studies demonstrate the role of CD38/cADPR signaling in ASM Ca2+ regulation. cADPR-mediated SR Ca2+ release in porcine ASM cells is agonist specific. 8-Br-cADPR inhibits Ach- and ET-1-, but not histamine-, induced Ca2+ release. CD38/cADPR signaling is coupled to M2 muscarinic receptors, suggesting receptor subtype specific activation. Heightened [Ca2+]i response to agonists is a feature of airway inflammatory diseases. The pathogenesis involves alterations in gene expression and activation of second messengers involved in Ca2+ regulation. Inflammatory cytokines, IL-1beta, TNF-alpha and IFN-gamma up regulate CD38 expression and ADP-ribosyl cyclase activity in human ASM cells, where the [Ca2+]i responses to BK, Ach, histamine and thrombin are augmented and 8-Br-cADPR attenuates this response. In anti-sense CD38 transfected cells exposed to TNF-alpha, the [Ca2+]i responses to BK are lower than in controls. These findings have provided new insights into the role of CD38-cADPR-SR Ca2+ release in ASM hyperresponsiveness. The goal of the proposed studies is to obtain evidence for CD38 activation by multiple contractile agonists and its role in altered [Ca2+]i regulation and contractility of ASM in inflammatory diseases such as asthma. The overall hypothesis of the proposed study is that contractile agonists activate CD38/cADPR signaling in ASM cells to elevate [Ca2+]i, and cytokines upregulate CD38 expression, cADPR mediated calcium mobilization and contraction contributing to airway hyperresponsiveness. Our findings will lead to the development of potential drugs that target this signaling pathway.