Dynamic Calcium Regulation in Airway Smooth Muscle

描述（由申请方提供）：在气道平滑肌（ASM）细胞中，乙酰胆碱（Ach）刺激期间肌浆网（SR）通过兰尼碱受体通道释放的Ca 2+由环ADP-核糖（cADPR）介导，cADPR是β-NAD+的代谢产物。 分别通过ADP-核糖基环化酶和cADPR水解酶合成和降解cADPR的酶与单一双功能蛋白CD 38相关。 在ASM中，RT-PCR和Western印迹分析揭示了CD 38的表达。 在ASM中，Ach引起CD 38活化，反映为ADP-核糖基环化酶活性和cADPR产生增加。 ASM与8-Br-cADPR（一种cADPR拮抗剂）预孵育导致对激动剂的Ca 2+反应和收缩减弱。 激动剂在用反义CD 38转染的ASM细胞和来自CD 38敲除小鼠的细胞中引起降低的Ca 2+应答。 这些研究证实了CD 38/cADPR信号在ASM Ca 2+调节中的作用。 猪ASM细胞中cADPR介导的SR Ca 2+释放是激动剂特异性的。 8-Br-cADPR抑制Ach-和ET-1-诱导的Ca ~（2+）释放，但不抑制组胺诱导的Ca ~（2+）释放。 CD 38/cADPR信号传导与M2毒蕈碱受体偶联，表明受体亚型特异性活化。 对激动剂的[Ca 2 +]i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Ca 2+调节的第二信使的激活。 炎性细胞因子IL-1 β、TNF-α和IFN-γ上调人ASM细胞中CD 38表达和ADP-核糖基环化酶活性，其中[Ca 2 +]i对BK、Ach、组胺和凝血酶的反应增强，而8-Br-cADPR减弱这种反应。 在暴露于TNF-α的反义CD 38转染细胞中，[Ca 2 +]i对BK的反应低于对照。 这些发现为CD 38-cADPR-SR Ca 2+释放在ASM高反应性中的作用提供了新的见解。 拟定研究的目的是获得多种收缩激动剂激活CD 38的证据，以及其在炎症性疾病（如哮喘）中改变[Ca 2 +]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CD 38/cADPR信号以升高[Ca 2 +]i，细胞因子上调CD 38表达，cADPR介导的钙动员和收缩有助于气道高反应性。 我们的研究结果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.