Dynamic Calcium Regulation in Airway Smooth Muscle

描述(申请人提供)：在呼吸道平滑肌(ASM)细胞中，在乙酰胆碱(Ach)刺激过程中，肌浆网(SR)钙离子通过兰尼定受体通道释放，由β-NAD+的代谢物环状ADP-核糖(CADPR)介导。分别通过ADP-核糖环化酶和cADPR水解酶合成和降解cADPR的酶与单一的双功能蛋白CD38相关。RT-PCR和Western印迹分析显示CD38在ASM中有表达。在ASM中，Ach引起CD38激活，表现为ADP-核糖环化酶活性增加和cADPR产生增加。CADPR拮抗剂8-BR-cADPR与ASM预先孵育可导致钙离子反应减弱和对激动剂的收缩。激动剂可降低反义CD38转基因ASM细胞和CD38基因敲除小鼠细胞的钙反应。这些研究证实了CD38/cADPR信号在ASM钙调节中的作用。CADPR介导的猪ASM细胞肌浆网钙释放是激动剂特异性的。8-溴-cADPR抑制Ach-和ET-1-诱导的钙释放，但不抑制组胺-诱导的钙释放。CD38/cADPR信号与M2受体偶联，提示受体亚型特异性激活。[Ca~(2+)]i对激动剂的高反应是呼吸道炎症性疾病的一个特征。其发病机制涉及参与钙调节的基因表达的改变和第二信使的激活。炎症细胞因子IL-1β、肿瘤坏死因子-α和干扰素-γ上调人ASM细胞CD38的表达和ADP-核糖环化酶活性，其中BK、Ach、组胺和凝血酶的[Ca~(2+)]i反应增强，而8-Br-cADPR减弱这种反应。在反义CD38转基因细胞中，暴露于肿瘤坏死因子-α后，[Ca~(2+)]i对BK的反应低于对照组。这些发现为CD38-cADPR-SR钙离子释放在ASM高反应性中的作用提供了新的见解。这项研究的目的是获得多种收缩激动剂激活CD38的证据，以及它在哮喘等炎症性疾病中改变ASM的[Ca2+]i调节和收缩能力中的作用。这项研究的总体假设是，收缩激动剂激活ASM细胞中的CD38/cADPR信号通路，使[Ca+]i升高，细胞因子上调CD38的表达，cADPR介导的钙动员和收缩参与了气道高反应性。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.