Dynamic Calcium Regulation in Airway Smooth Muscle

气道平滑肌的动态钙调节

• 批准号: 6832227
• 负责人: MATHUR S KANNAN
• 金额: $ 28.08万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6832227
• 关键词: ADP ribosylation; CD38 molecule; acetylcholine; calcium channel; calcium flux; confocal scanning microscopy; cytokine; human tissue; interleukin 13; laboratory mouse; muscle contraction; muscle function; pentosyltransferase; polymerase chain reaction; respiratory airflow disorder; respiratory muscles; sarcoplasmic reticulum; smooth muscle; swine; tissue /cell culture; western blottings; wild animals

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.

描述（由申请人提供）：在气道平滑肌（ASM）细胞中，肌质网（SR）Ca2+通过乙酰胆碱（ACH）刺激在乙酰胆碱（ACH）刺激过程中通过环状ADP-ribose（CADPR）介导的ryanodine受体通道释放，这是Beta-NAD+的代谢物。 通过ADP-核糖基环酶和CADPR水解酶合成和降解的酶分别与单个双功能蛋白CD38相关。 在ASM中，RT-PCR和Western印迹分析显示CD38表达。 在ASM中，ACH引起CD38激活，反映为增加的ADP-核糖基环化酶活性和CADPR的产生。 ASM与8-BR-CADPR的预留，CADPR拮抗剂会导致Ca2+反应减弱和对激动剂的收缩。 激动剂引起的ASM细胞中的Ca2+反应降低了抗Sense CD38和CD38基因敲除小鼠的细胞的Ca2+反应。 这些研究证明了CD38/CADPR信号在ASM Ca2+调节中的作用。 CADPR介导的猪ASM细胞中的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Ca2+释放。 CD38/CADPR信号传导与M2毒蕈碱受体耦合，表明受体亚型特异性激活。 对激动剂的反应提高[Ca2+] I反应是气道炎症性疾病的特征。 发病机理涉及基因表达的改变以及参与Ca2+调控的第二个使者的激活。 炎性细胞因子，IL-1BETA，TNF-α和IFN-gamma在人ASM细胞中调节CD38的表达和ADP-核糖基环化酶活性，其中[Ca2+] I对BK，ACH，HACH，组胺和凝血酶的反应增强，并增强了8--BR-CADPR，并且会减弱这种反应。 在暴露于TNF-Alpha的反义CD38转染的细胞中，[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超应答性中的作用提供了新的见解。 拟议的研究的目的是获得多种收缩激动剂激活CD38激活的证据，及其在改变[Ca2+] I ASM在炎症性疾病（例如哮喘）中的调节和收缩性中的作用。 拟议研究的总体假设是，收缩激动剂激活了ASM细胞中的CD38/CADPR信号传导，以升高[Ca2+] I，而细胞因子上调CD38表达，CADPR介导的钙动员钙动员和对气道超副反应有助于。 我们的发现将导致针对该信号通路的潜在药物的发展。