Dynamic Calcium Regulation in Airway Smooth Muscle

气道平滑肌的动态钙调节

基本信息

批准号：
6724027
负责人：
MATHUR S KANNAN
金额：
$ 28.04万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
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

项目摘要

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，乙酰胆碱 (Ach) 刺激期间，肌浆网 (SR) Ca2+ 通过兰尼碱受体通道释放，由环状 ADP-核糖 (cADPR)（β-NAD+ 的代谢物）介导。分别通过 ADP-核糖基环化酶和 cADPR 水解酶合成和降解 cADPR 的酶与单个双功能蛋白 CD38 相关。在 ASM 中，RT-PCR 和蛋白质印迹分析揭示了 CD38 的表达。在 ASM 中，Ach 引起 CD38 激活，表现为 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）预孵育会导致 Ca2+ 反应减弱以及对激动剂的收缩。激动剂会降低转染反义 CD38 的 ASM 细胞和来自 CD38 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。猪 ASM 细胞中 cADPR 介导的 SR Ca2+ 释放具有激动剂特异性。 8-Br-cADPR 抑制 Ach- 和 ET-1-，但不抑制组胺-诱导的 Ca2+ 释放。 CD38/cADPR 信号传导与 M2 毒蕈碱受体偶联，表明受体亚型特异性激活。 [Ca2+]i 对激动剂的反应增强是气道炎症性疾病的一个特征。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。炎症细胞因子、IL-1β、TNF-α 和 IFN-γ 上调人 ASM 细胞中的 CD38 表达和 ADP-核糖基环化酶活性，其中 [Ca2+]i 对 BK、Ach、组胺和凝血酶的反应增强，并且 8-Br -cADPR 减弱这种反应。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。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。拟议研究的目的是获得多种收缩激动剂激活 CD38 的证据，及其在哮喘等炎症性疾病中改变 [Ca2+]i 调节和 ASM 收缩性中的作用。该研究的总体假设是收缩激动剂激活 ASM 细胞中的 CD38/cADPR 信号传导以升高 [Ca2+]i，细胞因子上调 CD38 表达，cADPR 介导钙动员和收缩，导致气道高反应性。我们的研究结果将导致针对该信号通路的潜在药物的开发。