Calcium Regulation in Airway Smooth Muscle

描述（由申请人提供）：哮喘等疾病中的气道高反应性涉及由于细胞内Ca 2+（[Ca 2 +]i）增加和/或Ca 2+敏感性（给定[Ca 2 +]i的力）增加而增强的气道平滑肌（ASM）收缩。气道炎症是气道疾病的一个关键方面，暴露于几种炎症介质（如肿瘤坏死因子（TNF α）和白细胞介素-13（IL-13））会增加ASM收缩性。几项研究表明，细胞因子增加激动剂诱导的[Ca 2 +]i反应在ASM中，因此连接在当前的建议，我们将探索炎症诱导的变化，在[Ca 2 +]i的调节机制，导致整体增加[Ca 2 +]i反应。在ASM中，响应于SR耗竭的Ca 2+内流（钙池操作的Ca 2+内流; SOCE）被细胞因子增强。初步数据表明，Na+/Ca 2+交换（NCX）介导的流入细胞因子增强。这种SOCE和流入模式NCX的增强将导致[Ca 2 +]i水平增加。[Ca 2 +]i的降低通常通过质膜（PM）外排机制（可能包括NCX介导的外排）和通过SR ATP酶（SERCA）的SR Ca 2+再摄取来实现。细胞器如线粒体可以缓冲Ca 2+并改变SR再填充的Ca 2+可用性。通常，这些机制有助于将基础[Ca 2 +]i维持在低水平，而SR Ca 2+储存充满，直到激动剂刺激时[Ca 2 +]i随着SR储存耗尽而升高。初步研究表明，SERCA和线粒体Ca 2+缓冲受损的炎症。基于这些增强的Ca 2+内流，但减少的隔离或流出的对比初步研究结果，我们的中心假设是，炎症促进机制，增加[Ca 2 +]i，但削弱那些降低[Ca 2 +]i。这导致基础[Ca 2 +]i的总体增加以及对激动剂刺激的增强的[Ca 2 +]i响应。在这方面，我们提出，PM与细胞内机制在正常情况下在[Ca 2 +]i稳态中功能性地相互作用，并且这些机制的破坏及其与炎症的相互作用导致[Ca 2 +]i增加。我们的总体方法将是使用人ASM细胞或组织条来检查上述机制，有或没有暴露于促炎细胞因子（TNF α，IL-13）。研究将使用补充技术，包括分子生物学（siRNA;过表达），[Ca 2 +]i，[Na+]i和管腔（SR）Ca 2+的成像，荧光标记蛋白质的实时共聚焦成像，以及力测量来解决这些目标。我们的具体目标是：目的1：确定炎性细胞因子对人ASM调节中的STIM 1、STIM 2和Orai 1相互作用的影响;目的2：确定炎性细胞因子对NCX的影响及其在人ASM中的[Ca 2 +]i调节中的作用;目的3：确定炎性细胞因子对人ASM中的线粒体的影响及其在人ASM中的[Ca 2 +] i调节中的作用;目的4：确定炎性细胞因子对NCX的影响及其在人ASM中的[Ca 2 +]i调节中的作用。确定炎性细胞因子对SERCA的影响及其在人ASM中[Ca 2 +]i调节中的作用; [Ca 2 +]i目的5：确定炎性细胞因子对人ASM中[Ca 2 +]i调节机制对收缩性的总体贡献的影响。 公共卫生相关性：越来越多的人认识到，气道平滑肌收缩性的异常有助于在临床上重要的疾病如哮喘和慢性支气管炎中过度的气道狭窄和伴随的呼吸短促。气道收缩性高度依赖于细胞内钙水平。拟议的研究将检查人体气道平滑肌细胞内钙的控制机制。这些研究将为更好地了解气道疾病和开发新的治疗靶点奠定基础。

DESCRIPTION (provided by applicant): Airway hyperreactivity in diseases such as asthma involves enhanced airway smooth muscle (ASM) contraction due either to increased intracellular Ca2+ ([Ca2+]i) and/or increased Ca2+ sensitivity (force for a given [Ca2+]i). Airway inflammation is a key aspect of airways disease, and exposure to several inflammatory mediators (such as tumor necrosis factor (TNFa) and interleukin-13 (IL-13)) increase ASM contractility. Several studies showed that cytokines increase agonist-induced [Ca2+]i responses in ASM, thus linking In the current proposal, we will explore inflammation-induced changes in [Ca2+]i regulatory mechanisms that result in overall increased [Ca2+]i responses. In ASM, Ca2+ influx in response to SR depletion (store-operated Ca2+ entry; SOCE) is enhanced by cytokines. Preliminary data suggests that Na+/Ca2+ exchange (NCX)-mediated influx is enhanced by cytokines. Such enhancement of SOCE and influx-mode NCX would lead to increased [Ca2+]i levels. Reduction in [Ca2+]i is normally achieved by plasma membrane (PM) efflux mechanisms (perhaps including NCX-mediated efflux), and by SR Ca2+ reuptake via SR ATPase (SERCA). Organelles such as mitochondria can buffer Ca2+ and alter Ca2+ availability for SR refilling. Normally, these mechanisms help maintain basal [Ca2+]i at low levels, while SR Ca2+ stores are replete until agonist stimulation when [Ca2+]i rises as SR stores deplete. Preliminary studies suggest that SERCA and mitochondrial Ca2+ buffering are impaired by inflammation. Based on these contrasting preliminary findings of enhanced Ca2+ influx, but decreased sequestration or efflux, our central hypothesis is that inflammation promotes mechanisms that increase [Ca2+]i, but impairs those that decrease [Ca2+]i. This leads to an overall increase in basal [Ca2+]i as well as enhanced [Ca2+]i responses to agonist stimulation. In this regard, we propose that PM vs. intracellular mechanisms functionally interact in [Ca2+]i homeostasis under normal circumstances, and that disruption of these mechanisms and their interactions with inflammation leads to increased [Ca2+]i. Our overall approach will be to use human ASM cells or tissue strips to examine the above mechanisms with or without exposure to pro-inflammatory cytokines (TNFa, IL-13). Studies will use complementary techniques including molecular biology (siRNA; overexpression), imaging of [Ca2+]i, [Na+]i and luminal (SR) Ca2+, real-time confocal imaging of fluorescently- tagged proteins, as well as force measurements to address these aims. Our Specific Aims are: decrease [Ca2+]i Aim 1: To determine the influence of inflammatory cytokines on STIM1, STIM2 and Orai1 interactions in human ASM regulation; Aim 2: To determine the influence of inflammatory cytokines on NCX and its role in [Ca2+]i regulation in human ASM; Aim 3: To determine the influence of inflammatory cytokines on mitochondria and its role in [Ca2+]i regulation in human ASM; Aim 4: To determine the influence of inflammatory cytokines on SERCA and its role in [Ca2+]i regulation in human ASM; [Ca2+]i Aim 5: To determine the influence of inflammatory cytokines on the overall contribution of [Ca2+]i regulatory mechanisms to contractility in human ASM. PUBLIC HEALTH RELEVANCE: There is increasing recognition that abnormalities in airway smooth muscle contractility contribute to exaggerated airway narrowing and accompanying shortness of breath in clinically-important diseases such as asthma and chronic bronchitis. Airway contractility is highly dependent on intracellular calcium levels. The proposed studies will examine the mechanisms by which intracellular calcium is controlled in human airway smooth muscle. These studies will the foundation for better understanding of airway diseases, and potential development of new therapeutic targets.