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。

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.