Calcium Regulation in Airway Smooth Muscle

描述(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钙离子([钙]i)增加和/或钙敏感性增加(对给定的[钙离子]i的作用力)引起的气道平滑肌(ASM)收缩增强。呼吸道炎症是呼吸道疾病的一个重要方面，暴露于多种炎症介质(如肿瘤坏死因子(TNFa)和白介素13(IL-13))可增加ASM的收缩能力。一些研究表明，细胞因子增加了激动剂诱导的ASM中的[Ca~(2+)]_i反应，因此我们将结合当前的提议，探索炎症诱导的[Ca~(2+)]_i调节机制的变化，从而导致总体上[Ca~(2+)]_i反应的增加。在ASM中，响应SR耗竭的钙内流(存储操作的钙内流；SOCE)被细胞因子增强。初步数据表明，细胞因子可促进Na+/Ca~(2+)交换(NCX)介导的内流。SOCE和内流模式NCX的这种增强将导致[Ca~(2+)]i水平升高。[Ca~(2+)]i的降低通常通过质膜(PM)外流机制(可能包括NCX介导的外流)和SR通过SR-ATPase(SERCA)重摄取Ca~(2+)来实现。线粒体等细胞器可以缓冲Ca~(2+)并改变Ca~(2+)对SR再充盈的有效性。正常情况下，这些机制有助于将基础[Ca~(2+)]i维持在较低水平，而SR的Ca~(2+)存储在激动剂刺激之前是充满的，当SR存储耗尽时[Ca~(2+)]i上升。初步研究表明，炎症会损害SERCA和线粒体的钙缓冲。基于这些不同的初步发现，钙离子内流增加，但隔离或外流减少，我们的中心假设是炎症促进了升高[钙]i的机制，但削弱了降低[钙]i的机制。这导致基础[钙]i的总体增加以及对激动剂刺激的增强的[钙]i反应。在这方面，我们认为在正常情况下，PM和细胞内机制在功能上相互作用于[Ca~(2+)]i动态平衡，这些机制的破坏及其与炎症的相互作用会导致[Ca~(2+)]i升高。我们的总体方法将是使用人ASM细胞或组织条来研究上述机制，无论是否暴露于促炎症细胞因子(TNFa，IL-13)。研究将使用补充技术，包括分子生物学(siRNA；过表达)、[钙]i、[Na+]i和鲁米诺(SR)钙离子的成像、荧光标记蛋白质的实时共聚焦成像，以及强制测量以实现这些目标。我们的具体目标是：降低[Ca~(2+)]i；目的1：确定炎性细胞因子对人ASM调节中STIM1、STIM2和Orai1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[Ca~(2+)]i调节中的作用；目的3：确定炎性细胞因子对人ASM线粒体的影响及其在[Ca~(2+)]i调节中的作用；目的4：确定炎性细胞因子对SERCA的影响及其在人ASM[Ca~(2+)]i调节中的作用；[Ca~(2+)]i目的5：确定炎性细胞因子对[Ca~(2+)]_i调节机制对人ASM收缩功能的整体影响。

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.