Calcium Regulation in Airway Smooth Muscle

气道平滑肌中的钙调节

• 批准号: 8220908
• 负责人: Gary C. Sieck
• 金额: $ 38.51万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8220908
• 关键词: ATP phosphohydrolase; Address; Agonist; Asthma; Biology; Bradykinin; Buffers; Calcium; Cardiac; Cell membrane; Cells; Chronic Bronchitis; Color; Cyclic ADP-Ribose; Data; Development; Disease; Exposure to; Feedback; Foundations; Funding; Histamine; Homeostasis; Human; Image; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Interleukin-13; Investigation; Lead; Leukotriene D4; Leukotrienes; Link; Measurement; Mediating; Membrane Potentials; Mitochondria; Molecular; Molecular Biology; Muscle Contraction; Organelles; P(3)-1-(2-nitro)phenylethyladenosine 5' -triphosphate; Pathway interactions; Pharmacology; Production; Proteins; Regulation; Relative (related person); Reporting; Reticulum; Role; Ryanodine Receptors; STIM1 gene; Shortness of Breath; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; System; TNF gene; Techniques; Testing; Time; Tissues; Tumor Necrosis Factor-alpha; airway hyperresponsiveness; airway inflammation; airway remodeling; asthmatic patient; base; cell motility; cell type; cellular imaging; cytokine; flash photolysis; genetic regulatory protein; mitochondrial membrane; new therapeutic target; overexpression; protein expression; public health relevance; receptor; research study; respiratory smooth muscle; response; reuptake; tool; uptake

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.

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。 与公共卫生相关：越来越多的人认识到，在哮喘和慢性支气管炎等临床重要疾病中，呼吸道平滑肌收缩功能的异常会导致气道过度狭窄和伴随的呼吸短促。呼吸道收缩功能高度依赖于细胞内钙离子水平。这项拟议的研究将探讨人体呼吸道平滑肌细胞内钙离子的调控机制。这些研究将为更好地了解呼吸道疾病和潜在开发新的治疗靶点奠定基础。