Asthma and EC coupling in airway smooth muscle cells

气道平滑肌细胞中的哮喘和 EC 耦合

• 批准号: 6892010
• 负责人: SEAN M WILSON
• 金额: $ 3.01万
• 依托单位: UNIVERSITY OF MISSISSIPPI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2005-07-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6892010
• 关键词: allergens; asthma; biological signal transduction; calcium channel; calcium flux; confocal scanning microscopy; immunocytochemistry; laboratory rat; muscle contraction; polymerase chain reaction; receptor coupling; receptor sensitivity; respiratory airway pressure; respiratory hypersensitivity; respiratory muscles; smooth muscle

DESCRIPTION (provided by applicant): Allergic asthma is the result of airway sensitization and subsequent challenge with allergen, which induces a reduction in gas exchange at the alveoli. Contributing to this impaired gas exchange there is a marked proliferation of airway smooth muscle cells (ASMCs) that also become hypersensitive to both specific (allergen) and nonspecific (acetylcholine) challenge. Given that increases in the intracellular Ca2+ concentration ([Ca2+]i) are integral to airway smooth muscle (ASM) contraction, the central hypothesis is that alterations in Ca2+ signaling are a component of asthma-induced hypersensitivity of ASM. A number of reports indicate that inflammatory agents cause [Ca2+]I to increase in ASMCs. However, the interaction between inflammatory mediators and/or allergic cytokines and increases in ASM cytosolic Ca2+ is unresolved. The two specific aims outlined in the proposal are designed to examine the interaction between inflammatory stimulation, asthma, and cytosolic Ca2+. Specific Aim 1 tests the hypothesis that allergen sensitization and challenge induced ASMC hypersensitivity is due to enhanced intracellular Ca2+ release and extracellular Ca2+ entry. [Ca2+]i will be measured in isolated ASMCs from control and ovalbumin allergen sensitized and challenged Brown-Norway rats, which mimics allergen induced asthma in humans using global Ca2+ imaging and real-time laser scanning confocal microscopy techniques. Changes in basal [Ca2+]i, Kd of Ca2+ increases, change in the spatial and temporal aspects of Ca2+ signaling, or changes in the rate and routes of Ca2+ entry and cytosolic Ca2+ removal with serotonin exposure will be determined. Specific Aim 2 tests the hypothesis that allergen sensitization and challenge induced ASM hypersensitivity is the result of a change in expression of gene products that directly alter Ca2+ signaling. Quantitative RT-PCR and immunohistochemistry techniques will be used to determine if allergen sensitization and challenge changes the expression of non-selective cation channels as well as changes the expression and spatial location of ryanodine and IP3 receptors. Experimental results from these two aims will provide critical pilot data towards the long-term objectives that are to understand the role of changes in Ca2+ signaling to asthma induced hypersensitivity and to elucidate the cell signaling pathways that lead to airway hyperresponsiveness.

描述（由申请人提供）：过敏性哮喘是气道致敏和随后过敏原激发的结果，这会导致肺泡气体交换减少。 气道平滑肌细胞（ASMC）显着增殖，导致气体交换受损，这些细胞也对特异性（过敏原）和非特异性（乙酰胆碱）挑战变得过敏。 鉴于细胞内 Ca2+ 浓度 ([Ca2+]i) 的增加是气道平滑肌 (ASM) 收缩不可或缺的一部分，因此中心假设是 Ca2+ 信号传导的改变是哮喘诱发的 ASM 超敏反应的一个组成部分。 许多报告表明，炎症因子会导致 ASMC 中的 [Ca2+]I 增加。 然而，炎症介质和/或过敏细胞因子与 ASM 胞质 Ca2+ 增加之间的相互作用尚未解决。 该提案中概述的两个具体目标旨在检查炎症刺激、哮喘和胞质 Ca2+ 之间的相互作用。 具体目标 1 检验了以下假设：过敏原致敏和攻击诱导的 ASMC 超敏反应是由于细胞内 Ca2+ 释放和细胞外 Ca2+ 进入增强所致。 将在来自对照和卵清蛋白过敏原致敏和激发的 Brown-Norway 大鼠的分离 ASMC 中测量 [Ca2+]i，使用整体 Ca2+ 成像和实时激光扫描共聚焦显微镜技术模拟人类过敏原诱发的哮喘。 将确定基础 [Ca2+]i 的变化、Ca2+ 的 Kd 增加、Ca2+ 信号传导的空间和时间方面的变化，或随着血清素暴露，Ca2+ 进入和胞质 Ca2+ 去除的速率和途径的变化。 具体目标 2 测试了以下假设：过敏原致敏和攻击诱导的 ASM 超敏反应是直接改变 Ca2+ 信号传导的基因产物表达变化的结果。 定量 RT-PCR 和免疫组织化学技术将用于确定过敏原致敏和激发是否改变非选择性阳离子通道的表达以及兰尼碱和 IP3 受体的表达和空间位置。 这两个目标的实验结果将为长期目标提供关键的试点数据，即了解 Ca2+ 信号传导变化对哮喘引起的超敏反应的作用，并阐明导致气道高反应性的细胞信号传导途径。