Asthma and EC coupling in airway smooth muscle cells

描述（由申请人提供）：过敏性哮喘是气道致敏和随后的过敏原挑战的结果，导致肺泡气体交换减少。气道平滑肌细胞（ASMCs）的显著增殖也导致了这种气体交换受损，这些细胞对特异性（过敏原）和非特异性（乙酰胆碱）挑战都变得超敏感。考虑到细胞内Ca2+浓度（[Ca2+]i）的增加是气道平滑肌（ASM）收缩的组成部分，中心假设是Ca2+信号的改变是哮喘诱导的ASM超敏反应的一个组成部分。许多报道表明，炎症因子导致ASMCs中[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。提案中概述的两个具体目标旨在检查炎症刺激，哮喘和细胞质Ca2+之间的相互作用。特异性Aim 1验证了过敏原致敏和挑战诱导的ASMC超敏反应是由于细胞内Ca2+释放和细胞外Ca2+进入增强的假设。[Ca2+]i将在来自对照和卵清蛋白过敏原致敏和挑战的褐挪威大鼠的分离asmc中进行测量，褐挪威大鼠使用全局Ca2+成像和实时激光扫描共聚焦显微镜技术模拟过敏原诱导的人类哮喘。将确定基础[Ca2+]i的变化，Ca2+的Kd增加，Ca2+信号的时空变化，或Ca2+进入和胞质Ca2+去除随血清素暴露的速率和途径的变化。特异性目标2验证了过敏原致敏和挑战诱导的ASM超敏反应是直接改变Ca2+信号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刺激是否改变非选择性阳离子通道的表达以及ryanodine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，这些目标是了解Ca2+信号变化在哮喘诱导的超敏反应中的作用，并阐明导致气道高反应性的细胞信号通路。

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.