Molecular Mechanisms of Airway Smooth Muscle Relaxation

尽管吸入β-激动剂是治疗和预防急性支气管痉挛的一线治疗方法 哮喘会出现这种情况，但人们对其作用机制知之甚少。在此Roo应用程序中，我们建议 一系列假设驱动的实验，以及新方法的开发，以澄清 介导气道平滑肌(ASM)松弛的机制。拟议的研究将利用这两种方法 小鼠和人类组织和细胞，以探索两种相关药物，β-激动剂和 前列腺素E2(PGE2)，介导抑制ASM张力的产生。具体目标包括：(目标1) 确定β-激动剂和前列腺素E_2通过其抑制ASM钙流动和途径的特定靶点 以及(目标2)鉴定ISO和PGE2处理的ASM中的PKA底物以及这些基因 它的表达会因慢性激动剂治疗而改变。拟议的研究将提供更多 通过确定β-激动剂的特定细胞内靶点对I期结果的机械性洞察 拮抗钙离子流动和收缩的信号，以及这些靶点的调节如何受到影响 使用慢性激动剂治疗。其他目标包括：(目标3)建立慢病毒感染的应用 用于在培养的小鼠和人的呼吸道中异源表达；以及(目标4)产生初步的 基因型对初级结局影响的分析。后两个目标将开发新的、强大的工具 实现对ASM收缩调节的进一步机械性洞察，并确定 功能基因组学后续研究。在完成这些第二阶段研究后，私隐专员应已 在ASM领域进行高度相关研究的经验基础和强大的方法 生理学和生物学。通俗摘要：本提案中的研究将帮助我们了解 哮喘疗法是一种β-激动剂，在细胞和分子水平上起作用，从而提高我们的发育能力 类似的、更有效的疗法。

Although inhaled beta-agonist Is a first line therapy for treatment and prophylaxis of acute bronchospasm that occurs with asthma, its mechanisms of action are poorly understood. In this ROO application we propose of series of hypothesis-driven experiments, as well as the development of new approaches, to clarify mechanisms mediating airway smooth muscle (ASM) relaxation. The proposed studies will utilize both murine and human tissue and cells to explore mechanisms by which two relevant agents, beta-agonist and prostaglandin E2 (PGE2), mediate inhibition of ASM tension generation. Specific Aims include: (Aim 1) Identify the specific targets through which beta-agonist and PGE2 attenuate ASM calcium flux and ainway contraction; and (Aim 2) Identify the PKA substrates in ASM treated with ISO and PGE2, and those genes whose expression is altered by chronic agonist treatment. The proposed studies will provide greater mechanistic insight into the Phase I outcomes by identifying the specific intracellular targets of beta-agonist signaling that antagonize calcium flux and contraction, and how modulation of these targets is influenced with chronic agonist treatment. Additional aims include: (Aim 3) Establish the application of lentiviral infection for heterologous expression in murine and human airways in culture; and (Aim 4) Generate preliminary analyses of genotype effects on primary outcomes. The latter 2 Aims will develop new, powerful tools for enable further mechanistic insight into ASM contractile regulation, and identify genotype effects enabling subsequent studies in Functional Genomics. Upon completion of these Phase II studies, the PI should have both an empirical basis and powerful methodologies to pursue highly relevant research in the field of ASM physiology and biology. LAY SUMMARY: Studies in this proposal will help us understand how a common asthma therapy, beta-agonist, works at a cellular and molecular level thus improving our ability to develop similar, more effective therapies.