Molecular Mechanisms of Airway Smooth Muscle Relaxation

虽然吸入性β受体激动剂是治疗和预防急性支气管痉挛的一线疗法， 但其作用机制尚不清楚。在这个ROO应用程序中，我们建议 一系列假设驱动的实验，以及新方法的发展，以澄清 调节气道平滑肌（ASM）松弛的机制。拟议的研究将利用这两个 小鼠和人的组织和细胞，以探索两种相关药物，β-激动剂和 前列腺素E2（PGE 2）介导ASM张力产生的抑制。具体目标包括：（目标1） 确定β-激动剂和PGE 2通过其减弱ASM钙通量和通道的特定靶点 （目的2）鉴定用ISO和PGE 2处理的ASM中的PKA底物，以及那些基因 其表达被慢性激动剂治疗改变。拟议的研究将提供更多的 通过识别β-激动剂的特异性细胞内靶点，对I期结局的机制性见解 拮抗钙流动和收缩的信号传导，以及如何影响这些靶点的调节 慢性激动剂治疗。其他目的包括：（目的3）建立慢病毒感染的应用 用于在培养物中的鼠和人气道中的异源表达;以及（目的4）产生初步的 分析基因型对主要结果的影响。后两个目标将开发新的，强大的工具， 进一步深入了解ASM收缩调节机制，并确定基因型效应， 功能基因组学的后续研究。完成这些II期研究后，PI应 既有经验基础，又有强大的方法，可在ASM领域进行高度相关的研究 生理学和生物学。总结：这项建议的研究将帮助我们了解一个常见的 哮喘治疗，β-激动剂，在细胞和分子水平上起作用，从而提高我们的发展能力， 更有效的治疗方法。

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.