The NAD(P)H Oxidase in Airways Remodeling and Reactivity

描述（申请人提供）：目的：本项目的目标是 为了确定内源性NAD（P）H氧化酶在增殖中的作用， 反应和增强的收缩性，导致气道平滑肌（AWSM） 重塑和高反应性。假设：包容性假设 气道平滑肌NAD（P）H氧化酶高度调节， 在启动AWSM增殖中发挥独特作用的ROS产生酶 和收缩。具体目标：第一个具体目标将描述 NAD（P）H氧化酶（s）的气道平滑肌（AWSM）细胞，并测定其 有助于ROS的生成。这一目标将解决假设 NAD（P）H氧化酶4（Nox 4）与内质网膜结合， 是NAD（P）H氧化酶的催化亚基。的 第二个具体目标是确定NAD（P）H氧化酶在AWSM中的作用 增殖这一目标将检验特定生长因子 通过激活Nox 4氧化酶诱导AWSM增殖， 核因子-κ B（NF-κ B）的反式激活。第三个具体目标是 明确NAD（P）H氧化酶在AWSM收缩功能中的作用。这一目标 将检验由NAD（P）H氧化酶产生的ROS诱导AWSM的假设 但是收缩表型中氧化酶组分 与增殖性表型不同。研究：对于 第一个目标，我们的战略是定义和充分表征的结构， 负责AWSM NAD（P）H氧化酶活性的组分，并确定其 亚细胞定位和相互作用。我们还将描述活动的特征， AWSM NAD（P）H氧化酶的氧化还原中点电位和个体的作用 在ROS的生成过程中。对于第二个目标，我们将利用 纯化的细胞缺乏NAD（P）H氧化酶组分， 确定氧化酶在AWSM增殖中的重要性。重点将 为了确定氧化酶在NF-κ B反式激活中的作用， 平滑肌细胞增殖所必需的因子。对于第三个目标，我们将 再次利用纯化的细胞和具有NAD（P）H遗传缺陷的动物 氧化酶组分直接建立的重要性和特点， 调节呼吸道收缩功能的氧化酶。重点将 在研究氧化酶中是否存在表型转换时， 件.意义：这项工作将提供一个更好的理解 AWSM NAD（P）H氧化酶在调节气道平滑肌中作用 哮喘的重塑和哮喘状态下的收缩性， ROS在呼吸生理学和疾病中的重要性。

DESCRIPTION (provided by applicant): OBJECTIVE: The goals of this project are to identify the roles of endogenous NAD(P)H oxidases in the proliferative responses and enhanced contractility leading to airways smooth muscle (AWSM) remodeling and hyperreactivity in asthma. HYPOTHESIS: The inclusive hypothesis to be tested is that airway smooth muscle NAD(P)H oxidases are highly regulated ROS producing enzymes that play distinct roles in initiating AWSM proliferation and contraction. SPECIFIC AlMS: The first specific aim will characterize the NAD(P)H oxidase(s) of airway smooth muscle (AWSM) cells and determine its contribution to the generation of ROS. This aim will address the hypothesis that NAD(P)H Oxidase 4 (Nox4) bound to membranes in the endoplasmic reticulum is the catalytic subunit of the NAD(P)H oxidase in proliferating AWSM. The second specific aim will define the role of NAD(P)H oxidase(s) in AWSM proliferation. This aim will test the hypotheses that specific growth factors induce AWSM proliferation via activation of the Nox4 oxidase with resultant transactivation of nuclear factor-kappa B (NF-kB). The third specific aim will define the role of NAD(P)H oxidase(s) in AWSM contractile function. This aim will test the hypothesis that ROS generated by an NAD(P)H oxidase induce AWSM contraction, but that components of the oxidase in the contractile phenotype are distinct from those of the proliferative phenotype. RESEARCH PLAN: For the first aim, our strategy is to define and fully characterize the structure of the components responsible for AWSM NAD(P)H oxidase activity and pinpoint their subcellular location and interaction. We will also characterize the activity, the redox midpoint potential of AWSM NAD(P)H oxidase and the role of individual components in the generation of ROS. For the second aim we will utilize purified cells with deficiencies of NAD(P)H oxidase components to directly establish the importance of the oxidase in AWSM proliferation. Emphasis will be placed on establishing the role of the oxidase in transactivation of NF-kB, a factor essential for smooth muscle proliferation. For the third aim we will again utilize purified cells and animals with genetic deficiences of NAD(P)H oxidase components to directly establish the importance and characteristics of the oxidase that regulates airways contractile function. Emphasis will be placed upon investigating whether there is a phenotype switch in the oxidase components. SIGNIFICANCE: The work will provide a better understanding of the importance of AWSM NAD(P)H oxidase in mediating airways smooth muscle remodeling in asthma and contractility in the asthmatic state, with broad importance in the role of ROS in respiratory physiology and disease.