Redox regulation of alveolar fluid balance

肺泡液平衡的氧化还原调节

• 批准号: 8288111
• 负责人: My N. Helms
• 金额: $ 24.24万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8288111
• 关键词: AGTR2 gene; Alveolar; Alveolar Cell; Alveolus; Amiloride; Apical; Binding; Biological Assay; Biological Models; Breathing; Cell Death; Cells; Complex; Cyclic GMP-Dependent Protein Kinases; Data; Environment; Epithelial Cells; Epithelium; Equilibrium; Fatigue; Fluid Balance; Gases; Genes; Goals; Homeostasis; Ion Channel; Ion Transport; Lead; Length; Life; Liquid substance; Lung; Measurement; Measures; Mentors; Methods; Modeling; Molecular; Mus; Nitric Oxide; Oxidation-Reduction; Oxygen; Oxygen measurement, partial pressure, arterial; Peroxonitrite; Phase; Physiological; Play; Preparation; Production; Property; Protocols documentation; Rattus; Reactive Nitrogen Species; Reactive Oxygen Species; Regulation; Research; Research Design; Respiratory physiology; Role; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Slice; Small Interfering RNA; Sodium Chloride; Superoxide Dismutase; Superoxides; Surface; Techniques; Testing; Time; Tissues; Transgenic Mice; Water; Work; alveolar epithelium; cGMP production; career; cell type; clinically relevant; epithelial Na+ channel; epithelial amiloride-sensitive sodium channel; human NOS2A protein; in vivo; in vivo Model; inhibitor/antagonist; insight; novel strategies; patch clamp; pneumocyte

DESCRIPTION (provided by applicant): The alveolar epithelium in normal lungs is comprised of two morphologically distinct types of cells (type 1 and type 2) that are responsible for maintaining lung fluid balance. Tight regulation of alveolar fluid clearance is essential for maintaining a dry breathing space, and hence, proper gas exchange. It has been established that net ion transport through amiloride-sensitive epithelial sodium channels (ENaC), located on the apical surface of alveolar epithelial cells, play a critical role in fluid clearance in normal lung. However, the specific mechanisms regulating ENaC function are not completely understood. Within the alveoli, complex regulatory mechanisms must also be in place to balance the redox state of type 1 and type 2 cells, since inspired oxygen is converted to superoxide anions (O2-). Excessive O2- production, caused by high oxygen tensions, can lead to tissue damage and cell death, whereas insufficient oxygenation can result in anything from fatigue to life threatening conditions. In vivo, superoxides react quickly and irreversibly with nitric oxide (NO) to form peroxynitrite. We hypothesize that endogenous 02- binding to NO limits nitric oxide inhibition of ENaC function, thereby enhancing alveolar fluid clearance. Indeed, we have preliminary data suggesting that nitric oxide-unresponsive AT1 cells may have elevated levels of O2-, and that increasing O2- enhances Na transport. To investigate the role of redox signaling in alveolar fluid clearance, and more specifically, ENaC function, I will utilize single channel patch clamp analysis to examine ion transport in lung slice preparations, bio-molecular techniques to examine redox signaling in pneumocytes, and perform whole lung studies in vivo. My first aim, performed during the mentored phase, directly examines the role of O2- in alveolar fluid clearance. Successful completion of this aim will naturally transition into aims 2 and 3, which utilizes several protocols that will be established in the mentored phase, as well as incorporate new approaches to studying type 1 and type 2 cells. The second aim will determine the role of NO in lung function, and lastly, the third aim examines the putative reciprocal relationship between O2- and NO regulation of lung fluid balance. The studies proposed have real clinical relevance, and the potential for a very productive independent research career.

描述（由申请人提供）： 正常肺中的肺泡上皮由两种形态不同的细胞类型（1 型和 2 型）组成，负责维持肺液平衡。严格调节肺泡液清除率对于维持干燥的呼吸空间以及适当的气体交换至关重要。已经确定，通过位于肺泡上皮细胞顶端表面的阿米洛利敏感上皮钠通道 (ENaC) 的净离子转运在正常肺的液体清除中发挥着关键作用。然而，调节 ENaC 功能的具体机制尚不完全清楚。在肺泡内，还必须建立复杂的调节机制来平衡 1 型和 2 型细胞的氧化还原状态，因为吸入的氧气会转化为超氧阴离子 (O2-)。高氧张力引起的过量氧气产生会导致组织损伤和细胞死亡，而氧合作用不足会导致从疲劳到危及生命的状况。在体内，超氧化物与一氧化氮 (NO) 快速且不可逆地反应，形成过氧亚硝酸盐。我们假设内源性O2-与NO的结合限制了一氧化氮对ENaC功能的抑制，从而增强肺泡液清除率。事实上，我们的初步数据表明，一氧化氮无反应的 AT1 细胞可能具有升高的 O2- 水平，并且增加 O2- 会增强 Na 转运。为了研究氧化还原信号在肺泡液清除中的作用，更具体地说，ENaC 功能，我将利用单通道膜片钳分析来检查肺切片制剂中的离子传输，利用生物分子技术来检查肺细胞中的氧化还原信号，并进行整体分析。体内肺研究。我的第一个目标是在指导阶段进行的，直接检查 O2- 在肺泡液清除中的作用。成功完成这一目标将自然过渡到目标 2 和 3，其中利用将在指导阶段建立的多个方案，并纳入研究 1 型和 2 型细胞的新方法。第二个目标将确定 NO 在肺功能中的作用，最后，第三个目标检查 O2- 和 NO 调节肺液平衡之间假定的相互关系。所提出的研究具有真正的临床相关性，并且有可能成为非常富有成效的独立研究生涯。