Redox regulation of alveolar fluid balance

描述(由申请人提供)： 正常肺的肺泡上皮由两种形态不同的细胞(类型1和类型2)组成，它们负责维持肺液平衡。严格调节肺泡液体清除对于保持干燥的呼吸空间，从而进行适当的气体交换是必不可少的。现已证实，位于肺泡上皮细胞顶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起着关键作用。然而，调节ENaC功能的具体机制还不完全清楚。在肺泡内，还必须有复杂的调节机制来平衡1型和2型细胞的氧化还原状态，因为吸入的氧气被转化为超氧阴离子(O2-)。高氧压导致过多的O2-产生，会导致组织损伤和细胞死亡，而氧气不足则会导致从疲劳到危及生命的各种情况。在体内，超氧化物与一氧化氮(NO)迅速不可逆地反应生成过氧亚硝酸根。我们假设内源性02与NO的结合限制了一氧化氮对ENaC功能的抑制，从而增强了肺泡液的清除。事实上，我们有初步数据表明，一氧化氮反应迟钝的AT1细胞可能具有升高的O2-水平，并且O2-的增加加强了Na的运输。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，研究ENaC的功能，我将利用单通道膜片钳分析来检测肺切片制备中的离子传输，利用生物分子技术来检测肺细胞中的氧化还原信号，并进行体内的全肺研究。我的第一个目标是在指导阶段完成的，直接检查O2-在肺泡液清除中的作用。这一目标的成功完成将自然过渡到目标2和目标3，这两个目标利用了将在指导阶段建立的几个方案，以及纳入了研究类型1和类型2细胞的新方法。第二个目的将确定NO在肺功能中的作用，最后，第三个目的是研究O2-和NO调节肺液平衡的假定的相互关系。所提出的研究具有真正的临床意义，并有可能成为一个非常富有成效的独立研究生涯。

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.