Redox regulation of alveolar fluid balance

描述（由申请人提供）： 正常肺中的肺泡上皮由两种形态上不同类型的细胞（1型和2型）组成，其负责维持肺流体平衡。严格调节肺泡液体清除率对于维持干燥的呼吸空间以及因此的适当气体交换是必不可少的。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性上皮钠通道（ENaC）的净离子转运在正常肺中的液体清除中起关键作用。然而，调节ENaC功能的具体机制尚未完全了解。在肺泡内，复杂的调节机制也必须到位，以平衡1型和2型细胞的氧化还原状态，因为吸入的氧气转化为超氧阴离子（O2-）。由高氧张力引起的过量O2-产生可导致组织损伤和细胞死亡，而氧合不足可导致从疲劳到危及生命的任何情况。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ENaC功能的一氧化氮抑制，从而增强了肺泡液清除。事实上，我们有初步的数据表明，一氧化氮无反应的AT 1细胞可能有升高的水平的O2-，增加O2-增强钠转运。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备中的离子转运，生物分子技术来检查肺细胞中的氧化还原信号，并进行体内全肺研究。我的第一个目标，在指导阶段进行，直接检查肺泡液体清除中O2-的作用。这一目标的成功完成将自然过渡到目标2和3，这两个目标利用了将在指导阶段建立的几个协议，并结合了研究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，第三个目标检查肺液体平衡的O2-和NO调节之间的假定的相互关系。所提出的研究具有真实的临床相关性，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.