Regulation of lung epithelial sodium channels by cGMP

描述（由申请人提供）：为了以最佳方式进行气体交换，肺泡腔必须保持无液体。令人信服的证据表明，在体内的肺泡上皮细胞的主动Na+运输有助于胎儿液体的重吸收和维持成人肺中的无液体肺泡空间，特别是当肺泡对血浆蛋白的渗透性或肺静水压力增加时。Na+离子重吸收通过阿米洛利敏感的上皮Na+通道（ENaC））和表征不佳的阳离子通道发生。基于本申请中提供的令人兴奋的初步数据，我们假设一种新描述的蛋白（（ENaC）从根本上改变了（（ENaC）型通道的生物物理特性，并使它们对通过cGMP/PKG轴的调节敏感。本申请的主要目标是：1）充分表征的生物物理和药理学性质的（非洲爪蟾卵母细胞和人肺上皮细胞中的ENaC型通道; 2）研究ENaC型通道的调控。（卵母细胞和人肺泡细胞中cGMP的含ENaC通道;和3）使用最新研究结果评估ENaC对体内穿过安装在Ussing室中的人上皮细胞单层和小鼠肺的Na+载体转运的贡献。涉及生物化学、分子生物学、电生理学和生理学技术。将检验以下具体假设：1）（在非洲爪蟾卵母细胞中表达的含ENaC的同源和异源通道具有不同的生物物理特性; 2）cGMP调节含有ENaC的同源和异源通道，但在非洲爪蟾卵母细胞中不表达（（）通道;和3）ENaC有助于穿过原代人肺泡II型（ATII）和H441单层的非（ENaC）Na+电流以及小鼠肺中的肺泡液清除。这项研究的主题是及时和重要的。这些研究的结果可能会形成新的治疗策略（如cGMP输送），以打击肺水肿的发展的分子基础。

DESCRIPTION (provided by applicant): In order for gas exchange to occur in an optimum fashion, the alveolar space must remain free of fluid. Convincing evidence indicates that active Na+ transport across the alveolar epithelium in vivo contributes to the reabsorption of the fetal fluid and to the maintenance of fluid free alveolar spaces in adult lungs, especially when either alveolar permeability to plasma proteins or lung hydrostatic pressures are increased. Na+ ion reabsorption occurs through the amiloride sensitive epithelial Na+ channels (((( ENaC) and poorly characterized cation channels. Based on the exciting preliminary data presented in this application, we hypothesize that a newly described protein (( ENaC) fundamentally alters the biophysical properties of ((( ENaC-type channels and renders them sensitive to regulation via the cGMP/PKG axis. The main goals of this application are: 1) to fully characterize the biophysical and pharmacological properties of ( ENaC type channels in both Xenopus oocytes and human lung epithelial cells; 2) to investigate the regulation of ( ENaC-containing channels by cGMP in oocytes and human alveolar cells; and 3) to assess the contribution of ( ENaC to vectorial Na+ transport across human epithelial cell monolayers mounted in Ussing chambers and mouse lungs in vivo using state-of-the-art biochemical, molecular biological, electrophysiological and physiological techniques. The following specific hypotheses will be tested: 1) ( ENaC-containing homo- and heteromeric channels expressed in Xenopus oocytes have diverse biophysical properties; 2) cGMP regulates (ENaC-containing homo- and heteromeric channels but not ((( channels expressed in Xenopus oocytes; and 3) ( ENaC contributes to the non-((( ENaC Na+ currents across primary human alveolar type II (ATII) and H441 monolayers and to alveolar fluid clearance in mouse lungs. The subject matter of this research is timely and important. Results of these studies may form the molecular basis for development of new therapeutic strategies (such as cGMP delivery) to combat pulmonary edema.