Regulation of lung epithelial sodium channels by cGMP

说明（由申请人提供）：为了以最佳方式进行气体交换，肺泡空间必须保持无液体。令人信服的证据表明，体内通过肺泡上皮活跃的Na+转运有助于胎儿液的重吸收和成人肺中无液肺泡间隙的维持，特别是当肺泡对血浆蛋白的通透性或肺静水压力增加时。钠离子的重吸收通过阿米洛利敏感的上皮Na+通道(((（ENaC）和特征不明显的阳离子通道发生。基于本应用程序中提供的令人兴奋的初步数据，我们假设新描述的蛋白质（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。本应用的主要目的是：1)全面表征爪蟾卵母细胞和人肺上皮细胞中ENaC型通道的生物物理和药理学特性；2)研究cGMP对卵母细胞和人肺泡细胞中ENaC型通道的调节作用；3)利用最先进的生物化学、分子生物学、电生理学和生理学技术，评估ENaC对Na+在人上皮细胞单层和小鼠肺中的载体运输的贡献。以下具体假设将被验证：1)爪蟾卵母细胞中表达的含ENaC的同质和异质通道具有不同的生物物理特性；2)cGMP调节含ENaC的同质和异质通道，但不调节爪蟾卵母细胞中表达的ENaC通道；3)ENaC有助于非ENaC Na+电流穿过人类初级肺泡II型（ATII）和H441单层，并有助于小鼠肺部肺泡液清除。这项研究的主题是及时和重要的。这些研究结果可能为开发新的治疗策略（如cGMP递送）提供分子基础，以对抗肺水肿。

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.