Regulation of lung epithelial sodium channels by cGMP

描述(申请人提供)：为了以最佳的方式进行气体交换，肺泡腔必须保持无液体状态。令人信服的证据表明，体内活跃的Na+跨肺泡上皮细胞转运有助于胎儿液体的重吸收，并有助于维持成人肺的无液体肺泡空间，特别是当肺泡对血浆蛋白的通透性或肺静水压增加时。钠离子重吸收通过阿米洛利敏感的上皮性钠离子通道(ENaC)和特性不佳的阳离子通道。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质((ENaC)从根本上改变了(ENaC-型通道)的生物物理性质，并使它们对cGMP/PKG轴的调节敏感。本应用的主要目标是：1)全面了解非洲爪哇卵母细胞和人肺上皮细胞中ENaC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含ENaC通道的调节；以及3)利用先进的生化、分子生物学、电生理学和生理学技术，评估(ENaC)在体内通过安装在Ussing小室和小鼠肺上皮细胞单层的载体Na+转运的作用。将检验下列特定假设：1)(非洲爪哇卵母细胞中表达的含ENaC的同分和异构体通道具有不同的生物物理性质；2)cGMP调节(含ENaC的同分异构体通道，但不调节)((在非洲爪哇卵母细胞中表达的通道；以及3)(ENaC参与非((ENaC Na+电流穿过原始人肺泡II型(ATII)和H441单层以及小鼠肺组织中肺泡液的清除)。这项研究的主题是及时和重要的。这些研究的结果可能形成开发新的治疗策略(如cGMP传递)以对抗肺水肿的分子基础。

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.