LIQUID SECRETION IN THE ALVEOLAR WALL

描述(申请人提供)：目的：我们最近报道了肺泡壁液体(AWL)，接近肺泡上皮的液层是由肺泡上皮细胞(AEC)依赖氯离子的液体分泌建立的。肺泡囊性纤维化跨膜调节因子(CFTR)调节这种分泌。在这里，我们的总体目标是了解调节AWL分泌的因素。在这三个特定的目标中，我们将检验以下假设：(1)肺泡I型(AT1)而不是2型(AT2)可促进AWL的分泌；(2)在低氧条件下，过氧化氢(H_2O_2)可阻断AWL的分泌；(3)在肺部炎症中，一氧化氮(NO)可阻断AWL的分泌。程序：在所有特定的目标中，研究将通过对分离的小鼠肺进行双光子显微镜来进行。我们将通过实时荧光成像来确定AWL的分泌，并通过免疫染色、药物抑制、siRNA蛋白下调和转基因小鼠来确定调节AWL分泌的信号通路。在特定的目标1中，我们将在AT1或AT2细胞中产生定向CFTR缺失的小鼠，并通过光解去核诱导细胞特异性的钙离子增加。在特定目标2中，我们将AEC置于外源性H_2O_2和肺泡缺氧中。我们将通过荧光团和基于FRET的成像来测定过氧化氢。在具体目标3中，我们将在外源性NO供体和内毒素诱导的肺炎症的背景下，确定AEC NO对AWL分泌的影响。意义：尽管AWL在肺泡气体交换和肺泡免疫功能中的重要性是众所周知的，但AWL在肺部炎症中的作用仍然知之甚少。在我们最近的报告之前，没有关于成人肺中AWL形成的研究。我们的方法将第一次导致对完整肺中AWL形成的调节的理解。这些研究将为急性肺损伤的发生机制提供新的见解。 公共卫生相关性：该项目旨在确定通过主动跨肺泡氯转运形成肺泡壁液体的基本机制及其在肺泡缺氧和肺部炎症中的调节作用。这项研究的发现可能会影响对急性肺损伤机制的理解和相关治疗的发展。

DESCRIPTION (provided by applicant): Aims: We recently reported the alveolar wall liquid (AWL), the liquid layer adjacent to the alveolar epithelium is established by Cl--dependent liquid secretion from alveolar epithelial cells (AEC). The alveolar cystic fibrosis transmembrane regulator (CFTR) regulates this secretion. Here our overall objective is to understand factors regulating AWL secretion. In the three specific aims, we will test the hypotheses that (1) alveolar type I (AT1) but not type 2 (AT2) establish AWL secretion (2) in hypoxia, hydrogen peroxide (H2O2) blocks AWL secretion, and that (3) in lung inflammation, nitric oxide (NO) blocks AWL secretion. Procedures: In all specific aims, studies will be developed through two-photon microscopy of isolated mouse lungs. We will determine AWL secretion through real-time fluorescent imaging and determine signaling pathways regulating AWL secretion by immunostaining, pharmacological inhibition, siRNA protein knock-down and in genetically modified mice. In Specific Aim 1, we will generate mice with targeted CFTR deletion in AT1 or AT2 cells and induce cell-specific Ca2+ increases by photolytic uncaging. In Specific Aim 2, we will subject AEC to exogenous H2O2 and alveolar hypoxia. We will determine H2O2 by fluorophore and FRET based imaging. In Specific Aim 3, we will determine the effect of AEC NO on AWL secretion in the context of exogenous NO donors and LPS-induced lung inflammation. Significance: Despite the well-known importance of the AWL in alveolar gas exchange and alveolar immune function, the AWL role in lung inflammation remains poorly understood. Prior to our recent report, no studies addressed AWL formation in the adult lung. For the first time our approach will lead to an understanding of the regulation of AWL formation in the intact lung. These studies will provide new insights into mechanisms underlying the development of acute lung injury. PUBLIC HEALTH RELEVANCE: This project is to determine fundamental mechanisms underlying formation of the alveolar wall liquid by active trans-alveolar chloride transport and its regulation in alveolar hypoxia and lung inflammation. The findings of this research are likely to impact understanding of mechanisms of acute lung injury and the development of relevant therapy.