LIQUID SECRETION IN THE ALVEOLAR WALL

项目摘要 目的：我们最近报道了肺泡壁液体(AWL)，即肺泡壁附近的液层。 肺泡上皮细胞依赖氯离子分泌液体建立肺泡上皮 细胞(AEC)。肺泡囊性纤维化跨膜调节因子(CFTR)对此进行调节 分泌物。在这里，我们的总体目标是了解调节AWL分泌的因素。在 三个特定的目标，我们将检验以下假设：(1)肺泡I型(AT1)而不是2型 (2)在低氧条件下建立AWL分泌(2)，过氧化氢(H_2O_2)阻断AWL分泌， (3)在肺部炎症中，一氧化氮(NO)抑制AWL的分泌。 程序：在所有特定目标中，研究将通过双光子显微镜进行 隔离的小鼠肺。我们将通过实时荧光成像来确定AWL的分泌 并通过免疫染色确定调节AWL分泌的信号通路， 药理抑制、siRNA蛋白敲除和转基因小鼠。在……里面 具体目标1，我们将在AT1或AT2细胞中产生靶向CFTR缺失的小鼠，并 通过光解脱钙诱导细胞特异性钙离子升高。在具体目标2中，我们将 AEC对外源性H_2O_2和肺泡缺氧的影响。我们将用荧光团和荧光团测定过氧化氢 基于FRET的成像。在具体目标3中，我们将确定AEC NO对AWL的影响 在外源性NO供体和内毒素诱导的肺炎症背景下的分泌。 意义：尽管广为人知的AWL在肺泡气体交换和 在肺泡免疫功能方面，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。对于第一次 我们的方法将导致对AWL形成规则的理解 完整的肺部。这些研究将对潜在的机制提供新的见解。 发生急性肺损伤。

PROJECT ABSTRACT 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.