LIQUID SECRETION IN THE ALVEOLAR WALL

项目摘要 目的：我们最近报道了肺泡壁液（AWL），即邻近肺泡壁的液体层。 肺泡上皮细胞是由肺泡上皮细胞分泌Cl-依赖性液体而形成的 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 三个具体的目标，我们将测试的假设，（1）肺泡I型（AT 1），而不是2型 (AT2)建立AWL分泌（2）缺氧时，过氧化氢（H2 O2）阻断AWL分泌， （3）在肺部炎症中，一氧化氮（NO）阻断AWL的分泌。 程序：在所有具体目标中，将通过双光子显微镜进行研究， 分离的小鼠肺。 我们将通过实时荧光成像确定AWL分泌 并通过免疫染色确定调节AWL分泌的信号通路， 药理学抑制、siRNA蛋白质敲低和遗传修饰小鼠中。 在 具体目标1，我们将产生在AT 1或AT 2细胞中具有靶向CFTR缺失的小鼠， 通过光解释放诱导细胞特异性Ca 2+增加。 在具体目标2中，我们将 AEC对外源性H2 O2和肺泡缺氧的反应。 我们将通过荧光团测定H2 O2， 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LPS诱导的肺部炎症的背景下， 意义：尽管众所周知AWL在肺泡气体交换中的重要性， 肺泡免疫功能，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，没有研究涉及成人肺中AWL的形成。 第一 时间，我们的方法将导致对AWL形成的监管的理解， 完整的肺 这些研究将提供新的见解的机制， 急性肺损伤的发展。

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.