Effects of hypoxia on the alveolar epthelium

急性肺损伤患者会出现低氧血症和气体交换障碍，导致显著的发病率和死亡率。据报道，缺氧可通过抑制肺泡上皮细胞中的Na+通道和Na，K-ATP酶而损害肺清除水肿的能力。本申请的重点是确定1.5%或3%（约10或20 mm Hg）的严重缺氧1至4小时是否通过引起Na+泵经由活性氧（ROS）和蛋白激酶C（PKC）信号传导分子激活的特定途径内吞到细胞内区室中来抑制Na，K-ATP酶。我们将确定是否暴露于缺氧后，肺泡上皮细胞（AEC）的复氧的结果在招聘以前内吞的Na+泵回到细胞基底外侧膜（BLM）。我们将研究特布他林和毛喉素激活蛋白激酶A通路是否逆转缺氧对AEC Na+泵的影响，以及这是否增加肺液体清除率。我们还将确定在AEC暴露于长时间缺氧（>12小时）时，Na，K-ATP酶 蛋白质通过蛋白酶体或溶酶体途径进行泛素化和降解。因此，我们将从四个相互关联的目标研究缺氧对肺泡上皮细胞Na，K-ATP酶的调节机制：在具体目标# 1中，我们提出确定缺氧过程中产生的线粒体ROS对Na，K-ATP酶功能和蛋白质内吞的作用;在具体目标#2中，我们将研究在缺氧期间肺泡上皮Na，K-ATP酶是否被PKC磷酸化，从而触发Na+泵的内吞作用;在具体目标#3中，我们将确定缺氧介导的Na，K-ATP酶功能的抑制和Na+泵的内吞作用在再氧合时是否可逆，以及特布他林和毛喉素对PKA通路的激活是否阻止或逆转缺氧的作用;在具体目标#4中，我们提出确定AEC长时间暴露于缺氧是否通过蛋白体/溶酶体途径导致Na，K-ATP酶蛋白的泛素化和降解。针对每个具体目标进行了实验，初步结果支持了该建议的可行性。拟定研究的完成将提供有关缺氧对肺泡上皮细胞影响的新信息，特别是因为它涉及Na+泵的抑制和降解机制以及Na，K-ATP酶抑制的逆转途径，这对于设计新方法以增加肺水肿患者的水肿清除可能具有重要意义。

Patients with acute lung injury develop hypoxemia and gas exchange impairment which results in significant morbidity and mortality. It has been reported that hypoxia may impair the lung's ability to clear edema by inhibiting Na+ channels and the Na,K-ATPase in the alveolar epithelium. The focus of this application is to determine whether severe hypoxia of 1.5% or 3% (about 10 or 20 mm Hg) for 1 to 4 hours inhibits Na,K-ATPase by causing endocytosis of the Na+ pump into intracellular compartments via specific pathways activated by reactive oxygen species (ROS) and protein kinase C (PKC) signaling molecules. We will determine whether after exposure to hypoxia, reoxygenation of the alveolar epithelial cells (AEC) results in the recruitment of previously endocytosed Na+ pumps back into the cell basolateral membranes (BLM). We will study whether activation of the protein kinase A pathway by terbutaline and forskolin reverses the effects of hypoxia on AEC Na+ pumps and whether this increases lung liquid clearance. We will also determine whether in AEC exposed to prolonged hypoxia (>12 hours) the Na,K-ATPase proteins undergo ubiquitination and degradation via the proteasomal or lysosomal pathways. As such, we will study the effects of hypoxia on the alveolar epithelium by focusing on the mechanisms of Na,K-ATPase regulation in four interrelated aims: in Specific Aim # 1 we propose to determine the role of mitochondrial ROS generated during hypoxia on Na,K-ATPase function and protein endocytosis; in Specific Aim # 2 we will study whether the alveolar epithelial Na,K-ATPase is phosphorylated during hypoxia by PKC triggering the endocytosis of the Na+ pump; in Specific Aim # 3 we will determine whether hypoxia-mediated inhibition of the Na,K-ATPase function and endocytosis of the Na+ pump are reversible upon reoxygenation and whether activation of the PKA pathway by terbutaline and forskolin prevents or reverses the effects of hypoxia; in Specific Aim # 4 we propose to determine whether prolonged exposure of AEC to hypoxia leads to ubiquitination and degradation of the Na,K-ATPase proteins via the proteosomal/lysosomal pathways. Experiments have been conducted for each of the specific aims and the preliminary results support the feasibility of this proposal. Completion of the proposed studies will provide novel information on the effects of hypoxia on the alveolar epithelium, specifically as it pertains to mechanisms of inhibition and degradation of the Na+ pump as well as pathways of reversal of Na,K-ATPase inhibition, which may be of importance for the design of novel approaches to increase edema clearance in patients with pulmonary edema.