PATHOPHYSIOLOGY OF SUBLETHAL OXYGEN INJURED LUNGS

活性钠(Na+)跨肺泡上皮转运的作用 在维持肺液平衡方面起着重要作用，尤其是 在亚致死性高氧损伤血气屏障后，当 被动的Starling部队的效力被削弱。目前， Na+离子进入正常根尖膜和根尖膜的机制 氧损伤的肺泡上皮细胞，目前还没有阐明。 根据初步数据，我们假设肺泡II型细胞 (ATII)含有与阿米洛利亲和力低的Na+通道，并且 这些通道的属性和空间分布可通过以下方式更改 暴露在亚致命的高氧环境中。由于钠通道以 远远超过任何其他运输商，他们的活动可能是 在一些制剂的上调下，它们可能形成一条主要的途径 Na+离子进入肺泡上皮细胞。的总目标是 本研究项目旨在评估这些离子通道的分布。 在正常和高氧损伤大鼠的肺泡上皮细胞中， 在单细胞水平上描述它们的特性，并研究一些 他们的基本监管机制。亚致死性高氧损伤将 将大鼠暴露于100%O2的60h，并将其送回房间 在空气中停留24小时和72小时，这样的暴露时间会增加酶的活性 肺组织Na~+-K~+-ATPase活性和肺泡液体清除率 上皮组织。这些研究将在新隔离的和 培养的ATII细胞以评估这些通道特性的变化 随着时间的推移，在文化上。本申请的具体目的是：(1) 定义选择性、单离子电流、打开和关闭时间 概率、对阿米洛利和其他药理药物的敏感性 通过膜片钳技术；(2)确定存在 用Western blotting技术检测血管紧张素Ⅱ细胞中的阿米洛利结合蛋白(S) 并通过免疫细胞化学研究确定它们的空间定位 光学和电子显微镜水平；以及(3)研究 这些通道是否通过cAMP依赖蛋白磷酸化 激酶(PKA)与两者Na+转运的增加有关 单元级和单信道级。完成这些具体目标将 提供有关流体可能机制的新的基础知识 正常人和伤员对牙槽间隙的清除 哺乳动物的肺。

Active sodium (Na+) transport across the adult alveolar epithelium plays an important role in the maintenance of lung fluid balance, especially after sublethal hyperoxic injury to the blood-gas barrier, when the effectiveness of the passive Starling forces is diminished. Presently, the mechanisms by which Na+ ions enter the apical membranes of normal and oxygen-injured alveolar epithelial cells, have not been elucidated. Based on preliminary data, we hypothesize that alveolar type II cells (ATII) contain Na+ channels with low affinity to amiloride and that the properties and spatial distribution of these channels may be altered by exposure to sublethal hyperoxia. Since sodium channels conduct at rates far exceeding that of any other transporter, and their activities may be upregulated by a number of agents, they may form a major pathway for the entry of Na+ ions into alveolar epithelial cells. The overall goal of this research project is to assess the distribution of these ion channels in the alveolar epithelium of normal and hyperoxic-injured rats, characterize their properties at the single cell level and study some of their fundamental regulatory mechanisms. Sublethal hyperoxic injury will be induced by exposing rats to 60 h of 100% 02 and returning them to room air for 24 h and 72 h, an exposure period known to increase the activity of lung Na+-K+ ATPase, and the rate of fluid removal across the alveolar epithelium. These studies will be conducted in both freshly isolated and cultured ATII cells to assess changes in the properties of these channels with time in culture. The specific aims of this application are: (1) to define the selectivity, single ion current, open and close time probabilities, sensitivity to amiloride and other pharmacological agents by patch-clamp techniques; (2) to establish the existence of amiloride-binding protein(s) in ATII cells by Western blotting techniques and determine their spatial localization by immunocytochemical studies at both the light and electron microscopic level; and (3) to investigate whether phosphorylation of these channels via the cAMP-dependent protein kinase (PKA) correlated with increased Na+ transport at both the whole cell and single channel level. Completion of these specific aims will provide new and fundamental knowledge on the possible mechanisms of fluid clearance across the alveolar space of both the normal and injured mammalian lung.