PATHOPHYSIOLOGY OF SUBLETHAL OXYGEN INJURED LUNGS

亚致命氧损伤肺的病理生理学

• 批准号: 2750312
• 负责人: Sadis Matalon
• 金额: $ 26.1万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2750312
• 关键词: Xenopus; Xenopus oocyte; biological fluid transport; chimeric proteins; free radical oxygen; hyperoxia; laboratory rabbit; laboratory rat; lipid bilayer membrane; lung alveolus; lung injury; mutant; nitric oxide; nitrites; peroxides; protein structure function; respiratory airway clearance; respiratory epithelium; sodium channel; sodium ion; tissue /cell culture; toxicology

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.

活性钠 (Na+) 穿过成人肺泡上皮的转运发挥作用 对维持肺液平衡有重要作用，尤其是 血气屏障受到亚致死性高氧损伤后，当 被动的椋鸟部队的有效性被削弱。 目前， Na+离子进入正常和正常细胞顶膜的机制 氧损伤肺泡上皮细胞，尚未阐明。 根据初步数据，我们假设肺泡 II 型细胞 (ATII) 含有与阿米洛利低亲和力的 Na+ 通道，并且 这些通道的特性和空间分布可能会改变 暴露于亚致死的高氧环境。 由于钠通道的传导速率 远远超过任何其他运输者，他们的活动可能是 由许多药物上调，它们可能形成主要途径 Na+离子进入肺泡上皮细胞。 总体目标为 该研究项目是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中， 在单细胞水平上表征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 通过将大鼠暴露于 100% 02 中 60 小时并将其放回房间来诱导 空气中 24 小时和 72 小时，已知会增加活性的暴露时间 肺 Na+-K+ ATP 酶的浓度，以及穿过肺泡的液体清除率 上皮。 这些研究将在新分离的和 培养 ATII 细胞以评估这些通道特性的变化 随着时间的文化。 该应用程序的具体目标是：（1） 定义选择性、单离子电流、打开和关闭时间 对阿米洛利和其他药物的概率、敏感性 通过膜片钳技术； (2) 确定存在 通过蛋白质印迹技术检测 ATII 细胞中的阿米洛利结合蛋白 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； (3) 调查 这些通道是否通过 cAMP 依赖性蛋白磷酸化 激酶 (PKA) 与整个细胞中 Na+ 转运的增加相关 单元和单通道级别。 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 正常人和受伤者的牙槽间隙间隙 哺乳动物的肺。