ROLE AND REGULATION OF NA,K-ATPASE IN LUNG EPITHELIUM

NA,K-ATP酶在肺上皮细胞中的作用和调控

• 批准号: 2392686
• 负责人: Jacob I Sznajder
• 金额: $ 24.87万
• 依托单位: MICHAEL REESE HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392686
• 关键词: active transport; antisense nucleic acid; cell differentiation; enzyme activity; gene expression; genetic regulation; isolation perfusion; isozymes; laboratory rat; lung alveolus; protein isoforms; protein structure function; pulmonary edema; receptor expression; respiratory airway clearance; respiratory epithelium; respiratory insufficiency /failure; sodium potassium exchanging ATPase; tissue /cell culture; transfection /expression vector

Acute hypoxemic respiratory failure (AHRF) is a life-threatening condition with high morbidity and mortality. In the initial events of AHRF, lung edema accumulates and impairs alveolar oxygen exchange until mechanisms for edema clearance become effective. Lung edema is cleared predominantly by active Na+ transport; recent evidence suggests that accelerated edema clearance is associated with reduced mortality in patients with AHRF. It has been recently reported that alveolar epithelial type II (ATII) cells contain apical amiloride-sensitive Na+ channels and basolaterally located Na,K-ATPase (the alpha-1,beta-1 heterodimer); both are important contributors to Na+ transport across the alveolar epithelium. Until now Na,K-ATPase has not been localized to alveolar epithelial type I (ATI) cells. In this application we propose to study the role and regulation of the Na,K-ATPase in alveolar epithelial cells (AFC) which contributes to active Na+transport and thus lung edema clearance, by five specific aims. Specific aim # 1. To determine whether as ATII cells in different culture conditions differentiate phenotypically into ATII cells in different culture conditions differentiate phenotypically into ATI-like cells, they shift from predominantly expressing the Na,K-ATPase alpha-1,beta-1 heterodimer to the alpha-2,beta-1 heterodimer. Specific aim # 2. To determine whether this shift in isoform expression in cultured AEC is transcriptionally regulated. Specific aim #3. To determine mechanisms of Na,K-ATPase regulation in cultured ATII cells as they differentiate. Specific aim #4: To determine whether rat ATII and ATI cells express both Na,K-ATPase isozymes in situ, and if the ATI cells express predominantly the alpha-2, beta-1 Na,K-ATPase. Specific aim #5: To determine whether AEC Na,K-ATPase abundance and function can be reduced by transfecting AEC with isoform specific antisense constructs, or increased by overexpressing isoform specific sense constructs. Further, to determine whether these modulations will alter Na+ transport and lung liquid clearance. Preliminary studies have been conducted for each of the specific aims and the preliminary results support our hypotheses and the feasibility of the proposed studies. Completion of the proposed experiments will provide new information on the role and regulation of the NaK-ATPase in alveolar epithelial cells and eventually help with the design of new strategies to enhance lung edema clearance in patients with hypoxemic respiratory failure.

急性低氧性呼吸衰竭(AHRF)是一种危及生命的疾病。 发病率和死亡率都很高。在AHRF的最初事件中，肺 水肿会积聚并损害肺泡氧交换，直到 清除浮肿变得有效。肺水肿主要通过以下方式清除 主动的Na+转运；最近的证据表明加速的浮肿 清除与降低AHRF患者的死亡率有关。它 最近有报道称肺泡上皮II型(ATII)细胞 含有心尖部对阿米洛利敏感的Na+通道，并位于基底面 Na，K-ATPase(α-1，β-1异二聚体)；两者都很重要 Na+跨肺泡上皮运输的贡献者。到目前为止 Na，K-ATPase尚未定位于I型肺泡上皮(ATI) 细胞。在这一应用中，我们建议研究 肺泡上皮细胞(AFC)中的Na，K-ATPase参与 主动的Na+转运，从而清除肺水肿，通过五个特定的目标。 特异目的：1.确定不同培养条件下的ATII细胞 不同条件下分化为ATII细胞的表型 培养条件将表型分化为ATI样细胞，它们 从主要表达Na，K-ATPaseα-1，β-1的转变 杂二聚体转变为α-2，β-1杂二聚体。具体目标2.至 确定在培养的血管内皮细胞中这种异构体表达的变化是否 转录调控的。具体目标#3.确定 Na，K-ATPase在ATII细胞分化过程中的调节作用 特定目标#4：确定大鼠ATII和ATI细胞是否同时表达 Na，K-ATPase同工酶，以及ATI细胞是否主要表达 α-2，β-1Na，K-ATPase。具体目标5：确定 转染AEC可降低AEC Na，K-ATPase的丰度和功能 通过异构体特异性反义构建，或通过过度表达而增加 同形异构体的特定意义结构。此外，为了确定这些是否 调节会改变Na+转运和肺液清除。 已就每一项具体目标和 初步结果支持了我们的假设，并证明了 建议进行的研究。拟议实验的完成将提供新的 NAK-ATPase在肺泡发育中的作用和调控研究进展 并最终帮助设计新的策略来 提高低氧呼吸患者的肺水肿清除能力 失败了。