Interactions of surfactant protein A with alveolar cells

表面活性蛋白 A 与肺泡细胞的相互作用

• 批准号: 6564812
• 负责人: Aron B. FISHER
• 金额: $ 22.37万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-10 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564812
• 关键词: actins; alveolar macrophages; clathrin; confocal scanning microscopy; electron microscopy; human tissue; immunologic assay /test; laboratory rat; lipid metabolism; lipid transport; newborn animals; phosphatidylcholines; protein metabolism; protein transport; pulmonary surfactants; receptor mediated endocytosis; respiratory epithelium; secretion; tissue /cell culture

This project will investigate the mechanisms and pathways for the turnover of surfactant protein-A (AP-A) by the lung. The emphasis is on granular pneumocytes (alveolar epithelial type II cells) by also will evaluate the degradation of SP-A by alveolar macrophages. In addition, we will compare and contrast the routes for the uptake of SP-A and phosphatidylcholine (PC) by pneumocytes. We hypothesize that (a) SP- A is taken up the receptor-mediated endocytosis through clathrin-coated pits. Receptor numbers will be increased by secretagogue exposure. (b) PC uptake occurs through two different pathways, one clathrin mediated and the second via specific lipid binding sites on the type II cells membrane that represent lamellar body limiting membranes which have been inserted during the process of exocytosis. We propose that SP-A is not required to maintain normal rat of uptake of PC under basal condition, but uptake cannot be stimulated in the absence of SP-A. We speculate that SP-A reaches lamellar bodies directly from the endoplasmic reticulum and through an endocytic route. We further propose that SP-A is resecreted by type II cells and ultimately degraded by alveolar macrophages. Studies will be carried out with intact rodents, the isolated perfused lung, lung micropuncture, and primary cultures of type II cells and alveolar macrophages. Specific Aim 1 will examine the interactions of SP-A with the type II cell surface. Specific Aim 2 will investigate th mechanisms responsible for the uptake of SP-A by type II cells. Specific Aim 3 will determine the pathway for incorporation of SP-A into lamellar bodies via either direct transfer from the endoplasmic reticulum or endocytosis using biochemical techniques. Specific Aim 4 will evaluate the actin-mediated and clathrin-dependent pathways for uptake of dipalmitoylphosphophatidyl-choline (DPPC) by type II cells using inhibitors specific for each route with SP-A present (wild type) or absent (SP-A KO mouse) Since the clearance rate for SP-A is rapid, Specific Aim 5 will determine the criteria necessary for the clearance of SP-A from the isolated perfused lung by macrophages. These studies will provide evidence defining the pathways for the trafficking of surfactant components and clearance of the principle surfactant protein, SP-A.

本项目将研究肺表面活性蛋白-A（AP-A）周转的机制和途径。重点是颗粒肺细胞（肺泡上皮II型细胞），也将评估肺泡巨噬细胞对SP-A的降解。此外，我们将比较和对比肺细胞摄取SP-A和磷脂酰胆碱（PC）的途径。我们推测：（a）SP-A通过网格蛋白包被的小凹被受体介导的内吞作用摄取。受体数量将通过促分泌素暴露而增加。(b)PC摄取通过两种不同的途径发生，一种是网格蛋白介导的，第二种是通过II型细胞膜上的特异性脂质结合位点，该位点代表在胞吐过程中插入的板层体限制膜。我们认为，在基础条件下，SP-A不需要维持正常大鼠对PC的摄取，但在缺乏SP-A的情况下，摄取不能被刺激。我们推测SP-A直接从内质网通过内吞途径到达板层体。我们进一步提出SP-A被II型细胞再分泌，并最终被肺泡巨噬细胞降解。将使用完整啮齿动物、分离的灌注肺、肺微穿刺以及II型细胞和肺泡巨噬细胞的原代培养物进行研究。具体目标1将检查SP-A与II型细胞表面的相互作用。具体目标2将研究负责II型细胞摄取SP-A的机制。具体目标3将确定SP-A通过从内质网直接转移或使用生化技术的内吞作用掺入板层体的途径。具体目标4将使用SP-A存在（野生型）或不存在（SP-A KO小鼠）的每种途径的特异性抑制剂，评价II型细胞摄取二棕榈酰磷脂酰胆碱（DPPC）的肌动蛋白介导和网格蛋白依赖性途径。由于SP-A的清除率很快，具体目标5将确定巨噬细胞从分离的灌注肺中清除SP-A所需的标准。这些研究将提供定义表面活性剂组分运输和主要表面活性剂蛋白SP-A清除途径的证据。