Interactions of surfactant protein A with alveolar cells

本项目将研究表面活性剂蛋白a （AP-A）在肺中的转化机制和途径。重点是颗粒状肺细胞（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SP-A和磷脂酰胆碱（PC）被肺细胞摄取的途径。我们假设(a) SP- a通过网格蛋白包被的凹坑被受体介导的内吞作用所吸收。分泌素暴露会增加受体数量。(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，另一种是通过II型细胞膜上的特定脂质结合位点，这是在胞吐过程中插入的层状体限制膜。我们认为SP-A不需要在基础条件下维持正常大鼠对PC的摄取，但在缺乏SP-A的情况下不能刺激摄取。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由II型细胞重分泌，最终被肺泡巨噬细胞降解。研究将以完整的啮齿动物、分离的灌注肺、肺微穿刺、II型细胞和肺泡巨噬细胞的原代培养进行。特异性目的1将检查SP-A与II型细胞表面的相互作用。特异性目的2将研究II型细胞摄取SP-A的机制。特异性目标3将确定SP-A通过内质网直接转移或使用生化技术内吞作用进入板层体的途径。特异性Aim 4将评估肌动蛋白介导的和网状蛋白依赖的II型细胞摄取双palmitoylphosphophatiylcholine （DPPC）的途径，使用SP-A存在（野生型）或不存在（SP-A KO小鼠）时每种途径的特异性抑制剂。由于SP-A的清除率很快，特异性Aim 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。这些研究将为确定表面活性剂成分的运输途径和主要表面活性剂蛋白SP-A的清除提供证据。

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.