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.
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