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We evaluated the contribution of endocytotic pathways to pulmonary uptake of surfactant lipids from the alveolar space. Resting and stimulated 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) uptake of unilamellar liposomes labeled with either [(3)H]dipalmitoylphosphatidylcholine ([(3)H]DPPC) or 1-palmitoyl-2-[12-(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl-phosphatidylcholine (NBD-PC) was studied in isolated perfused rat lungs and isolated type II cells. Amantadine and phenylarsine oxide, inhibitors of clathrin-mediated endocytosis, each decreased [(3)H]DPPC uptake under resting conditions by approximately 40%; their combination had no additional effect. Cytochalasin D, an inhibitor of actin-dependent processes, reduced liposome uptake by 55% and potentiated the effect of either clathrin inhibitor alone. Relative inhibition for all agents was higher in the presence of 8-Br-cAMP. The effect of inhibitors was similar for liposomes labeled with [(3)H]DPPC or NBD-PC. By fluorescence microscopy, NBD-PC taken up by lungs was localized primarily to alveolar type II cells and was localized to lamellar bodies in both lungs and isolated cells. These studies indicate that both clathrin-mediated and actin-mediated pathways are responsible for endocytosis of DPPC-labeled liposomes by alveolar type II cells in the intact lung.

surfactant protein A (SP-A) is endocytosed by type II epithelial cells through clathrindependent uptake and targeted to lamellar bodies for resecretion. However, the mechanism for secretion of newly synthesized SP-A, whether regulated exocytosis of lamellar bodies or constitutive secretion, is unresolved. If it is the latter, lamellar body SP-A would represent endocytosed protein. Amantadine, an inhibitor of clathrincoated vesicle budding, was used to evaluate the role of endocytosis in accumulation of SP-A in lamellar bodies. In isolated rat lungs, amantadine (10 mM) inhibited uptake of endotracheally instilled 35 S-labeled biosynthesized surfactant proteins by Ͼ80%. To study trafficking of newly synthesized SP-A, lungs were perfused for up to 6 h with [35 S]methionine, and surfactant was isolated from lung lavage fluid and lamellar bodies were isolated from lung homogenate. With control lungs, the mean specific activity of [ 35 S]SP-A (disintegrations per minute per microgram of SP-A) increased linearly with time of perfusion: it was significantly higher in isolated lamellar bodies than in surfactant and was increased in both compartments by 50 -60% in the presence of 0.1 mM 8-bromo-cAMP. These results suggest a precursor-product relationship between lamellar body and extracellular [35 S]SP-A. Specific activities in both compartments were unaffected by addition of amantadine (10 mM) to the lung perfusate, indicating that uptake from the alveolar space was not responsible for the increase in lamellar body [35 S]SP-A. Thus the pathway for secretion of newly synthesized SP-A is by transfer from the site of synthesis to the storage/secretory organelle prior to lamellar body exocytosis. protein trafficking; surfactant secretion; alveolar epithelial type II cell; protein synthesis; clathrin-dependent endocytosis THERE IS GENERAL AGREEMENT that lung surfactant phospholipids and surfactant proteins B and C (SP-B and SP-C) are synthesized in the endoplasmic reticulum of lung alveolar type II epithelial cells and transported to the lamellar bodies for processing and storage prior to secretion into the alveolar space. However, there is less agreement concerning the intracellular trafficking of SP-A. Like SP-B and SP-C, SP-A is synthesized by type II cells and is secreted into the alveolar space, where it associates with the lung surfactant phospholipids (19,22,37). Extracellular SP-A appears to play crucial roles in the formation of tubular myelin, in the regulation of lamellar body exocytosis, and in the uptake of phospholipids by endocytosis and their subsequent remodeling (20,23,30,40). The uptake process is dependent on the association of phospholipids with SP-A, followed by binding of the SP-Aphospholipid complex to a specific cell membrane-localized SP-A receptor (4,5,16), and then internalization of the complex by a clathrin-dependent pathway (21,32,35). Although the pathway for uptake of SP-A seems to be fairly well understood, there is uncertainty regarding the pathway for secretion of newly synthesized SP-A by ...

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