Trafficking of newly synthesized surfactant protein A in isolated rat alveolar type II cells.
We examined the synthesis, transport, and localization of surfactant protein A (SP-A) in primary cultures of alveolar type II cells. In type II cells maintained in culture for 6 h, 39% of the SP-A pool detected with an enzyme-linked immunosorbent assay (ELISA) was found in lamellar bodies (LBs). After 24 h in culture, 53% of the cellular SP-A pool was found in LBs. The absolute amount of SP-A in the LB compartment was almost identical at 6 and 24 h of culture. In contrast to the results obtained with ELISA, 35S labeling of newly synthesized SP-A revealed that less than 7% of the cellular SP-A pool was in LBs at either 6 or 24 h of culture. In the 6-h cultures, 17% of the total (i.e., cells and media) [35S]SP-A pool was extracellular. In the 24-h cultures, 70% of the [35S]SP-A pool was extracellular. The secretion of [35S]SP-A was blocked by brefeldin A at all times. When medium containing newly secreted [35S]SP-A was incubated with alveolar type II cells maintained in culture for 24 h, the protein was taken up and incorporated into the LB fraction. More than 80% of the internalized SP-A was associated with the LB compartment after a 6 h incubation. The uptake of [35S]SP-A was blocked at 4 degrees C and was promoted by addition of unlabeled SP-A at 37 degrees C. These findings support a pathway of extracellular routing of SP-A prior to its accumulation in LBs in cultured type II cells.
Osanai
K
K
National Jewish Medical and Research Center; and Department of Medicine, Anna Perahia Adatto Clinical Research Center, Denver, Colorado, USA.
Mason
R J
RJ
Voelker
D R
DR
D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D008297:Male; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011510:Proteolipids / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000378:metabolism* / Q000648:ultrastructure; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013462:Sulfur Radioisotopes
D011500:Protein Synthesis Inhibitors; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D013462:Sulfur Radioisotopes; D020126:Brefeldin A