Mucus and surfactant synthesis and secretion by cultured hamster respiratory cells

Abstract

Procedures for the selective isolation and cultivation in monolayer of respiratory cells have been developed. This technique requires repeated protease treatment and gradient centrifugation of hamster tracheal or lung tissues and permits the establishment of proliferating cultures of epithelial cells with biologic specialization. Mucus synthesis was monitored in cultured tracheal cells by incorporation of 3H-labeled N-acetyl-D-galactosamine and 14C-serine into glycoprotein as determined by trichloroacetic acid precipitation of growth medium followed by acrylamide gel electrophoresis. For comparative purposes tracheal explants and several established cell lines were also examined. Synthesis and secretion of the glycoprotein macromolecule by tracheal cell monolayers appeared to be regulated by vitamin A since its addition to the culture medium significantly increased both the number of cell-associated granules and glycoprotein secretion. Lung-originated cell cultures were grown to confluence and radio-labeled with 3H-choline in serum-free medium for 24 hr to examine surfactant synthesis. Cell monolayers and growth medium were then extracted by the Folch method, and total radioactive phosphatidylcholine as well as disaturated phosphatidylcholine were determined by thin-layer chromatography and alumina gel fractionation of osmium tetroxide-reactive phospholipid, respectively. Data indicate that these cultures have a marked ability to synthesize and secrete surfactant when compared to other established cell lines. In addition, naturally transformed cells that arose during passage and senescence of the primary cultures were analyzed for their biosynthetic capabilities.