Density gradient analysis of secretions produced in vitro by human and canine airway mucosa: identification of lipids and proteoglycans in such secretions

Abstract

Human and canine airway mucosal explants synthesize and secrete high molecular weight glycoconjugates, incorporating 14C-glucosamine, a radioactive precursor to epithelial glycoprotein. Our examination of secretions produced by several individual specimens, however, did not reveal epithelial glycoprotein of typical buoyant density (1.5 g/ml in CsBr); only a high-density component with features of glycoprotein and proteoglycan. To provide sufficient material for characterization, secretions from several specimens of human and canine explants were separately pooled and subjected to DGU in CsBr. After removal of lipids and proteins, the glycoconjugates were recovered into five fractions of different density. 14C-glucosamine had been incorporated in all five fractions. Fractions 1-4 together accounted for 88% of the radiolabel but gas chromatography indicated that none of these contained epithelial glycoprotein. Their amino acid compositions were similar to those of proteoglycans and electrophoresis confirmed the presence of chondroitin sulfates A, B, C, heparan sulfate and hyaluronic acid. Sugars typical of epithelial glycoprotein were identified only in the glycoconjugate subfraction 5 of lowest density (and also lowest in yield) in which glycosaminoglycans were also identified. By addition of radioactive precursors, 14C acetate, 14C palmitate and 14C mevalonic acid to the culture medium and autoradiography of the secreted lipids we have shown that the tracheal explants actively synthesize lipids. Lipids accounted for a high proportion, almost half by weight, of the explant secretion. While neutral and phospholipids predominate, glycolipids were also identified.