Turnover of cell-surface macromolecules in cultured dog tracheal epithelial cells

• Published in: Biochimica et biophysica acta Vol. 966; no. 3; pp. 336 - 346
• Main Authors: Iwamoto, Itsuo, Nadel, Jay A., Varsano, Shabtai, Forsberg, Lennart S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 08.09.1988
• Subjects: Animals; Cell Membrane - enzymology; Cell Membrane - metabolism; Cell surface macromolecule; Cells, Cultured; Dog trachea; Dogs; Epithelial cell; Epithelial Cells; Epithelium - enzymology; Epithelium - metabolism; Glycocalyx; Kinetics; Macromolecular Substances; Membrane Proteins - metabolism; Protease Inhibitors; Serine - metabolism; Sulfated macromolecule; Sulfates - metabolism; Sulfur Radioisotopes; Temperature; Trachea - cytology; Trachea - enzymology; Trachea - metabolism; Trypsin; Cell surface macromolecule; Glycocalyx; Dog trachea; Epithelial cell; DME; Dulbecco's modified Eagle's medium; Sulfated macromolecule
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(88)90083-9

We studied the metabolism of sulfated cell-surface macromolecules in dog tracheal epithelial cells in primary culture. To examine the time-course and rate of appearance of sulfated macromolecules at the cell surface, the cells were pulsed with 35SO 4 for short periods (5–15 min), and the incubation medium was sampled for spontaneously released macromolecules (basal secretions) and for release induced by trypsin (trypsin-accessible secretions). Trypsin-accessible 35S-labeled macromolecules appeared on the cell surface within 5–10 min, increased linearly, and plateaued by 40 min; the median transit time for 35S-labeled macromolecules to reach the cell surface was 21 min. 35S-labeled macromolecules in basal secretions increased with a similar time-course, reaching a plateau by 40 min. Incorporation of [ 3H]serine into the protein moiety of trypsin-accessible macromolecules occured more slowly; trypsin-accessible 3H-labelled macromolecules were barely detectable at 1 h and increased to a maximum after 2 h, suggesting the presence of a preformed pool of nonsulfated core protein. Pretreatment with cycloheximide, an inhibitor of protein synthesis, decreased trypsin-accessible 35S-labeled macromolecules log-linearly depending on the duration of pretreatment providing an estimate of the rate of depletion of the core protein pool ( t 1 2 = 32 min ). During continuous exposure to 35SO 4, 35S-labeled macromolecules accumulated on the cell surface (trypsin-accessible compartment) for 16 h, at which point the cell-surface pool was saturated ( t 1 2 = 7.5 h ). After pulse-labeling the cells with 35SO 4 for 15 min, the 35S-labeled macromolecules disappeared continuously from the cell surface ( t 1 2 = 4.6 h ), and 79% of the radioactivity was recovered in the medium as nondialyzable macromolecules. Release of the 35S-labeled macromolecules from the cell surface was abolished at 4°C, indicative of an energy-dependent process, but multiple proteinase inhibitors did not affect the release. We conclude that sulfate is metabolized rapidly into epithelial cell-surface macromolecules, which accumulate continuously into a relatively large cell-surface pool, before they are released by an undefined energy-dependent mechanism.