Mucins Secreted by Rat Tracheal Explants in Culture: Characterization and Influence of Coal Dust

The mucus secreted in the respiratory tract provides the first barrier against inhaled particulate and gaseous toxicants. The major component of mucus which is responsible for the physical properties and protective functions of the respiratory mucus is mucin. We have initiated investigations to determine the effects of coal mine dust on the synthesis of respiratory mucin by rat trachea under in vitro and in vivo conditions. In this study we have established optimum conditions-:ro-r~ the asceptic growth and mucin secretion of rat tracheal explants in culture. The best outgrowth of ciliated epithelial cells and maximum mucin production was obtained in CMRL 1066 medium containing 10% fetal calf serum. The explants continued to secrete mucin even after three weeks in culture. For the estimation and characterization of mucins the tracheae were cultured in the presence of [3H] glucosamine and [35S] sulfate or [14C]leucine. The mucins were isolated from the medium by gel filtration, treatment with hyaluronidase, ion exchange chromatography and CsBr density gradient centrifugation. Precipitation of polyanionic material in the culture media with cetyl pyridinium chloride and fractional extraction with NaCl also yielded mucin free of glycosaminoglycans, but in low yields. The tritium label in the purified mucin was distributed in sialic acid (50%) galactosamine (37%) and glucosamaine (13%). The density of purified mucin as determined by CsBr gradient centrifugation was about l.43g per ml in comparison to l.46g per ml for human respiratory mucin with a carbohydrate content of about 80% by weight. Alkaline borohydride treatment released saccharides which contained about 80% of the radioactivity. The effect of adding varying amounts of coal dust to the culture medium is under investigation. (Supported by U.S. Bureau of Mines Grant G1135142, Project 4210).