Summary
Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm2 per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 μM glucosamine. A higher concentration of35SO4, 1000 μM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 μg/ml increased the rate of secretion twofold, whereas 0.1 to 100 μg/ml of hydrocortisone and 0.1 to 100 μg/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10−5 M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10−9 M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats.
Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistingushable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio ofN-acetylgalactosamine,N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations.
Similar content being viewed by others
References
Yeager, H.; Massaro, G.; Massaro, D. Glycoprotein synthesis by the trachea. Am. Rev. Respir. Dis. 103: 188–197; 1971.
Boat, T. F.; Cheng, P. W.; Iyer, R. N.; Carlson, D. M.; Polony, I. Human respiratory tract secretions. Arch. Biochem. Biophys. 177: 95–104; 1976.
Roussel, P.; Lamblin, G.; Degand, P.; Walker-Nasir, E.; Jeanloz, R. W. Heterogeneity of the carbohydrate chains of sulfated bronchial glycoproteins isolated from a patient suffering from cystic fibrosis. J. Biol. Chem. 250: 2114–2122; 1975.
Roberts, J. R. Isolation and characterization of glycoproteins from sputum. Eur. J. Biochem. 50: 265–280; 1974.
Zinn, A. B.; Planter, J. J.; Carlson, D. M. In nature of linkage between protein core and oligosaccharides in glycoconjugates. Harowitz, M.; Pigman, W. eds. New York: Academic Press; 1977; 69–85.
Havez, R.; Roussel, P.; Degand, P.; Randoux, A.; Biserte, G. In proteins of biological fluids. Vol. 16. Oxford: Pergamon Press; 1968: 343.
Sachdev, G. P.; Fox, O. F.; Wen, G.; Schroeder, T.; Elkins, R. C.; Carubelli, R. Isolation and characterization of glycoproteins from canine tracheal mucus. Biochem. Biophys. Acta 536: 184–196; 1978.
Meyrick, B.; Reed, L. In vitro incorporation of threonine and glucose by mucus and serous cells of human bronchial submucosal gland. J. Cell Biol. 67: 320–344; 1975.
Kent, P. W.; Daniel, P. F.; Gallager, J. T. Secretion of the mucosubstances by organ cultures of mammalian trachea. Biochem. J. 124: 59–60; 1971.
Boat, T. F.; Iyer, R. N.; McIntyre, M. N.; Carlson, D. M.; Matthews, L. W. Evaluation of a culture method for study of human tracheobronchial secretions. Am. Rev. Respir. Dis. 103: 915–921; 1971.
Barrett, A.; McDowell, E. M.; Frank, A. L.; Harris, C. C.; Trump, B. F. Long-term organ culture of bronchial epithelium. Cancer Res. 36: 1003–1008; 1976.
Gallagher, J. T.; Kent, P. W.; Passatore, M.; Phipps, R. J.; Richardson, P. S. The composition of tracheal mucus and the nervous control of its secretion in the cat. Proc. R. Soc. Long. [Biol. Sci.] 192: 49–72; 1975.
Gallagher, J. T.; Kent, P. W. Structure and metabolism of glycoproteins and glycosaminoglycans secreted by organ cultures of rabbit tracheas. Biochem. J. 148: 187–196; 1975.
Bonanni, F.; Levinson, S. S.; Wolf, G.; DeLuca, L. Glycoproteins from the hamster respiratory tract and their response to Vitamin A. Biochem. Biophys. Acta 297: 441–451; 1973.
Chakrin, L. W.; Baker, A. P.; Christian, P.; Wardell, J. R. Effect of cholinergic stimulation on the release of macromolecules by canine trachea in vitro. Am. Res. Respir. Dis. 108: 69–76; 1973.
Ellis, D. B.; Stahl, G. H. Biosynthesis of respiratory tract mucin. Biochem. J. 136 837–844; 1973.
Adler, K. B.; Brody, A. R.; Craighead, J. E. Studies on the mechanism of mucin secretions by cells of the porcine tracheal epithelium. Proc. Soc. Exp. Biol. Med. 166: 96–106; 1981.
Clark, N.; Marchok, C. The effect of vitamin A on cellular differentiation and mucus glycoprotein synthesis in long term rat tracheal organ cultures. Differentiation 14: 175–183; 1979.
Daniel, P. F.; Wolf, G. Glycoprotein biosynthesis by organ cultures of hamster trachea. Biochem. Biophys. Acta. 451: 184–192; 1976.
Stahl, G. H.; Ellis, D. B. A comparison of canine epithelial goblet cells and submucosal gland secretions. Biochem. J. 136: 845–850; 1973.
Mendicino, J.; Sivakami, S.; Davila, M.; chandrasekaran, E. V. Purification and properties of UDP-Gal:N-acetylgalactosaminide mucin: 1,3-galactosyltransferase from swine trachea mucosa. J. Biol. Chem. 257: 3987–3994; 1982.
Rao, A. K.; Mendicino, J. Influence of glycopeptide structure on the regulation of galactosyltransferase activity. Biochemistry 17: 5632–5638; 1978.
Ong, D. E.; Tsai, C. H.; Chytil, F. Cellular retinol-binding protein and retinoic acid-binding protein in rat testis. J. Nutr. 106: 204–213; 1976.
Lamb, A. J.; Apiwatanaporn, P.; Olson, J. A. Induction of rapid synchronous vitamin A deficiency in the rat. J. Nutr. 104: 1140–1148; 1974.
Thompson, J. N.; Erdody, P.; Brien, R.; Murray, T. K. Fluorometric determination of vitamin A in human blood and liver. Biochem. Med. 5: 67–73; 1971.
Jones, R.; Bolduc, P.; Reid, L. Goblet cell glycoprotein and tracheal gland hypertrophy in rat airways. Br. J. Exp. Pathol. 229: 239; 1973.
Mendicino, J.; Chandrasekaran, E. V.; Anumula, K. R.; Davila, M. Isolation and properties of-d-mannose:-1,2-N-acetylglucosaminyl transferase from trachea mucosa. Biochemistry 20: 967–976; 1981.
Lowry, O. H.; Rosebrough, N. J.; Farr A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275; 1951.
Mendicino, J.; Kratowich, N. Properties and subunit structure of fructose 1,6-diphosphatase from swine kidney. J. Biol. Chem. 247: 6643–6650; 1972.
Holden, K. G.; Yim, N. C. F.; Griggs, L. J.; Weisbach, J. A. Gel electrophoresis of mucus glycoproteins. Biochemistry 10: 3110–3113; 1971.
Estep, J. A.; Marin, M. G. Incorporation of35SO4 into sulfated mucin glycoproteins in dog trachea. Fed. Proc. 38: 1325; 1979.
Pearlman, K., In: Jacoby, W. B., and Pastan, I. A. eds. Methods in enzymology. Vol. LVIII. New York: Academic Press; 1979.
Hoorn, B. Organ cultures of ciliated epithelium for the study of respiratory virus, Acta Pathol. Microbiol. Scand. 66: 1–37; 1966.
Weisman, Z.; Sade, J. Effect of environmental CO2, O2 and pH on the growth of respiratory epitheliumin vitro. Ann. Otol. 88: 21–30; 1979.
Jennings, M.; Cross, C. E.; Last, J. A. Synthesis of mucin in tracheal explants. Comp. Biochem. Physiol. 57A; 317–320; 1977.
Harada, Y.; Sasaki, H.; Gaafar, H. Effect of an expectorant on the tracheal mucosa. Otorhinolaryngology 39: 113–121; 1979.
Hayeshi, M. Influence of estrous on the secretory cells of the respiratory tract. Chest 68: 414–418; 1975.
Jones, R.; Baskerville, A.; Reid, L. Histochemical identification of glycoproteins in pig bronchial epithelium. J. Pathol. 116: 1–11; 1975.
Sturgess, J.; Reid, L. An organ culture study of the effect of drugs on the secretory activity of the human bronchial submucosal gland. Clinical Sci. 43: 533–543; 1972.
Marchok, A. C.; Rhoton, J. C.; Griesemer, R. A.; Nettesheim, P. Increasedin vitro growth capacity of tracheal epithelium exposed to 7,12-dimethylbenz(a)anthracene. Cancer Res. 37: 1811–1821; 1977.
Sporn, M. B.; Clamon, G. H.; Dunlop, M. N.; Newton, D. L.; Smith, J. M.; Saffiotti, U. Activity of vitamin A analogues in cell cultures of mouse epidermis and organ cultures of hamster trachea. Nature 253: 47–49; 1975.
Clamon, G. H.; Sporn, M. B.; Smith, J. A.; Saffiotti, U. -and-retinyl acetate reverse metaplasias of vitamin A deficiency in hamster trachea in organ culture. Nature 250: 64–66; 1974.
Marchok, A.; Cone, V.; Nettesheim, P. Induction of squamous metaplasia (vitamin A-deficiency) and hypersecretory activity in tracheal organ cultures. Lab. Invest. 33: 451–460; 1975.
Hall, R. L.; Peatfield, A. C.; Richardson, P. S. Influence of serum on glycoprotein synthesis in cat trachea. J. Physiol. (Lond.) 282: 47–48; 1978.
Author information
Authors and Affiliations
Additional information
This investigation was supported by U.S. Public Health Service Grants HL 20868, HL 24688, and HL 24718 from the National Heart, Lung and Blood Institute, Bethesda, MD, and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, Bethesda, MD.
Rights and permissions
About this article
Cite this article
Lloyd, C., Kennedy, J.R. & Mendicino, J. Regulation of the synthesis of mucin glycoproteins in swine trachea explants. In Vitro 20, 416–432 (1984). https://doi.org/10.1007/BF02619588
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02619588