Skip to main content
Log in

Regulation of the synthesis of mucin glycoproteins in swine trachea explants

  • Published:
In Vitro Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Yeager, H.; Massaro, G.; Massaro, D. Glycoprotein synthesis by the trachea. Am. Rev. Respir. Dis. 103: 188–197; 1971.

    PubMed  CAS  Google Scholar 

  2. 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.

    Article  PubMed  CAS  Google Scholar 

  3. 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.

    PubMed  CAS  Google Scholar 

  4. Roberts, J. R. Isolation and characterization of glycoproteins from sputum. Eur. J. Biochem. 50: 265–280; 1974.

    Article  PubMed  CAS  Google Scholar 

  5. 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.

    Google Scholar 

  6. Havez, R.; Roussel, P.; Degand, P.; Randoux, A.; Biserte, G. In proteins of biological fluids. Vol. 16. Oxford: Pergamon Press; 1968: 343.

    Google Scholar 

  7. 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.

    PubMed  CAS  Google Scholar 

  8. 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.

    Article  PubMed  CAS  Google Scholar 

  9. 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.

    Google Scholar 

  10. 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.

    Google Scholar 

  11. 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.

    PubMed  CAS  Google Scholar 

  12. 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.

    Article  CAS  Google Scholar 

  13. 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.

    PubMed  CAS  Google Scholar 

  14. 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.

    PubMed  CAS  Google Scholar 

  15. 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.

    CAS  Google Scholar 

  16. Ellis, D. B.; Stahl, G. H. Biosynthesis of respiratory tract mucin. Biochem. J. 136 837–844; 1973.

    PubMed  CAS  Google Scholar 

  17. 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.

    PubMed  CAS  Google Scholar 

  18. 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.

    Article  PubMed  CAS  Google Scholar 

  19. Daniel, P. F.; Wolf, G. Glycoprotein biosynthesis by organ cultures of hamster trachea. Biochem. Biophys. Acta. 451: 184–192; 1976.

    PubMed  CAS  Google Scholar 

  20. Stahl, G. H.; Ellis, D. B. A comparison of canine epithelial goblet cells and submucosal gland secretions. Biochem. J. 136: 845–850; 1973.

    PubMed  CAS  Google Scholar 

  21. 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.

    PubMed  CAS  Google Scholar 

  22. Rao, A. K.; Mendicino, J. Influence of glycopeptide structure on the regulation of galactosyltransferase activity. Biochemistry 17: 5632–5638; 1978.

    Article  PubMed  CAS  Google Scholar 

  23. 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.

    PubMed  CAS  Google Scholar 

  24. Lamb, A. J.; Apiwatanaporn, P.; Olson, J. A. Induction of rapid synchronous vitamin A deficiency in the rat. J. Nutr. 104: 1140–1148; 1974.

    PubMed  CAS  Google Scholar 

  25. 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.

    Article  PubMed  CAS  Google Scholar 

  26. Jones, R.; Bolduc, P.; Reid, L. Goblet cell glycoprotein and tracheal gland hypertrophy in rat airways. Br. J. Exp. Pathol. 229: 239; 1973.

    Google Scholar 

  27. 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.

    Article  PubMed  CAS  Google Scholar 

  28. 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.

    PubMed  CAS  Google Scholar 

  29. Mendicino, J.; Kratowich, N. Properties and subunit structure of fructose 1,6-diphosphatase from swine kidney. J. Biol. Chem. 247: 6643–6650; 1972.

    PubMed  CAS  Google Scholar 

  30. Holden, K. G.; Yim, N. C. F.; Griggs, L. J.; Weisbach, J. A. Gel electrophoresis of mucus glycoproteins. Biochemistry 10: 3110–3113; 1971.

    Article  PubMed  CAS  Google Scholar 

  31. Estep, J. A.; Marin, M. G. Incorporation of35SO4 into sulfated mucin glycoproteins in dog trachea. Fed. Proc. 38: 1325; 1979.

    Google Scholar 

  32. Pearlman, K., In: Jacoby, W. B., and Pastan, I. A. eds. Methods in enzymology. Vol. LVIII. New York: Academic Press; 1979.

    Google Scholar 

  33. Hoorn, B. Organ cultures of ciliated epithelium for the study of respiratory virus, Acta Pathol. Microbiol. Scand. 66: 1–37; 1966.

    Google Scholar 

  34. Weisman, Z.; Sade, J. Effect of environmental CO2, O2 and pH on the growth of respiratory epitheliumin vitro. Ann. Otol. 88: 21–30; 1979.

    CAS  Google Scholar 

  35. Jennings, M.; Cross, C. E.; Last, J. A. Synthesis of mucin in tracheal explants. Comp. Biochem. Physiol. 57A; 317–320; 1977.

    Article  Google Scholar 

  36. Harada, Y.; Sasaki, H.; Gaafar, H. Effect of an expectorant on the tracheal mucosa. Otorhinolaryngology 39: 113–121; 1979.

    Google Scholar 

  37. Hayeshi, M. Influence of estrous on the secretory cells of the respiratory tract. Chest 68: 414–418; 1975.

    Google Scholar 

  38. Jones, R.; Baskerville, A.; Reid, L. Histochemical identification of glycoproteins in pig bronchial epithelium. J. Pathol. 116: 1–11; 1975.

    Article  PubMed  CAS  Google Scholar 

  39. 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.

    CAS  Google Scholar 

  40. 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.

    PubMed  CAS  Google Scholar 

  41. 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.

    Article  PubMed  CAS  Google Scholar 

  42. 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.

    Article  PubMed  CAS  Google Scholar 

  43. 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.

    PubMed  CAS  Google Scholar 

  44. 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.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

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

Reprints 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

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02619588

Key words

Navigation