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. 1996 Jun 15;316(Pt 3):943–951. doi: 10.1042/bj3160943

P2u purinoceptor regulation of mucin secretion in SPOC1 cells, a goblet cell line from the airways.

L H Abdullah 1, S W Davis 1, L Burch 1, M Yamauchi 1, S H Randell 1, P Nettesheim 1, C W Davis 1
PMCID: PMC1217440  PMID: 8670174

Abstract

The SPOC1 cell, a novel goblet cell line derived from rat trachea, was tested for its ability to exhibit regulated mucin secretion in response to purinergic (P2) agonists. High-molecular mass glycoconjugates (HMMGs) purified by CsCl-density-gradient centrifugation had a buoyant density of 1.45 g/ml. The purified HMMG material exhibited a single major band with an apparent molecular mass of greater than 1000 kDa in SDS/ polyacrylamide gels stained with silver or blotted and stained with soya-bean agglutinin. [3H]HMMG was resistant to proteoglycan-degrading enzymes, but was susceptible to neuraminidase. The HMMG was approx. 91% carbohydrate by weight, and the glycosides were O-linked. The HMMG amino acid composition was enriched in Ser and Thr (sum 27%). Thus SPOC1-cell HMMG possess the characteristics of mucin. Mucin secretion by SPOC1 cells, grown on permeable supports and perfused luminally, was stimulated by ATP, UTP and adenosine 5'-[gamma-thio]triphosphate (100 microM) 4-5-fold over a baseline of 4 ng/min. The three dose-effect relations were nearly identical (K0.5 approximately 4 microM). SPOC1 cells grown on plastic and rat tracheal epithelial primary cells responded similarly to ATP and/or UTP. SPOC1 cells failed to respond to other purinergic agonists, either luminally or serosally, and consequently seem to possess an apical membrane P2u purinoceptor. SPOC1-cell total RNA was probed for P2u purinoceptor mRNA. Using conserved primers for both reverse transcriptase and PCR, a single band of the predicted size was observed, which had a nucleotide base sequence identical with the rat P2u purinoceptor mRNA. Thus SPOC1 cells secrete mucin under the control of a P2u purinoceptor; they should prove useful in dissecting the associated cellular regulatory pathways.

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Selected References

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  1. Adler K. B., Akley N. J., Glasgow W. C. Platelet-activating factor provokes release of mucin-like glycoproteins from guinea pig respiratory epithelial cells via a lipoxygenase-dependent mechanism. Am J Respir Cell Mol Biol. 1992 May;6(5):550–556. doi: 10.1165/ajrcmb/6.5.550. [DOI] [PubMed] [Google Scholar]
  2. Adler K. B., Holden-Stauffer W. J., Repine J. E. Oxygen metabolites stimulate release of high-molecular-weight glycoconjugates by cell and organ cultures of rodent respiratory epithelium via an arachidonic acid-dependent mechanism. J Clin Invest. 1990 Jan;85(1):75–85. doi: 10.1172/JCI114436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boat T. F., Cheng P. W. Biochemistry of airway mucus secretions. Fed Proc. 1980 Nov;39(13):3067–3074. [PubMed] [Google Scholar]
  4. Brown H. A., Lazarowski E. R., Boucher R. C., Harden T. K. Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5'-nucleotide receptor in human airway epithelial cells. Mol Pharmacol. 1991 Nov;40(5):648–655. [PubMed] [Google Scholar]
  5. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  6. Davies J. R., Gallagher J. T., Richardson P. S., Sheehan J. K., Carlstedt I. Mucins in cat airway secretions. Biochem J. 1991 May 1;275(Pt 3):663–669. doi: 10.1042/bj2750663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis C. W., Dowell M. L., Lethem M., Van Scott M. Goblet cell degranulation in isolated canine tracheal epithelium: response to exogenous ATP, ADP, and adenosine. Am J Physiol. 1992 May;262(5 Pt 1):C1313–C1323. doi: 10.1152/ajpcell.1992.262.5.C1313. [DOI] [PubMed] [Google Scholar]
  8. Doherty M. M., Liu J., Randell S. H., Carter C. A., Davis C. W., Nettesheim P., Ferriola P. C. Phenotype and differentiation potential of a novel rat tracheal epithelial cell line. Am J Respir Cell Mol Biol. 1995 Apr;12(4):385–395. doi: 10.1165/ajrcmb.12.4.7535063. [DOI] [PubMed] [Google Scholar]
  9. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  10. Gum J. R., Jr Mucin genes and the proteins they encode: structure, diversity, and regulation. Am J Respir Cell Mol Biol. 1992 Dec;7(6):557–564. doi: 10.1165/ajrcmb/7.6.557. [DOI] [PubMed] [Google Scholar]
  11. Jourdian G. W., Dean L., Roseman S. The sialic acids. XI. A periodate-resorcinol method for the quantitative estimation of free sialic acids and their glycosides. J Biol Chem. 1971 Jan 25;246(2):430–435. [PubMed] [Google Scholar]
  12. Kaartinen L., Nettesheim P., Adler K. B., Randell S. H. Rat tracheal epithelial cell differentiation in vitro. In Vitro Cell Dev Biol Anim. 1993 Jun;29A(6):481–492. [PubMed] [Google Scholar]
  13. Kennedy C. P1- and P2-purinoceptor subtypes--an update. Arch Int Pharmacodyn Ther. 1990 Jan-Feb;303:30–50. [PubMed] [Google Scholar]
  14. Kim K. C., Lee B. C. P2 purinoceptor regulation of mucin release by airway goblet cells in primary culture. Br J Pharmacol. 1991 May;103(1):1053–1056. doi: 10.1111/j.1476-5381.1991.tb12299.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim K. C., Rearick J. I., Nettesheim P., Jetten A. M. Biochemical characterization of mucous glycoproteins synthesized and secreted by hamster tracheal epithelial cells in primary culture. J Biol Chem. 1985 Apr 10;260(7):4021–4027. [PubMed] [Google Scholar]
  16. Knowles M. R., Clarke L. L., Boucher R. C. Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. N Engl J Med. 1991 Aug 22;325(8):533–538. doi: 10.1056/NEJM199108223250802. [DOI] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Lethem M. I., Dowell M. L., Van Scott M., Yankaskas J. R., Egan T., Boucher R. C., Davis C. W. Nucleotide regulation of goblet cells in human airway epithelial explants: normal exocytosis in cystic fibrosis. Am J Respir Cell Mol Biol. 1993 Sep;9(3):315–322. doi: 10.1165/ajrcmb/9.3.315. [DOI] [PubMed] [Google Scholar]
  19. Lustig K. D., Shiau A. K., Brake A. J., Julius D. Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5113–5117. doi: 10.1073/pnas.90.11.5113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mason S. J., Paradiso A. M., Boucher R. C. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium. Br J Pharmacol. 1991 Jul;103(3):1649–1656. doi: 10.1111/j.1476-5381.1991.tb09842.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  22. O'Connor S. E., Dainty I. A., Leff P. Further subclassification of ATP receptors based on agonist studies. Trends Pharmacol Sci. 1991 Apr;12(4):137–141. doi: 10.1016/0165-6147(91)90530-6. [DOI] [PubMed] [Google Scholar]
  23. Parr C. E., Sullivan D. M., Paradiso A. M., Lazarowski E. R., Burch L. H., Olsen J. C., Erb L., Weisman G. A., Boucher R. C., Turner J. T. Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3275–3279. doi: 10.1073/pnas.91.8.3275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. RICHARDSON K. C., JARETT L., FINKE E. H. Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 1960 Nov;35:313–323. doi: 10.3109/10520296009114754. [DOI] [PubMed] [Google Scholar]
  25. Randell S. H., Liu J. Y., Ferriola P. C., Kaartinen L., Doherty M. M., Davis C. W., Nettesheim P. Mucin production by SPOC1 cells--an immortalized rat tracheal epithelial cell line. Am J Respir Cell Mol Biol. 1996 Feb;14(2):146–154. doi: 10.1165/ajrcmb.14.2.8630264. [DOI] [PubMed] [Google Scholar]
  26. Rice W. R., Burton F. M., Fiedeldey D. T. Cloning and expression of the alveolar type II cell P2u-purinergic receptor. Am J Respir Cell Mol Biol. 1995 Jan;12(1):27–32. doi: 10.1165/ajrcmb.12.1.7811468. [DOI] [PubMed] [Google Scholar]
  27. Rice W. R., Singleton F. M. P2-purinoceptors regulate surfactant secretion from rat isolated alveolar type II cells. Br J Pharmacol. 1986 Nov;89(3):485–491. doi: 10.1111/j.1476-5381.1986.tb11148.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rieves R. D., Goff J., Wu T., Larivee P., Logun C., Shelhamer J. H. Airway epithelial cell mucin release: immunologic quantitation and response to platelet-activating factor. Am J Respir Cell Mol Biol. 1992 Feb;6(2):158–167. doi: 10.1165/ajrcmb/6.2.158. [DOI] [PubMed] [Google Scholar]
  29. Roberton A. M., Mantle M., Fahim R. E., Specian R. D., Bennick A., Kawagishi S., Sherman P., Forstner J. F. The putative 'link' glycopeptide associated with mucus glycoproteins. Composition and properties of preparations from the gastrointestinal tracts of several mammals. Biochem J. 1989 Jul 15;261(2):637–647. doi: 10.1042/bj2610637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Robinson C. B., Wu R. Mucin synthesis and secretion by cultured tracheal cells: effects of collagen gel substratum thickness. In Vitro Cell Dev Biol Anim. 1993 Jun;29A(6):469–477. [PubMed] [Google Scholar]
  31. Rose M. C. Mucins: structure, function, and role in pulmonary diseases. Am J Physiol. 1992 Oct;263(4 Pt 1):L413–L429. doi: 10.1152/ajplung.1992.263.4.L413. [DOI] [PubMed] [Google Scholar]
  32. Sheehan J. K., Oates K., Carlstedt I. Electron microscopy of cervical, gastric and bronchial mucus glycoproteins. Biochem J. 1986 Oct 1;239(1):147–153. doi: 10.1042/bj2390147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sheehan J. K., Thornton D. J., Somerville M., Carlstedt I. Mucin structure. The structure and heterogeneity of respiratory mucus glycoproteins. Am Rev Respir Dis. 1991 Sep;144(3 Pt 2):S4–S9. doi: 10.1164/ajrccm/144.3_pt_2.S4. [DOI] [PubMed] [Google Scholar]
  34. Verdugo P. Goblet cells secretion and mucogenesis. Annu Rev Physiol. 1990;52:157–176. doi: 10.1146/annurev.ph.52.030190.001105. [DOI] [PubMed] [Google Scholar]
  35. Wu R., Martin W. R., Robinson C. B., St George J. A., Plopper C. G., Kurland G., Last J. A., Cross C. E., McDonald R. J., Boucher R. Expression of mucin synthesis and secretion in human tracheobronchial epithelial cells grown in culture. Am J Respir Cell Mol Biol. 1990 Nov;3(5):467–478. doi: 10.1165/ajrcmb/3.5.467. [DOI] [PubMed] [Google Scholar]

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