Skip to main content
SpringerLink
Log in

A quantitative analysis of lectin binding to adult rat hepatocyte cell surfaces

  • Regular Papers
  • Published:
In Vitro Cellular & Developmental Biology Aims and scope Submit manuscript

Summary

A quantitative evaluation of lectin binding to adult rat hepatocyte cell surfaces was done using cells isolated by two different collagenase perfusion methodologies and cultured as monolayers with two different tissue culture media formulations (protocol I vs. protocol II). The presence of α-D-mannosyl and α-D-glucosyl groups was detected by the binding of Concanavalin A (Con A), Lens culinaris agglutinin (LCA), and Pisum sativum agglutinin (PSA) to freshly isolated cells. Furthermore, β-D-galactose [Ricinus communis agglutinin (RCA)] and sialic acid residues [wheat germ (WGA)] were also found. Protocols I and II served as models for evaluation of: a) the stripping effect of collagenase separation procedures, b) the restoration in culture of collagenase-stripped sugar residues, c) the effect of the culture environment on cell viability [as measured by lactic acid dehydrogenase (LDH) leakage] and the protein content of hepatocytes, and d) the presence of cell surface sugar residues as a function of culture duration. The ultrastructural morphology of freshly isolated and cultured hepatocytes was also evaluated. These studies indicated that a decline in lectin binding invariably occurred earlier than a massive leakage of LDH and a decrease in the protein content of the cells in culture. Ultrastructurally, autophagocytosis was an early phenomenon in cells isolated and cultured by protocol I, which was also inferior to protocol II regarding the preservation of hepatocyte glycocalyces. Sugar residues lost due to the collagenase-stripping effect were restored, as shown by lectin binding, within the first 24 h of culture. This stripping effect was confirmed by quantitative evaluations of lectin binding to hepatocytes in culture after an incubation with collagenase. This study shows that the binding of peroxidase-labeled lectins is a useful tool for quantitative evaluation of the sugar composition of hepatocyte cultures.

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

Access this article

Log in via an institution

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. Virtanen, I.; Miettinen, A.; Wartiovaara, J. Lectin binding sites are found in rat liver cell plasma membrane only on its extracellular surface. J. Cell Sci. 29:287–296; 1978.

    PubMed  CAS  Google Scholar 

  2. Roth, J. Lens culinaris lectin receptors in the plasma membrane of rat liver cells: Comparative electron microscopic studies on normal cells, on cells in vivo transformed by diethylnitrosamine and on Zadjela ascites hepatoma cells. Exp. Pathol. Bd. 11:123–132; 1975.

    CAS  Google Scholar 

  3. Caron, M.; Picard, J.; Kern, P. Involvement of glycoconjugates in insulin-receptor interactions: Studies in liver plasma membranes of control and diabetic mice. Biochim. Biophys. Acta 512:29–40; 1978.

    Article  PubMed  CAS  Google Scholar 

  4. Capuzzi, D. M.; Sparks, C. E.; DeHoff, J. L. Effect of residual enzymes on degradation of radioiodinated VLDL by collagenase-dispersed hepatocytes. Biochem. Biophys. Res. Commun. 90:587–595; 1979.

    Article  PubMed  CAS  Google Scholar 

  5. Bonney, R. T.; Becker, J. E.; Walker, P. R., et al. Primary monolayer cultures of adult rat liver parenchymal cells suitable for study of the regulation of enzyme synthesis. In Vitro 9:399–413; 1974.

    Article  CAS  Google Scholar 

  6. Bernaert, D.; Wanson, J. C.; Drochmans P., et al. Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes. J. Cell Biol. 75:878–900; 1977.

    Article  Google Scholar 

  7. Goldfarb, S.; Barber, T. A.; Pariza, M. W., et al. Lipid synthesis and ultrastructure of adult rat hepatocytes during their first twenty four hours in culture. Exp. Cell Res. 117:39–46; 1978.

    Article  PubMed  CAS  Google Scholar 

  8. Driscoll, J. L.; Hayner, N. T.; Williams-Holland, R., et al. Phenolsulfonphthalein (phenol red) metabolism on primary monolayer cultures of adult rat hepatocytes. In Vitro 18:835–842; 1982.

    Article  PubMed  CAS  Google Scholar 

  9. Decad, G. M.; Hseih, D. P. H.; Byard, J. L. Maintenance of cytochrome P-450 and metabolism of aflatoxin B in primary hepatocyte cultures. Biochem. Biophys. Res. Commun. 78:279–287; 1977.

    Article  PubMed  CAS  Google Scholar 

  10. Bisell, D. M.; Guzelian, P. S. Phenotypic stability of adult rat hepatocytes in primary monolayer cultures. Ann. NY Acad. Sci. 349:85–98; 1980.

    Article  Google Scholar 

  11. Michalopoulos, G.; Pitot, H. C. Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations. Exp. Cell Res. 94:70–78; 1975.

    Article  PubMed  CAS  Google Scholar 

  12. Reid, L. M.; Gairmaitan, Z.; Arias, I., et al. Long term cultures of normal rat hepatocytes on liver biomatrix. Ann. NY Acad. Sci. 349:17–76; 1980.

    Article  Google Scholar 

  13. Michalopoulos, G.; Russell, F.; Biles, C. Primary cultures of hepatocytes on human fibroblasts. In Vitro 15:796–806; 1979.

    Article  PubMed  CAS  Google Scholar 

  14. Tarentino, A. L.; Galivan, J. Membrane characteristics of adult rat liver parenchymal cells in primary monolayer culture. In Vitro 16:833–846; 1980.

    Article  PubMed  CAS  Google Scholar 

  15. Horisberger, M.; Rosset, J.; Vonlanthen, M. Location of lectin receptors on rat hepatocytes by transmission and scanning electron microscopy. Experientia 34:274–276; 1978.

    Article  PubMed  CAS  Google Scholar 

  16. Starling, J. J.; Hixson, D. C.; Davis, E. M., et al. Surface properties of adult rat hepatocytes. Exp. Cell Res. 104:164–175; 1977.

    Google Scholar 

  17. Hixson, D. C.; Miller, M. F.; Maruyama, K., et al. A statistical evaluation of the binding of ferritin conjugated lectins to the surface of rat cells. Topographical variations as a function of lectin concentration and cell type. J. Histochem. Cytochem. 27:1618–1629; 1979.

    PubMed  CAS  Google Scholar 

  18. McMillan, P. N.; Ferayorni, L. S.; Gerhardt, C. O., et al. Light and electron microscope analysis of lectin binding to adult rat liver in situ. Lab Invest. 50:408–420; 1984.

    PubMed  CAS  Google Scholar 

  19. Hixson, D. C.; Allison, J. P.; Chesner J. E. Variations in the expression and structure of a subset of normal bile canalicular glycoproteins on transplantable hepatocellular carcinomas. Galeotti, T.; Cittadini, A.; Neri, G., eds. Membranes in Tumor Growth. New York: Elsevier Biomedical Press; 1982:19–21.

    Google Scholar 

  20. Berry, M. N.; Friend, D. S. High yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Cell Biol. 43:506–520; 1969.

    Article  PubMed  CAS  Google Scholar 

  21. Seglen, P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 13:391–398; 1973.

    Google Scholar 

  22. Jauregui, H. O.; McMillan, P. N.; Driscoll, J., et al. Attachment and long term survival of adult rat hepatocytes in primary monolayer cultures: comparison of different substrate and tissue culture media formulations. In Vitro 22:13–22; 1986.

    CAS  Google Scholar 

  23. Hayner, N.; Driscoll, J.; Ferayorni, L. S., et al. A sensitive method for protein determination in freshly isolated and cultured cells. J. Tissue Cult. Method. procedure 75181, 1983.

  24. Jauregui, H. O.; Hayner, N. T.; Driscoll, J. L., et al. Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—freshly isolated cells, cell suspensions and primary monolayer cultures. In Vitro 17:1100–1110; 1981.

    Article  PubMed  CAS  Google Scholar 

  25. Spurr, A. R. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26:31–43; 1969.

    Article  PubMed  CAS  Google Scholar 

  26. Arstila, A. U.; Jauregui, H. O.; Chang, J., et al. Studies on cellular autophagocytosis. Relationship between heterophagy and autophagy in HeLa cells. Lab Invest. 24:162–174; 1971.

    PubMed  CAS  Google Scholar 

  27. Chapman, G. S.; Jones, A. L.; Meyer, U. A., et al. Parenchymal cells from adult rat liver in nonproliferating monolayer culture. II. Ultrastructural studies. J. Cell Biol. 59:735–747; 1973.

    Article  PubMed  CAS  Google Scholar 

  28. Sattler, C. A.; Michalopoulos, G.; Sattler, G. L., et al. Ultrastructure of adult rat hepatocytes cultured on floating collagen membranes. Canc. Res. 38:1539–1549; 1978.

    CAS  Google Scholar 

  29. Guzelian, P. S.; Diegelman, R. F. Retention of clostridial collagenase by primary cultures of parenchymal cells prepared from adult rat liver. Life Sci. 24:513–518; 1979.

    Article  PubMed  CAS  Google Scholar 

  30. Seglen, P. O.; Grinde, B.; Solheim, A. E. Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur. J. Biochem. 95:215–225; 1979.

    Article  PubMed  CAS  Google Scholar 

  31. Inbar, M.; Huet, C.; Oseroff, A. R., et al. Inhibition of lectin agglutinability by fixation of the cell surface membrane. Biochim. Biophys. Acta 311:594–599; 1973.

    Article  PubMed  CAS  Google Scholar 

  32. Noonan, K.; Levine, A. J.; Burger, M. M. Cell cycle-dependent changes in the surface membrane as detected with (3H) concanavalin A. J. Cell Biol. 58:491–497; 1973.

    Article  PubMed  CAS  Google Scholar 

  33. Katzen, H. M. Carbohydrate inhibitors of concanavalin A that inhibit binding of insulin-sepharose to fat cells and antagonize and mimic insulin's bioactivity. A possible role for membrane carbohydrate in insulin's action. J. Biol. Chem. 254:2983–2992; 1979.

    PubMed  CAS  Google Scholar 

  34. Kornfeld, K.; Reitman, M. L.; Kornfeld, R. The carbohydrate-binding specificity of pea and lentil lectins. Fucose is an important determinant. J. Biol. Chem. 256:6633–6640; 1981.

    PubMed  CAS  Google Scholar 

  35. Hughes, R. C. Glycoproteins. Brammer, W. J.; Edidin, M., eds. Outline studies in biology. London: Chapman and Hall; 1983:36–53.

    Google Scholar 

  36. VanDenBosch, R. A.; Gruze, H. J.; Stroub, G. I. Presence of asialoglycoprotein receptors in the Golgi complex in the absence of proteins synthesis. Exp. Cell Res. 162:231–242; 1986.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departments of Pathology and Oncology, Rhode Island Hospital, 02902, Providence, Rhode Island

    Hugo O. Jauregui, Paul N. McMillan, Karen Hevey & Sharda Naik

  2. Division of Biology and Medicine, Brown University, 02902, Providence, Rhode Island

    Hugo O. Jauregui, Paul N. McMillan, Karen Hevey & Sharda Naik

Authors
  1. Hugo O. Jauregui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paul N. McMillan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karen Hevey
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sharda Naik
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This study was supported by grant I-ROI-AM 26520 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Bethesda, MD, and by W. R. Grace Corporation.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jauregui, H.O., McMillan, P.N., Hevey, K. et al. A quantitative analysis of lectin binding to adult rat hepatocyte cell surfaces. In Vitro Cell Dev Biol 24, 401–412 (1988). https://doi.org/10.1007/BF02628491

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02628491

Key words

Search

Navigation