Skip to main content
SpringerLink
Log in

The bicarbonate ion is essential for efficient DNA synthesis by primary cultured rat hepatocytes

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

Summary

Bicarbonate in the culture medium is essential for DNA synthesis of primary cultured rat hepatocytes stimulated by epidermal growth factor (EGF). When primary cultured hepatocytes in supplemented Leibovitz L15 medium were placed in a 100% air incubator, no increase in DNA synthesis was observed even after stimulation by EGF. However, when these cells were cultured with NaHCO3 and EGF and placed in a 5% CO2:95% air incubator, a stimulus of DNA synthesis more than 10-fold greater than in cultures in air only was seen, and many mitotic figures could be identified. Furthermore, NaHCO3 added to supplemented DMEM/F12 medium enhanced the DNA synthesis of primary cultured rat hepatocytes in this medium. The ideal pH of the medium for DNA synthesis of cultured hepatocytes was in the range of 7.6 to 8.0. A dose response of NaHCO3 in several media showed that DNA synthesis of the cells increased as the concentration of NaHCO3 increased and that 25 to 30 mM NaHCO3 in the medium was optimal for the replication of DNA by primary cultured rat hepatocytes.

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. Althaus, F. R.; Lawrence, S. D.; Sattler, G. L., et al. The effect of nicotinamide on unscheduled DNA synthesis in cultured hepatocytes. Biochem. Biophys. Res. Commun. 95:1063–1073; 1980.

    Article  PubMed  CAS  Google Scholar 

  2. Althaus, F. R.; Lawrence, S. D.; He, Y-Z, et al. Effects of altered [ADP-ribose]n metabolism on expression of fetal functions by adult hepatocytes. Nature 300:366–368; 1982.

    Article  PubMed  CAS  Google Scholar 

  3. Althaus, F. R.; Lawrence, S. D.; Sattler, G. L., et al. ADP-ribosyltransferase activity in cultured hepatocytes. J. Biol. Chem. 257:5528–5535; 1982.

    PubMed  CAS  Google Scholar 

  4. Bicz, W. The influence of carbon dioxide tension on the respiration of normal and leukemic human leukocytes. I. Influence on endogenous respiration. Cancer Res. 20:184–190; 1960.

    PubMed  CAS  Google Scholar 

  5. Brunk, C. F.; Jones, K. C.; James, T. W. Assay for nanogram quantities of DNA in cellular homogenate. Anal. Biochem. 92:497–500; 1979.

    Article  PubMed  CAS  Google Scholar 

  6. Ceccarini, C.; Eagle, H. pH as a determinant of cellular growth and contact inhibition. Proc. Natl. Acad. Sci. USA 68:229–233; 1971.

    Article  PubMed  CAS  Google Scholar 

  7. Chang, R. S.; Liepins, H.; Margolish, M. Carbon dioxide requirement and nucleic acid metabolism of HeLa and conjunctival cells. Proc. Soc. Exp. Biol. Med. 106:149–152; 1961.

    PubMed  CAS  Google Scholar 

  8. Eagle, H. The effect of environmental pH on the growth of normal and malignant cells. J. Cell. Physiol. 82:1–8; 1973.

    Article  PubMed  CAS  Google Scholar 

  9. Ehmann, U. K.; Misfeldt, D. S. CO2/bicarbonate stimulates growth independently of pH in mouse mammary epithelial cells. In Vitro Cell. Dev. Biol. 19:767–774; 1983.

    Article  CAS  Google Scholar 

  10. Geyer, R. P.; Chang, R. S. Bicarbonate as an essential for human cells in vitro. Arch. Biochem. Biophys. 73:500–506; 1958.

    Article  PubMed  CAS  Google Scholar 

  11. Geyer, R. P.; Neimark, J. M. Response of CO2-deficient human cells in vitro to normal cell extracts. Proc. Soc. Exp. Biol. Med. 99:599–601; 1958.

    PubMed  CAS  Google Scholar 

  12. Gwatkin, R. B. L.; Siminovitch, L. Multiplication of single mammalian cells in a nonbicarbonate medium. Proc. Soc. Exp. Biol. Med. 103:718–721; 1960.

    PubMed  CAS  Google Scholar 

  13. Harris, M. The role of bicarbonate for outgrowth of chick heart fibroblasts. J. Exp. Zool. 125:85–98; 1954.

    Article  CAS  Google Scholar 

  14. Hasegawa, K.; Koga, M. A high concentration of pyruvate is essential for survival and DNA synthesis in primary cultures of adult hepatocytes in a serum-free medium. Biomed. Res. 2:217–221; 1981.

    CAS  Google Scholar 

  15. Hasegawa, K.; Watanabe, K.; Koga, M. Induction of mitosis in primary cultures of adult rat hepatocytes under serum-free conditions. Biochem. Biophys. Res. Commun. 104:884–891; 1982.

    Article  Google Scholar 

  16. Hawley-Nelson, P.; Sullivan, J. E.; Kung, M., et al. Optimized conditions for the growth of human epidermal cells in culture. J. Invest. Dermatol. 75:176–182; 1980.

    Article  PubMed  CAS  Google Scholar 

  17. Houck, K. A.; Michalopoulos, G. K. Proline is required for the stimulation of DNA synthesis in hepatocyte cultures by EGF. In Vitro Cell. Dev. Biol. 21:121–124; 1985.

    Article  PubMed  CAS  Google Scholar 

  18. Itagaki, A.; Kimura, G. TES and HEPES buffers in mammalian cell cultures and viral studies: problems of carbon dioxide requirement. Exp. Cell Res. 83:351–361; 1974.

    Article  PubMed  CAS  Google Scholar 

  19. Kleeman, K. T.; Fryer, J. L.; Pilcher, K. S. Observed difference in CO2 requirements between mammalian and salmonid fish cell lines. J. Cell Biol. 47:796–798; 1970.

    Article  PubMed  CAS  Google Scholar 

  20. Kreamer, B. L.; Staecker, J. L.; Sawada, N., et al. Use of a low-speed, isodensity Percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell. Dev. Biol. 22:201–211; 1986.

    Article  PubMed  CAS  Google Scholar 

  21. Leibovitz, A. The growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere. Am. J. Hyg. 78:173–180; 1963.

    PubMed  CAS  Google Scholar 

  22. McGowan, J. A. Hepatocyte proliferation in culture. In: Guillouzo, A.; Guguen-Guillouzo, C., eds. Research in isolated and cultured hepatocytes. John Libbey Eurotext Letters; 1986:13–38.

  23. McGowan, J. A.; Bucher, N. L. R. Pyruvate promotion of DNA synthesis in serum-free primary cultures of adult rat hepatocytes. In Vitro Cell. Dev. Biol. 19:159–166; 1983.

    Article  CAS  Google Scholar 

  24. McKeehan, W. L.; McKeehan, K. A. Oxocarboxylic acids, pyridine nucleopeptide-linked oxidoreductases and serum factors in regulation of cell proliferation. J. Cell. Physiol. 101:9–16; 1979.

    Article  PubMed  CAS  Google Scholar 

  25. Michalopoulos, G. K. Liver regeneration: molecular mechanisms of growth control. FASEB J. 4:176–187; 1990.

    PubMed  CAS  Google Scholar 

  26. Michalopoulos, G. K.; Sattler, G. L.; Pitot, H. C. Maintenance of microsomal cytochromes b5 and p-450 in primary cultures of parenchymal liver cells on collagen membranes. Life Sci. 18:1139–1144; 1976.

    Article  PubMed  CAS  Google Scholar 

  27. Michalopoulos, G. K.; Pitot, H. C. Primary culture of parenchymal liver cells on collagen membranes. Exp. Cell Res. 94:70–78; 1975.

    Article  PubMed  CAS  Google Scholar 

  28. Michalopoulos, G. K.; Sattler, G. L.; Sattler, C. A., et al. Interaction of chemical carcinogens and drug-metabolizing enzymes in primary cultures of hepatic cells from the rat. Am. J. Pathol. 85:755–770; 1976.

    PubMed  CAS  Google Scholar 

  29. Mitaka, T.; Sattler, C. A.; Sattler, G. L., et al. Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor. Hepatology. 13:21–30; 1991.

    Article  PubMed  CAS  Google Scholar 

  30. Nakamura, T.; Teramoto, H.; Tomita, Y., et al.l-Proline is an essential amino acid for hepatocyte growth in culture. Biochem. Biophys. Res. Commun. 122:884–891; 1984.

    Article  PubMed  CAS  Google Scholar 

  31. Richman, R. A.; Claus, T. H.; Pilkis, S. J., et al. Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes. Proc. Natl. Acad. Sci. USA 73:3589–3593; 1976.

    Article  PubMed  CAS  Google Scholar 

  32. Rubin, H. pH and population density in the regulation of animal cell multiplication. J. Cell Biol. 51:686–702; 1971.

    Article  PubMed  CAS  Google Scholar 

  33. Sargent, L. M.; Xu, Y-H.; Sattler, G. L., et al. Ploidy and karyotype of hepatocytes isolated from enzyme-altered foci in two different protocols of multistage hepatocarcinogenesis in the rat. Carcinogenesis 10:387–391; 1989.

    Article  PubMed  CAS  Google Scholar 

  34. Sattler, C. A.; Sawada, N.; Sattler, G. L., et al. Electron microscopic and time lapse studies of mitosis in cultured rat hepatocytes. Hepatology 8:1540–1549; 1988.

    Article  PubMed  CAS  Google Scholar 

  35. Sawada, N.; Tomomura, A.; Sattler, C. A., et al. Effects of extracellular matrix components on the growth and differentiation of cultured rat hepatocytes. In Vitro Cell. Devel. Biol. 23:267–273; 1987.

    Article  CAS  Google Scholar 

  36. Sawada, N.; Tomomura, A.; Sattler, C. A., et al. Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture. Exp. Cell Res. 167:458–470; 1986.

    Article  PubMed  CAS  Google Scholar 

  37. Seglen, P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 13:29–83; 1976.

    Article  PubMed  CAS  Google Scholar 

  38. Seglen, P. O.; Gordon, P. B.; Schwarze, P. E. Autophagy and protein degradation in rat hepatocytes. In: Harris, R. A.; Cornell, N. W., eds. Isolation, characterization, and use of hepatocytes. New York: Elsevier; 1983:153–163.

    Google Scholar 

  39. Sirica, A. E.; Richards, W.; Tsukada, Y., et al. Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes. Proc. Natl. Acad. Sci. USA 76:283–287; 1979.

    Article  PubMed  CAS  Google Scholar 

  40. Staecker, J. L.; Sattler, C. A.; Pitot, H. C. Sodium butyrate preserves aspects of the differentiated phenotype of normal rat hepatocytes in culture. J. Cell. Physiol. 135:367–376; 1988.

    Article  PubMed  CAS  Google Scholar 

  41. Staecker, J. L.; Pitot, H. C. The effect of sodium butyrate on tyrosine aminotransferase induction in primary cultures of normal adult rat hepatocytes. Arch. Biochem. Biophys. 261:291–298; 1988.

    Article  PubMed  CAS  Google Scholar 

  42. Swim, H. E.; Parker, R. F. The role of carbon dioxide as an essential nutrient for six permanent strains of fibroblasts. J. Biophys. Biochem. Cytol. 4:525–529; 1958.

    Article  PubMed  CAS  Google Scholar 

  43. Taylor, C. A. Responses of cells to pH changes in the medium. J. Cell Biol. 15:201–209; 1962.

    Article  PubMed  CAS  Google Scholar 

  44. Tomomura, A.; Sawada, N.; Sattler, G. L., et al. The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: interactions of extracellular matrix components, epidermal growth factor, and the cell cycle. J. Cell. Physiol. 130:221–227; 1987.

    Article  PubMed  CAS  Google Scholar 

  45. Xu, Y.-H.; Sattler, G. L.; Pitot, H. C. A method for the comparative study of replicative DNA synthesis in GGT-positive and GGT-negative hepatocytes in primary culture isolated from carcinogen-treated rats. In Vitro Cell. Dev. Biol. 24:995–1000; 1988.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Toshihiro Mitaka

    Present address: Cancer Research Institute, Department of Pathology, Sapporo Medical College, Chuo-ku, S1, W17, 060, Sapporo, Japan

Authors and Affiliations

  1. McArdle Laboratory for Cancer Research, Department of Oncology, Medical School, University of Wisconsin, 1400 University Avenue, 53706, Madison, Wisconsin

    Toshihiro Mitaka, Gerald L. Sattler & Henry C. Pitot

Authors
  1. Toshihiro Mitaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gerald L. Sattler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Henry C. Pitot
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

The investigations described in this study were supported in part by grants CA-07175, CA-22484, and CA-45700 from the National Cancer Institute, Bethesda, MD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mitaka, T., Sattler, G.L. & Pitot, H.C. The bicarbonate ion is essential for efficient DNA synthesis by primary cultured rat hepatocytes. In Vitro Cell Dev Biol - Animal 27, 549–556 (1991). https://doi.org/10.1007/BF02631285

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02631285

Key words

Search

Navigation