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The γ-Glutamyl Transpeptidase Inhibitor Acivicin Preserves Glutathione Released by Astroglial Cells in Culture

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Abstract

The release of glutathione from astroglial cells was investigated using astroglia-rich primary cultures prepared from the brains of newborn rats. These cells release glutathione after onset of an incubation in a glucose-containing minimal medium. The amount of extracellular glutathione increased with the time of incubation, although the accumulation slowed down gradually. An elevated rate of increase of the glutathione concentration in the incubation medium was found if the astroglial ectoenzyme γ-glutamyl transpeptidase was inhibited by acivicin. The activity of γ-glutamyl transpeptidase in astroglia-rich primary cultures, which was found to be 1.9 ± 0.3 nmol/(min × mg protein), was markedly reduced if the cells had been incubated in the presence of acivicin. After 2 h of incubation with acivicin half-maximal and maximal inhibition of γ-glutamyl transpeptidase activity was found at concentrations of about 5 μM and 50 μM, respectively. In the presence of acivicin at a concentration above 10 μM the glutathione content found released from astroglial cells apparently increased almost proportional to time for up to 10 h. Under these conditions the average rate of release was 2.1 ± 0.3 nmol/(h × mg protein) yielding after a 10 h incubation an extracellular glutathione content three times that of the medium of cells incubated without inhibitor. Half-maximal and maximal effects on the level of extracellular glutathione were found at 4 μM and 50 μM acivicin, respectively. After a 10 h incubation with acivicin the intracellular content of glutathione was reduced to 75% of the level of untreated astroglial cultures. These results suggest that glutathione released from astroglial cells can serve as substrate for the ectoenzyme γ-glutamyl transpeptidase of these cells.

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REFERENCES

  1. Meister, A., Tate, S. S., and Griffith, O. W. 1981. γ-Glutamyl transpeptidase. Meth. Enzymol. 77:237–253.

    Google Scholar 

  2. Kakimoto, Y., Nakajima, T., Kanazawa, A., Takesada, M., and Sano, I. 1964. Isolation of γ-L-glutamyl-L-glutamic acid and γ-L-glutamyl-L-glutamine from bovine brain. Biochim. Biophys. Acta 93:333–338.

    Google Scholar 

  3. Kanazawa, A., Kakimoto, Y., Nakajima, T., and Sano, I. 1965. Identification of γ-glutamylserine, γ-glutamylalanine, γ-glutamylvaline and S-methylglutathione of bovine brain. Biochim. Biophys. Acta 111:90–95.

    Google Scholar 

  4. Reichelt, K. L. 1970. The isolation of gamma-glutamyl peptides from monkey brain. J. Neurochem. 17:19–25.

    Google Scholar 

  5. Sandberg, M., Li, X., Folestad, S., Weber, S. G., and Orwar, O. 1994. Liquid chromatographic determination of acidic β-aspartyl and γ-glutamyl peptides in extracts of rat brain. Anal. Biochem. 217:48–61.

    Google Scholar 

  6. Reyes, E., and Barela, T. D. 1980. Isolation and purification of multiple forms of γ-glutamyl transpeptidase from rat brain. Neurochem. Res. 5:159–170.

    Google Scholar 

  7. Tate, S. S., Ross, L. L., and Meister, A. 1973. The γ-glutamyl cycle in the choroid plexus: its possible function in amino acid transport. Proc. Natl. Acad. Sci. USA 7:1447–1449.

    Google Scholar 

  8. Okonkwo, P. O., Orlowski, M., and Green, J. P. 1974. Enzymes of the γ-glutamyl cycle in the choroid plexus and brain. J. Neurochem. 22:1053–1058.

    Google Scholar 

  9. Orlowski, M., Sessa, G., and Green, J. P. 1974. γ-Glutamyl transpeptidase in brain capillaries: possible site of a blood-brain barrier for amino acids. Science 184:66–68.

    Google Scholar 

  10. Ghandour, M. S., Langley, O. K., and Varga, V. 1980. Immunohistological localization of γ-glutamyltranspeptidase in cerebellum at light and electron microscope levels. Neurosci. Lett. 20:125–129.

    Google Scholar 

  11. Frey, A., Meckelein, B., Weiler-Güttler, H., Möckel, B., Flach, R., and Gassen, H. G. 1991. Pericytes of the brain microvasculature express γ-glutamyl transpeptidase. Eur. J. Biochem. 202: 421–429.

    Google Scholar 

  12. Shine, H. D., and Haber, B. 1981. Immunocytochemical localization of γ-glutamyl transpeptidase in the rat CNS. Brain Res. 217:339–349.

    Google Scholar 

  13. Philbert, M. A., Beiswanger, C. M., Manson, M. M., Green, J. A., Novak, R. F., Primiano, T., Reuhl, K. R., and Lowndes, H. E. 1995. Glutathione-S-transferases and γ-glutamyl transpeptidase in the rat nervous system: a basis for differential susceptibility to neurotoxicants. Neurotoxicology 16:349–362.

    Google Scholar 

  14. Shine, H. D., Hertz, L., de Vellis, J., and Haber, B. 1981. A fluorometric assay for γ-glutamyl transpeptidase: demonstration of enzymatic activity in cultured cells of neural origin. Neurochem. Res. 6:453–463.

    Google Scholar 

  15. Kaplowitz, N., Fernandez-Checa, J. C., Kannan, R., Garcia-Ruiz, C., Ookhtens, M., and Yu, J. R. 1996. GSH transporters: molecular characterization and role in GSH homeostasis. Biol. Chem. Hoppe-Seyler 377:267–273.

    Google Scholar 

  16. Anderson, M. E., Underwood, M., Bridges, R. J., and Meister, A. 1989. Glutathione metabolism at the blood-cerebrospinal fluid barrier. FASEB J. 3:2527–2531.

    Google Scholar 

  17. Zängerle, L., Cuenod, M., Winterhalter, K. H., and Do, K. Q. 1992. Screening of thiol compounds: depolarization-induced release of glutathione and cysteine from rat brain slices. J. Neurochem. 59:181–189.

    Google Scholar 

  18. Li, X., Orwar, O., Revesjö, C., and Sandberg, M. 1996. γ-Glutamyl peptides and related amino acids in rat hippocampus in vitro: effect of depolarization and γ-glutamyl transpeptidase inhibition. Neurochem. Int. 29:121–128.

    Google Scholar 

  19. Yudkoff, M., Pleasure, D., Cregar, L., Lin, Z.-P., Nissim, I., Stern, J., and Nissim, I. 1990. Glutathione turnover in cultured astrocytes: studies with [15N]glutamate. J. Neurochem. 55:137–145.

    Google Scholar 

  20. Juurlink, B. H. J., Schültke, E., and Hertz, L. 1996. Glutathione release and catabolism during energy substrate restriction in astrocytes. Brain Res. 710:229–233.

    Google Scholar 

  21. Sagara, J., Makino, N., and Bannai, S. 1996. Glutathione efflux from cultured astrocytes. J. Neurochem. 66:1876–1881.

    Google Scholar 

  22. Stole, E., Smith, T. K., Manning, J. M., and Meister, A. 1994. Interaction of γ-glutamyl transpeptidase with acivicin. J. Biol. Chem. 269:21435–21439.

    Google Scholar 

  23. Hamprecht, B., and Löffler, F. 1985. Primary glial cultures as a model system for studying hormone action. Meth. Enzymol. 109: 341–345.

    Google Scholar 

  24. Hamprecht, B., and Dringen, R. 1995. Energy metabolism. Pages 473–487, in Kettenmann, H., and Ransom, B. R. (eds.), Neuroglia, Oxford University Press, New York.

    Google Scholar 

  25. Baker, M. A., Cerniglia, G. J., and Zaman, A. 1990. Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal. Biochem. 190:360–365.

    Google Scholar 

  26. Tietze, F. 1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27: 502–522.

    Google Scholar 

  27. Dringen, R., and Hamprecht, B. 1996. Glutathione content as an indicator for the presence of metabolic pathways of amino acids in astroglial cultures. J. Neurochem. 67:1375–1382.

    Google Scholar 

  28. Vassault, A. 1983. Lactate dehydrogenase: UV-method with pyruvate and NADH. Pages 118–126, in Bergmeyer, H. U. (Ed.), Methods of Enzymatic Analysis, Vol. 3, Verlag Chemie, Weinheim, F. R. G.

    Google Scholar 

  29. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.

    Google Scholar 

  30. Stastny, F., Hilgier, W., Albrecht, J., and Lisy, V. 1988. Changes in the activity of γ-glutamyl transpeptidase in brain microvessels, astroglial cells and synaptosomes derived from rats with hepatic encephalopathy. Neurosci. Lett. 84:323–328.

    Google Scholar 

  31. Makar, T. K., Nedergaard, M., Preuss, A., Gelbard, A. S., Perumal, A. S., and Cooper, A. J. L. 1994. Vitamin E, ascorbate, glutathione, glutathione disulfide, and enzymes of glutathione metabolism in cultures of chick astrocytes and neurons: evidence that astrocytes play an important role in antioxidative processes in the brain. J. Neurochem. 62:45–53.

    Google Scholar 

  32. Smith, T. K., Ikeda, Y., Fujii, J., Taniguchi, N., and Meister, A. 1995. Different sites of acivicin binding and inactivation of γ-glutamyl transpeptidases. Proc. Natl. Acad. Sci. USA 92:2360–2364.

    Google Scholar 

  33. Hertz, L., Yager, J. Y., and Juurlink, B. H. J. 1995. Astrocyte survival in the absence of exogenous substrate: comparison of immature and mature cells. Int. J. Dev. Neurosci. 13:523–527.

    Google Scholar 

  34. Reinhart, P. H., Pfeiffer, B., Spengler, S., and Hamprecht, B. 1990. Purification of glycogen phosphorylase from bovine brain and immunocytochemical examination of rat primary cultures using monoclonal antibodies raised against this enzyme. J. Neurochem. 54:1474–1483.

    Google Scholar 

  35. Orwar, O., Li, X., Andine, P., Bergström, C.-M., Hagberg, H., Folestad, S., and Sandberg, M. 1994. Increased intra-and extracellular concentration of γ-glutamylglutamate and related dipeptides in the ischemic rat striatum: involvement of γ-glutamyl transpeptidase. J. Neurochem. 63:1371–1376.

    Google Scholar 

  36. Yang, C. S., Chou, S. T., Lin, N. N., Liu, L., Tsai, P. J., Kuo, J. S., and Lai, J. S. 1994. Determination of extracellular glutathione in rat brain by microdialysis and high-performance liquid chromatography with fluorescence detection. J. Chromatogr. B Biomed. Appl. 661:231–235.

    Google Scholar 

  37. Sagara, J., Miura, K., and Bannai, S. 1993. Maintenance of neuronal glutathione by glial cells. J. Neurochem. 61:1672–1676.

    Google Scholar 

  38. Guo, N., McIntosh, C., and Shaw, C. 1992. Glutathione: a new candidate neuropeptide in the central nervous system. Neuroscience 51:835–842.

    Google Scholar 

  39. Lanius, R. A., Shaw, C. S., Wagey, R., and Krieger, C. 1994. Characterization, distribution, and protein kinase C-mediated regulation of [35S]glutathione binding sites in mouse and human spinal cord. J. Neurochem. 63:155–160.

    Google Scholar 

  40. Varga, V., Janaky, R., Saransaari, P., and Oja, S. S. 1994. Endogeneous γ-L-glutamyl and β-L-aspartyl peptides and excitatory aminoacidergic neurotransmission in the brain. Neuropeptides 27: 19–26.

    Google Scholar 

  41. Ogita, K., Enomoto, R., Nakahara, F., Ishitsubo, N., and Yoneda, Y. 1995. A possible role of glutathione as an endogenous agonist at the N-methyl-D-aspartate recognition domain in rat brain. J. Neurochem. 64:1088–1096.

    Google Scholar 

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Authors and Affiliations

  1. Physiologisch-chemisches Institut der Universität, Hoppe-Seyler-Str. 4, D-72076, Tübingen, Germany

    Ralf Dringen, Oliver Kranich & Bernd Hamprecht

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  1. Ralf Dringen
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  2. Oliver Kranich
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  3. Bernd Hamprecht
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Correspondence to Ralf Dringen.

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Dringen, R., Kranich, O. & Hamprecht, B. The γ-Glutamyl Transpeptidase Inhibitor Acivicin Preserves Glutathione Released by Astroglial Cells in Culture. Neurochem Res 22, 727–733 (1997). https://doi.org/10.1023/A:1027310328310

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