Neurochemical Research

, Volume 19, Issue 2, pp 111–115 | Cite as

Pharmacological characterization of glutamate binding sites in cultured cerebellar granule cells and cortical astrocytes

  • I. Holopainen
  • Pirjo Saransaari
  • S. S. Oja
Original Articles


Membranes prepared from cerebellar granule cells and cortical astrocytes exhibited specific, saturable binding ofl-[3H]glutamate. The apparent binding constant K d was 135 nM and 393 nM and the maximal binding capacity Bmax 42 and 34 μmol/kg in granule cells and astrocytes, respectively. In granule cells the binding was strongly inhibited by the glutamate receptor agonists kainate, quisqualate, N-methyl-d-aspartate (NMDA),l-homocysteate and ibotenate, and the antagonistdl-5-aminophosphonovalerate. In astrocytes, only quisqualate among these was effective.l-Aspartate,l-cysteate,l-cysteinesulphinate and γ-d-glutamylglycine were inhibitors in both cell types. The binding was totally displaced in both cell types byl-cysteinesulphinate with IC50 in the micromolar range. In astrocytes the binding was also totally displaced by quisqualate, but in granule cells only partially by NMDA, kainate and quisqualate in turn. It is concluded from the relative potencies of agonists and antagonists in [3H]glutamate binding that cerebellar granule cells express the NMDA, kainate and quisqualate types of the glutamate receptor, while only the quisqualate-sensitive binding seems to be present in cortical astrocytes.

Key Words

Cerebellar granule cells cortical astrocytes excitatory amino acid binding glutamate receptor subtypes 


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  1. 1.
    Gallo, V., Giovannini, C., and Levi, G. 1989. Quisqualic acid modulates kainate responses in cultured cerebellar granule cells. J. Neurochem. 52:10–16.Google Scholar
  2. 2.
    Monaghan, D. T., Bridges, R. J., and Cotman, C. W. 1989. The excitatory amino acid receptors: Their classes, pharmacology, and distinct properties in the function of the central nervous system. Annu. Rev. Pharmacol. Toxicol. 29:365–402.Google Scholar
  3. 3.
    Bowman, C. L., and Kimelberg, H. K. 1984. Excitatory amino acids directly depolarize rat brain astrocytes in primary culture. Nature (Lond.) 311:656–659.Google Scholar
  4. 4.
    Kettenmann, H., and Schachner, M. 1985. Pharmacological properties of γ-aminobutyric acid-, glutamate-, and aspartate-induced depolarizations in cultured astrocytes. J. Neurosci. 5:3295–3301.Google Scholar
  5. 5.
    Brew, H., and Attwell, D. 1987. Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells. Nature (Lond.) 327:707–709.Google Scholar
  6. 6.
    Bridges, R. J., Nieto-Sampedro, M., Kadri, M., and Cotman, C. W. 1987. A novel chloride-dependent L-[3H]glutamate binding site in astrocyte membranes. J. Neurochem. 48:1709–1715.Google Scholar
  7. 7.
    Pearce, B., Albrecht, J., Morrow, C., and Murphy, S. 1986. Astrocyte glutamate receptor activation promotes inositol phospholipid turnover and calcium flux. Neurosci. Lett. 72:335–340.Google Scholar
  8. 8.
    Holopainen, I., and Kontro, P. 1990.d-Aspartate release from cerebellar astrocytes: modulation of the high K-induced release by neurotransmitter amino acids. Neuroscience 36:115–120.Google Scholar
  9. 9.
    Enkvist, M. O. K., Holopainen, I., and Åkerman, K. E. O. 1989. Glutamate receptor-linked changes in membrane potential and intracellular Ca2+ in primary rat astrocytes. Glia 2:397–402.Google Scholar
  10. 10.
    Holopainen, I., Malminen, O., and Kontro, P. 1987. Sodium-dependent high-affinity uptake of taurine in cultured cerebellar granule cells and astrocytes. J. Neurosci. Res. 18:479–483.Google Scholar
  11. 11.
    Holopainen, I. 1984. Modification of taurine and hypotaurine uptake systems in cultured primary astrocytes by serum-free medium and dibutyryl cyclic AMP treatment. Int. J. Dev. Neurosci. 2:529–534.Google Scholar
  12. 12.
    Nelder, J. A., and Mead, R. 1985. A simplex method for function minimization. Computer J. 7:308–313.Google Scholar
  13. 13.
    Holopainen, I., and Kontro, P. 1988. Glutamate release from cerebellar granule cells differentiating in culture: modulation of the K+-stimulated release by inhibitory amino acids. Neurochem. Int. 12:155–161.Google Scholar
  14. 14.
    Holopainen, I., Enkvist, M. O. K., and Åkerman, K. E. O. 1989. Glutamate receptor agonists increase intracellular Ca2+ independently of voltage-gated Ca2+ channels in rat cerebellar granule cells. Neurosci. Lett. 98:57–62.Google Scholar
  15. 15.
    Holopainen, I., Louve, M., Enkvist, M. O. K., and Åkerman, K. E. O. 1990. Coupling of glutamatergic receptors to changes in intracellular Ca2+ in rat cerebellar granule cells in primary culture. J. Neurosci. Res. 25:187–193.Google Scholar
  16. 16.
    Schoepp, D., Bockaert, J., and Sladeczek, F. 1990. Pharmacological and functional characteristics of metabotropic excitatory amino acid receptors. Trends Pharmacol. Sci. 11:508–515.Google Scholar
  17. 17.
    Holopainen, I., Louve, M., and Åkerman, K. E. O. 1991. Interactions of glutamate receptor agonists coupled to changes in intracellular Ca2+ in rat cerebellar granule cells in primary culture. J. Neurochem. 57:1729–1734.Google Scholar
  18. 18.
    Backus, K. H., Kettenmann, H., and Schachner, M. 1989. Pharmacological characterization of the glutamate receptor in cultured astrocytes. J. Neurosci. Res. 22:274–282.Google Scholar
  19. 19.
    Enkvist, M. O. K., Holopainen, I., and Åkerman, K. E. O. 1988. The effect of K+ and glutamate receptor agonists on the membrane potential of suspensions of primary cultures of rat astrocytes as measured with a cyanine dye, DiS-C2-(5). Brain Res. 462:67–75.Google Scholar
  20. 20.
    Holopainen, I., and Åkerman, K. E. O. 1990. Efflux of45calcium from cultured primary astrocytes: effects of glutamate receptor agonists and antagonists. Neuropharmacology 29:713–717.Google Scholar
  21. 21.
    Honoré, T., Drejer, J., Nielsen, M., and Braestrup, C. 1986. Differentiation of Cl/Ca2+-dependent and sodium dependent3H-glutamate binding to cortical membranes from rat brain by high energy radiation inactivation analysis. J. Neural Transm. 65:93–101.Google Scholar
  22. 22.
    Usowicz, M. M., Gallo, V., and Cull-Candy, S. G. 1988. Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids. Nature 339:380–383.Google Scholar
  23. 23.
    Ascher, P., and Nowak, L. 1988. The role of divalent cations in the N-methyl-d-aspartate responses of mouse central neurones in culture. J. Physiol. (Lond.) 399:247–266.Google Scholar
  24. 24.
    Ascher, P., and Nowak, L. 1988. Quisqualate- and kainate-activated channels in mouse central neurones in culture. J. Physiol. (Lond.) 399:227–245.Google Scholar
  25. 25.
    Foster, A. C., and Fagg, G. E. 1987. Comparison ofL-[3H]-glutamate,D-[3H]aspartate,DL-[3H]AP5 and [3H]NMDA as ligands for NMDA receptors in crude postsynaptic densities from rat brain. Eur. J. Pharmacol. 133:291–300.Google Scholar
  26. 26.
    Varga, V., Janáky, R., Holopainen, I., Saransaari, P., and Oja, S. S. 1992. Effect of magnesium on calcium influx activated by glutamate and its agonists in cultured cerebellar granule cells. Neurochem. Res. 17:1195–1200.Google Scholar
  27. 27.
    Fagg, G. E., Foster, A. C., Mena, E. E., and Cotman, C. W. 1983. Chloride and calcium ions separatel-glutamate receptor populations in synaptic membranes. Eur. J. Pharmacol. 88:105–110.Google Scholar
  28. 28.
    Fagg, G. E., and Lanthorn, T. H. 1986. Cl/Ca2+-dependentl-glutamate binding sites do not correspond to 2-amino-4-phosphonobutanoate-sensitive excitatory amino acid receptors. Br. J. Pharmacol. 86:743–751.Google Scholar
  29. 29.
    Cha, J.-H. J., Greenamyre, J. T., Nielsen, E. Ø., Penney, J. B., and Young, A. B. 1988. Properties of quisqualate-sensitivel-[3H]glutamate binding sites in rat brain as determined by quantitative autoradiography. J. Neurochem. 51:469–478.Google Scholar
  30. 30.
    Collingridge, G. L., and Lester, R. A. J. 1989. Excitatory amino acid receptors in the vertebrate central nervous system. Pharmacol. Rev. 40:143–210.Google Scholar
  31. 31.
    Bridges, R. J., Kesslak, J. P., Nieto-Sampedro, M., Broderick, J. T., Yu. J., and Cotman, C. W. 1987. Al-[3H]glutamate binding site on glia: an autoradiography study on implanted astrocytes. Brain Res. 415:163–168.Google Scholar
  32. 32.
    Johnson, J. W., and Ascher, P. 1987. Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325:529–531.Google Scholar
  33. 33.
    Monaghan, D. T., Olverman, H. J., Nguyen, L., Watkins, J. C., and Cotman, C. W. 1988. Two classes of N-methyl-d-aspartate recognition sites: differential distribution and differential regulation by glycine. Proc. Natl. Acad. Sci. USA, 85:9836–9840.Google Scholar
  34. 34.
    Do, K. Q., Mattenberger, M., Streit, P., and Cuénod, M. 1986. In vitro release of endogenous excitatory sulfur-containing amino acids from various rat brain regions. J. Neurochem. 46:779–786.Google Scholar
  35. 35.
    Monahan, J. B., and Michel, J. 1987. Identification and characterization of an N-methyl-d-aspartate-specificl-[3H]glutamate recognition site in synaptic plasma membranes. J. Neurochem. 48:1699–1708.Google Scholar
  36. 36.
    Patneau, D. K., and Mayer, M. L. 1990. Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-d-aspartate and quisqualate receptors. J. Neurosci. 10:2385–2399.Google Scholar

Copyright information

© Plenum Publishing Corporation 1994

Authors and Affiliations

  • I. Holopainen
    • 1
  • Pirjo Saransaari
    • 2
  • S. S. Oja
    • 2
  1. 1.Department of Biochemistry and PharmacyUniversity of Åbo AkademiÅboFinland
  2. 2.Department of Biomedical SciencesUniversity of TampereTampereFinland

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