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Neurochemical Research

, Volume 18, Issue 6, pp 647–654 | Cite as

Uptake and metabolism of glutamate and aspartate by astroglial and neuronal preparations of rat cerebellum

  • V. L. Raghavendra Rao
  • Ch. R. K. Murthy
Original Articles

Abstract

Astrocytes, neuronal perikarya and synaptosomes were prepared from rat cerebellum. Kinetics of high and low affinity uptake systems of glutamate and aspartate, nominal rates of14CO2 production from [U−14C]glutamate, [U−14C]aspartate and [1−14C]glutamate and activities of enzymes of glutamate metabolism were studied in these preparations. The rate of uptake and the nomial rate of production of14CO2 from these amino acids was higher in the astroglia than neuronal perikarya and synaptosomes. Activities of glutamine synthetase and glutamate dehydrogenase were higher in astrocytes than in neuronal perikarya and synaptosomes. Activities of glutaminase and glutamic acid decarboxylase were observed to be highest in neuronal perikarya and synaptosomes respectively. These results are in agreement with the postulates of theory of metabolic compartmentation of glutamate while others (presence of glutaminase in astrocytes and glutamine synthetase in synaptosomes) are not. Results of this study also indicated that (i) at high extracellular concentrations, glutamate/aspartate uptake may be predominantly into astrocytes while at low extracellular concentrations, it would be into neurons (ii) production of α-ketoglutarate from glutamate is chiefly by way of transamination but not by oxidative deamination in these three preparations and (iii) there are topographical differences glutamate metabolism within the neurons.

Key Words

Glutamate aspartate astrocytes neurons synaptosomes uptake CO2 production 

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References

  1. 1.
    Berl, S., Takagaki, G., Clarke, D. D., and Waelsch, H. 1962. Metabolic compartmentsin vivo. Ammonia and glutamic acid metabolism in brain and liver. J. Biol. Chem. 237:2562–2569.Google Scholar
  2. 2.
    Benjamin, A. M., and Quastel, J. H. 1972. Location of amino acids in brain slices from the rat: Tetrodotoxin-sensitive release of amino acids. Biochem. J. 128:631–646.Google Scholar
  3. 3.
    Benjamin, A. M., and Quastel, J. H. 1974. Fate of L-glutamate in the brain. J. Neurochem. 23:457–464.Google Scholar
  4. 4.
    Schousboe, A., Hertz, L., Svenneby, G., and Kvamme, E. 1979. Phosphate activated glutaminase activity and glutamine uptake in primary cultures of astrocytes. J. Neurochem. 32:943–950.Google Scholar
  5. 5.
    Ito, M. 1984. In: The Cerebellum and Neural Control. Raven Press, New York.Google Scholar
  6. 6.
    Subbalakshmi, G. Y. C. V., and Murthy, Ch. R. K. 1985. Isolation of astrocytes, neurons and synaptosomes of rat brain cortex: Distribution of enzymes of glutamate metabolism. Neurochem. Res. 10:239–250.Google Scholar
  7. 7.
    Farooq, M., and Norton, W. T. 1978. A modified procedure for isolation of astrocyte and neuron enriched fractions from rat brain. J. Neurochem. 31:887–894.Google Scholar
  8. 8.
    Rao, V. L. R., and Murthy, Ch. R. K. 1991. Hyperammonemic alterations in the uptake and release of glutamate and aspartate by rat cerebellar preparations. Neurosci. Lett. 130:49–52.Google Scholar
  9. 9.
    Rao, V. L. R., and Murthy, Ch. R. K. 1992. Ammonia-induced alterations in the metabolism of glutamate and aspartate in neuronal perikarya and synaptosomes of rat brain. Metab. Brain Dis. 7:51–61.Google Scholar
  10. 10.
    Cotman, C. W. 1974. Isolation of synaptosomes and synaptic plasma membrane fractions. Pages 445–452in Fleischer, S. and Packer, L. (eds.) Methods in Enzymology, Vol. 31. Academic Press, New York.Google Scholar
  11. 11.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurements with Folin-phenol reagent. J. Biol. Chem. 193:265–175.Google Scholar
  12. 12.
    Rao, V. L. R., and Murthy, Ch. R. K. 1992. Hyperammonemic alterations in the metabolism of glutamate and aspartate in rat cerebellar astrocytes. Neurosci. Lett. 138:107–110.Google Scholar
  13. 13.
    Lai, J. C. K., Murthy, Ch. R. K., Cooper, A. J. L., Hertz, E., and Hertz, L. 1989. Differential effects of ammonia and β-methylene-DL-aspartate on the metabolism of glutamate and related amino acids by astrocytes and neurons in primary cultures. Neurochem. Res. 14:377–389.Google Scholar
  14. 14.
    Bergmeyer, H. U., and Brent, E. 1974. Glutamate oxaloacetate transaminase and glutamate pyruvate transaminase. Pages 727–758 in Bergmeyer, H.U. (ed.) Methods of Enzymatic Analysis vol. II. Academic Press, New York.Google Scholar
  15. 15.
    Nimmo, G. A., and Tipton, K. F. 1979. The distribution of soluble and membrane bound forms of glutaminase in pig brain. J. Neurochem. 33:1083–1094.Google Scholar
  16. 16.
    McCarthy, A. D., Walker, J. M., and Tipton, K. F. 1980. Purification of glutamate dehydrogenase from ox brain and liver. Biochem. J. 191:605–611.Google Scholar
  17. 17.
    Rowe, W. B., Ronzio, R. A., Welner, V. P., and Meister, A. 1970. Glutamine synthetase (sheep brain). Pages 900–910in Tabor, H., and Tabor, C. W., (eds.,) Methods in Enzymology, Vol. XVIIA. Academic Press, New York.Google Scholar
  18. 18.
    Sytinsky, I. A., Guzikov, M., Gomanko, M. V., Eremin, V. P., and Konovalova, N. N. 1975. The GABA system in brain during acute and chronic ethanol intoxication. J. Neurochem. 25:43–48.Google Scholar
  19. 19.
    Sadasivudu, B., and Murthy, Ch. R. K. 1978. Effects of ammonia on monoamine oxidase and enzymes of GABA metabolism in mouse brain. Arch. Internat. Physiol. Biochem. 86:67–82.Google Scholar
  20. 20.
    Nandakumar, N. V., Murthy, Ch. R. K., Vijayakumari, D., and Swami, K. S. 1973. Axonal protein charges and succinate dehydrogenase activity in sheep medulla oblongata. Indian J. Exp. Biol. 11:525–528.Google Scholar
  21. 21.
    Ellman, G. L., Courtney, L. D., Andrews, V. Jr., and Featherstone, R. M. 1961. A new and rapid colorimetric determination of acetyl cholinesterase activity. Biochem Pharmacol. 7:88–95.Google Scholar
  22. 22.
    Shank, R. P., and Campbell, G. L.-M. 1984. Amino acid uptake, content and metabolism by neuronal and glial enriched cellular fractions from mouse brain. J. Neurosci. 4:58–69.Google Scholar
  23. 23.
    Gordon, R. J., and Balazs, R. 1983. Characterization of separated cell types from the developing rat cerebellum: transport of glutamate and asparate by preparations enriched in Purkinje cells, granule neurons, and astrocytes. J. Neurochem. 40:1090–1099.Google Scholar
  24. 24.
    Benjamin, A. M. and Quastel, J. H. 1976. Cerebral uptakes and exchange diffusion in vitro ofl- andd- glutamates. J. Neurochem. 26:431–441.Google Scholar
  25. 25.
    Campbell, G. L. M., and Shank, R. P. 1978. Glutamate and GABA uptake by cerebellar granule and glial enriched populations. Brain Res. 153:618–622.Google Scholar
  26. 26.
    Weiler, C. T., Nyström, B. and Hamberger, A. 1979. Characteristics of glutamate vs glutamine transport in isolated glia and synaptosomes. J. Neurochem. 32:559–565.Google Scholar
  27. 27.
    Hannuniemi, R., and Oja, S. S. 1981. Uptake of leucine, lysine, aspartic acid, and glycine into isolated neurons and astrocytes. Neurochem. Res. 6:873–884.Google Scholar
  28. 28.
    Hertz, L. Murthy, Ch. R. K., and Schousboe, A. 1988. Metabolism of glutamate and related amino acids. Pages 395–406in Norenberg, M. D., Hertz, L., and Schousboe, A. (eds.) The Biochemical Pathology of Astrocytes. Alan R. Liss, New York.Google Scholar
  29. 29.
    Yu, A. C. H., Schousboe, A., and Hertz, L. 1982. Metabolic fate of14C-labeled glutamate in astrocytes. J. Neurochem. 39:954–960.Google Scholar
  30. 30.
    Berl, S. 1973. Biochemical consequences of compartmentation of glutamate and associated metabolites. Pages 3–17in Balazs, R., and Cremer, J. E. (eds). Metabolic Compartmentation in the Brain, McMillan Press, New York.Google Scholar
  31. 31.
    Farinelli, S. E. and Nicklas, W. J. 1992. Glutamate metabolism in rat cortical astrocyte cultures. J. Neurochem. 58:1905–1915.Google Scholar
  32. 32.
    Yudkoff, M., Nissim, I., Nelson, D., Lin, Z.-P., and Erecinska, M. 1991. Glutamate dehydrogenase reaction as a source of glutamic acid in synaptosomes. J. Neurochem. 57:153–160.Google Scholar
  33. 33.
    Carter, C. J., Savasta, M., Fage, D., and Scatton, B. 1986. 2-Oxo-[14C]glutarate is taken up by glutamatergic nerve terminals in the rat striatum. Neurosci. Lett. 72:227–231.Google Scholar
  34. 34.
    Kihara, M., and Kubo, T. 1989. Aspartate aminotransferase for synthesis of transmitter glutamate in the medulla oblongata: Effect of aminooxyacetic acid and 2-oxoglutarate. J. Neurochem. 52:1127–1134.Google Scholar
  35. 35.
    Peng, L., Schousboe, A., and Hertz, L. 1991. Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells. Neurochem. Res. 16:29–34.Google Scholar
  36. 36.
    Norenberg, M. D. 1979. The distribution of glutamine synthetase in the rat central nervous system. J. Histochem. Cytochem. 27:756–762.Google Scholar
  37. 37.
    Kaneko, T., Urade, Y., Watanabe, Y., and Mizuno, N. 1987. Production, characterization and immunochemical application of monoclonal antibodies to glutaminase purified from rat brain. J. Neurosci. 7:302–309.Google Scholar
  38. 38.
    Ward, H. K., and Bradford, H. F. 1979 Relative activities of glutamine synthetase and glutaminase in mammalian synaptosomes. J. Neurochem. 33:339–342.Google Scholar
  39. 39.
    Yudkoff, M., Nissim, I., and Pleasure, D. 1988. Astrocyte metabolism of15N glutamine: implications for the glutamine-glutamate cycle. J. Neurochem. 51:843–850.Google Scholar
  40. 40.
    Murthy, Ch. R. K., and Hertz, L. 1988. Pyruvate decarboxylation in astrocytes and in neurons in primary cultures in the presence and the absence of ammonia. Neurochem. Res. 13:57–61.Google Scholar
  41. 41.
    Weiler, C. T., Nystrom, B., and Hamberger, A. 1979. Glutaminase and glutamine synthetase activity in synaptosomes, bulk isolated glia and neurons. Brain Res. 160:539–543.Google Scholar
  42. 42.
    Rani, B. U., Singh, N. I., Ray, A., and Rao, K. S. 1983. Isolation procedure of neuron and astrocyte enriched fractions from chick brain of different ages. J. Neurosci. Res. 10:101–105.Google Scholar

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • V. L. Raghavendra Rao
    • 1
    • 2
  • Ch. R. K. Murthy
    • 1
  1. 1.School of Life SciencesUniversity of HyderabadHyderabadIndia
  2. 2.Neuroscience Research Unit, André-Viallet Clinical Research Center, Hôpital Saint-LucUniversity of MontrealMontrealCanada

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