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Isolation of astrocytes, neurons, and synaptosomes of rat brain cortex

Distribution of enzymes of Glutamate Metabolism

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Abstract

A simplified method was developed for the bulk separation of neuronal perikarya and astroglial celis from adult rat brain without the involvement of density gradients. Activities of various enzymes involved in glutamate metabolism were estimated and compared with those of synaptosomes. The activities of glutamate dehydrogenase and aspartate aminotransferase were higher in synaptosomes than in neuronal perikarya or glia. Glutamine synthetase was distributed in all the three fractions while glutaminase activity was higher in astrocytes than in synaptosomes and was not detectable in neuronal perikarya. The significance of these results in relation to metabolic compartmentation was discussed.

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References

  1. Benjamin, A. M., andQuastel, J. H. 1972. Locations of amino acids in brain slices from the rat: Tetrodotoxin-sensitive release of amino acids. Biochem. J. 128:631–646.

    PubMed  Google Scholar 

  2. Benjamin, A. M., andQuastel, J. H. 1974. Fate ofl-glutamate in the brain J. Neurochem. 23:457–464.

    PubMed  Google Scholar 

  3. Bergmeyer, H. U., andBernt, E. 1974. Lactate dehydrogenase. Pages 574–579in Bergmeyer, H. U. (ed.) Methods of Enzymatic analysis vol II. Academic Press, New York.

    Google Scholar 

  4. Bergmeyer, H. U., andBernt, E. 1974. Glutamate oxaloacetate transaminase and glutamate pyruvate transaminase. Pages 727–758in Bergmeyer, H. U. (ed.) Methods of Enzymatic Analysis. Vol. II Academic Press, New York.

    Google Scholar 

  5. Berl, S. 1974. Cerebral amino acid metabolism in hepatic coma. Exptl. Biol. Med. 4:71–84.

    Google Scholar 

  6. Bradford, H. F., Ward, H. K., andThomas, A. J. 1978. Glutamine: a major substrate for nerve endings. J. Neurochem. 30:1453–1459.

    PubMed  Google Scholar 

  7. Bradford, H. F., andWard, H. K. 1976. On glutaminase activity in mammalian synaptosomes. Brain Res. 110:115–125.

    PubMed  Google Scholar 

  8. Bregnard, A., Keunzle, C. C., andRuche, F. 1971. Cytophotometric and autoradiographic evidence for post-natal DNA synthesis in neurons of the rat cerebral cortex. Extl. Cell. Res. 107:151–157.

    Google Scholar 

  9. Chee, P. Y., Dahl, J. L., andFahein, L. A. 1979. The purification and properties of rat brain glutamate dehydrogenase. J. Neurochem. 33:53–60.

    PubMed  Google Scholar 

  10. Cotman, C. W. 1974. Isolation of synaptosomal and synaptic plasma membrane fractions. Pages 445–452in Fleischer, S. andPacker, L. (eds.) Methods in Enzymology Vol. 31, Academic Press, New York.

    Google Scholar 

  11. Dunn, A. J., andBondy, S. C. 1974. Pages 34–35in Functional Chemistry of the Brain, John Wiley and Sons, New York.

    Google Scholar 

  12. Ellman, G. L., Courtney, L. D., Andrews, V. Jr., andFeatherstone, R. M. 1961. A new and rapid colorimetric determination of acetyl cholinesterase activity. Biochem. Pharmacol. 7:88–95.

    PubMed  Google Scholar 

  13. Farooq, M., andNorton, W. T. 1978. A modified procedure for isolation of astrocyte and neuron enriched fractions from rat brain. J. Neurochem. 31:887–894.

    PubMed  Google Scholar 

  14. Farooq, M., Ferszt, R., Moore, C. L., andNorton, W. T. 1977. The isolation of cerebral neurons with partial retention of processes. Brain Res. 124:69–81.

    PubMed  Google Scholar 

  15. Fonnum, F. 1975. A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24:407–409.

    PubMed  Google Scholar 

  16. Hamberger, A., Chiang, G. H., Nylen, E. S., Scheff, S. W., andCotman, C. W. 1979. Glutamate as a CNS transmitter. I. Evaluation of glucose and glutamine as precursors for the synthesis of preferentially released glutamate. Brain Res. 168:513–530.

    PubMed  Google Scholar 

  17. Hamberger, A., Chiang, G. H., Sandoval, E., andCotman, C. W. 1979. Glutamate as a CNS transmitter. Regulation of synthesis in the releasable pool. Brain Res. 168:531–541.

    PubMed  Google Scholar 

  18. Hertz, L. 1979. Functional interactions between neurons and astrocytes I. Turnover and metabolism of putative amino acid transmitters. Progr. Neurobiol. 13:277–323.

    PubMed  Google Scholar 

  19. Krebs, H. A., andRoughton, F. J. W. 1948. Carbonic anhydrase as a tool in studying the mechanism of reactions involving H2CO3, CO2 or HCO 3 Biochem. J. 43:550–555.

    Google Scholar 

  20. Kvamme, E., Svenneby, G., Hertz, L., andSchousboe, A. 1982. Properties of phosphate activated glutaminase in astrocytes cultured from mouse brain. Neurochem. Res. 7:761–770.

    Google Scholar 

  21. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., andRandall, R. J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265–275.

    PubMed  Google Scholar 

  22. Martinez-Hernandez, A., Bell, K. P., andNorenberg, M. D. 1977. Glutamine synthestase: glial localization in brain. Science 195:1356–1358.

    PubMed  Google Scholar 

  23. Nagata, Y., Mikoshiba, K., andTsukada, Y. 1974. Neuronal cell body enriched and glial cell enriched fractions from young and adult rat brains: Preparation and morphological and biochemical properties. J. Neurochem. 22:493–503.

    PubMed  Google Scholar 

  24. Nimmo, G. M., andTipton, K. F. 1979. The soluble and membrane bound forms of glutaminase in pig brain. J. Neurochem. 33:1083–1094.

    PubMed  Google Scholar 

  25. Norton, W. T., andPodusulo, S. E. 1971. Neuronal perikarya and astroglia of rat brain: Chemical composition during myelination. J. Lipid Res. 12:84–90.

    PubMed  Google Scholar 

  26. Palay, S. L., andPalay, V. C. 1977. General morphology of neurons and neuroglia. Pages 5–37,in Kandel, E. R. (ed.) Handbook of Physiology, Vol. I. Part 1, American physiological society, Bethesda.

    Google Scholar 

  27. Reubi, J. C., Van Den Berg, C., andCuenod, M. 1978. Glutamine as precursor for the GABA and glutamate transmitter pools. Nuerosci. Lett. 10:171–174.

    Google Scholar 

  28. Rose, S. P. R. 1967. Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells. Biochem. J. 102:33–43.

    PubMed  Google Scholar 

  29. Rowe, W. B., Ronzio, R. A., Wellner, V. P., andMeister, A. 1970. Glutamine synthetase (sheep brain) Pages 900–910,in Tabor, H. andTabor, C. W. (eds.), Methods in Enzymology, vol XVII A, Academic Press, New York.

    Google Scholar 

  30. Sadasivudu, B., Indira Rao, T. andRadhakrishna Murthy, Ch. 1977. Acute metabolic effects of ammonia in mouse brain. Neurochem. Res. 2:639–655.

    Google Scholar 

  31. Schmidt, G., andThannhauser, S. J. 1945. A method for the determination of deoxyribonucleic acid, ribonucleic acid and phosphoproteins in animal tissues. J. Biol. Chem. 161:83–89.

    Google Scholar 

  32. Schousboe, A., Hertz, L., Svenneby, G., andKvamme, E. 1979. Phosphate activated glutaminase activity and glutamine uptake in astrocytes in primary cultures. J. Neurochem. 32:943–950.

    PubMed  Google Scholar 

  33. Sellinger, O. Z., andAzcurra, J. M. 1974. Bulk separation of nueronal cell bodies and glial cells in the absence of added digestive enzymes. Pages 3–38,in Marks, N. andRodnight, R. (eds.) Research Methods in Neurochemistry, Plenum Press, New York.

    Google Scholar 

  34. Shank, R. P., andAprison, M. H. 1977. Glutamine uptake and metabolism by the isolated toad brain: Evidence pertaining to its proposed role as a transmitter precursor. J. Neurochem. 28:1189–1196.

    PubMed  Google Scholar 

  35. Shank, R. P. andAprison, M. H. 1981. Present status and significance of the glutamine cycle in neural tissues. Life Sci. 28:837–842.

    PubMed  Google Scholar 

  36. Subbalakshmi, G. Y. C. V. 1984. Studies on the enzymes of cerebral cellular glutamate metabolism in hyperammonemia., Ph.D. Thesis, University of Hyderabad, Hyderabad.

    Google Scholar 

  37. Subbalakshmi, G. Y. C. V., andMurthy, Ch. R. K. 1983. Acute metabolic effects of ammonia on the enzymes of glutamate metabolism in isolated astroglial cells. Neurochem. Intnl. 5:593–597.

    Google Scholar 

  38. Subbalakshmi, G. Y. C. V., andMurthy, Ch. R. K. 1983. Effects of methionine sulfoximine on the enzymes of glutamine metabolism in astrocytes of rat brain. Biochem. Pharmacol. 32:3695–3700.

    PubMed  Google Scholar 

  39. Ward, H. K., Thanki, C. M., andBradford, H. F. 1983. Glutamine and glucose as precursors of transmitter amino acids: Ex vivo studies. J. Neurochem. 40:855–860.

    PubMed  Google Scholar 

  40. Weiler, C. T., Nystrom, B., andHamberger, A. 1979. Glutaminase and glutamine synthetase activity in synaptosomes, bulk isolated glia and neurons. Brain Res. 160:539–543.

    PubMed  Google Scholar 

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Subbalakshmi, G.Y.C.V., Murthy, C.R.K. Isolation of astrocytes, neurons, and synaptosomes of rat brain cortex. Neurochem Res 10, 239–250 (1985). https://doi.org/10.1007/BF00964570

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