Abstract
Glutamine synthetase (GS) activity in cultured rat astrocytes was measured in extracts and compared to the intracellular rate of glutamine synthesis by intact control astrocytes or astrocytes exposed to 1 mM 8-bromo-cAMP (8Br-cAMP)+1 μM dexamethasone (DEX) for 4 days. GS activity in extracts of astrocytes treated with 8Br-cAMP+DEX was 7.5 times greater than the activity in extracts of control astrocytes. In contrast, the intracellular rate of glutamine synthesis by intact cells increased only 2-fold, suggesting that additional intracellular effectors regulate the expression of GS activity inside the intact cell. The rate of glutamine synthesis by astrocytes was 4.3 times greater in MEM than in HEPES buffered Hank's salts. Synthesis of glutamine by intact astrocytes cultured in MEM was independent of the external glutamine or ammonia concentrations but was increased by higher extracellular glutamate concentrations. In studies with intact astrocytes 80% of the original [U-14C]glutamate was recovered in the medium as radioactive glutamine, 2–3% as aspartate, and 7% as glutamate after 2 hours for both control and treated astrocytes. The results suggest: (1) astrocytes are highly efficient in the conversion of glutamate to glutamine; (2) induction of GS activity increases the rate of glutamate conversion to glutamine by astrocytes and the rate of glutamine release into the medium; (3) endogenous intracellular regulators of GS activity control the flux of glutamate through this enzymatic reaction; and, (4) the composition of the medium alters the rate of glutamine synthesis from external glutamate.
Similar content being viewed by others
References
Martinez-Hernandez, A., Bell, K. P., and Norenberg, M. D. 1977. Glutamine synthetase: glial localization in brain. Science 195:1356–1358.
Norenberg, M. D., and Martinez-Hernandez, A. 1979. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 161:303–310.
Lopes-Cardozo, M., and Warringa, R. A. J. 1987. complement-mediated cell lysis in cultured glial cells. J. Neurochem. 48:S175.
Cammer, W., Farooq, M., and Tansey, F. Glutamine synthetase in oligodendrocytes and astrocytes. Abstract 259, American Society for Neurochemistry Meeting, Phoenix, AZ, March 4–9, 1990.
Warringa, R. A., van Berlo, M. F., and Lopes-Cardoza, M. 1988. Cellular location of glutamine synthetase and lactate dehydrogenase in oligodendrocyte-enriched cultures from rat brain. J. Neurochem. 50:1461–1468.
Van den Berg, C. J. and Garfinkel, D. 1971. A simulation study of brain compartments-metabolism of glutamate and related substances in mouse brain. Biochem. J. 123:211–218.
Benjamin, A. M., and Quastel, J. H. 1975. Metabolism of amino acids and ammonia in rat brain cortex slices in vitro: a possible role of ammonia in brain function. J. Neurochem. 25:197–206.
Waniewski, R. A., and Martin, D. L. 1986. Exogenous glutamate is metabolized to glutamine and exported by rat primary astrocyte cultures. J. Neurochem. 47:304–313.
Bradford, H. F., Ward, H. K., and Thomas, H. J. 1978. Glutamine-a major substrate for nerve endings. J. Neurochem. 30:1453–1459.
Hamberger, A. C., Chiang, G. H., Nylen, E. S., Scheff, S. W., and Cotman, 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.
Battaglioli, G. and Martin, D. L. 1990. Stimulation of synaptosomal γ-aminobutyric acid synthesis by glutamate and glutamine. J. Neurochem. 54:1179–1187.
Perry, T. L. and Hansen, S. 1981. Amino acid abnormalities in epileptogenic foci. Neurology 31:872–876.
Plaitakis, A., Berl, S., and Yaho, M. D. 1982. Abnormal glutamate metabolism in an adult-onset degenerative neurological disease. Science 216:193–196.
Plaitakis, A. and Caroscio, J. T. 1987. Abnormal glutamate metabolism in amyotropic lateral sclerosis. Ann. Neurol. 22:575–579.
Maragos, W. F., Greenmyre, J. T., Penney, Jr., J. B., and Young, A. B. 1987. Glutamate dysfunction in Alzheimer's disease: an hypothesis. TINS 10:65–68.
Drejer, J., Benveniste, H., Diemer, N. H., and Schousboe, A. 1985. Cellular origin of ischemia-induced glutamate release from brain tissue in vivo and in vitro. J. Neurochem. 45:145–151.
Rothman, S. M., and Olney, J. W. 1986. Glutamate and the pathophysiology of hypoxic-ischemic brain damage. Ann. Neurol. 19:105–111.
Max, S. R., Mill, J., Mearow, K., Konagaya, M., Konagaya, Y., Thomas, J. W., Banner, C., and Vitković, L. 1988. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles. Am. J. Physiol. 255 (Endocrinol. Metab. 18) E397–E403.
Hallermayer, K., Harmening, C., and Hamprecht, B. 1981. Cellular localization and regulation of glutamine synthetase in primary cultures of brain cells from newborn mice. J. Neurochem. 37:43–52.
Kumar, S., Holmes, E., Scully, J. S., Birren, B. W., Wilson, R. H., and deVellis, J. 1986. The hormonal regulation of gene expression of glial markers: glutamine synthetase and glycerol phosphate dehydrogenase in primary cultures of rat brain and in C6 cell line. J. Neurosci. Res. 16:251–264.
Patel, A. J., Hunt, A., and Faraji-Shadan, F. 1986. Effect of removal of glutamine and addition of dexamethasone on the activities of glutamine synthetase, ornithine decarboxylase and lactate dehydrogenase in primary cultures of forebrain and cerebellar astrocytes. Devel. Brain Res. 26:229–238.
Browning, E. T. and Nicklas, W. J. 1982. Induction of glutamine synthetase by dibutyryl cyclic AMP in C-6 glioma cells. J. Neurochem. 39:336–341.
Zielke, H. R., Tildon, J. T., and Max, S. R. 1989. cAMP increeses glutamine synthetase activity in astrocytes. Transact. Am. Soc. Neurochem. 20:79A.
Max, S. R., Landry, M. E., and Zielke, H. R. 1990. Induction of glutamine synthetase by 8-bromo cyclic AMP in primary cultures of rat brain astrocytes. Neurochem. Res. 15:589–592.
Zielke, H. R., Tildon, J. T., Zielke, C. L., Baab, P. J., and Landry, M. E. 1989. Functional intracellular glutaminase activity in intact astrocytes. Neurochem. Res. 14:327–332.
Zielke, H. R. 1985. Determination of amino acids in the brain by high-performance liquid chromatography with isocratic elution and electrochemical detection. J. Chrom. 347:320–324.
Hertz, L., Juurlink, B. H. J., Fosmark, H., and Schousboe, A. 1982. Astrocytes in primary culture. Pages 175–186,in Pfeiffer, S. E. (ed.), Neuroscience Approached Through Cell Culture, Vol. 1, CRC Press, Boca Raton, Florida.
McCarthy, K. D., and DeVellis, J. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85:890–902.
Oyama, V. I., and Eagle, H. 1956. Measurement of cell growth in tissue culture with a phenol reagent (Folin-Ciocalteaux). Proc. Soc. Exp. Biol. Med. 91:305–307.
Hertz, L., and Schousboe, A. 1988. Metabolism of glutamate and glutamine in neurons and astrocytes in primary cultures. Pages 39–55.in Kvamme, E. (ed.) Glutamine and Glutamate in Mammals, Vol. II, CRC Press, Boca Raton, Florida.
Smith, R. J., Carson, S., Stred, S. E., and Durschlag, R. P. 1984. Regulation of glutamine synthetase and glutaminase activities in cultured skeletal muscle cells. J. Cell. Physiol. 120:197–203.
Feng, B., and Max, S. R. 1990. Post-transcriptional control of muscle glutamine synthetase expression by glutamine. Fed. Proc. 4:A916.
Waniewski, R. A. 1989. Glutamate and ammonia enhance glutamate synthesis by intact astrocytes. Transact. Am. Soc. Neurochem. 20:167A.
Yu., A. C. H., Schousboe, A., and Hertz, L. 1984. Influence of pathological concentration of ammonia on metabolic fate of14C-labeled glutamate in astrocytes in primary cultures. J. Neurochem. 42:594–597.
Yu, A. C., Schousboe, A., and Hertz, L. 1982. Metabolic fate of14C-labeled glutamate in astrocytes in primary cultures. J. Neurochem. 39:954–960.
Palaiologos, G., Hertz, L., and Schousboe, A. 1988. Evidence that aspartate aminotransferase activity and ketodicarboxylate carrier function are essential for biosynthesis of transmitter glutamate. J. Neurochem. 51:317–320.
Zielke, H. R., Tildon, J. T., Baab, P. J., and Hopkins, I. B. 1989. Synthesis of glutamate and glutamine in dibutyryl cyclic AMP-treated astrocytes. Neurosci. Lett. 97:209–214.
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.
Hundal, H. S., Rennie, M. J., and Watt, P. W. 1987. Characteristics ofl-glutamine transport in perfused rat skeletal muscle. J. Physiol. 393:283–305.
Farinelli, S. E. and Nicklas, W. J. 1989. Glutamate metabolism in cultured rat astrocytes. Transact. Am. Soc. Neurochem. 20:161A.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Zielke, H.R., Tildon, J.T., Landry, M.E. et al. Effect of 8-bromo-cAMP and dexamethasone on glutamate metabolism in rat astrocytes. Neurochem Res 15, 1115–1122 (1990). https://doi.org/10.1007/BF01101713
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01101713