Metabolic Brain Disease

, Volume 25, Issue 4, pp 413–418 | Cite as

Glutamine synthetase gene expression and glutamate transporters in C6-glioma cells

  • Zafeer Baber
  • Nasrin Haghighat
Original Paper


Glutamine synthetase (GS) is the major glutamate-forming enzyme of vertebrae and is accepted to be a marker of astroglial cells. Maturation of astroglial cells is characterized by an increase in GS activity, and the regulation of this enzyme is the topic of many publications. The amino acid glutamate is the major excitatory neurotransmitter in the brain and mediates normal excitatory synaptic transmission by interaction with postsynaptic receptors. Glutamate also acts as a potent neurotoxin when present at high concentration. Glutamate neurotoxicity plays an important role in the pathophysiology of many neurological disorders, such as Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis. In the normal condition, L-glutamate is predominantly taken up, metabolized and recycled by astrocytes through the glutamate transporters (GLAST/GLT1) and glutamine synthetase (GS) catalytic activity. Because of the fundamental role of these glutamate transporters and the glutamine synthetase enzyme in controlling cerebral glutamate level, regulation of GS and studying of the glutamate transporters in glial cells is important. Astrocytes are supportive cells and act as the site of detoxification of glutamate in the brain. However, their isolation from the brain is a tedious, costly and time consuming procedure. On the other hand, the C6-glioma cells are readily available on the market. They are well characterized and have been a useful model for CNS glia in many laboratories. For this study, we used the C6-glioma cell line as a model system. We examined the presence or absence of glial specific glutamate transporters (GLTI and GLAST) in C6-glioma cells, which was done by immunocytochemistry. We also examined glutamine synthetase gene expression in these cells by treatment of the C6-glioma cells with estrogen (17ß estradiol). The findings from this study provide useful information about C6-glioma cells which makes the study of the CNS tremendously inexpensive.


C6-glioma Glutamine synthetase Glutamate transporters GLT1 GLAST 



The authors wish to thank the medical student, Ms Nicole Gentile for her continuous help and support in the lab.


  1. Azcoitia I, Sierra A, Garcia-Segura LM (1999) Localization of estrogen receptor beta-immunoreactivity in astrocytes of the adult rat brain. Glia 26(3):260–267CrossRefPubMedGoogle Scholar
  2. Azcoitia I, Garcia-Ovejero D, Chowen JA, Garcia-Segura LM (2001) Astroglia plays a key role in the neuroprotective actions of estrogen. Prog Brain Res 132:469–478CrossRefPubMedGoogle Scholar
  3. Blutstein T, Devidze N, Choleris E, Jasnow AM, Pfaff DW, Mong JA (2006) Oestradiol up-regulates glutamine synthetase mRNA and protein expression in the hypothalamus and hippocampus: implications for a role of hormonally responsive glia in amino acid neurotransmission. J Neuroendocrinol 18(9):692–702CrossRefPubMedGoogle Scholar
  4. Buchanan CD, Mahesh VB, Brann DW (2000) Estrogen astrocyte-Luteinizing hormone releasing hormone signaling: a role for transforming growth factor ß1. Biol Reprod 62(6):1710–1721CrossRefPubMedGoogle Scholar
  5. Carrick T, Dunlop J (1999) Protein kinase C modulation of the human excitatory amino acid transporter 2 subtype of glutamate transporter. Soc Neurosci Abs 25:426Google Scholar
  6. Chio DW (1988) Glutamate neurotoxitcity and diseases of the nervous system. Neuron 1(8):623–634CrossRefGoogle Scholar
  7. Choi DW, Rothman SM (1990) The role of glutamate neurotoxicity in hypoxic-ischemic neurons death. Annu Rev Neurosci 13:171–182CrossRefPubMedGoogle Scholar
  8. Dowd LA, Coyle AJ, Rothstein JD, Pritchett DB, Robinson MB (1996) Comparison of Na+-dependent glutamate transport activity in synaptosomes, C6-glioma, and xenopus oocytes expressing excitatory amino acid carrier (EAAC1). Mol Pharmacol 49:465–473PubMedGoogle Scholar
  9. Gegelashvili G, Schousboe A (1997) High affinity glutamate transporters: regulation and expression and activity. Mol Pharmacol 52(1):6–15PubMedGoogle Scholar
  10. Gillessen T, Budd SL, Lipton SA (2002) Excitatory amino acid neurotoxicity. Adv Exp Med Biol 513:3–40PubMedGoogle Scholar
  11. Greenamyre JT, Toung AB (1989) Excitary amino acids and Alzheimer’s disease. Neurobiol Aging 10:593–602CrossRefPubMedGoogle Scholar
  12. Haghighat N (2005) Estrogen (17β-Estradiol) enhances glutamine synthetase activity in C6-glioma cells. Neurochem Res 30(5):661–667CrossRefPubMedGoogle Scholar
  13. Haghighat N, McCandless DW (1997a) Effect of 6-ammoniocotinamide on metabolism of astrocytes and C6-glioma cells. Metab Brain Dis 12(1):29–45CrossRefPubMedGoogle Scholar
  14. Haghighat N, McCandless DW (1997b) Effect of ammonium chloride on energy metabolism of astrocytes and C6-glioma cells in vitro. Metab Brain Dis 12(4):287–298PubMedGoogle Scholar
  15. Haghighat N, McCandless DW, Geraminejad P (2000) Responses in primary astrocytes and C6-glioma cells to ammonium chloride and Dibutyryl Cyclic-AMP. Metab Brain Dis 15(2):151–162CrossRefPubMedGoogle Scholar
  16. Haghighat N, Oblinger MM, McCandless DW (2004) Cytoprotective effect of estrogen on ammonium Chloride-treated C6-glioma cells. Neurochem Res 29(7):1359–1364CrossRefPubMedGoogle Scholar
  17. Hertz L, Dringen R, Schouboe A, Robinson SR (1999) Astrocytes: glutamate producers for neurons. J Neurosci Res 57:417–428CrossRefPubMedGoogle Scholar
  18. Hosli E, Jurasin K, Ruhl W, Luthy R, Hosli L (2001) Colocalization of androgen, estrogen and cholinergic receptors on cultured astrocytes of rat Pharmacokinetic/pharmacodynamic models for corticosteroid receptor down-regulation and glutamine synthetase induction in rat skeletal muscle by a receptor/gene-mediated mechanism. J Pharmacol Exp Ther 288:720–728Google Scholar
  19. Imura T, Shimohama S, Kageyama T, Kimura J (1999) Selective induction of glial glutamate transporter GLT1 by hypertonic stress in C6 glioma cells. Biochem Biophys Res Commun 265:240–245CrossRefPubMedGoogle Scholar
  20. Kalandadze A, Wu Y, Robinson MB (2002) Protein kinase C activation decreases cell surface expression of the GLT1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486. J Biol Chem 277:45741–45750CrossRefPubMedGoogle Scholar
  21. Meldrum BS (2000) Glutamate as a neurotransmitter in the brain: review of physiology and pathology. J Nutr 130:1007S–1015SPubMedGoogle Scholar
  22. Mong JA, Blutstein T (2006) Estradiol modulation of astrocytic form and function: implication for hormonal control of synaptic communication. Neuroscience 138(3):967–75CrossRefPubMedGoogle Scholar
  23. Murphy A, Vines A, McBean GJ (2008) Stimulation of EAAC1 in C6 glioma cells by store-operated calcium influx. Biochemica et Biophysica Acta (BBA)-Biomembranes 1788(2):551–558CrossRefGoogle Scholar
  24. Norenberg MD, Martinez-Hernandez A (1979) Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res 161:303–310CrossRefPubMedGoogle Scholar
  25. Palos TP, Ramachandran B, Boado R, Howard BD (1996) Rat C6 and human astrocytic tumor cells express a neuronal type of glutamate transporter. Brain Res 37:297–303CrossRefGoogle Scholar
  26. Perry TL, Hansen S (1990) What excitotoxin kills striatal neurons in Huntington’s disease? Clues from neurochemical studies. Neurology 40:20–24PubMedGoogle Scholar
  27. Rothstein JD, Van Kammen M, Levy ASI, Martin LJ, Kancl RW (1995) Selective loss of glial glutamate transporter GLUT-1 in amyotrophic lateral sclerosis. Ann Neurol 38:73–84CrossRefPubMedGoogle Scholar
  28. Santagati S, Melcangi RC, Celotti F, Martini L, Maggi A (1994) Estrogen receptor is expressed in different types of glial cells in culture. J Neurochem 63:2058–2064CrossRefPubMedGoogle Scholar
  29. Shaked I, Ben-Dror I, Vardimon L (2002) Glutamine synthetase enhances the clearance of extracellular glutamate by the neural retina. J Neurochem 83:574–580CrossRefPubMedGoogle Scholar
  30. Shughrue PJ, Komm B, Merchenthaler J (1996) The distribution of estrogen receptor β mRNA in the rat hypothalamus. Steroids 61:678–681CrossRefPubMedGoogle Scholar
  31. Su JD, Zhong YP, Li XY, Wang JW, Chen YZ (2001) Expression of estrogen receptor (ER)—alpha and beta immunoreactivity in hippo campal cell cultures with special attention to GABA nrgic neurons. J Neurosci Res 65(5):396–402CrossRefPubMedGoogle Scholar
  32. Susarla BTS, Robinson MB (2008) Internalization and degradation of the glutamate transporter GLT1 in response to phorbol ester. Neurochem Int 52(4–5):709–722CrossRefPubMedGoogle Scholar
  33. Vanhoutte N, Hermans E (2008) Glutamate-induced glioma cell proliferation is prevented by functional expression of the glutamate transporter GLT1. FEBS Lett 582:1847–1852CrossRefPubMedGoogle Scholar
  34. Whetsell WO Jr, Shapira NA (1993) Neuroexcitation, excitotoxicity and neurological disease. Lab Invest 68:372–387PubMedGoogle Scholar
  35. Zhou J, Sutherland ML (2004) Glutamate transporter cluster formation in astrocytic processes regulates glutamate uptake activity. J Neurosci 24:6301–6306CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Cell Biology and AnatomyChicago Medical SchoolNorth ChicagoUSA

Personalised recommendations