Glutathione peroxidase-1 contributes to the protection of glutamine synthetase in astrocytes during oxidative stress Article First Online: 06 February 2006 Received: 29 May 2005 Accepted: 12 September 2005 DOI:
Cite this article as: Knorpp, T., Robinson, S., Crack, P. et al. J Neural Transm (2006) 113: 1145. doi:10.1007/s00702-005-0389-y Summary.
Glutamine synthetase (GS) is an astrocytic enzyme that is essential for the glutamate–glutamine cycle between neurons and astrocytes. To measure the effects of oxidative stress on the activity of GS in astrocytes, astrocyte-rich primary cultures from the brains of wild-type and glutathione peroxidase-1 deficient mice (GPx1(−/−)) were exposed to a chronic hydrogen peroxide-generating system consisting of xanthine oxidase, hypoxanthine and superoxide dismutase. The specific activity of GS was strongly diminished by chronic exposure to hydrogen peroxide in astrocytes cultured from both mouse lines. After 60 min of oxidative stress in the presence of 5 mU/mL, 10 mU/mL and 20 mU/mL of xanthine oxidase, the specific GS activity of wild-type astrocytes was reduced to 47%, 22% and 13% of the initial activity, respectively. For all activities of xanthine oxidase applied, astrocytes from GPx1(−/−) mice experienced a significantly greater rate of GS inactivation compared to their wild-type counterparts. These results confirm that GS is sensitive to inactivation by chronic peroxide stress in viable astrocytes and show that glutathione peroxidase-1 helps to protect GS from inactivation by oxidative stress.
Keywords: Brain, glutamate–glutamine cycle, metabolism, oxidative stress. References Barnham, KJ, Masters, CL, Bush, AI 2004 Neurodegenerative diseases and oxidative stress Nat Rev Drug Discov 3 205 214 PubMed CrossRef Google Scholar Behrens, PF, Franz, P, Woodman, B, Lindenberg, KS, Landwehrmeyer, GB 2002 Impaired glutamate transport and glutamate–glutamine cycling: downstream effects of the Huntington mutation Brain 125 1908 1922 PubMed CrossRef Google Scholar Berr, C, Richard, MJ, Gourlet, V, Garrel, C, Favier, A 2004 Enzymatic antioxidant balance and cognitive decline in aging–the EVA study Eur J Epidemiol 19 133 138 PubMed CrossRef Google Scholar Boksha, IS, Tereshkina, EB, Burbaeva, GS 2000 Glutamine synthetase and glutamine synthetase-like protein from human brain: purification and comparative characterization J Neurochem 75 2574 2582 PubMed CrossRef Google Scholar Butterfield, DA, Hensley, K, Cole, P, Subramaniam, R, Aksenov, M, Aksenova, M, Bummer, PM, Haley, BE, Carney, JM 1997 Oxidatively induced structural alteration of glutamine synthetase assessed by analysis of spin label incorporation kinetics: relevance to Alzheimer’s disease J Neurochem 68 2451 2457 PubMed CrossRef Google Scholar Cooper, AJ, Lai, JC 1987 Cerebral ammonia metabolism in normal and hyperammonemic rats Neurochem Pathol 6 67 95 PubMed Google Scholar Crack, PJ, Taylor, JM, Flentjar, NJ, de Haan, J, Hertzog, P, Iannello, RC, Kola, I 2001 Increased infarct size and exacerbated apoptosis in the glutathione peroxidase-1 (Gpx-1) knockout mouse brain in response to ischemia/reperfusion injury J Neurochem 78 1389 1399 PubMed CrossRef Google Scholar Daikhin, Y, Yudkoff, M 2000 Compartmentation of brain glutamate metabolism in neurons and glia J Nutr 130 1026S 1031S PubMed Google Scholar De Haan, JB, Bladier, C, Griffiths, P, Kelner, M, O’Shea, RD, Cheung, NS, Bronson, RT, Silvestro, MJ, Wild, S, Zheng, SS, Beart, PM, Hertzog, PJ, Kola, I 1998 Mice with a homozygous null mutation for the most abundant glutathione peroxidase, Gpx1, show increased susceptibility to the oxidative stress-inducing agents paraquat and hydrogen peroxide J Biol Chem 273 22528 22536 PubMed CrossRef Google Scholar Derouiche, A, Heimrich, B, Frotscher, M 1993 Loss of layer-specific astrocytic glutamine synthetase immunoreactivity in slice cultures of hippocampus Eur J Neurosci 5 122 127 PubMed CrossRef Google Scholar Dodd, PR 2002 Excited to death: different ways to lose your neurones Biogerontology 3 51 56 PubMed CrossRef Google Scholar Dringen, R, Hamprecht, B 1997 Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells Brain Res 759 67 75 PubMed CrossRef Google Scholar Dringen, R, Kussmaul, L, Hamprecht, B 1998 Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay Brain Res Brain Res Protoc 2 223 228 PubMed CrossRef Google Scholar Dringen, R, Pawlowski, PG, Hirrlinger, J 2005 Peroxide detoxification by brain cells J Neurosci Res 79 157 165 PubMed CrossRef Google Scholar Felipo, V, Butterworth, RF 2002 Neurobiology of ammonia Prog Neurobiol 67 259 279 PubMed CrossRef Google Scholar Flentjar, NJ, Crack, PJ, Boyd, R, Malin, M, De Haan, JB, Hertzog, P, Kola, I, Iannello, R 2002 Mice lacking glutathione peroxidase-1 activity show increased TUNEL staining and an accelerated inflammatory response in brain following a cold-induced injury Exp Neurol 177 9 20 PubMed CrossRef Google Scholar Fraser, CM, Sills, GJ, Forrest, G, Thompson, GG, Brodie, MJ 1999 Effects of anti-epileptic drugs on glutamine synthetase activity in mouse brain Br J Pharmacol 126 1634 1638 PubMed CrossRef Google Scholar Fressinaud, C, Weinrauder, H, Delaunoy, JP, Tholey, G, Labourdette, G, Sarlieve, LL 1991 Glutamine synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors J Cell Physiol 149 459 468 PubMed CrossRef Google Scholar Gras, G, Chretien, F, Vallat-Decouvelaere, AV, Le Pavec, G, Porcheray, F, Bossuet, C, Leone, C, Mialocq, P, Dereuddre-Bosquet, N, Clayette, P, Le Grand, R, Creminon, C, Dormont, D, Rimaniol, AC, Gray, F 2003 Regulated expression of sodium-dependent glutamate transporters and synthetase: a neuroprotective role for activated microglia and macrophages in HIV infection? Brain Pathol 13 211 222 PubMed CrossRef Google Scholar Halliwell, B 2001 Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment Drugs Aging 18 685 716 PubMed CrossRef Google Scholar Hamprecht, B, Löffler, F 1985 Primary glial cultures as a model for studying hormone action Methods Enzymol 109 341 345 PubMed CrossRef Google Scholar Hansson, E, Ronnback, L, Persson, LI, Lowenthal, A, Noppe, M, Alling, C, Karlsson, B 1984 Cellular composition of primary cultures from cerebral cortex, striatum, hippocampus, brainstem and cerebellum Brain Res 300 9 18 PubMed CrossRef Google Scholar Hertz, L, Dringen, R, Schousboe, A, Robinson, SR 1999 Astrocytes: glutamate producers for neurons J Neurosci Res 57 417 428 PubMed CrossRef Google Scholar Hirrlinger, J, Hamprecht, B, Dringen, R 1999 Application and modulation of a permanent hydrogen peroxide-induced oxidative stress to cultured astroglial cells Brain Res Brain Res Protoc 4 223 229 PubMed CrossRef Google Scholar Ho, YS, Magnenat, JL, Bronson, RT, Cao, J, Gargano, M, Sugawara, M, Funk, CD 1997 Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia J Biol Chem 272 16644 16651 PubMed CrossRef Google Scholar Iqbal, K, Ottaway, JH 1970 Glutamine synthetase in muscle and kidney Biochem J 119 145 156 PubMed Google Scholar Khelil, M, Rolland, B, Fages, C, Tardy, M 1990 Glutamine synthetase modulation in astrocyte cultures of different mouse brain areas Glia 3 75 80 PubMed CrossRef Google Scholar Le Prince, G, Delaere, P, Fages, C, Lefrancois, T, Touret, M, Salanon, M, Tardy, M 1995 Glutamine synthetase (GS) expression is reduced in senile dementia of the Alzheimer type Neurochem Res 20 859 862 PubMed CrossRef Google Scholar Liddell, JR, Robinson, SR, Dringen, R 2004 Endogenous glutathione and catalase protect cultured rat astrocytes from the iron-mediated toxicity of hydrogen peroxide Neurosci Lett 364 164 167 PubMed CrossRef Google Scholar Liddell, JR, Hoepken, HH, Crack, PJ, Robinson, SR, Dringen, R 2005 Glutathione peroxidase-1 contributes to the rapid clearance, and protects against the iron-mediated toxicity, of hydrogen peroxide J Neurochem 94 92 Google Scholar Lowry, OH, Rosebrough, NJ, Farr, AL, Randall, RJ 1951 Protein measurement with the Folin phenol reagent J Biol Chem 193 265 275 PubMed Google Scholar Ma, YS, Chao, CC, Stadtman, ER 1999 Oxidative modification of glutamine synthetase by 2,2′-azobis(2-amidinopropane)dihydrochloride Arch Biochem Biophys 363 129 134 PubMed CrossRef Google Scholar Makino, N, Mochizuki, Y, Bannai, S, Sugita, Y 1994 Kinetic studies on the removal of extracellular hydrogen peroxide by cultured fibroblasts J Biol Chem 269 1020 1025 PubMed Google Scholar Meldrum, BS 2000 Glutamate as a neurotransmitter in the brain: review of physiology and pathology J Nutr 130 1007S 1015S PubMed Google Scholar Michaelis, ML 2003 Drugs targeting Alzheimer’s disease: some things old and some things new J Pharmacol Exp Ther 304 897 904 PubMed CrossRef Google Scholar Norenberg, MD 1979 Distribution of glutamine synthetase in the rat central nervous system J Histochem Cytochem 27 756 762 PubMed Google Scholar Norenberg, MD, Martinez-Hernandez, A 1979 Fine structural localization of glutamine synthetase in astrocytes of rat brain Brain Res 161 303 310 PubMed CrossRef Google Scholar Omar, RA, Chyan, YJ, Andorn, AC, Poeggeler, B, Robakis, NK, Pappolla, MA 1999 Increased expression but reduced activity of antioxidant enzymes in Alzheimer’s disease J Alzheimers Dis 1 139 145 PubMed Google Scholar Pike, CJ, Ramezan-Arab, N, Miller, S, Cotman, CW 1996 beta-Amyloid increases enzyme activity and protein levels of glutamine synthetase in cultured astrocytes Exp Neurol 139 167 171 PubMed CrossRef Google Scholar Pinteaux, E, Copin, JC, Ledig, M, Tholey, G 1996 Modulation of oxygen-radical-scavenging enzymes by oxidative stress in primary cultures of rat astroglial cells Dev Neurosci 18 397 404 PubMed Google Scholar Pow, DV, Robinson, SR 1994 Glutamate in some retinal neurons is derived solely from glia Neuroscience 60 355 366 PubMed CrossRef Google Scholar Rama Rao, KV, Jayakumar, AR, Norenberg, DM 2003 Ammonia neurotoxicity: role of the mitochondrial permeability transition Metab Brain Dis 18 113 127 PubMed CrossRef Google Scholar Robinson, SR 2000 Neuronal expression of glutamine synthetase in Alzheimer’s disease indicates a profound impairment of metabolic interactions with astrocytes Neurochem Int 36 471 482 PubMed CrossRef Google Scholar Robinson, SR 2001 Changes in the cellular distribution of glutamine synthetase in Alzheimer’s disease J Neurosci Res 66 972 980 PubMed CrossRef Google Scholar Santoro, JC, Harris, G, Sitlani, A 2001 Colorimetric detection of glutamine synthetase-catalyzed transferase activity in glucocorticoid-treated skeletal muscle cells Anal Biochem 289 18 25 PubMed CrossRef Google Scholar Sasaki, K, Bannai, S, Makino, N 1998 Kinetics of hydrogen peroxide elimination by human umbilical vein endothelial cells in culture Biochim Biophys Acta 1380 275 288 PubMed Google Scholar Schliess, F, Gorg, B, Fischer, R, Desjardins, P, Bidmon, HJ, Hermann, A, Butterworth, RF, Zilles, K, Haussinger, D 2002 Ammonia induced MK-801-sensitive nitration and phosphorylation of protein tyrosine residues in rat astrocytes FASEB J 16 739 741 PubMed Google Scholar Schor, NF 1988 Inactivation of mammalian brain glutamine synthetase by oxygen radicals Brain Res 456 17 21 PubMed CrossRef Google Scholar Taylor, JM, Iannello, RC, Hertzog, PJ, Crack, PJ 2005 Diminished Akt phosphorylation in neurons lacking Gpx1 leads to increased susceptibility to oxidative stress-induced cell death J Neurochem 92 283 293 PubMed CrossRef Google Scholar Tholey, G, Ghandour, MS, Bloch, S, Ledig, M, Mandel, P 1987 Glutamine synthetase and energy metabolism enzymes in cultivated chick neurons and astrocytes: modulation by serum and hydrocortisone Brain Res 428 73 81 PubMed Google Scholar Tiffany-Castiglioni, E, Roberts, JA, Sheeler-Gough, LV 1989 Reduction of glutamine synthetase specific activity in cultured astroglia by ferrous chloride J Neurosci Res 24 508 516 PubMed CrossRef Google Scholar Zwingmann, C, Butterworth, R 2005 An update on the role of brain glutamine synthesis and its relation to cell-specific energy metabolism in the hyperammonemic brain: further studies using NMR spectroscopy Neurochem Int 47 19 30 PubMed CrossRef Google Scholar