Oxidative neuronal death caused by glutamate uptake inhibition in cultured hippocampal neurons

J Neurosci Res. 2003 Mar 1;71(5):679-88. doi: 10.1002/jnr.10510.


Glutamate transporters are coupled with cystine/glutamate antiporters to supply cystine as a component of glutathione, an important antioxidant. We sought evidence that L-trans-pyrrolidine-2,4-dicarboxylate (PDC) enhances glutamate-induced neuronal damage not only via the N-methyl-D-aspartate (NMDA) receptor mediated pathway, but also through induction of oxidative stress. Cultured hippocampal cells were exposed to glutamate (100 microM) for 5 min, washed and incubated for 18 hr with PDC (200 microM). PDC, increasing the neuronal death to 147% of that induced by glutamate alone, depleted glutathione in the culture, and produced dichloro-dihydro-fluorescein-diacetate-positive reactive oxygen species in neurons. N-acetylcysteine (2 mM) not only reduced PDC-enhanced neuronal death but also recovered glutathione and abolished the reactive oxygen species in these neurons. Threo-beta-benzyloxyaspartate, another type of glutamate transporter inhibitor, also induced glutathione depletion in the glutamate-preloaded cells, suggesting the involvement of glutamate transporter blocking in glutathione depletion. The NMDA receptor antagonist MK-801, although partially effective in reducing PDC toxicity, slightly recovered glutathione level but did not reduce the reactive oxygen species even at a high concentration (100 microM). N-acetylcysteine, dimethylsulfoxide, alpha-phenyl-N-butyl nitrone and glutathione ethylester prevented neuronal death enhanced by PDC, but superoxide dismutase and catalase did not. Our study provides evidence that the block of glutamate uptake by PDC exerts toxicity on glutamate-pretreated neurons not only through the accumulation of extracellular glutamate and subsequent activation of the NMDA receptor but also through depletion of glutathione and generation of reactive oxygen species.

MeSH terms

  • Acetylcysteine / pharmacology
  • Amino Acid Transport System X-AG / antagonists & inhibitors
  • Animals
  • Antioxidants / pharmacology
  • Aspartic Acid / pharmacology
  • Cell Death / drug effects
  • Cell Death / physiology
  • Cells, Cultured
  • Dicarboxylic Acids / antagonists & inhibitors
  • Dicarboxylic Acids / pharmacology
  • Dizocilpine Maleate / pharmacology
  • Drug Interactions
  • Excitatory Amino Acid Antagonists / pharmacology
  • Fluorescent Dyes
  • Glutamic Acid / metabolism
  • Glutamic Acid / pharmacokinetics*
  • Glutathione / deficiency
  • Glutathione / metabolism
  • Hippocampus / cytology*
  • Neurons / cytology
  • Neurons / drug effects
  • Neurons / metabolism*
  • Neurotransmitter Uptake Inhibitors / pharmacology
  • Oxidative Stress / drug effects
  • Oxidative Stress / physiology*
  • Pyrrolidines / antagonists & inhibitors
  • Pyrrolidines / pharmacology
  • Rats
  • Rats, Wistar
  • Reactive Oxygen Species / metabolism


  • Amino Acid Transport System X-AG
  • Antioxidants
  • Dicarboxylic Acids
  • Excitatory Amino Acid Antagonists
  • Fluorescent Dyes
  • Neurotransmitter Uptake Inhibitors
  • Pyrrolidines
  • Reactive Oxygen Species
  • benzyloxyaspartate
  • Aspartic Acid
  • Glutamic Acid
  • Dizocilpine Maleate
  • pyrrolidine-2,4-dicarboxylic acid
  • Glutathione
  • Acetylcysteine