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The Glutamate Transport Inhibitor L-trans-pyrrolidine-2,4-dicarboxylate Indirectly Evokes NMDA Receptor Mediated Neurotoxicity in Rat Cortical Cultures

Authors

  • Rachel Blitzblau,

    1. Department of Neurology and Program in Neuroscience, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA
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  • Shalini Gupta,

    1. Department of Neurology and Program in Neuroscience, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA
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  • Sina Djali,

    1. Departments of Pediatrics and Pharmacology, Children's Hospital and the University of Pennsylvania Medical School, Philadelphia, PA 19104, USA
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  • Michael B. Robinson,

    1. Departments of Pediatrics and Pharmacology, Children's Hospital and the University of Pennsylvania Medical School, Philadelphia, PA 19104, USA
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  • Paul A. Rosenberg

    Corresponding author
    1. Department of Neurology and Program in Neuroscience, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA
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Dr Paul A. Rosenberg, Enders Research Building, Department of Neurology, Children's Hospital, 300 Longwood Avenue, Boston, MA 02215, USA

Abstract

Because of the well-documented importance of glutamate uptake in protecting neurons against glutamate toxicity, we were interested in testing the effects of L-trans-pyrrolidine-2,4- dicarboxylate (PDC) on rat cortical cultures. This compound is a substrate for glutamate transporters and is a potent glutamate transport inhibitor that does not interact significantly with glutamate receptors. Using a 30 min exposure, and assessing neuronal survival after 20-24 h, PDC was neurotoxic in conventional astrocyte-rich cortical cultures, with an EC50 in these cultures of 320 ± 157 μM. In astrocyte-poor cultures, an EC50 for PDC of 50 ± 5 μM was determined. The neurotoxicity of PDC in both astrocyte-rich and astrocyte-poor cultures was blocked by the NMDA antagonist MK-801, but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). We tested the possibility that the neurotoxicity of PDC might be due to release of excitatory amino acids using several approaches. After pre-loading cells with the non-metabolizable analogue of glutamate, [3H]-D-aspartate, first we demonstrated that PDC caused significant efflux of [3H]-D-aspartate. This effect of PDC was dependent upon extracellular sodium. In contrast with glutamate neurotoxicity, PDC neurotoxicity was inhibited by removal of extracellular sodium. In the presence of 1 mM PDC, sodium caused neurotoxicity with an EC50 of 18 ± 7.6 mM. Tetrodotoxin had no effect on either PDC neurotoxicity or on PDC-evoked [3H]-D-aspartate release. PDC-evoked release of [3H]-D-aspartate was demonstrable in astrocyte cultures with no neurons present. PDC also evoked release of endogenous glutamate. Finally, the neurotoxicity of PDC was blocked by coincubation with glutamate-pyruvate transaminase plus pyruvate to degrade extracellular glutamate. These results demonstrate the neurotoxicity of PDC, and suggest that the mechanism of this toxicity is the glutamate transporter-dependent accumulation of glutamate in the extracellular space.

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