The glutamate transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate indirectly evokes NMDA receptor mediated neurotoxicity in rat cortical cultures.
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 microM. In astrocyte-poor cultures, an EC50 for PDC of 50 +/- 5 microM 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.
Blitzblau
R
R
Department of Neurology, Children's Hospital, Boston, MA 02115, USA.
Gupta
S
S
Djali
S
S
Robinson
M B
MB
Rosenberg
P A
PA
D000818:Animals; D001253:Astrocytes / Q000187:drug effects; D002452:Cell Count / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects*; D003998:Dicarboxylic Acids / Q000494:pharmacology*; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D009498:Neurotoxins / Q000494:pharmacology*; D011759:Pyrrolidines / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists*; D013237:Stereoisomerism
D003998:Dicarboxylic Acids; D018690:Excitatory Amino Acid Agonists; D009498:Neurotoxins; D011759:Pyrrolidines; D016194:Receptors, N-Methyl-D-Aspartate; C083486:pyrrolidine-2,4-dicarboxylic acid