Dual effect of glycine on NMDA-induced neurotoxicity in rat cortical cultures

Abstract

To examine the roles of glycine in neurotoxicity caused by NMDA, primary rat cortical cultures were exposed to 100-300 μM NMDA plus glycine (0-3000 μM) or other glycine analogs in a simple saline solution, and toxicity was assessed by the amount of lactate dehydrogenase (LDH) released from the cultures. NMDA-induced neurotoxicity was abolished by 100 μM D-2-amino-5-phosphonovaleric acid (D-APV), phencyclidine (IC50, 4.1 μM), and Mg (IC50, 7.5 mM), or by reducing [Ca]o to 0.1 mM. NMDA-induced neurotoxicity could also be abolished by 7-chlorokynurenic acid (IC50, 8.6 μM), suggesting the presence of residual glycine in the culture medium (confirmed by high-performance liquid chromatography measurement). Moreover, in the presence of 30 μM 7-chlorokynurenic acid, glycine, D-serine, D-alanine, β-fluoro-D-alanine, and 1-aminocyclopropanecarboxylic acid could restore the neurotoxic action of NMDA, and their relative potencies and relative efficacies were the same as measured in electrophysiological assays in Xenopus oocytes or cultured neurons. The addition of >100 MM glycine doubled the excitotoxic effect of NMDA. The potency of glycine was low (EC50, 27 μM), and this effect was not due to a direct action on the NMDA receptor. The above-mentioned agonists were unable to substitute for glycine, even at high concentrations (1 mM). On the other hand, β-alanine, taurine, and GABA (1 mM) did potentiate NMDA neurotoxicity, and strychnine (IC50, 550 nM) could greatly reduce neurotoxicity in the presence of 1 mM glycine plus 300 μM NMDA. In addition, replacement of 67% of the bath chloride by methanesulfonate, which should limit chloride entry through amino acid-receptor channels, prevented the potentiation of neurotoxicity by glycine. These data are consistent with a requirement for glycine in NMDA neurotoxicity. They also suggest that the rise in cytoplasmic chloride concentration that follows activation of the "inhibitory" glycine (and GABA) receptors potentiates NMDA-induced neurotoxicity, perhaps by causing neuronal swelling or by diverting energy stores for chloride transport that would otherwise be used to clear cytosolic calcium. These findings emphasize a metabolic coupling between the NMDA receptor and inhibitory amino acid receptors.