NMDA receptor antagonists that bind to the strychnine-insensitive glycine site and inhibit NMDA-induced Ca2+ fluxes and [3H]GABA release.

We have examined the actions of putative antagonists of the strychnine-insensitive glycine-mediated modulation of the N-methyl-D-aspartate (NMDA) receptor using [3H]MK801 binding, Ca2+ influx and [3H]GABA release assays. Kynurenic acid and HA-966 inhibited [3H]MK801 binding, NMDA and glycine induced Ca2+ influx measured using fura-2 and NMDA and glycine simulated [3H]GABA release. The effects of kynurenic acid could be partially overcome by the addition of excess glutamate and glycine, indicating limited selectivity for the glycine binding site. In addition, a component of the action of kynurenic acid was insensitive to agonist concentration, indicating a third action of kynurenic acid at high concentrations. In contrast, HA-966 was 100-fold selective for the glycine compared to the NMDA site. HA-966 only partially inhibited [3H]MK801 binding (IC50 19.7 microM), NMDA-induced Ca2+ influx and neurotransmitter release. The failure of HA-966 to completely block NMDA responses, even at high concentrations, suggests that glycine may not be an absolute requirement for the activation of NMDA receptors under these experimental conditions.