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-foled selective for the glycine compared to the NMDA site. HA-966 only partially inhibited [³H]MK801 binding (IC₅₀ 19.7 μM), 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.