[H-3] MDL-105,519, A HIGH-AFFINITY RADIOLIGAND FOR THE N-METHYL-D-ASPARTATE RECEPTOR-ASSOCIATED GLYCINE RECOGNITION SITE

MDL 105,519 arboxyethenyl)-4,6-dichloro-1H-indole-2-carboxylic acid] i s a potent ligand at the N-methyl-D-aspartate (NMDA) receptor-associat ed glycine recognition site and is a noncompetitive inhibitor of NMDA receptor-mediated responses in vitro and in vivo. For purposes of char acterizing its action at the glycine binding site, a halogenated analo g was reduced with tritium to form radiolabeled MDL 105,519. [H-3]MDL 105,519 bound to rat brain membranes with high affinity (K-d = 3.77 nM ) and capacity (B-max = 12.1 pmol/mg protein). Isolation of bound liga nd by filtration gave identical levels of specific binding as did cent rifugation techniques. The kinetics of the binding reaction were studi ed. Association was monophasic with K-on equal to 7.0 x 10(7) M(-1) mi n(-1). Dissociation was also monophasic with the K-off value calculate d from association experiments (0.257 min(-1)) being similar to that m easured directly in dissociation experiments (0.232 min(-1)). A kineti cally derived value for the equilibrium dissociation constant was calc ulated with the two values for K-off and the association rate constant . The respective values (K-d = 3.67 arid 3.31 nM, respectively) agreed well with that obtained from the saturation experiments. The pharmaco logy of the site labeled by [H-3]MDL 105,519 matched that of the glyci ne recognition site labeled by [H-3]glycine. A strong relationship exi sted between the pK(i) values of a series of glycine site agonists, pa rtial agonists and antagonists obtained by use of these two radioligan ds (r = 0.90; P < .0005; slope = 0.997). No effect on specific binding of [H-3]MDL 105,519 was observed with ligands (10 mu M) interacting w ith other sites on the NMDA receptor complex or with non-NMDA glutamat e recognition sites.