MDL 105519

Complex polyamine effects on [3H]MDL 105,519 binding to the NMDA receptor glycine site

Several studies have suggested that polyamines modulate the interaction of glycine with the NMDA receptor. We have further investigated the effects of polyamines using the NMDA receptor glycine site antagonist [(E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1H-indole-2-carbox ylic acid] ([3H]MDL 105,519). [3H]MDL 105,519 binding assays were performed using well washed membranes prepared from frozen rat brains. The polyamines spermine and spermidine increased the fraction of non-specific binding (determined by the addition of 1 mM glycine) in the [3H]MDL 105,519 binding assay from 40-60% when spermine or spermidine concentration was increased from 1 to 100 microM. Polyamine agonists spermine, spermidine and 1,5-(diethylamino)piperidine (30 microM) did not have a significant effect on displacement of [3H]MDL 105,519 binding by glycine or glycine site antagonists. Similarly, the polyamine antagonist arcaine did not have a significant effect on displacement of [3H]MDL 105,519 binding by glycine or glycine site antagonists. However, spermidine significantly depressed the potency of MDL 105,519 in displacing [3H]dizocilpine binding. These data suggest that [3H]MDL 105,519 may preferentially bind to a polyamine insensitive form of the NMDA receptor.

[3H]MDL 105,519 binds with equal high affinity to both assembled and unassembled NR1 subunits of the NMDA receptor

[3H]MDL 105,519 (((E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1[3H]-indole-2-ca rboxylic acid) is a novel radioligand which binds with high affinity, Kd = 2.5 nM, to the glycine site of adult rodent forebrain, N-methyl-D-aspartate subtype of glutamate receptors. As with other glycine site antagonists, the major determinants for high-affinity binding of [3H]MDL 105,519 resides upon the NRI subunit, and not the NR2 subunits. [3H]MDL 105,519 binds with equal affinity, Kd = 3 nM, to both NR1-1a or NR1-4b splice variants, as well as the NRI-1a/NR2A receptor expressed in human embryonic kidney (HEK) 293 cells. One percent Triton X-100/1 M NaCl solubilises with a recovery of 15+/-3%, a mixed pool of assembled and unassembled forebrain NR1 subunit polypeptides. In this preparation, the recovery of [3H]MK801 ((+)-5-[3H]methyl-10,11-dihydrodibenzo[alpha,d]cyclohepten-5 ,10-imine binding activity (7+/-1%) reflects the amount of assembled NR1 subunits whereas [3H]MDL 105,519 binds quantitatively, with a recovery of 19+/-4% and Kd = 3 nM, to both assembled and unassembled NRI subunits. Therefore, [3H]MDL 105,519 should prove a useful ligand, in conjunction with immunopurification approaches, to address the question of NMDA receptor subunit stoichiometry.

[3H]MDL 105,519, a high-affinity radioligand for the N-methyl-D-aspartate receptor-associated glycine recognition site

MDL 105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4, 6-dichloro-1H-indole-2-carboxylic acid] is a potent ligand at the N-methyl-D-aspartate (NMDA) receptor-associated glycine recognition site and is a noncompetitive inhibitor of NMDA receptor-mediated responses in vitro and in vivo. For purposes of characterizing its action at the glycine binding site, a halogenated analog was reduced with tritium to form radiolabeled MDL 105,519. [3H]MDL 105,519 bound to rat brain membranes with high affinity (Kd = 3.77 nM) and capacity (Bmax = 12.1 pmol/mg protein). Isolation of bound ligand by filtration gave identical levels of specific binding as did centrifugation techniques. The kinetics of the binding reaction were studied. Association was monophasic with Kon equal to 7.0 x 10(7) M-1.min-1. Dissociation was also monophasic with the Koff value calculated from association experiments (0.257 min-1) being similar to that measured directly in dissociation experiments (0.232 min-1). A kinetically derived value for the equilibrium dissociation constant was calculated with the two values for Koff and the association rate constant. The respective values (Kd = 3.67 and 3.31 nM, respectively) agreed well with that obtained from the saturation experiments. The pharmacology of the site labeled by [3H]MDL 105,519 matched that of the glycine recognition site labeled by [3H]glycine. A strong relationship existed between the pKi values of a series of glycine site agonists, partial agonists and antagonists obtained by use of these two radioligands (r = 0.90; P < .0005; slope = 0.997). No effect on specific binding of [3H]MDL 105,519 was observed with ligands (10 microM) interacting with other sites on the NMDA receptor complex or with non-NMDA glutamate recognition sites.

Pharmacological characterization of MDL 105,519, an NMDA receptor glycine site antagonist

MDL 105,519, (E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1 H-indole-2-carboxylic acid, is a potent and selective inhibitor of [3H]glycine binding to the NMDA receptor. MDL 105,519 inhibits NMDA (N-methyl-D-aspartate)-dependent responses including elevations of [3H]N-[1,(2-thienyl)cyclohexyl]-piperidine ([3H]TCP) binding in brain membranes, cyclic GMP accumulation in brain slices, and alterations in cytosolic CA2+ and NA(+)-CA2+ currents in cultured neurons. Inhibition was non-competitive with respect to NMDA and could be nullified with D-serine. Intravenously administered MDL 105,519 prevented harmaline-stimulated increases in cerebellar cyclic GMP content, providing biochemical evidence of NMDA receptor antagonism in vivo. This antagonism was associated with anticonvulsant activity in genetically based, chemically induced, and electrically mediated seizure models. Anxiolytic activity was observed in the rat separation-induced vocalization model, but muscle-relaxant activity was apparent at lower doses. Higher doses impair rotorod performance, but were without effect on mesolimbic dopamine turnover or prepulse inhibition of the startle reflex. This pattern of activities differentiates this compound from (5R,10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) and indicates a lower psychotomimetic risk.

Binding of the radiolabeled glycine site antagonist [3H]MDL 105,519 to homomeric NMDA-NR1a receptors

We have characterized the binding of [3H]MDL 105,519 ((E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1 H-indole-2-carboxylic acid), a NMDA receptor glycine recognition site antagonist, to homomeric NMDA subunit 1a (NR 1a) receptors. Chinese hamster ovary cells (CHO-K1) were transfected with the rat NR 1a gene and cell lines stably expressing the receptor were isolated from amongst clones resistant to the neomycin analog G418. Saturation analysis indicated that the radioligand bound to the homomeric receptor with a similar high affinity (Kd = 1.8 nM) to that reported for the native receptor. The binding capacity (Bmax) was 370 fmol/mg protein reflecting approximately 110000 receptors per cell. The radioligand interacted with a single class of binding sites as indicated by linear Scatchard transformation of the saturation data and a unitary Hill slope in competition experiments. Thus, the MDL 105,519 recognition site is present on the NR 1a subunit and has similar radioligand binding properties to the native brain-derived receptor. However, pharmacologic characterization of [3H]MDL 105,519 binding indicated that agonists were weaker competitors at the homometric receptor relative to the native receptors. In contrast, representative of three distinct chemical classes of glycine site antagonists exhibited similar potencies at both types of binding sites.