title: Characterization of the binding of [3H]L-689,560, an antagonist for the glycine site on the N-methyl-D-aspartate receptor, to rat brain membranes.
abstract: The binding characteristics of [3H]L-689,560 [(+/-)-4-(trans)-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino -1,2,3,4- tetrahydroquinoline], a selective antagonist for the glycine site on the N-methyl-D-aspartate receptor, have been evaluated using rat cortex/hippocampus P2 membranes. Specific [3H]L-689,560 binding was saturable, having a Kd of 2.97 nM and a Bmax of 4.15 pmol/mg of protein. The Bmax value was not significantly different from that obtained for [3H]glycine in the same membrane preparation, and L-689,560 and glycine were found to be mutually competitive. The specific binding of [3H]L-689,560 (1 nM) represented 96 +/- 0.02% (four experiments) of total binding. Association experiments at 4 degrees revealed that [3H]L-689,560 reached equilibrium in 120 min, with a t1/2 of 40 min. The dissociation of [3H]L-689,560 was slow at 4 degrees (t1/2 = 118 min), allowing the use of filtration to separate free from bound radioactivity. Both association and dissociation curves were best fitted by a double-exponential function, suggesting the presence of two components. Comparison of IC50 values obtained using [3H]glycine and [3H]L-689,560 binding for 21 glycine site ligands (including agonists, partial agonists, and antagonists, with affinities spanning 5 orders of magnitude) showed a 1:1 correlation, with a correlation coefficient of 0.97. This suggests that efficacy does not have a large influence on the affinity of glycine site ligands when an agonist or antagonist radioligand is used. Ligands for other amino acid recognition sites did not directly inhibit [3H]L-689,560 binding. [3H]L-689,560 is an improved radioligand for the glycine site that will facilitate further investigations of its properties.
authors.affiliation: Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, UK.
authors.forename: S
authors.initials: S
authors.lastname: Grimwood
authors.forename: A M
authors.initials: AM
authors.lastname: Moseley
authors.forename: R W
authors.initials: RW
authors.lastname: Carling
authors.forename: P D
authors.initials: PD
authors.lastname: Leeson
authors.forename: A C
authors.initials: AC
authors.lastname: Foster
mesh_terms: D000634:Aminoquinolines / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D005998:Glycine / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008297:Male; D011804:Quinolines; D051381:Rats; D011919:Rats, Inbred Strains; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014316:Tritium
chemical_list: D000634:Aminoquinolines; D002413:Cations, Divalent; D007473:Ion Channels; D011804:Quinolines; D016194:Receptors, N-Methyl-D-Aspartate; D014316:Tritium; C074617:4-trans-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline; D005998:Glycine