title: Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes.
abstract: We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.
authors.affiliation: Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, England. Sarah_Grimwood@Merck.com
authors.forename: S
authors.initials: S
authors.lastname: Grimwood
authors.forename: P
authors.initials: P
authors.lastname: Richards
authors.forename: F
authors.initials: F
authors.lastname: Murray
authors.forename: N
authors.initials: N
authors.lastname: Harrison
authors.forename: P B
authors.initials: PB
authors.lastname: Wingrove
authors.forename: P H
authors.initials: PH
authors.lastname: Hutson
mesh_terms: D000495:Allosteric Site / Q000187:drug effects; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive / Q000187:drug effects; D001923:Brain Chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008297:Male; D051379:Mice; D010880:Piperidines / Q000378:metabolism*; D016548:Prosencephalon / Q000378:metabolism; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D018765:Dopamine Uptake Inhibitors; C409466:NR1 NMDA receptor; C121001:NR2B NMDA receptor; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; C010739:ifenprodil