title: Unravelling the complex dissociation of [(3)H]-rimonabant from plated CB(1) cannabinoid receptor-expressing cells.
abstract: The dissociation profile of the antagonist [(3)H]-rimonabant from recombinant CB(1) cannabinoid receptors expressed in plated HEK293 cells followed a complex pattern when measured in medium only. After a rapid decline, the specific binding levelled off at about 20% below the initial value. To unravel the responsible mechanism(s), we examined the relative contribution of binding to cells and walls of the culture wells respectively. Washout was also performed in the presence of an excess of unlabelled ligand and/or bovine serum albumin (BSA). The findings suggest that dissociated [(3)H]-rimonabant molecules not only undergo rebinding to the same or neighbouring receptors but also partition in the cell membranes and fix to the walls. As these non-receptor associations still occur in presence of unlabelled ligand, they can be erroneously regarded to represent 'specific binding'. While the unlabelled ligand was most effective in preventing receptor rebinding, BSA was most effective in preventing non-receptor associations. To measure receptor-dissociation only, washout is best performed in presence of unlabelled ligand and BSA or any other protein that can pick-up free radioligand molecules. Yet, washout in medium only could hint at mechanisms that affect the in vivo residence time of the drug in question.
authors.affiliation: AstraZeneca R&D, Mölndal, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Wennerberg
authors.forename: A
authors.initials: A
authors.lastname: Balendran
authors.forename: J C
authors.initials: JC
authors.lastname: Clapham
authors.forename: Georges
authors.initials: G
authors.lastname: Vauquelin
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D010880:Piperidines / Q000378:metabolism*; D011485:Protein Binding; D011720:Pyrazoles / Q000378:metabolism*; D011869:Radioligand Assay / Q000379:methods*; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism*; D000077285:Rimonabant; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D012710:Serum Albumin, Bovine; D000077285:Rimonabant