In vitro receptor binding characteristics of the new dopamine D2 antagonist [125I]NCQ-298: methodological considerations of high affinity binding.
The substituted benzamide [125I]NCQ-298 is a recently developed radioligand that has been shown to bind with high affinity and selectivity to dopamine D2 receptors. The present studies were designed to optimize the in vitro receptor binding method of [125I]NCQ-298 and to determine whether it labels the same receptor population as the widely used benzamide [3H]raclopride. Rat striatal D2 receptors and cloned human D2 and D3 receptors were used. We found that due to the high affinity of [125I]NCQ-298 (Kd approximately 20 pmol/l), long incubation time (4 hrs at 30 degrees C) and low receptor concentration (approximately 2 pmol/l) were necessary in order to reach equilibrium and avoid ligand depletion. The optimal composition of the incubation buffer for rat striatal [125I]NCQ-298 binding assays was (in mM): 50 Tris-HCI, 120 NaCl, 5 KCl, 1 MgCl2, 0.01 pargyline, 0.1 EDTA, 0.05 protease inhibitors (PMSF and bacitracin) and 0.01% ascorbic acid. It is concluded that, when studied under correct experimental conditions, [125I]NCQ-298 is an excellent high-affinity D2 receptor radioligand which labels the same receptor population as [3H]raclopride (Bmax values; 32 +/- 3 and 36 +/- 1 pmol/g w.w., respectively).
Department of Neuropharmacology, CNS Preclinical R&D, Sodertalje, Sweden.
E
E
Jerning
A
A
Malmberg
N
N
Mohell
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000378:metabolism; D006224:Cricetinae; D018492:Dopamine Antagonists / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D020891:Raclopride; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000378:metabolism*; D012457:Salicylamides / Q000378:metabolism*
D018492:Dopamine Antagonists; D017448:Receptors, Dopamine D2; D012457:Salicylamides; D020891:Raclopride; C064246:NCQ 298