IN-VITRO RECEPTOR-BINDING CHARACTERISTICS OF THE NEW DOPAMINE D-2 ANTAGONIST [I-125] NCQ-298 - METHODOLOGICAL CONSIDERATIONS OF HIGH-AFFINITY BINDING

The substituted benzamide [I-125]NCQ-298 is a recently developed radioligand that has been shown to bind with high affinity and selectivity to dopamine D-2 receptors. The present studies were designed to optimize the in vitro receptor binding method of [I-125]NCQ-298 and to determine whether it labels the same receptor population as the widely used benzamide [H-3]raclopride. Rat striatal D-2 receptors and cloned human D-2 and D-3 receptors were used. We found that due to the high affinity of [I-125]NCQ-298 (K-d approximate to 20 pmol/l), long incubation time (4 hrs at 30 degrees C) and low receptor concentration (approximate to 2 pmol/l) were necessary in order to reach equilibrium and avoid ligand depletion. The optimal composition of the incubation buffer for rat striatal [I-125]NCQ-298 binding assays was (in mM): 50 Tris-HCl, 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, [I-125]NCQ-298 is an excellent high-affinity D-2 receptor radioligand which labels the same receptor population as [H-3]raclopride (B-max values; 32 +/- 3 and 36 +/- 1 pmol/g w.w., respectively).