In Vitro Receptor Binding Characteristics of the New Dopamine D₂ Antagonist [¹²⁵I]NCO-298: Methodological Considerations of High Affinity Binding

Abstract

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 (K_d = 20 pmol/l), long incubation time (4 hrs at 30°C) and low receptor concentration (= 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-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, [¹²⁵I]NCQ-298 is an excellent high-affinity D₂ receptor radioligand which labels the same receptor population as [³H]raclopride (B_max values; 32 ± 3 and 36 ± 1 pmol/g w.w., respectively).