[3H]Fluphenazine Binding to Brain Membranes: Simultaneous Measurement of D‐1 and D‐2 Receptor Sites

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Abstract
[3H]Fluphenazine was used to label both D‐1 and D‐2 dopamine receptors in mouse striatal membranes. The D‐1 and D‐2 specific binding of [3H]fluphenazine was discriminated by the dopamine antagonists SCH‐23390 (D‐1 selective) and spiperone (D‐2 selective). Saturation analyses of these two sites yielded a D‐1 receptor density in mouse striatum of 1,400 fmol/mg of protein and a D‐2 receptor density of 700 fmol/mg of protein. The affinity of [3H]fluphenazine for the D‐2 site was slightly greater than for the D‐1 site; the equilibrium dissociation constant (KD) was 0.7 versus 3.2 nM, respectively. Assay conditions are described that reduce nonspecific binding of [3H]fluphenazine to acceptable levels (35% of total binding at 1 nM [3H]fluphenazine). By comparison of displacement curves from a series of dopaminergic and nondopaminergic ligands, the pharmacological specificity of [3H]fluphenazine binding in mouse striatum was demonstrated to be dopaminergic. Only small amounts of dopamine‐specific (apomorphine‐sensitive) [3H]fluphenazine binding were found in other brain regions. However, chlorpromazine displaced considerable [3H]fluphenazine from all brain regions, including cerebellum, suggesting the presence of a [3H]fluphenazine binding site with a phenothiazine specificity.