Biochemical and Pharmacological Characterization of [3H]GBR 12935 Binding In Vitro to Rat Striatal Membranes: Labeling of the Dopamine Uptake Complex

Abstract

Abstract: Binding of the selective dopamine (DA) uptake inhibitor [3 H]GBR 12935 to rat striatal membranes was characterized biochemically and pharmacologically. [3 H]‐GBR 12935 binding at 0°C was reversible and saturable and Scatchard analysis indicated a single binding site with a KD of 5.5 n M and a B max of 760 pmol/mg tissue. [3 H]GBR 12935 labeled two binding sites. One binding site was identified as the classic DA uptake site, since methylphenidate, cocaine, diclofensine, and Lu 19–005 potently inhibited [3 H]GBR 12935 binding to it. Binding to the second site was inhibited by high concentrations of the above compounds. IC 50 values for inhibition of [3 H]GBR 12935 binding to the DA uptake site were proportional to IC50 values for inhibition of DA uptake. However, substrates of DA uptake, e.g., DA and 1‐methyl‐4‐phenylpyridine, and DA releasers, e.g., the amphetamines, inhibited [3 H]GBR 12935 binding less than DA uptake. Rate experiments excluded the possibility that these “weak” inhibitors affected the binding by alloste‐ric coupled binding sites. The second binding site was not a noradrenergic, serotonergic, or GABAergic uptake site. Neither was it a dopaminergic, acetylcholinergic, histaminic, serotonergic, or adrenergic receptor. However, [3 H]GBR 12935 was potently displaced from it by disubstituted piper‐azine derivatives, i.e., flupentixol and piflutixol. DA uptake and the DA uptake binding site of [3 H]GBR 12935 were located primarily in the striatum, but the piperazine acceptor site was distributed uniformly throughout the brain. Also only the DA uptake binding site was destroyed by 6‐OH‐DA. Thus, [3 H]GBR 12935 labels the classic DA uptake site in rat striatum and also a piperazine acceptor site. Substrates for DA uptake and releasers of DA inhibited [3 H]GBR 12935 binding with low potency, but did not alter the rate constants for [3 H]GBR 12935 binding. Therefore inhibitors of DA uptake label the carrier site and prevent the carrier process.