STUDIES OF THE BIOGENIC-AMINE TRANSPORTERS .3. DEMONSTRATION OF 2 BINDING-SITES FOR [H-3] GBR12935 AND [H-3] BTCP IN RAT CAUDATE MEMBRANES

The present study addressed the hypothesis that there exist multiple s ites/states associated with the dopamine (DA) transporter ligands. The authors used [H-3](1-[2-(diphenylmethoxy) ethyl]-4-(3-phenylpropyl)pi perazine) (GBR12935) and [H-3]-N-{1-(2-benzo[b]thiophenyl)cyclohexyl } piperidine ([H-3]BTCP) to label binding sites present in striatal memb ranes and conducted experiments under nearly identical assay condition s, i.e., 18- to 24-hr incubations at 4 degrees C in 55.2 mM sodium pho sphate buffer, pH 7.4, with a protease inhibitor and antioxidant cockt ail. To obtain data suitable for quantitative curve fitting, it was ne cessary to repurify the [H-3]ligands periodically by high-performance liquid chromatography. Under these conditions, greater than 90% specif ic binding was observed. The method of binding surface analysis was us ed to characterize the interaction of GBR12935, BTCP, mazindol and 2 b eta-carbomethoxy-3 beta-(4-fluorophenyl) tropane with binding sites la beled by the [H-3]ligands. Nonlinear least-squares curve fitting of th e data to one- and two-site binding models demonstrated that, for both [H-3]ligands, the two-site model fit the data far better than did the one-site model. The results indicated that [H-3]GBR12935 labeled two binding sites, with higher (GBR site 1) and lower affinity (GBR site 2 ) for BTCP (K-i values of 5.84 nM and 1394 nM). [H-3]BTCP labeled two sites with high affinity (K-d values of 9.3 nM and 6.3 nM) at which GB R12935 also had high affinity (K-i values of 8.9 nM and 0.98 nM). Intr astriatal 6-hydroxy-DA lesions decreased the density of both [H-3]GBR1 2935 binding sites but affected site 1 much more than site 2, which in dicated that a greater portion of GBR site 1 is localized on striatal nerve terminals. By contrast, the 6-hydroxy-DA lesions decreased BTCP site 2 without significantly decreasing BTCP site 1, which indicated t hat BTCP site 1 is not located on DA nerve terminals. The i.c.v. admin istration of 5,7,- dihydroxytryptamine did not decrease GBR site 1 or 2, which indicated that neither is located on serotonin striatal nerve terminals. Viewed collectively with other reports, these data support the hypothesis that DA transporter ligands label multiple binding sit es in caudate membranes. The identification of selective agents for th ese sites may be valuable tools for identifying drugs that might modul ate the effects of cocaine.