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A characterization of [3H]ketanserin ([3H]KTS) binding in the frontal cortex (fCTX) and neostriatum (caudate-putamen, CPU) of rabbit was carried out to determine whether this ligand labels a non-serotoninergic receptor. The association and dissociation kinetics in fCTX were rapid, and could be fitted to two-site models, suggesting [3H]KTS is labeling two cortical sites. Using the serotonin-2 (5-HT2) antagonist mianserin to determine nonspecific binding, the saturation curves revealed a single high-affinity binding site. In contrast, when unlabeled ketanserin was used for nonspecific counts, the Scatchard plots were best fitted to a two-site model but the binding parameters of the high-affinity site were similar to that obtained in the presence of mianserin. The 5-HT2 antagonists mianserin, methysergide and ritanserin inhibited [3H]KTS binding in fCTX at nanomolar concentrations, however, the curves were best fitted to two-site models. In contrast, [3H]KTS binding to membrane preparations from the CPU could only be inhibited by high (micromolar) concentrations of these antagonists. Low micromolar concentrations of the monoamine uptake blockers GBR12909, desipramine, nomifensine, cocaine and fluoxetine competed with [3H]KTS in both fCTX and CPU. This study demonstrates that [3H]KTS labels a non-serotoninergic recognition site in the rabbit fCTX and CPU similar to that found in the rat neostriatum, i.e.: probably a monoamine transport site.

Receptor binding studies were performed in rabbit neostriatum (caudate-putamen) using the dopamine D2 antagonist [3H]raclopride. Treatment of the membrane preparations with the reducing agent L-dithiothreitol (L-DTT) as well as with the alkylating compoundN-ethylmaleimide (NEM), produced dose-dependent decreases of specific [3H]raclopride binding; the IC50 values were of 3.1 and 1.2 mM, respectively. Saturation experiments showed that the reduction of disulfide (-S-S-) bonds by L-DTT (1 mM) decreased the number of binding sites, with only a slight increase in the affinity. On the other hand, alkylation of sulfhydryl (-SH) groups by NEM (1mM) decreased both receptor number and affinity. The properties of the remaining binding sites were examined in competition curves with the physiological substrate dopamine and the dopaminergic antagonist (+)butaclamol. The IC50 values for (+)butaclamol in control and in L-DTT and NEM treated membranes were between 3.4 and 4.8 nM, with Hill coefficients (nH) of 1, indicating that the remaining binding sites conserved a high affinity for antagonist binding. In the case of dopamine, the curves were shallow (nH 0.45–0.64) and both compounds increased the IC50 from 0.7 µM (control) to 8 µM and 11 µM, for L-DTT and NEM respectively. Iterative analysis revealed that L-DTT produced a very important (>60%) decrease in the number of high-affinity (RH) binding. After NEM, there was a decrease in both the number of (RH) and the affinity (KH) of the high-affinity binding sites, and in the affinity (KL) of the low-affinity sites. These results demonstrate the participation of-S-S- and-SH groups in the agonist conformation of theprimary ligand recognition site of the dopamine D2 receptor. Alternatively,-S-S-and-SH groups could be related to the coupling of the primary ligand recognition protein with adenylate cyclase by means of an inhibitory type ofG protein.