Kinetic and pharmacological profiles of the in vitro binding of the potent dopamine agonist [ 3H]N,N-dipropyl-5,6-dihydroxy-2-aminotetralin to rat striatal membranes

The in vitro binding of the new tritiated dopaminergic ligand [ 3H]N,N-dipropyl-5,6-dihydroxy-2-aminotetralin to rat striatal membranes is described. Binding assays were performed in 50 mM Tris-HCl pH 7.5 containing 1 mM EDTA and 0.01% ascorbic acid at 25°C for 30 min. Specific binding at 0.5 nM [ 3H]DP-5,6-ADTN and 5 mg/ml membrane suspension was very high (87–93%) and was found to be linearly related with homogenate concentration over the range of 0.5–10 mg/ml (r = 0.9968). (+)Butaclamol 1 μM was used to define specific binding. Specific binding disappeared completely within 20 min when the tissue was incubated at 55°C. Association and dissociation curves (obtained after addition of 1 μM(−)-DP-5,6-ADTN) were converted to linear logarithmic plots and the kinetic constants were computed (k 1 = 0.054 min −1 and k −1 = 0.0188 min −1) as were the t 1 2 for association (3.52 min) and dissociation (20.8 min). This resulted in an apparent K D of 0.34 nM. Increasing concentrations of [ 3H]DP-5,6-ADTN (0.05–3.0 nM) were found to saturate the receptor site. Linear transformation of the saturation curves and presentation of the curves as Eadie-Hofstee plots showed that only one set of receptors was labeled (n Hill was 0.995 ± 0.07) with a high affinity constant K D of 0.57 ± 0.10 nM and a maximum number of binding sites B max of 18.6 ± 2.4 pmol/g tissue. Various compounds were tested for their potency to displace the specific binding of [ 3H]DP-5,6-ADTN to striatal membranes (2.5 mg/ml). From the K i values it can be concluded that binding was selective for dopaminergic receptors and was stereoselective. The high affinity of both DA agonists and DA antagonists and the relative inactivity of the D-1 antagonists SCH 23390 and the D-1 agonist SK&F 38393 suggests that [ 3H]DP-5,6-ADTN labels the high affinity state of the D-2 receptor in rat striatal homogenates.