Characteristics of 1251-lodocyanopindolol Binding to 8-Adrenergic and Serotonin-1B Receptors of Rat Brain: Selectivity of 19-Adrenergic Agents

The present study was designed to examine the specificity of (3-adrenergic antagonists for al-, 192-adrenergic and 5HT1B-serotonergic receptors by the compe titive interaction with 1251-iodocyanopindolol (1251-ICYP) as a radioligand. The 81 -adrenoceptors were preferred by acebutolol, atenolol, betaxolol, practolol, and I-, dl and d-metoprolol, while butoxamine and ICI-118,551 preferred 82-adre noceptors. The selectivities of these 81 and (32-antagonists are well-known, but alprenolol which is known as a non-selective antagonist was 7.2-fold more selective for the (32-adrenoceptors in the present study. All (3-antagonists used were more selective towards ,3-adrenoceptors as compared with 5HT1B-receptors. Good correlations were observed between the potencies of 19-adrenoceptor antagonists for inhibition of 1251-ICYP binding to 81 and /32-adrenoceptor sites and their potencies for inhibiting the binding of the same radioligand to 5HT1B-serotonergic receptor sites. These results suggest that 19-adrenoceptor antagonists can bind to /3-adrenoceptors and 5 HT1 B-receptors. In our previous reports (1-3), the Scat chard plots of 1251-iodocyanopindolol (1251 ICYP) binding to (3-adrenoceptors in the rat cerebral cortex were shown to be biphasic and suggested that the high and low-affinity sites in the biphasic Scatchard plots ap parently correspond to (3 and 5HT1B-re ceptors, respectively. Furthermore, we found that the selectivities of 1251-ICYP were in the order of (32'>-(31 >5HT1 B-receptors when dis placement experiments were done with the competitor I-metoprolol (3). In addition, we found that some (9-adrenoceptor antagonists displaced 1251-ICYP bound to 5HT1B-sero tonergic receptors (3). Thus, the present study was designed to examine the preference of j9 adrenoceptor antagonists for (31-, (32-adreno ceptors and 5HT1B-serotonergic receptors by radioligand binding assay using displacement experiments. Materials and Methods Materials: (-)-1251-ICYP (2200 Ci/mmole) was purchased from New England Nuclear Co., Ltd. Preparation of the membrane-enriched fractions: The membrane-enriched fraction from the rat cerebral cortex was prepared by using the previously described method (1). The membrane-enriched fraction was frozen in liquid nitrogen, stored at -80'C and diluted to appropriate concentrations immediately before use. Protein concentrations were deter mined by the methods of Lowry et al. (4) using bovine serum albumin as the standard. Binding assay: (a) Direct Ki determina tions of the (3-antagonists for the three binding sites (311-, (32 and 5HT1B receptors) were carried out by duplicate displacement experiments with 20 pM of 1251-ICYP in the presence of various concentrations of ,3 antagonists such as I-metoprolol. The non specific binding was determined in the presence of 10 W I-propranolol. The ap parent percentage of 31-, (32 and 5HT1B receptors observed in this experiment were 32, 19 and 50% of the binding sites, respec tively. (b) K; determinations for the 8-antagonists of 5HT1B-receptors by displacement experi ments was also carried out in duplicate with 20 pM of 251-ICYP in the presence of various concentrations of /3-antagonists such as I metoprolol. These experiments were carried out in the presence of 300 /M I-metoprolol because of the complete inhibition of 1251 ICYP binding to 8-adrenoceptors as de scribed previously (3). Incubation was started by the addition of 0.25 ml of membrane suspension (0.1 mg of protein) from rat cerebral cortex for 60 min in a total volume of 0.5 ml containing 60 mM Tris-HCI and 20 mM MgCl2 (pH 7.2). At the end of the incubation period, the sample was immediately filtered through a GF/C glass fiber filter using our improved method (5). The radioactivity was counted with an auto well gamma counter (Aloka ARC-500). The difference in mean values between the total and non-specific binding was taken as the specific binding. Kinetic analyses: All kinetic analyses were carried out on an NEC PC-9801 computer with iterative non-linear regression (1-3, 6). The previous results (3) showed that 1251 ICYP bound to a1-, 82 and 5HT1B-receptors with preferences in the order of 82>81 > 5HT1B-receptors. In the present study, the displacement curves were used to analyze the three binding site model, using the following equation: where either Bi and Bo are the concentrations of the radioligand bound with or without the cold ligand, respectively; x and L are the con centration of cold ligand and radioligand, respectively; and Kit, Ki2 and Ki3 are the in hibition constants of a cold ligand for re ceptors 1, 2 and 3, respectively. LTA, LT2 and LT3 are the proportion of receptors 1, 2 and 3 (LT1+LT2+LT3=1), respectively: and Kdl, Kd2 and Kd3 are the dissociation constants between a radioligand and receptors 1, 2 and 3, respectively. When receptors 1, 2 and 3 respectively corresponded to 81-, 82 and 5HT1B-receptors, the values of LT, (0.05), LT2 (0.09), LT3 (0.86), Kdi (30.9 pM), Kd2 (44.4 pM), Kd3 (369 pM) were determined as described previously (3), and the determined values were substituted into this equation. By means of these substitutions, the Ki values for the three receptor sites: K;1 for 81 adrenoceptors, Ki2 for Q2-adrenoceptors, K;3 for 5HT1B-receptors were directly determined by this equation. In addition, the determinations of Ki values of various drugs for 5HT1B-receptors were also carried out using the one receptor-one ligand model as follows: where IC50 is an apparent inhibition con stant (concentration of the cold ligand which inhibits the radioligand binding by 50%). K; was determined by the equation of Cheng and Prusoff as follows: