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Home , Arotinolol, Iodocyanopindolol

Characteristics of 1251-lodocyanopindolol Binding to 8- and -1B Receptors of Rat Brain: Selectivity of 19-Adrenergic Agents Hiroshi TSUCHIHASHI,Yasuo NAKASHIMA,Junji KINAMI and Takafumi NAGATOMO Departmentof Pharmacology,Niigata College of Pharmacy, 5-13-2Kamishin'ei-cho, Niigata 950-21, Japan AcceptedOctober 9, 1989

Abstract-The present study was designed to examine the specificity of (3-adrenergic antagonists for al-, 192-adrenergic and 5HT1B- receptors by the compe titive interaction with 1251- (1251-ICYP) as a radioligand. The 81 -adrenoceptors were preferred by , , , , and I-, dl and d-, while butoxamine and ICI-118,551 preferred 82-adre noceptors. The selectivities of these 81 and (32-antagonists are well-known, but 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 HT1B-receptors.

In our previous reports (1-3), the Scat Co., Ltd. chard plots of 1251-iodocyanopindolol (1251 Preparation of the membrane-enriched ICYP) binding to (3-adrenoceptors in the rat fractions: The membrane-enriched fraction cerebral cortex were shown to be biphasic and from the rat cerebral cortex was prepared by suggested that the high and low-affinity using the previously described method (1). sites in the biphasic Scatchard plots ap The membrane-enriched fraction was frozen parently correspond to (3 and 5HT1B-re in liquid nitrogen, stored at -80'C and diluted ceptors, respectively. Furthermore, we found to appropriate concentrations immediately that the selectivities of 1251-ICYP were in the before use. Protein concentrations were deter order of (32'>-(31>5HT1 B-receptors when dis mined by the methods of Lowry et al. (4) placement experiments were done with the using bovine serum albumin as the standard. competitor I-metoprolol (3). In addition, we Binding assay: (a) Direct Ki determina found that some (9-adrenoceptor antagonists tions of the (3-antagonists for the three displaced 1251-ICYP bound to 5HT1B-sero binding sites (311-, (32 and 5HT1B receptors) tonergic receptors (3). Thus, the present study were carried out by duplicate displacement was designed to examine the preference of j9 experiments with 20 pM of 1251-ICYP in the adrenoceptor antagonists for (31-, (32-adreno presence of various concentrations of ,3 ceptors and 5HT1B-serotonergic receptors by antagonists such as I-metoprolol. The non radioligand binding assay using displacement specific binding was determined in the experiments. presence of 10 W I-. The ap parent percentage of 31-, (32 and 5HT1B Materials and Methods receptors observed in this experiment were Materials: (-)-1251-ICYP (2200 Ci/mmole) 32, 19 and 50% of the binding sites, respec was purchased from New England Nuclear tively. (b) K; determinations for the 8-antagonists fiber filter using our improved method (5). of 5HT1B-receptors by displacement experi The radioactivity was counted with an auto ments was also carried out in duplicate with well gamma counter (Aloka ARC-500). The 20 pM of 251-ICYP in the presence of various difference in mean values between the total concentrations of /3-antagonists such as I and non-specific binding was taken as the metoprolol. These experiments were carried specific binding. out in the presence of 300 /M I-metoprolol Kinetic analyses: All kinetic analyses were because of the complete inhibition of 1251 carried out on an NEC PC-9801 computer ICYP binding to 8-adrenoceptors as de with iterative non-linear regression (1-3, 6). scribed previously (3). The previous results (3) showed that 1251 Incubation was started by the addition of ICYP bound to a1-, 82 and 5HT1B-receptors 0.25 ml of membrane suspension (0.1 mg of with preferences in the order of 82>81 > protein) from rat cerebral cortex for 60 min in 5HT1B-receptors. In the present study, the a total volume of 0.5 ml containing 60 mM displacement curves were used to analyze the Tris-HCI and 20 mM MgCl2 (pH 7.2). At the three binding site model, using the following end of the incubation period, the sample was equation: immediately filtered through a GF/C glass

where either Bi and Bo are the concentrations where IC50 is an apparent inhibition con of the radioligand bound with or without the stant (concentration of the cold ligand which cold ligand, respectively; x and L are the con inhibits the radioligand binding by 50%). K; centration of cold ligand and radioligand, was determined by the equation of Cheng and respectively; and Kit, Ki2 and Ki3 are the in Prusoff as follows: hibition constants of a cold ligand for re K;=IC50/(1+L/Kd) ceptors 1, 2 and 3, respectively. LTA, LT2 and The values of Kd (369 PM) were determined LT3 are the proportion of receptors 1, 2 and as described previously (3) and used in this 3 (LT1+LT2+LT3=1), respectively: and Kdl, study. Kd2 and Kd3 are the dissociation constants In these non-linear or linear regression between a radioligand and receptors 1, 2 and analyses, the parameter fitting method, termi 3, respectively. When receptors 1, 2 and 3 nation of iteration, and justification of the respectively corresponded to 81-, 82 and models were carried out by the methods 5HT1B-receptors, the values of LT, (0.05), described previously (1, 6). The values were LT2 (0.09), LT3 (0.86), Kdi (30.9 pM), Kd2 sometimes expressed as pK; (-log K;) values (44.4 pM), Kd3 (369 pM) were determined as in this report. described previously (3), and the determined values were substituted into this equation. By Results means of these substitutions, the Ki values The preference of (3-antagonists for 81 for the three receptor sites: K;1 for 81 and Q2-adrenergic receptors were initially adrenoceptors, Ki2 for Q2-adrenoceptors, K;3 examined by displacement analysis, and the for 5HT1B-receptors were directly determined values of pK; of 9-antagonists were directly by this equation. determined numerically by the model of three In addition, the determinations of Ki values binding sites. Table 1 summarizes the results of various drugs for 5HT1B-receptors were of (3-adrenergic antagonists for 81 and 82 also carried out using the one receptor-one adrenoceptors in 1251-ICYP binding to rat ligand model as follows: cerebral cortical membranes. Acebutolol, atenolol, betaxolol, practolol, and I-, dl and d-metoprolol bound Q1 -adrenoceptors selec tively, whereas butoxamine and ICI-118,551 Table 1. Selectivity of (3-adrenergic agents for 13-adrenergic receptors

Each value in parenthesis shows the number of experiments. Data are the mean values±S.E. Sig nificance of difference between values of pK; to (31 and (32-sites was determined by Student's t-test. A: P<0.05, B: P<0.02, C: P<0.01, D: P<0.001, and N: not significant. were selective towards Q2-adrenoceptors. In in the rat myocardium determined by the addition, alprenolol which is well-known as a binding assay using 3H-CGP12177 as a non-selective antagonist preferred 132-adreno radioligand, which were described previously ceptors. , , YM-09538, ox (7). A good correlation (pK; values for 131 prenolol, , , and d-, dl and I (r=0.97) and 132-adrenoceptors (r=0.96)) was propranolol were not selective towards 81 observed between the pK; values in this study and (32-adrenoceptors. The most (32-selective and those obtained in the previous study. antagonist was dl-metoprolol, and the most The relationship between the pK; values of 131-selective antagonist was ICI-1 18,551. these 13-adrenergic antagonists for 131 and Table 2 summarizes pK; values of these (3 / 2-adrenoceptors determined in this study adrenergic antagonists used for 5HT1B and their pK; values (A values in Table 2) for serotonergic receptors, determined by the 5HT1B-serotonergic receptors determined in three binding site model and the one receptor the present study is shown in Fig. 2. A good one ligand model carried out in the presence correlation (131 vs. 5HT1B; 0.87 and / 2 vs. of the (3-adrenoceptor competitor 30 ,uM I 5HT1B; 0.88) was also observed between the metoprolol. The values obtained from these pK; values for 131 and 132-adrenoceptors and two models were identical. All these 9 those for 5HT1B-receptors. adrenergic antagonists displaced 1251-ICYP binding to 5HT1B-receptors; arotinolol was Discussion the most potent of the drugs, and d-meto The selectivities of 1251-ICYP binding to prolol was the least potent. F31-, 132-adrenoceptors and 5HT1B-serotoner Figure 1 depicts the relationship between gic receptors were in the order of 132--'131 the K; values of these drugs for 131 and 132 5HT1B-receptors as described previously (3). adrenoceptors determined by the binding The preferences of 13-antagonists for 81-, 132 assays in the current study and the K; values and 5HT1B-receptors were assessed by the of these drugs for X31 and 132-adrenoceptors radioligand binding assay using 1251-ICYP as Table 2. pK; values of p-antagonists for 5HTla receptors

The pKi values of A and B are the results obtained by the displacement experiments using the one site model in the presence of 30 pM 1-metoprolol and the three site model, respectively. Each value in parenthesis shows the number of experiments. Data are the mean values±S.E. The pK; values of j3 antagonists to R, and 92-adrenergic receptors were those in Table 1, and these values were compared with the values (A) for 5HT1B-receptors. The values indicated by * were obtained from the report pub lished by Tsuchihashi et al. (3). a radioligand. When the preferences of these tivity towards 5HT,B-receptors, whereas the antagonists for 81 and Q2-adrenoceptors selective antagonists showed 33-8300-fold were compared, acebutolo!, atenolol, betax higher selectivity towards 31 and 82-ad olol, practolol, and I-, dl and d-metoprolol renoceptors than 5HT,B-receptors. These preferred 81-adrenoceptors; alprenolol, but results suggested that all (3-adrenoceptor oxamine and ICI-118,551 preferred 82-ad antagonists tested in this study displaced renoceptors; and arotinolol, pindolol, YM 1251-ICYP bound to 5HT,B-serotonergic re 09538, , labetalol, sotalol, and d-, ceptors. dl and I-propranolol were non-selective Furthermore, it is possibile that these re towards d, and a2-adrenoceptors. Alprenolol ceptors may exist in the brain, heart, lung and is well-known as a non-selective antagonist, spleen (2, 3). In the present study, a good cor but the present study showed that it has 7.2 relation was observed between the pK; values fold higher affinity for Q2-adrenoceptors of l3-antagonists for l3-adrenoceptors and (Table 1), suggesting that the methods in this those of 5HT,B-receptors, suggesting that (3 study are useful for determining the pre adrenoceptors antagonists may regulate the ferences of (3-antagonists with low affinity function of 5HT1B-receptors. Although the differences between Q, and Q2-adreno existence of an interaction between the func ceptors. tion and structure of (3-adrenoceptors and All 8-antagonists displaced the binding of those of 5HT,B-receptors is yet to be proven, 1251-ICYPfromthe 5HT,B-receptors . The non it would be of interest to examine the function selective antagonists for 8-adrenoceptors had and distribution of 5HT1B-receptors in the 18 to 300-fold preferences towards l3 body. adrenoceptors as compared to their selec A good correlation was also observed be Fig.2. Relationship between the potencies (pKi) of the chemicals for inhibition of 1251-ICYP binding to Fig. 1. Relationship between the potencies (pKi) j, (A) and j32-adrenoceptors (B) and those to of the chemicals for inhibition of 1251-ICYP binding 5HTis-receptors (A values in the Table 2). Numbers to (3, (A) and j92-adrenoceptors (B) obtained by the refer to individual drugs in Table 1. (A) y=0.67x+ present study in the rat cerebral cortex and those 4.02, r=0.87 (P<0.001). (B) y=0.90x+2_.37, r=0.88 obtained by the binding of 3H-CGP12177 in the rat (P<0.001). myocardium, which were reported previously (7). Numbers refer to individual drugs in Table 1. (A) for the assessing the relative potencies of the y=0.91x+0.90, r=0.97 (P<0.001). (B) y=0.96x+ chemicals as Q-adrenoceptor antagonists. 0.39, r=0.96 (P<0.001 ). The radioligand 1251-ICYP was used as a model ligand in competitive binding experi tween the pK; values of 8-antagonists for ments. The present results quantitated the 31 and 82-adrenoceptors obtained in the affinity of Q-adrenoceptor antagonists for 8, -, present study and those obtained by the 82 and 5HT1B-serotonergic receptors and binding of 3H-CGP12177 in the rat myocar found that the Q-adrenoceptors may be re dium. In our previous study (5), we found that lated to 5HT1B-receptors. there was a difference in the IC50 values be tween rat heart and brain; especially, the af References finity of arotinolol for (9-adrenoceptors in the 1 Tsuchihashi, H. and Nagatomo, T.: Biphasic brain was much lower than those in the heart. binding of 1251-iodocyanopindolol to S-adren However, the present results did not demon ergic receptors in rat cerebral cortical mem strate such differences in affinity of arotinolo! branes. I. Assessment by the use of . in the rat myocardium and cerebral cortex, Chem. Pharm. Bull. (Tokyo) 35, 2966-2972 indicating that binding methods may be useful (1987) 2 Tsuchihashi, H. and Nagatomo, T.: Binding Binding characteristics of [3H] characteristics of 1251-iodocyanopindolol to to R-adrenergic receptors of rat brain: Com adrenergic receptors: Biphasic Scatchard plots. parison with those of rat heart treated with 11.Effects of selective antagonists. Chem. Phar n. neuraminidase. Chem. Pharm. Bull. (Tokyo) 33, Bull. (Tokyo) 35, 3424-3432 (1987) 3972-3976 (1985) 3 Tsuchihashi, H., Nagatomo, T. and Imai, S.: 6 Tsuchihashi, H. and Nagatomo, T.: Characteriza Three binding sites of 1251-iodocyanopindolol to tion of 3H-dihydroalprenolol binding to /3 p,, fi2-adrenergic and 5HT1B-receptors in rat adrenergic receptors of rat brain: Two binding brain determined by the displacement and sites of racemic propranolol in displacement Scatchard analysis. J. Pharmacobiodyn. 12, 509 experiments. Chem. Pharm. Bull. (Tokyo) 35, 516 (1989) 2979-2984 (1987) 4 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and 7 Tsuchihashi, H., Yokoyama, H. and Nagatomo, Randall, R.J.: Protein measurement with the T.: Binding characteristics of 3H-CGP12177 to Folin phenol reagent. J. Biol. Chem. 193, 265 (3-adrenoceptors in rat myocardial membranes. 275 (1951) Japan. J. Pharmacol. 49, 11-19 (1989) 5 Tsuchihashi, H., Sasaki, M. and Nagatomo, T.: