Japan. J. Pharmacol. 45, 349-356 (1987) 349

Effects of Bunitrolol on and Serotonergic Receptors

Hiroshi TSUCHIHASHI, Junichiro AONO, Takafumi NAGATOMO, Tomie KAWADA*,Hideo OHTA* and Shoichi IMAI* Departmentof Pharmacology,Niigata College of Pharmacy, 5-13-2 Kamishin'ei-cho,Niigata 950-21, Japan *Departmentof Pharmacology, NiigataUniversity School of Medicine, 757, Asahimachidori1, Niigata951, Japan Accepted July 27, 1987

Abstract-To assess the importance of anti-adrenergic and anti-serotonergic activities of bunitrolol for its efficacy as an antihypertensive and agent, effects of this substance on the binding of adrenergic and serotonergic agents to the respective receptors of the rat brain, rat heart, dog brain, and/or dog aorta were examined using the radioligand binding assay methods. In addition, the pA2 values of bunitrolol as an antagonist against the positive chronotropic and inotropic actions (Q1-adrenoceptor) of isoproterenol were also determined by pharmacological methods using the isolated guinea pig atria. To assess the specificity, pA2 values were also obtained in the isolated trachea (132-adrenoceptor) using isoproterenol as an agonist and in the isolated aorta from the guinea pig and the rat using as an agonist (a,-adrenoceptor). A strong inhibition by bunitrolol of 3H- (3H-DHA) binding to /3-adrenoceptors was observed, while the inhibition of 3H- binding to a,-adrenoceptors, 3H-serotonin binding to 5HT,-receptors. 3H-p-aminoclonidine binding to a2-adrenoceptors, and 3H- binding to 5HT2-receptors were found to be very weak. The rank order of antagonistic potencies of bunitrolol against the adrenergic receptors as asessed with pA2 values were 31>82>a,. From these two different types of experiments, it is clear that the antihypertensive and antianginal effects of bunitrolol are mainly due to its ,3-blocking actions, with the a,-blocking action of this drug playing a minor role.

Bunitrolol has been clinically used because of its anti hypertensive and anti Materials and Methods anginal efficacy. As is well-known, bunitrolol Materials: 3H-DHA (90 Ci/mmole), 3H has a very potent (3-adrenergic blocking prazosin 80.9 Ci/mmole), 3H-p-amino action (1-3). However, it was reported that, (42.2 Ci/mmole), 3H-5-hydroxy in addition, bunitrolol possessed a weak a, tryptamine creatine sulfate (3H-serotonin; blocking action (4). To assess the relative 24.3 Ci/mmole), and 3H-ketanserin (95Ci/ importance of these two actions for clinical mmole) were purchased from New England efficacy, in the present study, we attempted Nuclear Co., Ltd. Bunitrolol, 0-[2-hydroxy-3 to determine the antagonistic effects of (tert.-butylamino)propoxy]benzonitrile HCI, bunitrolol on a and (3-adrenergic receptors was kindly donated by Nippon Boehringer with radioligand binding methods as well as Ingelheim Co., Ltd. by pharmacological assessment. Furthermore, Animals: In these experiments, male the effect of bunitrolol on serotonergic Wistar rats weighing 200-350 g, mongrel receptors was examined, as serotonin is also dogs of either sex weighing 10-25 kg, and important as a regulator of the blood pres male Hartley guinea pigs weighing 350-550 sure in the body (5-9). g were used. 350 H. Tsuchihashi et al.

Preparation of the membrane-enriched Tris-HCI buffer and then incubated for 10 min fraction: The membrane-enriched fractions at 37'C. The suspension was centrifuged at from the rat brain and heart used for 3H-DHA 40,000 g for 30 min. Both pellets from the dog binding were prepared by the methods brain and aorta were homogenized using a described previously (10-12). The mem glass homogenizer. brane-enriched fractions from the rat brain Binding assays: Q-Adrenoceptor binding and dog aorta used for 3H-prazosin binding assays using the rat brain and heart treated were prepared by the methods described with neuraminidase were carried out by the previously (13). The membrane-enriched methods described previously (10-12). In fractions from the dog brain used for 3H-DHA the case of dog brain, the membrane sus binding, those from the brain used for 3H pension (0.25 mg of protein) was incubated prazosin binding, and those from the dog and with constant shaking for 30 min at 23 °C rat brain used for 3H-p-aminoclonidine with 1.0 nM 3H-DHA in a total volume of binding were prepared by the following 0.5 ml containing 60 mM Tris-HCI, 20 mM methods. After removal, the brain was im MgC12 buffer (pH 7.4), and the indicated mediately frozen in liquid nitrogen and concentration of unlabelled drugs. al stored at -80°C until used. The brain tissue Adrenoceptor binding assay using the rat was defrosted at room temperature and was brain and the dog aorta was carried out by minced with small scissors in 10 vol. of the methods described previously (13). The 0.25 M sucrose, 10 mM Tris-HCI buffer (pH membrane suspensions (0.25 mg of protein) 7.4). The suspension was homogenized from dog brain were incubated for 60 min at using a glass homogenizer and filtered 23°C with 0.2 nM 3H-prazosin in a total through 4 layers of gauze. The filtrate was volume of 1 ml containing 15 mM Tris-HCI, centrifuged at 40,000 g for 30 min. The 5 MM MgC12 buffer (pH 7.4). In the case of pellet obtained was homogenized using a a2-adrenoceptor binding, the membrane sus glass homogenizer in an incubation buffer pension (0.25 mg of protein) from the rat and for the radioligand binding assay described dog brain was incubated for 60 min at 23°C below. The membrane-enriched fraction from with 0.6 nM 3H-p-aminoclonidine in a total the dog brain used for 3H-serotonin and 3H volume of 1 ml containing 15 mM Tris-HCI, ketanserin binding was prepared by the 5 MM MgC12 buffer (pH 7.4). Binding of 3H following method: The brain defrosted at serotonin (1.0 nM) to the dog brain (0.5 mg room temperature was minced with small of protein) was determined at 37°C for 30 min scissors and homogenized with a glass by incubating with 30 mM Tris-HCI, 5 mM homogenizer in 10 vol. 0.25 M sucrose, 1 mM MgC12, 2 mM CaC12, 2.85 mM ascorbic acid, MgC12, 5 mM Tris-HCI buffer (pH 7.4). The 4 aM pargyline buffer (pH 7.4) in a final homogenate was centrifuged at 18,000 g for volume of 1 ml of the reaction mixture. 3H 15 min. The pellet was suspended in 10 vol. Ketanserin binding to the membrane fraction of 60 mM Tris-HCI buffer (pH 7.4) and then (0.25 mg of protein) from the brain and the incubated for 10 min at 37'C. The suspension aorta was determined at 37'C for 30 min in an was again centrifuged at 18,000 g for 15 min. incubation medium containing 50 mM Tris In the case of the aorta, the frozen tissue was HCI, 2.85 mM ascorbic acid, 5 uM pargyline crushed into a fine powder using a mortar buffer (pH 7.4). Reactions of all the binding and pestle in liquid nitrogen and suspended assays were started by adding the membrane in 10 vol. 0.25 M sucrose, 1 MM MgC12, suspension. After incubation, the reaction 5 mM Tris-HCI buffer (pH 7.4). The sus was terminated by a rapid filtration under a pension was homogenized using a Polytron vacuum through GF/C glass fiber filters using homogenizer, twice for 10 sec, at setting 8. an Automatic Cell Harvester Labomash (LM The homogenate was filtered through 1 101, Labo Science) (10). Five ml of scintil layer of gauze and then centrifuged at 1,000 g lation fluid (Scintisol EX-H, Dojindo La for 10 min. The supernatant obtained was boratories) was added to the filters, and the again centrifuged at 40,000 g for 30 min. The radioactivity was counted using a scintil pellet was suspended in 10 vol. of 60 mM lation counter (Aloka LSC-700). The Bunitrolol and Adrenergic and Serotonergic Receptors 351 specific binding of 3H-DHA and 3H-prazosin a straingauge transducer (Toyo-Baldwin T7 to receptors was defined by the previously 30-240) and a carrier amplifier (San-ei described methods (10-13). The specific 1829). Isolated tracheal smooth muscle binding of 3H-p-aminoclonidine, 3H preparations of the guinea pig were used to serotonin, and 3H-ketanserin to receptors in assess the antagonistic effects towards the the brain or aorta was defined as the amount Q2-adrenoceptor. Isoproterenol was used as of radioligand bound in the absence of an agonist. Seven tracheal rings removed competing drug minus the amount in the from guinea pigs were sutured together and presence of 0.1 mM I- (3H mounted vertically in a 5 ml organ bath. The p-aminoclonidine binding), 0.1 mM serotonin contractile tension of the preparation was (3H-serotonin binding), 0.1 mM methy recorded on a potentiometric recorder sergide (3H-ketanserin binding to the brain), (Watanabe SR6204). Drugs were admin and 10 /1M cinancerin (3H-ketanserin istered in a cumulative fashion, and pA2 binding to the aorta). Protein was determined values of each of the chemicals were calcu using the method of Lowry et al. (14). lated using the equation described previously Kinetic analysis: All kinetics analyses were (12, 20). The bathing solution used was carried out on a N EC PC-9801 F computer Krebs-Henseleit solution (containing in mM: system that performs iterative non-linear NaCI, 118; KCI, 4.7; CaCI2, 2.5; NaHC03, regression, which is based on the theory of 25.0; MgSO4, 1.2; KH2PO4, 1.2; glucose, 12) Munson and Rodbard (15). The goodness which was aerated with a mixture of 95% of fit was evaluated on a model having only 02 and 5% C02. The temperature of the one receptor subtype and a model having the solution was maintained at 32±0.30°C. two receptor subtypes by a Scatchard Isolated preparations of the rat and guinea analysis as described previously (16). In the pig aorta were used to assess the antagonistic displacement analysis, the goodness of fit effects towards the a, -adrenoceptor. The was evaluated on a general model for the descending aorta was cut into rings of ap interactions of one ligand with one class of proximately 2 mm width, and the preparations receptor site (17). The values of inhibition were mounted in a 10 ml organ bath. The constants (Ki) were calculated by the contractile tension of these preparations methods previously described (17) and ex was recorded on a potentiometric recorder pressed as pKi values in this report. In order (Watanabe SR6204) using a strain gauge to quantify the mode of saturation, Hill transducer (Toyo-Baldwin T7-30-240) and numbers were determined by Hill plots (18). a carrier-amplifier (San'ei 1829). The bathing Pharmacological observations: (9-Blocking solution used was the Krebs-Henseleit actions were determined as described pre solution as described above which was viously (12, 19), and a,-blocking actions aerated with a mixture of 95% 02 and 5% were studied as described previously (13, 20). C02. The temperature of the solution was The right and left atria of the guinea pig were maintained at 37±1 °C. used for the assessment of the antagonistic potencies against the positive chronotropic Results and inotropic actions of isoproterenol (81 Table 1 summarizes the dissociation effect). The rate of the spontaneous con constants (Kd) and the capacity of binding traction of the right atria was recorded on a sites (Borax) of the membrane preparations thermo-stylus oscillograph (Watanabe Mark of the various tissues for adrenergic and V) to test the positive chronotropic ac serotonergic radioligands. All Scatchard tions. The left atria stimulated electrically plots were of uniphasic character, and the by a squarewave pulse stimulator (Nihon Hill coefficients were equal to unity. Kohden MSE-40) at the frequency of 1 Hz Table 2 summarizes the pKi values of with voltages 30% above the threshold were bunitrolol derived from the displacement used to evaluate the inotropic effects. Their experiments. Bunitrolol exhibited a higher contractile tension was recorded on a thermo affinity to i9-adrenoceptors than dl-pro stylus oscillograph (Watanabe Mark V) with pranolol and it had negligibly lower af Table 1. Binding affinities (K,i) and capacities (Bmax) of various tissues for adrenergic and serotonergic radioligands

Values are the means±S.E. Numbers in parentheses represent the numbers of experiments. The values marked as 0, 1) and I') were obtained from the reports described previously: :1) Ref. 11 , b) Ref. 10, 0 Ref. 13. Bunitrolol and Adrenergic and Serotonergic Receptors 353

Table 2. pKi values of bunitrolol for adrenergic and serotonergic receptors of various tissues

Values are the means+S.E. Numbers in parentheses represent the numbers of experiments.

Table 3. Comparison of adrenoceptor blocking activities of bunitrolol, dl- and prazosin

Values are the means+S.E. Numbers in parentheses represent the numbers of experiments. 354 H. Tsuchihashi et al.

Fig. 1. Assessments of pharmacological activities of bunitrolol using the guinea pig trachea (A), left (B) and right atria (C) and aorta (D), and the rat aorta (E). Dose-response curves were obtained in the absence (0) or presence of bunitrolol (•) of the following concentrations: trachea, left and right atria, 1 X10-$ M; guinea pig aorta, 1 X10-4 M; rat aorta, 3X10-6 M. The data shown are means+S.E. from five or six experiments as indicated in Table 3. finities for al-, a2-, 5HT1 and 5HT2-re much the same in different tissues, while the ceptors than prazosin, clonidine, serotonin BmaX values (fmoles/g tissues) were dif and ketanserin, respectively. ferent depending on the tissues. For example, Table 3 and Fig. 1 show the results of the the BmaXvalues expressed as fmoles/g tissue pharmacological assessment of antagonistic of the binding of 3H-prazosin, 3H-p-amino effects of bunitrolol against adrenergic clonidine and 3H-ketanserin were lower in receptors. The rank order of antagonistic the aorta than in the brain. This is probably potency of bunitrolol was: a, >32>>ai . due to the lower protein yields ascribable to the technical difficulties inherent in the Discussion preparation of membranes. As regards the In the present study, it was found by BmaX there was also a species difference. Scatchard analyses that Kd values of the For example, the numbers of Q-adrenoceptors binding of a radioligand to a receptor were were greater in the rat brain than in the dog

In our previous work, affinities of ;3 antagonists to receptors as assessed by K; values were lower in the brain than in the heart; especially marked was the lower af finity of S-596 towards Q-receptor in the brain as compared with that in the heart (10). In the present study, the pK; values of bunitrolol obtained in the rat brain for 3H DHA binding were lower than those in the rat heart. Thus, the blocking activities of chemicals against the same type of receptors are not necessarily the same in different preparations. Based on pharmacological studies, buni trolol is admittedly a 9-blocker with a weak a,-blocking action (1-4). This was con firmed in the present study, in which the antagonistic potencies were assessed both by the binding assay method and by phar macological observations. We have already reported that 3 and a,-blocking potencies of compounds as assessed from K; or IC50 values obtained from the radioligand binding assays correlate well with those assessed with pA2 values obtained by pharmacological methods (13, 15). The result obtained in the present study with these two methods is another example of a good correlation between pA2 and K;. The antihypertensive effects of (3-adren ergic blocking drugs are well documented in man. Recently, several Q-adrenergic block ing drugs with an a, -adrenergic blocking effect have been developed. I n the case of , a prototype (3-blocker with a, blocking activities, an acute hypotensive effect was observed, indicating that not only the 8-blocking effect but also the a, block ing effect may be responsible for the anti hypertensive effects of this compound (21). However, in the case of bunitrolol, the a, blocking activities may not be important in the antihypertensive effect of the compound, for the a,-blocking activities were found in the present study to be extremely weak as compared with those of labetalol. The present study clearly showed the ex istence of 5HT, and 5HT2-receptors in the dog brain and 5HT2-receptors in the dog aorta. Central serotonergic neurons were shown to influence the blood pressure (8).

Bunitrolol and Adrenergic and Serotonergic Receptors 355

brain. Furthermore, the hypertension produced by serotonin has been shown to be mediated through 5HT2-receptors in the peripheral blood vessels (5-7). Serotonin receptors have been demonstrated in the various tissues, and highly significant correlations were found between the concentration of the compound necessary for half-maximal contraction of vessels (ED50) and the binding affinity for binding sites (8). Ketanserin has been developed as an antihypertensive agent because this drug was found to antagonize the vascular effect of serotonin via 5HT2 receptor in vivo (9). However, the possible contribution of the serotonergic blocking effects of bunitrolol to its antihypertensive effects was excluded in the present study; practically no inhibition of 3H-serotonin and 3H-ketanserin binding was observed with bunitrolol.

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