Displays a Unique Pharmacological Profile of Affinities

for the Different ƒ¿1-Adrenoceptor Subtypes

Junji Kinami, Hiroshi Tsuchihashi, Keiko Maruyama, Keiko Sasaki and Takafumi Nagatomo

Department of Pharmacology, Niigata College of Pharmacy, 5-13-2 Kamishin'ei-cho, Niigata 950-21, Japan

Received May 25, 1992 Accepted November 6, 1992

ABSTRACT-The selectivity of antagonistic effects of nipradilol, its four isomers and denitronipradilol, a major metabolite of nipradilol, on ƒ¿1-adrenoceptor subtypes in rat heart, brain and spleen were examined by radioligand binding assay with [3H]-. Pharmacological characteristics of these compounds were determined in isolated aortae from rats and guinea pigs. The order of the pKi values for ƒ¿1High-affinity sites in the heart, spleen and brain was SR > nipradilol •† RR •† SS ≈RS•âdenitronipradilol, but the order of the

pKi values for the ƒ¿1LOW -affinity sites was different in the heart and brain. There were good correlations between the pKi values of these compounds for the ƒ¿1High -affinity sites and the pA2 values for the contractile inhibition of the -induced response in rat aorta. There was no correlation between the pKi values of these compounds for the ƒ¿1LOW -affinity sites and the pA2 values. These results indicate that: 1) ƒ¿1High -Affinity sites are related to vasoconstriction mediated by ƒ¿1- adrenoceptors; 2) Nipradilol and its isomers possess low affinity to ƒ¿1 -adrenoceptors; and 3) The nitroxy group in nipradilol is important for its -blocking activity . ƒ¿1

Keywords: [3H]-Prazosin, Nipradilol, ƒ¿1-Adrenoceptor subtype, ƒ¿1- and ƒÀ-•@Blocking activity, Optical isomer

Our recent studies on the binding characteristics of (ICYP) and [3H]-CGP 12177, respectively; [3H]-prazosin for the a,-adrenoceptor subtypes revealed and the results showed that nipradilol had a higher affin that there are two binding sites having different affinities, ity for (3-adrenoceptors (with no selectivity for f3, and (32 high and low-affinity sites, for prazosin in the rat ven adrenoceptors) than for 5HT,B-receptors (5). The rank tricular muscle, brain and spleen membranes; and these orders of potency of the isomers for 8-adrenoceptors and sites are designated as a,High and a1L0 -affinity sites (1, 2). 5HT,B-receptors were SR > nipradilol > SS > RR > RS and We also found that rat ventricular muscle and brain pos SS > SR >_nipradilol > RS > RR, respectively (5). sess both a,High and a1L0 affinity sites, whereas rat spleen had only a,High-affinity sites (1, 2). Most a,-adrenergic an tagonists had different affinities for each site in these tissues, suggesting that there may exist five different a, adrenoceptors (a,High-sites in the ventricular muscle, brain and spleen and a1LoW-sitesin the ventricular muscle and brain). Recently, a, •~-blockers possessing antagonistic po tency for both a, and j3-adrenoceptors have been shown to be useful for the clinical treatment of patients with hypertension and angina. Nipradilol, one of these a, ~ blockers, possesses two asymmetric carbon atoms in its chemical structure and is a mixture of four isomers, as shown in Fig. 1 (3, 4). We previously reported the binding characteristics of nipradilol for (3,-, (32-adrenergic and 5HT,B-serotonergic binding sites by using [125I]-iodo Fig. 1. Chemical structure of nipradilol and its isomers. In this study, we examined the selectivity of nipradilol, in the presence or absence of unlabelled ligand. The con its isomers and its major metabolite denitronipradilol (6) centrations of [3H]-prazosin were 0.04, 0.1 and 0.2 nM for a,-adrenoceptor subtypes and compared them with for assessing a,High-affinity sites in the brain, heart and those of other a, • n-blockers, , and spleen membrane, respectively. The affinity of the a,LoW . affinity sites for unlabelled ligand were determined with 0.5 nM [3H]-prazosin in the presence of 0.1 pM phenoxy MATERIALS AND METHODS benzamine in the brain membrane, or with 0.6 nM [3H] prazosin in the presence of 1 pM in Materials the heart membrane. Phenoxybenzamine inhibited the [3H]-Prazosin (76.6 Ci/mmole) was purchased from a,High-affinity sites completely (1, 2). After the incuba New England Nuclear/Dupont, Ltd., Boston, MA, tion period of 45 min, the medium was immediately U.S.A. Nipradilol, 3,4-dihydro-8-(2-hydroxy-3-isopropyl filtered through a GF/C glass fiber filter and washed with amino)propoxy-3-nitroxy-2H-1-benzopyran, its isomers the incubation buffer according to previously described (Fig. 1) and denitronipradilol were kindly donated by methods (8). The radioactivity on the filter was counted Kowa Co., Ltd. by a Packard 2200 Tri-Carb Scintillation Analyzer. The specific binding was determined by subtracting the non Animals specific binding in the presence of 10 pM of Male Wistar rats weighing 200-350 g were used. from the total binding.

Preparation of membrane-enriched fractions Kinetic analysis The membrane-enriched fractions from rat heart, brain All kinetic analyses were performed on an NEC PC and spleen were prepared as described previously (1, 2). 9801 computer system with an iterative non-linear regres Protein was determined by the method of Lowry et al. sion program (9-12). The data were fitted to models hav (7). ing only one or two receptor binding sites (9 -12). To quantitate the displacement characteristics, the slope fac Binding assay tor (nH) for the displacement curves was determined as Displacement analysis for a,-adrenoceptor subtypes described previously (9-12). Most K; values of various was performed in duplicate with [3H]-prazosin as ligands are expressed as pK; ( log K;) in this report. described previously (1, 2). In brief, the membrane sus pension (0.1 mg of heart and brain and 0.25 mg of spleen Pharmacological observations membrane proteins) was incubated for 45 min at 231C in a The contractile tension of the rat and guinea pig aorta total volume of 0.5 ml containing 60 mM Tris-HC1 (pH was determined as described previously (13, 14). Briefly, 7.4) with an appropriate concentration of [3H]-prazosin aortae were cut into rings and freed of excess tissues.

Table 1. pK; values of nipradilol and its optical isomers for the a,-adrenoceptor subtypes

Values in parentheses are the numbers of experiments. Data are the mean values ±S.E. The slope factors (nH) of the displacement curves of all ligands used in the present study were equal to one. *Some of these data were reported previously (11). These rings, about 2 mm in width, were mounted in 12-m1 Table 2. pA2 values of nipradilol and its optical isomers for a, organ baths. The Krebs-Henseleit solution had the follow adrenoceptors in rat and guinea pig aorta ing composition: 118 mM NaCI, 4.7 mM KCI, 2.5 mM CaC12, 25 mM NaHCO3, 1.2 mM MgSO4i 1.2 mM KH2PO4, and 11 mM glucose. The temperature of the so lution was maintained at 37 ± 1 V and aerated with a mix ture of 95% 02 and 5% CO2. The contractile tension of these preparations was recorded on a potentiometric recorder (Hitachi APD-74) with a strain gage transducer and a carrier amplifier (Nihon Kohden RP-3 or San'ei Instrument Co., Ltd. 6M52). The aorta was always stretched to 0.5 -1 g to obtain the optimum response. Concentration-response curves were determined for phenylephrine before and after addition of each com Values in parentheses are the numbers of experiments. Data are the pound, and their pA2 values were calculated by the previ means ± S.E. ously described equation (15). heart a,LoW, heart alHigh brain alHigh? spleen alHigh• RESULTS Table 2 also summarizes the pA2 values of the contrac tile tension of rat and guinea pig aorta. a1-Adrenoceptor Table 1 summarizes the pKi values of nipradilol, its subtypes in the rat and guinea pig aorta were alHigh and isomers and denitronipradilol for a1-adrenoceptor sub a1Low-affinity sites, respectively, as determined by the types in [3H]-prazosin binding to brain, heart and spleen pA2 values of prazosin (10.80 in rat aorta and 8.30 in membranes. Both alHigh and a1Low-affinity sites were guinea pig aorta) (16). All compounds antagonized the found in the brain and heart membranes, while only phenylephrine-induced contractile response in a competi alHigh-affinity sites existed in the spleen membranes (1, 2). tive manner when analyzed by Schild plots (data are not These five a1-adrenoceptors had different affinities for shown). The pA2 value of nipradilol in the rat aorta was various antagonists (1, 2). The binding affinity of lower than the value of amosulalol and labetalol, and it labetalol for these subtypes were distinguishable among was higher than that of arotinolol. On the other hand, the the compounds used in the present study: brain alHigh> pA2 values of SS, RS and denitronipradilol were lower heart alHigh = spleen alHigh > brain a1Low> heart a1Low• than the value of arotinolol. The order of pA2 values of The pKi values of the other a1 •n-blockers, arotinolol and these compounds was SR= nipradilol= RR > SS RS amosulalol, for alHigh-affinity sites were higher than those denitronipradilol in the rat aorta. The pA2 of nipradilol for a1Low-affinity sites. On the other hand, the pKi values in the guinea pig aorta was lower than that of amosulalol of nipradilol for various subtypes were similar except for and was higher than that of arotinolol. On the other heart alHigh > heart a1Lo,. The pKi values of nipradilol hand, the pA2 values of RS and denitronipradilol were and related compounds for the alHigh-affinity site were sig lower than the value of arotinolol. The order of pA2 nificantly lower than those of prazosin, amosulalol, values of these compounds was SR >_nipradilol RR >_ labetalol and arotinolol. The order of potency of SS denitronipradilol > RS in guinea pig aorta. The pA2 nipradilol, its isomers and denitronipradilol for alHigh values of these compounds in the guinea pig aorta were affinity sites was SR > nipradilol RR > SS = RS > similar to those in the rat aorta except for those of denitronipradilol in the heart, brain and spleen. Thus, the denitronipradilol. orders of potency were similar for the alHigh-affinity sites Figure 2 shows the relationship between the pKi values in these three tissues. On the other hand, the orders of of these compounds for al High-affinitysites in the three tis potency for the a1Lowaffinity sites was not consistent in sues and the pA2 values for the antagonistic potency in the heart and brain. The rank order of the pKi values of the contractile response to phenylephrine of rat and denitronipradilol was heart a1Low> heart alHigh > spleen guinea pig aorta. These results suggested a good correla alHigh brain alHigh ? brain a1Low.The rank order of the tion between the pKi values of the alHigh-affinity sites and pKi values of the optical isomers of nipradilol for these the pA2 values in rat aorta (r = 0.90 0.94, P < 0.001) . The subtypes was different for each compound: SR, brain pA2 values in the guinea pig aorta showed less correlation a1Low brain alHigh >_ heart alHigh = spleen alHigh > heart (r=0.74-0.79, P<0.01) with the pKi values than those alHigh SS, heart a1Low> brain a1Low? heart alHigh brain obtained in the rat aorta. There was no correlation be alHigh ? spleen alHigh; RR, brain a1Low > heart alHigh tween the pKi values of the a1Low-affinitysites and the pA2 heart a1Low? brain alHigh > spleen alHigh; RS, brain a1Low values in both rat and guinea pig aorta (heart vs. rat Fig. 2. Relationship between the pK; values of nipradilol and its isomers for ajHigh-affinity sites in the rat heart, brain and spleen and the pA2 values in the rat and guinea pig aorta. 1, Nipradilol; 2, SR; 3, SS; 4, RR; 5, RS; 6, amosulalol; 7, labetalol; 8, arotinolol; 9, denitronipradilol.

aorta, r=0.26; brain vs. rat aorta, r=0.56; heart vs. the isomers and denitronipradilol was also different for guinea pig aorta, r=0.28; brain vs. guinea pig aorta these subtypes. Denitronipradilol and SS were selective r=0.43). for a,Low-affinity sites in the heart, but RR was selective for a,L0 -affinity sites in the brain. These results suggested DISCUSSION that these subtypes have different affinities for various a, adrenoceptor antagonists and that the configuration of Nipradilol is a potent R-adrenergic blocking agent that the 3 position of the benzopyran ring and the 2' position has direct vasodilating and a,-blocking properties. It pos of the aryloxypropanolamine residue was important for sesses two asymmetric carbon atoms and is a mixture of the recognition of the a,-adrenoceptor subtypes. four isomers (3, 4). There is one asymmetric carbon atom The order of potency of these compounds for the aiHigh at the 3 position of the benzopyran ring and another at affinity sites in the heart, brain and spleen was generally the 2' position of the aryloxypropanolamine group. SR > nipradilol >_ RR >_ SS RS > denitronipradilol. Denitronipradilol, which has weak a,-adrenergic activity, Therefore, the results suggest that the R configuration of is a major metabolite (6). We demonstrated that a, the nitroxy group of the benzopyran ring and the S adrenoceptor subtypes exist in various tissues and that configuration of the 2' position at the aryloxypropanola most of the a,-adrenergic antagonists had various affin mine residue were important for the a,-antagonistic ac ities for each subtype in these tissues (1, 2). The present tions of these drugs. We previously reported that study also showed that nipradilol, its isomers and nipradilol and its isomers were not selective for (3, and P2 denitronipradilol had different affinities for various a, adrenoceptors and that the order of potency of these com adrenoceptor subtypes. The rank order of pK; values of pounds for (3-adrenoceptor subtypes and 5HT,B-receptors was SR > nipradilol > SS > RR > RS and SS > SR >_nipr correlation between the pKi values for a1Low affinity sites adilol > RS > RR, respectively (5). Previous reports (14, and the pA2 values in the rat and guinea pig aorta.

17-20) have also shown that p-blockers possessing the S Muramatsu et al. (25) demonstrated that the rat and configuration are more potent than those of the R configu guinea pig aorta contained a1H and a1L-adrenoceptor sub ration among aryloxypropanolamines. On the other types, respectively. The pA2 values of prazosin for these hand, the R configuration of the nitroxy group of a1H and a1L-subtypes were 9.89 and 8.45, respectively. nipradilol played an important role in the a1-blocking ac The a1H-subtype in the rat aorta has high affinity for WB tivity. These results suggested that these three receptors, 4101 and is sensitive to CEC (25, 26), and the a1L-subtype a1-, (3 and 5HT1B-receptors, had different characteristics in the guinea pig aorta has low affinity for WB-4101 and is in the recognition sites for the optical isomers of insensitive to CEC (25). The classification of alHigh and nipradilol. a1Low affinity sites is based on the different affinities for

Based on recently available data, an increasing number prazosin (1, 2), suggesting that the a1H and a1L-subtypes of complex classification schemes for the a-adrenoceptor may be identical to the alHigh and a1Low-subtypes. We also subtypes are being published. In blood vessels, there are showed that the pA2 values of prazosin for the subtypes two distinct subtypes (a1H & a1L) of a1-adrenoceptors that of the rat and guinea pig aorta were 10.80 and 8.30, are distinguished by their affinities for prazosin and respectively (16). On the other hand, the present study yohimbine (21). Our results also show that there are two showed that denitronipradilol had a higher affinity for distinct receptor subtypes (alHigh& a1LoW)that are distin guinea pig aorta than rat aorta. Our results suggest that guished by their affinities for [3H]-prazosin in the rat the a1H1gh-affinity sites for [3H]-prazosin binding were simi brain, spleen, and heart (1, 2). On the other hand, Mor lar to the vascular a1H (alHigh)-subtypes, but that the a1Low row and Creese (22) reported that rat brain contains two affinity binding sites were different from the vascular alL subtypes (ala & alb) with similar affinities for prazosin but (a1Low)-subtypes. On the other hand, for the alHigh-affinity different affinities for phentolamine. Han et al. (23, 24) sites, the pKi values of some compounds were not correlat proposed a similar classification (a1A & a1B) in rat brain, ed with the pA2 values in the rat aorta when the pKi values spleen, heart and other tissues. The a!A-subtype has a of all compounds were compared with the pA2 values. high affinity for WB-4101, while the a1B-subtype has a low These observations suggest that al-adrenoceptors in the affinity for this drug and is susceptible to chlorethylcloni rat and guinea pig aorta may be new types of alHigh and dine (CEC); these subtypes have similar affinities for a1Low affinity sites, respectively. Furthermore, the prazosin. The relationship between alHigh and a1LoW-sub present findings imply that the alHigh and a1Low affinity types and a1A and a1B-subtypes in the rat brain, spleen sites are involved in vasoconstriction, while the alLow and heart has not yet been clarified (1, 2). Muramatsu et affinity binding sites in the heart and brain were different al. (25) recently proposed that the al-adrenoceptors in from the a1Low subtypes in guinea pig aorta. blood vessels can be divided into three subtypes (alH, alL Recently, al • (3-blockers have been used for the treat

& a1N) by their antagonist affinity and susceptibility to ment of hypertension or ischemic heart diseases. The CEC. The alH-subtype has a high affinity for prazosin, affinities of a1 • p-blockers, amosulalol, labetalol, susceptible to CEC and includes alB-subtypes in the dog arotinolol and nipradilol, for (3-adrenoceptors were 0.6 carotid artery and atypical a1A-subtypes in the rat aorta to 170-, 3 to 90-, 1300 to 10000 and 1200 to 3000-fold (26). The a1L-subtype has a low affinity for prazosin in the higher than those for al-adrenoceptor subtypes, respec guinea pig aorta, and the a1N-subtype has a higher affinity tively (5, 11). Amosulalol, labetalol and arotinolol had for HV-723 and WB-4101 than for prazosin. On the other higher selectivity for alHigh-affinity sites than a1Low affinity hand, Han and Minneman (27) classified the subtype in sites, while nipradilol had similar affinities for alHigh and the rat aorta as a1B. Four subtypes (alA, a1B, aic & a1D) alLow-affinity sites. Thus nipradilol has a unique pharma have been cloned (28-31), and the a1B, aic and a1D-sub cological profile of selectivity towards the a1-adrenocep types are susceptible to CEC. These classifications (cloned tor subtypes. a1A, a1B, aic & a1Di ala & alb, and a1A & a1B) do not in clude the subtypes with low affinities for prazosin. REFERENCES It was revealed that there was a good correlation be tween the pKi values obtained for the alHigh-affinity sites 1 Tsuchihashi, H., Maruyama, K., Baba, S., Mano, F., Kinami, and the pA2 values in the rat aorta (r=0.90-0.94, J. and Nagatomo, T.: Comparison of ƒ¿1-adrenoceptors be tween rat brain and spleen. Japan. J. Pharmacol. 56, 523-530 P<0.001). There was a significant but lower correlation (1991) (r=0.74-0.79, P<0.01) between the pKi values for the 2 Kinami, J., Tsuchihashi, H., Baba, S., Mano, F., Maruyama, alHigh-affinity sites and the pA2 values in guinea pig K. and Nagatomo, T.: al-Adrenoceptor subtypes in the rat ven aorta, as compared to that in the rat aorta. There was no tricular muscle. J. Pharm. Pharmacol. 44, 97-100 (1992) 3 Shiratsuchi, M., Kawamura, K., Akashi, T., Ishihama, H. and serotonergic receptors. Japan. J. Pharmacol. 45, 349-356

Uchida, Y.: Synthesis and hypertensive and hypotensive activity (1987) of benzopyran derivatives. Chem. Pharm. Bull. (Tokyo) 35, 17 Howe, R. and Shanks, R.G.: Optical isomers of .

632-641 (1987) Nature 210, 1336-1338 (1966) 4 Shiratsuchi, M., Kawamura, K., Akashi, T., Ishihama, H., 18 Weinstock, L.M., Mulvery, D.M. and Tull, R.: Synthesis of the

Nakamura, M. and Takenaka, F.: Synthesis and activity of opti -adrenergic blocking agent timolol from optically active precur ƒÀ

cal isomers of nipradilol. Chem. Pharm. Bull. (Tokyo) 35, sors. J. Org. Chem. 41, 3121-3125 (1976) 3691-3698 (1987) 19 Dukes, M. and Smith, L.H.: jS-Adrenergic blocking agents. 9.

5 Tsuchihashi, H., Nakashima, Y., Yokoyama, H., Kinami, J. Absolute configuration of propranolol and a number of related

and Nagatomo, T.: Assessments of nipradilol and its isomers by aryloxypropanolamines and arylethanolamine. J. Med. Chem.

radioligand binding assay using 125I and 3H 14, 326-328 (1971)

CGP-12177 for ƒÀ1-,ƒÀ2-adrenoceptors and 5HT1B-serotonergic - 20 Danilewicz, J.C. and Kemp, J.E.G.: Absolute configuration

receptors. Asia Pacific J. Pharmacol. 5, 33-38 (1990) by asymmetric synthesis of (+)-1-(4-acetamidophenoxy)-3

6 Ebihara, A., Kondoh, K. and Ohashi, K.: Pharmacodynamics (isopropylamino)-propan-2-ol ().- J. Med. Chem. 16, and pharmacological effects of nipradilol (K-351) in healthy 168-171 (1973)

volunteers -Comparison with propranolol-. Rinsyo Yakuri 21 Flavahan, N.A. and Vanhoutte, P.M.: ƒ¿1-Adrenoceptor sub

(Japan. J. Pharmacol. Ther.) 17, 391-401 (1986) (Abs. in En classification of vascular smooth muscle. Trends Pharmacol.

glish) Sci. 7, 347-349 (1986) 7 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.: 22 Morrow, A.L. and Creese, I.: Characterization of ƒ¿1-adrenergic

Protein measurement with the Folin phenol reagent. J. Biol. receptor subtypes in rat brain: A reevaluation of [3H]WB4101

Chem. 193, 265-275 (1951) and [3H]prazosin binding. Mol. Pharmacol. 29, 321-330 (1986) 8 Tsuchihashi, H., Sasaki, M. and Nagatomo, T.: Binding charac 23 Han, C., Abel, P.W. and Minneman, K.P.: ƒ¿1-Adrenoceptor

teristics of [3H]-dihydroalprenolol to ƒÀ-adrenergic receptors of subtypes linked to different mechanisms for increasing intra

rat brain: Comparison with those of rat heart treated with neu cellular Ca2+ in smooth muscle. Nature 329, 333-335 (1987) raminidase. Chem. Pharm. Bull. (Tokyo) 33, 3972-3976 (1985) 24 Han, C., Abel, P.W. and Minneman, K.P.: Heterogeneity of

9 Tsuchihashi, H. and Nagatomo, T.: Characterization of 3H-di -adrenergic receptors revealed by chlorethylclonidine. Mol. ƒ¿1

ydroalprenolol binding to ƒÀ-adrenergic receptors of rat hbrain: Pharmacol. 32, 505-510 (1987) Two binding sites of racemic propranolol in displacement ex 25 Muramatsu, I., Ohmura, T., Kigoshi, S., Hashimoto, S. and

periments. Chem. Pharm. Bull. (Tokyo) 35, 2979-2984 (1987) Oshita, M.: Pharmacological subclassification of ƒ¿1-adrenocep 10 Tsuchihashi, H., Nagatomo, T. and Imai, S.: Three binding tors in vascular smooth muscle. Br. J. Pharmacol. 99, 197-201

sites of 125I-iodocyanopindolol, i.e. ƒÀ1,ƒÀ2-adrenergic and (1990) 5HT1B-serotonergic receptors in rat brain determined by the dis 26 Muramatsu, I., Kigoshi, S. and Ohmura, T.: Subtypes of ƒ¿1-

placement and Scatchard analysis. J. Pharmacobiodyn. 12, adrenoceptors involved in noradrenaline-induced contractions 509-516 (1989) of rat thoracic aorta and dog carotid artery. Japan. J. Pharma

11 Tsuchihashi, H., Yokoyama, H. and Nagatomo, T.: Binding col. 57, 535-544 (1991) characteristics of 3H-CGP12177 to ƒÀ-adrenoceptors in rat myo 27 Han, C., Li, J. and Minneman, K.P.: Subtypes of ƒ¿1-adrenocep

cardial membranes. Japan. J. Pharmacol. 49, 11-19 (1989) tors in the rat blood vessels. Eur. J. Pharmacol. 190, 97-104

12 Tsuchihashi, H., Nakashima, Y., Kinami, J. and Nagatomo, (1990) T.: Characteristics of 125I-iodocyanopindolol to ƒÀ-adrenergic 28 Lomasney, J.W., Cotecchia, S., Lorenz, W., Leung, W.-Y.,

and serotonin-1B receptors of rat brain: Selectivity of ƒÀ-adren Schwinn, D.A., Yang Feng, T.L., Brownstein, M., Lefkowitz,

ergic agents. Japan. J. Pharmacol. 52, 195-200 (1990) R.J. and Caron, M.G.: Molecular cloning and expression of the

13 Nagatomo, T., Tsuchihashi, H., Sasaki, S., Nakagawa, Y., cDNA for the ƒ¿1A-adrenergic receptor. The gene for which is lo

Nakahara, H. and Imai, S.: Displacement by ƒ¿-adrenergic cated on human chromosome 5. J. Biol. Chem. 266, 6365-6369

agonists and antagonists of 3H-prazosin bound to the ƒ¿ (1991) adrenoceptors of the dog aorta and the rat brain. Japan.- J. 29 Cotecchia, S., Schwinn, D.A., Randall, R.H., Lefkowitz, R.J.,

Pharmacol. 37, 181-187 (1985) Caron, M.G. and Kobilka, B.: Molecular cloning and expres

14 Nakagawa, Y., Shimamoto, N., Nakazawa, M. and Imai, S.: sion of the cDNA for the hamster ƒ¿1-adrenergic receptor. Proc.

Alpha and beta-blocking activities of racemates of labetalol. Natl. Acad. Sci. U.S.A. 85, 7159-7163 (1988)

Japan. J. Pharmacol. 30, 743-745 (1980) 30 Schwinn, D.A., Lomasney, J.W., Lorenz, W., Szklut, P.J.,

15 Nagatomo, T., Tsuchihashi, H., Sasaki, M., Nakagawa, Y., Fremeau, R.T., Yang-Feng, T.L., Caron, M.G., Lefkowitz,

Nakahara, H. and Imai, S.: Beta-receptor blocking potencies R.J. and Cotecchia, S.: Molecular cloning and expression of the

of the three newly synthesized (3-adrenergic antagonists (S-596, cDNA for a novel ƒ¿1-adrenergic receptor subtype. J. Biol.

K-351, N-696) as assessed with the radioligand binding assay Chem. 265, 8183-8189 (1990)

method in cardiac muscle membrane treated with neuramini 31 Perez, D.M., Piascik, M.T. and Graham, R.M.: Solution dase. Japan. J. Pharmacol. 34, 249-254 (1984) phase library screening for the identification of rare clones: Iso 16 Tsuchihashi, H., Aono, J., Nagatomo, T., Kawada, T., Ohta, lation of an ƒ¿1D-adrenergic receptor cDNA. Mol. Pharmacol. H. and Imai, S.: Effects of on adrenergic and 40, 876-883 (1991)