J. Smooth Muscle Res. 33 : 99-106. 99

The )32 and [33-Adrenoceptor-Mediated Relaxation Induced by and in Guinea Pig Taenia Caecum Katsuo KOIKE, Tsukasa IcHiNo, Takahiro HORINOUCHI and Issei TAKAYANAGI Departmentof Chemical Pharmacology, Toho University School of PharmaceuticalSciences, 2-2-1, Miyama,Funabashi, Chiba 274, Japan

Abstract

To understand the receptor subtypes responsible for /3adrenoceptormediated relaxa tion of guinea pig taenia caecum, we investigated the effects of isoprenaline and salbutamol . Isoprenaline and salbutamol caused dose-dependent relaxation of the guinea pig taenia caecum. , bupranolol and butoxamine produced shifts of the concentration response curves for isoprenaline and salbutamol. Schild regression analyses carried out for propranolol against isoprenaline and salbutamol gave pA2 values of 8.43 and 8.88, respective ly. Schild regression analyses carried out for butoxamine against isoprenaline and sal butamol gave pA2 values of 6.46 and 6.68, respectively. Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 8.60 and 8.69, respectively. However, in the presence of 3 x 10' M , 10-4 M butoxamine and 10-6 M to block the fir , /32 and a -adrenoceptor effects, respectively, Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 5.77 and 5.97, respectively. These results suggest that the relaxant responses to isoprenaline and salbutamol in the guinea pig taenia caecum are mediated by both the /.32 and the A-adrenoceptors.

Key words : f2-adrenoceptor, A-adrenoceptor, isoprenaline, salbutamol , guinea pig taenia caecum

Introduction

The /3adrenoceptors were subclassified as and /32subtypes based on the agonist potency and tissue localization. Lands et al. (1967) examined the relative potency of sympath omimetic amines on fatty acid mobilization (lipolysis), cardiac stimulation , vasodepression, and bronchodilation and designated the receptors mediating lipolysis and cardiac stimulation as /31, whereas those linked to bronchodilation and vasodilation as fl2. Recently, existence of the /33 adrenoceptor has been suggested in different tissues by binding and functional studies (Arch and

Correspondence to : Katsuo Koike, Depatment of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, 2-2-1, Miyama, Funabashi, Chiba 274, Japan Phone : 0474-72-1435 Fax : 0474-72-1448 100 K. KOIKE et al.

Kaumann, 1993). A large body of evidence indicates that the ƒÀ3-adrenoceptors occur in the intestinal smooth muscle of different species, including humans, with a function of inhibiting muscle contractility (Bianchetti and Manara, 1990; De Ponti et al., 1996).

We had demonstrated that the ƒÀ2- and ƒÀ3-adrenoceptors are involved in the ƒÀ-adrenoce ptor-mediated relaxation of the guinea pig taenia caecum (Koike at al., 1994; 1995a; 1995b), although the ƒÀ1-adrenoceptors are not involved (Koike et al., 1994). Our previous studies also showed that the relaxant responses to BRL37344, CGP12177 and in the guinea pig taenia caecum are mediated by ƒÀ3-adrenoceptors (Koike et al., 1995b; 1995c; 1997). However, it is not known whether the ƒÀ3-subtype is involved in the ƒÀ-adrenoceptor-mediated relaxation induced by isoprenaline, a typically ƒÀ-adrenoceptor agonist, and salbutamol, a ƒÀ2- adrenoceptor selective agonist. Therefore, we have studied in detail the ƒÀ-adrenoceptor mediated relaxation of guinea pig taenia caecum by measuring the potencies of isoprenaline and salbutamol.

Materials and methods

Mechanical responses

Male guinea pigs weighing 300-500g were killed by cervical dislocation and a 2 to 3-cm piece of the taenia caecum was isolated and suspended in a 20-ml organ bath filled with a Ringer-Locke solution (NaCl, 154; KCl, 5.6; CaCl2, 2.2; MgCl2, 2.1; NaHCO3, 5.9 and glucose,

2.8mM) kept at 32•Ž and bubbled with a mixture of 95% O2 and 5% CO2. The mechanical responses of the smooth muscle preparations were recorded isotonically under a tension of 0.7 g. The experiments were started after the preparations had been allowed to develop their

spontaneous tone for 2h. The concentration-response curves for the agonists were obtained

cumulatively and the relaxation induced by these drugs was expressed as a percentage of the

maximal relaxation produced by 3•~10-7M isoprenaline, the reference drug. To test the

antagonism, one of the antagonists was added to the bath 30min before the addition of the

agonist. The concentration-response curves for the agonist were then obtained in the presence

of an antagonist. The time interval between two consecutive curves was usually set at 60min.

The spontaneous smooth muscle tone was reproducible when taenia caecum pieces were

without the load. In our previous experiments, after the control concentration-response

curves were determined, two or three successive cumulative concentration-response curves for

isoprenaline were determined. The curves were nearly superimposable and changes in sensi

tivity (sensitization or desensitization) were slight (data not shown). Six or more concentra

tion-response curves could be made in succession. In some experiments, atenolol (3•~10-4M),

butoxamine (10-4M) and phentolamine (10-6M) were added to inhibit the ƒÀ1-, ƒÀ2- and ƒ¿-

adrenoceptors, respectively. Agonistic potency was expressed as the pD2 value (Van Rossum,

1963). The competitive antagonistic potency was expressed as the pA2 value. It was calcu

lated according to the method of Tallarida et al. (1979), which was originally described by

Arunlakshana and Schild (1959). ƒÀ2- and ƒÀ3-Receptor-mediated Relaxation 101

Data analysis

Numerical results are expressed as means•}S.E. and statistical analyses wereperformed with the Newman-Keuls test when appropriate. A P value of less than 0.05 was considered significant.

Drugs

The drugs used were obtained from the following sources: isoprenaline hydrochloride , salbutamol hemisulfate salt, butoxamine hydrochloride, propranolol hydrochloride (Sigma

Chemical Co., St. Louis, MO, U.S.A.); bupranolol hydrochloride (Looser; Kaken Seiyaku Co.,

Tokyo, Japan); atenolol (Research Biochemicals, Natik, MA , U.S.A.); hydrochloride, yohimbine hydrochloride (Wako Pure Chemical Industries, Osaka, Japan); and phentolamine mesylate (Ciba Geigy, Basel, Switzerland). All the drugs were dissolved in distilled water . The other chemicals used were of analytical grade.

Results

Responses to isoprenaline

Isoprenaline caused graded relaxation of the guinea pig taenia caecum piece in which the tone had been raised spontaneously, with the pD2 value of 8.14•}0.06 (Fig.1). Propranolol

(10-8-10-7M) competitively antagonized the relaxant responses to isoprenaline (Fig.1). The

Schild plot of the data gave the pA2 value of 8.43•}0.11 and the slope of the regression line

(1.17•}0.07) was not significantly different from unity. Bupranolol (10-8-10-7M) caused com petitive antagonism of the relaxant responses (Fig.2). The Schild plot of the data gave the pA2 value of 8.60•}0.08 and the slope of the regression line (1.06•}0.02) was not significantly different

Fig. 1. Antagonism of isoprenaline-induced relaxation by propranolol. Control (•œ), propranolol 10-8M (•›), 3•~10-8M (•¢), 10-7M (• ). Ordinate: relaxation (%), expressed as percentage of the maximum relaxation induced by isoprenaline, and abscissa: concentration (M) of isoprenaline. Each point represents the mean•}S.E. of six experiments. 102 K. KOIKE et al.

Fig. 2. Antagonism of isoprenaline-induced relaxation by bupranolol. Control (•œ), bupranolol 10-8 M (•›), 3•~10-8M (•¢), 10-7M (• ). Ordinate: relaxation (%), expressed as percentage of the maximum relaxation induced by isoprenaline, and abscissa: concentration (M) of isoprenaline. Each point represents the mean•}S.E. of six experiments.

from unity. Butoxamine (10-6-10-5M), a ƒÀ2-selective antagonist, caused competitive antago nism of the relaxant responses (data not shown). The Schild plot of the data gave the pA2 value of 6.46•}0.06 and the slope of the regression line (0.99•}0.13) was not significantly different from unity. In the presence of 3•~10-4M atenolol, 10-4M butoxamine and 10-6M phentolamine to block the ƒÀ1, ƒÀ2 and ƒ¿-adrenoceptor effects, respectively, bupranolol (3•~ 10-6-3•~10-5M) also caused competitive antagonism of the relaxant responses (Fig.3). How

Fig. 3. Antagonism of isoprenaline-induced relaxation by bupranolol in the presence of 3•~10-4M atenolol, 10-4M butoxamine and 10-6M phentolamine. Isoprenaline, in the absence of blockers (•œ), isoprenaline in the presence of blockers (•¡), bupranolol 3•~10-6M (•›), 10-5M

(•¢), 3•~10-5M (• ). Ordinate: relaxation (%), expressed as percentage of the maximum relaxation induced by isoprenaline, and abscissa: concentration (M) of isoprenaline. Each

point represents the mean•}S.E. of six experiments. ƒÀ2- and ƒÀ3-Receptormediated Relaxation 103 ever, the Schild plot of the data gave the pA2 value of 5.77•}0.04 and the slope of the regression line (1.14•}0.09) was not significantly different from unity. The difference between the pA2 values for bupranolol in the presence and in the absence of the ƒÀ1-, ƒÀ2- and ƒ¿-blockers was statistically significant (P<0.05).

Responses to salbutamol

Salbutamol caused graded relaxation of the guinea pig taenia caecum piece in which the tone had been raised spontaneously, with the pD2 value of 7.21•}0.05 (Fig.4). Propranolol

(10-8-10-7M) competitively antagonized the relaxant responses to salbutamol (Fig.4). The

Schild plot of the data gave the pA2 value of 8.88•}0.04 and the slope of the regression line

(1.12•}0.08) was not significantly different from unity. Bupranolol (10-8-10-7M) caused com petitive antagonism of the relaxant responses (Fig.5). The Schild plot of the data gave the pA2 value of 8.69•}0.07 and the slope of the regression line (1.27•}0.13) was not significantly different from unity. Butoxamine (10-6-10-5M), a ƒÀ2-selective antagonist, caused competitive antago nism of the relaxant responses (data not shown). The Schild plot of the data gave the pA2 value of 6.68•}0.09 and the slope of the regression line (1.09•}0.06) was not significantly different from unity. In the presence of 3•~10-4M atenolol, 10-4M butoxamine and 10-6M phentolamine to block the ƒÀ1-, ƒÀ2- and ƒ¿-adrenoceptor effects, respectively, bupranolol (3•~ 10-6-10-5M) also caused competitive antagonism of the relaxant responses (Fig.6). However, the Schild plot of the data gave the pA2 value of 5.97•}0.05 and the slope of the regression line

(1.10•}0.08) was not significantly different from unity. Thedifference between the pA2 values for bupranolol in the presence and in the absence of the ƒÀ1-, ƒÀ2- and ƒ¿-blockers was statisti cally significant (P<0.05).

Fig. 4. Antagonism of salbutamol-induced relaxation by propranolol. Control (•œ), propranolol

10-8M (•›), 3•~10-8M (•¢), 10-7M (• ). Ordinate: relaxation (%), expressed as percentage of the maximum relaxation induced by isoprenaline, and abscissa: concentration (M) of salbutamol. Each point represents the mean•}S.E. of six experiments. 104 K. KOIKE et al.

Fig. 5. Antagonism of salbutamol-induced relaxation by bupranolol. Control (•œ), bupranolol 10-8M (•›), 3•~10-8M (•¢), 10-7M (•¢). Ordinate: relaxation (%), expressed as percentage of the maximum relaxation induced by isoprenaline, and abscissa: concentration (M) of salbutamol. Each point represents the mean•}S.E. of six experiments.

Fig. 6. Antagonism of salbutamol-induced relaxation by bupranolol in the presence of 3•~10-4M atenolol, 10-4M butoxamine and 10-6M phentolamine. Salbutamol in the absence of blockers (•œ), salbutamol in the presence of blockers (•¡), bupranolol 3•~10-6M (•›), 10-5M

(•¢). Ordinate: relaxation (%), expressed as percentage of the maximum relaxation induced by isoprenaline, and abscissa: concentration (M) of salbutamol. Each point represents the mean•}S.E. of six experiments.

Discussion

Previously, we have demonstrated that both the ƒÀ2- and the ƒÀ3-adrenoceptors are involved in the ƒÀ-adrenoceptor-mediated relaxation of the guinea pig taenia caecum (Koike et al., 1994;

1995a; 1995b). In the present studies, isoprenaline and salbutamol-induced relaxations of the ƒÀ2 and ƒÀ3-Receptormediated Relaxation 105 guinea pig taenia caecum were antagonized in a concentration-dependent manner by butox amine, a ƒÀ2-selective antagonist. The pA2 values for butoxamine calculated from the Schild plot (6.46 and 6.68, respectively) are in good agreement with the generally accepted value (6.20)

(Koike et al., 1994), indicating a predominance of ƒÀ2-adrenoceptors.

Propranolol and bupranolol antagonized the responses to isoprenaline and salbutamol in a concentration-dependent manner. The pA2 values for propranolol and bupranolol are in good agreement with the generally accepted value. Moreover, in the presence of 3•~10-4M atenolol,

10-4M butoxamine and 10-6M phentolamine to block the ƒÀ1-, ƒÀ2- and ƒ¿-adrenoceptor effects, respectively, bupranolol also shifted the concentration-response curves for isoprenaline and salbutamol. However, a Schild regression analysis carried out for bupranolol against iso prenaline and salbutamol gave pA2 values of 5.77 and 5.97, respectively, which were significantly different from the values (8.60 and 8.69, respectively) obtained in the absence of the blockers. Bupranolol was used in our study since it acts as an antagonist at the ƒÀ3-adrenoce ptors (Arch and Kaumann, 1993; Blin et al., 1993), although at concentrations much higher than those necessary for the blockade of the ƒÀ1- or the ƒÀ2-adrenoceptors (Kaumann, 1996; Kaumann and Molenaar, 1996; Malinowska and Schlicker, 1996). It has been suggested that, at low concentrations (nM), bupranolol is a non-selective ƒÀ1 and ƒÀ2-adrenoceptor antagonist and that at high concentrations (ƒÊM), bupranolol is a selective ƒÀ3-adrenoceptor antagonist (Kaumann,

1989). These results suggest that the relaxant response of the guinea pig taenia caecum to isoprenaline and salbutamol may be mediated by the ƒÀ3-adrenoceptors in the presence of ƒÀ1-, ƒÀ

2- and ƒ¿-blockers.

The main objective of the present studies was to clarify whether ƒÀ3-subtype is involved in the ƒÀ-adrenoceptor-mediated relaxation induced by isoprenaline and salbutamol in the guinea pig taenia caecum. Our results suggest that the relaxant responses to isoprenaline and salbutamol in the guinea pig taenia caecum are mediated by both the ƒÀ2- and the ƒÀ3-adrenoce ptors.

References

Arch, J.R.S. and Kaumann, A.J. (1993). ƒÀ3- and atypical ƒÀ-adrenoceptors. Med. Res. Rev. 13: 663

729. -

Arunlakshana, O. and Schild, H.O. (1959). Some quantitative uses of drug antagonists. Br. J.

Pharmacol. Chemother. 14: 48-58.

Bianchetti, A. and Manara, L. (1990). In vitro inhibition of intestinal motility by phenyleth

anolaminotetralines: evidence of atypical beta-adrenoceptors in rat colon. Brit. J. Phar

macol. 100: 831-839.

Blin, N., Camoin, L., Maigret, B. and Strosberg, A.D. (1993). Structural and conformational features

determining selective signal transduction in the ƒÀ3- receptor. Mol. Pharmacol. 44:

1094-1104.

De Ponti, F., Gibelli, G., Croci, T., Arcidiaco, M., Crema, F. and Manara, L. (1996). Functional

evidence of atypical ƒÀ3-adrenoceptors in the human colon, using the ƒÀ3 selective adrenoceptor

antagonist, SR59230A. Brit. J. Pharmacol. 117: 1374-1376.

Kaumann, A.J. (1989). Is there a third heart ƒÀ-adrenoceptor? Trends Pharmacol. Sci. 10: 316-320.

Kaumann, A.J. (1996). (-)-CGP12177-induced increase of human atrial contraction through a 106 K. KOIKE et al.

putative third fl-adrenoceptor. Br. J. Pharmacol. 117 : 93-98. Kaumann, A.J. and Molenaar, P. (1996). Differences between the third cardiac fl-adrenoceptor and the colonic A-adrenoceptor in the rat. Br. J. Pharmacol. 118 : 2085-2098. Koike, K., Takayanagi, I., Muramatsu, M., Ohki, S. and Horinouchi, T. (1994). Involvement of /33 adrenoceptor in the relaxation response in guinea pig taenia caecum. Jpn. J. Pharmacol. 66 : 213-220. Koike, K., Horinouchi, T. and Takayanagi, I. (1995a). Signal transduction pathway involved in J33 adrenoceptor-mediated relaxation in guinea pig taenia caecum. Jpn. J. Pharmacol. 68 : 41 46. Koike, K., Horinouchi, T. and Takayanagi, I. (1995b). Possible mechanisms of /3-adrenoceptor mediated relaxation induced by noradrenaline in guinea pig taenia caecum. Eur. J. Phar macol. 279 : 159-163. Koike, K., Horinouchi, T. and Takayanagi, I. (1995c). Effect of bupranolol on CGP12177-induced relaxation and cAMP accumulation in the guinea pig taenia caecum. Gen. Pharmacol. 26 : 1791-1794. Koike, K., Takayanagi, I., Ichino, T., Koshikawa, H. and Nagatomo, T. (1997). A-adrenoceptor mediate relaxation of guinea pig taenia caecum by BRL37344A and BRL35135A. Eur. J. Pharmacol. 334 : 217-221 Lands, A.M., Arnold, A., McAnliff, J.P., Luduena, F.P. and Brown, T.G. (1967). Differentiation of the receptor systems activated by sympathomimetic amines. Nature 214 : 597-598. Malinowska, B. and Schlicker, E. (1996). Mediation of the positive chronotropic effect of CGP12177 and in the pithed rat by atypical fl-adrenoceptors, different from A-adrenoce ptors. Brit. J. Pharmacol. 117 : 943-949. Tallarida, R.J., Cowan, A. and Adler, M.W. (1979). PA2 and receptor differentiation : a statistical analysis of competitive antagonism. Life Sci. 25 : 637-654. Van Rossum, J.M. (1963). Cumulative dose-response curves, II. Technique for the making of dose response curves in isolated organs and the evaluation of drug parameters. Arch. Mt. Pharmacodyn. 143 : 299-330.

(Received September 1, 1997 Accepted October 1, 1997)