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A beta-adrenoceptor blocking agent, befunolol as a partial agonist in isolated organs
Gen. Pharmac. Vol. 16, No. 3, pp. 265-267, 1985 Printed in Great Britain. All rights reserved
0306-3623/85$3.00+ 0.00 Copyright © 1985 Pergamon Press Ltd
A BETA-ADRENOCEPTOR BLOCKING AGENT, BEFUNOLOL AS A PARTIAL AGONIST IN ISOLATED ORGANS I. TAKAYANAGI and K. KOIKE Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba 274, Japan Tel. (0474) 72-I141 (Received 6 August 1984)
Abstract--1. A beta-adrenoceptor blocking agent, befunolol, which is clinically used in the treatment of glaucoma in Japan, was found to have intrinsic sympathomimetic activities in isolated right atria, trachea and taenia caecum of guinea pig (intrinsic activities are 0.22-0.28). 2. The pD2-values of befunolol estimated in the isolated organs were significantly different from its pA2-values against isoprenaline. 3. A possible explanation of our findings would to be as follows: befunolol interacts with the beta-adrenoceptor where there may be two different sites: one site for agonistic action and the other for competitive antagonistic action. Both sites are supposed to be mutually exclusive through unknown mechanisms. The intrinsic activity of befunolol may be equal to its selectivity for both the sites.
INTRODUCTION It is well known that partial agonists have an affinity to their receptors, an intermediate intrinsic activity and that the partial agonists have little receptor reserve (Stephenson, 1956; Nickerson, 1956). Betaadrenergic partial agonists (fl-adrenoceptor blocking agents with intrinsic sympathomimetic activity) also have little spare receptor (Koike and Takayanagi, 1983; Takayanagi and Koike, 1983; Takayanagi et al., 1984). However, p D : v a l u e s (indexes for agonistic activity) of some fl-adrenergic partial agonists are significantly different from their pA2-values (indexes for competitive antagonistic activity) against isoprenaline, suggesting that interactions of the partial agonists with the fl-adrenoceptor cannot be understood by the classical receptor theory (Takayanagi and Koike, 1983; Koike and Takayanagi, 1984; Takayanagi et al., 1984). A fl-adrenoceptor blocking agent, befunolol [2acetyl-7-(2-hydroxy-3-isopropylaminopropoxy) benzofuran hydrochloride] is used clinically in the treatment of glaucoma in Japan. As befunolol was found to have an intrinsic sympathomimetic activity in this study, we investigated an interaction of befunolol with the fl-adrenoceptor in isolated cardiac, tracheal and intestine muscles in order to study its mechanisms of action. METHODS AND MATERIALS Male guinea pigs, weighing 250-350 g, were killed by a blow on the head. The heart, trachea and taenia caecum were removed as quickly as possible. 1. Right atria An isolated heart was placed in Ringer-Locke solution gassed with carbogen and kept at 37°C. All other tissue was cut away until nothing was left except the right atria. The preparation was suspended in Ringer-Locke solution at 37°C, through which a brisk stream of carbogen was blown. Chronotropic and inotropic effects were recorded isometrically under a resting tension of 0.5 g.
2. Tracheal preparation and taenia caecum A tracheal preparation was made according to Takagl and Takayanagi (1958). A piece of taenia caecum was isolated and used as the intestinal preparation. The tracheal preparation or a piece (about 4 cm) of taenia caecum was suspended in the 20 ml organ bath filled with Ringer-Locke solution kept at 37°C and gassed with carbogen. Responses to drugs were recorded isotonically under a tension of 0.7 g. The experiments were started after the preparations were allowed to develop their spontaneous tone for 1 hr. A composition of Ringer-Locke used was 154 mM NaCI, 5.6mM KC1, 2.2mM CaCI2, 2.1mM MgCI2, 5.9raM NaHCOa and 2.8 mM glucose. Concentration-action curves of drugs were obtained cumulatively. In order to test antagonism between an agonist and antagonist, the preparations were preincubated with the antagonist for 5 min. Agnnistic and competitive antagonistic activities of befunolol were expressed as PD2- and PA2-values, which were calculated according to the tables of Van Rossum (1963). The intrinsic activity was expressed as a ratio between the maximum response to a test drug and that to isoprenaline, a reference drug. Drugs used Befunolol hydrochloride (Kaken-Seiyaku), isoprenaline hydrochloride (Sigma) and propranolol hydrochloride (Sumitomo), all in powder form.
RESULTS 1. Right atria
Befunolol increased chronotropic and inotropic actions. Concentration-action curves of befunolol were shown in Figs 1 and 2. Each maximum response to befunolol was abolished by propranolol 0 0 -6 M) (data not shown) suggesting that befunolol had intrinsic sympathomimetic activity. Isoprenaline increased the chronotropic and inotropic actions greatly. Both the maximum responses to befunolol were much less than those to isoprenaline (Table 1). Both the concentration-action curves of isoprenaline , were shifted in parallel by befunolol (10 -s M). T h e / concentration 0 0 -8 M) used of befunolol induced a/
265
I. TAKAYANAGIand K. KOIKE
266 100
¢/'"
/ ,~ • "o ~ o ca
10-10
10 9
10-8
/.,.o0r.oo n. oon.
l
/ 50
M
~ ~,~::,_---o
f
/
[
Oil ",
0 Isoprer~line
-e with befuno,o, 10 M V Befunolol alone
/
10-7
10-9
10-8
10-7
10-6
10-5
M
Fig. 1. Concentration chronotropic action curves of isoprenaline in the absence and presence of befunolol, and of befunolol alone. Ordinate: percent of the maximum heart rates induced by isoprenaline. Abscissa: concentration (M) of drug. O: isoprenaline alone, O: isoprenaline with befunolol (10 -~ M), and ~ : befunolol alone. Each point is presented as a mean +_.SE of 6 experiments with guinea pig right atria.
lOO
-~
/
E
tJ " S
/--°
,~
• .~
//
~ 50
g
/
/
//
/
renaline alone
0 Isopreneline
R
with befunolol IO--M
D[
V Befunolol (]lone
./
10 5
to pt i
oR
o /10~0
10_6
IO_eM
n II
10C
Fig. 3. Concentration relaxation curves of isoprenaline in the absence and presence of befunolol, and of befunolol alone. Ordinate: percent of the maximum relaxation induced by isoprenaline and abscissa: concentration (M) of drug. O: isoprenaline alone, O: isoprenaline with befunolol (10 -~ M) and ~ : befunolol alone. Each point is presented as a means ___SE of 6 experiments with guinea pig tracheal preparation. was much less than that to isoprenaline in each muscle (Table 2). The concentration-action curve of isoprenaline in each muscle was shifted in parallel by 10 -8 M of befunolol, which induced a slight relaxation (Fig. 3), and by propranolol (10 -7 M) (data not shown). The pD2- and pA2-values estimated in the tracheal preparation and taenia caecum are summarized in Table 2.
0
10-10
10-9
10--8
10--7
10--6
10--5
M
Fig. 2. Concentration inotropic action curves of isoprenaline in the absence and presence of befunolol, and of befunolol alone. Ordinate: percent of the maximum tension increased by isoprenaline. Abscissa: concentration (M) of drug. O: isoprenaline alone, ©: isoprenaline with befunolol (10 -8 M), and ~ : befunolol alone. Each point is presented as a mean +_SE of 6 experiments with the guinea pig right atria. slight sympathomimetic action in the right atria (Figs 1 and 2). The PA2-values of befunolol against isoprenaline, which were calculated from the parallel shift of each curve, the pD2-values and intrinsic activities of befunolol are summarized in Table l. Propranolol (3 × 10 8 M) was competitively antagonized by the chronotropic and inotropic actions of isoprenaline (data not shown). The pD2-values of isoprenaline and the pA2-values of propranolol were also summarized in Table 1.
2. Tracheal preparation and taenia caecum Befunolol relaxed the spontaneously contracted tracheal preparation (Fig. 3) and taenia caecum. Each maximum response to befunolol, which was abolished by propranolol (10 -6 M) (data not shown)
DISCUSSION Befunolol had low intrinsic activities and antiisoprenaline actions in all the organs used (Tables 1 and 2). These results indicate that befunolol is the partial agonist (the /~-adrenoceptor blocking agent with intrinsic sympathomimetic activity) on the /~-adrenoceptor. Some fl-adrenergic partial agonists have already been found to exert agonistic effects in concentrations higher than those required for/~-adrenoceptor blockade in cardiac muscle ( K a u m a n n and Blinks, 1980), tracheal preparation and taenia caecum (Takayanagi and Koike, 1983; Koike and Takayanagi, 1984; Takayanagi et el., 1984). In the present study, the PD2-values of befunolol were significantly different from its pA2-values in all the test organs. The data cited above were confirmed in the present experiments with befunolol. If befunolol, a partia ! agonist, interacted with one site in the B-adrenoceptor, the pD2-values would be equal to the pA2-values of befunolol, which had little receptor reserve. However, the pD2- and pA2-values of befunolol were significantly different from each other. A possible interaction of befunolol with the fl-adrenoceptor would be as follows. The
Table 1. The intrinsic activities, pD2- and pA2-valuesfor the test drugs estimated in the guinea pig right atria (chronotropic and inotropic actions). Right atria Chronotropic Inotropic Intrinsic Intrinsic activity pD2.value pA2-value activity pD2-value PA2-value Befunolol 0.22 + 0.02 7.08 + 0.17 9.01 _ 0.08 0.24 + 0.02 7.07 __+0.11 8.99 4-0.04 Isoprenaline 1.00 8.76 4-0.02 1.00 8.75 + 0.04 Propranolol 0.00 8.72 4-0.21 0.00 8.62 + 0.32 Mean 4- SE of 6 experiments.
Befunolol as r-blocker with ISA
267
Table 2. The intrinsic activities,pD2-and pA2-valuesfor the test drugs estimated in the guinea pig tracheal preparation and taenia caecum Tracheal preparation Taenia caecum Intrinsic Intrinsic activity pDz-value pA2-value activity pD2-value pA2-value 7.21+_0.13 9.13+_0.11 0.28+_0.03 7.06+_0.16 9.22+_0.10 Befunolol 0.28 +_0.06 8.62 +_0.18 1.00 8.59 +_0.06 Isoprenaline 1.00 8.43 +_0.06 0.00 8.65 +_0.40 Propranolol 0.00 Mean +_SE of 6 experiments. fl-adrenoceptor has two binding sites: one binding site for agonistic action and the other site for antagonistic action. Both the binding sites are supposed to be mutually exclusive and produce their actions separately through unknown mechanisms (Takayanagi and Koike, 1983; Koike and Takayanagi, 1984; Takayanagi et al., 1984). The findings of Kaum a n n and Blinks (1980) also suggested that more than one type of fl-adrenoceptor might be involved in the interaction of fl-adrenergic partial agonists with their receptors in cardiac muscle. Therefore the intrinsic activity of befunolol may be equal to its selectivity for both the sites in the fl-adrenoceptor. Acknowledgement--The authors are grateful to KakenSeiyaku company for the generous supply of befunolol. REFERENCES
Kaumann A. J. and Blinks J. B. (1980) fl-Adrenoceptor blocking agents as partial agonists in isolated heart muscle: dissociation of stimulation and blockade. Naunyn-Schmiedebergs Archs Pharmac. 311, 237-248. Koike K. and Takayanagi I. (1983) Relationship between
intrinsic activity of fl-adrenoceptor agonist and amount of spare receptors in guinea pig taenia caecum. Jap. J. Pharmac. 33, 327-333. Koike K. and Takayanagi L (1984) Interaction of fl-adrenergic partial agonist with its receptor in guineapig taenia caecum. Gen. Pharmac. 15, 47-50. Nickerson M. (1956) Receptor ocupancy and tissue response. Nature 178, 697-698. Stephenson R. P. (1956) A modification of receptor theory. Br. J. Pharmac. 11, 379-393. Takagi K. and Takayanagi I. (1958) Chemicopharmacological studies on antispasmodic action XV. Non-specific antispasmodic action on tracheal muscle. Chem. Pharm. Bull. 6, 716-720. Takayanagi I. and Koike K. (1983) A possible mechanism of action of a beta-adrenergic partial agonist (carteolol) in guinea pig taenia caecum. J. Pharm. Dyn. 6, 56-59. Takayanagi I., Koike K. and Sato T. (1984) Interactions of some partial agonists with beta-adrenoceptor in the isolated taenia caecum and tracheal smooth muscle of guinea pig. Jap. J. Pharmac. 35, 301-305. Van Rossum J. M. (1963) Cumulative dose-response curve II. Techniques for the making of dose response curve in isolated organs and evaluation of drug parameters. Archs int. Pharmacodyn. Ther. 143, 299-330.
0306-3623/85$3.00+ 0.00 Copyright © 1985 Pergamon Press Ltd
A BETA-ADRENOCEPTOR BLOCKING AGENT, BEFUNOLOL AS A PARTIAL AGONIST IN ISOLATED ORGANS I. TAKAYANAGI and K. KOIKE Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba 274, Japan Tel. (0474) 72-I141 (Received 6 August 1984)
Abstract--1. A beta-adrenoceptor blocking agent, befunolol, which is clinically used in the treatment of glaucoma in Japan, was found to have intrinsic sympathomimetic activities in isolated right atria, trachea and taenia caecum of guinea pig (intrinsic activities are 0.22-0.28). 2. The pD2-values of befunolol estimated in the isolated organs were significantly different from its pA2-values against isoprenaline. 3. A possible explanation of our findings would to be as follows: befunolol interacts with the beta-adrenoceptor where there may be two different sites: one site for agonistic action and the other for competitive antagonistic action. Both sites are supposed to be mutually exclusive through unknown mechanisms. The intrinsic activity of befunolol may be equal to its selectivity for both the sites.
INTRODUCTION It is well known that partial agonists have an affinity to their receptors, an intermediate intrinsic activity and that the partial agonists have little receptor reserve (Stephenson, 1956; Nickerson, 1956). Betaadrenergic partial agonists (fl-adrenoceptor blocking agents with intrinsic sympathomimetic activity) also have little spare receptor (Koike and Takayanagi, 1983; Takayanagi and Koike, 1983; Takayanagi et al., 1984). However, p D : v a l u e s (indexes for agonistic activity) of some fl-adrenergic partial agonists are significantly different from their pA2-values (indexes for competitive antagonistic activity) against isoprenaline, suggesting that interactions of the partial agonists with the fl-adrenoceptor cannot be understood by the classical receptor theory (Takayanagi and Koike, 1983; Koike and Takayanagi, 1984; Takayanagi et al., 1984). A fl-adrenoceptor blocking agent, befunolol [2acetyl-7-(2-hydroxy-3-isopropylaminopropoxy) benzofuran hydrochloride] is used clinically in the treatment of glaucoma in Japan. As befunolol was found to have an intrinsic sympathomimetic activity in this study, we investigated an interaction of befunolol with the fl-adrenoceptor in isolated cardiac, tracheal and intestine muscles in order to study its mechanisms of action. METHODS AND MATERIALS Male guinea pigs, weighing 250-350 g, were killed by a blow on the head. The heart, trachea and taenia caecum were removed as quickly as possible. 1. Right atria An isolated heart was placed in Ringer-Locke solution gassed with carbogen and kept at 37°C. All other tissue was cut away until nothing was left except the right atria. The preparation was suspended in Ringer-Locke solution at 37°C, through which a brisk stream of carbogen was blown. Chronotropic and inotropic effects were recorded isometrically under a resting tension of 0.5 g.
2. Tracheal preparation and taenia caecum A tracheal preparation was made according to Takagl and Takayanagi (1958). A piece of taenia caecum was isolated and used as the intestinal preparation. The tracheal preparation or a piece (about 4 cm) of taenia caecum was suspended in the 20 ml organ bath filled with Ringer-Locke solution kept at 37°C and gassed with carbogen. Responses to drugs were recorded isotonically under a tension of 0.7 g. The experiments were started after the preparations were allowed to develop their spontaneous tone for 1 hr. A composition of Ringer-Locke used was 154 mM NaCI, 5.6mM KC1, 2.2mM CaCI2, 2.1mM MgCI2, 5.9raM NaHCOa and 2.8 mM glucose. Concentration-action curves of drugs were obtained cumulatively. In order to test antagonism between an agonist and antagonist, the preparations were preincubated with the antagonist for 5 min. Agnnistic and competitive antagonistic activities of befunolol were expressed as PD2- and PA2-values, which were calculated according to the tables of Van Rossum (1963). The intrinsic activity was expressed as a ratio between the maximum response to a test drug and that to isoprenaline, a reference drug. Drugs used Befunolol hydrochloride (Kaken-Seiyaku), isoprenaline hydrochloride (Sigma) and propranolol hydrochloride (Sumitomo), all in powder form.
RESULTS 1. Right atria
Befunolol increased chronotropic and inotropic actions. Concentration-action curves of befunolol were shown in Figs 1 and 2. Each maximum response to befunolol was abolished by propranolol 0 0 -6 M) (data not shown) suggesting that befunolol had intrinsic sympathomimetic activity. Isoprenaline increased the chronotropic and inotropic actions greatly. Both the maximum responses to befunolol were much less than those to isoprenaline (Table 1). Both the concentration-action curves of isoprenaline , were shifted in parallel by befunolol (10 -s M). T h e / concentration 0 0 -8 M) used of befunolol induced a/
265
I. TAKAYANAGIand K. KOIKE
266 100
¢/'"
/ ,~ • "o ~ o ca
10-10
10 9
10-8
/.,.o0r.oo n. oon.
l
/ 50
M
~ ~,~::,_---o
f
/
[
Oil ",
0 Isoprer~line
-e with befuno,o, 10 M V Befunolol alone
/
10-7
10-9
10-8
10-7
10-6
10-5
M
Fig. 1. Concentration chronotropic action curves of isoprenaline in the absence and presence of befunolol, and of befunolol alone. Ordinate: percent of the maximum heart rates induced by isoprenaline. Abscissa: concentration (M) of drug. O: isoprenaline alone, O: isoprenaline with befunolol (10 -~ M), and ~ : befunolol alone. Each point is presented as a mean +_.SE of 6 experiments with guinea pig right atria.
lOO
-~
/
E
tJ " S
/--°
,~
• .~
//
~ 50
g
/
/
//
/
renaline alone
0 Isopreneline
R
with befunolol IO--M
D[
V Befunolol (]lone
./
10 5
to pt i
oR
o /10~0
10_6
IO_eM
n II
10C
Fig. 3. Concentration relaxation curves of isoprenaline in the absence and presence of befunolol, and of befunolol alone. Ordinate: percent of the maximum relaxation induced by isoprenaline and abscissa: concentration (M) of drug. O: isoprenaline alone, O: isoprenaline with befunolol (10 -~ M) and ~ : befunolol alone. Each point is presented as a means ___SE of 6 experiments with guinea pig tracheal preparation. was much less than that to isoprenaline in each muscle (Table 2). The concentration-action curve of isoprenaline in each muscle was shifted in parallel by 10 -8 M of befunolol, which induced a slight relaxation (Fig. 3), and by propranolol (10 -7 M) (data not shown). The pD2- and pA2-values estimated in the tracheal preparation and taenia caecum are summarized in Table 2.
0
10-10
10-9
10--8
10--7
10--6
10--5
M
Fig. 2. Concentration inotropic action curves of isoprenaline in the absence and presence of befunolol, and of befunolol alone. Ordinate: percent of the maximum tension increased by isoprenaline. Abscissa: concentration (M) of drug. O: isoprenaline alone, ©: isoprenaline with befunolol (10 -8 M), and ~ : befunolol alone. Each point is presented as a mean +_SE of 6 experiments with the guinea pig right atria. slight sympathomimetic action in the right atria (Figs 1 and 2). The PA2-values of befunolol against isoprenaline, which were calculated from the parallel shift of each curve, the pD2-values and intrinsic activities of befunolol are summarized in Table l. Propranolol (3 × 10 8 M) was competitively antagonized by the chronotropic and inotropic actions of isoprenaline (data not shown). The pD2-values of isoprenaline and the pA2-values of propranolol were also summarized in Table 1.
2. Tracheal preparation and taenia caecum Befunolol relaxed the spontaneously contracted tracheal preparation (Fig. 3) and taenia caecum. Each maximum response to befunolol, which was abolished by propranolol (10 -6 M) (data not shown)
DISCUSSION Befunolol had low intrinsic activities and antiisoprenaline actions in all the organs used (Tables 1 and 2). These results indicate that befunolol is the partial agonist (the /~-adrenoceptor blocking agent with intrinsic sympathomimetic activity) on the /~-adrenoceptor. Some fl-adrenergic partial agonists have already been found to exert agonistic effects in concentrations higher than those required for/~-adrenoceptor blockade in cardiac muscle ( K a u m a n n and Blinks, 1980), tracheal preparation and taenia caecum (Takayanagi and Koike, 1983; Koike and Takayanagi, 1984; Takayanagi et el., 1984). In the present study, the PD2-values of befunolol were significantly different from its pA2-values in all the test organs. The data cited above were confirmed in the present experiments with befunolol. If befunolol, a partia ! agonist, interacted with one site in the B-adrenoceptor, the pD2-values would be equal to the pA2-values of befunolol, which had little receptor reserve. However, the pD2- and pA2-values of befunolol were significantly different from each other. A possible interaction of befunolol with the fl-adrenoceptor would be as follows. The
Table 1. The intrinsic activities, pD2- and pA2-valuesfor the test drugs estimated in the guinea pig right atria (chronotropic and inotropic actions). Right atria Chronotropic Inotropic Intrinsic Intrinsic activity pD2.value pA2-value activity pD2-value PA2-value Befunolol 0.22 + 0.02 7.08 + 0.17 9.01 _ 0.08 0.24 + 0.02 7.07 __+0.11 8.99 4-0.04 Isoprenaline 1.00 8.76 4-0.02 1.00 8.75 + 0.04 Propranolol 0.00 8.72 4-0.21 0.00 8.62 + 0.32 Mean 4- SE of 6 experiments.
Befunolol as r-blocker with ISA
267
Table 2. The intrinsic activities,pD2-and pA2-valuesfor the test drugs estimated in the guinea pig tracheal preparation and taenia caecum Tracheal preparation Taenia caecum Intrinsic Intrinsic activity pDz-value pA2-value activity pD2-value pA2-value 7.21+_0.13 9.13+_0.11 0.28+_0.03 7.06+_0.16 9.22+_0.10 Befunolol 0.28 +_0.06 8.62 +_0.18 1.00 8.59 +_0.06 Isoprenaline 1.00 8.43 +_0.06 0.00 8.65 +_0.40 Propranolol 0.00 Mean +_SE of 6 experiments. fl-adrenoceptor has two binding sites: one binding site for agonistic action and the other site for antagonistic action. Both the binding sites are supposed to be mutually exclusive and produce their actions separately through unknown mechanisms (Takayanagi and Koike, 1983; Koike and Takayanagi, 1984; Takayanagi et al., 1984). The findings of Kaum a n n and Blinks (1980) also suggested that more than one type of fl-adrenoceptor might be involved in the interaction of fl-adrenergic partial agonists with their receptors in cardiac muscle. Therefore the intrinsic activity of befunolol may be equal to its selectivity for both the sites in the fl-adrenoceptor. Acknowledgement--The authors are grateful to KakenSeiyaku company for the generous supply of befunolol. REFERENCES
Kaumann A. J. and Blinks J. B. (1980) fl-Adrenoceptor blocking agents as partial agonists in isolated heart muscle: dissociation of stimulation and blockade. Naunyn-Schmiedebergs Archs Pharmac. 311, 237-248. Koike K. and Takayanagi I. (1983) Relationship between
intrinsic activity of fl-adrenoceptor agonist and amount of spare receptors in guinea pig taenia caecum. Jap. J. Pharmac. 33, 327-333. Koike K. and Takayanagi L (1984) Interaction of fl-adrenergic partial agonist with its receptor in guineapig taenia caecum. Gen. Pharmac. 15, 47-50. Nickerson M. (1956) Receptor ocupancy and tissue response. Nature 178, 697-698. Stephenson R. P. (1956) A modification of receptor theory. Br. J. Pharmac. 11, 379-393. Takagi K. and Takayanagi I. (1958) Chemicopharmacological studies on antispasmodic action XV. Non-specific antispasmodic action on tracheal muscle. Chem. Pharm. Bull. 6, 716-720. Takayanagi I. and Koike K. (1983) A possible mechanism of action of a beta-adrenergic partial agonist (carteolol) in guinea pig taenia caecum. J. Pharm. Dyn. 6, 56-59. Takayanagi I., Koike K. and Sato T. (1984) Interactions of some partial agonists with beta-adrenoceptor in the isolated taenia caecum and tracheal smooth muscle of guinea pig. Jap. J. Pharmac. 35, 301-305. Van Rossum J. M. (1963) Cumulative dose-response curve II. Techniques for the making of dose response curve in isolated organs and evaluation of drug parameters. Archs int. Pharmacodyn. Ther. 143, 299-330.