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8-Phenyltheophylline: A potent P1-purinoceptor antagonist
European Journal of Pharmacology, 75 ( 1981 ) 61-64 Elsevier/North-Holland Biomedical Press
61
Short communication
8-PHENYLTHEOPHYLLINE: A POTENT PI-PURINOCEPTOR ANTAGONIST SUSAN G. GRIFFITH, PARVIZ MEGHJI, CATHERINE J. MOODY and GEOFFREY BURNSTOCK *
Department of Anatomy and Embryology, and Centre for Neuroscience, University College London, Gowerr Street, London WCI E 6BT, U.K. Received 21 July 1981, accepted 19 August 1981
S.G. GRIFFITH, P. MEGHJI, C.J. MOODY and G. BURNSTOCK, 8-Pheto,Itheophylline: a potent Pt'purin°cept°r antagonist, European J. Pharmacol. 75 (1981) 61-64. 8-Phenyltheophylline was more potent than theophylline in antagonizing the inhibitory effects of adenosine in guinea-pig driven left atrium, rabbit basilar artery and electrically stimulated guinea-pig ileum preparations. In guinea-pig atrium and ileum, the antagonism of adenosine responses by the methylxanthines is of a competitive nature, but in rabbit basilar artery it does not appear to be so. In all three preparations, the effects of theophylline and 8-phenyltheophyllin could be reversed by washing. It is concluded that 8-phenyltheophylline is a more potent Pl'purin°cept°r antagonist than theophylline. 8-Phenyltheophylline
Theophylline
PI -purinoceptor
Adenosine
1. Introduction
2. Materials and methods
Theophylline and other methylxanthines act as Pl-purinoceptor antagonists in a wide variety of tissues (see Burnstock, 1978; Burnstock and Meghji, 1981). The antagonism of responses to adenosine by theophylline has been shown to be competitive, but pA 2 values (Arunlakshana and Schild, 1959) for theophylline are low compared with values for the weU-recognised antagonists of other receptors, and theophylline has other known actions besides the antagonism of responses to adenosine (see Burnstock, 1978). Recently, it has been reported that 8phenyltheophylline (8-PT) is more potent than theophylline in antagonising adenosine-induced accumulation of cyclic AMP in guinea-pig cerebral cortex (Smellie et al., 1979) and in human fibroblasts (Bruns, 1981). In the study reported here, the potencies of theophylline and 8-PT as antagonists of the actions of adenosine are compared in three different isolated preparations: guinea-pig left atrium and rabbit basilar artery where adenosine acts postsynaptically, and in guinea-pig ileum where adenosine has a presynaptic mode of action.
Guinea-pigs (300-600 g) of either sex and male rabbits (2.5-3.5 kg) were killed by a blow to the back of the neck and were exsanguinated.
* To whom proofs should 15e addressed.
2.1. Guinea-pig atrium Left atria were set up in tissue baths and the muscle was electrically stimulated at 2.5 Hz using 5 msec pulses of twice threshold voltage, as described previously (Bumstock and Meghji, 1981). 2.2. Rabbit basilar artery Ring segments of basilar arteries, 5 mm long, were mounted in tissue baths containing KrebsRinger bicarbonate solution (De Mey et al., 1979) at 37°C, which was gassed with 95% 02 and 5% CO 2. Dipyridamole (0.2 #M) was routinely present. The vessels were supported by two wires inserted into the lumen, one anchored and the other connected to a Grass FT.03C force transducer for measurement of the isometric responses. A Grass 79D polygraph recorded the output from the transducers. Optimal passive stretch was applied to the arterial segments which were allowed
0014-2999/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
62
to equilibrate for 2h. The vessels were preconstricted with 0.5 /zM histamine before testing responses to adenosine.
2.3. Guinea-pig ileum Sections of approximately 3 cm in length were taken from the mid-region of the ileum. Tissues were mounted in organ baths containing modified Krebs solution (Btilbring, 1953) at 37°C, which was gassed with 95% 02 and 5% CO 2. The intramural cholinergic nerves were electrically stimulated with biphasic pulses of 25-30 V, 0.5 msec duration and at 0.2 Hz from a Grass SD9 stimulator, using the method of Paton (1955). Responses were recorded as described for the other tissues. The ileal segments were allowed to equilibrate for lh. Log (adenosine concentration) response curves were obtained by adding the drug directly to the tissue baths and allowing the responses to plateau. Theophylline (3-100 /~M) or 8-PT (0.1-10 /~M) were allowed to equilibrate with the tissues for 20-30 min before their effects on adenosine responses were investigated. (Only concentrations of up to 10/~M 8-PT could be used as 8-PT precipitated out of solution at higher concentrations.) Reversibility of the action of the methylxanthines was examined by washing the drugs from the tissue baths and re-testing the responses to adenosine. ECso values were estimated from mean log (adenosine concentration) response curves and were used to calculate the concentration ratio (CR) at different antagonist doses. Each dose of antagonist was tested on at least four tissues. Apparent pA 2 values were obtained from regression lines of log ( C R - 1 ) versus - l o g (methylxanthine concentration) (Arunlakshana and Schild, 1959). Adenosine, histamine dihydrochloride and theophyUine were obtained from Sigma and aqueous stock solutions were used. Dipyridamole was obtained from Boehringer-Ingelheim. A stock solution of 10 mM 8-PT (Calbiochem) was made up in 80% v / v methanol containing 0.2 M NaOH, and aqueous dilutions of this were used. Results were tested for significance using Student's t-test (unpaired). A probability level P ~<0.05 was considered to be significant.
3. Results Adenosine produced a negative inotropic effect in guinea-pig driven left atria, relaxed rabbit basilar arteries pre-constricted with histamine, and reduced the twitch height elicited by intramural cholinergic nerve stimulation of guinea-pig ileum (fig. 1). The effect of adenosine was concentrationdependent in each preparation. Theophylline and 8-PT slightly enhanced the contractile responses of guinea-pig atria and ileum to electrical stimulation of the muscle and intramural nerves respectively, but the effects were not significant. The methylxanthines generally produced no change in the contractile response to 0.5 /~M histamine in rabbit basilar arteries, although high concentrations of theophylline ( > 3 0 /~M) often produced a pronounced relaxation, and in
T HEOPHYLLINE
8-PHENYLTHEOPHYLLINE
a.Guinea-pig atrium
L,I, L T°L,mk IO#M
20 sec
IO#M 8-PT ~
b. Rabbit basilar artery
o.f,o\ \ T
4rain c. Guinea-I~ ileum
t
lO/zM 8-PT $
Fig. 1. Comparison of the potencies of theophylline (Theo) and 8-phenyltheophylline (8-PT) in antagonizing the responses to adenosine (applied at O) in: (a) guinea-pig driven left atrium (30/~M adenosine), (b) rabbit basilar artery preconstricted with 0.5 /~M histamine (0.7 /~M adenosine), (c) electrically stimulated guinea-pig ileum (10 /~M adenosine). Note that 8phenyltheophylline was more potent than theophylline in the three tissues studied. The antagonism of the responses to adenosine by the methylxanthines could be reversed by washing (arrow).
63
these cases antagonism of the adenosine responses could not be tested. Fig. 1 shows that 8-PT was more potent than theophylline in antagonising the responses to adenosine, and that the antagonism by the methylxanthines was reversed by washing the drugs from the tissue baths. 8-PT (0.3-10 /~M) displaced the log (adenosine concentration) response curves to the right in all three preparations. Parallel shifts in log (adenosine contraction) response curves were also obtained with theophylline but only at concentrations exceeding 5/~M. The antagonistic effects of the two methylxanthines on adenosine responses were concentration-dependent. The apparent pA 2 values, calculated from Schild plots, were higher for 8-PT than theophylline in all tissues (table 1). Potency ratios calculated from the pA 2 values were approximately 8, 20 and more than 100 for ileum, atrium and basilar artery respectively, when 8-PT was compared with theophylline. The slopes of the regression lines of the Schild plots for theophylline and 8-PT did not differ significantly from - 1 in guinea-pig atrium and ileum suggesting that the antagonism of adenosine responses is competitive in these tissues. However, although both methylxanthines produced parallel shifts in the log (adenosine concentration) response curves in rabbit basilar artery, the slopes of the Schild plots were significantly different from - 1 (P < 0.05).
TABLE I Comparison of apparent pA 2 (calculated from Schild plots) for theophylline and 8-phenyltheophylline in three different tissues, using adenosine as the agonist. Tissue
Guinea-pig left atrium Rabbit basilar artery Guinea-pigileum
Theophylline
8-Phenyltheophylline
Slope a
Apparent pA2
Slope a
Apparent pA2
- 1.03
4.92
-0.85
6.24
- 0 . 4 9 h 5.09 -- 1.13 5.20
- 0 . 5 8 b 7.16 --0.80 6.12
a Slope of regression line of Schild plot. h The slopes of the lines are significantly different from - I (P<0.05).
4. Discussion
8-PT was found to be a more potent P~purinoceptor antagonist than theophylline, having effects at both postsynaptic (guinea-pig atrium and rabbit basilar artery) and presynaptic (cholinergic nerve terminals in guinea-pig ileum) sites. The antagonism of the inhibitory responses to adenosine by 8-PT and theophylline was reversible in all three preparations. In this study, the antagonism appears to be of a competitive nature in guinea-pig atrium and ileum, but the difference from - 1 of the slopes of the Schild plots for both methylxanthines in rabbit basilar artery suggests that the antagonism may not be competitive in this tissue. However, uptake systems (not blocked by 0.2 /~M dipyridamole), the existence of multiple receptors for adenosine, or other actions of the antagonists, may result in the failure of the experimental data to fit the theoretical slope of a Schild plot (see Waud, 1975). It has been reported previously that 8-PT is a more potent antagonist of adenosine-induced inhibition of cyclic AMP accumulation than theophylline in guinea-pig cerebral cortex (Smellie et al., 1979) and in human fibroblasts (Bruns, 1981). In addition, studies using the derivative 8-(pbromophenyl)theophylline show that it is a more potent adenosine antagonist than 8-PT (Bruns, 1981). Thus, it can be postulated that 8-(pbromophenyl)theophylline may be a more potent Pl-purinoceptor antagonist than 8-PT in the preparations studied here. Theophylline is known to have phosphodiesterase inhibitory activity (Amer and Kreighbaum, 1975) and this action may explain the relaxation produced in basilar arteries by high concentrations of theophylline. 8-PT however, has been shown to have relatively low activity as a phosphodiesterase inhibitor (Smellie et al., 1979), and therefore its adenosine antagonistic activity is relatively selective. Although 8-PT is much less soluble in aqueous solutions than theophylline, its greater potency combined with its low phosphodiesterase inhibitory activity make its use as a P~-purinoceptor antagonist preferable to that of theophylline.
64
References Amer, M.S. and W.E. Kreighbaum, 1975, Cyclic nucleotide phosphodiesterases: properties, activators, inhibitors, structure-activity relationships, and possible role in drug development, J. Pharm. Sci. 64, 1. Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 48. Bruns, R.F., 1981, Adenosine antagonism by purines, pteridines and benzopteridines in human fibroblasts, Biochem. Pharmacol. 30, 325. Bialbring, E., 1953, Measurements of oxygen consumption in smooth muscle, J. Physiol. (London) 122, 111. Burnstock, G., 1978, A basis for distinguishing two types of purinergic receptor, in: Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, eds. L. Bolis and R.W. Straub (Raven Press, New York) p. 107.
Burnstock, G. and P. Meghji, 1981, Distribution of PI and P2 purinoceptors in the guinea-pig and frog heart, Br. J. Pharmacol. (in press). De Mey, J., G. Burnstock and P.M. Vanhoutte, 1979, Modulation of the evoked release of noradrenaline in canine saphenous vein via presynaptic receptors for adenosine but not ATP, European I. Pharmacol. 55, 401. Paton, W.D.M., 1955, The response of the guinea-pig ileum to electrical stimulation by coaxial electrodes, J. Physiol. (London) 127, 40P. Smellie, F.W., C.W. Davis, J.W. Daly and J.N. Wells, 1979, Alkylxanthines: inhibition of adenosine-elicited accumulation of cyclic AMP in brain slices and of brain phosphodiesterase activity, Life Sci. 24, 2475. Waud, D.R., 1975, Analysis of dose-response curves, in: Methods in Pharmacology, Vol. 3, Smooth Muscle, eds. E.E. Daniel and D.M. Paton (Plenum Press, New York) p. 471.
61
Short communication
8-PHENYLTHEOPHYLLINE: A POTENT PI-PURINOCEPTOR ANTAGONIST SUSAN G. GRIFFITH, PARVIZ MEGHJI, CATHERINE J. MOODY and GEOFFREY BURNSTOCK *
Department of Anatomy and Embryology, and Centre for Neuroscience, University College London, Gowerr Street, London WCI E 6BT, U.K. Received 21 July 1981, accepted 19 August 1981
S.G. GRIFFITH, P. MEGHJI, C.J. MOODY and G. BURNSTOCK, 8-Pheto,Itheophylline: a potent Pt'purin°cept°r antagonist, European J. Pharmacol. 75 (1981) 61-64. 8-Phenyltheophylline was more potent than theophylline in antagonizing the inhibitory effects of adenosine in guinea-pig driven left atrium, rabbit basilar artery and electrically stimulated guinea-pig ileum preparations. In guinea-pig atrium and ileum, the antagonism of adenosine responses by the methylxanthines is of a competitive nature, but in rabbit basilar artery it does not appear to be so. In all three preparations, the effects of theophylline and 8-phenyltheophyllin could be reversed by washing. It is concluded that 8-phenyltheophylline is a more potent Pl'purin°cept°r antagonist than theophylline. 8-Phenyltheophylline
Theophylline
PI -purinoceptor
Adenosine
1. Introduction
2. Materials and methods
Theophylline and other methylxanthines act as Pl-purinoceptor antagonists in a wide variety of tissues (see Burnstock, 1978; Burnstock and Meghji, 1981). The antagonism of responses to adenosine by theophylline has been shown to be competitive, but pA 2 values (Arunlakshana and Schild, 1959) for theophylline are low compared with values for the weU-recognised antagonists of other receptors, and theophylline has other known actions besides the antagonism of responses to adenosine (see Burnstock, 1978). Recently, it has been reported that 8phenyltheophylline (8-PT) is more potent than theophylline in antagonising adenosine-induced accumulation of cyclic AMP in guinea-pig cerebral cortex (Smellie et al., 1979) and in human fibroblasts (Bruns, 1981). In the study reported here, the potencies of theophylline and 8-PT as antagonists of the actions of adenosine are compared in three different isolated preparations: guinea-pig left atrium and rabbit basilar artery where adenosine acts postsynaptically, and in guinea-pig ileum where adenosine has a presynaptic mode of action.
Guinea-pigs (300-600 g) of either sex and male rabbits (2.5-3.5 kg) were killed by a blow to the back of the neck and were exsanguinated.
* To whom proofs should 15e addressed.
2.1. Guinea-pig atrium Left atria were set up in tissue baths and the muscle was electrically stimulated at 2.5 Hz using 5 msec pulses of twice threshold voltage, as described previously (Bumstock and Meghji, 1981). 2.2. Rabbit basilar artery Ring segments of basilar arteries, 5 mm long, were mounted in tissue baths containing KrebsRinger bicarbonate solution (De Mey et al., 1979) at 37°C, which was gassed with 95% 02 and 5% CO 2. Dipyridamole (0.2 #M) was routinely present. The vessels were supported by two wires inserted into the lumen, one anchored and the other connected to a Grass FT.03C force transducer for measurement of the isometric responses. A Grass 79D polygraph recorded the output from the transducers. Optimal passive stretch was applied to the arterial segments which were allowed
0014-2999/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
62
to equilibrate for 2h. The vessels were preconstricted with 0.5 /zM histamine before testing responses to adenosine.
2.3. Guinea-pig ileum Sections of approximately 3 cm in length were taken from the mid-region of the ileum. Tissues were mounted in organ baths containing modified Krebs solution (Btilbring, 1953) at 37°C, which was gassed with 95% 02 and 5% CO 2. The intramural cholinergic nerves were electrically stimulated with biphasic pulses of 25-30 V, 0.5 msec duration and at 0.2 Hz from a Grass SD9 stimulator, using the method of Paton (1955). Responses were recorded as described for the other tissues. The ileal segments were allowed to equilibrate for lh. Log (adenosine concentration) response curves were obtained by adding the drug directly to the tissue baths and allowing the responses to plateau. Theophylline (3-100 /~M) or 8-PT (0.1-10 /~M) were allowed to equilibrate with the tissues for 20-30 min before their effects on adenosine responses were investigated. (Only concentrations of up to 10/~M 8-PT could be used as 8-PT precipitated out of solution at higher concentrations.) Reversibility of the action of the methylxanthines was examined by washing the drugs from the tissue baths and re-testing the responses to adenosine. ECso values were estimated from mean log (adenosine concentration) response curves and were used to calculate the concentration ratio (CR) at different antagonist doses. Each dose of antagonist was tested on at least four tissues. Apparent pA 2 values were obtained from regression lines of log ( C R - 1 ) versus - l o g (methylxanthine concentration) (Arunlakshana and Schild, 1959). Adenosine, histamine dihydrochloride and theophyUine were obtained from Sigma and aqueous stock solutions were used. Dipyridamole was obtained from Boehringer-Ingelheim. A stock solution of 10 mM 8-PT (Calbiochem) was made up in 80% v / v methanol containing 0.2 M NaOH, and aqueous dilutions of this were used. Results were tested for significance using Student's t-test (unpaired). A probability level P ~<0.05 was considered to be significant.
3. Results Adenosine produced a negative inotropic effect in guinea-pig driven left atria, relaxed rabbit basilar arteries pre-constricted with histamine, and reduced the twitch height elicited by intramural cholinergic nerve stimulation of guinea-pig ileum (fig. 1). The effect of adenosine was concentrationdependent in each preparation. Theophylline and 8-PT slightly enhanced the contractile responses of guinea-pig atria and ileum to electrical stimulation of the muscle and intramural nerves respectively, but the effects were not significant. The methylxanthines generally produced no change in the contractile response to 0.5 /~M histamine in rabbit basilar arteries, although high concentrations of theophylline ( > 3 0 /~M) often produced a pronounced relaxation, and in
T HEOPHYLLINE
8-PHENYLTHEOPHYLLINE
a.Guinea-pig atrium
L,I, L T°L,mk IO#M
20 sec
IO#M 8-PT ~
b. Rabbit basilar artery
o.f,o\ \ T
4rain c. Guinea-I~ ileum
t
lO/zM 8-PT $
Fig. 1. Comparison of the potencies of theophylline (Theo) and 8-phenyltheophylline (8-PT) in antagonizing the responses to adenosine (applied at O) in: (a) guinea-pig driven left atrium (30/~M adenosine), (b) rabbit basilar artery preconstricted with 0.5 /~M histamine (0.7 /~M adenosine), (c) electrically stimulated guinea-pig ileum (10 /~M adenosine). Note that 8phenyltheophylline was more potent than theophylline in the three tissues studied. The antagonism of the responses to adenosine by the methylxanthines could be reversed by washing (arrow).
63
these cases antagonism of the adenosine responses could not be tested. Fig. 1 shows that 8-PT was more potent than theophylline in antagonising the responses to adenosine, and that the antagonism by the methylxanthines was reversed by washing the drugs from the tissue baths. 8-PT (0.3-10 /~M) displaced the log (adenosine concentration) response curves to the right in all three preparations. Parallel shifts in log (adenosine contraction) response curves were also obtained with theophylline but only at concentrations exceeding 5/~M. The antagonistic effects of the two methylxanthines on adenosine responses were concentration-dependent. The apparent pA 2 values, calculated from Schild plots, were higher for 8-PT than theophylline in all tissues (table 1). Potency ratios calculated from the pA 2 values were approximately 8, 20 and more than 100 for ileum, atrium and basilar artery respectively, when 8-PT was compared with theophylline. The slopes of the regression lines of the Schild plots for theophylline and 8-PT did not differ significantly from - 1 in guinea-pig atrium and ileum suggesting that the antagonism of adenosine responses is competitive in these tissues. However, although both methylxanthines produced parallel shifts in the log (adenosine concentration) response curves in rabbit basilar artery, the slopes of the Schild plots were significantly different from - 1 (P < 0.05).
TABLE I Comparison of apparent pA 2 (calculated from Schild plots) for theophylline and 8-phenyltheophylline in three different tissues, using adenosine as the agonist. Tissue
Guinea-pig left atrium Rabbit basilar artery Guinea-pigileum
Theophylline
8-Phenyltheophylline
Slope a
Apparent pA2
Slope a
Apparent pA2
- 1.03
4.92
-0.85
6.24
- 0 . 4 9 h 5.09 -- 1.13 5.20
- 0 . 5 8 b 7.16 --0.80 6.12
a Slope of regression line of Schild plot. h The slopes of the lines are significantly different from - I (P<0.05).
4. Discussion
8-PT was found to be a more potent P~purinoceptor antagonist than theophylline, having effects at both postsynaptic (guinea-pig atrium and rabbit basilar artery) and presynaptic (cholinergic nerve terminals in guinea-pig ileum) sites. The antagonism of the inhibitory responses to adenosine by 8-PT and theophylline was reversible in all three preparations. In this study, the antagonism appears to be of a competitive nature in guinea-pig atrium and ileum, but the difference from - 1 of the slopes of the Schild plots for both methylxanthines in rabbit basilar artery suggests that the antagonism may not be competitive in this tissue. However, uptake systems (not blocked by 0.2 /~M dipyridamole), the existence of multiple receptors for adenosine, or other actions of the antagonists, may result in the failure of the experimental data to fit the theoretical slope of a Schild plot (see Waud, 1975). It has been reported previously that 8-PT is a more potent antagonist of adenosine-induced inhibition of cyclic AMP accumulation than theophylline in guinea-pig cerebral cortex (Smellie et al., 1979) and in human fibroblasts (Bruns, 1981). In addition, studies using the derivative 8-(pbromophenyl)theophylline show that it is a more potent adenosine antagonist than 8-PT (Bruns, 1981). Thus, it can be postulated that 8-(pbromophenyl)theophylline may be a more potent Pl-purinoceptor antagonist than 8-PT in the preparations studied here. Theophylline is known to have phosphodiesterase inhibitory activity (Amer and Kreighbaum, 1975) and this action may explain the relaxation produced in basilar arteries by high concentrations of theophylline. 8-PT however, has been shown to have relatively low activity as a phosphodiesterase inhibitor (Smellie et al., 1979), and therefore its adenosine antagonistic activity is relatively selective. Although 8-PT is much less soluble in aqueous solutions than theophylline, its greater potency combined with its low phosphodiesterase inhibitory activity make its use as a P~-purinoceptor antagonist preferable to that of theophylline.
64
References Amer, M.S. and W.E. Kreighbaum, 1975, Cyclic nucleotide phosphodiesterases: properties, activators, inhibitors, structure-activity relationships, and possible role in drug development, J. Pharm. Sci. 64, 1. Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 48. Bruns, R.F., 1981, Adenosine antagonism by purines, pteridines and benzopteridines in human fibroblasts, Biochem. Pharmacol. 30, 325. Bialbring, E., 1953, Measurements of oxygen consumption in smooth muscle, J. Physiol. (London) 122, 111. Burnstock, G., 1978, A basis for distinguishing two types of purinergic receptor, in: Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, eds. L. Bolis and R.W. Straub (Raven Press, New York) p. 107.
Burnstock, G. and P. Meghji, 1981, Distribution of PI and P2 purinoceptors in the guinea-pig and frog heart, Br. J. Pharmacol. (in press). De Mey, J., G. Burnstock and P.M. Vanhoutte, 1979, Modulation of the evoked release of noradrenaline in canine saphenous vein via presynaptic receptors for adenosine but not ATP, European I. Pharmacol. 55, 401. Paton, W.D.M., 1955, The response of the guinea-pig ileum to electrical stimulation by coaxial electrodes, J. Physiol. (London) 127, 40P. Smellie, F.W., C.W. Davis, J.W. Daly and J.N. Wells, 1979, Alkylxanthines: inhibition of adenosine-elicited accumulation of cyclic AMP in brain slices and of brain phosphodiesterase activity, Life Sci. 24, 2475. Waud, D.R., 1975, Analysis of dose-response curves, in: Methods in Pharmacology, Vol. 3, Smooth Muscle, eds. E.E. Daniel and D.M. Paton (Plenum Press, New York) p. 471.