EUROPEAN JOURNAL OF PHARMACOLOGY 24 (1973) 211-217. NORTH-HOLLAND PUBLISHING COMPANY
OF ISOETHARINE ON AIRWAYS RESISTANCE, HEART RATE, AND CONTRACTIONS OF
THE SOLEUS MUSCLE OF THE ANAESTHETISED
I.W. RODGER Department o f Pharmacology, University o f Strathclyde, Glasgow, GI 1XW, U.K.
Received 6 September 1972
Accepted 28 May 1973
I.W. RODGER, Effects of isoetharine on airways resistance, heart rate and contractions of the soleus muscle of the anaesthetised cat, European J. Pharmacol. 24 (1973)211-217, The actions of the sympathomimetic bronchodilator, isoetharine, were compared with those of laevoisoprenaline, racemic isoprenaline and salbutamol, on the heart and lungs and on contractions of the soleus muscle of cats under chloralose anaesthesia. Isoetharine and salbutamol injected i.v. were approximately equipotent in all tests, and were about 8 times less potent than laevoisoprenaline both in decreasing the tension and degree of fusion of incomplete tetanic contractions of the cat soleus muscle, and in opposing the bronchoconstrictor action of 5-hydroxytryptamine. They were about 22 times less potent than laevoisoprenaline in increasing heart rate. In all tests, racemic isoprenaline was about 2 times less potent than laevoisoprenaline. The results suggest that the configuration of soleus /3-receptors is closely similar to that of/3-receptors in the lungs, and that the systemic administration of isoetharine might produce muscle tremor as a side-effect in man. lsoetharine Salbutamol
Airways resistance Isoprenaline
1. Introduction Isoetharine is a sympathomimetic bronchodilator which was first described by Lands et al. (1950) and by Lands and Tainter (1953). It was included by Lands et al. (1967) in a series o f sympathomimetic amines that they tested on isolated tissues, and was also studied on isolated hearts by Brittain et al. (1970). F r o m the results obtained, these authors concluded that isoetharine exhibits some selectivity for /32-adrenoceptors (in the blood vessels and lungs) compared with ~l-adrenoceptors (in the heart and intestine). Ekue et al. (1971) studied the effects of isoetharine on the cardiovascular system o f anaesthetised dogs. They confirmed that, like salbutamol, isoetharine was more p o t e n t in causing vasodilation (/32-receptors) than in stimulating the heart (/31-receptors). Potency comparisons made on different tissues obtained from different species are difficult to interpret in relation to receptor specificity. In the experiments described in this paper, the effects of iso-
Muscle tremor Heart rate
etharine have therefore been simultaneously examined on the heart rate, on the respiratory system and on contractions of the soleus muscle o f the anaesthetised cat. Sympathomimetic bronchodilators may produce muscle tremor as an unwanted side-effect (Beumer, 1971; Prime, 1971; Legge et al., 1971; Minette, 1971; Schumann and Herxheimer, 1971), and their effects on the contractions o f the soleus muscle were investigated in this study because this preparation is believed to provide a useful model for detecting the likelihood o f this side-effect in man (Bowman and Nott, 1970). The effects of isoetharine were compared with those of racemic isoprenaline, laevoisoprenaline and salbutamol.
2. Materials and methods 2.1. General
Experiments were made on 26 adult cats o f either
1. I¢. Rodger, Isoetharine in the cat
sex which were anaesthetised by the i.p. injection of a mixture of a-chloralose (80 mg/kg) and sodium pentobarbitone (6 mg/kg). The trachea was cannulated but, except in those experiments involving measurement of airways resistance, the cat was allowed to breathe spontaneously. In all experiments general arterial blood pressure was recorded from a cannulated common carotid artery by means of a Statham (model P23AC) pressure transducer, All records were made on a Grass 6-channel curvilinear polygraph (model 7 WC 12 PA). It was not possible to test all the sympathomimetic amines in any one cat. In all experiments, therefore, laevoisoprenaline was used as the standard against which the other drugs were compared. In each experiment a series of doses of laevoisoprenaline and the same number of doses of one, or at the most two, other sympathomimetic amines were injected in a ramdom order, which differed in each experiment, Dose-response curves were then constructed, and ratios of effective doses(laevoisoprenaline = 1.0)determined for each cat. 2.2. Airways resistance
The method employed in the 15 cats in which airways resistance was measured, was identical with that described by Bowman and Rodger (1972) and Gwee et al. (1972). The cats were artificially ventilated by positive pressure at a rate of 2 7 - 3 0 breaths/ min and a stroke volume of 13 ml/kg body weight. At intervals of 20 min a constant degree of submaximal bronchoconstriction was induced by the i.v. injection of 5-hydroxytryptamine (5-HT, 1-15/ag/kg in different experiments),
In all experiments on the respiratory system, heart rate and contractions of the soleus muscle were recorded simultaneously. 2.3. Heart rate
Heart rate was recorded in all 26 cats by means of a Grass (model 7P 4C) tachograph triggered by the general arterial pulse. In some experiments in which blood pressure, heart rate and soleus muscle contractions only were being recorded, a method of cumulative injection, similar to that described by Nott and Raper (1972) for skeletal muscle, was employed. 2.4. Soleus muscle
In 24 of the cats the tendon of insertion of a soleus muscle was cut and attached to a Grass (model FT 10 C) force transducer. Subtetanic contractions of the soleus muscle were evoked by stimulating the motor nerve at frequencies of 6 - 1 0 H z for 1 sec every 10 sec (the frequency was constant throughout any one experiment). The method was identical with that described by Bowman and Nott (1970). 2.5. Injection method
In all experiments drugs were injected through a cannula in a femoral vein. 2. 6. Statistics
Relative effective doses of the sympathomimetic amines were determined at the 50% of maximal effect
Table 1 Summary of experiments performed. Parameters measured in experiments
Blood pressure, heart rate, airways resistance and soleus muscle contractions
Laevoisoprenaline + isoetharine Laevoisoprenaline + racemic isoprenaline Laevoisoprenaline + salbutamol
Blood pressure, heart rate, airways resistance
Laevoisoprenaline + isoetharine
Blood pressure, heart rate and soleus muscle contractions
Laevoisoprenaline + isoetharine Laevoisoprenaline + racemic isoprenaline Laevoisoprenaline + salbutamol
Number of cats
1. W. Rodger, lsoetharine in the cat
~H2CH3 HO ~
CH~CH~NH~CIq I OH
Ho Fig. 1. Structure of isoetharine, level for each individual cat and the means and standard errors of these dose ratios were calculated by standard procedures, 2. 7. Drugs
The drugs used were: isoetharine hydrochloride (3M-Riker), laevoisoprenaline bitartrate (Wyeth), racemic isoprenaline sulphate and bethanidine sulphate (Burroughs Wellcome), 5-hydroxytryptamine' creatinine sulphate (British Drug Houses), salbutamol hydrochloride (Allen and Hanburys) and propranolol hydrochloride (Imperial Chemical Industries). The doses quoted refer to the bases,
(mean -+ S.E.M.; 10.0-+ 2.9) times less potent than laevoisoprenaline in different experiments. Racemic isoprenaline was 1.6-2.4 (mean -+ S.E.M.; 1.95 _ + 0.17) times and salbutamol 5.5-10.0 (mean + S.E.M.; 6,8 + 0.73) times less potent than laevoisoprenaline. All estimates of relative potency were made on a weight basis. It was not feasible to produce maximal increases in airways resistance and so the mean dose of laevoisoprenaline quoted in the text, is that which reduced an arbitrary degree of bronchoconstriction by 50%. Thus, for differing degrees of bronchoconstriction produced by different doses of 5-HT, the doses of the amines differed accordingly. The greater the bronchoconstriction the higher the dose of amine required to cause a 50% reduction in 5-HT effect. For this reason, the degrees of bronchoconstriction produced in different cats were kept closely similar. The design of these experiments also precluded accurate assessments of the durations of the drugs' actions. Nevertheless, the protective effect afforded by isoetharine was clearly longer lasting than that of laevoisoprenaline and racemic isoprenaline but shorter than that of salbutamol. After either laevo- or racemic isoprenaline the control response to 5hydroxytryptamine returned within 20 rain, whereas,
3.1. Airways resistance 100
In assessing the bronchodilating activity of the sympathomimetic amines, the increase in airways resistance produced by 5-hydroxytryptamine in their presence was expressed as a percentage reduction of that produced by 5-hydroxytryptamine alone. The values for the increases in airways resistance were calculated from the airflow and transpulmonary pressure records at isovolumic points (on the tidal volume trace), as described by Amdur and Mead (1958). All the sympathomimetics were capable of reducing or abolishing the bronchoconstriction resulting from 5hydroxytryptamine administration. The graph of fig. 2 illustrates dose-response curves from a typical experiment in which isoetharine and laevoisoprenaline were compared. In all experiments the dose-response curves for all 4 drugs were close to parallel. In the 15 experiments performed, the mean dose of laevoisoprenaline required to reduce the 5-HT-induced increase in airways resistance by 50% was 0.022 + 0.006tag/kg. Isoetharine was 5 - 1 5
~ "~" ~ ~0.E _ .-~ ,0~ _ 20
I,o/kg Fig. 2. Dose-response curves from a typical experiment in which the effect of i.v. laevoisoprenaline(') were compared with those of isoetharine (e) in reducing the bronchoconstriction produced by 5-hydroxytryptamine. The responses are expressedas the percentage reduction of the degree of bron-
choconstriction produced by 5-hydroxytryptamine (12#g/ kg).
1. W. Rodger, Isoetharine in the cat
after isoetharine 2 0 - 4 0 min elapsed before control values were re-established. The effects of salbutamol persisted for up to 60 min after injection. The tension and fusion of the soleus muscle contractions were always decreased by effective bronchodilating doses of the sympathomimetics, maximal soleus depression being produced by doses which almost abolished the 5-hydroxytryptamine-induced bronchoconstriction. The doses of the sympathomimetics required to abolish the 5-hydroxytryptamine effects produced significant but submaximal rises in heart rate. In doses just sufficient to abolish 5-hydroxytryptamine bronchoconstriction, laevoisoprenaline ( 0 . 1 0 - 0 . 3 0 /ag/kg) produced increases in heart rate ranging from 37 to 82 beats/min, isoetharine (0.90-3.0/~g/kg) from 29 to 58 beats/min, and salbutamol ( 0 . 8 - 2 . 5 /ag/kg) from 25 to 61 beats/rain in different experimerits, 3.2. Heart rate
In all experiments the sympathomimetics caused increases in heart rate. In those experiments where the sympathomimetics were injected cumulatively to produce maximal increases in heart rate (increases of 65 to 118 beats/min in different experiments), the maxima for all drugs were closely similar. The time to half-recovery from maximal effects after laevo and racemic isoprenaline was 2.5-4.5 min; after isoethafine, 5 - 1 8 min and after salbutamol, more than 90 min. The rank order of the amines for duration of action was always laevoisoprenaline = racemic isoprenaline < isoetharine < salbutamol. Propranolol (0.2 mg/kg) administered at any time during the salbutamol-induced tachycardia caused a fall in heart rate to control resting levels within 10 rain. The graphs of fig. 3 illustrate curves from a typical experiment. In all experiments the dose-response curves were close to parallel. In the 18 experiments where maximal increases in heart rate were induced the mean dose of laevoisoprenaline necessary to produce 50% of the maximal effect was 0.107 + 0.029 ttg/kg. As estimated at this 50% maximum effect level, isoetharine was 1 5 - 2 5 (mean + S.E.M.; 24.3 -+ 0.90) times less potent than laevoisoprenaline. Racemic isoprenaline was 1.5-2.5 (mean +- S.E.M.; 1.97 -+ 0.26) times and salbutamol 1 0 - 2 5 (mean +- S.E.M.; 20.0 -+
1°0 ~ ,: .~ ._= ~ ~ .~ .E_ ~ E
00 / /
60 // 6° 5, i 00,
VQ/kg Fig. 3. Cumulative dose-response curves from a typical experiment in which the effects of i.v. laevoisoprenaline (A), racemic isoprenaline (Q), isoetharine (e) and salbutamol (o) on heart rate were compared. The responses are expressed as the percentage of the maximal increase in heart rate. In this experiment resting heart rate varied from 82 to 92 beats/rain; and this increased to maxima of 160 to 172 beats/rain after cumulative injection of the drugs.
1.89) times less potent than laevoisoprenaline in different experiments. All estimates of relative potency were made on a weight basis. In two experiments the relative potencies were redetermined after bilateral vagotomy and the administration of bethanidine ( 2 6 mg/kg). The relative potencies for the amines under these conditions maintained the same ratios as those quoted above. 3. 3. Soleus muscle
All the sympathomimetics decreased the tension and fusion of incomplete tetanic contractions of the soleus muscle. Fig. 4 illustrates the results from an experiment in which the amines were injected cumulatively to produce maximum depression of soleus muscle contractions. In all experiments the curves were close to parallel. The maximal effects for all the amines in any one experiment were closely similar (in different experiments the decreases in tension ranged from 45 to 63%). The mean dose of laevoisoprenaline required to produce 50% maximal soleus depression, in 21 experiments, was 0.032 +- 0.009/ag/kg. Isoetharine was 7 - 1 4 (mean + S.E.M.; 10.5 -+ 0.84) times
1. W. Rodger, lsoetharine in the cat
1,a "~ ,g '~i e, ,,
~g/ko Fig. 4. Cumulative dose-response curves, from a typical experiment in which the effects of i.v. laevoisoprenaline (A), racemic isoprenaline (D), salbutamol (o) and isoetharine (.) on incomplete tetanic contractions of the soleus muscle were compared. The responses are expressed as the percentage of the maximal depression of contractions.
less potent than laevoisoprenaline, on a weight basis, as determined at this 50% maximum effect level. The relative potency ranges for racemic isoprenaline and salbutamol, with respect to laevoisoprenaline, were respectively 1 . 5 - 2 . 2 (mean -+ S.E.M.; 1.8 -+ 0.18) and 4 - 1 0 (mean + S.E.M.; 6.5 -+ 1.10) times less potent in different experiments, These relative potency values for the amines on the soleus muscle correspond very closely to those determined during airways resistance studies. After
injection of a dose of laevoisoprenaline just sufficient to produce a maximal depression of the soleus muscle contractions, the time to half-recovery was 3 - 4 min. Racemic isoprenaline had an almost identical duration of action. After. isoetharine and salbutamol halfrecovery occurred in 5 - 2 0 min, but in any o n e e x periment, salbutamol was always slightly longer lasting than isoetharine. The effects of all the sympathomimetic amines on the lungs, the heart and the soleus muscle were prevented by the previous i.v. injection of propranolol (0.2 mg/kg). The mean dose ratios of all of the sympathomimetic amines for their effects on all 3 systems are presented in table 2.
4. Discussion The results, summarised in table 2, showed that in the cat, isoetharine and salbutamol were approximately equipotent in their effects on the lungs, the heart and the soleus muscle. Both were 7 - 1 0 times less potent than laevoisoprenaline in antagonising 5HT-induced bronchoconstriction and in decreasing the tension and fusion of the soleus muscle contractions, and 2 0 - 2 5 times less potent than laevoisoprenaline in increasing heart rate. The results therefore confirm, that in comparison with isoprenaline, isoetharine shows greater selectivity for bronchial (~2) than cardiac ( ~ ) adrenoreceptors. The effects of all the amines on heart rate were due to direct actions
Table 2 Mean dose ratios (5 S.E.M.) of the sympathomimetic amines on airways resistance, heart rate and contractions of the soleus muscle. Laevoisoprenaline was used as reference compound in each experiment. The mean doses (~g/kg, i.v.) of laevoisoprenaline required to produce 50% of the maximal increase in heart rate and depression of soleus and 50% reduction in the arbitrary, but constant degree of bronchoconstriction, are quoted in square brackets. These values are taken as unity, the dose ratios being determined in individual cats at these 50% levels. The means quoted are those for the numbers of experiments indicated in the small brackets. Relative potencies are the reciprocals of the means given. Drug
Antagonism of 5-HT-induced increase in airways resistance
[0.022 ± 0.006
Increase in heart rate
Depression of soleus muscle contractions
[0.107 ± 0.029
1.95 ± 0.17
1.97 ± 0.26
1.8 ± 0.18
10.0 ± 2.90
10.5 ± 0.84
6.8 ± 0.73
20.0 ± 1.89
6.5 ± 1.10
L ~ Rodger, Isoetharine in the cat
on cardiac /3-receptors; they were not the result o f reflex activity consequent upon their depressor actions since they were unaltered by bilateral vagotomy and treatment with bethanidine or by lowering the blood pressure with anaesthetic to a level at which no further fall in blood pressure was produced, Since maximal effects were not produced in all 3 systems it is invalid to make direct comparisons of the drugs in terms of 'selectivity ratios'. It is, however, valid to compare the safety margins which isoetharine and salbutamol have over laevoisoprenaline. At equiactive bronchodilator doses, both isoetharine and salbutamol were less effective than isoprenaline or laevoisoprenaline in stimulating the heart, and in order to produce a comparable degree of cardiac stimulation, the dose of isoetharine or salbutamol had to be increased 2- to 3-fold. These results with salbutamol confirm those o f a previous study (Bowman and Rodger, 1972). The surprising finding is that in the cat, selectivity o f isoetharine for the bronchi compared with the heart, was equal to that of salbutamol. In the cat, the safety margin over isoprenaline for both compounds is small compared with that reported for salbutamol in other laboratory animals (Cullum et al., 1969; Daly et al., 1971). Possibly/31- and /32-receptors are less well differentiated in the cat. The important question is which species resembles man most closely. There are reports of systemically administered salbutamol producing tachycardia in man (Gibson and Coltart, 1971; Paterson, 1971; Svedmyr and Thiringer, 1971), y e t this would be unlikely to occur if the extremely wide safety margin reported for some laboratory animals also held in man. The cat may therefore provide the best model for experiments concerning adrenoceptor specificity with respect to bronchodilator and cardiac stimulant actions of/3-receptor agonists, Like all other sympathomimetics so far examined in this laboratory, isoetharine decreased the tension and degree of fusion of incomplete tetanic contractions of the soleus muscle in doses comparable to those producing bronchodilatation. This again supports the view (Bowman and Rodger, 1972) that the configuration of soleus /3-receptors is closely similar to that of/3-receptors in the lungs, and suggests that, should isoetharine be administered systemically, it would produce muscle tremor in man.
Acknowledgements I am grateful to Mr. J. Houston for technical assistance and to Allen & Hanburys Ltd., Burroughs WeUcome & Co., Ltd., Imperial Chemical Industries Ltd., and John Wyeth & Brother Ltd., for generous gifts of drugs. I am also indebted to the WeUcome Trust for a grant towards apparatus, to the Medical Research Council for a grant towards the cost of animals, and to Professor W.C. Bowman for invaluable criticism. References
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I. W. Rodger, lsoetharine in the cat
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