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The effects of some autonomic blocking agents on the heart rates of anaesthetized and pithed rats
EUROPEAN JOURNAL OF PHARMACOLOGY 15 (1971) 267-273. NORTH-HOLLAND PUBLISHING COMPANY
THE EFFECTS OF SOME AUTONOMIC BLOCKING AGENTS ON THE HEART RATES OF ANAESTHETIZED
AND PITHED RATS
A.M. B A R R E T T *
Department o f Pharmacology, L C.L Ltd., Pharmaceuticals Division, AIderley Park, Macclesfield, Cheshire
Received 29 July 1970
Accepted 21 May 1971
A.M. BARRETT, The effect o f some autonomic blocking agents on the heart rates o f anaesthetized and pithed rats, European J. Pharmacol. 15 (1971) 267-273. The effect of anaesthesia induced by pentobarbitone and urethane on the heart rate responses to autonomic blocking drugs was studied in rats. Under pentobarbitone anaesthesia cholinergic and ¢~-adrenergicantagonists had little effect on heart rate except for those agents possessing partial agonist or general depressant 'properties. There was no response to pain or hypothalamic stimulation and t3-adrenergic blockade to exogenous catecholamines was easily demonstrated. Under urethane anaesthesia, marked effects were seen in response to autonomic antagonists, to pain and to hypothalamic stimulation./3-adrenergic blockade was difficult to establish. Responses in pithed rats closely paralleled those in rats anaesthetized with pentobarbitone. It was concluded that whereas pentobarbitone depressed autonomic function urethane increased it. No evidence was found to support the view that the cardiovascular responses of the rat were different from those of other mammals. A warning was given of the dangers of misinterpreting results obtained from animals anaesthetized with urethane. Urethane Pentobarbitone
Autonomic blockade Differences between rat and other s p e c i e s
1. INTRODUCTION Early experiments with pronethalol appeared to show that the rat behaved differently from other species in the context of 13-adrenoceptor blockade. The drug failed to block an isoprenaline-induced tachycardia (contrary to the effect in mice, guineapigs, rabbits, cats, dogs, monkeys and men) yet produced a marked bradycardia somewhat greater than that seen in the other species. Atropine significantly reduced but did not abolish the bradycardia produced by pronethalol in rats (Black et al., 1962). Concurrent experiments by the present author in a different part of the same laboratory demonstrated that pronethalol produced a conventional d o s e dependent inhibition of an isoprenaline-induced * Present address: Dept. of Pharmacology, School of Medicine, The University, Leeds 2.
Atropine Propranolol
tachycardia in rats and that the drug alone raised heart rate. A survey of the differences in technique of the two investigators revealed that the principal variable was the choice of anaesthetic. It was found that the anaesthetic used could have a dramatic effect on the heart rate response to a number of autonomic blocking drugs, thereby making direct transposition of results into a clinical context potentially misleading. In addition, it was shown that there was not a unique difference in the cardiovascular responses of the rat to/3-adrenoceptor blockade.
2. MATERIALS AND METHODS The animals used were male albino rats ( 1 9 0 - 2 1 0 g) of the Wistar Strain bred at Alderley Park under s.p.f, conditions. They were maintained on a cubed diet and water ad libitum. Anaesthesia
268
A.M.Barrett, Anaesthesia and autonomic blockade
was induced by intraperitoneal injections of pentobarbitone (55 mg/kg) or urethane (diethylcarbamate) (1.5 g/kg) as a 25% solution. Some rats were pithed according to the method o f Shipley and Tilden (1947). Heart rate was monitored by a cardiotachometer triggered by the QRS complex of the electrocardiogram. Blood pressure was monitored from a cannula in the left carotid artery by means of a Condon manometer. Intravenous injections were made in an exposed saphenous vein in volumes of 0.1 ml/100 g b o d y weight. Autonomic blocking drugs were given by i.p. injection 15 min after the establishment of a steady heart rate under anaesthesia. The change in rate was monitored continuously but since a plateau response was achieved at 15 rain, the effect at that time has been used for these experiments. For catecholamines the peak response was recorded which usually occurred at 90 sec. In all experiments, the mean value for 6 rats was recorded together with the standard error of the mean. In some experiments an electrical crocodile clip was applied to the tail under anaesthesia as a " p a i n " stimulus. Some rats were prepared with indwelling electrodes in the anterior and posterior hypothalamus selecting the appropriate coordinates for use with a stereotaxic instrument, l_eads were exteriorized and cemented to the skull. An electrical stimulus was applied ( 7 - 1 0 V, 3 0 - 4 0 pulses/sec, duration 10msec) for 3 0 s e c under anaesthesia. The same animals were tested under halothane, pentobarbitone and urethane anaesthesia in that sequence at 4-day intervals. The animals did not recover consciousness from urethane and were killed. The position of the electrodes was checked microscopically post-mortem. The drugs used were adrenaline bitatrate, isoprenaline sulphate (Burroughs Welcome), hexamethonium (May & Baker), atropine sulphate (B.D.H.) and pronethalol, propranolol, practolol (I.C.I. 50,172) and sotalol (MJ 1999) all prepared in the chemical laboratories o f I.C.I. Ltd. The doses of c o m p o u n d used refer to the base.
3. RESULTS
3.1. Effects o f pronethalol and atropine on heart rate under urethane and pentobarbitone anaesthesia The effect o f a single dose of pronethalol on heart rate was compared in rats anaesthetized with either urethane or pentobarbitone. The resting heart rate under urethane was generally lower than that under pentobarbitone but the difference did not always reach statistical significance. The response under the two anaesthetics to pronethalol was quite different. Whereas a significant bradycardia (p < 0.01) occurred in animals anaesthetized with urethane, pronethalol induced a small, but statistically significant Co < 0.05), tachycardia in rats anaesthetized with pentobarbitone. Administration of atropine also produced different results under the two experimental conditions. In rats anaesthetized with pentobarbitone, heart rate was not significantly altered but a highly significant rise in heart rate (p < 0.01) was observed under urethane anaesthesia. The results, together with those for saline controls, are summarized in table 1. 3.2. Effect o f pronethalol on an isoprenaline-induced
tachycardia anaesthesia
under
urethane and pentobarbitone
The sensitivity of rats anaesthetized with pentobarbitone or urethane to isoprenaline stimulation of heart rate was found to be markedly different. Using
Table 1 Effect of anaesthetic on heart rate response to atropine and pronethalol (beats/min ± S.E.M., n = 6).
Anaesthetic
Treatment
Urethane
Saline Pronethalol Saline Atropine
Pentobarbitone Saline Pronethalol Saline Atropine
Dose
(mg/kg)
Change in heart rate 15 min after treatment
20
+ 8± 6 - 62 ± 10
5
+ 15 t 11 +102 ± 21
20
+ 4± 4 + 29 ± 11
5
- 5 -+ 3 - 14 ± 8
A.M. Barrett, Anaesthesia and autonomic blockade pentobarbitone, 0.1 #g/kg isoprenaline i.v. was found to give a sub-maximal response of about 100 beats/rain. When urethane was employed it was necessary to raise the dose of isoprenaline to 2 /~g/kg i.v., to produce a response of similar magnitude. In pentobarbitone anaesthetized rats the dose of pronethalol required to produce a 50% block of the isoprenaline response averaged 0.82 mg/kg, whereas in urethane anaesthetized animals an average of 16.5 mg/kg of pronethalol was required. Since in the earlier experiments of Black et al. (1962) the respective doses used were 10 gg/kg of isoprenaline and 25 mg/kg of pronethalol it is probable that failure to block the tachycardia (in animals anaesthetized with urethane) was due to the use of a supra-maximal dose of agonist and an inadequate dose of pronethalol under their particular experimental conditions. i
3.3. E f f e c t o f atropine and vagotomy on heart rate response to pronethalol under urethane anaesthesia It was of interest to determine whether or not the bradycardia produced by pronethalol under urethane anaesthesia was sensitive to atropine. Since cardiac rate is modified by both sympathetic and parasympathetic nerve fibres, blockade of one is bound to accentuate the effects of the other. The effect of a single dose of pronethalol was therefore compared in groups of rats which had been pretreated with saline, atropine or bilateral vagotomy, all under urethane anaesthesia. As shown in table 2, pretreatment with atropine or by surgical vagotomy resulted in a highly significant rise in heart rate (p < 0.001). This pretreatment did not, however, abolish the bradycardia consequent upon pronethalol administration. It was observed that the final heart rate in the control group was considerably lower than that in the atropine or vagotomy groups thus showing the full extent of vagal dominance as compared to the inhibition of the sympathetic component only in the latter groups. 3.4. E f f e c t o f anaesthetic on heart rate response to pain It was noted that handling of animals under urethane anaesthesia sometimes produced irregular fluctuations in heart rate. The application of a painful stimulus produced quite different results with the two anaesthetics. Under urethane there was a marked tachycardia which was substantially reduced by hexa-
269
Table 2 Effect of pronethalol (20 mg/kg) on heart rate in rats anaesthetized with urethane, pre-treated with atropine (5 mg/kg) or by vagotomy (beats/min -+S.E.M., n = 6). Pre-
treatment
Heart rate Treatment Initial
15 rain postChange treatment
Saline
Saline Pronethalol
291_+17 290_+38
291_+23 229-+46
0-+ 8 61-+ 8
Atropine (5mg/kg)
Saline Pronethalol
419,25 411-+10
411-+22 358_+ 2
- 8-+ 5 - 5 2 -+ 9
Bilateral Saline vagotomy Pronethalol
415-+11 440-+17
412-+17 353 -+29
- 3-+ 8 -87 -+12
methonium treatment. No significant changes were observed under pentobarbitone (table 3). 3.5. Effect o f anaesthesia on heart rate response to hypothalamic stimulation It was possible that the apparent differences between responses in rats anaesthetized with urethane and pentobarbitone was due to ganglion blocking activity of the latter agent. Attempts were made therefore to stimulate the sympathetic nerves to the heart preganglionically but were abandoned for technical reasons. As an alternative, indwelling electrodes were placed in the anterior and posterior regions of the hypothalamus for the application of an electrical stimulus. Stimulation of the anterior region produced a significant bradycardia in rats anaesthetized with halothane or urethane, but was without effect in animals receiving pentobarbitone. Posterior hypo-
Table 3 Effect of pain on heart rate under pentobarbitone or urethane anaesthesia before and after hexamethonium (beats/min -+ S.E.M., n = 6).
Control heart rate Response to pain Response to pain 15 min post-hexamet honium (5 mg/kg)
Pentobarbitone
Urethane
415 -+ 12 + 9 -+ 3
400 -+ 17 +85 -+ 5
+14-+ 9
+17 -+ 7
A.M.Barrett, Anaesthesia and autonomic blockade
270
Table 4 Change in heart rate produced by hypothalamlc stimulation under halothane, urethane and pentobarbitone anaesthesia (beats/min ± S.E.M., n = 6). Position of electrodes Anterior hypothalamus Posterior hypothalamus
Halothane
Urethane
Pentobarbitone
- 7 2 ± 12
-47 ± 7
+2 ± 3
+36 ± 10
+82 ± 9
-2 ± 3
p r o p e r t y in more detail and relate it to other /3-adr e n o c e p t o r antagonists. In addition to urethane and p e n t o b a r b i t o n e anaesthetized animals, studies were also made in pithed rats. The results (table 5) showed that the behaviour o f these agents in rats anaesthetized with p e n t o b a r b i t o n e , or those which were pithed, was very similar. In all cases p r o n e t h a l o l was significantly less stimulant than dichloroisoprenaline b u t nevertheless significantly m o r e so than propranolol or sotalol. Had e x p e r i m e n t s only been carried out in rats anaesthetized with urethane, however, then only dichloroisoprenaline w o u l d have appeared to possess partial agonist properties. The origin o f the bradycardia in rats anaesthetized w i t h p e n t o b a r b i t o n e following propranolol or sotalol at 20 m g / k g was puzzling. Administration o f atropine b r o u g h t about no increase in the low rate and one is t e m p t e d to conclude some other effect than/3-adrenoc e p t o r blockade.
thalamus stimulation p r o d u c e d a tachycardia following h a l o t h a n e or urethane but n o t p e n t o b a r b i t o n e . The results are s u m m a r i z e d in table 4. 3.6. Demonstration o f intrinsic sympathomflnetic ac-
tivity U n d e r p e n t o b a r b i t o n e anaesthesia, p r o n e t h a l o l p r o d u c e d a small b u t reproducible i n c r e m e n t in heart rate. Since the selection o f this c o m p o u n d for clinical evaluation derived f r o m its apparent lack o f symp a t h o m i m e t i c properties (Black and Stephenson, 1962), it was o f interest to explore this stimulant
3.7. Effect o f anaesthetic on intrinsic sympathomimetic activity and H-blocking activity An a t t e m p t was m a d e to d e t e r m i n e the effect o f p e n t o b a r b i t o n e or urethane on the estimation o f
Table 5 Effect of various #-adrenoceptor blocking drugs (20 mg/kg) on heart rate in rats anaesthetized with pentobarbitone or urethane and in pithed animals (beats/min ± S.E.M., n = 6). Heart rate Anaesthetic
Treatment
(20 mg/kg)
Before treatment
15 min after treatment
Change
Pentobarbitone
Sa.qne Dichloroisoprenaline Pronethalol Propranolol Sotalol
371 365 356 381 392
± 20 ± 8 ± 8 ± 22 ± 19
365211 441± 8 390 ± 12 318 ± 20 304 ± 14
- 6± +76± +35 ± -63 ± -88 ±
Urethane
Saline Dichloroisoprenaline Pronethalol Propranolol Sotalol
328 322 335 322 337
± 16 ± 12 ± 39 ± 21 ± 19
343 351 245 250 280
+16 +29 -90 -72 -63
Pithed rats
Saline Dichloroisoprenaline Pronethalol Propranolol Sotalol
299 270 270 288 276
± ± ± ± ±
288± 332 ± 307 ± 272 ± 242±
18 9 7 12 10
± 23 ± 24 ± 12 ± 10 ± 7 12 9 17 12 7
9 12 14 12 13
± 7 ± 17 ± 26 ± 15 ± 20
-11± 4 +63 ± 8 +38 ± 20 - 1 6 ± 10 - 3 3 ±11
271
A.M.Barrett, Anaesthesia and autonomic blockade
Table 6 The effect of adrenergic #-receptor antagonists on (a) heart rate at various doses and (b) the response to adrenaline infusion in rats anaesthetized with pentobarbitone (P) or urethane (U) (beats/min ± S.E.M., n = 6). Dose (mg/kg)
Saline P
U
Dichloroisoprenaline
Pronethalol
Propranolol
Sotalol
P
U
P
U
P
U
P
U
+ 47±11 +118±17 4±15
+53±15 +28±12 + 4±16
- 9±15 -22±19 -84±18
-18±15 -14±13 -78±10
-27±19 -52±23 -82±12
-29± 9 -57± 6 -78±10
-18±16 -48±21 -47± 8
(a) Change in heartrate 15 minafterantagonist 1 5 25
- 4± 5 + 12± 6 + 3± 3
+ 13± 6 8± 8 6±16
+ 89±10 +100± 8 +115±16
(b) C h a n g e i n r a t e a ~ e r a d r e n f l m e i n f u s i o n ( 2 g ~ k ~ m i n ) f o r l 5 min, theinfusion c o m m e n c m g i m m e d m t e l y a f t e r t h e t a k i n g of readmgsgwenin(a) 1 5 25
+108±13 +100± 8 +138±30
+ 97±11 +163± 8 +116± 7
+ 10± 6 8± 8 - 39±27
+ 50±16 + 67±19 +109±19
+ 7± 9 +20±12 -31±32
i n t r i n s i c s y m p a t h o m i m e t i c a c t i o n a n d t h e ability to b l o c k a n a d r e n a l i n e - i n d u c e d t a c h y c a r d i a ( t a b l e 6). T h e e f f e c t o f various doses o f t h e f o u r / ~ - a d r e n o c e p t o r a n t a g o n i s t s p e r se is s u m m a r i z e d in t a b l e 6a. U n d e r p e n t o b a r b i t o n e , d i c h l o r o i s o p r e n a l i n e raised h e a r t rate in a d o s e - d e p e n d e n t m a n n e r b u t w i t h a v e r y s h a l l o w
+47±16 +60±17 +68± 4
+14±16 -75±25 - 4±18
+39±12 +37± 7 +27± 5
+64± 9 -48±16 -44±32
+18±11 +50±23 + 1±18
slope. C o n v e r s e l y , the e f f e c t o f p r o n e t h a l o l was an inverse r e l a t i o n s h i p in r e l a t i o n to t a c h y c a r d i a . Since s y m p a t h e t i c activity is l o w u n d e r p e n t o b a r b i t o n e this p r e s u m a b l y reflects the d o m i n a n c e o f t h e direct d e p r e s s a n t a c t i o n o f p r o n e t h a l o l over its partial agonist p r o p e r t i e s w h e r e a s t h e relatively greater agonist
200
150
Blood
Blood
175 Pressure
Pressure 150
100
Imm. HO)
I mm Hg ]
75'
125
F
, ~
Heart j Rate 400~(beats/mini /
350 L.
Heart Rate 350 ( beats/rain. 1 300
................
t
400
t
Adrenaline infusion [ 21~g.I kg./min. )
T Pronethalot 25 mgJk9, i.p
I I I I I I I I I I I i i i i i i i i
I
t Adrenaline infusion
Fig. 1. Blood pressure and heart rate responses to adrenaline infusion before and after pronethalol in a rat anaesthetized with pentobarbitone (55 mg/kg).
272
A.M.Barrett, Anaesthesia and autonomic blockade
potency of dichloroisoprenaline remains dominant even at the highest dose level. Under urethane anaesthesia the agonist activity of dichloroisoprenaline was less apparent, there being an equilibrium between that effect and blockade of endogenous catecholamines. Under urethane, pronethalol demonstrated a dose-dependent bradycardia. Both propranolol and sotalol decreased heart rate under both anaesthetics. If the marked effect of propranolol at 25 mg/kg was due to a direct depressant action, under pentobarbitone it was puzzling to observe the same effect with sotalol, which is apparently devoid of direct action on cardiac muscle (Lish et al., 1965). Recently, however, prolongation of the cardiac action potential with sotalol has been reported both in guinea pigs (Singh and Vaughan Williams, 1970) and dogs (Strauss et al., 1970). Infusion of adrenaline, 15 rain after various blocking drugs did not produce a uniform response (table 6b). Under pentobarbitone anaesthesia, all four drugs either abolished or significantly reduced the tachycardia, sotalol being the least effective antagonist. At higher doses, adrenaline usually resulted in a bradycardia. The reason for this is illustrated in fig. 1. Whereas the normal response of the rat to adrenaline infusion is an increase in heart rate and blood pressure, following t3-adrenoceptor blockade the pressor response is potentiated as a result of blockade of peripheral vasodilator receptors. In the presence of cardiac adrenergic blockade the activation of the baro-receptor reflex results in a vagal stimulation. The bradycardia to adrenaline following 13-adrenergic blockade was always abolished by atropine. In marked contrast to the results using pentobarbitone as an anaesthetic, it was much more difficult to demonstrate ¢3-blockade under urethane and in no case was bradycardia observed following adrenaline infusion. Limited experiments with pithed rats showed total blockade to the adrenaline tachycardia at 25 mg/kg for all the antagonists without the appearance of any bradycardia.
4. DISCUSSION The results of the present experiments demonstrate unequivocally that the choice of anaesthetic agent in acute experiments may exert most important
influences on the information obtained, especially with autonomic blocking drugs. The observed response of heart rate to either cholinergic or/3-adrenergic blockade is highly dependent on the pre-existing degree of autonomic activity. In the absence of any information concerning this, the observed response cannot be used in a valid manner to determine the exact or comparative effect of any given drug. If autonomic tone is absent, as in pithed animals, or at a very low level as in pentobarbitone anaesthesia, then antagonists devoid of other properties will show no effect, unless a challenge is applied. Conversely, if autonomic activity appears to be high, as apparently is the case with urethane anaesthesia, then marked effects of either cholinergic or/3-adrenergic blockade will be expected. If partial agonist blocking drugs are studied against a high endogenous stimulus such effects are minimal, but accentuated in the absence of endogenous drive. It was of interest to note that although pentobarbitone anaesthesia abolished the responses to hypothalamic stimulation, it did not prevent reflex responses to elevations in blood pressure. The pattern of results reported under urethane anaesthesia was quite different from those obtained when pentobarbitone was used. Blockade of endogenous catecholamines predominated over partial agonist properties and antagonism of exogenous catecholamines was less easy to establish. This difference in response to sympathetic stimulation and blockade under urethane and pentobarbitone affords a complete explanation of the earlier differences in results reported by Black et al. (1962) and the present author. These results also help to explain the findings of Dasgupta (1968) and Yamamoto and Sekiya (1969), who found that propranolol produced a pressor response in rats. Both groups used urethane as the anaesthetic and showed the pressor response to be absent in pithed or spinal rats. The pressor response was not abolished by ganglionic or a-adrenergic blockade but prevented by pre-treatment with another /3-adrenoceptor antagonist, pronethalol. Taking all the data into consideration, it is reasonable to assume that the pressor response derives from blockade of the peripheral vasodilator actions of catecholamines, raised to an artificially high level by the choice of urethane as the anaesthetic. The evidence does not support the view that the cardiovascular responses of the rat, as a species, are unique.
A.M.Barrett, Anaesthesia ana autonomic blockade
Supportive documentation for the present analysis comes from the work of Reinert (1964), who showed marked hyperglycaemia following urethane anaesthesia in rats, subsequently confirmed by Barrett et al. (1966), not apparent with pentobarbitone. Reinert concluded that urethane produced a hypothalamic (or general limbic) activation resulting in increased descending sympathetic discharge and enhanced catecholamine secretion from the adrenal medulla. Other workers have shown that the frequency of discfiarge of spindle motor fibres is increased by urethane but depressed by barbiturates such as thiopentone and hexobarbitone (Diete-Spiff and Pascoe, 1959). In conclusion it is evident that great care must be taken in the interpretation of results derived from experiments in anaesthetized animals, especially when a response appears to be unique in any given respect. In particular, careful attention should be given to experiments utilizing urethane which, despite its technical convenience, may create a situation irrelevant to that found with other anaesthetics and in the conscious state.
ACKNOWLEDGEMENT The invaluable technical assistance of Miss Maureen Wain is gratefully acknowledged.
273
REFERENCES Barrett, A.M., J. Raventos and R.A. Siddall, 1966, The influence of some anaesthetics on pharmacologically stimulated gastric secretion in the rat, Brit. J. Pharmacol. 28,51. Black, J.W., A.C. Dornhorst, T.R.E. Pilkington, B.F. Robinson and R.D. Lowe, 1962, Laboratory and clinical experiences with a new adrenergic beta-receptor antagonist, Conference Abstracts. IV World Congress on Cardiology, Mexico City. Black, J.W. and J.S. Stevenson, 1962, Pharmacology of a new adrenergic beta-receptor blocking compound (Nethalide), Lancet I, 1080. Dasgupta, N.K., 1968, On the mechanism of the pressor response due to propranolol, Brit. J. Pharmacol. 34, 200 P. Diete-Spiff, K. and J.E. Pascoe, 1959, The spindle motor nerves to the gastroenemius muscle of the rabbit, J. Physiol. (Lond.) 149, 120. Lish, P.M., J.H. Weikel and K.W. Dungan, 1965, Pharmacological and toxicological properties of two new ~-adrenergic receptor antagonists, J. Pharmacol. Exptl. Therap. 149,161. Reinert, H., 1964, Urethane hyperglycaemia and hypothalamic activation, Nature (Lond.) 204,889. Shipley, R.E. and J.J. Tilden, 1947, Pithed rat preparation suitable for assaying pressor substances, Proc. Soc. Exptl. Biol. 64,453. Singh, B.N. and E.M. Vaughan Williams, 1970, A third class of anti-arrhythmic action. Effects on atrial and ventricular intracellular potentials, and other pharmacological actions on cardiac muscle, of MJ 1999 and AH 3474, Brit. J. Pharmacol. 39,675. Strauss, H.C., T.H. Bigger and B.F. Hoffman, 1970, Electrophysiological and beta-receptor blocking effects of MJ 1999 on dog and rabbit cardiac tissue. Circulation Res. 26, 661. Yamamoto, J. and A. Sekiya, 1969, On the pressor action of propranolol in the rat, Arch. Intern. Pharmacodyn. 179, 372.
THE EFFECTS OF SOME AUTONOMIC BLOCKING AGENTS ON THE HEART RATES OF ANAESTHETIZED
AND PITHED RATS
A.M. B A R R E T T *
Department o f Pharmacology, L C.L Ltd., Pharmaceuticals Division, AIderley Park, Macclesfield, Cheshire
Received 29 July 1970
Accepted 21 May 1971
A.M. BARRETT, The effect o f some autonomic blocking agents on the heart rates o f anaesthetized and pithed rats, European J. Pharmacol. 15 (1971) 267-273. The effect of anaesthesia induced by pentobarbitone and urethane on the heart rate responses to autonomic blocking drugs was studied in rats. Under pentobarbitone anaesthesia cholinergic and ¢~-adrenergicantagonists had little effect on heart rate except for those agents possessing partial agonist or general depressant 'properties. There was no response to pain or hypothalamic stimulation and t3-adrenergic blockade to exogenous catecholamines was easily demonstrated. Under urethane anaesthesia, marked effects were seen in response to autonomic antagonists, to pain and to hypothalamic stimulation./3-adrenergic blockade was difficult to establish. Responses in pithed rats closely paralleled those in rats anaesthetized with pentobarbitone. It was concluded that whereas pentobarbitone depressed autonomic function urethane increased it. No evidence was found to support the view that the cardiovascular responses of the rat were different from those of other mammals. A warning was given of the dangers of misinterpreting results obtained from animals anaesthetized with urethane. Urethane Pentobarbitone
Autonomic blockade Differences between rat and other s p e c i e s
1. INTRODUCTION Early experiments with pronethalol appeared to show that the rat behaved differently from other species in the context of 13-adrenoceptor blockade. The drug failed to block an isoprenaline-induced tachycardia (contrary to the effect in mice, guineapigs, rabbits, cats, dogs, monkeys and men) yet produced a marked bradycardia somewhat greater than that seen in the other species. Atropine significantly reduced but did not abolish the bradycardia produced by pronethalol in rats (Black et al., 1962). Concurrent experiments by the present author in a different part of the same laboratory demonstrated that pronethalol produced a conventional d o s e dependent inhibition of an isoprenaline-induced * Present address: Dept. of Pharmacology, School of Medicine, The University, Leeds 2.
Atropine Propranolol
tachycardia in rats and that the drug alone raised heart rate. A survey of the differences in technique of the two investigators revealed that the principal variable was the choice of anaesthetic. It was found that the anaesthetic used could have a dramatic effect on the heart rate response to a number of autonomic blocking drugs, thereby making direct transposition of results into a clinical context potentially misleading. In addition, it was shown that there was not a unique difference in the cardiovascular responses of the rat to/3-adrenoceptor blockade.
2. MATERIALS AND METHODS The animals used were male albino rats ( 1 9 0 - 2 1 0 g) of the Wistar Strain bred at Alderley Park under s.p.f, conditions. They were maintained on a cubed diet and water ad libitum. Anaesthesia
268
A.M.Barrett, Anaesthesia and autonomic blockade
was induced by intraperitoneal injections of pentobarbitone (55 mg/kg) or urethane (diethylcarbamate) (1.5 g/kg) as a 25% solution. Some rats were pithed according to the method o f Shipley and Tilden (1947). Heart rate was monitored by a cardiotachometer triggered by the QRS complex of the electrocardiogram. Blood pressure was monitored from a cannula in the left carotid artery by means of a Condon manometer. Intravenous injections were made in an exposed saphenous vein in volumes of 0.1 ml/100 g b o d y weight. Autonomic blocking drugs were given by i.p. injection 15 min after the establishment of a steady heart rate under anaesthesia. The change in rate was monitored continuously but since a plateau response was achieved at 15 rain, the effect at that time has been used for these experiments. For catecholamines the peak response was recorded which usually occurred at 90 sec. In all experiments, the mean value for 6 rats was recorded together with the standard error of the mean. In some experiments an electrical crocodile clip was applied to the tail under anaesthesia as a " p a i n " stimulus. Some rats were prepared with indwelling electrodes in the anterior and posterior hypothalamus selecting the appropriate coordinates for use with a stereotaxic instrument, l_eads were exteriorized and cemented to the skull. An electrical stimulus was applied ( 7 - 1 0 V, 3 0 - 4 0 pulses/sec, duration 10msec) for 3 0 s e c under anaesthesia. The same animals were tested under halothane, pentobarbitone and urethane anaesthesia in that sequence at 4-day intervals. The animals did not recover consciousness from urethane and were killed. The position of the electrodes was checked microscopically post-mortem. The drugs used were adrenaline bitatrate, isoprenaline sulphate (Burroughs Welcome), hexamethonium (May & Baker), atropine sulphate (B.D.H.) and pronethalol, propranolol, practolol (I.C.I. 50,172) and sotalol (MJ 1999) all prepared in the chemical laboratories o f I.C.I. Ltd. The doses of c o m p o u n d used refer to the base.
3. RESULTS
3.1. Effects o f pronethalol and atropine on heart rate under urethane and pentobarbitone anaesthesia The effect o f a single dose of pronethalol on heart rate was compared in rats anaesthetized with either urethane or pentobarbitone. The resting heart rate under urethane was generally lower than that under pentobarbitone but the difference did not always reach statistical significance. The response under the two anaesthetics to pronethalol was quite different. Whereas a significant bradycardia (p < 0.01) occurred in animals anaesthetized with urethane, pronethalol induced a small, but statistically significant Co < 0.05), tachycardia in rats anaesthetized with pentobarbitone. Administration of atropine also produced different results under the two experimental conditions. In rats anaesthetized with pentobarbitone, heart rate was not significantly altered but a highly significant rise in heart rate (p < 0.01) was observed under urethane anaesthesia. The results, together with those for saline controls, are summarized in table 1. 3.2. Effect o f pronethalol on an isoprenaline-induced
tachycardia anaesthesia
under
urethane and pentobarbitone
The sensitivity of rats anaesthetized with pentobarbitone or urethane to isoprenaline stimulation of heart rate was found to be markedly different. Using
Table 1 Effect of anaesthetic on heart rate response to atropine and pronethalol (beats/min ± S.E.M., n = 6).
Anaesthetic
Treatment
Urethane
Saline Pronethalol Saline Atropine
Pentobarbitone Saline Pronethalol Saline Atropine
Dose
(mg/kg)
Change in heart rate 15 min after treatment
20
+ 8± 6 - 62 ± 10
5
+ 15 t 11 +102 ± 21
20
+ 4± 4 + 29 ± 11
5
- 5 -+ 3 - 14 ± 8
A.M. Barrett, Anaesthesia and autonomic blockade pentobarbitone, 0.1 #g/kg isoprenaline i.v. was found to give a sub-maximal response of about 100 beats/rain. When urethane was employed it was necessary to raise the dose of isoprenaline to 2 /~g/kg i.v., to produce a response of similar magnitude. In pentobarbitone anaesthetized rats the dose of pronethalol required to produce a 50% block of the isoprenaline response averaged 0.82 mg/kg, whereas in urethane anaesthetized animals an average of 16.5 mg/kg of pronethalol was required. Since in the earlier experiments of Black et al. (1962) the respective doses used were 10 gg/kg of isoprenaline and 25 mg/kg of pronethalol it is probable that failure to block the tachycardia (in animals anaesthetized with urethane) was due to the use of a supra-maximal dose of agonist and an inadequate dose of pronethalol under their particular experimental conditions. i
3.3. E f f e c t o f atropine and vagotomy on heart rate response to pronethalol under urethane anaesthesia It was of interest to determine whether or not the bradycardia produced by pronethalol under urethane anaesthesia was sensitive to atropine. Since cardiac rate is modified by both sympathetic and parasympathetic nerve fibres, blockade of one is bound to accentuate the effects of the other. The effect of a single dose of pronethalol was therefore compared in groups of rats which had been pretreated with saline, atropine or bilateral vagotomy, all under urethane anaesthesia. As shown in table 2, pretreatment with atropine or by surgical vagotomy resulted in a highly significant rise in heart rate (p < 0.001). This pretreatment did not, however, abolish the bradycardia consequent upon pronethalol administration. It was observed that the final heart rate in the control group was considerably lower than that in the atropine or vagotomy groups thus showing the full extent of vagal dominance as compared to the inhibition of the sympathetic component only in the latter groups. 3.4. E f f e c t o f anaesthetic on heart rate response to pain It was noted that handling of animals under urethane anaesthesia sometimes produced irregular fluctuations in heart rate. The application of a painful stimulus produced quite different results with the two anaesthetics. Under urethane there was a marked tachycardia which was substantially reduced by hexa-
269
Table 2 Effect of pronethalol (20 mg/kg) on heart rate in rats anaesthetized with urethane, pre-treated with atropine (5 mg/kg) or by vagotomy (beats/min -+S.E.M., n = 6). Pre-
treatment
Heart rate Treatment Initial
15 rain postChange treatment
Saline
Saline Pronethalol
291_+17 290_+38
291_+23 229-+46
0-+ 8 61-+ 8
Atropine (5mg/kg)
Saline Pronethalol
419,25 411-+10
411-+22 358_+ 2
- 8-+ 5 - 5 2 -+ 9
Bilateral Saline vagotomy Pronethalol
415-+11 440-+17
412-+17 353 -+29
- 3-+ 8 -87 -+12
methonium treatment. No significant changes were observed under pentobarbitone (table 3). 3.5. Effect o f anaesthesia on heart rate response to hypothalamic stimulation It was possible that the apparent differences between responses in rats anaesthetized with urethane and pentobarbitone was due to ganglion blocking activity of the latter agent. Attempts were made therefore to stimulate the sympathetic nerves to the heart preganglionically but were abandoned for technical reasons. As an alternative, indwelling electrodes were placed in the anterior and posterior regions of the hypothalamus for the application of an electrical stimulus. Stimulation of the anterior region produced a significant bradycardia in rats anaesthetized with halothane or urethane, but was without effect in animals receiving pentobarbitone. Posterior hypo-
Table 3 Effect of pain on heart rate under pentobarbitone or urethane anaesthesia before and after hexamethonium (beats/min -+ S.E.M., n = 6).
Control heart rate Response to pain Response to pain 15 min post-hexamet honium (5 mg/kg)
Pentobarbitone
Urethane
415 -+ 12 + 9 -+ 3
400 -+ 17 +85 -+ 5
+14-+ 9
+17 -+ 7
A.M.Barrett, Anaesthesia and autonomic blockade
270
Table 4 Change in heart rate produced by hypothalamlc stimulation under halothane, urethane and pentobarbitone anaesthesia (beats/min ± S.E.M., n = 6). Position of electrodes Anterior hypothalamus Posterior hypothalamus
Halothane
Urethane
Pentobarbitone
- 7 2 ± 12
-47 ± 7
+2 ± 3
+36 ± 10
+82 ± 9
-2 ± 3
p r o p e r t y in more detail and relate it to other /3-adr e n o c e p t o r antagonists. In addition to urethane and p e n t o b a r b i t o n e anaesthetized animals, studies were also made in pithed rats. The results (table 5) showed that the behaviour o f these agents in rats anaesthetized with p e n t o b a r b i t o n e , or those which were pithed, was very similar. In all cases p r o n e t h a l o l was significantly less stimulant than dichloroisoprenaline b u t nevertheless significantly m o r e so than propranolol or sotalol. Had e x p e r i m e n t s only been carried out in rats anaesthetized with urethane, however, then only dichloroisoprenaline w o u l d have appeared to possess partial agonist properties. The origin o f the bradycardia in rats anaesthetized w i t h p e n t o b a r b i t o n e following propranolol or sotalol at 20 m g / k g was puzzling. Administration o f atropine b r o u g h t about no increase in the low rate and one is t e m p t e d to conclude some other effect than/3-adrenoc e p t o r blockade.
thalamus stimulation p r o d u c e d a tachycardia following h a l o t h a n e or urethane but n o t p e n t o b a r b i t o n e . The results are s u m m a r i z e d in table 4. 3.6. Demonstration o f intrinsic sympathomflnetic ac-
tivity U n d e r p e n t o b a r b i t o n e anaesthesia, p r o n e t h a l o l p r o d u c e d a small b u t reproducible i n c r e m e n t in heart rate. Since the selection o f this c o m p o u n d for clinical evaluation derived f r o m its apparent lack o f symp a t h o m i m e t i c properties (Black and Stephenson, 1962), it was o f interest to explore this stimulant
3.7. Effect o f anaesthetic on intrinsic sympathomimetic activity and H-blocking activity An a t t e m p t was m a d e to d e t e r m i n e the effect o f p e n t o b a r b i t o n e or urethane on the estimation o f
Table 5 Effect of various #-adrenoceptor blocking drugs (20 mg/kg) on heart rate in rats anaesthetized with pentobarbitone or urethane and in pithed animals (beats/min ± S.E.M., n = 6). Heart rate Anaesthetic
Treatment
(20 mg/kg)
Before treatment
15 min after treatment
Change
Pentobarbitone
Sa.qne Dichloroisoprenaline Pronethalol Propranolol Sotalol
371 365 356 381 392
± 20 ± 8 ± 8 ± 22 ± 19
365211 441± 8 390 ± 12 318 ± 20 304 ± 14
- 6± +76± +35 ± -63 ± -88 ±
Urethane
Saline Dichloroisoprenaline Pronethalol Propranolol Sotalol
328 322 335 322 337
± 16 ± 12 ± 39 ± 21 ± 19
343 351 245 250 280
+16 +29 -90 -72 -63
Pithed rats
Saline Dichloroisoprenaline Pronethalol Propranolol Sotalol
299 270 270 288 276
± ± ± ± ±
288± 332 ± 307 ± 272 ± 242±
18 9 7 12 10
± 23 ± 24 ± 12 ± 10 ± 7 12 9 17 12 7
9 12 14 12 13
± 7 ± 17 ± 26 ± 15 ± 20
-11± 4 +63 ± 8 +38 ± 20 - 1 6 ± 10 - 3 3 ±11
271
A.M.Barrett, Anaesthesia and autonomic blockade
Table 6 The effect of adrenergic #-receptor antagonists on (a) heart rate at various doses and (b) the response to adrenaline infusion in rats anaesthetized with pentobarbitone (P) or urethane (U) (beats/min ± S.E.M., n = 6). Dose (mg/kg)
Saline P
U
Dichloroisoprenaline
Pronethalol
Propranolol
Sotalol
P
U
P
U
P
U
P
U
+ 47±11 +118±17 4±15
+53±15 +28±12 + 4±16
- 9±15 -22±19 -84±18
-18±15 -14±13 -78±10
-27±19 -52±23 -82±12
-29± 9 -57± 6 -78±10
-18±16 -48±21 -47± 8
(a) Change in heartrate 15 minafterantagonist 1 5 25
- 4± 5 + 12± 6 + 3± 3
+ 13± 6 8± 8 6±16
+ 89±10 +100± 8 +115±16
(b) C h a n g e i n r a t e a ~ e r a d r e n f l m e i n f u s i o n ( 2 g ~ k ~ m i n ) f o r l 5 min, theinfusion c o m m e n c m g i m m e d m t e l y a f t e r t h e t a k i n g of readmgsgwenin(a) 1 5 25
+108±13 +100± 8 +138±30
+ 97±11 +163± 8 +116± 7
+ 10± 6 8± 8 - 39±27
+ 50±16 + 67±19 +109±19
+ 7± 9 +20±12 -31±32
i n t r i n s i c s y m p a t h o m i m e t i c a c t i o n a n d t h e ability to b l o c k a n a d r e n a l i n e - i n d u c e d t a c h y c a r d i a ( t a b l e 6). T h e e f f e c t o f various doses o f t h e f o u r / ~ - a d r e n o c e p t o r a n t a g o n i s t s p e r se is s u m m a r i z e d in t a b l e 6a. U n d e r p e n t o b a r b i t o n e , d i c h l o r o i s o p r e n a l i n e raised h e a r t rate in a d o s e - d e p e n d e n t m a n n e r b u t w i t h a v e r y s h a l l o w
+47±16 +60±17 +68± 4
+14±16 -75±25 - 4±18
+39±12 +37± 7 +27± 5
+64± 9 -48±16 -44±32
+18±11 +50±23 + 1±18
slope. C o n v e r s e l y , the e f f e c t o f p r o n e t h a l o l was an inverse r e l a t i o n s h i p in r e l a t i o n to t a c h y c a r d i a . Since s y m p a t h e t i c activity is l o w u n d e r p e n t o b a r b i t o n e this p r e s u m a b l y reflects the d o m i n a n c e o f t h e direct d e p r e s s a n t a c t i o n o f p r o n e t h a l o l over its partial agonist p r o p e r t i e s w h e r e a s t h e relatively greater agonist
200
150
Blood
Blood
175 Pressure
Pressure 150
100
Imm. HO)
I mm Hg ]
75'
125
F
, ~
Heart j Rate 400~(beats/mini /
350 L.
Heart Rate 350 ( beats/rain. 1 300
................
t
400
t
Adrenaline infusion [ 21~g.I kg./min. )
T Pronethalot 25 mgJk9, i.p
I I I I I I I I I I I i i i i i i i i
I
t Adrenaline infusion
Fig. 1. Blood pressure and heart rate responses to adrenaline infusion before and after pronethalol in a rat anaesthetized with pentobarbitone (55 mg/kg).
272
A.M.Barrett, Anaesthesia and autonomic blockade
potency of dichloroisoprenaline remains dominant even at the highest dose level. Under urethane anaesthesia the agonist activity of dichloroisoprenaline was less apparent, there being an equilibrium between that effect and blockade of endogenous catecholamines. Under urethane, pronethalol demonstrated a dose-dependent bradycardia. Both propranolol and sotalol decreased heart rate under both anaesthetics. If the marked effect of propranolol at 25 mg/kg was due to a direct depressant action, under pentobarbitone it was puzzling to observe the same effect with sotalol, which is apparently devoid of direct action on cardiac muscle (Lish et al., 1965). Recently, however, prolongation of the cardiac action potential with sotalol has been reported both in guinea pigs (Singh and Vaughan Williams, 1970) and dogs (Strauss et al., 1970). Infusion of adrenaline, 15 rain after various blocking drugs did not produce a uniform response (table 6b). Under pentobarbitone anaesthesia, all four drugs either abolished or significantly reduced the tachycardia, sotalol being the least effective antagonist. At higher doses, adrenaline usually resulted in a bradycardia. The reason for this is illustrated in fig. 1. Whereas the normal response of the rat to adrenaline infusion is an increase in heart rate and blood pressure, following t3-adrenoceptor blockade the pressor response is potentiated as a result of blockade of peripheral vasodilator receptors. In the presence of cardiac adrenergic blockade the activation of the baro-receptor reflex results in a vagal stimulation. The bradycardia to adrenaline following 13-adrenergic blockade was always abolished by atropine. In marked contrast to the results using pentobarbitone as an anaesthetic, it was much more difficult to demonstrate ¢3-blockade under urethane and in no case was bradycardia observed following adrenaline infusion. Limited experiments with pithed rats showed total blockade to the adrenaline tachycardia at 25 mg/kg for all the antagonists without the appearance of any bradycardia.
4. DISCUSSION The results of the present experiments demonstrate unequivocally that the choice of anaesthetic agent in acute experiments may exert most important
influences on the information obtained, especially with autonomic blocking drugs. The observed response of heart rate to either cholinergic or/3-adrenergic blockade is highly dependent on the pre-existing degree of autonomic activity. In the absence of any information concerning this, the observed response cannot be used in a valid manner to determine the exact or comparative effect of any given drug. If autonomic tone is absent, as in pithed animals, or at a very low level as in pentobarbitone anaesthesia, then antagonists devoid of other properties will show no effect, unless a challenge is applied. Conversely, if autonomic activity appears to be high, as apparently is the case with urethane anaesthesia, then marked effects of either cholinergic or/3-adrenergic blockade will be expected. If partial agonist blocking drugs are studied against a high endogenous stimulus such effects are minimal, but accentuated in the absence of endogenous drive. It was of interest to note that although pentobarbitone anaesthesia abolished the responses to hypothalamic stimulation, it did not prevent reflex responses to elevations in blood pressure. The pattern of results reported under urethane anaesthesia was quite different from those obtained when pentobarbitone was used. Blockade of endogenous catecholamines predominated over partial agonist properties and antagonism of exogenous catecholamines was less easy to establish. This difference in response to sympathetic stimulation and blockade under urethane and pentobarbitone affords a complete explanation of the earlier differences in results reported by Black et al. (1962) and the present author. These results also help to explain the findings of Dasgupta (1968) and Yamamoto and Sekiya (1969), who found that propranolol produced a pressor response in rats. Both groups used urethane as the anaesthetic and showed the pressor response to be absent in pithed or spinal rats. The pressor response was not abolished by ganglionic or a-adrenergic blockade but prevented by pre-treatment with another /3-adrenoceptor antagonist, pronethalol. Taking all the data into consideration, it is reasonable to assume that the pressor response derives from blockade of the peripheral vasodilator actions of catecholamines, raised to an artificially high level by the choice of urethane as the anaesthetic. The evidence does not support the view that the cardiovascular responses of the rat, as a species, are unique.
A.M.Barrett, Anaesthesia ana autonomic blockade
Supportive documentation for the present analysis comes from the work of Reinert (1964), who showed marked hyperglycaemia following urethane anaesthesia in rats, subsequently confirmed by Barrett et al. (1966), not apparent with pentobarbitone. Reinert concluded that urethane produced a hypothalamic (or general limbic) activation resulting in increased descending sympathetic discharge and enhanced catecholamine secretion from the adrenal medulla. Other workers have shown that the frequency of discfiarge of spindle motor fibres is increased by urethane but depressed by barbiturates such as thiopentone and hexobarbitone (Diete-Spiff and Pascoe, 1959). In conclusion it is evident that great care must be taken in the interpretation of results derived from experiments in anaesthetized animals, especially when a response appears to be unique in any given respect. In particular, careful attention should be given to experiments utilizing urethane which, despite its technical convenience, may create a situation irrelevant to that found with other anaesthetics and in the conscious state.
ACKNOWLEDGEMENT The invaluable technical assistance of Miss Maureen Wain is gratefully acknowledged.
273
REFERENCES Barrett, A.M., J. Raventos and R.A. Siddall, 1966, The influence of some anaesthetics on pharmacologically stimulated gastric secretion in the rat, Brit. J. Pharmacol. 28,51. Black, J.W., A.C. Dornhorst, T.R.E. Pilkington, B.F. Robinson and R.D. Lowe, 1962, Laboratory and clinical experiences with a new adrenergic beta-receptor antagonist, Conference Abstracts. IV World Congress on Cardiology, Mexico City. Black, J.W. and J.S. Stevenson, 1962, Pharmacology of a new adrenergic beta-receptor blocking compound (Nethalide), Lancet I, 1080. Dasgupta, N.K., 1968, On the mechanism of the pressor response due to propranolol, Brit. J. Pharmacol. 34, 200 P. Diete-Spiff, K. and J.E. Pascoe, 1959, The spindle motor nerves to the gastroenemius muscle of the rabbit, J. Physiol. (Lond.) 149, 120. Lish, P.M., J.H. Weikel and K.W. Dungan, 1965, Pharmacological and toxicological properties of two new ~-adrenergic receptor antagonists, J. Pharmacol. Exptl. Therap. 149,161. Reinert, H., 1964, Urethane hyperglycaemia and hypothalamic activation, Nature (Lond.) 204,889. Shipley, R.E. and J.J. Tilden, 1947, Pithed rat preparation suitable for assaying pressor substances, Proc. Soc. Exptl. Biol. 64,453. Singh, B.N. and E.M. Vaughan Williams, 1970, A third class of anti-arrhythmic action. Effects on atrial and ventricular intracellular potentials, and other pharmacological actions on cardiac muscle, of MJ 1999 and AH 3474, Brit. J. Pharmacol. 39,675. Strauss, H.C., T.H. Bigger and B.F. Hoffman, 1970, Electrophysiological and beta-receptor blocking effects of MJ 1999 on dog and rabbit cardiac tissue. Circulation Res. 26, 661. Yamamoto, J. and A. Sekiya, 1969, On the pressor action of propranolol in the rat, Arch. Intern. Pharmacodyn. 179, 372.