Brit. J. Pharmacol. (1960), 15, 117. THE ACTION OF SYMPATHETIC BLOCKING AGENTS ON ISOLATED AND INNERVATED ATRIA AND VESSELS BY S. HUKOVIC* From the Department of Pharmacology, University of Oxford (RECEIVED JULY 31, 1959) A preparation is described of isolated rabbit atria with both vagus and sympathetic nerves. The action on it of bretylium and of choline 2,6-xylyl ether bromide (TM10) was studied. A concentration of breylium sufficient to abolish the response to sympathetic stimulation also depressed the response to vagal stimulation. The effect was reversible, though more easily with choline xylyl ether. Both drugs abolished the accelerating action of acetylcholine in the presence of atropine, but they augmented the action of tyramine, and did not reduce that of amphetamine. In the vessels of the perfused rabbit ear they abolished the constrictor effect of nervous stimulation and of acetylcholine, but increased that of tyramine. The substance choline 2,6-xylyl ether (TM10; hereafter called choline xylyl ether) was synthesized by Hey, and Willey discovered that it blocked the response to stimulation of postganglionic adrenergic fibres without affecting the actions of added adrenaline or noradrenaline (Hey and Willey, 1954). This action was studied in greater detail by Exley (1957). Recently bretylium, N-obromobenzyl-N-ethyl-NN-dimethylammonium p- toluene sulphonate, has been introduced by Boura, Green, McCoubrey, Laurence, Moulton, and Rosenheim (1959) as a substance having a similar action which can be used clinically. The action of these two substances on the isolated atria of the rabbit heart has been examined using in some experiments the sympathetically innervated preparation which has been recently described (Hukovid, 1959), and, in some, a doubly innervated preparation so that the vagal fibres or the sympathetic fibres could be stimulated at will. The substances have also been examined for their action on the perfused vessels of the rabbit ear. METHODS Tn all experiments on the atria the solution used was: NaCl 9.0 g., KCl 0.42 g., CaCI2 0.24 g., dextrose 2.0 g. and NaHCO3 0.5 g. in 1 litre. The temperature of the bath was 30. The bath was bubbled with 02. Rabbit's and guinea-pig's atria were set up in the isolated organ bath, as described by Burn (1952). Rabbit atria with the vagus nerve (McEwen, 1956) *Present address: Department of Pharmacology, University of Sarajevo, Yugoslavia. and with the sympathetic nerve were prepared as follows. Rabbits were stunned and killed, and quickly tied out on a table. The skin was removed from the thorax and neck. The vagus nerve and the sympathetic chain of the right side were identified and tied. The trachea and the oesophagus were divided as they entered the thorax. The ventral wall of the chest was removed without disturbing the thymus and neighbouring tissues. The vertebral column was divided with strong scissors between the cervical and thoracic vertebrae. The main vessels were divided in front of the 3rd thoracic vertebra, and the vertebral column was again divided between the 2nd and 3rd thoracic vertebrae. All skeletal muscles were removed and the preparation was immersed in oxygenated solution at 30. The preparation was left in this solution at least 5 min. The preparation was then cleaned again and fixed on the short part of an L-shaped rod, and then was again immersed in the solution where it remained. The proximal part of the vagus nerve was found and tied. The cervical sympathetic nerve was tied and freed of connective tissue as far as the stellate ganglion by blunt dissection, freeing the ganglion as well as possible. The vagus nerve was placed in one pair of electrodes approximately in the middle of its length. The second pair of electrodes were placed around the stellate ganglion itself. The electrodes were of a pattern described by Burn and Rand (1959) with a channel in the holder through which a small amount of solution was constantly siphoned from the bath so that the portion of nerve stimulated was continually irrigated by fully oxygenated solution. The other end of the atria was tied to a spring lever. The stimulus applied was 0.5 ma strength, at a frequency of 20 pulses/sec. Each stimulus was 5 msec. duration, and stimulation was maintained for 15 to 30 sec.
118 S. HUKOVIC If I v I FIG. 1.-Isolated rabbit atria with sympathetic and vagus nerves. S, sympathetic stimulation, showing increase in rate (beats/min.; figures above the tracing) and amplitude. V, vagus stimulation, showing inhibition. B, addition of bretylium, the concentration per ml. being given. Note that in the concentration of 40 ug./ml., bretylium nearly abolished the effect of sympathetic stimulation and in addition reduced the effect of vagal stimulation. W, washing out of bath many times with recovery of effect of sympathetic and vagal stimulation. W1P11N FIG. 0 _ 7 0 Rabbit ears were perfused with Locke solution of the composition described above with half the amount of dextrose. The arrangement was that described by Burn (1952) using Stephenson's recorder (1948). Stimulation was applied by a pair of electrodes to the great auricular nerve at the base of the pinna. RESULTS Bretylium in Doubly Innervated Preparation. The effect of bretylium in isolated rabbit atria innervated by the vagi and by the sympathetic 2.-Isolated rabbit atria, showing block of effect of sympathetic stimulation (S) by choline xylyl ether (TMI0). After 3 min. in bath 10 itg./ml. had no effect (second panel). A further 10 fg. jml. was added 5 min. later, making 20,tg./ml. in all; 3 min. after this second' addition, the effect of sympathetic stimulation was diminished but not abolished (not shown). After 5 min. a third addition of 10 pg./ml. was made, making 30,tg./ml. in all. The effect of nerve stimulation was now abolished (third panel). After four washings (W) over 20 min., the effect of nerve stimulation was restored (last panel). nerves is shown in Fig. 1. Stimulation of the sympathetic nerves increased the amplitude and also increased the rate from 88 to 116 beats/min. Stimulation of the vagus caused atrial arrest. Bretylium added in a concentration of 10 jig./ml. had no effect on sympathetic stimulation, but diminished the effect of vagal stimulation; in a concentration of 20,ug. /ml., however, it diminished the effect of sympathetic stimulation without much further effect on vagal stimulation. Finally
bretylium in a concentration of 40,ug. /ml. nearly abolished the effect of sympathetic stimulation; whereas the initial rise in rate was 28 beats/min., it was reduced to 6 beats/min. However, the effect of vagal stimulation was also smaller. After removing the bretylium from the bath, the initial effects both of sympathetic and of vagal stimulation were restored after many washings during 1 hr. SYMPATHETIC BLOCKING AGENTS ON ATRIA 119 Choline Xylyl Ether on Sympathetic Stimulation.- The effect of choline xylyl ether on sympathetic stimulation is shown in Fig. 2. At first stimulation increased the rate by 28 beats/min. In the presence of 10,.Lg./ml. stimulation was unaffected, but a higher concentration, FIG. 3.-The effecct of bretylium (B) in preventing the increase in rate and 30 /Ag./ml., blocked the effect amplitude due to acetylcholine (ACh) in the presence of atropine. Atropine of stimulation. After washing (Atr) 5 pg./ml. acetylcholine 200,ug./ml., bretylium 20 pg./ml. W, washing out the drug, the initial effect out. of stimulation was restored much more readily than when bretylium was used. The Response to Acetylcholine.-In the presence of atropine, acetylcholine causes stimulation of the isolated rabbit atria as shown in Fig. 3. The concentration of atropine was 5 /Ag. /ml., and the concentration of acetylcholine was 200,ug./ml. This caused the rate to increase by 27 beats/min. in two successive trials. Bretylium (20 4g./ml.) was then added and in its presence acetylcholine was without effect. On washing out the bretylium the effect of acetylcholine was quickly restored. The action of choline xylyl ether was similar to that of bretylium; it also blocked the action of acetylcholine. The Response to Tyramine and Amphetamine.-Concentrations of choline xylyl ether and of bretylium which blocked the accelerator action of acetylcholine had no blocking action on the accelerating action of tyramine. This is illustrated in Fig. 4. A concentration of tyramine, 2,ug./ml., applied twice caused a rise in rate of 16 and of 14 beats/min.; in the presence of bretylium, 20 gug./ml., tyramine, 2,ug./ml., caused a rise in rate of 23 beats/min. ~~~~~~~~ FIG. 4.-The effect of bretylium (B), 20 pg./ml., in increasing the effect of tyramine (Tyr), 2 pg./ml., on the rate and amplitude of the atria. W, washing out.
120 S. HUKOVIC FIG. 5.-Record of outflow from veins of rabbit ear perfused with Locke solution at room temperature. a, Fall in outflow due to stimulation of great auricular nerve at the base of the ear (st); also fall in outflow due to injection of 30,g. acetylcholine (ACh) after 8,g. of atropine (Atr) into the solution entering the artery. b, No change in outflow after the injection of 1,g. tyramine. c, Fall in outflow after injection of 4 plg. tyramine. The ear was then perfused overnight with Locke solution containing I jig./ml. b-etylium. d, Rise in outflow following stimulation of the nerve. e, Rise in outflow following injection of 30 jig. acetylcholine after 8,g. atropine. f, I all in outflow after injection of I tg. tyramine. After washing out the bretylium a further application of tyramine caused a rise in rate of 17 beats/ min. Thus bretylium increased the action of tyramine. The action of amphetamine in increasing the amplitude and rate of the atria was likewise not blocked by bretylium. Since the action of amphetamine is prolonged, a comparison was made between the action of amphetamine in one atrial preparation with its action in the presence of bretylium in another preparation. No difference was observed. In the presence of bretylium, 20 Ag. /ml., amphetamine, 5 jug. /ml., increased the rate from 86 to 132 beats/min. Vessels of Rabbit Ear.-Similar observations were made on the vessels of the rabbit ear. Bretylium and choline xylyl ether when added to the perfusion fluid blocked the constrictor effect of stimulating the great auricular nerve and also that of acetylcholine in the presence of atropine. However, they increased the effect of tyramine. Fig. 5 illustrates these actions of bretylium when it was present in the perfusion fluid in a ;lg./ml. and had been perfused concentration of 1 throughout the night. The dilator effect of stimulation in the presence of bretylium may have been due to stimulation of sensory fibres in the mixed nerve. The dilator action of acetylcholine in the presence of bretylium may have been due, to the administration of insufficient atropine. DISCUSSION The use of an isolated atrial preparation with both vagal and sympathetic nerves intact made
SYMPATHETIC BLOCKING AGENTS ON ATRIA 121 it possible to compare the action of bretylium on the two innervations. In a concentration which almost completely blocked the effect of stimulating sympathetic fibres there was a considerable diminution in the effect of vagal stimulation also. Hence the selective action on the sympathetic terminations was only partial. In a previous paper (Hukovic, 1959) it has been shown that when atrial preparations were made from rabbits treated with reserpine, stimulation of the sympathetic fibres often caused inhibition, and that this inhibitory effect was increased by eserine and abolished by atropine. This showed that cholinergic fibres were present. As Burn and Rand (1959) have suggested, such cholinergic fibres would normally have an adrenergic effect, since the acetylcholine they liberate would in turn liberate noradrenaline from the store in the neighbourhood of the nerve endings. These cholinergic fibres are indeed only recognized as cholinergic when treatment with reserpine has dispersed the store, and the liberated acetylcholine then exerts its own effect. Choline xylyl ether and bretylium inhibit the accelerating action of acetylcholine seen in the presence of atropine. This action has been shown to be exerted by release of noradrenaline from the store, since it is absent in preparations from rabbits treated with reserpine. Choline xylyl ether and bretylium do not appear to affect the store of noradrenaline itself, since they do not reduce the action of tyramine, and indeed they increase it. It is suggested, therefore, that they inhibit the accelerating action of acetylcholine mentioned above by preventing the liberation of noradrenaline from the store. They would also be expected to inhibit the noradrenaline-liberating action of cholinergic nerve fibres. This work has been done during the tenure of a Fellowship from the British Council. I am indebted to Miss Roneen Hobbs for the experiments on the perfused rabbit's ear. This work was suggested and supervised by Professor J. H. Burn, to whom I am very grateful. REFERENCES Boura, A. L. A., Green, A. F., McCoubrey, A., Laurence, D. R., Moulton, R., and Rosenheim, M. L. (1959). Lancet, 2, 17. Burn, J. H. (1952). Practical Pharmaco.ogy. Oxford: Blackwell Scientific Publications. - and Rand, M. J. (1959). Nature (Lond.), 184, 163. Exley, K. A. (1957). Brit. J. Pharmacol., 12, 297. Hey, P., and Willey, G. L. (1954). Ibid., 9,471. Hukovid, S. (1959). Ibid., 14, 372. McEwen, L. M. (1956). J. Physiol. (Lond.), 131, 678. Stephenson, R. P. (1948). Ibid., 107, 162.