INTRODUCTION. IN a previous paper(l) we have been able to show that adrenaline may

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1 REVERSAL OF THE ACTION OF ADRENALINE. BY B. A. McSWINEY AND G. L. BROWN. (From the Department of Physiology, University of Manchester.) INTRODUCTION. IN a previous paper(l) we have been able to show that adrenaline may have either an augmentor or an inhibitory action on excised strips of muscle of certain regions of the rabbit's stomach. It had an inhibitory action on the pyloric sphincter, antrum and cardiac sphincter, and an augmentor action on the lower body and the circular fibres of the upper body and fundus. On strips taken from the longitudinal fibres of the upper body and fundus, it had in some preparations an augmentor, in others an inhibitor action. If an augmentor effect was elicited, addition of pilocarpine reversed the action of adrenaline, subsequent doses of adrenaline causing not a contraction, but a pronounced relaxation. These results suggest that the condition of "tonus" of the preparations determined whether adrenaline had an augmentor or an inhibitor effect. Many references are found in the literature to the reversed action of adrenaline on smooth muscle. Cannon and Lyman(2) have obtained depressor effects with adrenaline, when the blood-pressure was high, a phenomenon which Dale and Richards (3) attributed to capillary dilatation. Kolm and Pick(4) demonstrated a reversal of the action of adrenaline on the heart by giving the drug subsequently to acetyl choline, but as the effect was abolished by atropine, they believed it to be due to adrenaline acting on the parasympathetic endings. Similar effects have been obtained on the heart and blood vessels by perfusing with fluids of varying ph and salt content. Cow(5) has recorded a reversal of the action of adrenaline in surviving strips of the guinea-pig's uterus, after the tissue had been " sensitised " by pituitrin. It appeared desirable to investigate further the reversal of the action of adrenaline obtained after pilocarpine and to determine under what conditions it may occur. The shortening of the muscle fibres obtained by the addition of pilocarpine would appear to predispose to an inhibitor response with adrenaline, but the possibility of a chemical interaction between pilocarpine and adrenaline had first to be excluded. To show

2 ADRENALIN REVERSAL. the relationship between the length of the muscle fibre and the action of adrenaline, we have first studied the effect of other stimulating drugs, choline, histamine, pituitrin and barium chloride, on the reaction of the tissue to adrenaline. Method. Rabbits, cats and dogs have been used in these experiments. Strips were excised from the stomach and suspended in Tyrode's solution at 380 C., through which a constant stream of oxygen was passed. The apparatus was similar to that previously described, except that the inner chamber was made of glass. 1 c.c. of the drug solution was added to 250 c.c. of Tyrode's solution in the chamber, giving a dilution of 1: 250,000; a 1: 1000 solution of adrenaline (Parke Davis) was employed. Time intervals on the tracings equal 60 seconds. EXPERIMENTS. 1. Reversal by pilocarpine. Pilocarpine brings about reversal in the longitudinal strips from the fundus and upper body of the rabbit, a typical record of this action being shown in Fig. 3. In many experiments, interesting mixed effects were obtained, the augmentor response being preceded by a slight inhibition: after pilocarpine the preliminary inhibition becomes the predominant feature and is followed by a slight augmentor action. In some experiments the pilocarpine did not cause complete reversal, simply converting a pure augmentor effect into an augmentor response preceded by a slight inhibition. In the other preparations of the rabbit's stomach, i.e. the lower body and circular fibres of the upper body, we were unable to obtain a definite reversal, the contraction produced by adrenaline being superimposed upon that caused by the pilocarpine. The same persistence of the augmentor response was observed by us in the cardia of the cat and dog (6). 2. Reversal by other drugs. (a) Choline. Doses of 40 mg. of choline chloride were used, as preliminary experiments had shown that this amount of the drug was approximately equivalent to 1 mg. of pilocarpine. The effect of choline on these preparations resembled closely that of pilocarpine. In the rabbit's fundus and upper body, addition of the drug was followed by a marked permanent shortening of the muscle fibres, but rhythmic contractions were little changed. In the lower body, rhythmic contractions were much augmented and there was slight permanent shortening. Choline was found to reverse the response to adrenaline in those regions in which pilocarpine had this effect. Choline, however, differed from pilocarpine as the relaxation produced by adrenaline seemed to be permanent, the muscle fibres not returning to 53

3 54 B. A. McSWINEY AND G. L. BROWN. their former length. In many instances a complete reversal was not obtained, the preliminary inhibition being followed by an augmentor effect. Reversal was not obtained from strips of the lower body, the circular fibres of the body, or the cardia of the cat and dog. (b) Histamine. Doses of 2 mg. histamine (6 mg. histamine phosphate) dissolved in saline were employed. This drug had an augmentor action on all the strips, which resembled the records obtained with adrenaline: a marked and rapid shortening of the muscle and some increase in amplitude of rhythmic movements were recorded. The effect passed off rapidly, the tissue resuming its previous length in approximately minutes. During the augmentor stage, the action of adrenaline was reversed, addition of the drug producing a relaxation. The inhibition was not always followed by recovery, since the effect of histamine passes off during the inhibition. The reversal was only elicited from the longitudinal fibres of the upper body and fundus of the rabbit's stomach. Fig. 1. Fundus of rabbit's stomach, longitudinal strip; records showing reversal of response to adrenaline after addition of pituitrin.

4 ADRENALIN REVERSAL. (c) Pituitrin. The action of pituitrin closely resembled that of histamine, but in contradistinction to histamine, rhythmic contractions were usually diminished. The augmentor effect passed off gradually and the strips resumed their previous length in some 20 minutes. Reversal of the adrenaline action was recorded in the longitudinal fibres of the upper body and fundus, but not in the other regions. In many experiments, incomplete reversal was produced, a diminished augmentor effect being preceded by a rapid inhibition. (d) Barium chloride. This drug caused in the preparations used a slow but sustained contraction of the muscle. In the majority of our experiments, the contraction was well maintained, the lever showing no tendency to return to the base line. Addition of adrenaline, which previously caused an augmentor effect, produced after the addition of barium chloride a well-marked inhibitor response. The reversal effects obtained after the use of either pituitrin or barium chloride are of interest, as these drugs are described as acting directly on the muscle fibres. 55. SI. Fig. 2. Fundus of rabbit's stomach, longitudinal strip; reversal of response to adrenaline after addition of barium chloride. if, as in many experiments, the contraction produced by pituitrin and histamine was less than that obtained with the first dose of adrenaline, then the second dose of adrenaline seldom caused a definite

5 56 B. A. McSWINEY AND G. L. BROWN. inhibitor response, the record showing at the most a slight preliminary inhibition. (e) Atropine. By the use of atropine, further evidence may be advanced to show the relation between the length of the muscle fibre and the response to adrenaline. If the shortening of the muscle fibres predisposes to an inhibitor response, lengthening of the muscle by atropine might be expected to bring about a return of the augmentor response. Atropine was added to strips in which reversal had been produced by either pilocarpine, histamine or pituitrin, and if the relaxation recorded was sufficiently great, addition of adrenaline then caused an augmentor effect. Thus it was possible to obtain in the one strip with equal doses of adrenaline, an augmentor action, then an inhibitor action, and finally, an augmentor action. Fig. 3. Fundus of rabbit's stomach, longitudinal strip; reversal of response to adrenaline after contraction with pilocarpine; recovery of augmentor effect of adrenaline after relaxation with atropine. We found the augmentor reaction of a strip to adrenaline to be in no way impaired by a dose of atropine sufficient to neutralise the action of subsequent doses of as much as 4 mg. of pilocarpine. If adrenaline be considered as acting on a motor and inhibitor mechanism of the neuro-muscular apparatus, then the reversal effects obtained could be accounted for on the assumption that the motor mechanism is thrown out of action either by the physical changes

6 ADRENALIN REVERSAL. accompanying the shortening of the muscle fibres or by the production of some substance in the muscle by the stimulating drugs. There is, further, the possibility, suggested by Langley7, that reversal action may be due to the displacement of one absorbed substance by another. The ability, however, of adrenaline to reverse its own action would appear to disprove the latter suggestion. 57 Fig. 4. Fundus of rabbit's sitomach, longitudinal strip; inhibitor effect of adrenaline when given during contraction produced by previous dose. Thus adrenaline was added to strips from the longitudinal fibres of the fundus, and during the contraction produced, a second dose of the drug was given: this was followed in many experiments by a typical inhibitory response such as is obtained after the use of stimulating drugs. Ergotamine was employed in our later experiments, as this drug, or ergotoxine, as shown by Dale(8, has the property of paralysing the motor endings of the sympathetic. After a motor response had been obtained with adrenaline, 10 mg. of ergotamine (Sandoz) were added. The addition of the drug caused no change in the activity of the preparation, but on addition of adrenaline, a marked inhibitor response was recorded which persisted even after a relaxation had been obtained with atropine. In the regions of the lower body, where rhythmic activity was well seen with but little or no tonus change, adrenaline after ergo-

7 58 B. A. McSWVINEY AND G. L. BROWN. Fig. 5. Fundus of rabbit's stomach, longitudinal strip; reversal of action of adrenaline by ergotamine; persistence of inhibitor response after relaxation with atropine. Fig. 6. Lower body of rabbit's stomach; reversal of action of adrenaline by ergotamine; augmentor and inhibitor effects of adrenaline involving rhythmic contractions only.

8 ADRENALIN REVERSAL. tamine produced a marked decrease in the frequency of contraction, but no change in base line. Fundus of the dog's stomach. In our previous experiments on the fundus of the dog's stomach, an inhibitor response was always obtained with adrenaline, though Smith(9) and Kuroda(lo) recorded an augmentor effect. We have recently been able to obtain a similar effect by keeping the preparation for some hours at 00 C. before suspending it in the bath at a temperature of 380 C. An inhibitor effect was observed witt adrenaline when the muscle was tonically contracted. Guinea-pig's uterus. We have repeated the experiments described by Cow on the isolated uterus of the guinea-pig and have obtained an augmentor response with adrenaline instead of the usual inhibitor effect, by leaving the preparation in Ringer-Locke solution containing 1 in 5000 pituitrin for 1-2 hours at room temperature. The presence of pituitrin is not essential as the same augmentor effect is obtained on leaving the tissue in cold Ringer-Locke solution alone. The experiments were performed with strips of the cornu. Oxygen was bubbled into the solution as in our previous experiments, the temperature of the bath being kept constant at 380 C. 59 Fig. 7. Guinea-pig's uterus, non-pregnant, left in Ringer-Locke solution, containing 1 : 5000 pituitrin, for 1 hour at room temperature; augmentor response to adrenaline reversed after contraction with pituitrin.

9 60 B. A. McSWINEY AND G. L. BROWN. Reversal of the effects of adrenaline was obtained in these experiments where a preliminary response had been recorded with adrenaline. The addition of 0.1 c.c. of pituitrin caused a marked shortening of the muscle fibres with some augmentation of rhythmic movement. The addition of adrenaline now elicited, in the great majority of experiments, a relaxation of the muscle fibres. The action of histamine in doses of 1 mg. closely resembled that of pituitrin. The effect of adrenaline on the muscle was reversed, a wellmarked inhibitor response being recorded. 0 Rabbit's uterus. Preparations were made of the cornu of the uterus of the rabbit, circular and longitudinal strips being used. Addition of adrenaline caused the usual augmentor effect, the tracing showing a marked increase of tone and augmentation of movement. Pituitrin in doses of 0.1 c.c. produced a marked rise of the lever, the tissue remaining in a state of sustained contraction for some 4-5 minutes before rhythmic movements returned with gradually increasing amplitude. A reversed response to adrenaline was never obtained, further addition of the drug causing either a continuation of the tonic contraction or a further Fig Rabbit's uterus, non-pregnant; reversal of response to adrenaline after contraction with histamine.

10 ADRENALIN REVERSAL. increase in tone. Histamine added in doses of 1 mg. had a similar action to pituitrin, but the reaction to adrenaline was reversed, the drug causing inhibition of rhythmic contractions and in most experiments a marked fall in tone. Ergotamine. The addition of ergotamine in doses of 5 mg. caused no appreciable change in tone or rate of rhythmic contractions in either the rabbit or guinea-pig's uterus; with the subsequent addition of adrenaline, a relaxation of the muscle fibres and/or an inhibition of rhythmic movements. The type of tracing depended on the existing condition of tonus. Oxygen supply. We have performed experiments to demonstrate the action of adrenaline on the various preparations after the oxygen supply to the bath had been cut off. The fundus of the rabbit's stomach relaxed on cessation of bubbling and addition of adrenaline invariably caused an increase of tone. Strips of the cornu of the rabbit's uterus relaxed if tone was high and rhythmic movements were usually inhibited if tone was low; rhythmic movements only were affected. The action of adrenaline under these conditions was always augmentor. With preparations of the guinea-pig's uterus, cessation of bubbling caused a rise in tone and the muscle fibres became tonically contracted. Adrenaline now caused an inhibitor effect, a reversal of the action of the drug; as in preliminary treatment of the strip, an augmentor effect was obtained. 61 Fig. 9. Guinea-pig's uterus, pregnant; augmentor action of adrenaline; reversal of adrenaline action after muscle contracted on cessation of oxygen supply.

11 62 B. A. McSWINEY AND G. L. BROWN. From these experiments, lack of oxygen in itself would appear to have no connection with the response obtained with adrenaline. DISCUSSION. A relationship clearly exists between the condition of the tissue and the nature of the response elicited by adrenaline, an inhibitor effect being obtained with the contracted or tonic muscle, and an augmentor response from the long or atonic strip. As these effects are obtained after the use of diverse drugs, some of which are regarded as acting on the parasympathetic system, others directly on the muscle, it would appear probable that the reversal effects recorded are due to changes accompanying variations in the condition of the muscle substance. By the use of ergotamine, the inhibitor response to adrenaline may be obtained in the preparations described, which previously gave an augmentor response. The presence of an inhibitor mechanism demonstrated by ergotamine does not necessarily imply that reversal of the action of adrenaline can be produced merely by shortening the muscle fibres by drugs. In certain preparations which normally give a motor response to adrenaline, namely, the retractor penis of the dog and the cardia of the cat, we have been unable to obtain results similar to those described for the fundus of the rabbit's stomach. Edmunds(11) has, however, obtained relaxation of the retractor penis after addition of ergotamine, and we have recorded similar results with preparations of the cat's cardia. It is possible that, under certain conditions, relaxation may be obtained with adrenaline after shortening of the muscle fibres by other means, just as in experiments on the rabbit's uterus, an inhibitor effect was only observed after the use of histamine. In the region of the lower body of the rabbit's stomach, an inhibitor effect can only be obtained after ergotamine. Accumulation of metabolites or any other results due to cessation of oxygen bubbling had no specific effect on the relction of the tissue to adrenaline. With tissues which relaxed on stopping the oxygen supply, augmentor response was recorded from the relaxed muscle, while on the other hand, with preparations that contracted on cessation of oxygen bubbling, an inhibitor response with adrenaline was obtained from the contracted strip. In view of the recent suggestion by Burn and Dale(l2) that the relaxation of the capillaries obtained with a second injection of adrenaline is due to the production of histamine or some such substance in the body, it is interesting to note that we have obtained relaxation of the

12 ADRENALIN REVERSAL. rabbit's fundus with a second dose of adrenaline when the muscle remained tonically contracted after the first dose. The reversal of the normal response of strips of the dog's stomach and the uterus of the guinea-pig on keeping the preparation in cold Ringer or Tyrode's solution is of interest, as Cow only obtained the augmentor response of the guinea-pig's uterus with adrenaline after "sensitising" the preparation with pituitrin. He argued that there is normally an interaction of the secretion of the pituitary and suprarenal glands in the body during pregnancy. Our experiments suggest that the mechanism controlling the augmentor and inhibitor response to adrenaline may be present in all preparations of smooth muscle capable of active contraction and relaxation. SUMMARY. 1. In certain regions of the rabbit's stomach, adrenaline elicits an augmentor response. 2. Strips of the fundus of the dog's stomach and the guinea-pig's uterus are contracted by adrenaline if the preparations are left in cold Ringer-Locke solution for 2-3 hours. 3. If the muscle fibres of the rabbit's fundus are shortened by pilocarpine, choline, histamine, pituitrin or barium chloride, the rabbit's uterus by histamine, the guinea-pig's uterus by pituitrin or histamine, an inhibitor response is obtained with adrenaline. 4. An inhibitor response may be changed into an augmentor response by certain agencies causing a lengthening of the muscle fibres. 5. Ergotamine, which in the doses used does not cause any change in the length of the muscle fibres, brings about a reversal of the action of adrenaline. 6. The type of response produced by adrenaline is related to the condition of tonus of the gastric musculature of the rabbit, an inhibitor response being obtained in the tonic or contracted muscle, an augmentor response in the atonic or relaxed muscle. The expenses of this investigation were defrayed by a grant from the Government Grants Committee of the Royal Society, to whom our best thanks are due. 63

13 64 B. A. McSWINEY AND G. L. BROWN. REFERENCES. 1. Brown and McSwiney. This Journ. 61. p Cannon and Lyman. Amer. Journ. Physiol. 31. p Dale and Richards. This Journ. 52. p Kolm and Pick. Arch. f. d. ges. Physiol p Cow. This Journ. 52. p Brown and McSwiney. Quart. Journ. Exp. Physiol. 16. p Langley. Autonomic Nervous System. Heffer, Cambridge, Dale. This Journ. 46. p Smith. Amer. Journ. Physiol. 46. p Kuroda. Zeit. f. d. ges. exp. Med. 39. p Edmunds. Journ. Pharm. and Exp. Ther. 15. p Burn and Dale. This Journ. 61. p