QUARTERLY JOURNAL OF EXPERIMENTAL PHYSIOLOGY

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1 0 1.5(-.I QUARTERLY JOURNAL OF EXPERIMENTAL PHYSIOLOGY THE ACTION OF THE SKIN SECRETION OF THE SOUTH AFRICAN CLAWED TOAD. By J. W. C. GUNN. From the Department of Pharmacology, University of Cape Town. (With five figures in the text.) (Received for publication 24th July 1929.) IT was known that the toad was poisonous long before VULPIAN in 1854 discovered that the skin secretion had an action similar to that of digitalis. Many observations have since been made on this action as well as on the congenital tolerance of toads to digitalis bodies. ABEL and MACHT (1) showed that the "parotid gland" secretion of Bufo agua contains adrenaline as well as the "digitalis" substance, bufagin. The South African clawed toad, Xenopus lcevis, is in many ways more like Rana than Bufo. In habitat it is aquatic. It is active in its movements and muscular responses. Its skin is smooth, moist, and not "warty." It shows no tolerance to the digitalis group (2). In physiological experiments in South Africa it is used in place of the frog. The average weight of the animals used in the laboratories is 40 to 50 grm., but specimens weighing 80 to 100 grm. are frequently seen. From the skin, when irritated by mechanical, electrical, or chemical stimuli, a white, viscid secretion exudes; mainly on the back and extensor aspects of the limbs. This is also seen after the inhalation of irritating gases, the injection of certain drugs, or on direct stimulation of the central nervous system. Sections of the skin show that the secreting glands are of a simple saccular type, lined with one layer of columnar epithelium. There is no evidence of the presence of any tissue similar to suprarenal gland, and no massing of glands into a "parotid gland" as is the case in Bufo agua and in several other species of toad. THE SKIN SECRETION. For experimental purposes the most convenient method of obtaining the secretion is to place the toad under a small bell-jar and produce VOL. XX., NO

2 2 Gunn slight ansesthesia with ether. The secretion is then scraped away and used either at once or after being dried in vacuo. The amount obtained from each animal is 0,1 c.c. to 0 3 c.c. It consists chiefly of water and albuminous material, but contains a small amount of substances which are pharmacologically active. When dried, it seems to retain its activity indefinitely. One specimen tested from time to time showed no deterioration at the end of six years. A weighed amount of the dried secretion was used in most of the experiments. When Ringer's solution is added the powder swells into small insoluble albuminous masses. An amount of the active principles can, however, be extracted even from small quantities of the dried secretion sufficient to produce striking effects. The secretion always contains at least one active principle. The action of this will be described first. Isolated Mammalian Heart.-The isolated mammalian heart (cat and rabbit), when perfused through the coronary vessels by the method of Langendorff, shows a marked acceleration of rate and an increase in amplitude of the beat. The coronary outflow is diminished. The effect is reversible, the normal beat being quickly restored when Locke's solution is again perfused. Mammalian Heart in situ.-the movements of the heart of the rabbit or cat were recorded by means of the Cushny myocardiograph. The rate and amplitude of the beat is increased and the blood-pressure rises rapidly on injection of the secretion. There may then be a brief lessening of the acceleration, but this is absent after atropine or section of the vagi. Fatal doses lead to fibriuation of the ventricle and acute dilatation of the heart. This is shown in fig. 1. Blood-vessels.-Records of the blood-pressure and intestinal volume show that the increased blood-pressure is associated with diminution of the intestinal volume. Blood-pressure.-The rise in blood-pressure ia due to the effect on the heart and to constriction of the vessels. The activity of different specimens of the dried secretion varies slightly, probably due to differences in the rapidity or completeness of desiccation, but a dose of 10 mg. per kilogram of body-weight produces a marked result in both rabbits and cats. Reversal of Blood-pressure Effect.-If sufficient doses of ergotoxine are previously given, injection of the secretion causes a fall in bloodpressure instead of a rise. Intestinal Movements.-The movements of the intestine, isolated and in situ, are inhibited. This is shown in fig. 2. Uterine Movements.-The effects on the movements of the uterus are similar to those caused by sympathetic stimulation. The uterus of the guinea-pig and non-pregnant cat is inhibited; that of the rabbit and pregnant cat stimulated. The effect on the uterine and intestinal movements of the pregnant cat are shown in fig. 2. The tracing

3 The Action of the Skin Secretion of the South African Clw(hel Toa(l Fu;. I..Slhow ill the eft&ft of 'I to\lc (dose of Xc ploi)s/ secretiml 11 the henrt iiid bloml -p)l1cs'1su. Fi( 2. 2-Effect (o11 hblo(1d)-ressllu. a111(1 11()\'toemetnts of the Ilteruts I(Id iflt&-stilln (of the pre-nant cat.

4 4 Gunn shows an immediate rise of blood-pressure of short duration, relaxation of the intestine and contraction of the uterus. As the blood-pressure falls the muscular tone and movements of the uterus and intestine gradually return to normal. Fig. 3 shows the effect on the uterus of the non-pregnant cat and a comparison with the effect of adrenaline. A tracing showing the action on the uterus of the rabbit has already been published (3). These effects are obtained both on the intact animal and on the excised uterus. Fic;. :3. Comparison of the action of skin secretioni and adrenialinie on- the bloodpressure and uterus of the non-pregnant, cat. The following method of recording the movements of the intestine and uterus in situ has been found satisfactory. After being anaesthetised, the animal is immersed in a saline bath at body temperature. The intestine or uterus is exposed, and two points about an inch apart are attached with threads to C and D (fig. 4). A thread from the mid-point between them is taken directly to a lever. In the device shown in fig. 4 the brass rod A and the limb C are screwed rigidly into the block B. The limb ID is bent at right angles and passes into a hole in B. The distance between C and D can be altered from 1 to 5 cm. When a suitable adjustmnent has been made D is fixed with the screw E, the side of D being flattened to give greater security. By means of the universal joint F the apparatus is attached firmly to G, which is clamped to a stand or to the side of the bath. As the points to which the organ is attached are rigid, respiratory movements do not interfere with the record of intestinal or uterine movements.

5 The Action of the Sklin Secretion of the South Africain Clawed Toad Effect on MIela nophore8 ojf Chvnmeleoni. Sul)cutaneosls injection inlto the chameleon produces contraction of the melanophores over an area which gradually exten(ds outwards from the site of injection. This is sinmilar to the effect of small doses of adrenaline. Nature of Actioen.-The effects above recorded are all the samle as those pro- (Illce( by stimlulation of the sympathetic system or bv adrenialine. It is therefore concluded that the active principle present in the secretion is a symj)athomimetic suibstance. The secretion does not give the colour reaction.s of adrenaline, and therefore the substance is probablv niot adrenaline itself but closely related to it. A OTIIER S9UBSTANCES PRESEN-T. In soime cases there is aw fall of 1lo)0(1- c pressure anid slowing of the lecart before D the effects of sympathetic stimulaltion becomie noticeable. In one case, for ex- Fi Device uised in aimiple. the blood-pressure fell from 120 Utile 111(1 intestil re(a(rdiil- Ilmm. to 90 ml-m. anid subsequently rose to 3 3() mm. rlie general effect w-as very similar to that of nicotine tbis 5 l -).-. A recordl of the utleti(ll11 (A) 1iidl ventriculllar (x-) heats of the isolalted hi'alrtsho-xw-itig iii this e.ase, at inkarked preliminarx inhibit ion. suggested that the site of actioin might be on the ga iglia. The slowincr

6 6 The Action of the Skin Secretion of the South African Clawed Toad of the heart, however, stihl occurs after the ganglia have been completely paralysed with nicotine, but is entirely prevented by a sufficient dose of atropine. The cardiac slowing is, therefore, almost certainly due to stimulation of the vagal terminations. The fact that this parasympathetic effect varies considerably in degree and may be absent suggests that it may be due to a decomposition product similar to choline. Fig. 5 shows a similar action on the isolated mammalian heart, namely, a short period of inhibitory slowing, followed by a typical adrenaline-like effect. No convincing evidence was obtained of any digitalis-like action. When this investigation was begun it was expected that the active substance would prove to be like digitalis, since the presence of such a substance in a susceptible animal would have an important bearing on the usually accepted explanation of the congenital tolerance of the toad. ABEL and MACHT (1) found that Bufo agua reacted normally to adrenaline, but the circulatory system of Xenopus shows little response to the skin secretion or to adrenaline. This is being investigated further (e.g. the action on blood-sugar, etc.) to determine how far there is a natural tolerance to adrenaline. It is noteworthy that in Xenopus there are no suprarenal glands. The secretion is bitter to taste, and thus probably has a protective action. It has been noticed, for instance, that if Xenopus levis is picked up by a dog it promptly secretes and is almost immediately dropped. An adrenaline-like substance alone would seem to have little value as a protective. An extract of the entire skin has the same action as the secretion. SUMMARY. The main active principle in the skin secretion of the South African clawed toad is a sympatho-mimetic substance similar to but not identical with adrenaline. In addition there is a varying proportion of a principle which stimulates the parasympathetic nerve-endings. The expenses of this research have been defrayed by the Council of the University of Cape Town. REFERENCES. (1) ABEL and MACHT, Journ. Pharm. Exper. Therap., 1912, iii (2) GuNN, J. W. C., Trans. Roy. Soc. S.A., 1921, x. 53. (3) GUNN and GOLDBERG, Journ. Pharm. Exper. Therap., 1922, xix. 201.