(Received June 7, 1934.)

Transcription

1 121 6I :6I2.OI8 THE CHEMICAL TRANSMISSION OF SECRETORY IMPULSES TO THE SWEAT GLANDS OF THE CAT. BY H. H. DALE AND W. FELDBERG. (From the National Institute for Medical Research, Hampstead, London, N.W. 3.) (Received June 7, 1934.) IT was recently suggested by one of us [Dale, 1933] that the anomalous response to drugs of the sweat glands of the cat probably indicates that their secretory nerve fibres, though originating in ganglia of the sympathetic chain, are cholinergic, and thus present an exception to the general rule that postganglionic sympathetic fibres are adrenergic. The experiments here described were undertaken to test this suggestion. A preliminary note on them has already been published [Dale and Feldberg, 1934]. METHODS. The object of the experiments was to collect the venous blood or perfusion fluid from the hairless pads of the cat's foot, and to discover whether stimulation of the sympathetic nerves, in such a way as to evoke a secretion of sweat, caused the appearance of acetylcholine in the venous effluent. A preliminary trial showed that with natural circulation it would be very difficult to obtain uncomplicated evidence. Stimulation of the abdominal sympathetic chain causes such an intense vasoconstriction in the foot that the venous outflow becomes extremely slow. A satisfactory comparison of the venous fluid, with and without stimulation, can only be made if the rate of flow is kept approximately constant. This could be achieved by using the adjustable Dale-Schuster pump, and so increasing or diminishing the stroke, as vaso-constriction appeared or passed away, as to keep the average rate of outflow within narrow limits. We have accordingly used artificial perfusion of the vessels of the foot with warm oxygenated Locke-Ringer solution, to which eserine sulphate

2 122 H. H. DALE AND W. FELDBERG. was added to the extent of 1 part in 660, ,000. The detailed procedure was as follows: The cat was anaesthetized with ether followed by chloralose. The abdomen was opened in the middle line and the abdominal portion of the alimentary canal completely removed, with ligature of the vessels. The abdominal sympathetic chain, on one or, in some experiments, on both sides, was then laid bare. The rami of the 4th, 5th, 6th and 7th lumbar ganglia were severed, the chain being tied and cut above the 4th lumbar ganglion and dissected clear, so that it could, when required, be raised out of the groove alongside the aorta and laid on platinum electrodes for faradic stimulation. The external iliac arteries were laid bare near their origins from the aorta, so that either of these, or the abdominal aorta itself, could be quickly clamped. It was found most convenient to dissect the artery and vein used for perfusion on the dorsum of the foot. The artery chosen was the anterior tibial, a small cannula being inserted into it just before the point at which it dips between the second and third metatarsal bones to become the plantaris profunda. Small branches leaving the artery before this point were tied. The area perfused from this artery includes, of course, much beside the toes and their hairless pads. It was found, however, that, long before the branches to the pads are reached, the artery has divided into branches so small that their use for perfusion would be impracticable. Similarly, the vein used for insertion of the outflow cannula was the saphena interna, just after its formation by the two veins running along the dorsum of the foot. Branches coming from tissues proximal to the toes were, so far as possible, tied. The external saphenous vein was also tied, so that the plantar venous arch drained into the dorsal veins uniting to form the saphena interna. Just before the cannule were inserted the blood of the animal was rendered incoagulable by intravenous injection of "chlorazine fast pink," 1 c.c. per kg. of an 8 p.c. solution of the dye being injected. This obviated the clotting in the arterial cannula of any blood which entered it. When the perfusion was started the fluid collected from the vein contained varying amounts of blood, entering the perfused system through anastomoses. The clamping of the external iliac artery, on the side of the foot perfused, usually eliminated this, and enabled a clean saline perfusion to be obtained. Occasionally, however, it was necessary to clamp the aorta, before this anastomotic contamination with blood could be completely eliminated. A very slight contamination could usually be neglected, since eserine was present in sufficient amount to protect any acetylcholine which might appear.

3 MECHANISM OF SWEAT SECRETION. 123 The perfusion was adjusted to give a moderate outflow of c.c. per min. before stimulation of the sympathetic chain. An assistant watched the steady rate of drops from the venous cannula, and, as soon as the retarding effect of stimulation was apparent, increased the stroke of the pump, endeavouring to restore the resting rate. Almost invariably he somewhat overshot the appropriate point, so that the short period of retardation before adjustment was compensated by a short period of acceleration, before the resting rate was regained. In no case was there such a difference between the average rates of flow during collection of the "resting " and " stimulation " samples as to have any significance for the result. Further, a retardation of the rate apart from nerve stimulation had no effect on the activity of the venous fluid. The venous effluent was tested for acetylcholine on strips of the anterior end of the back of the leech. The strip was suspended in a 2 c.c. bath containing diluted Locke solution to which eserine in a concentration of 1 in 200, ,000 was added. The Locke solution was diluted by adding four parts of distilled water to ten parts of Locke solution in order to render the solution isotonic to the cold-blooded muscle. The venous effluent was diluted in the same proportion before testing it on the leech. The minimal concentration of acetylcholine causing definite contraction of the leech muscle varied in the different experiments between 1: 5 x 108 to 1: 2 x 109, or from 05-2y per litre. The venous fluid could also be tested, after similar adjustment of its tonicity, on the isolated heart of a frog, attached to a Straub's cannula containing the unperfused eserinized Locke's solution similarly diluted. In one experiment the venous effluent collected during a long stimulation of the sympathetic chain was acidified to a ph of about 4 by adding a few drops of diluted HCl, and the fluid was then concentrated and freed from excess of salts in the way described in an earlier paper [D ale and Feldberg, 1934]. The concentrated fluid was tested on the arterial blood-pressure of a cat in chloralose anesthesia. RESULTS. Four experiments were made and their uniform results can be shortly described. When the perfusion had become well established, and anastomotic admixture so excluded that the venous effluent was practically clean Locke's solution, no activity corresponding to that of acetylcholine could be detected in it. When the sympathetic chain was stimulated, so that beads of sweat appeared on the hairless pads, the venous fluid

4 124 H. H. DALE AND W. FELDBERG. collected during the stimulation acquired a stimulant action on the leech muscle corresponding to a content of 2.5-lOy of acetylcholine per litre. In one experiment the control fluid (C) and that collected during venous stimulation (S) were tested also on the isolated frog's heart, with the result shown in Fig. 1. After the end of the stimulation period, the venous fluid gradually became free from acetylcholine, which reappeared when the stimulation was renewed. C9 Fig. 1. Fig. 2. Fig. 1. Isolated frog's heart. control venous fluid; S=venous fluid during sympathetic stimulation. Fig. 2. Eserinized leech preparation. For description see text. In the fourth experiment we performed a control, designed to verify the connection between the output of acetylcholine and the transmiission of impulses to the sweat glands. Both sympathetic chains were isolated and prepared for stimulation. The vessels of the right hind foot were then prepared for perfusion. Before this was undertaken, however, the hairless pads were transfixed with ligatures, which were tied tightly round the base of each pad, so as to stop its circulation completely. The cannulae were then inserted and the perfusion of the foot, with the exception of the pads, carried out in the ordinary way. Stimulation of the right sympathetic chain produced the usual intense vaso-constriction in the foot, compensated as usual by increasing the stroke of the pump. The

5 MECHANISM OF SWEAT SECRETION. 125 venous fluid, however, remained as free from acetylcholine as that collected before stimulation was applied (Fig. 2, CI and Si). The left foot, with the pads intact, was then similarly perfused, and gave, prior to stimulation, the usual inactive perfusate (Fig. 2, CII). Stimulation of the left sympathetic chain, causing vigorous secretion of sweat, was accompanied by the usual appearance of acetylcholine in the venous fluid (Fig. 2, SII). In this experiment a further and more prolonged stimulation was given, during which sufficient venous fluid was collected for concentration. The quantity so obtained was 12 c.c., and its activity on the leech preparation corresponded to that of about 7y of acetylcholine per litre. After evaporation and elimination of excess of salts it was made up with Ringer solution to a volume of 3 c.c. Tested on the blood-pressure of a cat under chloralose, its depressor action was approximately equal to that of a solution containing 20y of acetylcholine per litre. The activity, in terms of acetylcholine, was apparently about 30 p.c. less than that corresponding to the assay of the unconcentrated fluid on the leech preparation-a difference which, in relation to the errors of the measurements and the inevitable loss in concentration, is not really significant. This depressor activity disappeared from a sample of the solution made distinctly alkaline, and allowed to stand at room temperature for 30 min. It was also found that after an intravenous injection of 0-12 mg. eserine per kg. into the cat, the depressor effect of the active concentrate was much increased, and that the blood-pressure of the cat no longer showed any response to it after 01 mg. of atropine had further been injected. The substance appearing in the venous fluid, during stimulation of the sympathetic nerve supply to the sweat glands, therefore corresponds in all its reactions to acetylcholine, and there is no reason to doubt that it is that substance. DISCUSSION. This is the first case in which it has been possible to collect and to test separately the transmitter of the effects of postganglionic sympathetic fibres which, as an exception to the generally valid rule, appeared from previous evidence to be cholinergic; and it has thus been proved that the chemical transmitter of the action of such fibres is, indeed, acetylcholine. It should be noted that, although the concentrations of acetylcholine found in the venous fluid during activity of the glands were low in comparison with those obtained from some other active organs, the fluid from the glands was inevitably diluted by several times its volume of effluent from the rest of the foot, this extra-glandular circulation

6 126 H. H. DALE AND W. FELDBERG. remaining free from demonstrable acetylcholine. There can be no reason, therefore, for suspecting that the concentration in the fluid from the glands alone would not represent an output fully adequate to account for the transmission of the secretory stimulus. A further deduction is justified, namely, that the sweat glands of the human skin, similarly sensitive to pilocarpine and atropine and indifferent to adrenaline [Elliott, 1905], are supplied by cholinergic sympathetic fibres. In other animals the sympathetic fibres to the sweat glands appear to conform to the more normal type in chemical function, being adrenergic in the horse [Muto, 1916; Habersang, 1921; Langley and Bennett, 1923; Feldberg, 1925; Bacq, 1932], and in the sheep [Muto, 1916]. The observations of Young [1932] on the responses of the pupil in certain fishes to nerve stimulation and to neuromimetic substances suggest that localized mechanisms of chemical transmission, in these lower vertebrates, may even show a relation which is the reverse of that familiar in the mammal. In the eye of a teleostean, Uranoscopus, he found that the pupil was caused to constrict by stimulation of the sympathetic nerve supply or by application of acetylcholine, both these effects being abolished by atropine, whereas stimulation of the oculomotor nerve or application of adrenaline caused dilatation of the pupil in this species. The direct evidence here given concerning the sweat glands of the cat reinforces other less direct indications of the existence even in mammals, among the predominantly adrenergic fibres from sympathetic ganglia, of some which are cholinergic. The buccofacial dilatation produced by stimulating the cervical sympathetic nerve in the dog [D astre and Morat, 1880] is not reproduced by adrenaline [Elliott, 1905]. Rogowicz [1885] showed that it is associated with a contracture of motor-denervated facial muscles, resembling the contracture of the motor-denervated tongue in response to stimulation of the chordalingual nerve [Philippeau 'and Vulpian, 1865]; and Euler and Gaddum [1931], finding that the Rogowicz contracture is potentiated by eserine, conclude that the fibres responsible for it probably act by release of acetylcholine, though they showed them to arise from cells in the superior cervical ganglion. They were clearly dealing with another case of sympathetic fibres with a cholinergic mechanism. Recently Biilbring and Burn [1934] have given evidence that a particular vaso-dilator effect in the leg muscles, which they had observed on stimulating the abdominal sympathetic chain in the dog, was similarly due to a cholinergic component in the postganglionic sympathetic fibres.

7 MECHANISM OF SWEAT SECRETION. 127 The existence of a number of cases such as these makes it desirable to emphasize the fact that they are relatively rare exceptions to the generally adrenergic nature of peripheral sympathetic effects. Even the vaso-dilator effects of sympathetic nerves, as seen in the Carnivora after ergotoxine, are predominantly adrenergic, being produced by small injestions of adrenaline even more readily than by stimulation of the nerves, though these may in some cases contain cholinergic fibres, also of true sympathetic origin. A good example of the complicated physiological picture which may result, and of the readiness with which it can now be interpreted, is given by Langley's [1923] description of the vaso-motor changes in the pads of the cat's foot, in response to stimulation of the sympathetic nerve supply. The predominant feature of the response was vaso-constrictor pallor; but this was often followed, or might be preceded, interrupted, or even replaced, by flushing. Atropine removed the vasodilator component, leaving an uncomplicated vaso-constrictor effect. Langley attributed this atropine-sensitive vaso-dilatation to the escape of some metabolic product of the activity of the sweat glands. We can have little hesitation now in attributing it to leakage on to the blood vessels of acetylcholine, released to transmit the secretory sympathetic impulses. Langley confirmed the observation of Dale that after ergotoxine sympathetic stimulation caused uncomplicated flushing of the pads; and he found that under these conditions a vaso-dilator action persisted after further injection of atropine. Ergotoxine, accordingly, revealed an adrenergic vaso-dilator component in the sympathetic effect on the blood vessels, distinct from the cholinergic effect secondary to the transmission of stimuli to the gland cells. The general rule that postganglionic fibres in the parasympathetic parts of the system are cholinergic, and that those in the sympathetic part of the system are adrenergic, still holds; but there are exceptions to this rule, and the recognition of these will clear up a number of outstanding anomalies.

8 128 H. H. DALE AND W. FELDBERG. REFERENCES. Bacq, Z. M. (1932). C.R. Soc. Biol., Paris, 110, 568. Builbring, E. and Burn, J. H. (1934). J. Physiol. 81, 42P. Dale, H. H. (1933). Ibid. 80, lop. Dale, H. H. and Feldberg, W. (1934). Ibid. 81, 40P. Dastre, A. and Morat, J. P. (1880). C. R. Acad. Sci. Paris, 91, 393. Elliott, T. R. (1905). J. Phyeiol. 32, 401. Euler, U. S. v. and Gaddum, J. H. (1931). Ibid. 72, 74. Feldberg, W. (1925). Inaug. Diss., Berlin, quoted from Schilf, E., Das autonome Nervensystem, Verlag Thieme, Leipzig, 1926, p Habersang, 0. (1921). 8schr. prakt. Tierheilk. 32, 127. Langley, J. N. (1923). J. Physiol. 58, 70. Langley, J. N. and Bennett, S. (1923). Ibid. 57, 71P. Muto, K. (1916). Mitt. Med. Univ. Tokyo, 15, 365. Philippeau, J. M. and Vulpian, A. (1865). C.R. Acad. Sci. Paris, 56, Rogowicz, N. (1885). Pfluigers Arch. 36, 1. Young, J. Z. (1932). Proc. Roy. Soc. B, 112, 228.