SCHMIEDEBERG and Koppe, working on the action of muscarine on

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1 THE ACTION OF ATROPINE, PILOCARPINE AND PHYSOSTIGMINE. BY ARTHUR R. CUSHNY. (From the Pharmacological Laboratory, University College, London.) SCHMIEDEBERG and Koppe, working on the action of muscarine on the heart, were struck by the absence of effect when atropine had been injected previously, and on the ground that atropine was known to paralyse the vagus they formulated the theory that muscarine stimulates this nerve. Subsequently pilocarpine proved to resemble muscarine so closely in its action that it has generally been descrited as of the same nature. The exact point of action of these alkaloids on the path of the nerve impulse has been located differently by different workers, but the more recent supporters of this view are unanimous in placing it in the "terminations" of the post-ganglionic fibres. The later investigations of the behaviour of the heart under these alkaloids have only confirmed more exactly the resemblance between it and that under stimulation of the inhibitory fibres'. The action on the iris and on the salivary and sweat glands also resembles that of stimulation of the motor and secretory fibres, and the antagonism of atropine is so complete that here also the action on the terminations of the post-ganglionic fibres has been generally accepted. It is true that Anderson has shown that pilocarpine continues to contract the pupil after degeneration of the post-ganglionic fibres of the motor oculi, but this merely pushes the seat of action further towards the periphery, indicating that it affects not the anatomical terminations of the nerve fibres but some receptive substance (Langley) interpolated between them and the final contractile substance. The receptive substances for pilocarpine, muscarine and atropine have thus been localised in the extremities of the path of nerve impulse on the same grounds as have applied in the cases of adrenaline, nicotine and curarine. 1 See M aclean, Biochem. Journ. iii. p ; and iv. p

2 234 A. R. CUSHNY. The active movements of the intestine and other abdominal organs under muscarine and pilocarpine and the antagonism of atropine suggested a similar mechanism here, and it was generally assumed that pilocarpine stimulated and atropine paralysed the terminations of the motor nerves of the intestine. Some discrepancies had been noted, but this view was first discredited by Bayliss and Starling', who showed that while atropine antagonises the effects of pilocarpine on the bowel as completely as on the heart and iris, it does not prevent the effect on the bowel of stimulation of the motor and inhibitory nerves. Some doubt has been expressed by Frohlich and Loewi2 as to the correctness of this interpretation of Starling and Bayliss' experiments, and I may therefore state that soon after the appearance of their paper, their results with atropine were confirmed in a series of experiments performed in my laboratory by Ide and Torrey. From these unpublished experiments I select a tracing in which the movement of the dog's small intestine was recorded by means of a balloon inserted in the lumen and connected with a piston recorder (Fig. 1). The reaction to stimulation of the left vagus is shown in A before, and in B after 2 mg. of atropine. The effect of stimulation is practically identical while pilocarpine injected afterwards had no result. The reflex reaction to mechanical stimulation of the bowel by pinching was unchanged by atropine. These results, in confirmation of Bayliss and Starling, indicate that the violent movement of the intestine induced by pilocarpine may be antagonised by atropine without interruption of the paths of nerve impulse to the bowel. A.1 Fig. 1. Similar observations have been made in regard to other organs. Thus De Zilwa3 found muscarine cause a marked increase in the tone of the retractor penis, which was abolished by atropine, yet the muscle continued to contract on nerve stimulation. The violent contractions 1 This Journal, xxn. p Arch. f. exp. Path. u. Pharm. LIX. p This Jowrnal, xxvii. p

3 A TROPINE, PILOCA RPINE, PH YSOSTIGMINE. 235 of the rabbit's uterus under pilocarpine are arrested by atropine though stimulation of the hypogastric nerves continues to be effectivel. Prof. Langley informs me that he has recently observed a similar effect in the bladder, atropine preventing the pilocarpine contraction but not preventing that due to stimulation of the pelvic nerves in the cat. A somewhat analogous observation has been made in the voluntary muscles, in which the twitching induced by physostigmine is arrested by atropine in quantities which are incapable of affecting the passage of impulses from the nerves to the contractile substances. In a considerable number of cases in which atropine fails to interrupt the nerve impulse path, therefore minute quantities are sufficient to antagonise the effects of pilocarpine, and it is probable that these instances will increase when the action of pilocarpine has been more fully investigated. At intervals in the last few years, I have recorded the effects of pilocarpine and atropine on the uterus as opportunity offered, the method employed being that already described8. The animals used were cats, in which the effects of stimulation of the hypogastric nerves have been shown to vary; as a general rule in the pregnant cat or in one recently pregnant hypogastric stimulation causes contraction of the uterus, while in the non-pregnant state it inhibits contraction and reduces the tone of the muscle. There are occasional exceptions to this rule however; thus in one or two cats in late pregnancy, hypogastric stimulation was followed by marked inhibition, and in others in which there were no signs of recent pregnancy and the uterus was of small size, contraction was induced. I am therefore inclined to regard the change in the reaction of the uterus in pregnancy not as anything specific to that condition, but rather as due to the organ being in a more irritable condition and therefore prepared to respond more readily to the motor impulses than normally. The inhibitory fibres are not in any way reduced in power, for if the augmentor fibres be put out of action by ergotoxine the uterus responds by inhibition on hypogastric stimulation (Dale). As long as both sets of fibres are in functional connection with the uterus however, it generally responds more readily to the augmentor in pregnancy, while in the non-pregnant cat the inhibitory fibres prevail, unless the organ is in an unusual state of irritability. In every case the injection of 05-1 mg. pilocarpine nitrate was 1 This Journal, xxxv. p Edmunds and Roth. Amer. Journ. Physiol. xxiii. p This Journal, xxxv. p

4 236 A. R. CUSHNY. followed by the same changes as arose from stimulation of the hypogastric. In some experiments in addition to electrical stimulation, nicotine or adrenaline was injected and the movements of the uterus were found to be the same from all three alkaloids. In Fig. 2 the r<~~~

5 ATROPINE, PILOCARPINE, PHYSOSTrGM[NE. 237 tracings from the uterus of a pregnant cat are recorded'. Electrical stimulation of the hypogastrics had previously caused contraction. Nicotine (N), 1 mg., caused powerful contraction. Pilocarpine (P) also induced powerful prolonged contraction, which was released by 1 mg. atropine (A). After this, nicotine (N) was again followed by contraction of shorter duration, but pilocarpine had no effect even in large doses. Electrical stimulation of the hypogastrics also induced contraction. In Fig. 3, the tracing was obtained from a non-pregnant cat. Hypogastric stimulation (E) induced relaxation, and the same result followed the injection of pilocarpine (P), the uterus beginning to contract feebly after a time, but only returning to its normal activity after atropine (A) had been injected. Hypogastric stimulation again induced relaxation and adrenaline (S) had the same effect, but pilocarpine was now inactive. In this experiment as in many others, pilocarpine induced inhibition of the uterus exactly resembling the effects of nerve stimulation, while in the pregnant cat from which Fig. 2 was derived its injection was followed by contraction, again resembling the effects of nerve stimulation. In two experiments in which hypogastric stimulation caused a short contraction followed by inhibition, pilocarpine had the same effect. The action of pilocarpine on the uterus therefore follows exactly that of nerve stimulation and might be ascribed to its affecting some point on the path of the nervous impulse in the same way as the action of nicotine or adrenaline. Against this however is the antagonism of atropine which prevents the action of pilocarpine while permitting that of nerve stimulation, nicotine, or adrenaline. And the difference between pilocarpine and these is not merely a quantitative one, for while 1 mg. of atropine is sufficient to neutralise the effect of 10 mg. pilocarpine, the quantity of adrenaline necessary to cause a uterine contraction is not appreciably altered by this amount of atropine, nor is the minimal strength of current required to stimulate the hypogastrics changed in any way. Adrenaline and pilocarpine thus act in the same way as hypogastric stimulation on the uterus, but pilocarpine differs from the others in its being completely antagonised by small quantities of atropine, which have no effect whatever on the reaction to the others. Dale has shown that ergotoxine antagonises the motor effect of adrenaline on the uterus, and it was therefore of interest to determine whether it could also antagonise that of pilocarpine. Dr Dale kindly sent me a preparation of ergotoxine phosphate for this purpose. 1 In all the tracings from the uterus the downward movement indicates contraction, the upward relaxation.

6 238 A. R. CUSHNY. Exp. 1. A cat in the last weeks of pregnancy, anwsthetised with paraldehyde; tracheotomy and carotid cannula for blood-pressure, levers attached to one horn of the uterus in the space between two fcetal sacs Adrenaline injected intravenously caused strong contraction followed by a return to rest after about two minutes Pilocarpine nitrate (0-5 mg.) was followed by a series of rather weak contractions lasting until 3.24 and then by one powerful contraction (spontaneous?) the organ coming to rest at Pilocarpine nitrate (0 5 mg.) caused a powerful contraction followed by a series of movements in which no complete relaxation was reached. These still continuing, A solution of ergotoxine phosphate dissolved by the aid of a trace of sodium hydrate was injected. A strong contraction set in with a series of secondary movements The uterus still in contraction: injection of adrenaline Relaxation of uterus; small weak contractions cause undulations in the tracing, but the stronger movements are inhibited until 3.54 when a slow, strong, fairly regular rhythm sets in Pilocarpine i mg. arrests this rhythm, the uterus remaining motionless in complete relaxation for 5i minutes. The movements then recommence Pilocarpine J mg. again arrests these movements. 4.15i. Atropine 1 mg. injected Movements recommence. Subsequently pilocarpine had no effect. A similar effect is seen in Fig. 5, a, b, c, obtained from a cat in advanced pregnancy. In Fig..5 (a) a short contraction is seen from a very small dose of adrenaline (A), and prolonged activity from the subsequent injection of 0-5 mg. pilocarpine nitrate (P). A large dose of ergotoxine phosphate (about 21 mgs.) was now injected. Later, in Fig. 5 (b), pilocarpine (P) 0-5 mg., induced inhibition, which was more marked after a second injection of the same amount. Atropine (R) 1 mg. now injected was followed by contractions after a short time and increased tone of the uterus. In Fig. 5 (c) the effect of a small dose of adrenaline injected subsequently is shown to be inhibition resembling that of pilocarpine. The change from rest to activity as the prevailing phase during the experiment may have been due to the ergotoxine, but occurs usually during a long experiment. This experiment was repeated on other animals and gave the same result. The contractor effect of pilocarpiue on the pregnant cat's uterus was found to disappear after ergotoxine and to be replaced by an inhibitor action. I was not able to arrest by ergotoxine the movements of the uterus induced by pilocarpine in such a striking way as by atropine, because ergotoxine itself induces contraction in the beginning of its action and often causes a persistent rhythm of the

7 AY'ROPINE, PILOCARPINE, PHYSOSTIGMINE. 239 organ afterwards. But the subsequent injection of pilocarpine, instead of causing contraction as before the ergotoxine, now induced marked inhibition. The effects of pilocarpine on the blood-pressure and pulse rate were not changed by the previous injection of ergotoxine. Exp. 2. Cat, late in pregnancy, anesthetised with paraldehyde, levers attached as in Exp. 1. Stimulation of the hypogastric caused powerful contraction of the uterus. The injection of 1*5 mg. ergotoxine phosphate was followed by strong contraction followed by rapid contractions and partial relaxations for six minutes. The blood-pressure fell rapidly and artificial respiration was necessary. Hypogastric stimulation caused a weak contraction followed by inhibition, then on the third stimulation relaxation alone. Subsequent stimulation gave no further contraction, btut pure inhibition. Pilocarpine (1 mg.) injected now inhibited the uterus which remained in the relaxed position for 14 minutes. Atropine (1 mg.) was now injected and 2i minutes later contractions returned, although they were very slow. Stimulation of the hypogastric nerves now caused no contraction, the uterus remaining in a fully relaxed condition. A large dose of adrenaline still caused some rise in blood-pressure, and a contraction of the uterus occurred immediately after the injection, but this may have been spontaneous. It was followed by full relaxation lasting for nine minutes, and a new injection of adrenaline caused no contraction but a similar prolonged period of inhibition. In this experiment hypogastric stimulation at first caused contraction of the uterus, but this effect was reversed by the injection of ergotoxine. The nerve stimulation now caused inhibition, and adrenaline and pilocarpine had the same effect. These experiments indicate that the motor effects of pilocarpine on the pregnant uterus are antagonised by ergotoxine injected previously, while the inhibitory action is unimpaired by ergotoxine. In this again pilocarpine resembles adrenaline exactly, and in fact differs from it only in the former being antagonised by atropine, which has no effect on the latter. And not only is the motor action of pilocarpine abolished by atropine, but its effects on the inhibitory functions also disappear under it. When pilocarpine causes contraction of the uterus, atropine at once releases it. When it induces inhibition either directly or after ergotoxine, atropine reinstates the contractions only after some time, sometimes as long as 2-3 minutes elapsing before any movement occurs. This slow recovery from inhibition is not peculiar to pilocarpine, however, for even short stimulation of the hypogastric nerves with a weak current often induces inhibition lastiug for many minutes afterwards. The fact that the action of the pilocarpine on the uterus varies exactly with that of the hypogastric nerves suiggests that the alkaloid affects some point on the path of the nervous impulse. And strong confirmation is afforded by the antagonism of large doses of ergotoxine

8 240 A. R. CUSHNY. to the motor effects of pilocarpine and of nerve stimulation. On the other hand atropine, which antagonises pilocarpine completely, does not influence the passage of the nerve impulse or the action of adrenaline in any way; on the prevailing view that atropine antagonises pilocarpine by paralysing the point at which the latter acts, the nerve impulse would also fail to pass through this functionally inactive part of its course. Straub' has recently advanced the conception that pilocarpine exercises its influence on the contractile substance only in the course of its permeation into the cell and that atropine antagonises pilocarpine by retarding its permeation and thus reducing the potential of the inward current to suibliminal dimensions. This would appear consistent with the fact that atropine antagonises pilocarpine in the uterus without interfering with the passage of the nerve impulse, but on the other hand fails to explain its simultaneous antagonism to pilocarpine and interference with the passage of the impulse in other organs such as the heart. And other physiochemical conceptions of the action of these alkaloids seem to promise no more satisfactory explanation of this curious complex at the present time. Returning to the view that pilocarpine affects some specific receptive substance and that atropine prevents its effects by acting on this substance either chemically or physically, the close analogy offered in my experiments between pilocarpine, adrenaline and nerve stimulation suggests that the receptive substances for pilocarpine are closely associated with the path of nerve impulse. And the antagonism of ergotoxine to all these agents as far as the contractor action is concerned indicates that these receptive substances must either be identical with those for ergotoxine or must discharge through the latter to affect the contractile substance proper. On the other hand the antagonism of atropine to pilocarpine alone seems to imply that the receptors for pilocarpine are not actually on the path of nerve impulse. The simplest mechanism which can satisfy the conditions observed appears to be a set of receptors for adrenaline and ergotoxine lying on the nerve impuilse path and a separate and independent receptor for atropine and pilocarpine associated with the impulse path but not on the direct course to the contractile substance. This receptor can act on the contractile substance only as long as the adrenaline-ergotoxine receptive substance is intact and may be said to discharge through it. The relationship may be visualised by the nerve impulse path being represented as being joined above the adrenaline-ergotoxine receptor by a side branch on which 1 Pfll2ger's Arch. cxix. p

9 ATROPINE, PILOCA RPINE, PHYSOSTIGMINE. 241 the pilocarpine-atropine receptor lies. On the other hand, the receptor for atropine and pilocarpine in many other organs such as the heart, iris and salivary glands appears to lie on the nerve impulse path for the antagonism of pilocarpine by atropine involves the interruption of this path. It may be added that pilocarpine in some instances appeared to act less strongly on the inhibitory function of the uterus than adrenaline; for example it was observed in two experiments that while adrenaline brought the uterus to complete standstill for several minutes, pilocarpine merely caused marked relaxation but did not arrest the movements altogether. The effect of pilocarpine in slowing the heart and reducing the blood-pressure was of course unaffected by the previous injection of ergotoxine in sufficient quantities to prevent any contraction of the pregnant uterus. In several experiments in which the uterine movements were being recorded, special attention was paid to the changes in the movements of the intestine under pilocarpine; these were not recorded graphically but merely watched in the bath. The marked peristalsis under pilocarpine is of course well known; after ergotoxine this pilocarpine peristalsis occurred in the same way as in the norvial animal. Thus in experiments in which pilocarpine caused contractions of the pregnant uterus, intestinal peristalsis and slowing of the heart, the first only was eliminated by the subsequent injection of ergotoxine while the intestinal and cardiac changes persisted. A fresh injection of pilocarpine caused further slowing of the heart and more active peristalsis of the bowel, but the uterus was now inhibited. Atropine then accelerated the heart, induced contraction of the uterus and arrested the peristalsis of the bowel. Nothing could illustrate better the difficulties standing in the way of referring the effects of atropine (and of pilocarpine) to action on the ends of a definite group of nerves, as Loewi and Frohlich have recently attempted. From a review of the effects of atropine, these observers limit the action of this alkaloid to paralysing the augmentor nerves of the cranial and sacral autonomic system, and in their final statement admit only one exception, in the sweat nerves. In the particular experiment to which I have last referred, no one of the three features of atronine action comes under this rubric. The action of atropine and pilocarpine does not seem to me to be susceptible to definition by any such general rule. It is true that many of the most striking effects of atropine are due to its depressing the " terminations " of some of the augmentor cranial and sacral PH, XLI. 16

10 242 A. R. CUSHNY. autonomic nerves; but its sphere of action is not defined by these for it affects inhibitory cranial nerves (cardiac vagus) while it fails to paralyse cranial augmentor nerves (intestinal vagus), and it affects motor and inhibitory sympathetic functions (uterus, sweat-glands). It is true that in the case of some of the last it does not actually interrupt the path of nerve impulses, but it is impossible to dissociate entirely the action of atropine on the uterine mechanism from that on the heart and other organs. The effect of atropine in preventing certain forms of motor excitation in the intestine, uterus and bladder, while not interfering with the normal movements and innervation of these organs, affords an explanation of its use in therapeutics to allay too powerful or too frequent contractions of these organs; the cause of these may perhaps be some abnormal product acting in the same way as pilocarpine and muscarine on the receptors which are affected by atropine. In a number of experiments physostigmine was injected'. Kurdinowski2 found that physostigmine caused powerful contractions of the uterus in the rabbit. In pregnant and non-pregnant cats I found active contraction induced by it. On the other hand I have not observed physostigmine cause any relaxation of the uterus either in pregnant or non-pregnant animals. And in some instances in which pilocarpine caused definite inhibition, physostigmine injection was followed by contractions. Thus in Fig. 4, Es indicates the injection of physostigmine salicylicate which is followed by powerful contraction of the uterus. Later pilocarpine (P) causes relaxation of the uterus, which is released by atropine (A) after which the organ assumed the same position as before the injection of physostigmine. The effects of physostigmine on the uterus are antagonised by atropine, and I have not succeeded in restoring the contraction by subsequent injections of physostigmine. This seems to indicate that it acts not on the ultimate contractile substance but on a receptor similar to that affected by pilocarpine and perhaps identical with it. In one experiment, physostigmine failed to cause any movement after a large dose of ergotoxine, while pilocarpine was followed by relaxation and inhibition. And in no instance could I determine that physostigmine is capable of stimutlating the inhibitory mechanism in the way described under pilocarpine. This indicates that while the receptors for pilocarpine are associated with both motor and inhibitory impulse paths to the uterus, 1 Eserine salicylate (Merck) was employed. 2 Archivf. Gynaekoloaie, LXXVIII. p. 575, 196.

11 ATROPINE, PILOCARPINE, PHYSOSTIGMINE. 243 those for physostigmine are associated with the motor path only or at any rate predominatingly. The peripheral effects of physostigmine in other organs, except the striated muscles, seem generally to resemble those of pilocarpine closely, the differences being rather in degree than in kind. In the heart however physostigmine rarely causes such inhibition as is commonly seen from pilocarpine, though it apparently augments the effects of electrical stimulation of the vagus1. Here again the inhibitory impulse path seems to be less susceptible to the action of physostigmine than a \~~~~ A I b Pp.?~~~~~~~~~ I c A I I. 11. Fig. 5. to that of pilocarpine. Loewi and Mansfield2 have recently suggested that the peripheral action of physostigmine consists rather in augmenting the irritability of the terminations of the cranial and sacral autonomic nerves for normally subliminal stimuli than in acting as a stimulus itself; the grounds on which this view is based are that the effects of 1 Winterberg. Zt8chr. f. exp. Path. u. Pharm. Iv. p Arch.f. exp,. Path. u. Pharm. LXI. p

12 244 A. R. CUSHNY. electrical nerve stimulation are augmented by the previous injection of physostigmine, while not infrequently no actual augmentor action is seen from physostigmine injection without suibsequent electrical stimulation. The numerous exceptions to this observation they explain by a peripheral tone existing in certain organs. But this qualification of the theory seems to nullify the whole. It is undoubtedly true that not infrequently the injection of physostigmine is not followed by marked inhibition of the heart or by augmented salivary secretion, although the vagus and chorda nerves are found to respond by increased excitability; but this does not seem sufficient basis for the view that physostigmine differs from pilocarpine and muscarine qualitatively; for the difference in action appears to be equally explicable on their acting in the same way but in different degrees. I would suggest therefore that the increased irritability of the vagus and chorda manifested after physostigmine is really the lower phase of the stimulant action of the drug, which culminates in actual stimulation in more susceptible organs or in more favourable conditions. The fact that the inhibition of the uterus and of the heart is less marked under physostigmine than under pilocarpine, while the movement of contraction in uterus and bowel seems equally marked under each alkaloid suggests that the receptors for physostigmine are less developed in connection with the inhibitory impulse path than with the motor. Dale has shown that ergotoxine and tyramine similarly act less on the inhibitory than on the motor side compared with adrenaline, so that this aspect of physostigmine action is not without analogy. SUMMARY. (1) Pilocarpine causes contraction or inhibition of the uterus in the cat according to the condition of the organ, in this resembling exactly the effects of adrenaline and hypogastric stimulation. Its contractor action is also antagonised by ergotoxine, as is that of adrenaline and electric stimulation. (2) Pilocarpine differs from adrenaline in being antagonised completely by atropine whether it contracts or inhibits the uterus, while the effects of adrenaline or of hypogastric stimulation are not changed in any way by atropine. (3) Physostigmine causes contraction of the uterus, and has no apparent effect in causing inhibition, It is antagonised by atropine completely.

13 ATROPINE, PILOCARPINE, PHYSOSTIGMINE. 245 (4) It is suggested that pilocarpine and atropine act on receptive substances which are associated with the nerve impulse path to the uterus, but do not actually lie on this path. Physostigmine may probably affect the receptors associated with the augmentor path only. (5) In some other abdominal and pelvic organs, e.g. intestine, the receptor substances for atropine and pilocarpine appear to bear the same relation to the augmentor impulse path as in the uterus, while in other organs they must be regarded as lying on the impulse path, since this is interrupted by atropine.