By Long Lasting Depolarization of the Motor End-Plates

238

J. Physiol. (I953) I22, 238-251

MOTOR END-PLATE DIFFERENCES AS A DETERMINING FACTOR IN THE MODE OF ACTION OF NEUROMUSCULAR BLOCKING SUBSTANCES By ELEANOR J. ZAIMIS From the Department of Pharmacology, School of Pharmacy, 17 Bloomsbury Square, W.C. 1 (Received 26 February 1953) Work on cat, human, avian and frog skeletal muscle (Paton & Zaimis, 1952) led to the conclusion that true neuro-muscular block is mainly produced by two different processes: (a) by competition with acetylcholine at the motor end-plates; and (b) by long lasting depolarization of the motor end-plates. Experiments carried out upon other mammalian species reveal the possibility of a third mode of neuromuscular block which combines the two processes mentioned above. These experiments show the characteristics of such a neuromuscular block and provide evidence that differences in the motor end-plates are a determining factor in the mode of action of a substance at the neuromuscular junction. A preliminary account of this work has already appeared (Zaimis, 1952). METHODS The mammalian species used have been the cat, monkey, dog, rabbit and hare. Chloralose alone was used as an anaesthetic for the cats, the dose given being 80 mg/kg. To the other species between 100 and 120 mg/kg of chloralose was given, but in a few experiments it proved necessary to supplement this anaesthetic with a small dose of pentobarbitone sodium. The injections of chloralose were made into the saphenous vein. Twitches and tetani of the tibialis, soleus and flexor digitorum sublimis muscles were excited by twice-maximal shocks of 02 msec duration, applied to the tied sciatic nerve in the thigh. In some experiments records of the mechanical response of two muscles in the same leg were recorded on the same drum. Injections were made intravenously or intra-arterially. For the intra-arterial injection the external iliac of the non-operated leg was cannulated towards the bifurcation of the aorta. The aorta was ligatured below the origin of the external iliac arteries. Under such conditions the dose injected was carried to the operated leg. Respiration was recorded by the apparatus described by Paton (1949). For experiments on chicks, birds a few days old were used and injections were made into the jugular vein. NEUROMUSCULAR BLOCK AND SPECIES DIFFERENCES 239

RESULTS Decamethonium Monkeys. The investigations with which this paper deals started with the surprising observation that while a first intravenous dose of 03 mg/kg of decamethonium into a monkey produced a transitory 95 % paralysis of the tibialis muscle, a subsequent dose injected after the complete recovery of the twitch was without effect (Fig. 1 a-c). This is in contrast with the effect of

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Fig. 1. a-c, monkey, 3-3 kg. Contractions of tibialis excited by supramaximal shocks to the sciatic nerve, every 10 sec. At (1) and 30 min later at (2), intravenous injection of 1 mg of decamethonium iodide; 10 min later at (3) and (4) injections of 1 mg of decamethonium iodide. At (5) tetanic stimulus to motor nerve, 50/sec. d, from another experiment. Monkey, 8 kg. Chloralose. Tibialis: nerve shock every 10 sec. At (1) intravenous injection of 10 mg of decamethonium iodide (following three smaller doses of decamethonium). At (2) 2 mg neostigmine methylsulphate I.v. At (3) tetanic stimulus to motor nerve, 50/sec. decamethonium on the tibialis muscle of the cat, where a further dose produces a greater paralysis. But this is not the end of the story, for in a monkey the block is rarely preceded by potentiation of the twitch and then only to a very slight degree; the tetanus is not well maintained and antagonizes the block which is also antagonized by neostigmine (Fig. 1 d). In short, the characteristics 240 ELEANOR J. ZAIMIS of this block differ from those of a block produced by pure depolarization, which is usually preceded by a brief period of fasciculation of the muscle and potentiation of the single twitches, and in which the tension of a tetanus is well maintained during the block while neostigmine and tetanus are not antagonists. If one records simultaneously the contractions of the tibialis and soleus muscles in a cat, results show that the tibialis is much more sensitive to decamethonium than the soleus. The same muscles differ in sensitivity to 1 2 3 4 56~~~~~~~~kg7 X. ]~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

Fig. 2. Dog, 10 kg. Simultaneous records of supramaximal motor nerve twitches of tibialis (upper record) and flexor digitorum sublimiis (lower record). At (1), (2), (4) and (5) intravenous injection of 1 mg of decamethonium. At (3) 1 mg neostigmine methylsuiphate I.V. At (6) and (7) tetanic stimulus to motor nerve, 50/sec.

D-tubocurarine but in the opposite way (Paton & Zaimis, 1951). If one now records the contractions of the tibialis and soleus muscles in a monkey the first dose of decamethonium causes a paralysis of tibialis, but has little effect on soleus. When the doses are progressively increased it is found that tibialis becomes less and less sensitive but soleus is increasingly affected. When the paralysis of the soleus starts spontaneous respiration stops. Briefly we start with the picture as in a cat given by decamethonium and finish with that given by D-tubocurarine. On the other hand, the block produced in the monkey by D-tubocurarine has all the well-known characteristics of that produced by competition with acetylcholine; it is not preceded by stimulation and is antagonized by a tetanus or neostigmine. During the block the muscles cannot maintain a tetanus and show an increasing sensitivity to subsequent doses of D-tubocurarine. Dogs, rabbits and hares. A number of tests were also made upon dogs and rabbits. The results show that the neuromuscular block produced by decamethonium in these species has the same characteristics as that in the monkey. This is clearly indicated in Figs. 2 and 3. The muscles show the same decreasing sensitivity and under the influence of decamethonium they cannot maintain NEUROMUSCULAR BLOCK AND SPECIES DIFFERENCES 241 a tetanus. Moreover, the block is antagonized by tetanus and by neostigmine, and potentiated by D-tubocurarine. The only difference between the dog and rabbit is that the block produced by the first dose of decamethonium is always preceded in the rabbit (Fig. 3, 1) by potentiation of the twitch.

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Fig. 3. Rabbit, 3 kg. Contractions of tibialis excited by supramaximal shocks to the sciatic nerve, every 10 sec. At (1), (3) and (4) intravenous injection of 0-6 mg of decamethonium iodide. At (2) and (7), tetanic stimulus to motor nerve, 50/sec. At (5) 2 mg atropine sulphate i.v. At (6) 1-8 mg decamethonium iodide i.v. At (8) and (11) intravenous injection of 05 mg neostigmine methylsulphate. At (9) 1 mg decamethonium iodide and at (10) 0 5 mg D-tubocurarine chloride i.v.

The following phenomenon deserves attention as it frequently occurs and is difficult to explain. It has been observed that whereas a first dose of neostigmine given during a neuromuscular block antagonizes the block effectively a second dose given during a subsequent dose is a far less effective antagonizer. This is clearly indicated in Fig. 3. The same phenomenon occurs when the block is produced by D-tubocurarine. Results of an experiment carried out on a single wild hare are illustrated in Fig. 4 and are sufficiently clear-cut to be worth recording. Here again the block produced by decamethonium has the same peculiar characteristics: decreasing sensitivity of the muscles to subsequent doses, the tetanus very poorly sustained, neostigmine effectively antagonizing the block and no sign of stimulation preceding it.

PH. CXXII. 16 242 ELEANOR J. ZAIMIIS

Other depolarizing substances An attempt was then made to study the actions of succinylcholine, a neuromuscular blocking substance believed to produce its actions in the cat and in avian muscle by pure depolarization. The results obtained with this substance in the monkey and the dog are shown in Figs. 5 and 6. It is clear that in these species succinylcholine produces a block similar to that produced by decame-

2 3 4

7 8 Fig. 4. Hare, 3 kg. Simultaneous records of supramaximal motor nerve twitches of tibialis (upper record) and soleus (lower record). At (1) intravenous injection of 0 5 mg, followed at (2) by 1 mg decamethonium iodide. At (3) and (4) tetanic stimulus to motor nerve, 50/sec. 30 min later at (5) decamethonium iodide, 1 mg i.v. At (6) 1 mg neostigmine methylsulphate. At (7) 0-2 mg D-tubocurarine chloride and at (8) 1 mg neostigmine methylsulphate. NEUROMUSCULAR BLOCK AND SPECIES DIFFERENCES 243 thonium, that is to say, a block differing in many ways from one due to long- lasting depolarization only.

1 2 3 4 5 Fig. 5. Monkey 8 kg. Simultaneous records of supramaximal nerve twitches of tibialis (upper record) and soleus (lower record). At (1) and (2), 10 mg. and, 10 min later, at (3) and (4) 30 mg succinylcholine dibromide. At (5) tetanic stimulus to motor nerve, 50/sec. kg I 1.0

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I _ Fig. 6. Dog, 10 kg. Simultaneous records of supramaximal nerve twitches of tibialis (upper record) and flexor digitorum sublimis (lower record). At (1) and 20 min later at (2), intravenous injection of 1 mg of succinylcholine dibromide.

Result of interposition of a dose of D-tubocurarine between doses of decamethonium The impression resulting from the consideration of these facts is that the picture obtained from the monkey, dog and hare is similar to that obtained in the cat when an injection of D-tubocurarine is interposed between the doses of decamethonium, as all substances raising the end-plate threshold to acetylcholine diminiish the activity of any substance producing a neuromuscular block by long-lasting depolarization. Such an experiment is illustrated in 16-2 244 ELEANOR J. ZAIMIS Fig. 7. A first dose of 0.1 mg of decamethonium producing a 50% block, is followed 15 min later by a similar dose. The effect now produced is much greater. When the twitch returns to its normal height a dose of 1 mg of D-tubocurarine is injected producing a 70% block. When the muscle contrac-

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Fig. 7. Cat, 3-5'kg. Tibialis: nerve shock every 10 sec. At (1) and 15 min later at arrow (2), 01 mg of decamethonium iodide i.v. At (3) 1 mg of D-tubocurarine chloride and 25 min later at (4) 0*1 mg of decamethonium. At (5) and (6) 0 3 mg of decamethoniuim. tion fully recovers a dose of decamethonium is again injected, but does not now produce any effect. In order to produce a similar block to that produced before the injection of D-tubocurarine the dose has to be increased sixfold. Tridecamethonium in the bird and cat It would thus appear possible that in these species there must be a new mechanism of neuromuscular block where a substance begins its action by depolarization but becomes a competitor with acetylcholine during the blocking process. This idea arose from a study of the effects produced by tridecamethonium, a higher member of the polymethylene bistrimethyl- NEUROMUSCULAR BLOCK AND SPECIES DIFFERENCES 245 ammonium series, on chicks. In birds, D-tubocurarine produces the usual paralysis while decamethonium and succinylcholine, like acetylcholine, produce a pure contracture characterized by extension of the limbs and retraction of the head (Buttle & Zaimis, 1949). If the dose of decamethonium is small, recovery is abrupt; if large, the animal dies in contracture and never exhibits paralysis. But when an injection of tridecamethonium is given,

Fig. 8. The effect produced by an intravenous injection of 0.1 mg/kg of tridecamethonium iodide on a 4-day-old chick (see text). a completely different picture is produced. First contracture appears but slowly, whilst the legs are still extended the head drops forward in paralysis and finally the paralysis extends to the leg muscles so that the whole animal becomes flaccid. From this result it is obvious that tridecamethonium acts to begin with as a depolarizing substance but changes during the blocking process to a competitive inhibitor. Fig. 8 illustrates this sequence of events. On observing these results in the chicks it was decided that it might be useful to test tridecamethonium in the cat. The effect produced by tridecamethonium on the tibialis muscle of the cat is similar to that obtained with decamethonium and succinylcholine on the tibialis muscle of the monkey, dog and hare. The muscle shows the same decreasing sensitivity (Fig. 9) in contrast with the increasing sensitivity displayed when decamethonium is injected instead. In the experiment illustrated by Fig. 10 both tibialis and soleus muscles were simultaneously recorded. A first I.v. injection of decamethonium was made, producing once more a block which had the characteristics of a depolarization block. A similar injection of decamethonium was made later, given this time after the interposition of a dose of tridecamethonium. This second dose of decamethonium was very definitely less effective, acting as if a dose of a competitive inhibitor substance had been interposed between the two injections 246 ELEANOR J. ZAIMIS of decamethonium. It thus appears that tridecamethonium exhibits in the bird and the cat the same mechanism of action as that produced by decamethonium and succinylcholine in the monkey, dog and hare.

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Fig. 9. Kitten, 0 6 kg. Tibialis: supramaximal motor nerve twitches. At arrows intravenous injection of 0.3 mg tridecamethonium iodide.

Variation ofpotency of decamethonium with species The observations just described seem to provide an explanation for the varying sensitivity of different species to decamethonium, in contrast to their relatively uniform reaction to D-tubocurarine (Fig. 11). Sensitivity is at its highest where depolarization is the only mode of action of decamethonium. But immediately the dual mode of action appears the muscles become more resistant to decamethonium, most probably because these two modes of action are antagonistic. DISCUSSION The evidence which results from the present experiments shows that, contrary to expectation, not all mammalian species react in the same way to neuromuscular blocking substances. In the human being and the cat, decamethonium is known as the best representative of drugs producing depolarization block. Another drug equally good is succinylcholine. These results led us to believe that deca- NEUROMUSCULAR BLOCK AND SPECIES DIFFERENCES 247 methonium and succinylcholine produce their neuromuscular block by pure depolarization in all mammals (Paton & Zaimis, 1952). When consideration is given to the results obtained in the monkey, dog, rabbit and hare with the same substances there is no doubt that this generaliza- tion is no longer valid. If one studies carefully the characteristics of the block

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Fig. 10. Cat, 3 kg. Simultaneous records of supramaximal nerve twitches of soleus (upper record) and tibialis (lower record). At (1) and (4), 0 09 mg of decamethonium iodide i.v. At (2) tetanic stimulus to motor nerve, 50/sec and at (3) intravenous injection of tridecamethonium iodide. produced by decamethonium and succinylcholine in the monkey, dog, rabbit and hare it becomes apparent that one is confronted with a new type of block which has some of the characteristics of a depolarization block, some of the characteristics of a competitive block and some characteristics entirely absent in either of these two mechanisms when they appear in their pure form. The existence of some stimulant action preceding the block and the greater sensitivity of tibialis muscle, in contrast to that of the soleus, to the first injected doses of decamethonium and succinylcholine are characteristics of a depolarization block. On the other hand, a tetanus poorly sustained during the block and the antagonism of the block by a tetanus and by neostigmine 248 ELEANOR J. ZAIMIS are characteristics of a block produced by competitive inhibition. But the striking decreasing sensitivity of the muscles to subsequent doses of decamethonium or succinylcholine is a totally new feature in contrast to the well- known increased sensitivity of muscles to subsequent doses of a depolarizing substance or of a substance blocking by competition with acetylcholine.

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Decamethonium 1 D-Tubocurarine 1-5

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0 Fig. 11. Relative potency of decamethonium and D-tubocurarine in different species. The dose indicated in mg/kg is that required to produce a 90-100% neuromuscular block in all species but mouse where it represents the ED50 using the righting reflex test. These findings clearly indicate that we are dealing with a mechanism ofneuromuscular block which is qualitatively different from those previously described. The impression obtained is that when a block with such characteristics is produced, the neuromuscular-blocking substance starts its action as a depolarizing agent but subsequently changes into a competitive inhibitor. The molecules when first injected apparently adhere in the specific way necessary to produce a depolarization at the motor end-plate. As a result they produce some stimulant actions and they affect tibialis more than soleus. However, their grip changes so that they now become molecules competing with acetylcholine and in doing so they raise the threshold to any depolarizing substance. NEUROMUSCULAR BLOCK AND SPECIES DIFFERENCES 249 A second dose of decamethonium or succinylcholine will consequently be antagonized, as these substances again begin their action by depolarization. This idea was forcefully suggested by the action of tridecamethonium in the chick where the two sharply differentiated-processes are clearly seen to follow each other. In the light of this interpretation one can easily explain all the characteristics of this neuromuscular block. Hall & Parkes (1953) report the same findings with decamethonium in the guinea-pig. Two examples can be quoted which support with direct electrical evidence the idea that depolarization block may lead on to competitive block by the same drug: (a) Paton & Perry (1953) have shown that a transition from the picture of depolarization block to that of competitive block can be seen with nicotine in the superior cervical ganglion. With nicotine the depolarization is always transient, ending much before the actual block. During the period of block, in the absence of depolarization, a further similar dose of nicotine produces only a small fraction of the original depolarization. They therefore conclude that nicotine has a dual mode of action, being initially a depolarizing and later a competitive blocking drug. (b) On the muscle (cat's gracilis) a similar situation is obtained with the higher members of the alkyltrimethyl ammonium series studied for the first time by Philippot & Dallemagne (1951). Here an injection may first produce a typical end-plate depolarization, which then wanes, although block persists (Paton, personal communication). In reviewing past work with decamethonium one comes across accounts of experiments on many varieties ofspecies which cannot be explained in terms of a single mode of action. Some of the authors have given inadequate or un- acceptable explanations, but others have in fact suggested a dual mode of action. Bovet, Bovet-Nitti, Guarino, Longo & Fusco (1951) noted that in the rabbit the block produced by decamethonium can be antagonized by eserine; and they state that an absolute distinction between a depolarization block and a competitive block cannot be drawn. Jarcho, Berman, Eyzaguirre & Lilienthal (1951), experimenting in rats, noted that 'in normally innervated rat muscle, decamethonium appeared to act in some ways like acetylcholine, in others, like curare'. Working on the same species, but using the isolated diaphragm preparation, Brand (1952) deduced that the action of decamethonium thereon is curare-like. Philippot & Dallemagne (1952) reached the same conclusion for work done on dogs, finding that neostigmine and adrenaline antagonize a decamethonium block. At the same time they were unable to detect any reciprocal antagonism between decamethonium and D-tubocurarine. These observations, so far asthey go, are in agreement with the present findings in the dog. But the more extensive analyses here carried out led to the conclusion already discussed that 250 ELEANOR J. ZAIMIS in the dog we are not confronted with a pure curare-like action but with a dual mode of action of decamethonium. Jenden, Kamilo & Taylor (1951) using a new isolated mammalian nerve- muscle preparation, the medial lumbrical of the rabbit's hind foot, observed with decamethonium what they describe as a 'biphasic' action. An initial block of rapid onset and short duration, associated with depression of the muscle, was followed by recovery of neuromuscular transmission. The second phase followed in which block without muscular depression slowly increased over several hours to a steady state. In the first phase anticholinesterases, potassium and a fall of temperature had negligible antagonizing effects. In the second phase all three were powerful antagonists. The non-uniform responses of the cat, monkey, dog, rabbit and hare muscles to the same neuromuscular blocking substance suggests that there must be distinct physical differences between the muscle membranes of these mammalian species in spite of the apparent similarity of their reaction to acetylcholine, differences brought to light only by such pharmacological analyses as are here described. These normally occurring differences in certain species may appear as pathological changes in the muscles of any one species. Churchill-Davidson & Richardson (1952) have obtained convincing proof that the neuromuscular block produced by decamethonium in normal human beings is due to long- lasting depolarization of the motor end-plates but that the block produced by the same substance in myasthenics is the result of a dual mode of action. That means that in the myasthenic, as in the monkey, we are confronted with a transition from the picture of a depolarization block to that of a competitive block. Consequently, a change has occurred in the muscle membrane of the myasthenic patient. If this change can transform the Dure depolarization action of an injected substance into a dual mode of action, with a competitive blocking element in it, may it not also cause acetylcholine to exhibit a similar effect? This interpretation of the myasthenic syndrome may come closer to the truth than any other so far put forward.

SUMMARY 1. Decamethonium and succinylcholine do not produce their neuromuscular block in all mammals by pure depolarization. 2. On the tibialis and soleus muscles of the monkey, rabbit and hare and on the tibialis and flexor digitorum sublimis of the dog they produce a type of block which has some of the characteristics of a competitive block, some of the characteristics of a depolarization block and some characteristics absent from both these mechanisms. 3. It is suggested that in the above species decamethonium and succinylcholine exhibit a dual mode of action. Apparently their action begins by NEUROMUSCULAR BLOCK AND SPECIES DIFFERENCES 251 depolarization but changes, during the blocking process, to competition with acetylcholine. 4. Tridecamethonium, a higher member of the polymethylene bistrimethyl- ammonium series, produces on the tibialis of the cat an effect similar to that obtained with decamethonium and succinyl-choline on the tibialis of the monkey, dog, rabbit and hare. 5. The two sharply differentiated processes, depolarization and competition with acetylcholine, are clearly seen to follow each otherwhen tridecamethonium is injected into chicks. 6. The existence of a dual mode of action seems to provide an explanation for the varying sensitivity of different species to decamethonium.

I should like to thank the Medical Research Council for a grant towards the expenses of this work. My thanks are also due to Mr D. Lewin for skilled assistance.

REFERENCES BOVET, D., BOVET-NITTI, F., GuARrNo, S., LONGrO, V. G. & Fusco, R. (1951). Recherches sur les poisons curarisants de synthese. Arch. int. Pharmacodyn. 88, 1-50. BRAND, H. (1952). A propos dumode d'action du decamethonium et de l'amyl-trimethylammonium sur la pr6paration isolee nerf phr6nique-diaphragme du rat. Experientia, 8, 273. BUTrLE, G. A. H. & ZAmns, E. J. (1949). The action of decamethonium iodide in birds. J. Pharm., Lond., 1, 991-992. CHURCHTLL-DAVIDSON, H. C. & RIcHARDSON, A. T. (1952). Motor end-plate differences as a determining factor in the mode of action ofneuromuscular blocking substances. Nature, Lond., 170, 617-618. HALL, R. A. & PARKES, M. W. (1953). The effect of drugs upon neuromuscular transmission in the guinea-pig. J. Physiol. 122, 274-281. JARCHO, L. W., BERMAN, B., EYZAGUIRRE, C. & LILIENTHAL, J. L., JR. (1951). Curarization of denervated muscle. Ann. N. Y. Acad. Sci. 54, 337-346. JENDEN, D. J., KAmaLo, K. & TAYLOR, D. B. (1951). The action of decamethonium (C 10) on the isolated rabbit lumbrical muscle. J. Pharmacol. 103, 348. PATON, W. D. M. (1949). Respiration recorder. J. Physiol. 108, 57P. PATON, W. D. M. & PERRY, W. L. M. (1953). The relationship between depolarization and block in the cat's superior cervical ganglion. J. Physiol. 119, 43-57. PATON, W. D. M. & ZAms, E. J. (1951). The action of D-tubocurarine and of decamethonium on respiratory and other muscles in the cat. J. Physiol. 112, 311-331. PATON, W. D. M. & ZAms, E. J. (1952). The methonium compounds. Pharmacol. Rev. 4, 219-253. PHILPPOT, E. & DALLEMAGNE, M. J. (1951). Synergies et antagonismes C la junction neuro- musculaire. Action de sels d'alkyltrimethyl-ammonium. Arch. int. Physiol. 59, 357-373. PmLIPPOT, E. & DALLEMAGNE, M. J. (1952). Les inhibiteurs de la transmission neuro-musculaire 6tudi6s chez le chien. Experientia, 8, 273-274. ZAIMIS, E. J. (1952). Motor end-plate differences as a determining factor in the mode of action of neuromuscular blocking substances. Nature, Lond., 170, 617.