neuromuscular block in different species

Acta anaesth. scand. 1972, 16, 132-139

Neuromuscular Block in Different Species

ANIS BARAKA

Department of Anesthesiology, American University of Beirut, Beirut, Lebanon

The effects of tubocurarine and suxamethonium on the muscular twitch response were investigated on the phrenic nerve-diaphragm preparation of various species (rat, cat, dog and guinea pig). Tubocurarine produced a typical competitive (antidepolarizing) neuromuscular block in the species investigated: no initial stimulation, direct muscular response not affected, and the neuromuscular block adequately reversed with neostigmine. Although the block was qualitatively the same in the four species, the blocking dose of tubocurarine varied: the rat was the most sensitive (0.5 pg/ml), and the cat was the most resistant (1 pg/ml). This might reflect different safety margins of neuromuscular transmission.

In contrast with tubocurarine which showed only a quantitative dif- ference, suxamethonium showed both quantitative and qualitative differences among the different species. In the cat diaphragm, suxamethonium ( 2 kg/ml) produced a typical depolarizing block characterized by initial potentiation of the twitch response to nerve stimulation followed by neuromuscular block associated with partial depression of the direct muscular response. In the rat, no initial stimulation was observed, and a large dose of suxamethonium ( 10 pg/ml) was required to block neuromuscular transmission without any depression of direct muscular contractility. In the dog and guinea pig, a diphasic response was observed. These qualitative and quantitative differences were attributed to different degrees of suxamethonium-induced depolarization in the different species. I t is postulated that suxamethonium might act by two opposing mechanisms on the endplate: it competes with acetylcholine for the cholinergic receptors, and at the same time it can directly depolarize the endplate. The resulting neuromuscular block would be determined by a balance between the competitive and the depolarizing effects. There must be a broad spectrum where different proportions of both mechanisms co-exist.

Received August 2, 1971

The neuromuscular blocking agents in clin- ical use are classified according to their effect on the endplate, as “anti-depolarizing,” e.g. tubocurarine, or “depolarizing,” e. g. suxamethonium and decamethonium.

Antidepolarizing agents produce neuromuscular block by combining reversibly with the acetylcholine receptors without activat- ing them significantly, and prevent acetylcholine from doing so. The antagonism of tubocurarine is competive. ( PATON 1956).

Depolarizing agents not only combine with

the acetylcholine receptors but also activate them. The mechanism of their block was attributed either to a decrease in the sensitivity of the endplate to the transmitter substance (THESLEFF 1955) or to an area of altered sensitivity on the adjacent muscle membrane (BURNS & PATON 1951). These divergent opinions might be attributed to the use of different species (ZAIMIS 1953, BARAKA 1962, 1965).

The main purpose of the investigation presented here was to elucidate the mode of

NEUROMUSCULAR BLOCK IN DIFFERENT SPECIES 133

Fig. 1.-Kymographic trac- ing of twitch response of the rat phrenic nerve-diaphragm preparation to both nerve stimulation ( N ) , and

Tubocurarine (0.5 pglml) TUBOCURARINE produced complete neuromuscular block. Direct muscle response was not depressed.

muscle stimulation ( M ) . 4

action of suxamethonium in various mammalian species, and to compare its effect with that of tubocurarine.

MATERIALS AND METHODS The rat phrenic nerve-diaphragm preparation (Bur.- DRINO 1946) was selected for this investigation. This preparation was also selected by DEACOCK & DAVIES (1958) to provide a preparation of mammalian muscle which can be easily stimulated directly or indirectly via the phrenic nerve. The nerve is long and can easily be dissected free. Because of the large surface area and thin nature of the muscle, it is readily perfused and the effects of changes in the perfusion fluid are readily apparent.

Isolated phrenic nerve-diaphragm preparations from the kitten, the guinea pig, and the newly born dog were also used in order to study the effects of species variation. The animal was killed by a blow on the head and then bled to death. The hemidiaphragm with its costal origin and the phrenic nerve on the left side were then dissected and removed. The preparation was bathed in 50- 60 ml of Krebs' solution (KREBS & HENSELEIT 1932) at 37"C, through which was bubbled a mixture of 95 % oxygen and 5 % carbon dioxide. The p H was constant a t 7.4. The muscle was secured by its costal origin to an electrode. The insertion into the central tendon was fastened to a fine silver wire serving both as a second electrode and as attachment to a recording lever

Fig. 2.-Kymographic trac- ing of twitch response of the rat phrenic nerve-diaphragm preparation to both nerve stimulation ( N ) and muscle stimulation ( M ) . Tubocurarine (0.5 pg/ml) produced complete neuromuscular block, which could be completely reversed with neostigmine (0.2 pg/ml) .

writing on a smoked drum. The phrenic nerve rested across another pair of electrodes. Direct muscle stimulation through the first pair of electrodes was carried out by supramaximal square- wave pulses delivered by an electrode stimulator. This was found to give the most constant results and avoided any effects that might occur secondary to the mechanical effects of bubbling on the nerve. Each preparation was always left to rest for 30 min before any investigation was carried out. This allowed the preparation to recover from the trauma of dissection and to reach a steady state. Stimulation was then carried out every 5 s in order to avoid fatigue. When a steady twitch response occurred, the neuromuscular effects of suxamethonium in different species were investigated and compared to the effects of tubocurarine. Each ex- periment was repeated five times.

RESULTS Effect of tubocurarine Stimulating the nerve by a pulse wave of 0.1 ms duration and 40 V intensity produced maximal response. The addition of 25-50 pg tubocurarine to the bath perfusing the rat phrenic nerve-diaphragm gradually depressed the indirect response until complete neuromuscular block was reached in about 10 min. In no case was initial potentiation of the response observed. When complete neu-

134 ANIS BARAKA

10

CAT DOG RAT

Fig. 3.-A histogram showing the minimal dose of n " tubocurarine (pgiml) necessary to produce com- CAT RAT plete neuromuscular in the phrenic nerve- Fig. 4,-A histogram showing the marked resistance

was the resistant ( 2 pdm1)3 the as with the sensitivity of the cat dia- of the rat diaphragm to suxamethonium (10 pg/ml)

phragm ( 2 pg/ml).

diaphragm preparation of various species. The cat

rat was the most sensitive (0.5 pg/ml), the dog and guinea pig showed an intermediate response (1

romuscular block was achieved, the muscle was directly stimulated by a stimulus of 2 ms duration and 80 V intensity. The response obtained was equal to the original indirect response before the addition of tubocurarine (Fig. 1 ) .

The addition of neostigmine 10 pg to the perfusion bath (0.2 pg/ml) of a preparation just blocked with tubocurarine 25 pg could reverse neuromuscular block completely. Re- versal started within half a minute, and reached its maximum within 5 min (Fig. 2) . However, if an overdose of tubocurarine, e. g. 100 pg, was added to the perfusion bath, addition of neostigmine could not reverse the block.

The characteristics of tubocurarine block in the other three species (cat, dog and guinea pig) were qualitatively the same; yet the dose necessary to produce complete neuromuscular block varied. The rat diaphragm was the most sensitive (25-50 pg); the cat diaphragm was the most resistant (100-150 pg), while the dog and guinea pig diaphragms showed an intermediate response (50-75 pg) . The minimal blocking doses are shown in Fig. 3.

Effect of suxamethonium There was a marked species variation in the dose of suxamethonium necessary to produce complete block. The addition of 100 pg to the perfusion bath ( 2 pg/ml) of the kitten, guinea pig, or dog preparation, could produce a complete block. On the other hand, the diaphragm of the rat showed marked resistance to the effect of the drug, a dose of 500 pg suxamethonium (10 pg/ ml) being required (Fig. 4 ) .

In contrast with tubocurarine, the action of suxamethonium varied not only quantitatively but also qualitatively in different species:

Kitten. Administration of suxamethonium ( 2 pg/ml) produced initial potentiation of the response to be followed by subsequent depression. When maximum block was reached, direct maximal stimulation of the diaphragm produced a response which was slightly less than the original indirect response. The addition of neostigmine (0.2 pg/ml) to the perfusion bath did not reverse the block (Fig. 5).

NEUROMUSCULAR BLOCK I N DIFFEKENT SPECIES 135

Fig. 5.-Kymographic trac- ing of the twitch response of the cat phrenic nerve- diaphragm preparation to both nerve stimulation ( N ) and muscle stimulation ( M ) . Suxamethoniurn (2 pgiml) produced initial potentiation of the twitch response followed by complete neuromuscular block, which was not reserved with neostigmine (0.2 pgiml).

Rat. The rat diaphragm preparation was very resistant to suxamethonium; 10 pg/ml was needed to produce complete block. I n contrast with the effect of suxamethonium in the cat, there was no initial potentiation of the twitch. Also, when complete neuromuscular block was reached, direct muscle response was always equal to the original indirect response. The block was not antagonized when neostigmine (0.2 &ml) was added to the perfusion bath (Fig. 6) .

Dog. Addition of suxamethonium ( 2 p g f ml) to the perfusion bath produced a diphasic block :

1) Initial potentiation of the indirect response was observed before the onset of block. When complete neuromuscular block was reached, direct stimulation produced a smaller response than the original indirect one.

2 ) Within 10-20 min, the direct response was completely restored to normal, i. e. it

N M N Fig. 6.-Kymographic trac- ing of the twitch response of the rat phrenic nerve- diaphragm preparation to both nerve stimulation ( N ) and muscle stimulation ( M ) . Suxamethonium (10 pgiml) produced neuromuscular block without any initial potentiation of the twitch response. Direct muscle response was not depressed. Neostigmine (0.2 pg/ml) did not reverse the t block. Suxamet honium

became equal to the indirect response before the addition of suxamethonium. The response to indirect stimulation was partially restored. The partial recovery of indirect response could be potentiated by the addition of neostigmine (0.2 pg/ml) (Fig. 7 ) .

Guinea Pig. Similar to the dog, suxamethonium ( 2 pg/ml) produced a diphasic response in the guinea pig. Initially a contracture was observed to be followed by neuromuscular block. When complete block was reached, direct stimulation of the diaphragm produced a smaller response than the original one. Within 10-20 min the direct response increased, and the neuromuscular block could be completely reversed with neostigmine 0.2 pg/ml (Fig. 8).

DISCUSSION Neuromuscular blocking agents are usually classified according to their effect on the

M N M

Neostigmine

136 A N I S BARAKA

Fig. 7.-Kymographic trac- ing of the twitch response of the dog phrenic nerve- diaphragm preparation to both nerve stimulation ( N ) and muscle stimulation ( M ) . Suxamethonium ( 2 pg/ml) produced a diphasic response. The twich was initially potentiated, to be followed by complete neuro-

muscular block. Direct muscle response was partially depressed. After 10 min, the direct muscle response was conlpletely restored, and the indirect response to nerve stimulation showed partial recovery, which was enhanced with neostigmine (0.2 pgiml).

endplate as “competitive” (antidepolarizing) , e. g. tubocurarine, and “depolarizing,” e. g. suxamethonium and decamethonium (Fig. 9 ) .

The mechanism of tubocurarine neuromuscular block is qualitatively the same in the three species investigated. Antidepolariz- ing agents compete with acetylcholine for the cholinergic receptors at the endplate. The endplate potential will not reach the threshold level (- 45 mV) required to stim- ulate the adjacent muscle membrane and to initiate an action potential. The resting transmembrane potential at the endplate and the muscle fibre is normal (BURNS & PATON 1951). Response to direct muscular stimulation is therefore not affected. The reversal of tubocurarine neuromuscular block achieved by neostigmine depends on the degree of block at the time of reversal. Satisfactory

reversal is achieved when doses of tubocurarine do not exceed the blocking concentra- tion. Overdose of tubocurarine is probably the most important single factor contribut- ing to the so-called “neostigmine-resistant curarization” ( BARAKA 1967).

The dose of tubocurarine required to produce neuromuscular block varies in different species. This might be attributed to different safety margins of neuromuscular transmission. In the cat muscle the safety margin is great, and about 75-80 % of the endplate receptors must be occupied with tubocurarine before blocks appear ( PATON & WAUD 1967). In the rat diaphragm this safety margin is probably small, and much less tubocurarine is required to block neuromuscular transmission. The dog and guinea pig diaphragms show an intermediate response.

N M N M M N M M M M Fig. 8.-Kymographic trac- ing of the twitch response of the guinea pig phrenic nerve-diaphragm preparation to both nerve stimulation ( N ) and muscle stimulation ( M ) . Similar to the dog preparation, suxamethonium ( 2 pg/ml) produced a diphasic response. Initial contracture was observed, to be followed by

complete neuromuscular block. Direct muscle response was partially depressed. After 15 min, the direct muscle response showed recovery, and the neuromuscular block could be completely reversed with neostigmine (0.2 pgiml).

t Neostigrnine

t Suxamethoniurn

NEUROMUSCULAR BLOCK I N DIFFERENT SPECIES 137

Jy ;& action potential

A 45mV threshold pot. 90mV resting pot.

( K)o Normal neuromuscular transmission

B

C

L A n ti depolarizi ng block

. . . . * JL Depolariiing block

ENDPLATE POTENTIAL Fig. 9.-A diagram showing the endplate potential during normal neuromuscular transmission as compared with that during antidepolarizing and depolarizing neuromuscular block.

In contrast with tubocarine block, the mechanism of suxamethonium block is more complex, and varies both quantitatively and qualitatively according to the species investigated. In the isolated phrenic nerve-diaphragm preparation of the cat, suxamethonium produces a typical depolarizing block which is characterized by initial potentiation of the twitch response. This is followed by complete block of indirect response and partial block of direct response. These effects can be explained by the electromydgra- phic findings of BURNS & PATON (1951). In the cat’s gracilis muscle, depolarizing agents have induced marked endplate depolarization. This initially fires up repetitive action potentials. However, when endplate depolarization persists, it will spread within sec- onds to the adjacent muscle membrane di- minishing its electrical excitability; the endplate potential can no longer excite the muscle fibre. The electrical inexcitability at the endplate region and adjacent muscle mem-

brane has also prevented an action potential elicited by direct stimulation from crossing throughout the whole length of muscle fibre.

The effect of suxamethonium on the isolated phrenic nerve-diaphragm preparation of the rat differed from its effect in that of the cat. Such preparation showed marked resistance to suxamethonium. No initial potentiation was observed, and the resulting block was not associated with direct muscle depression.

THESLEFF ( 1955) has investigated the neuromuscular block produced in the isolated nerve-diaphragm preparation of the rat by decamethonium and suxamethonium; the drugs cause partial depolarization of the endplates from about -95 mV to only -65 mV; no significant repolarization occurs during the neuromuscular block. The block has been attributed to diminished response of the endplate to the chemical transmitter ACh and not due to muscular inexcitability which needs further depolarization.

The critical level of depolarization at which a muscle becomes electrically inexcitable is about - 52 mV (JENERICK & GERARD 1953). In contrast with a marked resistance to suxamethonium, the rat diaphragm show marked sensitivity to the antidepolarizing block of tubocurarine. Resistance to suxamethonium and sensitivity to tubocurarine have been also observed in myasthenic muscles and denote a low safety margin for neuromuscular transmission (BARAKA et al. 1971).

In the phrenic nerve-diaphragm preparations of the dog and guinea pig, suxamethonium produces a diphasic block. Such diphasic response has been described by JEN-

DEN, KAMIJO & TAYLOR (1954) in the rabbit lumbrical muscles, and by CREESE et al. (1957) in the isolated human intercostal muscles. I t has also been observed in the frog sartorius muscle by GISSEN & NASTUK ( 1966), who correlated the tension output curve with changes of the membrane potential. The early appearing neuromuscular block was associated with a reduction in the membrane potential below -55 mV, at which point the muscle fibres become electrically inexcitable. A depolarization-induced

138 A N I S BARAKA

depression of electrical excitability provides an adequate basis of Phase 1 block. During sustained drug application, the endplate is desensitized, and the membrane gradually repolarizes. When the membrane potential is reduced below -52 mV, the muscle becomes electrically excitable once again. How- ever, at this stage, Phase 2 block develops because nerve stimulation produces endplate potentials which gradually become smaller in amplitude, and depending on the depth of block, may disappear altogether.

Species variation is not the only factor determining desensitization. Both the con- centration and the structure of the quarter- nary ammonium depolarizer affect the rate of desensitization. In vitro experiments have shown that desensitization resulting from decamethonium appears earlier, lasts longer and is more resistant to corrective proce- dures than that produced by suxamethonium. High concentrations favor a rapid rate of desensitization (GISSEN & NASTUK 1970). CREESE et al. (1963) attributed the process of desensitization to the penetration of the depolarizing molecules intracellularly during the phase of depolarization. It has also been attributed to the electrochemical changes induced by prolonged depolarization ( WAUD 1968). Although this desensitiz- ing blcck might manifest curare-like characteristics, it is not always reversed with anticholinesterases ( VICKERS 1963).

I t is probable that suxamethonium might act by two opposing mechanisms on the endplate: it competes with acetylcholine for the cholinergic receptors, and at the same time it directly depolarizes the endplate. The resulting neuromuscular block would be determined by a balance between the two effects. In the cat diaphragm, the depolarizing effect predominated, and a small dose of suxamethonium produces a typical depolarizing block. On the other hand, in the rat diaphragm, the competitive effect predominates over the depolarizing effect. In between these two extremes, most species in- cluding man show a diphasic response: initially a depolarizing block predominates, but gradually repolarization occurs, and the com-

petitive nature of the drug shows up. There must be a broad spectrum of depolarization where different proportions of both mechanisms co-exist.

ZUSAMMENFASSUNG Am Phrenicus-Diaphragma-Praparat verschiedener Tierarten (Ratte, Katze, Hund und Meerschwein- chen) wurden die Wirkungen von Tubocucarin und Suxamethonium auf induzierte Muskelzuckungen untersucht. Tubocucarin erzeugte einen typischen kompetitiven (antidepolarisierenden) neuromuskularen Block bei den untersuchten Tierarten: keine initiale Stimulation, keine Beeinflussung der direkten Reizantwort des Muskels und angemessene Umkehr des neuromuskularen Blocks durch Neo- stigmin. Wenngleich der Block qualitativ bei den vier Tierarten gleich war, variierte jedoch die Do- sis, die zum Block fuhrte: Die Ratte war am ernp- findlichsten (0,5 pg/ml), die Katze am resistente- sten ( 1 pg/ml). Das konnte der Ausdruck unter- schiedlicher Sicherheitsgrenzen fur die neuromusku- Iare Ubertragung sein.

Im Gegensatz zu Tubocucarin, das nur quantitative Unterschiede zeigte, waren die Unterschiede der Suxamethoniumwirkung bei den verschiedenen Tierarten sowohl quantitativ als auch qualitativ. Am Katzenzwerchfell erzeugte Suxamethonium ( 2 pg/ml) einen typischen Depolarisationsblock, charakterisiert durch initiale Potenzierung der Mus- kelzuckung auf Nervreizung, gefolgt von einem neuromuskularen Block und gleichzeitiger Depres- sion des Effektes der direkten Muskelreizung. Bei der Ratte wurde keine initiale Stimulation beobachtet und eine groBe Dosis von Suxamethonium (10 pg/ml) war erforderlich, um die neuromuskulare Ubertragung zu blockieren, wobei keine Ver- minderung der direkten Muskelkontraktilitat ein- trat. Beim Hund und beim Meerschweinchen wurde eine zweiphasige Reaktion beobachtet. Diese quali- tativen und quantitativen Unterschiede werden durch verschiedene Grade der suxamethoniumbe- dingten Depolarisation bei den verschiedenen Tier- arten erklart. Es konnten zwei einander entgegen- gesetze Wirkungsmechanismen des Suxamethoniurns an der Endplatte angenommen werden: einerseits steht es im Wettstreit mit Azetylcholin urn die cholinergischen Rezeptoren, anderseits kann es gleichzeitig die Endplatte direkt depolarisieren. Der resultierende neuromuskulare Block wird durch das Gleichgewicht zwischen kompetitiven und de- polarisierenden Effekten bestimmt. Dabei mu13 es einen breiten Bereich geben, in dem verschiedene Anteile beider Mechanismen co-existieren.

REFERENCES BARAKA, A. (1962) Factors modifying the action of

tubocurarine on neuromuscular transmission. M. D. Thesis, Cairo University.

NEUROMUSCULAR BLOCK IN DIFFERENT SPECIES 139

BARAKA, A., EL-ZAYAT, F., ABDEL-RAHMAN, Y . & AWAD, E. (1965) Mode of action of tubocurarine and suxamethonium in different species, U . A . R. 3. physiol. Sci. 1, 107.

BARAKA, A. ( 1967 ) Irreversible tubocurarine neuromuscular block in the human. Brit. 3. Anaesth. 39, 89 1.

BARAKA, A,, AFIFI, A,, MUALLEM, M., KACHACHI, T. & FREIHA, F. (1971) Neuromuscular effects of halothane, suxamethonium and tubocurarine in a myasthenic undergoing thymectomy. Brit. 3. Anaesth. 43, 91.

BULBRING, E. ( 1946) Observations on the isolated phrenic nerve diaphragm preparation of the rat. Brit. 7. Pharmacol. 1, 38.

BURNS, B. D. & PATON, W. D. M. ( 1951 ) Depolari- zation of motor endplate by decamethonium and acetylcholine. 3. Physiol. 115, 41.

WALLA, D. J., SCHNEIDER, D. B., TAYLOR, D. B. & ZINN, D. E. (1957) The effect of neuromuscular blocking agents on isolated human intercostal muscles. 3. Pharmacol. exp. T h e r . 119, 485.

CREESE, R., TAYLOR, D. V. & TILSON, B. (1963) The influence of curare on the uptake and re- lease of a neuromuscular blocking agent labelled with radioactive iodine. 3. Pharmacol. exp. Therap. 139, 8.

DEACOCK, A. R. DE C. & DAVIES, T . D. W. ( 1958) The influence of certain ganglionic blocking agents on neuromuscular transmission. Brit. 3. Anaesth. 30, 217.

GISSEN, A. J. & NASTUK, W. L. (1966) The mechanisms underlying neuromuscular block following prolonged exposure to depolarizing agents. New York Acad. Sci. 135, 184.

GISSEN, A. J. & NASTUK, W. L. (1970) Succinyl- choline and decamethonium: Comparison of

CREESE, R., DILLON, J. B., MARSHALL, P. B., S A B A -

depolarization and desensitization. Anesthesio- logy 33, 611.

JENDIN, D. J., KAMIJO, K. & TAYLOR, D. B. (1954) The action of decamethonium on the isolated rabbit lumbrical muscle. 3. Pharmacol. exp. Therap . 111, 229.

JENERICK, H. P. & GERARD, R. W. (1953) Mem- brane potential and threshold of single muscle fibers. 3. cell. comp. Physiol. 42, 79.

KREBS, H. A. 6r HENSELEIT, K. (1932) Untersuch- ungen iiber die harnstoffbildung im tierkorper. Hoppe-Seyler’s Z . physiol. Chem. 210, 33.

PATON, W. D. M. (1956) Mode of action of neuromuscular blocking agents. Brit. 3. Anaesth. 28, 470.

PATON, W. D. M. & WAUD, D. R. (1967) The margin of safety of neuromuscular transmission. 3. Physiol. 191, 59.

THESLEFF, S. (1955) Effects of acetylcholine, decamethonium and succinylcholine on neuromuscular transmission in rat. Acta physiol. scand. 34, 386.

VICKERS, M. D. A. (1963) The mismanagement of suxamethonium apnea. Brit. 3. Anaesth. 35, 260.

WAUD, D. R. (1968) The nature of depolarization block. Anesthesiology 29, 1014.

ZAIMIS, E. (1953) Motor endplate differences as determining factors in mode of action of neuromuscular blocking substances. 3. Physiol. 122, 238.

Address: Anis Baraka, M . D . Associate Professor of Anesthesiology Department of Anesthesiology American University of Beirut Beirut Lebanon

neuromuscular block in different species

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