THE NEUROMUSCULAR INTERACTION OF PROPANIDID WITH

Brit. J. Anaesth. (1968), 40, 818

THE NEUROMUSCULAR INTERACTION OF PROPANIDID WITHSUXAMETHONIUM AND TUBOCURARINE

BT

F. RICHARD ELLIS

SUMMARY

Twelve rat phrenic nerve-diaphragm preparations were used to investigate the neuro-muscular interactions of propanidid, suxamethonium, tubocurarine and mipafox (ananticholinesterase compound). The potentiation of suxamethonium by propanidid wasdemonstrated to be a neuromuscular effect, and was increased by chemical inactivationof cholinesterase. A complex interaction between tubocurarine and propanidid wasfound. A hypothesis for these actions of propanidid is propounded.

Suxamethonium produces a more prolongedperiod of apnoea after a propanidid induction ofanaesthesia than after the same dose of suxameth-onium following a barbiturate induction (Howellset al., 1964; Clarke, Dundee and Daw, 1964).

The site of this potentiation has not so far beenconclusively demonstrated and it is the purpose ofthis paper to present pharmacological evidenceof a probable site and mechanism of action.

Harnik (1964) reported the biphasic effect ofpropanidid on ventilation which is characterizedby a period of hyperventilation followed by avariable period of respiratory depression proceed-ing frequently to complete apnoea. This findingcould be explained by either a central or a peri-pheral site of action of propanidid.

An increase in spinal reflexes in cats to whompropanidid had been given (FBA Pharmaceuticals,1964, personal communication) similarly mayresult from a central or a peripheral site of actionof the drug.

Putter (1965) has shown that propanidid israpidly destroyed in vitro by non-specific ester-ases and so it would be reasonable to postulatesome anticholinesterase action in order to explainthe potentiation of suxamethonium by a reducedrate of breakdown of the suxamethonium whenin the presence of propanidid.

A direct action of the barbiturates on striatedmuscle has been demonstrated (Kraatz and Gluck-man, 1954) and an increased muscular responseto electrical stimulation shown. A similar effectwas reported with propanidid using a nervemuscle preparation in vitro (Ellis, 1967).

The present paper reports the interaction ofsuxamethonium and propanidid, and the effect ofan anticholinesterase compound on this inter-action, in order to determine anticholinesteraseaction of propanidid at the neuromuscular junc-tion.

Similar experiments using tubocurarine andpropanidid were also performed and the type anddegree of interaction between these drugs in theregion of the neuromuscular junction is reported.

METHODS

Twelve rat phrenic nerve-diaphragm preparationssimilar to those described by Bulbring (1946)were used.

Wistar rats weighing between 250 and 400 gwere killed by a blow on the head, and the dissec-tion and assembly of the left hemidiaphragm andphrenic nerve was completed in approximately 10minutes. The preparation was continually gassedwith 5 per cent carbon dioxide in oxygen andintermittently perfused with Kreb's solution of thefollowing composition (mM): NaCl 118.4, K Q4.7, Cad 2 2.6, KH.PO, 1.2, MgSO41.2, NaHCO,25.0, d-glucose 10.1. The Kreb's solution washeated to 37°C±0.5°C before entering the tissuebath (see figs. 1 and 2). The tissue bath had avolume which allowed 15 ml of solution to coverthe tissue completely.

The diaphragm was made to contract 12 timesa minute by applying rectangular electrical im-F. RICHARD ELLIS, M.B., CH.B., F.F.A.R.C.S., Departmentof Pharmacology and Anaesthesia, University of Man-chester, England.

Present address: Department of Anaesthesia, Uni-versity of Leeds, England.

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NEUROMUSCULAR INTERACTION OF PROPANIDK) 819

Brodi* L«v«r

Phrenic Narve

Suction

FIG. 1Sagittal section of organ bath containing rat dia-phragm-phrenic nerve preparation. Organ bath isattached to water bath maintained at 37 °C. Positionof indirect stimulating electrodes on phrenic nerve

shown.

pulses either through platinum electrodes to thephrenic nerve, or directly to the muscle throughthe two inverted platinum hooks used to securethe lower (costal) portion of the preparation in thetissue bath.

The resulting muscular twitch caused a down-ward displacement of a Brodie lever whichmarked the smoked drum of a kymograph.

The drug solutions used were all standardizedand had the following compositions:

(1) propanidid 100 mg in 100 ml of freshKreb's solution;

(2) suxamethonium 20 mg in 100 ml of dis-tilled water;

(3) tubocurarine, 10 mg pure compound in 100ml distilled water;

(4) mipafox 10 mg to 1 litre of Kreb's solutiongiving a final concentration of 10 yug/ml(mipafox is a powerful irreversible organo-phosphorus anticholinesterase compound).

FIG. 2Front view of organ bath (front plate detached to showassembly). Position of rat diaphragm preparationmarked by dotted line; position of direct stimulating

electrodes shown.

In most of the experiments a routine procedurewas adopted. A dose of relaxant, measured byvolume of the standard solution, was added atzero time to the tissue bath and a recording ofthe gradual fade in twitch height due to the devel-oping neuromuscular block was made. The per-centage reductions in twitch height at 2, 4, 6 and8 minutes were measured and calculated. Thiscontrol rate of fall was compared with that pro-duced by adding the same dose of relaxant withpropanidid. The doses of propanidid used were200 fig, 400 /tg and 800 pg. There were, therefore,four possible treatments and each treatment wasassessed four times in a Latin square format.

Between treatments the tissue was washed fourtimes with fresh Kreb's solution allowing 2minutes for each wash. After this the tissue wasleft for 12 minutes before the next treatment.

Throughout the whole cycle the electrical stimu-lation of the preparation was continued.

The statistical analysis was by means of a one-way analysis of variance (Moroney, 1963) and thesignificance of the F ratio was taken from tables(Fisher and Yates, 1953).


820 BRITISH JOURNAL OF ANAESTHESI

RESULTS

(1) A neuromuscular potentiation of suxametho-nium by propanidid was conclusively shown.Figure 3 was taken from a typical experimentaltrace; at each arrow suxamethonium 200 fig wasadded, together with doses of propanidid whichvaried from 0 to 800 jug. The control rate ofdevelopment of neuromuscular blockade due to200 fig alone is shown on the right. The degreeof potentiation can be appreciated in figure 4which summarizes the results. The percentageinhibition at 2, 4, 6 and 8 minutes after the addi-tion to the tissue bath of suxamethonium 200 figand varying doses of propanidid is shown and thecontrol curve produced by suxamethonium 200/<g alone is in the lowest position. The shift of thepercentage inhibition-time curve upwards and tothe left is an indication of potentiation of theneuromuscular blockade. The shift can be seen tobe related to the dose of propanidid (P<0.01).

(2) In the presence of mipafox the dose ofsuxamethonium was reduced to 100 jug to producea similar degree of blockade as before. Potentiationof suxamethonium by propanidid was found to besimilar to that which occurred in the first experi-ment and figure 5 shows a typical experimentaltrace. The mipafox was dissolved in the Kreb'sperfusate and the suxamethonium and propanididwere added to the tissue bath as in the first experi-ment as indicated. Increase in neuromuscularblockade is shown in figure 6 by a shift of thepercentage inhibition-time curve upwards and tothe left. The neuromuscular blockade increasesproportionately to the dose of propanidid

A comparison of figures 4 and 6 reveals a moremarked potentiation of suxamethonium by pro-panidid 200 fig when in the presence of mipafox,as in figure 6, the curve is moved further to theleft. There is no significant difference between the400 /tg and the 800 fig propanidid curves in figure6, whereas in the absence of mipafox (fig. 4) thereis a significant difference (P<0.01) between the400 fig and the 800 jug propanidid curves.

(3) Doses of propanidid (0-800 fig) whencombined with tubocurarine 20 fi% produced vary-ing degrees of neuromuscular blockade. Figure 7was taken from an experimental trace and thedoses of propanidid and tubocurarine and thetimes of administration are marked.

There is little difference between the first threerecords but propanidid 800 fig produced a signifi-cantly greater neuromuscular blockade.

Th: small differences between drug doses canbe better appreciated in figure 8 in which the per-centage inhibition is plotted against time as before.The control curve produced by tubocurarine 20jug is the lower continuous line and does not signi-ficantly differ from the propanidid 400 jug withtubocurarine 20 fig curve. Potentiation of tubo-curarine as shown by a shift of the curve upwardsand to the left is produced by propanidid 800 fig,the upper continuous line in figure 8, and alsobut to a lesser extent by propanidid 200 fig, butnot by the intermediate dose of 400 fig of pro-panidid.

A comparison of figures 8 and 4 will demon-strate a smaller potentiation of tubocurarine 20fig than suxamethonium 200 /tg by the same doseof 800 fig of propanidid.

• DISCUSSION

The concentrations of propanidid chosen for theseexperiments are broadly similar to those whichcould be expected in vivo shortly after inductionof anaesthesia, assuming that a normal clinicaldose of propanidid is distributed throughout theextracellular fluid.

Findings with suxamethonium confirm the clini-cal results but also show that suxamethonium ispotentiated peripherally by a mechanism actingat the neuromuscular junction.

The chosen dose of mipafox for these experi-ments completely inactivates cholinesterase at theneuromuscular junction as in this concentration(10 /tg/ml) neostigmine was found not to antag-onize the neuromuscular blocking properties oftubocurarine.

If propanidid had possessed anticholinesteraseactivity which could have accounted for its poten-tiation of suxamethonium, inactivation of theneuromuscular cholinesterase with mipafox wouldreduce or abolish the potentiation of suxameth-onium by propanidid. This was not found and sopropanidid has no demonstrable anticholinesteraseactivity.

The presence of mipafox was associated with anincreased potency of suxamethonium and alsoan increased potentiation of suxamethonium bypropanidid.


/NEUROMUSCULAR INTERACTION OF PROPANIDID 821

FIG. 3Potentiation of suxamethonium(sux) by propanidid (prop).Smoked drum recording of ratdiaphragm twitch followingstimulation of phrenic nerve.Increasing rate of twitch fadewith increasing dose ofpropanidid.

X

* . <

zoh-OXz

zUJ

U 10

SUX. 200pg.

PROP. 800(jg.

SUX. 200 pa

PROP.

SUX. 200pg.

PROP 200pa-

SUX. 200pg.

FIG. 4Potentiation of effect ofsuxamethonium by pro-panidid. Rat diaphragmpreparation. Graph showingincrease in percentageinhibition of twitch withtime for 200 jug suxameth-onium (sux) alone and incombination with varyingdoses of propanidid (prop).

TIME IN MINUTES


822 BRITISH JOURNAL OF ANAESTHESIA

FIG. 5Relationship betweenpropanidid, mipafoxand suxamethonium(sux) on the ratdiaphragm. Prepara-tion similar to fig. 3but treated withmipafox in additionto suxamethoniumand propanidid.

FIG. 6Potentiation of suxameth-onium by propanidid in thepresence of mipafox. Ratdiaphragm. Graph showingincrease in percentageinhibition of twitch withtime for 100 /ig suxameth-onium (S) alone and incombination with varyingdoses of propanidid (P);preparation treated withmipafox (M).

TIME MINUTES


NEUROMUSCULAR INTERACTION OF PROPANIDID m

FIG. 7Effect of propanidid on tubo-curarine block. Rat diaphragm.Preparation similar to fig. 3 buttreated with tubocurarine (dtc)and propanidid.

ft ••

I*t a

-(top) dtc. » « » PROP «00 KS

,t • PROP 200 pg

n + PROP. 400 pg

FIG. 8Effect of combining tubocurarine and propanidid onmuscle twitch height. Rat diaphragm preparation.Graph showing increase in percentage inhibition oftwitch with time for 200 /ig fubocurarine (dtc) aloneand in combination with varying doses of propanidid

(prop).

The increased activity of propanidid in thepresence of mipafox can be explained by a re-duced breakdown of propanidid following theinactivation of the cholinesterase.

From investigations of the site of action of pro-panidid (Ellis, 1967) it was suggested that thedrug acts on the general muscle cell membrane. Ithas been recognized for some time (Burns andPaton, 1951) that depolarizing relaxants, such asdecamethonium, unlike tubocurarine, do not re-main at the myoneural junction but depolarize themuscle cell membrane in the vicinity of theendplate. Thus propanidid could potentiate suxa-methonium on the muscle cell membrane.

The action of propanidid on the muscle cellmembrane mimics the effect produced by increas-ing extracellular ionic potassium (Ellis, 1968, inpreparation) which partially depolarizes the mem-brane. The most likely effect of propanidid is,therefore, to depolarize the muscle cell membranepartially, and this would potentiate the depolariza-tion produced by suxamethonium in the vicinityof the endplate.

In clinical practice Clarke, Dundee and Hamil-ton (1967) found that higher doses of tubocurarinewere required following propanidid than followingthiopentone. They suggested that this finding waidue to curare-like activity of thiopentone at theendplate (Gross and Cullen, 1943; Kraatz andGluckman, 1954).

The present results are only partially in agree-ment with clinical observations. Low concentra-tions of propanidid (fig. 8) potentiate tubocurarine(P<0.05) as do high concentrations of propanidid(P<0.01). The intermediate concentration used,however, did not potentiate tubocurarine and(after 2 minutes of interaction) even appeared to


824 BRITISH JOURNAL OF ANAESTHESIA

reverse the activity of the tubocurarine (P<0.05).This finding suggests that propanidid may have

two separate actions at the neuromuscular level,and a crucial experiment currently being per-formed is to determine the effects of propanididon cell membrane electrical potentials.

ACKNOWLEDGEMENTS

I would like to thank Professor H. Schnieden of theDepartment of Pharmacology, University of Man-chester, for helpful criticism of this work and forproviding the facilities, and Dr. A. R. Hunter forcontinued interest.

As this work was done as a senior registrar in anaes-thesia in Manchester, thanks are due to Dr. T. H.Chadwick and the Manchester Regional HospitalBoard for release from clinical duties.

REFERENCES

Bulbring, E. (1946). Observations on the isolatedphrenic nerve diaphragm preparation of the rat.Brit. J. Pharmacol., 1, 38.

Burns, B. D., and Paton, W. D. M. (1951). Depolarisa-tion of the motor-end-plate by decamethoniumand acetylcholine. J. Physiol. (Land.), 115, 41.

Clarke, R. S. J., Dundee, J. W., and Daw, R. H.(1964). Clinical studies of induction agents. XI:The influence of some intravenous anaesthetics onthe respiratory effects and sequelae of suxa-methonium. Brit. J. Anaesth., 36, 307.

Hamilton, R. C. (1967). Interaction between induction agents and muscle relaxants.Anaesthesia, 22, 235.

Ellis, F. R. (1967). The neuromuscular effects of pro-panidid. Brit. J. Anaesth., 39, 515.

Fisher, R. A., and Yates, F. (1953). Statistical Tablesfor Agricultural, Biological and Medical Research.Edinburgh: Oliver and Boyd.

Gross, E. G., and Cullen, S. C. (1943). The effects ofanesthetic agents on muscular contraction. J.Pharmacol, exp. Ther., 78, 358.

Harnik, E. (1964). A study of the biphasic ventilatoryeffects of propanidid. Brit. J. Anaesth., 36, 655.

Howells, T. H., Odell, J. R., Hawkins, T. J., andSteare, P. A. (1964). An introduction toFBA.1420: a new non-barbiturate intravenousanaesthetic. Brit. J. Anaesth., 36, 295.

Kraatz, C P., and Gluckman, M I. (1954). The actionof barbiturates on the contractions of voluntarymuscle. J. Pharmacol, exp. Ther., I l l , 120.

Moroney, M. J. (1963). Facts from Figures. Harmonds-worth, Middlesex: Penguin.

Putter, J. (1965). Uber den fermentativen Abban desPropanidid; in Horatz, K., Frey, R., and Zindler,M., Die intravenose Kurznarkose mit dam nevenPhenoxyessigsaurederivat Propanidol {EpontoT).Berlin: Springer.

L'INTERACTION NEUROMUSCULAIRE DUPROPANIDID AVEC SUXAMETHONIUM ET

TUBOCURARINE

SOMMAIRE

Douze preparations nerf phrinique-diaphragme dcrats ont eti utilised pour dtudier l'interaction netirc-musculaire de propanidid, suxamethonium, tubo-curarine et rnipafox (une substance anticholinesterase).La potentialisation de suxamethonium par le pro-panidid a tt& prouvee etre un effet neuromusculaire,intensify par l'inactivation chimique de la cholin-esterase. On a note" une interaction complexe entretubocurarine et propanidid. L'auteur avance unehypothese au sujet de ces effets de propanidid.

DIE NEUROMUSKULARE WECHSELWIRKUNGVON PROPANIDID MIT SUXAMETHONIUM

UND TUBOCURARIN

ZUSAMMENFASSUNG

Zur Untersuchung der neuromuskularen Wechsel-wirkungen von Propanidid, Suxamethonium, Tubo-curarin und Mipafox (Anticholinesterase-Verbindung)wurden zwolf Nervus phrenicus-Diaphragma-Praparatevon Ratten verwendet. Bei der Potenzierung von Suxa-methonium durch Propanidid handelte es sich, wicgezeigt wurde, um einen neuromuskularen EffekLDieser wurde gesteigert durch chemische Inaktivierungder Cholinesterase. Es wurde eine komplexe gegen-seitige Beeinflussung von Tubocurarin und Propanididgefunden. Zur Erklarung dieser Wirkungen vonPropanidid wird eine Hypothese vorgelegt.


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THE NEUROMUSCULAR INTERACTION OF PROPANIDID WITH