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TUBOCURARINE AND PANCURONIUM INHIBIT EVOKED RELEASE OF ACETYLCHOLINE FROM THE MOUSE HEMIDIAPHRAGM PREPARATION
Br. J. Anaesth. (1987), 59, 226-231
TUBOCURARINE AND PANCURONIUM INHIBIT EVOKED RELEASE OF ACETYLCHOLINE FROM THE MOUSE HEMIDIAPHRAGM PREPARATION E. S. VIZI, G. T. SOMOGYI, H. NAGASHIMA, D. DUNCALF, I. A. CHAUDHRY, O. KOBAYASHI, P. L. GOLDINER AND F. F. FOLDES
E. S. VIZI, M.D., PH.D.; D . DUKCALF, M.D.; I. A. CHAUDHRY, D.V.M.; O. KOBAYASHI, M.D.; F. F. FOLDES, M.D.; H. NAGA-
SHIMA, M.D.; Department of Anesthesiology, Montefiore Medical Center, Albert Einstein College of Medicine, 111 East 210thStreet,Bronx,NewYorkl0467,U.S.A.G. T. SOMOGYI,
M.D., Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest, Hungary. Accepted for Publication: December 9, 1985. Correspondence to E.S.V.
SUMMARY Using a sensitive radioactive method that measures selectively the evoked release of acetylcholine, it was demonstrated that, when stimulating at 50 Hz, tubocurarine or pancuronium 2x.10~* mo I litre~l or hexamethonium 10~3 mol litre~l significantly decreased the evoked release of acetylcholine in the mouse in vitro phrenic nerve-hemidiaphragm preparation.
Baker, 1984). Bowman and Webb (1976) and Bowman (1980) suggested that the increase in the potency of non-depolarizing neuromuscular blockers at high rates of stimulation is, in part, the result of the inhibition of a positive, nicotinic feedback mechanism of acetylcholine release. According to Bowman (1980), the acetylcholine released by the nerve impulse acts, not only on the nicotinic (cholinergic) receptors of the postjunctional membrane, but also on nicotinic receptors of the motor nerve terminal. The interaction of acetylcholine with the latter results in an increase in the evoked release of acetylcholine. Because of the great safety margin of neuromuscular transmission (Paton and Waud, 1967), inhibition of this positive feedback mechanism only becomes important at relatively high (> 2 Hz) rates of stimulation when the margin of safety is decreased to a level where any effect on the release of acetylcholine affects chemical neurotransmission (Foldes, Yun et al., 1981). Until now, attempts to demonstrate the inhibitory effect of non-depolarizing neuromuscular blocking drugs on the evoked release of acetylcholine by neurochemical methods (assay of the
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Liley and North (1953) demonstrated that, in the rat hemidiaphragm paralysed by tubocurarine, repetitive stimulation of the phrenic nerve caused a gradual decrease in the amplitude of the end-plate potential (EPP). They assumed that this was the result of a decreased release of transmitter, and that it was a physiological response to high frequency stimulation which was unmasked, but not produced, by tubocurarine (Otsuka, Endo and Nonomura, 1962). However, other workers (Hubbard, Llinas and Quastel, 1969; Blaber, 1970, 1973; Hubbard and Wilson, 1973; Bowman and Webb, 1976; Magleby, Pallotta and Terrar, 1981), using different methods, showed that the rate of attenuation of the EPP in high frequency trains was greater in the presence than in the absence of tubocurarine—a decrease attributed to the reduction of acetylcholine release (Hutter, 1952; Otsuka and Endo, 1960; Gibb and Marshall, 1984). In agreement with the above findings, it has been reported that the neuromuscular blocking effects of tubocurarine and other non-depolarizing neuromuscular blocking drugs are greater during high frequency than during low frequency electrical stimulation in vitro (Foldes, Chaudhry et al., 1981) and in vivo both in animals (Paton and Zaimis, 1952; Bowman and Webb, 1976; Foldes, Chaudhry et al., 1981) and in man (Stance and
INHIBITION OF RELEASE OF ACETYLCHOLINE
MATERIAL AND METHODS
Male Swiss—Webster mice of 25-30 g body weight were lightly anaesthetized with ether and decapitated. The middle portions of both hemidiaphragms (about 2-3 mm to the right and left of the insertions of the phrenic nerves), together with the attached rib segments, were suspended
in organ baths of 2 ml volume in Krebs' solution (NaCl 113, KC1 4.7, CaCl, 2.5, MgSO4 1.2, NaHCO3 25, KH2PO« 1.2 and glucose 11.5 mmol litre"1). The bath temperature was maintained at 37 °C. The pH of the solution aerated with a 95% oxygen-5% carbon dioxide gas mixture was 7.38-7.42. The rib end of the preparation was attached with a ligature to the bottom of the bath and its tendon to an FT03 transducer. Stimulation. After 2 g resting tension was applied, the preparations were stimulated with a Grass S-88 stimulator by 0.8-s, 0.1-s or 1-s trains of 50-Hz, supramaximal impulses of 0.2 ms duration, every 10 s or 20 s through platinum electrodes placed above the proximal and below the distal parts of the preparation (field stimulation). Peak tetanic tension. Tetanic fade. The isometric contractions were measured with force displacement transducers and continuously monitored on a polygraph recorder. The peak tetanic tension and tetanic fade were recorded as described by Bowman and Webb (1976). Incubation. After a 15-min equilibration period, the preparations were loaded with 3H-choline 5 uCi ml"1 for 60 min. The preparations were stimulated during this 75 min as described above. This was done to facilitate the incorporation of 3 H-choline into the acetylcholine pool of the motor nerve terminal. For the next 20 min, the preparations were allowed to rest. At the end of this period, in seven preparations the radioactivity of the muscles, consisting partly of 3 Hcholine and partly of 3H-acetylcholine, was 7.54 ± 1.0 x 10* Bq g"1. After being washed three times to remove excess 3H-choline, each preparation was transferred to another bath and superfused with Krebs' solution at the rate of 2 ml min"1 for 60 min. From then on, throughout the investigations, Krebs' solution containing hemicholinium-3 5 x 10~6 mol litre"1 was superfused at a rate of 0.5 ml min"1. Hemicholinium-3 was added to prevent reuptake of 3H-choline that diffused out as such from the muscle or was liberated by hydrolysis of 3H-acetylcholine released during the subsequent stimulation periods.
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released acetylcholine) have been unsuccessful. Various investigators reported that tubocurarine had little (Beani, Bianchi and Ledda, 1964) or no (Dale, Feldberg and Vogt, 1936; Emmelin and Mclntosh, 1956; Straughan, 1960; Kmjevic and Mitchell, 1961; Cheymol, Bourilet and Ogura, 1962; Chang, Cheng and Chen, 1967; Fletcher and Forrester, 1975) effect on the evoked release of acetylcholine. In all of these studies the total (quantal + non-quantal) acetylcholine was measured in the presence of an anticholinesterase. The failure to demonstrate any changes in the evoked quantally released acetylcholine by tubocurarine may have been because most of the acetylcholine released from mammalian muscle and measured by bioassay is released in a non-quantal form. At rest, about 98 %, and during electrical stimulation about 70%, of the released acetylcholine is non-quantal (Vizi and Vyskocil, 1979). To resolve the contradiction between the results of the pharmacological and neurophysiological studies on the one hand and those of the direct measurement of acetylcholine on the other, the effect of non-depolarizing neuromuscular blockade on the evoked release of acetylcholine was re-examined with a sensitive radioactive method (Foldes et al., 1984). Radioactive choline was used to label the acetylcholine stores of the mouse hemidiaphragm preparation. It was shown previously that the newly synthetized 3H-acetylcholine is preferentially released in response to electrical stimulation in brain (Szerb, 1975), myenteric plexus (Vizi et al., 1984), and also in the mouse hemidiaphragm preparation (E. S. Vizi, unpublished observations). Furthermore, using this method, measurement of acetylcholine release can be accomplished without cholinesterase inhibition (Szerb, 1975; Kilbinger and Wessler, 1980; Foldes et al., 1984; Vizi et al., 1984). This is advantageous because cholinesterase inhibitors are known to affect the release of acetylcholine (Fatt and Katz, 1952; Boyd and Martin, 1956; Foldes et al., 1984).
227
228
Calculation. In the mouse phrenic nerve— hemidiaphragm preparation, similar to the situation in the myenteric plexus-longitudinal muscle preparation of the guineapig ileum (Szerb and Somogyi, 1973; Kilbinger and Wessler, 1980; Vizi et al., 1984), electrical stimulation increases only the release of 3H-acetylcholine and the outflow of 3H-choline remains unchanged (E. S. Vizi, unpublished observations). Consequently, the evoked release of sH-acetylcholine can be calculated from the difference between the expected spontaneous and the actually measured outflow of radioactivity caused by 3 min of electrical stimulation. Spontaneous release was assumed to follow first-order kinetics. It was determined by the "least square" fitting to an exponential curve method of the Bq g"1 concentrations of two 3-ml effluents collected before, and that of two others collected after, every period of stimulation. The expected spontaneous outflow was obtained by interpolation between the fitted points. The radioactivity of the samples was measured in a Packard Scintillation Beta Spectrometer. There was considerable variation in the controls from one preparation to the other, in the amount of radioactivity released during the corresponding periods of stimulation. In contrast, the ratios of radioactivity measured during the first two consecutive stimulation periods, 8,/Sj, were constant. Results are presented as mean values ±SEM. Statistical analysis. The statistical significance of the results was determined by one-way analysis of variance of the logarithmically transformed data, followed by Dunn's test. P < 0.05 was considered significant.
TABLE I. Inhibition of the evoked release of radioactivity from mouse hemidiaphragm preparations, loaded with *H-choline, by tubocurarine, pancuronium or hexamethonium. The inhibition of the evoked release of radioactivity could be attributed to the inhibition of the release of 'H-acetylcholine (Jor explanation see text). * Ratio of the evoked release of radioactivity during the second and first stimulation periods (mean±SEM). Significant differences from control determined by one-taay analysis of variance followed by Dunn's test Compound
n
Control Tubocurarine 2 x 10~5 mol litre"1 Pancuronium 2 x 10-» litre"1 Hexamethonium 10-' mol litre"1
14 6
S./S, Ratio*
P
0.79 ±0.02** 0.42 ±0.08
<0.01
8
0.51 ±0.08
<0.01
6
0.51 ±0.07
<0.01
Materials. The materials used were 3 Hmethylcholine chloride (Amersham, 78 Ci mmol litre"1, tetrodotoxin (Sigma), eserine sulphate (Sigma), tubocurarine (BDH), pancuronium (Organon) and hexamethonium (BDH). RESULTS
Resting release
In six investigations, the release of radioactivity at rest remained relatively constant (1513 ± 154 Bq g"1 min"1) throughout. Tetrodotoxin 5 x 10"' mol litre"1, pancuronium 2 x 10"6 mol litre"1 and hexamethonium 3 x 10"* mol litre"1 had no effect on resting release. Evoked release
When trains of 40 stimuli (50 Hz) were delivered every 10 s for 3 min (720 stimuli were applied), the release of radioactivity was significantly enhanced above spontaneous release by 2685 ± 180 Bq g"1 (n = 8). The output of radioactivity was very constant; the ratio between the amounts of radioactivity released by consecutive stimulation periods (S,/Sj, S,/S,) did not differ significantly from each other: Sj/S! was 0.83 ±0.09 and S s /S, was 0.72 ±0.08 (n = 8 and 8, P > 0.05). Tetrodotoxin 5 x 10"7 mol litre"1, added to the bath 10 min before stimulation, completely inhibited the stimulation evoked release of radioactivity. Table I shows the effect of different antinicotinic agents (hexamethonium, tubocurarine and pancuronium) on the release of radioactivity in response to stimulation. All these agents signifi-
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Collection. During superfusion at the rate of 0.5 ml min"1, 3-min effluents were collected continuously with a fraction collector into scintillation vials throughout the rest of the experimental period. The preparations were stimulated, as described, three times for 3 min each starting at the 10th (S t ), 52nd (Sj) and 82nd (S3) min. In 14 control experiments, no drug was added to the perfusing solution. In six, eight and six other studies, respectively, tubocurarine 2 x 10~6 mol litre"1, pancuronium 2 x 10~5 mol litre"1 or hexamethonium 10~s mol litre"1 was added to the perfusing Krebs'-hemicholinium-3 solution at 17 min.
BRITISH JOURNAL OF ANAESTHESIA
INHIBITION OF RELEASE OF ACETYLCHOLINE
229
TABLE II. Effect of different anlmkotmic agents on neuromuscular transmission of and "C-acetylcholme releasefrommousehemidiaphragmpreparation. * Phrenic nerve was stimulated and trams of 50 Hzifioe shocks) were delivered in every 20 s. The contractions were recorded isometrically. t The release of radioactivity was measured(see Methods). Trains of 50 Hz (40 shocks) were applied in every 10 s. X The contractures produced by trains of 50 Hz (100 shocks) were recorded and the effect of drugs in different concentrations was studied on the maintenance. ICU is the concentration needed to produce 25% reduction of maintenance Inhibition of Postjunctional Peak tetanic tension* (IQ,) (junol litre ')
'H-ACh release (threshold concn) (jimol litre"')t
(jimol litre ')
0.86 ±0.01 0.42 ±0.02 2430 ±200
0.5 0.25 50
0.28 ±0.02 0.18±0.03 260±12
cantly reduced the stimulation-evoked release of radioactivity. The threshold concentrations were found to be 0.5 umol litre"1 for tubocurarine, 0.25 umol litre"1 for pancuronium and 50 umol litre"1 for hexamethonium, respectively (table II). They produced a significant reduction of 3H-acetylcholine release: the S J / S J ratios were 0.601 ±0.03, 0.621 ±0.03 and 0.641 ±0.02, respectively (n = 3 and 3, P < 0.01). These data indicate that the effects of the neuromuscular blockers are concentration-dependent. The IC50 values for the inhibition of the peak tetanic tension of the mouse phrenic nerve-hemidiaphragm preparation were 0.86 ±0.01 umol litre"1 for tubocurarine, 0.42 ±0.02 umol litre"1 for pancuronium and 2430 ± 200 umol litre"1 for hexamethonium (table II). DISCUSSION
The sensitive radioisotope method (Foldes et al., 1984) used in this study revealed the significant inhibitory effect of tubocurarine on the evoked release of acetylcholine that could not be demonstrated by other methods (Dale et al., 1936; Emmelin and Mclntosh, 1956; Krnjevic and Mitchel, 1961; Chang, Cheng and Chen, 1967; Fletcher and Forrester, 1975). The data presented are in agreement with the results of the electrophysiological (Hubbard, Llinas and Quastel, 1969; Blaber, 1970, 1973; Bowman and Webb, 1972; Hubbard and Wilson, 1973) and pharmacological (Paton and Zaimis, 1952; Bowman, 1980) studies which indicated that tubocurarine inhibits evoked release of acetylcholine. Pharmacological evidence
Tetanic fade*
(IC«)
has been obtained (Bowman, 1980) that an extra facilitated release by a feedback mechanism occurs at high frequencies of nerve impulses. Both tubocurarine and hexamethonium (Paton and Zaimis, 1952) are known to inhibit the pharmacological effects of acetylcholine at nicotinic receptor sites of autonomic ganglia. Consequently, the inhibition of the evoked release of acetylcholine by these compounds supports the assumption of a positive nicotinic feedback mechanism of evoked acetylcholine release (Bowman, 1980). It would be incorrect, however, to attribute the tetanic fade solely to the inhibition of this positive feedback mechanism by non-depolarizing neuromuscular blockers. It is conceivable that, in much higher than the threshold concentrations in which they inhibit evoked release of acetylcholine, neuromuscular blocking drugs inhibit some other unknown mechanism of stimulated acetylcholine release. Inhibition of the refilling of the readily available acetylcholine stores of acetylcholine (Elmquist and Quastel, 1965) may be such a mechanism. However, the finding (table II) that the threshold concentrations of neuromuscular blockers required to decrease the evoked release of acetylcholine and tetanic fade were slightly less than those that inhibited peak tetanic tension and tetanic fade indicate that even a marginal decrease of acetylcholine release might affect the integrity of neuromuscular transmission at higher stimulation rates. Decrease of the evoked release of acetylcholine per stimulus (volley output of acetylcholine) during tetanic stimulation was observed in both neurophysiological (Liley and North, 1953) and neurochemical (Foldes et al.,
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Tubocurarine Pancuronium Hcxamethonium
Prejunctional
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BRITISH JOURNAL OF ANAESTHESIA
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1978) studies. The prejunctional inhibition of elicited by single impulses (at 0.1 Hz) had evoked release of acetylcholine by neuromuscular returned to or above 95 % of control, the T4/T1 blockers is also involved in the production of ratio was only 0.55 (Foldes, Yun et al., 1981). tetanic fade, train-of-four fade and tetanic reduc- Similarly, tetanic fade was found to be more tion of EPP. Even a small reduction in the output sensitive to non-depolarizing neuromuscular may produce a devastating "fade" of mechanical blockers than the single twitch (Stanec and responses, when a proportion of the postjunctional Baker, 1984). receptors is occluded. Hence, a neuromuscular blocking drug, by occluding some of the postREFERENCES junctional receptors, will exaggerate the conseBeani, L., Bianchi, C , and Ledda, F. (1964). The effect of quences of its own prejunctional action to tubocurarine on acetylcholine release from motor nerve diminish release. terminals. J. Physiol. {Lond.), 174, 172. There is considerable variation between the Blaber, I. C. (1970). The effect of facilitatory concentrations of decamethonium on the storage and release of transmitter at relative potencies of tubocurarine, pancuronium the neuromuscular junction of the cat. J. Pharmacol. Exp. and hexamethonium at pre- and postjunctional Ther., 175, 664. receptor sites (table II). The ICS0 values of (1973). The prejunctional actions of some non1 tubocurarine (0.86 ±0.01 ^mol litre" ) and hexadepolarizing blocking drugs. Br. J. Pharmacol., 47, 109. 1 methonium (2430 ± 200 nmol litre" ) in the Boyd, J. A., and Martin, A. R. (1956). Spontaneous subthreshold activity at mammalian neuromuscular junctions. mouse phenic nerve-hemidiaphragm preparation J. Physiol. {Lond.), 132, 61. required for the inhibition of peak tetanic Bowman, W. C. (1980). Prejunctional and postjunctional tension elicited by short trains of tetani (50 Hz, cholinoceptors at the neuromuscular junction. Arusth. five shocks, every 20 s) were higher than those Analg., 59, 935. Webb, S. N. (1972). Acetylcholinesterase drugs in required to decrease evoked acetylcholine release neuromuscular blocking and stimulation agents; in Interor produce tetanic fade. These findings indicate national Encyclopedia of Pharmacology and Therapeutics that there are important differences between pre(sec. 14). Vol. II (ed. J. Cheymol), p. 427. Oxford: Pergaand postjunctional nicotinic receptors. This mon Press. assumption has been already made by Gibb and (1976). Tetanic fade during partial transmission failure produced by nondepolarizing neuromuscular Marshall (1984). The order of relative selectivity blocking drugs in the cat. Clin. Exp. Pharmac. Physiol., 3, at prejunctional site is hexamethonium > tubocu545. rarine > pancuronium. This is in good agreement Chang, C. C , Cheng, H. C , and Chen, T. F. (1967). Does with the in vivo observation of Bowman and Webb d-tubocurarine inhibit the release of acetylcholine from (1976) who demonstrated in cats that pancuronium motor nerve endings ? Jap. J. Physiol., 17, 505. affected primarily the peak tetanic tension, Cheymol, J., Bourilet, F., and Ogura, Y. (1962). Action de quelques paralysants neuromusculaires sur la liberation de tubocurarine affected both peak tetanic tension l'acetylcholine au niveau des terminaisons nerveuses motand tetanic maintenance, and hexamethonium rices. Art. Int. Pharmacodyn. Ther., 139, 187. influenced primarily tetanic maintenance. Dale, H. H., Feldberg, W., and Vogt, M. (1936). Release of acetylcholine voluntary motor nerve endings. J. Physiol. Since all voluntary muscle movements in man (Lond.), 86, 353. and other mammals are elicited by short trains of Elmquist, D., and Quastel, D. M. J. (1965). A quantitative tetani (Zierler, 1974), the presynaptic inhibition study of endplate potentials in isolated human muscle. J. Physiol. {Lond.), 178, 505. of the evoked release of acetylcholine may have considerable physiological and clinical signifi- Emmelin, N. G., and Mclntosh, F. C. (1956). The release of acetylcholine from perfused sympathetic ganglia and skeletal cance. This assumption is supported by findings muscles. J. Physiol. {Lond.), 131, 477. in laboratory animals and by clinical observa- Fan, P., and Katz, B. (1952). Spontaneous subthreshold tions. Thus, for example, the blocking potency of activity at motor endings. J. Physiol. {Lond.), 117, 109. non-depolarizing neuromuscular blocking drugs Fletcher, P., and Forrester, T. (1975). The effect of curare on the release of acetylcholine from mammalian motor nerve and aminoglycoside- or polypeptide-type antibiterminals and an estimate of quantum content. J. Physiol. otics is much greater in in vivo and in vitro nerve{Lond.), IS, 131. muscle preparations stimulated with short trains Foldes, F. F., Chaudhry, I., Ohta, Y., Amaki, Y., Nagashima, of 50-Hz tetani than in those stimulated with single H., and Duncalf, D. (1981). The influence of stimulation parameters on the potency and reversibility of neuroimpulses at 0.1 Hz (Foldes, Chaudhry et al., muscular blocking agents. J. Neural Transm., 52, 227. 1981). In anaesthetized patients recovering from Kuze, S., Vizi, E. S., and Deery, A. (1978). The influence the effects of non-depolarizing neuromuscular of temperature on neuromuscular performance. J. Neural blockade, at the time when the twitch tension Transm., 43, 27.
INHIBITION OF RELEASE OF ACETYLCHOLINE
neuromuscular depression in the frog. Nature (Lond.), 188, 501. Otsuka, M., Endo, M., and Nonomura, Y. (1962). Presynaptic nature of neuromuscular depression. J. Physiol. (Lond.), 12, 573. Paton, W. D. M., and Waud, D. R. (1967). The margin of safety of neuromuscular transmission. J. Physiol. (Lond.), 191, 59. Zaimis, E. J. (1952). The methonium compounds. Pharmacol. Rev., 4, 219. Stance, A., and Baker, T. (1984). Prejunctional and postjunctional effects of tubocurarine and pancuronium in man. Br. J. Anaesth., 56, 607. Straughan, D . W. (1960). Release of acetylcholine from mammalian motor nerve endings. Br. J. Pharmacol. Chtmother., 15, 417. Szerb, J. C. (1975). Endogenous acetylcholine release and labelled acetylcholine formation from (*H)-choline m the myenteric plexus of the guinea-pig ileum. Can. J. Physiol. Pharmacol., 53, 566. Somogyi, G. T. (1973). Depression of acetylcholine release from cerebral cortical slices by cholinesterase inhibition and by oxotremorine. Nature (Lond.), 241, 121. Vizi, E. S., Ono, K., Adam-Vizi, V., Duncalf, D., and Foldes, F. F. (1984). Presynaptic inhibitory effect of metenkephalin on [14C] acetylcholine release from the myenteric plexus and its interaction with muscarinic negative feedback inhibition. J. Pharmacol. Exp. Ther., 230, 493. Vyskocil, F. (1979). Changes in total and quanta] release of acetylcholine in the mouse diaphragm during activation and inhibition of membrane ATPase. J. Physiol. (Lond.), 286,1. Zierler, K. L. (1974). Mechanism of muscle contraction and its energetics; in Medical Physiology, Vol. 1 (ed. V. B. Mountcastle), p. 84. St Louis: The Mosby Co.
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Foldes, F. F., Somogyi, G. T., Chaudhry, I. A., Nagashima, H., and Duncalf, D. (1984X Assay of 'H-acetylcholine release from mouse diaphragm without cholinesterase inhibition. Anesthesiology, 61, A395. Yun, H., Radnay, P. A., Badola, R. P., Kaplan, R., and Nagashima, H. (1981). Antagonism of the NM effe« of Org-NC45 by cdrophonium. Anesthesiology, 95, A201. Gibb, A. J., and Marshall, I. G. (1984). Pre and postjunctional effects of tubocurarine and other nicotinic antagonists during repetitive stimulation in the rat. J. Physiol. (.Land.), 351,275. Hubbard, J. E., Llinas, R., and Quastel, D. M. J. (1969). EUctrophysiological Analysis of Synaptic Transmission. London: Edward Arnold. Hubbard, J. I., and Wilson, D . F. (1973). Neuromuscular transmission in a mammalian preparation in the absence of blocking drugs and the effect of d-tubocurarine. J. Physiol. (Lond.), 228, 307. Hutter, O. F. (1952). Post-tetanic restoration of neuromuscular transmission blocked by d-tubocurarine. J. Physiol. (.Lond.), 118, 216. Kilbinger, H., and Wessler, I. (1980). Inhibition by acetylcholine of the stimulation evoked release of *Hacetylcholine from the guinea pig myenteric plexus. Neurosdenct, 5, 1331. Krnjevic, K., and Mitchell, J. F. (1961). The release of acetylcholine in the isolated rat diaphragm. J. Physiol. (Lond.), 155, 246. Liley, A. W., and North, K. A. K. (1953). An electrical investigation of effects of stimulation on mammalian neuromuscular junction. J. Nettrophysiol., 16, 509. Magleby, K. L., Pallotta, B. S., and Terrar, D. (1981). The effect of ( + )-tubocurarine on neuromuscular transmission during repetitive stimulation in the rat, mouse and frog. J. Physiol. (Lond.), 312, 97. Otsuka, M., and Endo, M. (1960). Presynaptic nature of
231
TUBOCURARINE AND PANCURONIUM INHIBIT EVOKED RELEASE OF ACETYLCHOLINE FROM THE MOUSE HEMIDIAPHRAGM PREPARATION E. S. VIZI, G. T. SOMOGYI, H. NAGASHIMA, D. DUNCALF, I. A. CHAUDHRY, O. KOBAYASHI, P. L. GOLDINER AND F. F. FOLDES
E. S. VIZI, M.D., PH.D.; D . DUKCALF, M.D.; I. A. CHAUDHRY, D.V.M.; O. KOBAYASHI, M.D.; F. F. FOLDES, M.D.; H. NAGA-
SHIMA, M.D.; Department of Anesthesiology, Montefiore Medical Center, Albert Einstein College of Medicine, 111 East 210thStreet,Bronx,NewYorkl0467,U.S.A.G. T. SOMOGYI,
M.D., Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest, Hungary. Accepted for Publication: December 9, 1985. Correspondence to E.S.V.
SUMMARY Using a sensitive radioactive method that measures selectively the evoked release of acetylcholine, it was demonstrated that, when stimulating at 50 Hz, tubocurarine or pancuronium 2x.10~* mo I litre~l or hexamethonium 10~3 mol litre~l significantly decreased the evoked release of acetylcholine in the mouse in vitro phrenic nerve-hemidiaphragm preparation.
Baker, 1984). Bowman and Webb (1976) and Bowman (1980) suggested that the increase in the potency of non-depolarizing neuromuscular blockers at high rates of stimulation is, in part, the result of the inhibition of a positive, nicotinic feedback mechanism of acetylcholine release. According to Bowman (1980), the acetylcholine released by the nerve impulse acts, not only on the nicotinic (cholinergic) receptors of the postjunctional membrane, but also on nicotinic receptors of the motor nerve terminal. The interaction of acetylcholine with the latter results in an increase in the evoked release of acetylcholine. Because of the great safety margin of neuromuscular transmission (Paton and Waud, 1967), inhibition of this positive feedback mechanism only becomes important at relatively high (> 2 Hz) rates of stimulation when the margin of safety is decreased to a level where any effect on the release of acetylcholine affects chemical neurotransmission (Foldes, Yun et al., 1981). Until now, attempts to demonstrate the inhibitory effect of non-depolarizing neuromuscular blocking drugs on the evoked release of acetylcholine by neurochemical methods (assay of the
Downloaded from http://bja.oxfordjournals.org/ at University of Sydney on May 2, 2015
Liley and North (1953) demonstrated that, in the rat hemidiaphragm paralysed by tubocurarine, repetitive stimulation of the phrenic nerve caused a gradual decrease in the amplitude of the end-plate potential (EPP). They assumed that this was the result of a decreased release of transmitter, and that it was a physiological response to high frequency stimulation which was unmasked, but not produced, by tubocurarine (Otsuka, Endo and Nonomura, 1962). However, other workers (Hubbard, Llinas and Quastel, 1969; Blaber, 1970, 1973; Hubbard and Wilson, 1973; Bowman and Webb, 1976; Magleby, Pallotta and Terrar, 1981), using different methods, showed that the rate of attenuation of the EPP in high frequency trains was greater in the presence than in the absence of tubocurarine—a decrease attributed to the reduction of acetylcholine release (Hutter, 1952; Otsuka and Endo, 1960; Gibb and Marshall, 1984). In agreement with the above findings, it has been reported that the neuromuscular blocking effects of tubocurarine and other non-depolarizing neuromuscular blocking drugs are greater during high frequency than during low frequency electrical stimulation in vitro (Foldes, Chaudhry et al., 1981) and in vivo both in animals (Paton and Zaimis, 1952; Bowman and Webb, 1976; Foldes, Chaudhry et al., 1981) and in man (Stance and
INHIBITION OF RELEASE OF ACETYLCHOLINE
MATERIAL AND METHODS
Male Swiss—Webster mice of 25-30 g body weight were lightly anaesthetized with ether and decapitated. The middle portions of both hemidiaphragms (about 2-3 mm to the right and left of the insertions of the phrenic nerves), together with the attached rib segments, were suspended
in organ baths of 2 ml volume in Krebs' solution (NaCl 113, KC1 4.7, CaCl, 2.5, MgSO4 1.2, NaHCO3 25, KH2PO« 1.2 and glucose 11.5 mmol litre"1). The bath temperature was maintained at 37 °C. The pH of the solution aerated with a 95% oxygen-5% carbon dioxide gas mixture was 7.38-7.42. The rib end of the preparation was attached with a ligature to the bottom of the bath and its tendon to an FT03 transducer. Stimulation. After 2 g resting tension was applied, the preparations were stimulated with a Grass S-88 stimulator by 0.8-s, 0.1-s or 1-s trains of 50-Hz, supramaximal impulses of 0.2 ms duration, every 10 s or 20 s through platinum electrodes placed above the proximal and below the distal parts of the preparation (field stimulation). Peak tetanic tension. Tetanic fade. The isometric contractions were measured with force displacement transducers and continuously monitored on a polygraph recorder. The peak tetanic tension and tetanic fade were recorded as described by Bowman and Webb (1976). Incubation. After a 15-min equilibration period, the preparations were loaded with 3H-choline 5 uCi ml"1 for 60 min. The preparations were stimulated during this 75 min as described above. This was done to facilitate the incorporation of 3 H-choline into the acetylcholine pool of the motor nerve terminal. For the next 20 min, the preparations were allowed to rest. At the end of this period, in seven preparations the radioactivity of the muscles, consisting partly of 3 Hcholine and partly of 3H-acetylcholine, was 7.54 ± 1.0 x 10* Bq g"1. After being washed three times to remove excess 3H-choline, each preparation was transferred to another bath and superfused with Krebs' solution at the rate of 2 ml min"1 for 60 min. From then on, throughout the investigations, Krebs' solution containing hemicholinium-3 5 x 10~6 mol litre"1 was superfused at a rate of 0.5 ml min"1. Hemicholinium-3 was added to prevent reuptake of 3H-choline that diffused out as such from the muscle or was liberated by hydrolysis of 3H-acetylcholine released during the subsequent stimulation periods.
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released acetylcholine) have been unsuccessful. Various investigators reported that tubocurarine had little (Beani, Bianchi and Ledda, 1964) or no (Dale, Feldberg and Vogt, 1936; Emmelin and Mclntosh, 1956; Straughan, 1960; Kmjevic and Mitchell, 1961; Cheymol, Bourilet and Ogura, 1962; Chang, Cheng and Chen, 1967; Fletcher and Forrester, 1975) effect on the evoked release of acetylcholine. In all of these studies the total (quantal + non-quantal) acetylcholine was measured in the presence of an anticholinesterase. The failure to demonstrate any changes in the evoked quantally released acetylcholine by tubocurarine may have been because most of the acetylcholine released from mammalian muscle and measured by bioassay is released in a non-quantal form. At rest, about 98 %, and during electrical stimulation about 70%, of the released acetylcholine is non-quantal (Vizi and Vyskocil, 1979). To resolve the contradiction between the results of the pharmacological and neurophysiological studies on the one hand and those of the direct measurement of acetylcholine on the other, the effect of non-depolarizing neuromuscular blockade on the evoked release of acetylcholine was re-examined with a sensitive radioactive method (Foldes et al., 1984). Radioactive choline was used to label the acetylcholine stores of the mouse hemidiaphragm preparation. It was shown previously that the newly synthetized 3H-acetylcholine is preferentially released in response to electrical stimulation in brain (Szerb, 1975), myenteric plexus (Vizi et al., 1984), and also in the mouse hemidiaphragm preparation (E. S. Vizi, unpublished observations). Furthermore, using this method, measurement of acetylcholine release can be accomplished without cholinesterase inhibition (Szerb, 1975; Kilbinger and Wessler, 1980; Foldes et al., 1984; Vizi et al., 1984). This is advantageous because cholinesterase inhibitors are known to affect the release of acetylcholine (Fatt and Katz, 1952; Boyd and Martin, 1956; Foldes et al., 1984).
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Calculation. In the mouse phrenic nerve— hemidiaphragm preparation, similar to the situation in the myenteric plexus-longitudinal muscle preparation of the guineapig ileum (Szerb and Somogyi, 1973; Kilbinger and Wessler, 1980; Vizi et al., 1984), electrical stimulation increases only the release of 3H-acetylcholine and the outflow of 3H-choline remains unchanged (E. S. Vizi, unpublished observations). Consequently, the evoked release of sH-acetylcholine can be calculated from the difference between the expected spontaneous and the actually measured outflow of radioactivity caused by 3 min of electrical stimulation. Spontaneous release was assumed to follow first-order kinetics. It was determined by the "least square" fitting to an exponential curve method of the Bq g"1 concentrations of two 3-ml effluents collected before, and that of two others collected after, every period of stimulation. The expected spontaneous outflow was obtained by interpolation between the fitted points. The radioactivity of the samples was measured in a Packard Scintillation Beta Spectrometer. There was considerable variation in the controls from one preparation to the other, in the amount of radioactivity released during the corresponding periods of stimulation. In contrast, the ratios of radioactivity measured during the first two consecutive stimulation periods, 8,/Sj, were constant. Results are presented as mean values ±SEM. Statistical analysis. The statistical significance of the results was determined by one-way analysis of variance of the logarithmically transformed data, followed by Dunn's test. P < 0.05 was considered significant.
TABLE I. Inhibition of the evoked release of radioactivity from mouse hemidiaphragm preparations, loaded with *H-choline, by tubocurarine, pancuronium or hexamethonium. The inhibition of the evoked release of radioactivity could be attributed to the inhibition of the release of 'H-acetylcholine (Jor explanation see text). * Ratio of the evoked release of radioactivity during the second and first stimulation periods (mean±SEM). Significant differences from control determined by one-taay analysis of variance followed by Dunn's test Compound
n
Control Tubocurarine 2 x 10~5 mol litre"1 Pancuronium 2 x 10-» litre"1 Hexamethonium 10-' mol litre"1
14 6
S./S, Ratio*
P
0.79 ±0.02** 0.42 ±0.08
<0.01
8
0.51 ±0.08
<0.01
6
0.51 ±0.07
<0.01
Materials. The materials used were 3 Hmethylcholine chloride (Amersham, 78 Ci mmol litre"1, tetrodotoxin (Sigma), eserine sulphate (Sigma), tubocurarine (BDH), pancuronium (Organon) and hexamethonium (BDH). RESULTS
Resting release
In six investigations, the release of radioactivity at rest remained relatively constant (1513 ± 154 Bq g"1 min"1) throughout. Tetrodotoxin 5 x 10"' mol litre"1, pancuronium 2 x 10"6 mol litre"1 and hexamethonium 3 x 10"* mol litre"1 had no effect on resting release. Evoked release
When trains of 40 stimuli (50 Hz) were delivered every 10 s for 3 min (720 stimuli were applied), the release of radioactivity was significantly enhanced above spontaneous release by 2685 ± 180 Bq g"1 (n = 8). The output of radioactivity was very constant; the ratio between the amounts of radioactivity released by consecutive stimulation periods (S,/Sj, S,/S,) did not differ significantly from each other: Sj/S! was 0.83 ±0.09 and S s /S, was 0.72 ±0.08 (n = 8 and 8, P > 0.05). Tetrodotoxin 5 x 10"7 mol litre"1, added to the bath 10 min before stimulation, completely inhibited the stimulation evoked release of radioactivity. Table I shows the effect of different antinicotinic agents (hexamethonium, tubocurarine and pancuronium) on the release of radioactivity in response to stimulation. All these agents signifi-
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Collection. During superfusion at the rate of 0.5 ml min"1, 3-min effluents were collected continuously with a fraction collector into scintillation vials throughout the rest of the experimental period. The preparations were stimulated, as described, three times for 3 min each starting at the 10th (S t ), 52nd (Sj) and 82nd (S3) min. In 14 control experiments, no drug was added to the perfusing solution. In six, eight and six other studies, respectively, tubocurarine 2 x 10~6 mol litre"1, pancuronium 2 x 10~5 mol litre"1 or hexamethonium 10~s mol litre"1 was added to the perfusing Krebs'-hemicholinium-3 solution at 17 min.
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INHIBITION OF RELEASE OF ACETYLCHOLINE
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TABLE II. Effect of different anlmkotmic agents on neuromuscular transmission of and "C-acetylcholme releasefrommousehemidiaphragmpreparation. * Phrenic nerve was stimulated and trams of 50 Hzifioe shocks) were delivered in every 20 s. The contractions were recorded isometrically. t The release of radioactivity was measured(see Methods). Trains of 50 Hz (40 shocks) were applied in every 10 s. X The contractures produced by trains of 50 Hz (100 shocks) were recorded and the effect of drugs in different concentrations was studied on the maintenance. ICU is the concentration needed to produce 25% reduction of maintenance Inhibition of Postjunctional Peak tetanic tension* (IQ,) (junol litre ')
'H-ACh release (threshold concn) (jimol litre"')t
(jimol litre ')
0.86 ±0.01 0.42 ±0.02 2430 ±200
0.5 0.25 50
0.28 ±0.02 0.18±0.03 260±12
cantly reduced the stimulation-evoked release of radioactivity. The threshold concentrations were found to be 0.5 umol litre"1 for tubocurarine, 0.25 umol litre"1 for pancuronium and 50 umol litre"1 for hexamethonium, respectively (table II). They produced a significant reduction of 3H-acetylcholine release: the S J / S J ratios were 0.601 ±0.03, 0.621 ±0.03 and 0.641 ±0.02, respectively (n = 3 and 3, P < 0.01). These data indicate that the effects of the neuromuscular blockers are concentration-dependent. The IC50 values for the inhibition of the peak tetanic tension of the mouse phrenic nerve-hemidiaphragm preparation were 0.86 ±0.01 umol litre"1 for tubocurarine, 0.42 ±0.02 umol litre"1 for pancuronium and 2430 ± 200 umol litre"1 for hexamethonium (table II). DISCUSSION
The sensitive radioisotope method (Foldes et al., 1984) used in this study revealed the significant inhibitory effect of tubocurarine on the evoked release of acetylcholine that could not be demonstrated by other methods (Dale et al., 1936; Emmelin and Mclntosh, 1956; Krnjevic and Mitchel, 1961; Chang, Cheng and Chen, 1967; Fletcher and Forrester, 1975). The data presented are in agreement with the results of the electrophysiological (Hubbard, Llinas and Quastel, 1969; Blaber, 1970, 1973; Bowman and Webb, 1972; Hubbard and Wilson, 1973) and pharmacological (Paton and Zaimis, 1952; Bowman, 1980) studies which indicated that tubocurarine inhibits evoked release of acetylcholine. Pharmacological evidence
Tetanic fade*
(IC«)
has been obtained (Bowman, 1980) that an extra facilitated release by a feedback mechanism occurs at high frequencies of nerve impulses. Both tubocurarine and hexamethonium (Paton and Zaimis, 1952) are known to inhibit the pharmacological effects of acetylcholine at nicotinic receptor sites of autonomic ganglia. Consequently, the inhibition of the evoked release of acetylcholine by these compounds supports the assumption of a positive nicotinic feedback mechanism of evoked acetylcholine release (Bowman, 1980). It would be incorrect, however, to attribute the tetanic fade solely to the inhibition of this positive feedback mechanism by non-depolarizing neuromuscular blockers. It is conceivable that, in much higher than the threshold concentrations in which they inhibit evoked release of acetylcholine, neuromuscular blocking drugs inhibit some other unknown mechanism of stimulated acetylcholine release. Inhibition of the refilling of the readily available acetylcholine stores of acetylcholine (Elmquist and Quastel, 1965) may be such a mechanism. However, the finding (table II) that the threshold concentrations of neuromuscular blockers required to decrease the evoked release of acetylcholine and tetanic fade were slightly less than those that inhibited peak tetanic tension and tetanic fade indicate that even a marginal decrease of acetylcholine release might affect the integrity of neuromuscular transmission at higher stimulation rates. Decrease of the evoked release of acetylcholine per stimulus (volley output of acetylcholine) during tetanic stimulation was observed in both neurophysiological (Liley and North, 1953) and neurochemical (Foldes et al.,
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Tubocurarine Pancuronium Hcxamethonium
Prejunctional
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1978) studies. The prejunctional inhibition of elicited by single impulses (at 0.1 Hz) had evoked release of acetylcholine by neuromuscular returned to or above 95 % of control, the T4/T1 blockers is also involved in the production of ratio was only 0.55 (Foldes, Yun et al., 1981). tetanic fade, train-of-four fade and tetanic reduc- Similarly, tetanic fade was found to be more tion of EPP. Even a small reduction in the output sensitive to non-depolarizing neuromuscular may produce a devastating "fade" of mechanical blockers than the single twitch (Stanec and responses, when a proportion of the postjunctional Baker, 1984). receptors is occluded. Hence, a neuromuscular blocking drug, by occluding some of the postREFERENCES junctional receptors, will exaggerate the conseBeani, L., Bianchi, C , and Ledda, F. (1964). The effect of quences of its own prejunctional action to tubocurarine on acetylcholine release from motor nerve diminish release. terminals. J. Physiol. {Lond.), 174, 172. 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