DOES D-TUBOCURARINE INHIBIT the RELEASE of ACETYLCHOLINE from MOTOR NERVE ENDINGS ?

The Japanese Journal of Physiology 17: pp.505-515, 1967

DOES d-TUBOCURARINE INHIBIT THE RELEASE OF ACETYLCHOLINE FROM MOTOR NERVE ENDINGS ?

C. C. CHANG,H. C. CHENGAND T. F. CHEN

Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China

d-Tubocurarine has been used for many years in the study of the physiology of neuromuscular transmission as an agent to immobilize isolated nerve- muscle preparations. It is generally agreed that d-tubocurarine does not have any effect of the presynaptic event of junctional transmission since the experiment of DALE et al. (1936) which showed that d-tubocurarine paralysed skeletal muscles without inhibition of the release of acetylcholine (ACh) from motor nerve endings. This concept, however, is challenged by STANDAERT(1964) basing on the evidence that the post-tetanic antidromic discharges in motor nerves were abolished by d-tubocurarine at doses much smaller than that needed for the blockage of neuromuscular transmission. On the other hand, BEANI et al. (1964) claimed to have demonstrated that d-tubocurarine inhibits the release of ACh from motor nerve endings under certain conditions using diisopropylphosphorofluoridate as anticholinesterase agent. They suggested that, in the curarized preparation, the presynaptic effect might be a contributory factor in Wedensky inhibition. In the view that many of conclusions concerning the physiology of motor nerve endings, especially on the problem of ACh stores and release, have been drawn out from experiments using curarized preparations and that there exists some contradictory results, it was felt necessary to repeat the experiment of BEANI et al. (1964) and to settle whether or not d-tubocurarine inhibits the release of ACh from the motor nerve endings. Some electrophysiological experiments were also done in order to see whether d-tubocurarine affects the presynaptic events of the neuromuscular transmission.

METHODS Guinea pig diaphragms for estimation of ACh. All the experiments for the study of

Received for publication January 7, 1967 張傅燗,鄭憲章,陳増福

505 506 C. C. CIEANG, H. C. CHENG AND T. F. CHEN

FIG.1. The response of guinea pig ileum to ACh. ACh, 1, 2, 3, and 4 ng, was added for 30 sec to guinea pig ileum previously treated with Mipafox.

effects on ACh release were carried out on phrenic nerve-diaphragm preparations of

guinea pigs, weighing 300-500g. Both sides of the hemidiaphragm were used after treatment with an anticholinesterase agent as described below. The organ bath contained 4ml of Krebs' bicarbonate solution, in which half of the NaHCO3 was replaced

with equimolar amount of NaCl in order to reduce the pII and to minimize the spontane- ous hydrolysis of ACh. The bath fluid was kept at 33•Ž and was oxygenated with 95%

2+5% CO2. The nerves were stimulated with a pair of platinum electrodes by rect-O angular pulses of 0.2 msec duration at a twice-threshold voltage. For the purpose of

estimation of ACh release, the diaphragm preparations were stimulated for 10min at

50 cps and the bath fluid was removed for assay at 5min after the completion of

stimulation. After washing, the preparations were kept unstimulated for another 5min before proceeding to the next period of stimulation. These procedures were essentially

similar to those reported by BEANI et al. (1964) as that d-tubocurarine had the most marked effect on ACh release.

Treatment with anticholinesterase agents. Diisopropylphosphorofluoridate (DFP), 500

ug/ml, N, N' diisopropylphosphorodiamidic fluoride (Mipafox), 1000ƒÊg/ml, Diethoxy-

phosphoryl-thiocholine iodide (Echothiophate), 15ƒÊg/ml, and neostigmine methylsulfate, 5ƒÊg/ml were used as anticholinesterase. The guinea pig diaphragm preparation was

pretreated with each of DFP, Mipafox, and Echothiophate for 150min at 37•Ž. The anticholinesterase agent was then washed out repetitively for 30min. When reversible

anticholinesterase neostigmine was used, 5ƒÊg/ml of the agent was added to the Krebs'

solution throughout the experiments.

Assay of ACh. The content of ACh in the samples was assayed against standards on

guinea pig terminal ileum which was previously treated with Mipafox, 10ƒÊg/ml, for 70min at 37•Ž as described by JOHNSON (1963). The ileum thus treated could be stored in a refrigerator for two days without loss of ACh sensitivity. For assay of ACh, the CURARE AND ACh RELEASE 507

preparation was kept in 5ml of oxygenated Krebs' solution at room temperature (26- 29•Ž). Pyribenzamine (5ng/ml) and morphine sulfate (50ng/ml) were added. Addition

of 1 to 4 ng of ACh to the organ bath usually gave a rather linear response as shown

in FIG. 1. The amounts of ACh in the samples were therefore calculated by simple interpolation. An aliquot of samples (0.4ml) obtained from the diaphragm preparations

pretreated with DFP, Mipafox and Echothiophate were directly added to the ileum for the ACh assay, while the samples containing neostigmine were run through a column

of Amberlite CG-120, 4mm•~6mm, before assay of ACh in order to remove the anti-

cholinesterase agent.

Antidromic activity. The method described by RANDIC and STRAUGIIAN (1964) for the recording of antidromic activity of the isolated rat phrenic nerve-diaphragm preparation

was followed. To 30ml of modified Tyrode solution, which contained 3.6mM CaCl2 and

0.12mM MgCl2, neostigmine methylsulfate was added to a final concentration of 0.3ƒÊg/ml. The temperature was kept at 22•‹1-0.5•Ž. In these conditions it was possible to record

the antidromic repetitive discharges of the nerve after single nerve volleys for more than 2hr if the stimulus frequency was kept at 0.05/sec or less.

Terminal nerve spikes. Extracellular microelectrode recordings of the terminal nerve spike of rat phrenic nerve-diaphragm preparations were made according to the method

described by HUBBARD and SCHMIDT (1963). MgCl2 was added to a concentration of 11-12mM in order to immobilize the preparation. The organ bath contained 30ml of

oxygenated modified Krebs' solution kept at 33•Ž.

Drugs. d-Tubocurarine chloride (Allied Biochemical Laboratories), DFP (Merck, Sharp

and Dohme Research Laboratories), Mipafox (Medical Research Council, England),

Echothiophate (Ayerst Laboratories), Neostigmine methylsulf ate (Shionogi Pharmaceuti- cal Co.), and Hemicholinium-3 (Aldrich Chemical Co.) were used.

RESULTS

Effects on the repetitive antidromic discharges. Curare has been shown to

abolish the repetitive antidromic discharges evoked by orthodromic nerve

volley in the presence of anticholinesterases at a dose lower than that required

for neuromuscular blockade (FENG and IA, 1941; RIKER et al., 1959). Since the

antidromic activities of motor nerve fibres have been shown not to be induced

by muscle activities (STANDAERT, 1963, RANDIe and STRAUGHAN, 1964) but

probably by a presynaptic effect of the neurotransmitter (HUBBARD et al., 1965)

it was suggested that this presynaptic effect of curare might be the cause of

blockade of neuromuscular transmission (STANDAERT, 1964). It was of interest

therefore to see whether curare also has this effect on the isolated phrenic

nerve-diaphragm preparations. FIG. 2 shows that in the presence of neostigmine

the nerve impulse-induced repetitive discharges were abolished by d-tubo-

curarine at a concentration (0.5pg/ml) which did not completely block neuro-

muscular transmission, indicating that d-tubocurarine does have an effect on

the presynaptic side of the rat diaphragm.

Effect on axonal conduction. Under certain conditions, such as pretreatment 508 C. C. CHANG, H. C. CHENG AND T. F. CHEN

FIG.2. Effect of d-tubocurarine on the repetitive antidromic discharges of isolated rat phrenic nerve-diaphragm preparations. The upper tracing of each panel shows the extracellularly monitored muscle action potential, and the lower, discharges of the phrenic nerve induced by a nerve volley. Left panel, before, and right panel, 20min after administration of d-tubocurarine, 0.5pg/ml.

FIG.3. Effect of d-tubocurarine on the compound action potentials of the rat phrenic nerve. The preparation was pretreated with DFP (500pg/ml)

for 150min and then washed. Left panel, before, and right panel, 30min after addition of d-tubocurarine (5ƒÊg/ml). Twenty superimposed potentials

elicited at 50 cps are shown.

FIG.4. Effect of d-tubocurarine on nerve terminal spikes and end-plate potentials of rat diaphragms. MgCl2 (11mM) was added

to immobilize the preparation. Twenty superimposed spikes and end-plate potentials elicited by stimulation of the phrenic nerve at

50 cps are shown. Left panel, before, and right panel, 30min after addition of d-tubocurarine (5ƒÊg/ml). CURARE AND ACh RELEASE 509

with snake venoms or surface active agents, curare has been shown to affect the axonal conduction in frog sciatic nerve (WALSH and DEAL, 1959) or in squid giant axon (ROSENBERG, 1961; ROSENBERG and EHRENPREIS, 1961). Ex- periments were designed therefore to see whether d-tubocurarine has such an effect on the DFP-pretreated preparation, since under this condition d-tubocurarine has been reported to inhibit the release of ACh (BEANI et al., 1964).

FIG. 3 shows that, at a concentration of 5ƒÊg/ml, d-tubocurarine did not affect the antidromically recorded compound action potentials of the rat phrenic nerve which was pretreated for 150min with 500ƒÊg/ml of DFP at 37•Ž and then washed. The conduction of repetitive impulses at 50 cps also was not impaired in the presence of d-tubocurarine.

Effect on nerve terminal spikes. The terminal nerve spike of frog sartorius

was shown not affected by d-tubocurarine at concentrations causing complete

neuromuscular blockade (KATZ and MILEDI, 1965). In the present study

d-tubocurarine was tested on the rat diaphragm preparation. The results

confirmed the powerful postsynaptic blocking action of the drug. While the

end-plate potentials were quickly abolished by d-tubocurarine (5ƒÊg/ml) the

terminal nerve spikes were not affected (FIG.4). The nerve terminal was still

able to conduct repetitive impulses at 50 cps without decline of the spikes.

Effects of anticholinesterases On the indirect contraction. All of the anticholine-

sterase agents used augmented the contraction height of guinea pig diaphragms

soon after addition of the drug. DFP and Mipafox then caused paralysis of the preparation in about 20 and 50min respectively when tested by single

shock. Washings after treatment with organophosphorus compounds restored

the single indirect contraction, while Wedensky inhibition was prominent on

repetitive stimulation in all preparations. On the other hand, neostigmine

(5ƒÊg/ml) and Echothiophate (15ƒÊg/ml) did not block the response to single

stimulations. After repeated washings of Echothiophate-treated diaphragms, the response to repetitive stimulation became more sustained, suggesting a

progressive recovery of cholinesterase. Addition of smal amount of Echothio-

phate (2ƒÊg/ml) into Krebs' solution for washing prevented this phenomenon.

Release of ACh in the controls. The output of ACh from guinea pig dia-

phragms stimulated at 50 cps for 10min was shown in TABLE 1. No signifi- cant difference in the amount of ACh released was found between each anti-

cholinesterase agent used. There was variation in the output of ACh from

preparation to preparation. The output from one preparation, however, was rather constant during the period of experiment of about 5hr except in the

Echothiophate-pretreated preparations, in which the ACh output declined as

the washing of the preparation was repeated, probably because of recovery 510 C. C. CHANG, H. C. CHENG AND T. F. CHEN

TABLE1. ACh output from guinea pig diaphragm preparations treated with various anticholinesterase agents.

of cholinesterase. It was found that this can be prevented by addition of

small amount of Echothiophate (2ƒÊg/ml) into the Krebs' solution. Therefore,

when Echothiophate was used as an anticholinesterase agent, the effect of d-tubocurarine on ACh output was studied using Krebs' solution containing

2ƒÊg/ml of Echothiophate instead of complete washing out.

Effect of d-tubocurarine on ACh release. d-Tubocurarine chloride at concent-

ration of 5ƒÊg/ml completely blocked the neuromuscular transmission of guinea.

pig diaphragms either treated with neostigmine or organophosphorus com-

pounds in about 5-15min. The amounts of ACh released by stimulation at 50 cps for 10min in the presence of d-tubocurarine were compared with the

control levels in FIG. 5. It is evident from the results that d-tubocurarine

has no appreciable effect on the release of ACh from motor nerve endings no

matter which anticholinesterase agent was used. It is interesting to note that

the conditions used in the present experiment were that BEANI et al. (1964)

reported to have the most marked inhibition of ACh output by d-tubocurarine.

Effect of Hemicholinium-3 on ACh release. The effect of Hemicholinium-3 on

the ACh release was examined for comparison in order to cheque the system

used in the present experiment. On addition of 150 figiml of Hemicholinium-3

to the neostigmine-treated guinea pig diaphragm preparations, the contractile

response was rapidly blocked within 10min. The release of ACh, on the

other hand, was progressively reduced to less than 20% of control levels in

50min (FIG.6). After washing out of Hemicholinium-3, the contractile response to single stimulation was partially restored in 40min while no recovery of

ACh release was observed. A similar result was obtained by CHEYMOL et al.

(1962) with rat diaphragm preparations.

Trains of end-plate potentials. FIG. 7 shows the end-plate potentials intracellulary recorded in rat diaphragm preparations treated with d-tubocurarine CURARE AND ACh RELEASE 511

FIG.5. Effect of d-tubocurarine on the

release of ACh from guinea pig diaphragm

preparations. The amount of ACh was ex-

pressed as per cent•}S. D. of control levels at the first period of stimulation. The third

period of stimulation began after 30min of incubation with d-tubocurarine (5ƒÊg/ml).

Hatched columns show the output of ACh in the presence of d-tubocurarine. For the

treatments with anticholinesterases see "Methods" .

FIG.6. Effect of Hemicholinium-3 on the release of ACh from guinea pig diaphragm preparations. Same experiments as in FIG. 5 using neostigmine (5ƒÊg/ml) as anticholinesterase.

The third period of stimulation began at 10min after ad-

dition of Hemicholinium-3, 150ƒÊg/ml, when complete blockade of neuromuscular transmission occurred. Hatched columns

show the output of ACh in the presence of Hemicholinium-3. 512 C. C. CHANG, H. C. CHENG ANI) T. F. CHEN

Tubocurarine HemichoIinium-3

FIG. 7. Train of end-plate potentials of rat diaphragms paralysed with d -tubo-

curarine or Hemicholinium-3. Intracellulary recorded end-plate potentials obtained from muscle blocked by d-tubocurarine (1ug/ml) or Hemicholinium-3 (150ug/ml).

Ten repetitive pulses at 100 cps were given.

(1 ug/ml) or Hemicholinium-3 (150 ug/ml). The size of successive end-plate potentials of the Hemicholinium-3-treated muscle appeared to decline more rapidly than that of d-tubocurarine-treated preparations. It is conceivable that, in the presence of an inhibitor of ACh synthesis, the available ACh may be more rapidly exhausted at high frequency of stimulation because of reduc- tion in size of the ACh stores.

DISCUSSION

The biological substance released upon nerve stimulation from the guinea

pig phrenic nerve-diaphragm preparation was ACh is evident from the follow-

ing evidences: (1) the response of the guinea pig ileum to the sample was blocked by low concentration of atropine (0.02 jig/ml) but not by pyribenzamine

(5 ng/ml); (2) the active substance was inactivated by an acetylcholinesterase obtained from the venom of Bungarus multicinctus (CHANG and LEE, 1955); and (3) the release of the substance from the diaphragm preparation was inhibited on treatment with inhibitors of ACh synthesis such as Hemicholini-

um-3 (FIG. 6) and bretylium (CHANG et al., 1967) or inhibitor of ACh release,

-Bungarotoxin (CHANG and LEE ƒÀ , 1963).

The electrophysiological data show that d-tubocurarine does not interfere

with the impulse conduction up to the nerve terminals. Since the nerve fibers were still able to conduct repetitive impulses at high frequency of stimulation

in the presence of d-tubocurarine it may be infered that the Wedensky inhibi-

tion generally observed in curarized muscle is not due to a conduction failure.

Our results clearly show that d-tubocurarine does not have any detectable effect on the release of ACh even at high frequency of stimulation. This CURARE AND ACh RELEASE 513 findings is in agreement with that obtained by DALE et al. (1936), KRNJEVIC and MITCHELL (1961) and CHEYMOL et al. (1962), but is contradictory to that obtained by BEANI et al. (1964), although the latter authors used the same anticholinesterase, DFP, and the same preparation. It might be that the inhibition of acetylcholinesterase was incomplete in the experiment of BEANI et al. (1964) and thus partial recovery of enzyme activity occurred on repetitive washings especially in the curare-treated preparations, just as we have observed with Echothiophate. Since d-tubocurarine did not show any inhibitory effect upon the release of ACh by using any of the various anticholinesterase agents with entirely different chemical structures, it may be further inferred that d- tubocurarine is probably also without effect on the ACh release mechanism in the absence of anticholinesterase agent. It would appear therefore that the phenomenon of Wedensky inhibition or the decline of successive end-plate potentials on repetitive stimulation in the curarized muscle (LILEY and NORTH, 1953; LUNDBERG and QUILISCH, 1953; OTSUKA et al., 1962; HUBBARD, 1963; THIES, 1965) is not due to an inhibition of the release of ACh by d-tubocurarine as postulated by BEANI et al. (1964). It is interesting in this regard that Hemicholinium-3, the competitive inhibitor of ACh synthesis, caused more pronounced decline of end-plate potentials, an indication that "available ACh" is exhausted. Our results therefore support the general view that d-tubocurarine blocks the neuromuscular transmission at post-synaptic site. Abolition of the repetitive antidromic discharges in motor nerves by low concentrations of d-tubocurarine may not participate in the blockade of neuromuscular transmission.

SUMMARY

The effect of d-tubocurarine on the release of ACh from guinea pig phrenic nerve-diaphragm preparations was reinvestigated at high frequency of stimulations (50 cps). Various types of anticholinesterases, including DFP, Mipafox, Echothiophate and neostigmine, were used as cholinesterase inhibitors. The results show that in no case d-tubocurarine appeared to reduce the ACh release. Thus, our result confirms the earlier finding of DALE et al. (1936) but is contradictory to that of BEANI et al. (1964) although essentially similar procedure was followed. In contrast, Hemicholinium-3 reduced the ACh output to less than 20 per cent of control levels. Nerve terminal spikes of rat diaphragm preparations recorded with extracellular microelectrodes were not affected by d-tubocurarine whether with single or repetitive stimulations. The size of successive end-plate potentials evoked by repetitive stimulation at 100 cps in the preparation treated with Hemicholinium-3 declined more rapidly than that of curarized muscle. It is concluded that d-tubocurarine does not influence the presynaptic event of neuromuscular transmission even at high 514 C. C. CHANG, H. C. CHENG AND T. F. CHEN frequency of stimulation and that the Wedensky inhibition in curarized muscles is not due to a pharmacological effect of d-tubocurarine.

The authors wish to thank Dr. J. M. BARNES of the Medical Research Council of England for the donation of Mipafox and Merck, Sharp and Dohme Research Labora- tories for DFP.

REFERENCES

BEANI, L. BIANCHI, C. AND LEDDA, F.: The effect of tubocurarine on acetylcholine release from motor nerve terminals. J. Physiol. (Lond.) 174: 172-183, 1964. CHANG, C. C. AND LEE, C. Y.: Cholinesterase and anticholinesterase activities in snake venoms. J. Formosan med. Assoc. 54: 103-112, 1955. CHANG, C. C. AND LEEC. Y.: Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action. Arch. int. Phar- macodyn. 144: 241-257, 1963. CHANG, C. C., CHEN, T. F. AND CHENG, H. C.: On the mechanism of neuromuscular blocking action of bretylium and guanethidine. J. Pharmac. exp. Ther. 158: in press, 1967. CHEYMOL, J., BEURILLET, F. AND OGURA, Y.: Action de quelques paralysants neuro- musculaires sur la liberation de l'acetylcholine an niveau des terminaisons nerve- nses mortices. Arch. int. Pharmacodyn. 139: 187, 197, 1962. DALE, H. H., FELDBERG, W. AND VOGT, M.: Release of acetylcholine at voluntary motor nerve endings. J. Physiol. (Lond.) 86: 353-380, 1936. FENG, T. P. AND LI, T. H.: Studies on neuromuscular junction. XXIII A new aspect of the phenomena of eserine potentiation and posttetanic facilitation in mammalian muscles. Chin. J. Physiol. 16: 37-50, 1941. HUBBARD, J. I.: Repetitive stimulation at the mammalian neuromuscular junction, and the mobilization of transmitter. J. Physiol. (Lond.) 169: 641-662, 1963. HUBBARD, J. I. AND SCHMIDT, R. F.: An electrophysiological investigation of mammalian motor nerve terminals. J. Physiol. (Lond.) 166: 145-167, 1963. HUBBARD, J. I., SCHMIDT, R. F. AND YOKOTA, T.: The effect of acetylcholine upon mammalian motor nerve terminals. J. Physiol. (Lond.) 181: 810-829, 1965. JOHNSON, E. S.: A note on the relation between the resting release of acetylcholine and increase in tone of the isolated guinea-pig ileum. J. Pharmacy Pharmac. 15: 69-72, 1963. KATZ, B. AND MILEDI, R.: Propagation of electric activity in motor nerve terminals. Proc. R. Soc. B. 161: 453-482, 1965. KRNJEVIC:, K. AND MITCHELL, J. F.: The release of acetylcholine in the isolated rat diaphragm. J. Physiol. (Lond.) 155: 246-262, 1961. LILEY, A. W. AND NORTH, K. A. K.: An electrical investigation of repetitive stimulation on mammalian neuromuscular junction. J. Neurophysiol. 16: 509-527, 1953. LUNDBERG, A. AND QUILISCH, H.: Presynaptic potentiation and depression of neuromuscular transmission in frog and rat. Acta Physiol. Scand. 30: Suppl. 111, 111-120, 1953. OTSUKA, M., ENDO, M. AND NONOMURA, Y.: Presynthetic nature of neuromuscular depression. Jap. J. Physiol. 12: 573-584, 1962. RANDI, M. AND STRAUGIIAN, D. M.: Antidromic activity in the rat phrenic nerve- diaphragm praparation. J. Physiol. (Lond.) 173: 130-148, 1964. RIKER, W. F., WERNER, G., ROBERTS, J. AND KUPERMAN, A.: The presynaptic element CURARE AND ACh RELEASE 515

in neuromuscular transmission. Ann. N. Y. Acad. Sci. 81: 328-344, 1959. ROSENBERG, P.: Reversible block of axonal conduction by curare after treatment with cobra venom. Biochem. Pharmac. 8: 192-206, 1961. ROSENBERG, P. AND EHRENPREIS, A.: Reversible block of axonal conduction by curare after treatment with cobra venom and a detergent. Nature (Lond.) 190: 728-729, 1961. STANDAERT, F. G.: The action of d-tubocurarine on the motor nerve terminal. J. Pharmac. exp. Ther. 143: 181-186, 1964. THIES, R. F.: Neuromuscular depression and the apparent depletion of transmitter in mammalian muscle. J. Neurophysiol. 28: 427-442, 1965. WALSH, R. R. AND DEAL, S. F.: Reversible conduction block produced by lipid-insoluble quaternary ammonium ions in cetyltrimethyl ammonium bromide-treated nerves. Am. J. Physiol. 197: 547-550, 1959.