Effects of ganglionic blocking agents on the neuromuscular junction

EUROPEAN JOURNAL OF PHARMACOLOGY 12 (1970) 342-347. NORTH-HOLLANDPUBLISHING COMPANY

EFFECTS OF GANGLIONIC BLOCKING AGENTS ON THE NEUROMUSCULAR

JUNCTION

C.Y. CHIOU Department of Pharmacology and Therapeutics, University o f Florida, College o f Medicine, Gainesville, Florida, 32601, U.S.A.

Accepted 29 June 1970

Received 9 June 1970

C.Y. Chiou, Effects o f ganglionic blocking agents on the neuromuscular ]unction, European J. Pharmacol. 12 (1970) 342-347. Effects of ganglionic blocking agents on neuromuscular transmission and on responses of nicotinic agents have been studied using the baby chick biventer cervicis nerve muscle preparation. Responses of nicotinic agents such as nicotine, tetramethylammonium, dimethylphenylpiperazinium and carbachol were markedly inhibited by 10 mg % of hexamethonium (C6) and 100 mg % of tetraethylammonium (TEA) and almost completely abolished by 100 mg % of C6 and 1 g % of TEA. However, responses induced by ACh were not significantly affected by these ganglionic blocking agents, indicating that nicotinic agents were blocked by these ganglionic blocking agents at a site other than the ACh receptor, probably at the motor nerve endings. Neuromuscular transmission was also blocked by C6 and TEA without affecting ACh responses suggesting that C6 and TEA might block the uptake of choline by nerve tissue across the membrane and thus inhibit ACh synthesis and thereby block neuromuscular transmission. Initial facilitation and concomitant contraction of the biventer cervicis muscle preceded neuromuscular blockade induced by TEA indicating that part of the TEA effect might be due to the release of endogenous ACh. Ganglionic blocking agents Nicotonic agents Autonomic ganglia Hexamethonium

Dimethylphenylpiperazinium Carbachol Neuromuscular junction

1. INTRODUCTION It is generally held that nicotinic agents produce their effects via direct interaction with nicotinic receptors which are blocked by hexamethonium (C6) at autonomic ganglia and d-tubocurarine (d-Tc) at neuromuscular junctions. However, recent findings indicated that most nicotinic agents act indirectly through release of acetylcholine (ACh) from storage sites of the nerve terminals. They are taken into intracellular sites of the nerve endings, releasing endogenous ACh, and are blocked by ganglionic blocking agents at the membrane site of the nerve terminals

Chick biventer cervicis Acetylcholine receptor Tetramethylammonium

(Chiou and Long, 1969a, 1969b; Chiou, 1970; Chiou et al., 1970). If this is true, then ganglionic blocking agents should be capable of (a) blocking nicotinic agents at nerve terminals of preganglionic fibers and those of motor nerves and (b) blocking neuromuscular transmission through blockade of the uptake of choline by nerve endings inhibiting ACh synthesis. On the other hand, they should not block the effect of ACh which is k n o w n to interact with the receptor site at the postjunctional membrane. The baby chick biventer cervicis nerve muscle preparation was used to test this hypothesis. The

C Y. Chiou, Effects of ganglionic blocking agents

results obtained in this study met all the criteria stated above and supported this new hypothesis.

2. METHODS Baby chicks ( 1 5 0 - 2 5 0 g ) were sacrificed with chloroform. The biventer cervicis nerve muscle preparation was dissected and mounted in an organ bath of 40 ml capacity (Chiou, 1970; Long and Chiou, 1970). The Tyrode solution (NaC1, 8.0; KC1, 0.2; CaClz, 0.2; MgC12, 0.1; NaHCO3, 1.0; NaHPO4,0.05; and dextrose, 2.0 g/l) was oxygenated with 95% O : - 5 % CO 2 at 37°C. The tendon with motor nerve was passed through a hole of an electrode and attached to an isometric force transducer (Myograph B, E & M Inst. Co., Houston, Texas). The tension developed by contraction of the muscle was measured in grams and recorded on a physiograph (Four A, E & M Inst. Co.). Twelve single shocks per min of 5 msec duration and 2 0 - 4 0 V were applied to the nerve from a stimulator (E & M Inst. Co.). Drug solutions were freshly prepared in distilled water. A wide range of doses for C6 'and tetraethylammonium (TEA) were administered to determine the minimum dose required to block completely the neuromuscular transmission within 15 rain. The drug was washed away after 15 min of interaction with the neuromuscular preparation to determine whether or not the neuromuscular blockade was reversible. ACh (100/ag/ml) was administered before and after the neuromuscular blockade to determine the effect of these ganglionic blocking agents on ACh responses. A superfusion technique was also used to study the dose-response curves of drug responses on the chick biventer cervicis nerve muscle preparation. The isolated preparation was mounted on a superfusion assembly described previously (Chiou and Long, 1969a; Long and Chiou, 1970). The preparation was superfused with Tyrode solution oxygenated with 95% 0 2 - 5 % CO2 at 37°C. The rate of flow of superfusion fluid was 3 - 4 ml/min maintained by a Polystalic pump (Buchler, Fort Lee, N.J.). Drug solutions of not more than 0.1 ml were injected into the stream of the superfusion fluid. Contractions of the muscle were measured and recorded as described above. The dose-response curves were determined before and after the treatment of the preparation

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with 10 mg % and 100 mg % of C6 and 100 mg % and 1 g % of TEA. The percentage response to drugs calculated using the maximum response to ACh as 10(~o was plotted against log dose of the drugs injected. The drugs studied included acetylcholine iodide (ACh), nicotine tartrate, tetramethylammonium bromide (TMA), 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), carbachol chloride, hexamethodium chloride (C6) and tetraethylammonium iodide (TEA).

3. RESULTS 3.1. Effects of C6 and TEA on responses induced by ACh The dose-response curves of ACh-induced contractions of the biventer cervicis muscle were not significantly altered by 10 min % and 100 mg % of C6 (fig. 1) nor by 100 mg % of TEA (fig. 2). Although responses induced by low concentrations of ACh were slightly inhibited by 1 g% of TEA, those induced by high concentrations of ACh were not affected by the same concentration of TEA (fig. 2). These results indicate that ganglionic blocking agents such as C6 and TEA do not block ACh at the receptor site of neuromuscular junctions. IOO-

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Fig. 1. Effects of hexamethonium (C6) on the dose-response curve of acetylcholine (ACh)-induced contractions of chick biventer cervicis muscle. ~-------o: control responses; ~ : responses after 10rag % of C6; <9 o: responses after I 0 0 mR % of C 6. Each point is a mean of 4 values and bars represent standard errors. Note the dose-response curve o f ACh was n o t significantly affected by C6 up to 100 mg %.

C.Y.Chiou, Effects of ganglionic blocking agents

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into nerve endings, which leads to inhibition of ACh synthesis and blockade of neuromuscular transmission (Chiou and Long, 1969a).

3.2. Effects o f C6 and TEA on the neuromuscular transmission

3.3. Effects o f C 6 and TEA on responses induced by nicotinic agents

Neuromuscular transmission elicited by electrical stimulation was blocked partially by 10 mg % of C6 and 100 mg % of TEA and completely by 100 mg % of C6 and 1 g % of TEA within 15 min after these ganglionic blocking agents were added to the organ bath (figs. 3 and 4). There was no initial facilitation nor was the base line elevated when C6 was added into the organ bath. However, initial facilitation of the fast muscle and concomitant contraction of the slow muscle of biventer cervicis proceded the neuromuscular blockade to TEA. This is probably due to the release of endogenous ACh by TEA but not due to the direct effect on ACh receptor because TEA itself did not affect responses to ACh (fig. 5B). The site of neuromuscular blockade produced by C6 and TEA was not at the ACh receptor because ACh responses were not affected by these ganglionic blocking agents while the neuromuscular transmission was completely blocked (fig. 5). These results suggest that C6 and TEA act at a site other than ACh receptor of postjunctional membranes, probably at nerve terminals, by blocking the uptake of choline

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C. Y. Chiou, Effects of ganglionic blocking agents

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Fig. 5. Effects of hexamethonium (C6) (A) and tetraethylammonium (TEA) (B) on neuromuscular transmission and acetylcholine (ACh) responses. W, washing; C 6, 100 g %; TEA, 1 g %; ACH, 100 #g/ml. The nerve was stimulated with 12 single shocks per min of 5 msec and 2 0 - 4 0 V. Note ACh responses were not altered by C6 and TEA while the neuromuscular transmission was completely blocked by these ganglionic blocking agents.

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Fig. 6. Effects of hexamethonium (C6) on the dose-response curves of nicotine, tetramethylammonium (TMA), dimethylphenylpiperazinium (DMPP) and carbachol. H : control responses; ~ : responses after 10 mg % of C6; © ©: responses after 100 mg % of C6. Each point is a mean of 5 values and bars represent standard errors.

346

C.Y. Chiou, Effects o f ganglionic blocking agents

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report (Chiou and Long, 1969a). They were markedly inhibited by 10 mg % of C6 and 100 mg % of TEA (figs. 6 and 7). These responses were almost completely abolished by 100 mg % of C6 and 1 g % of TEA (figs. 6 and 7) which did not affect the dose response curves of ACh (figs. 1 and 2). These results support the idea that ganglionic blocking agents block the uptake of nicotinic agents at the nerve endings and thus block the indirect release of endogenous ACh induced by nicotinic agents (Chiou and Long, 1969a; 1969b; Chiou, 1970; Chiou et al., 1970).

4. DISCUSSION Conventionally, ganglionic blocking agents such as

C6 and TEA are thought to block the effects of nicotinic agents located on the postganglionic membrane. This concept was questioned recently by the author and others because most nicotinic agents act at nerve terminals to release ACh (Volle, 1962; McKinstry et al., 1963; Henderson et al., 1968; Takagi et al., 1968; Chiou and Long, 1969a, 1969b). If this is the case, the site where ganglionic blocking agents work to block the effects of nicotinic agents must be at the nerve terminals rather than at the postganglionic membrane. It has been suggested that most nicotinic agents are taken into the nerve tissue across the membrane where they release endogenous ACh. The released ACh is blocked by atropine at the postganglionic membrane and by d-Tc at the postjunctional mem-

C.Y.Chiou, Effects of ganglionic blocking agents

brane, whereas uptake of nicotinic agents is blocked by ganglionic blocking agents at membrane sites on nerve endings (Chiou and Long, 1969a; 1969b, Chiou et al., 1970). Since there is great structural and functional similarity between the preganglionic fiber and the motor nerve fiber, it seems highly probable that ganglionic blocking agents would block the effects of nicotinic agents at the neuromuscular junction as well. This concept was supported by the fact that responses of nicotine, TMA, DMPP and carbachol were markedly depressed or entirely abolished by C6 and TEA at concentration levels which did not affect responses to ACh. The results indicate that C6 and TEA act at a site other than ACh receptors, possibly at the membrane site of the motor nerve endings to block the uptake of nicotinic agents across the membrane, thereby blocking their effect. The same mechanism was proposed for the effect of triethylcholine by Chiou and Long (1969a). It is established that hemicholinium (HC-3)-like compounds are capable of inhibiting ACh synthesis in cholinergic neurons by competing with choline for the carrier mechanism responsible for the transport of extracellular choline to the intracellular site (Bowman et al., 1967). It has also been shown that choline and other nicotinic agents release endogenous ACh at autonomic ganglia and at neuromuscular junctions. This release is blocked by HC-3-1ike compounds, such as triethylcholine (Chiou and Long, 1969a; 1969b; Chiou et al., 1970). Since neuromuscular transmission was blocked by C6 and TEA at concentrations which blocked the effects of nicotinic agents without affecting ACh response, there is reason to believe that ganglionic blocking agents block uptake of choline, just as HC-3-1ike compounds do, to inhibit ACh synthesis and block neuromuscular transmission. It is concluded that ganglionic blocking agents block nicotinic effects not only at the autonomic ganglia but also at the neuromuscular junction. They do not act at ACh receptors located on the postjunctional membrane as classically believed. Rather, they

347

block nicotinic effects at nerve terminals of motor nerve fibers.

ACKNOWLEDGEMENTS The author would like to thank Mrs. J.Y. Huang for her excellent technical assistance. This work was supported in part by research grants from College of Medicine, University of Florida and NIH (GM 16934-01) U.S.A.

REFERENCES Bowman, W.C., B.A. Hemsworth and M.J. Rand, 1967, Effects of analogs of choline on neuromuscular transmission, Ann. N.Y. Acad. Sei. 144,471-482. Chiou, C.Y., 1970, Effects of halogenoacetylcholines on neuromuscular junction, European J. Pharmacol., submitted for publication. Chiou, C.Y. and J.P. Long, 1969a, Acetylcholine releasing effects of some nicotinic agents on chick biventer cervicis nerve muscle preparation. Proc. Soc. Expl. Biol. Med. 132, 732-737. Chiou, C.Y. and J.P. Long, 1969b, Studies of cholinergic inhibition using guinea-pigileum, Arch. Intern. Pharmacodyn. 182, 269-278., Chiou, C.Y., J2. Long, R.F. Potrepka and J.L. Sptatt, 1970, The ability of various nicotinic agents to release acetylcholine from synaptic vesicles, Arch. Intern. Pharmacodyn., in press. Henderson, P.Th., E.J. Ati~ns and A.M. Simons, 1968, Differentiation of various types of cholinergic and spasmogenic actions on the isolated guinea-pig ileum, European J. Pharmacol. 4, 62-70. Eong, J.P. and C.Y. Chiou, 1970, Pharmacological testing methods for drugs acting on peripheral nervous system, J. Pharm. Sci. 59, 133-148. McKinstry, D.N., E. Koenig, W.A. Koelle and G.B. Koelle, 1963, The release of acetylcholine from a sympathetic ganglion by carbachol, Can. J. Bioehem. Physiol. 41, 2599-2609. Takagi, K., I. Takayangi and Y. Maezima, 1968, An analysis of the sites of action of some partial agonists, European J. Pharmacol. 3, 52-57. Voile, R.L., 1962, Enhencement of postgengiionic responses to stimulating agents, following repetitive pregangiionie stimulation, J. Pharmacol. Expfl. Therap. 136, 68-74.