Inhibitory Actions of Vecuronium Bromide on Acetylcholine and Glutamate Responses at the Frog and Crayfish Neuromuscular Junction Haruhiko SHINOZAKI and Michiko ISHIDA The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan Accepted November 7, 1983 Abstract-Effects of vecuronium bromide, an analog of pancuronium, on the cholinergic and glutamatergic neuromuscular junction were investigated. Vecuronium depressed the postsynaptic response of the frog end-plate at lower concentrations than 10-6 g/ml without affecting the presynaptic events. Vecuronium decreased the amplitude of the double ACh potential, but the second potential was more markedly reduced than the first. In analogy with d-tubocurarine, this suggests that vecuronium may act in part as an open channel blocker at the frog end-plate. Vecuro nium depressed both the glutamate response and the excitatory junctional potential at the crayfish neuromuscular junction, although high concentrations were required. The drug increased the decay rate of extracellularly recorded excitatory functional potentials at the crayfish neuromuscular junction. The reduction of the crayfish synaptic response caused by vecuronium can be explained by the open channel blocking action at this functional site. The problem that cholinergic antagonists possess a property of channel blocking at the other transmitter system was discussed. Vecuronium is an analog of pancuronium, The neuromuscular blocking action of pan but is not the bis-quarternary ammonium, one curonium has been well documented (2). quarternary moiety of pancuronium being The comparison of pharmacological activities replaced by a tertiary amine (Fig. 11. Although of vecuronium to pancuronium was already it is known that replacement of one quar done in some earlier studies (3-6), and ternary moiety of the bis-quarternary am the effectiveness of both drugs is almost monium structure by a primary amine group the same, however, electrophysiological results in a considerable loss of potency (1), studies are lacking. In the present study, the vecuronium possesses a powerful neuro inhibitory action of vecuronium on the frog muscular blocking action. Its principal value end-plate potential was investigated using an is expected to be as an adjunct to anaesthesia. electropharm 3cological technique. In the last decade, electrophysiological experimentation has revealed the electrical event associated with the opening and closing of the individual receptor channels associated with activation by an agonist. This was achieved by statistical analysis of membrane potential or conductance fluctu ations during continuous administration of Fig. 1. Chemical structure of vecuronium bromide: agonist (7, 8) and more recently, by direct 1-[(2~, 3a, 5a, 16/3, 17j3-3,17-bis(acetyloxy)-2 recording from a single receptor channel with (1 -piperidinyl)-androstane-1 6-yl]-1 -methyl a suction electrode on denervated skeletal piperidinium bromide. muscle (9). Since the rate of decay of synaptically evoked currents is thought to microelectrode to record the end-plate current reflect the average channel open time, (e.p.c.). The clamp method was similar to measurement of the end-plate current (e.p.c.) that described by Takeuchi and Takeuchi (21). is useful for elucidating agonist-receptor In order to measure the input resistance of kinetics in the presence of an antagonist (8, the muscle fiber, two intracellular micro 10-12). Recently, some competitive neuro electrodes were inserted separately into the muscular blockades and ganglion blocking middle of a muscle fiber less than 50 icm agents were found to act in part as an open apart, one for recording and the other for channel blocker at the vertebrate cholinergic passing hyperpolarizing current. In an experi nicotinic system (13-15) and to depress the ment, ACh was injected electrophoretically glutamate response as well at the insect (16, by applying a positive pulse to a micropipette 17) and crayfish neuromuscular junctions containing 1 M ACh (>100 MSS). Test (18, 19). It is of great interest that cholinergic samples were administered by bath antagonists possess a property of channel application. blocking at the other transmitter systems. Crayfish experiment: The methods used The crayfish neuromuscular junction provides were essentially similar to those reported an excellent model for studying the mecha previously (18). nism of action of drugs on synaptic trans The least squares procedures were mission. In the present study, whether performed with a desk-top computer (YH P vecuronium acted as an open channel 9845B). Experiments were made at a constant blocker at the crayfish neuromuscular junction bath temperature of about 22'C. The results or not was also examined by analyzing the are presented as the mean values±S.E.M. time course of the e.j.c. for n experiments. Differences were analyzed using Student's t-test to determine significant Materials and Methods differences. Drugs used: Acetylcholine chloride (ACh, Daiich Seiyaku), vecuronium bromide (Nip Results pon Organon), pancuronium bromide (San Vecuronium depresses the postsynaptic kyo) and monosodium L-glutamate (Wako response of the frog end-plate without Junyaku). affecting the presynaptic events: When the Frog experiment: The methods used were muscle fiber of the frog was treated with similar to those described in earlier papers vecuronium bromide (2 x 10-6 g/ml), the (20). The nerve -sartorius preparation of the resting membrane potential (-89.5±1.3 mV, frog (Rana catesbeiana) was used, immersed n=6) was not affected by the drug. Vecuro in an isotonic solution containing (mM) nium at a concentration of 2 x 10-6 g/ml did NaCI, 113; KCI, 2; CaCl2, 1.8; NaHCO3, 1 not produce a detectable change in amplitude and glucose, 2.8 (pH 6.9). In some experi of the electrotonic potential evoked by ments, the bathing medium was modified constant and hyperpolarizing current pulses according to the nature of the experiment. A (200 msec, 7 nA), suggesting that the drug nerve bundle to the muscle was exposed and did not affect the membrane resistance. After stimulated with a suction electrode to record the saline had been replaced by the one the end-plate potential (e.p.p.). Potential containing vecuronium (2x`10-6 g/ml), the changes of the muscle membrane were amplitude of the spontaneous miniature end recorded with a 3 M KCI-filled microelectrode. plate potential (m.e.p.p.) was markedly The usual procedure was to locate a suitable reduced, and the spontaneous m.e.p.p. com spot with the internal electrode and record pletely disappeared at higher concentrations spontaneous potentials on moving film. The of vecuronium, but recovery was complete amplitude of the potentials were measured, and very prompt after washing the pre and their distribution displayed in a histogram. paration. Figure 2 shows records of spon In some experiments, the membrane potential taneous m.e.p.ps in the absence and presence of muscle fibers was clamped at the resting of vecuronium (10-6 g/ml). Figure 3 re membrane potential with an intracellular presents histograms of the amplitude distri bution of the spontaneous m.e.p.p. in different concentrations of vecuronium. The values of the mean amplitude of spontaneous m.e.p.ps were lowered as the concentration of vecuronium increased. On the other hand, the frequency of spontaneous m.e.p.ps was not affected by the drug, even when the concen tration of vecuronium was increased up to 2x10-6 mg/ml (Table 1). When the nerve-muscle preparation was immersed in an isotonic solution containing of CaCl2 and MgCl2, adjusted so as to reduce the amplitude of e.p.ps to any desired level, vecuronium depressed the amplitude of the e.p.p. to about a half of the control at the concentration of 10-6 g/ ml (Fig. 4). When various concentrations of vecuronium were added to the bathing medium, the amplitude of the e.p.p. was reduced in a dose-dependent Fig. 3. Distribution of amplitudes of spontaneous m.e.p.ps in the absence and presence of vecuronium bromide at various concentrations. A: in normal Ringer, B: after addition of 10-6 g/ml vecuronium bromide, C: 2X10-6 g/ml. The resting membrane Fig. 2. Effect of vecuronium bromide on the size potential was -89 mV. The mean values of ampli of the m.e.p.p. The resting membrane potential was tudes of spontaneous m.e.p.ps in A, B and C are -89 mV . A: in normal Ringer, B: after addition of 0.39 mV ±0.01 mV, 0.227 mV +0.004 mV and 10-6 g/ml vecuronium bromide. The size of the 0.143 mV ±0.001 mV, respectively. Numbers of m.e.p.p. was reduced by vecuronium bromide without observations in A, B and C are 442, 409 and 438 for affecting its frequency. a time of 148 sec, respectively. Table 1. Effects of vecuronium bromide on spontaneous m .e.p.ps Fig. 5. A: Effect of vecuronium bromide on the successively induced ACh potentials. Successive ACh potentials were induced by a train pulse at a Fig. 4. Effect of vecuronium bromide on the e.p.p. interval of 50 msec. Resting potential: -94 mV. a: in and the ACh potential. A: e.p.p. B: ACh potential. normal Ringer, b: in the presence of 10-6 g/ml Upper traces: monitored injection currents. Lower vecuronium bromide. Upper traces: monitored traces: responses. a: control, b: after addition of it jection currents. Lower traces: responses. B: A 10-6 g/ml vecuronium. In A, the concentrations of slight increase in the decay rate of the e.p.c. a: Ca2+ and Mg2+ were adjusted to 0.9 mM and 5 mM, control, b: after addition of 2 X10-6 g/ml vecuronium. respectively. Each e.p.p. shown in the records is the The concentrations of Ca2+ and Mg2+ were adjusted one whose amplitude is almost equivalent to the to 0.9 mM and 5 mM, respectively.