THE ACTION OF CHLORPHENESIN CARBAMATE ON THE FROG SPINAL CORD

Transcription

1 Japan. J. Pharmacol. 30, (1980) 29 THE ACTION OF CHLORPHENESIN CARBAMATE ON THE FROG SPINAL CORD Hironaka AIHARA, Michio KURACHI, Sadao NAKANE, Michitada SASAJIMA and Masahiro OHZEKI Research Laboratories, Taisho Pharmaceutical Co., Ltd., Yoshino-cho 1-403, Omiya, Saitama 330, Japan Accepted September 10, 1979 Abstract-Studies were carried out to elucidate the mechanism of action of chlorphenesin carbamate (CPC) and to compare the effect of the drug with that of mephenesin on the isolated bullfrog spinal cord. Ventral and dorsal root potentials were recorded by means of the sucrose-gap method. CPC caused marked hyperpolarizations and depressed spontaneous activities in both of the primary afferent terminals (PAT) and motoneurons (MN). These hyperpolarizations were observed even in high-mg" and Ca2+-free Ringer's solution, suggesting that CPC has direct actions on PAT and MN. Various reflex potentials (dorsal and ventral root potentials elicited by stimulating dorsal and ventral root, respectively) tended to be depressed by CPC as well as by mephenesin. Excitatory amino acids (L-aspartic acid and L-glutamic acid) caused marked depolarizations in PAT and MN, and increased the firing rate in MN. CPC did not modify the depolarization but abolished the motoneuron firing induced by these amino acids. However, mephenesin reduced both the depolarization and the motoneuron firing. The dorsal and ventral root potentials evoked by tetanic stimulation (40 Hz) of the dorsal root were depressed by the drugs. These results indicate that CPC has an apparent depressing action on the spinal neuron, and this action may be ascribed to the slight hyperpolarization and/or the prolongation of refractory period. The skeletal muscle relaxant actions of mephenesin have been attributed to its depressant action on interneurons of polysynaptic reflex arcs in the spinal cord and at supraspinal levels (1, 2). Chlorphenesin carbamate (CPC), a drug structurally related to mephenesin (Fig. 1), has been reported to be a selective blocker of polysynaptic pathways at the spinal and supraspinal level (3). However, there are reports indicating that mephenesin and CPC depress monosynaptic as well as polysynaptic reflex (4, 5). Furthermore, these drugs were reported to have no effect on the arousal response evoked through polysynaptic pathways (6). Thus, it is unlikely that these drugs are selective blockers of polysynaptic pathways. As there is little direct evidence for specific actions of these drugs on spinal interneurons, FIG. 1. Chemical structures of CPC and mephenesin.

2 30 H. AIHARA ET AL. we studied the effect of CPC on the frog spinal cord in parallel with that of mephenesin. The mechanism of muscle relaxant activities of these drugs is also discussed. MATERIALS AND METHODS Bullfrog (Rana catesbeiana) was cooled in ice to an anaesthetic state, and the spinal cord with 9th or 10th ventral and dorsal roots was carefully isolated. As soon as possible, a glass cannula was inserted into the anterior spinal artery and the spinal cord was continuously perfused with oxygenated Ringer's solution (ph , Ž, perfusion velocity; 0.3 ml/min) consisted of NaCl120 mm, KCl2.5 mm, CaCl2 1.8 mm, Tris (hydroxymethyl) aminomethane 1 mm and glucose 5.6 mm. In some experiments, 12 mm MgCl2 was added to and CaCl2 was deleted from Ringer's solution. The potential changes occurring at the dorsal root nerve terminals and at the motoneurons in the spinal cord were recorded by means of the sucrose-gap method, the procedure being much the same as that reported by Kudo et al. (7). A schematic drawing of the experimental arrangement is shown in Fig. 2. Potential differences between the spinal cord and the peripheral stumps of ventral root or dorsal root were detected by calomel electrodes and then amplified by a DC-amplifiers (San-ei Sokki 6L5), and the outputs were connected with a two pen DC-recorder (Tohshin). In some experiments, the rate of discharges from the ventral root was recorded by a meter of our own design. Stimuli were delivered to the appropriate root (Nihonkohden MSE-3R) via bipolar platinum wires. All drugs were dissolved in Ringer's solution, and applied by means of exchanging the perfusate for the drug containing Ringer's solution or the injection of the drug containing Ringer's solution into the polyethylene tube (Fig. 2). Drugs used were L-glutamate monosodium salt (Wako), strychnine HNO3 (Sanko), picrotoxin (Tokyo Kasei), mephenesin (Sigma) and chlorphenesin carbamate (Taisho Pharm.). The following abbreviations were used; DR-DRP, the dorsal root potential induced by the stimulation of the dorsal root; VR-DRP, the dorsal root potential induced by the stimulation of the ventral root; DR-VRP, the ventral root potential induced by the stimu- FIG. 2. Diagram of sucrose-gap method for recording the potential changes occurring at the dorsal root nerve terminals and motoneurons in the isolated perfused spinal cord of the bullfrog. DR: dorsal root, VR: ventral root, S: stimulater, c.e.: calomel electrode.

3 CHLORPHENESIN CARBAMATE AND SPINAL CORD 31 lation of the dorsal root. RESULTS Effects on potential changes of the dorsal and ventral roots: When 0.3 ml of CPC (10-3 M) was applied to the spinal cord through a polyethylene tube inserted into the cannula (injection velocity; 30 sec/0.3 ml), a hyperpolarization was produced in the dorsal and the ventral roots (Fig. 3A). The slow spontaneous depolarizations in the dorsal and the ventral roots were diminished and the spontaneous discharges (not illustrated) in the ventral root were abolished by the injection of CPC. The rates of discharge in the ventral root caused by the stimulation of dorsal root were reduced by the application of CPC. Reflex potentials (DR-VRP, DR-DRP and VR-DRP) were depressed slightly by a one shot application of CPC (10-3 M, 0.3 ml, Fig. 3B) and were reduced markedly by the continuous application of CPC (not illustrated). A similar effect was seen with mephenesin (Fig. 3C). These effects were reversible. Effects of CPC in the high-mg2+ and Ca2+-free Ringer's solution: Exposure of the spinal cord to a drug solution could affect the dorsal and the ventral roots indirectly by activating pathways which synapse onto those neurons. To determine the indirect synaptic effects, the responses obtained in a high-mg2+ and Ca2+-free Ringer's solution were compared A B C FIG. 3. Effects of CPC and mephenesin on the DRPs and VRPs. A: spontaneous occurring potentials. B and C: the potentials elicited by stimulating dorsal or ventral root, and indicate the effects of CPC (10-3 M) and mephenesin (10-3 M), respectively. Filled circles indicate the injection of drugs.

4 32 H. AIHARA ET AL. with responses in normal Ringer's solution. Firstly, it was confirmed that the addition of MgCl2 to and the deletion of CaCl2 from Ringer's solution resulted in the disappearance of spontaneous activity and electrically evoked potentials. The hyperpolarization of dorsal and ventral roots by CPC occurred consistently in the high-mg2+ and Cas+-free Ringer's solution (Fig. 4), suggesting a direct effect of this drug on dorsal and ventral roots. Effects of strychnine and picrotoxin on the CPC hyperpolarization: Mimicking the FIG. 4. Effect of high-mg2+ and Ca2+-free Ringer's solution on the CPC response. Left and right traces represent CPC responses in Ringer's solution and high-mg2+ and Ca2+-free Ringer's solution, respectively. A B FIG. 5. Effects of strychnine and picrotoxin on CPC hyperpolarization. A: CPC hyperpolarization was unaffected by strychnine (10-4 M). B: CPC hyperpolarization was augmented by the application of picrotoxin (10-4 M).

5 CHLORPHENESIN CARBAMA TE AND SPINAL CORD 33 action of inhibitory amino acids could produce hyperpolarization in the dorsal and ventral roots. Thus, we attempted to determine whether CPC hyperpolarization was blocked by strychnine or picrotoxin, antagonists of inhibitory amino acids (8-12). The addition of strychnine (10-4 M) to the Ringer's solution had no effect on the CPC hyperpolarization (Fig. 5A), while picrotoxin (10-4 M) augmented CPC hyperpolarization (Fig. 5B). Accordingly, it was concluded that CPC did not mimick the action of inhibitory amino acids. Effects of CPC on L-glutamate responses: Acidic amino acids, L-glutamate and L- aspartate are considered to be candidates for excitatory neurotransmitters in the vertebrate central nervous system. Therefore, the application of drugs antagonizing these amino acids might result in the depression at the central nervous system. When L-glutamate (10-s M, 0.3 ml) or L-aspartate (10-3 M, 0.3 ml, not illustrated) was applied to the spinal cord, depolarizing potentials were recorded in the dorsal and the ventral roots, and the burst of discharges in the ventral root was observed. It was thus confirmed that repetitive applications of these acidic amino acids resulted in responses of identical size and constant firing rates. As shown in Fig. 6A, continuous application (for 10 min) of CPC (10-s M) did not affect the depolarization induced by L-glutamate, while occasionally a longer application (for 40 min) of CPC did affect it. The bursts in ventral root evoked by amino acid were abolished, despite the same size in depolarization seen in the control. On the other hand, mephenesin (10-3 M) slightly depressed the depolarization caused by L-glutamate in the dorsal and the ventral roots, and completely blocked the burst in the ventral root by L-glutamate (Fig. 6B). Effects of CPC on the potentials evoked by multiple stimuli: As the centrally acting muscle relaxants may depress the repetitive activity in motoneurons arising in muscle tonus, we investigated the effects of CPC on the potentials evoked by multiple stimuli. When multiple stimuli (pulse duration; 0.05 msec, frequency; 40 Hz, intensity; 4V, for 5 sec) were A B FIG. 6. Effects of CPC and mephenesin on L-glutamate responses. A: CPC (10-3 M). B: mephenesin (10-3 M). a: rate of discharges in motoneuron. 1 and 2: before and after perfusion with CPC or mephenesin, respectively. 3: the recovery in Ringer's solution. Note disappearance of discharge rates (a2) after perfusion with drugs.

6 34 H. AIHARA ET AL. A B FIG. 7. Effects of CPC and mephenesin on the potentials evoked by tetanic stimuli for dorsal root. A: CPC (10-3 M). B: mephenesin (10-3 M). Tetanic stimuli: pulse duration; 0.05 msec, frequency; 40 Hz, intensity; 4V, for 5 sec. Note that declination is increased after application of drugs. delivered to the dorsal root, the potential changes shown in Fig. 7 were observed in the dorsal and ventral roots. Usually, these potentials reached a peak immediately after the onset of stimulation, and were maintained at much the same levels or were reduced slightly during the delivery of stimulation. When CPC (10-8 M) or mephenesin (10-3 M) was applied continuously to the spinal cord, the peak potentials were diminished, and the evoked potentials, especially in VRPs, declined during multiple stimuli. DISCUSSION CPC produced a depressant action on the spinal activity in in vitro preparations, that is, caused hyperpolarization and depressed markedly the spontaneous occurring potentials in the dorsal and ventral roots. This CPC hyperpolarization was observed consistently even after synaptic transmission was blocked by the application of high-mg2+ and Ca2+-free Ringer's solution to the spinal cord. Thus, it would appear that CPC has a direct action on primary afferent nerve terminals and motoneurons. Reflex potentials (DR-DRP, VR- DRP and DR-VRP) were depressed by application of the drugs, particularly in higher concentrations. Mephenesin also had a depressing action on the reflex potentials and such actions are probably due to the hyperpolarization of the membranes. Hyperpolarizing action of CPC was not antagonized by strychnine or picrotoxin, suggesting that there is no interaction between CPC and inhibitory neurotransmitters. When picrotoxin was applied to the spinal cord, convulsive and oscillatory potential changes were usually observed in both dorsal and ventral roots. These potential changes which suggest clonic convulsion, disappeared completely during CPC hyperpolarization. This

7 CHLORPHENESIN CARI3AMATE AND SPINAL CORD 35 may support findings of the effective action of CPC against picrotoxin (6). Slight augmentation of the hyperpolarizing action of CPC during application of picrotoxin may be ascribed to the small depolarizations induced by picrotoxin in the dorsal and the ventral roots. CPC did not affect L-glutamate and L-aspartate evoked depolarizations but did abolish the firing of motoneurons. Therefore, the ions related to CPC hyperpolarization are probably different from those concerned with depolarizations by excitatory amino acids, and here slight hyperpolarization by CPC results in the abolition of neuron firing. If such is indeed the case, it may be that CPC and mephenesin depress the polysynaptic reflex which passes through plural neurons rather than the monosynaptic reflex. Mephenesin reduced the depolarization induced by L-glutamate and a similar effect has also been reported by Shirasawa and Koketsu (13). However, at present we have no adequate explanation for the difference between the actions of these muscle relaxants on L-glutamate induced depolarization. Prolonged application of CPC and also mephenesin diminished the peak potentials and declined the evoked potentials, especially in VRPs, induced by the delivery of multiple stimuli. Since mephenesin prolongs the refractory period of skeletal muscle (14, 15), the effect of CPC and mephenesin on the potential changes evoked by the multiple stimuli is perhaps due to the prolongation of the refractory period. Furthermore, this effect may explain why CPC and mephenesin predominantly depress polysynaptic pathways. CPC had a depressant action on spinal neurons and this action can probably be ascribed to the slight hyperpolarization and/or the prolongation of the refractory period. Accordingly, this probably explains why CPC depresses the polysynaptic reflex having a plural transmission. Acknowledgement: We thank Dr. I. Tanaka, Director of Research Laboratories, Taisho Pharmaceutical Co., Ltd., for encouragement throughout the study. REFERENCES 1) KING, E.E. AND UNNA, K.R.: The action of mephenesin and other interneuron depressants on the brain stem. J. Pharmacol. exp. Ther. 111, (1954) 2) BHARGAVA, K.P. AND SRIVASTAVA, P.K. Antitetanus activity of central muscle relaxants. Brit. J. Pharmacol. 25, (1965) 3) MATTHEWS, R.J., DAVANZO, J.P., COLLINS, R.J. AND VANDER BROOK, Mi.: The pharmacology of chlorphenesin carbamate, a centrally active muscle relaxant. Archs int. pharmacodyn. Then 143, (1963) 4) CRANKSHAW, D.P. AND RAPER, C.: Mephenesin, methocarbamol, chlordiazepoxide and diazepam: actions on spinal reflexes and ventral root potentials. Brit. J. Pharmacol. 38, (1970) 5) FUKUDA, H., KUDO, Y., ONO, H. AND KOKUBO, M.: Pharmacological study on a centrally acting muscle relaxant (chlorphenesin carbamate) with special reference to the effects on motor systems. Folia pharmacol. japon. 70, (1974) (Abs. in English) 6) KING, E.E.: Differential action of anesthetics and interneuron depressants upon EEG arousal and recruitment responses. J. Pharmacol. exp. Ther. 116, (1956) 7) KUDO, Y., ABE, N., GOTO, S. AND FUKUDA, H.: The chloride-dependent depression by GABA in the frog spinal cord. Europ. J. Pharmacol. 32, (1975) 8) DAVIDOFF, R.A., APRISON, M.H. AND WERMAN, R.: The effects of strychnine on the inhibition of interneurons by glycine and ć-aminobutyric acid. Int. J. Neuropharmacol.

8 36 H. AIHARA ET AL. 8, (1969) 9) BARKER, J.L. AND NICOLL, R.A.: The pharmacology and ionic dependency of amino acid responses in the frog spinal cord. J. Physiol. 228, (1973) 10) BARKER, J.L. AND NICOLL, R.A.: Gamma-aminobutyric acid: Role in primary afferent depolarization. Science 176, (1972) 11) BARKER, J.L., NICOLL, R.A. AND PADJAEN, A.: Studies on convulsants in the isolated frog spinal cord. 1. Antagonism of amino acid responses. J. Physiol. 245, (1975) 12) NICOLL, R.A., PADJEN, A. AND BARKER, J.L.: Analysis of amino acid responses on frog motoneurons. Neuropharmacology 15, (1976) 13) SHIRASAWA, Y. AND KOKETSU, K.: Action of 5-hydroxytryptamine on isolated spinal cord of bullfrogs. Japan. J. Pharmacol. 27, (1977) 14) ROSENBERG, F.J. AND COOKE, W.J.: A peripheral component of centrally acting muscle relaxants: chlormezanone and mephenesin. J. Pharmacol. exp. Ther. 155, (1967) 15) CRANKSHAW, D.P. AND RAPER, C.: Some studies on peripheral actions of mephenesin, methocarbamol and diazepam. Brit. J. Pharmacol. 34, (1968)