Anti-Botulismic Effect of Toosendanin and Its Facilitatory Action on Miniature End-Plate Potentials

Short Communication

Japanese Journal of Physiology, 33, 677-680,1983

Anti-botulismic Effect of Toosendanin and Its Facilitatory Action on Miniature End-plate Potentials

Yu-liang SHIN and Ke Hsu

Shanghai Institute of Physiology, Academia Sinica, 320 Yo-Yang Road, Shanghai, 200031 China

Summary The time for blocking neuromuscular transmission by botulinum toxin is lengthened in phrenic nerve-diaphragm preparation of rats sacrificed at various times after subcutaneous injection of toosendanin (7 mg/kg). This effect of toosendanin manifests itself 15 min after injection and lasts for 3 days. During this period the frequency of miniature end-plate potentials is increased.

Key Words: toosendanin, botulinum toxin miniature end-plate potential.

It is well known that botulinum toxin (BoTX)is a potent poison for vertebrates due to its presynaptic blocking action on the neuromuscularjunction of skeletal muscles. For a long time investigatorshave searchedfor effectiveantidotes against BoTX but without much success. Both CHANGet al. (1973b, 1974) and STERN et al. (1975) found a moderate antagonism between BoTX and some other presynaptic blockers, i. e. 13-bungarotoxin(13-BuTX) and black widow spider venom (BWSV). Although the mechanism of this antagonism is still unknown, it is in- teresting to note that both ~3-BuTX(LEE and CHANG,1966; CHANGet al., 1973a) and BWSV(LoNGENECKER et al., 1970; CLARKet al., 1972)increase the frequency of miniature end-plate potentials (MEPP) in the initial phase of their action, whereas BoTX decreases it immediately. We found that toosendanin, a triterpenoid derivative (C30H33011)extracted from the bark of Melia toosendanwhich has been used as an ascaris vermifugein traditional Chinese medicine, is a moderately potent presynaptic blocker with an initial facilitatoryaction on MEPP discharge(SHIN et a!., 1981a,b,1982). We also found that toosendanin lengthened the time for blocking neuromuscular transmission by BoTX in the isolated rat phrenic nerve-diaphragm preparation (SHIN and Hsu, 1983). In this work we try to answer the question whether toosendanin

Received for publication March 14, 1983 L *l

677 678 Y.-L. SHIH and K. HSU has the anti-botulismic effect in situ, and if so, whether it is related to its facilitatory action on MEPP discharge. Rats were divided into experimental and control groups. Toosendanin was dissolved in 60 % propane-l,2-diol. It was injected subcutaneously (7 mg/kg) to rats in the experimental group. Some toxic symptoms such as motionlessness, refusing to take food and to drink, paresis of the hind legs developed gradually to their maximum at a time varying from 1 to 24 hr and then disappeared within 72 hr after injection. Rats in the control group were injected with the solvent alone. The animals were sacrificed at 15 min, 2, 6 hr, and 1, 2, 3, 4, 5, 7 days after injection. The left and the right phrenic nerve-diaphragm preparations were made from each animal and mounted separately in different chambers filled with the Krebs solution ventilated by 95% 02+5% CO2 at 37°C. One preparation was used for MEPP recording and the other for recording the muscle twitch to determine the paralysis time by BoTX, i. e. the time taken for the twitch to decrease to 20% of the original. A type of botulinum toxin (2><10~ mouse LD50/ml) was used. As shown in Table 1, the lengthening of the BoTX paralysis time and the increase in MEPP frequency started to occur as early as 15 min after toosendanin injection. These changes reached their maximum within several hours and stayed at a high level for as long as 3 days (Table 1). During this period the distribution of MEPP frequency among different junctions became much dispersed (Fig. 1), but the MEPP size remained unchanged or showed some tendency to decrease. These results indicate that the anti-botulismic effect of toosendanin is probably

Fig, 1. Frequency distribution of MEPP in the phrenic nerve-diaphragm preparations prepared from rats sacrificed at various times after subcutaneous injection of toosendanin (7 mg/kg).

Japanese Journal of Physiology ANTI-BOTULISMIC EFFECT OF TOOSENDANIN 679

Vol. 33, No. 4, 1983 680 Y.-L. SHIH and K. HSU related to its facilitatory action on the frequency of MEPP discharge. In a previous work it was shown that the spontaneous transmitter release became more dependent on the extracellular Ca2+ concentration during the facilitatory phase of the toosendanin action (SHINet a!., 1982). It has been interpreted as due to an increase in Ca2+ conductance in the presynaptic membrane by toosendanin. We suggest that the antagonistic action of toosendanin against botulinum poisoning may be due to the same mechanism.

REFERENCES

CHANG, C. C., CHEN, T. F., and LEE, C. Y. (1973a) Studies of the presynaptic effect of (3- bungarotoxin on neuromuscular transmission. J. Pharmacol. Exp. Ther.,184: 339-345. CHANG, C. C. and HUANG, M. C. (1974) Comparison of the presynaptic actions of botulinum toxin and j3-bungarotoxin on neuromuscular transmission. Naunyn Schmiedebergs Arch. Pharmacol., 282: 129-142. CHANG, C. C., HUANG, M. C., and LEE, C. Y. (1973b) Mutual antagonism between botulinum toxin and j9-bungarotoxin. Nature, 243: 166-167. CLARK, A. W., HURLBUT,W. P., and MAURO, A. (1972) Changes in the fine structure of the neuromuscular junction of the frog caused by black widow spider venom. J. Cell Biol., 52: 1-14. LEE, C. Y. and CHANG, C. C. (1966) Modes of actions of purified toxins from elapid venoms on neuromuscular transmission. Mem. Inst. Butantan Simp. Int., 33: 555-572. LONGENECKER,H. E., HURLBUT, W. P., MAURO, A., and CLARK,A. W. (1970) Effects of black widow spider venom on the frog neuromuscular junction. Nature, 225: 701-703. SHIN, Y. L, and Hsu, K. (1983) The antagonistic effect of toosendanin against botulinum toxin in isolated phrenic nerve-diaphragm preparation of the rat. Kexue Tongbao, 28: 885-887 (in Chinese). SHIN, Y. L., WANG, W. P., and Hsu, K. (1981a) Electrophysiological analysis on the presynaptic blocking effects of toosendanin on neuromuscular transmission. Acta Physiol. Sin., 33: 259-265. SHIN, Y. L., WANG, W. P., YAN, S. C., and Hsu, K. (1982) Effects of calcium ions and nerve impulses on changes in miniature end-plate potential frequency produced by toosendanin. Acta Physiol. Sin., 34: 304-309. SHIN, Y. L., YANG, Y. Q., WANG, W. P., and Hsu, K. (1981b) Influence of stimulus frequency, temperature and Ca2+ on onset of toosendanin paralysis of isolated neuromuscular preparations of rats. Acta Physiol. Sin., 33: 141-146. STERN, P., VALJEVAC,K., DURSUM,K. D., and Ducic, V. (1975) Increased survival time in botulinum toxin poisoning by treatment with a venom gland extract from widow spider. Toxicon,13: 197-198.

Japanese Journal of Physiology