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Direct action of Physalia toxin on frog nerve and muscle
Toxlcon, 1970, Vol. 8, py. 21-23. Peraamon Prou . Printed in Grcat Britain
DIRECT ACTION OF
TOXIN ON FROG NERVE AND MUSCLE* PHYSALIA
J. B. LARSExt and CIiARLE3 E. LAxs Institute of Marine and Atmospheric Sciences, University of Miami, Miami, Florida, U.S.A . (Accepted forpublication 1 Octolxr
1969)
Abtá~act-Isolated frog sciatic-gastrocnemius preparations were used to investigate the actions of P.physalts toxin on the conduction of norn impulses andtin muscle contraction . Se»ments of sciatic nerves treated for 10 min with toxin solutions (1~0 mg per ml) failed to wnduct impulses, although stimulation of the nerve distal to the treated segments elicited normal muscle contractions. Direct electrical stimulation of curarized gastrocnemius muscles was ineffective when toxin was present in the Riclger's solution bathing the muscle at a concentration of 1~0 mg per ml . INTRODUCTION
TIC NsultoroxlC nature of the nematocyst toxin of Physalia physalis has been confirmed in
numerous experiments, both in vlvo and in vitro (LArt1: and DoDG$, 1958 ; LArrE, 1960, 1961, 1967 ; LANE and LARSEN, 1965; LARS1?rr and LArr1?, 1966 ; HASTINGS et al., 1967; GARRIOTT and LANs, 1969). However, the direct effects of this toxin on nerve and vertebrate skeletal muscle have not been investigated . Accordingly, we report here on experiments utilizing isolated frog sciati~gastrocnemius preparations to assess such effects. MATERIALS AND METHODS
P. physalis nematocyst toxin was prepared as described previously (LANE and DODGE,
1958), except that sea water remaining in the frozen nematocyst preparation was removed, after partial thawing, by washing each portion several times with glass distilled water. Nematocysts were reconcentrated between each wash by slow speed centrifugation . No discharge was observed as a result of these procedures . Since pH has been shown to affect the stability ofthis toxin, it was maintained at pH=6~6 during homogenization by using 0067 M Sörensen's phosphate buffer (PRINGLE, 1967). Lyophilized toxin was later redissolved in the same buffer for experimental use. Sciati~gastrocnemius preparations were dissected from freshly pithed frogs (Rana pipiens) collected locally. For nerve conduction studies, the gastrocnemius was suspended by the Achilles tendon from a force displacement transducer and immobilized by clamping the femur. Muscle contractions were monitored on a Polygraph. The nerve was immersed in Ringer's solution on a glass slide and threaded through a short length of polyethylene capillary tubing fixed to the slide. The muscle was irrigated periodically with the same Ringer's solution . Solutions 'Contribution No . 1160 . Institute of Marine and Atmospheric Sciences, University of Miami. Supported by USPHS Graat H1154ß9. tPresent address : Department of Biology, Hamfine University, Saint Paul, Minnesota, U.S .A . 21
22
J. B. LARSEN and CHARLES E. LANE
to be tested were placed in contact with a segment of the sciatic by filling the polyethylene tubing using a finely drawn Pasteur pipette and were removed by suction using a similar pipette. Surface tension prevented contamination of the adjacent Ringer's . Under these conditions electrical stimulation of the nerve was possible, both proximally and distally to the treated segment . Stimulus pulses of 0~ 1 cosec duration which ranged from 250 to 350 mV in amplitude were supplied through platinum electrodes from a square wave stimulator . In several experiments the epineurium of the treated segment was teased open to provide the toxin more direct access to the axons. The preparations were arranged in similar fashion for muscle contraction studies, except that the gastrocnemius, in an organ bath, was immersed in Ringer's solution aerated with compressed air. The muscle was stimulated directly using square wave stimulus pulses of 0~1 cosec duration which ranged from 2~5 to 3~5 V in amplitude. In these experiments ntubocurarine chloride (Nutritional Biochemicals Corporation) at a final concentration of 3~0 wg per ml was used to block conduction across neuromuscular junctions. Toxin solutions were added to the perfusion bath for testing only after competent muscles failed to react upon stimulation of the sciatic. RESULTS Stimulation of the sciatic nerve proximal to the segment treated with P. physalis toxin failed to elicit any response, while stimulation distal to the treated segment was still effective. In five experiments the average minimum effective toxin concentration was 1 ~0 mg per ml in the intact nerve and 025 mg per ml in the teased nerve. Blockade was generally complete after 10 min in the intact nerve, and after 5 min in the teased nerve. The blockade was not reversed by 30 min washing in several changes of Ringer's solution . Sörensen's phosphate buffer at pH=6~6 (0067 M), tested alone, had no effect on nerve conduction . Muscle preparations, with all neuromuscular junctions blocked with curare failed to contract in response to direct stimulation after ca. l0 min when the final toxin concentration was 1 ~0 mg per ml . DISCUSSION AND CONCLUSIONS The inhibition of nerve impulse conduction and muscle contraction demonstrated in these experiments is consistent with the results of previous investigations already cited . The toxin affects any treated segment of a nerve trunk, but its effects are restricted to the site of application . Its effects on muscle contraction, which do not depend on functional neuromuscular junctions, supplement earlier data which revealed no competition between this toxin and acetylcholine in perfused rat ileum (LANE, 1967). The stimulating action of P. physalis toxin on the isolated intestine is not due to the presence of histamine, acetylcholine, nor serotonin, and is not ganglionic in origin (GARRIOTT and LANE, 1969). All these results are consistent with previous suggestions (LAxseN and LANE, 1966 ; HASTINGS et al., 1967) that the toxin may act by depolarizing cell membranes. Since the toxin concentration, as well as the time, required to block nerve conduction was reduced in teased preparations, it follows that toxin penetration was impeded by the connective tissue which surrounds nerve and muscle. This would account for the unusually high concentration of toxin necessary to produce the effects reported here .
Neuromuscular Effects of Physalia Toxin
23
REFERENCES
GARRIOTI', J. C. andLave, C. E. (1%9) Some autonomic effects of Physalia toxin. Toxicon 6, 281 . HASTDVGS, S. G., LARSerr, J. B. and LAxE, C. E. (1967) Effects of nematocyst toxin of Physalia physalis
(Portuguese Mao-of-War) on the canine cardiovascular system . Proc. Soc . exp. Biol. Med, N Y. 125, 41 . LAive, C. E. (1960) The toxin of Physalia nematocysts . Ann . N.Y. Acad. Sci. 90, 742. LAxe, C. E. (1961) Physalia nematocysts and their toxin. In : The Biology ojHydra and ojSome Other Cuelenterares (LENHOFF, H. M. and Looms, W. F., Eds.) . Coral Gables, Florida: University of Miami Press. LANH, C. E. (1967) Recent observations on the pharmacology of Physalia toxin. In : Animal Toxins (RUSSELL, F. E. and SAVrroERS, P. R., Eds.) . Pergamon Press : Oxford . LANE, C. E. and llonce, E. (1958) The toxicity of Physalia nematocysts. Biol. Bull. nrar. Biul. Lab ., Wuods Hole 115, 219. LAxe, C. E. 8IId LARSEN, J. B. (1965) Some effects of the toxin of Physalia physalis on the heart of the land crab, Cardisoma guanhumi (L .atreille) . Toxicon 3, 69 . LARSEN, J. B. and LArre, C. E. (1966) Some effects of Physalia physalis toxin on the cardiovascular system of the rat. Toxicon 4, 199. PRINGLE, M. E. (1967) Activity and stability of Physalia physalls toxin. M.S . Thesis, University of Miami.
DIRECT ACTION OF
TOXIN ON FROG NERVE AND MUSCLE* PHYSALIA
J. B. LARSExt and CIiARLE3 E. LAxs Institute of Marine and Atmospheric Sciences, University of Miami, Miami, Florida, U.S.A . (Accepted forpublication 1 Octolxr
1969)
Abtá~act-Isolated frog sciatic-gastrocnemius preparations were used to investigate the actions of P.physalts toxin on the conduction of norn impulses andtin muscle contraction . Se»ments of sciatic nerves treated for 10 min with toxin solutions (1~0 mg per ml) failed to wnduct impulses, although stimulation of the nerve distal to the treated segments elicited normal muscle contractions. Direct electrical stimulation of curarized gastrocnemius muscles was ineffective when toxin was present in the Riclger's solution bathing the muscle at a concentration of 1~0 mg per ml . INTRODUCTION
TIC NsultoroxlC nature of the nematocyst toxin of Physalia physalis has been confirmed in
numerous experiments, both in vlvo and in vitro (LArt1: and DoDG$, 1958 ; LArrE, 1960, 1961, 1967 ; LANE and LARSEN, 1965; LARS1?rr and LArr1?, 1966 ; HASTINGS et al., 1967; GARRIOTT and LANs, 1969). However, the direct effects of this toxin on nerve and vertebrate skeletal muscle have not been investigated . Accordingly, we report here on experiments utilizing isolated frog sciati~gastrocnemius preparations to assess such effects. MATERIALS AND METHODS
P. physalis nematocyst toxin was prepared as described previously (LANE and DODGE,
1958), except that sea water remaining in the frozen nematocyst preparation was removed, after partial thawing, by washing each portion several times with glass distilled water. Nematocysts were reconcentrated between each wash by slow speed centrifugation . No discharge was observed as a result of these procedures . Since pH has been shown to affect the stability ofthis toxin, it was maintained at pH=6~6 during homogenization by using 0067 M Sörensen's phosphate buffer (PRINGLE, 1967). Lyophilized toxin was later redissolved in the same buffer for experimental use. Sciati~gastrocnemius preparations were dissected from freshly pithed frogs (Rana pipiens) collected locally. For nerve conduction studies, the gastrocnemius was suspended by the Achilles tendon from a force displacement transducer and immobilized by clamping the femur. Muscle contractions were monitored on a Polygraph. The nerve was immersed in Ringer's solution on a glass slide and threaded through a short length of polyethylene capillary tubing fixed to the slide. The muscle was irrigated periodically with the same Ringer's solution . Solutions 'Contribution No . 1160 . Institute of Marine and Atmospheric Sciences, University of Miami. Supported by USPHS Graat H1154ß9. tPresent address : Department of Biology, Hamfine University, Saint Paul, Minnesota, U.S .A . 21
22
J. B. LARSEN and CHARLES E. LANE
to be tested were placed in contact with a segment of the sciatic by filling the polyethylene tubing using a finely drawn Pasteur pipette and were removed by suction using a similar pipette. Surface tension prevented contamination of the adjacent Ringer's . Under these conditions electrical stimulation of the nerve was possible, both proximally and distally to the treated segment . Stimulus pulses of 0~ 1 cosec duration which ranged from 250 to 350 mV in amplitude were supplied through platinum electrodes from a square wave stimulator . In several experiments the epineurium of the treated segment was teased open to provide the toxin more direct access to the axons. The preparations were arranged in similar fashion for muscle contraction studies, except that the gastrocnemius, in an organ bath, was immersed in Ringer's solution aerated with compressed air. The muscle was stimulated directly using square wave stimulus pulses of 0~1 cosec duration which ranged from 2~5 to 3~5 V in amplitude. In these experiments ntubocurarine chloride (Nutritional Biochemicals Corporation) at a final concentration of 3~0 wg per ml was used to block conduction across neuromuscular junctions. Toxin solutions were added to the perfusion bath for testing only after competent muscles failed to react upon stimulation of the sciatic. RESULTS Stimulation of the sciatic nerve proximal to the segment treated with P. physalis toxin failed to elicit any response, while stimulation distal to the treated segment was still effective. In five experiments the average minimum effective toxin concentration was 1 ~0 mg per ml in the intact nerve and 025 mg per ml in the teased nerve. Blockade was generally complete after 10 min in the intact nerve, and after 5 min in the teased nerve. The blockade was not reversed by 30 min washing in several changes of Ringer's solution . Sörensen's phosphate buffer at pH=6~6 (0067 M), tested alone, had no effect on nerve conduction . Muscle preparations, with all neuromuscular junctions blocked with curare failed to contract in response to direct stimulation after ca. l0 min when the final toxin concentration was 1 ~0 mg per ml . DISCUSSION AND CONCLUSIONS The inhibition of nerve impulse conduction and muscle contraction demonstrated in these experiments is consistent with the results of previous investigations already cited . The toxin affects any treated segment of a nerve trunk, but its effects are restricted to the site of application . Its effects on muscle contraction, which do not depend on functional neuromuscular junctions, supplement earlier data which revealed no competition between this toxin and acetylcholine in perfused rat ileum (LANE, 1967). The stimulating action of P. physalis toxin on the isolated intestine is not due to the presence of histamine, acetylcholine, nor serotonin, and is not ganglionic in origin (GARRIOTT and LANE, 1969). All these results are consistent with previous suggestions (LAxseN and LANE, 1966 ; HASTINGS et al., 1967) that the toxin may act by depolarizing cell membranes. Since the toxin concentration, as well as the time, required to block nerve conduction was reduced in teased preparations, it follows that toxin penetration was impeded by the connective tissue which surrounds nerve and muscle. This would account for the unusually high concentration of toxin necessary to produce the effects reported here .
Neuromuscular Effects of Physalia Toxin
23
REFERENCES
GARRIOTI', J. C. andLave, C. E. (1%9) Some autonomic effects of Physalia toxin. Toxicon 6, 281 . HASTDVGS, S. G., LARSerr, J. B. and LAxE, C. E. (1967) Effects of nematocyst toxin of Physalia physalis
(Portuguese Mao-of-War) on the canine cardiovascular system . Proc. Soc . exp. Biol. Med, N Y. 125, 41 . LAive, C. E. (1960) The toxin of Physalia nematocysts . Ann . N.Y. Acad. Sci. 90, 742. LAxe, C. E. (1961) Physalia nematocysts and their toxin. In : The Biology ojHydra and ojSome Other Cuelenterares (LENHOFF, H. M. and Looms, W. F., Eds.) . Coral Gables, Florida: University of Miami Press. LANH, C. E. (1967) Recent observations on the pharmacology of Physalia toxin. In : Animal Toxins (RUSSELL, F. E. and SAVrroERS, P. R., Eds.) . Pergamon Press : Oxford . LANE, C. E. and llonce, E. (1958) The toxicity of Physalia nematocysts. Biol. Bull. nrar. Biul. Lab ., Wuods Hole 115, 219. LAxe, C. E. 8IId LARSEN, J. B. (1965) Some effects of the toxin of Physalia physalis on the heart of the land crab, Cardisoma guanhumi (L .atreille) . Toxicon 3, 69 . LARSEN, J. B. and LArre, C. E. (1966) Some effects of Physalia physalis toxin on the cardiovascular system of the rat. Toxicon 4, 199. PRINGLE, M. E. (1967) Activity and stability of Physalia physalls toxin. M.S . Thesis, University of Miami.