Ciguatoxin enhances quantal transmitter release from frog motor nerve terminals

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Abstract
1 Ciguatoxin (CTX), a marine toxin produced by the benthic dinoflagellate Gambierdiscus toxicus, is responsible for a complex endemic disease in man known as ciguatera fish poisoning. In the present study we have investigated the effects of purified CTX extracted for Gymnothorax javanicus moray-eel liver on frog isolated neuromuscular preparations with conventional electrophysiological techniques. 2 CTX (1–2.5 nm) applied to cutaneous pectoris nerve-muscle preparations induced, after a short delay, spontaneous fibrillations of the muscle fibres that could be suppressed with 1 μm tetrodotoxin (TTX) or by formamide to uncouple excitation-contraction. 3 In preparations treated with formamide, CTX (1–2.5 nm) caused either spontaneous or repetitive muscle action potentials (up to frequencies of 60–100 Hz) in response to a single nerve stimulus. Recordings performed at extrajunctional regions of the muscle membrane revealed that during the repetitive firing a prolongation of the repolarizing phase of the action potential occurred. At junctional sites the repetitive action potentials were triggered by repetitive endplate potentials (e.p.ps). 4 CTX (2.5 nm) caused a TTX-sensitive depolarization of the muscle membrane. 5 In junctions equilibrated in solutions containing high Mg2+ + low Ca2+, addition of CTX (1.5 nm) first induced an average increase of 239 ± 36% in the mean quantal content of e.p.ps. Subsequently CTX reduced and finally blocked nerve-evoked transmitter release irreversibly. 6 CTX (1.5–2.5 nm) increased the frequency of miniature endplate potentials (m.e.p.ps) in junctions bathed either in normal Ringer, low Ca2+-high Mg2+ medium or in a nominally Ca2+-free solution containing EGTA. Extensive washing with toxin-free solutions did not reverse the effect. Furthermore, Cd2+ (0.1 mm), a potent calcium channel blocker, neither antagonized nor abolished the increase in transmitter release caused by CTX. 7 TTX (1 μm) completely prevented the effect of CTX (2.5 nm) on m.e.p.p. frequency. This effect was independent of the presence of extracellular Ca2+. TTX, when added after CTX (2.5 nm) exposure, antagonized the increase in m.e.p.p. frequency. The antagonism was complete in Ca2+-free medium. These results strongly suggest that increased permeability of the nerve terminal to Na+ is responsible for the increase in m.e.p.p. frequency caused by CTX. It is likely that CTX may trigger calcium release from internal stores due to an increase of intraterminal Na+ concentration. 8 It is concluded that CTX exerts, in the nanomolar concentration range, a selective action on sodium channels of the neuromuscular junction causing both pre- and postsynaptic effects.