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The actions of a chromatographically identified extract of the marine dinoflagellate Ostreopsis lenticularis, named ostreotoxin-3 (OTX-3), were studied on frog isolated neuromuscular preparations. OTX-3 (1–10μg ml−1) applied to cutaneous pectoris nerve-muscle preparations depolarized skeletal muscle fibres and caused spontaneous contractions. The depolarization was neither reversed by prolonged washing nor by (+)-tubocurarine. OTX-3 decreased the amplitude of miniature end plate potentials (m.e.p.ps) but did not affect their frequency. Extracellular recording of compound action potentials revealed that OTX-3 affected neither excitability nor conduction along intramuscular nerve branches. End-plate potentials (e.p.ps) elicited by nerve stimulation were reduced in amplitude by OTX-3 and even showed reversed polarity in junctions deeply depolarized by the toxin. Membrane depolarization induced by OTX-3 was decreased about 70% in muscles pretreated for 30min with 10μm tetrodotoxin. In contrast, muscles pretreated with 5μm μ-conotoxin GIIIA were completely insensitive to OTX-3-induced depolarization. OTX-3 did not affect e.p.p. amplitude and the quantal content of e.p.ps in junctions in which muscle depolarization was abolished by μ-conotoxin GIIIA. OTX-3 is a novel type of sodium-channel activating toxin that discriminates between nerve and skeletal muscle membranes.

The contractility of guinea pig ileum and frog skeletal muscle is inhibited in solutions containing 0.4 to 2.5 M formamide (FMD). Contrary to mammalian vis ceral muscle, this blocking action is not reversed when frog muscles are transferred back to isotonic Ringer's after FMD treatment. Under these conditions the water content of the skeletal muscles is markedly increased and electronmicrographs show a swellingof the transverse tubules. These changes are not observed when frog muscles are transferred to ethylene glycol solutions that are isosmotic with the FMD containing Ringer's solution. In addition, over 50% of the contractility is recovered in these muscles. These observations provide direct evidence of the occurrence of an osmotic shock in frog muscles transferred from FMD solutions to isotonic Ringer's. It is concluded that the resulting alterations in the triad structure and function are re sponsible for the irreversibility of the FMD uncoupling action in these muscles.

Succinic anhydride (SA) dissolved in Ringer solution buffered with N-tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid (SA-TES solution) potentiates the depolarizing action of acetylcholine (ACh, 10-40 microM) on frog muscle and the tension induced by bath application of this agonist. Applied from one side of a double-barrelled micropipette, SA-TES increases the amplitude of iontophoretically elicited ACh potentials. The potentiation of the effects of ACh by SA-TES does not involve changes in either the activity of the ACh esterase or the input resistance of the muscle membrane. For depolarizations of frog sartorius muscle, dose-response relationships obtained for ACh concentrations from 0.5 to 20 microM indicate that SA-TES increases the apparent affinity of ACh by a factor of 3. SA-TES exerts an "accelerating' effect on the responses elicited by bath-applied ACh; i.e., it increases the rate of depolarization when ACh is added to the bath and the rate of repolarization upon washing out. These effects are particularly marked in preparations treated with neostigmine (3 microM). SA-TES does not potentiate the depolarizing action of agonists which do not contain an ester group. Moreover, the time course of the responses elicited by these compounds is not influenced by SA-TES. SA-TES fails to influence significantly the effects of the neurally released transmitter. Only a 10% increase in the average amplitude of the endplate potentials was observed. SA hydrolyzes in about 30 min at room temperature; however the SA-TES solution retains its activity for several weeks. Succinate is inactive, and so is SA in Ringer buffered with phosphate. The SA-TES solution contains seven succinyl-TES derivatives, which were separated by ion-exchange chromatography and paper chromatography. At concentrations between 1 to 150 microM, these succinyl-TES derivatives affected the ACh-induced contraction of frog rectus abdominus muscle. The most abundant derivative potentiated the action of high doses of ACh, but was inhibitory at lower ones. The other derivatives were mostly inhibitory. These results are discussed in terms of two hypotheses. One postulates the presence of a diffusion barrier formed by groups that bind ACh and are saturated by SA-TES. The other assumes that SA-TES acts directly on the ACh receptor exerting its potentiating effect through a cooperative mechanism.

INACTIVATION of acetylcholinesterase (AChE) causes the half-decay times of the endplate currents (e.p.cs) and miniature e.p.cs (m.e.p.cs) to increase from about 1.5–2.0ms to 3.0–10.00 ms, times far too long to be accounted for by removal of the transmitter by free diffusion1–3. To explain this, Katz and Miledi1 postulated that, when the AChE is blocked, each acetylcholine (ACh) molecule undergoes numerous collisions with the post-synaptic membrane, leading to several attachments with the ACh receptors and a consequent delay in transmitter removal. This was supported by the observation that ( + )-tubocurarine, which reduces the number of free receptors, markedly decreases the half-decay of m.e.p.cs and e.p.cs after treatment with neostigmine1,3. In addition to this delaying mechanism, due to interaction with the ACh receptors the results described below suggest the existence of a second diffusion barrier for the transmitter at the frog neuromuscular junction. This barrier seems to be formed by non-depolarising sites which bind the ester moiety of ACh. Saturation of these sites with substances which possess –CO–O– or –CO–O–CO– groups accelerates the diffusion of externally applied ACh towards the ACh receptor, enhancing its depolarising effects. The barrier also hinders the outward diffusion of neurally released ACh in neostigmine-treated muscles, but this effect can only be demonstrated if the motor nerve is stimulated at high frequencies (200 Hz).