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1. 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. 2. OTX-3 (1-10 microg 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. 3. OTX-3 decreased the amplitude of miniature end plate potentials (m.e.p.ps) but did not affect their frequency. 4. Extracellular recording of compound action potentials revealed that OTX-3 affected neither excitability nor conduction along intramuscular nerve branches. 5. 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. 6. Membrane depolarization induced by OTX-3 was decreased about 70% in muscles pretreated for 30 min with 10 microM tetrodotoxin. In contrast, muscles pretreated with 5 microM mu-conotoxin GIIIA were completely insensitive to OTX-3-induced depolarization. 7. 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 mu-conotoxin GIIIA. 8. OTX-3 is a novel type of sodium-channel activating toxin that discriminates between nerve and skeletal muscle membranes.

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.