Enhancement of acetylcholine release by homoanatoxin-a from Oscillatoria formosa

The strain NIVA-CYA 92 of Oscillatoria formosa Bory ex Gormont produces phycotoxins with neurotoxic properties. Chemical analysis by gas chromatography/mass spectrometry of a water extract of lyophilized material of the organism showed the presence of only homoanatoxin-a. The mechanism of action of homoanatoxin-a on peripheral cholinergic nerves is so far not known. The neurotoxicity of O. formosa containing homoanatoxin-a was investigated in rat bronchi, rat brain synaptosomes and in GH 4C 1 cells. The water extract of lyophilized material of the organism produced a concentration-dependent reversible increase in the release of [ 3H]acetylcholine from both K + (51 mM) depolarised and non-depolarised cholinergic nerves of the rat bronchial smooth muscle. The K +-evoked release of [ 3H]acetylcholine was enhanced by about 75% by a water extract from 15–20 mg/ml of lyophilized algal material. The enhanced release of [ 3H]acetylcholine was substantially reduced by the L-type Ca 2+-channel blocker verapamil (100 μM) and not by the N-type Ca 2+-channel blocker ω-conotoxin GVIA (1.0 μM) or the P-type Ca 2+-channel blocker ω-agatoxin IV-A (0.2 μM). Chelation of intra-cellular Ca 2+ by 1,2-bis-(aminofenoxi)etan- N,N,N′,N′-tetraacidic acid/acetoxymethyl (BAPTA/AM) (30 μM) had no effect on the phycotoxin-induced release of [ 3H]acetylcholine, indicating that an extracellular pool of Ca 2+ was important for the action of the phycotoxin on the release of [ 3H]acetylcholine from peripheral cholinergic nerves. In rat brain synaptosomes the algal extract enhanced the influx of 45Ca 2+ in a tetrodotoxin (1.0 μM) and ω-conotoxin MVIIC (blocker of N-, P- and Q-type Ca 2+ channels) (1.0 μM) insensitive manner. Patch-clamp studies showed that the phycotoxin opened endogenous voltage dependent L-type Ca 2+ channels in neuronal GH 4C 1 cells. These Ca 2+ channels and the effect of the toxin on the channels were blocked by the L-type Ca 2+-channel antagonist gallopamil (200 μM). The present results suggest, therefore, that the investigated strain of O. formosa contains homoanatoxin-a, which enhances the release of acetylcholine from peripheral cholinergic nerves through opening of endogenous voltage dependent neuronal L-type Ca 2− channels.