Effects of toxic extracts and purified borbotoxins from Prorocentrum borbonicum (Dinophyceae) on vertebrate neuromuscular junctions

Benthic dinoflagellates of the genus Prorocentrum are common in tropical an d subtropical water and several species produce phycotoxins potentially inv olved in human toxic outbreaks. The toxic dinoflagellate Prorocentrum borbo nicum collected at La Reunion Island (France) was cultured in laboratory. A crude extract of the organism displayed significant toxicity in mice chara cterized by progressive limb paralysis, severe dyspnea, and death, and the toxicity was retained, after partition, in the extract's butanol-soluble fr action (BSF). Electrophysiological experiments characterizing the fraction' s effect on isolated vertebrate neuromuscular preparations revealed that it depolarizes the muscle membrane and reduces the driving force for endplate potentials (EPPs) evoked by nerve stimulation, blocking directly- and indi rectly-elicited muscle twitches. The depolarization induced by P. borbonicu m BSF was not due to Na+ influx through voltage-dependent Na+ channels, sin ce tetrodotoxin neither prevented nor suppressed the depolarization. Howeve r, ouabain, a specific ligand of the Na/K ATPase, reduced the depolarizatio n. These results suggest the presence of palytoxin-like compounds in the fr action. HPLC-MS and MSIMS analysis showed the presence of several toxins ha ving identical UV absorbance, among which two new isomeric toxins, borbotox in-A and -B, of molecular mass of 1037.6 Da were isolated. The purified bor botoxin-A, had no effect on the resting membrane potential of muscle fibers and did not affect directly-elicited muscle twitches. However, the toxin r educed nerve-evoked muscle twitches, in a dose-dependent manner, reduced EP Ps' amplitudes and completely blocked miniature endplate potentials. These observations suggest that the main action of borbotoxin-A is to block post- synaptic nicotinic ACh receptors. (C) 2001 Elsevier Science Ltd. All rights reserved.