Marine algal pinnatoxins E and F cause neuromuscular block in an in vitro hemidiaphragm preparation

Abstract

Members of the cyclic imine group of toxins, gymnodimine and spirolides, have been found to be potent antagonists of both muscle type and neuronal nicotinic acetylcholine receptors. These toxins exhibit fast acting toxicity in vivo, causing death within minutes by respiratory depression. This toxicity is shared by the novel cyclic imine pinnatoxins E and F, produced by marine dinoflagellates and recently isolated from New Zealand shellfish. However, there is currently very little data available regarding the mechanism of action for any of the pinnatoxins, and no data at all on the novel pinnatoxins E and F. The aim of the current study was to investigate potential antagonism of nicotinic acetylcholine receptors by pinnatoxins E and F using two in vitro tissue preparations. Compound muscle action potentials elicited by stimulation of the phrenic nerve were recorded from the hemidiaphragm in order to test effects on muscle type heteromeric nicotinic receptors, while effects on α7 homomeric neuronal nicotinic receptors were investigated by recording gamma oscillations in response to tetanic stimulation of the CA1 region of the hippocampus. Both a crude extract containing a mixture of pinnatoxins E and F, as well as pure pinnatoxin F, had no effect on gamma oscillation spectral density or spike count at any concentrations. Conversely, at these same concentrations, both crude and pure pinnatoxin caused an almost complete abolition of nerve-evoked hemidiaphragm action potential responses, without any effect on electrically-evoked (direct) responses. This neuromuscular block could not be reversed by neostigmine. These results show that pinnatoxins E and F block neuromuscular transmission and suggest that observed in vivo muscle paralysis by pinnatoxin is due to selective antagonism of muscle type nicotinic acetylcholine receptors.