The effects of two phospholipase A 2 inhibitors on the neuromuscular blocking activities of homologous phospholipases A 2 from the venom of Pseudechis australis, the Australian king brown snake

Abstract

Previous studies have shown that homologous phospholipases A 2 (PLA 2) (Pa-3, Pa-9C, Pa-10F and Pa-11) from the venom of the Australian king brown snake, Pseudechis australis, significantly reduce the resting membrane potentials and quantal contents of endplate potentials recorded from endplate regions of mouse triangularis sterni nerve-muscle preparations. It is not clear whether PLA 2 activity is essential for their neuromuscular activities. Therefore, pharmacological studies were carried out to determine whether neuromuscular activity of the toxins changed after treatment with the phospholipase A 2 inhibitors 7,7-dimethyl-eicosadienoic acid (DEDA) and manoalide. After incubation of the toxins with manoalide (120nM), or DEDA (50 μM), no PLA 2 activity against 1-stearoyl 2-[ 3H]arachidonoylglycerophosphocholine was detected. After incubation with manoalide and/or DEDA, the toxins did not depolarize muscle fibre membranes up to 60 min after administration. However, manoalide and DEDA had different influences on the inhibitory effect of these toxic enzymes on acetylcholine release from nerve terminals. Manoalide abolished the inhibitory effect of the toxins on evoked release of acetylcholine. In contrast, DEDA was not able to prevent the reduction of quantal content of endplate potentials induced by the toxins. This study provides evidence that the depolarizing action and the inhibitory effect on release of acetylcholine exerted by these toxic PLA 2 from king brown snake are independent phenomena. The evidence for this conclusion was that inhibition of enzymatic activity with an arachidonic acid analogue (DEDA) abolished the depolarizing effect of the toxins but not the effects on the quantal release of acetylcholine from mouse motor nerve terminals. The data suggest that the depolarizing effect of these toxins is probably due to the enzymatic activity. Since manoalide interacts with lysine residues of PLA 2 polypeptides, and, as shown here, manoalide prevented inhibition of neurotransmitter release, lysine residues may play an important role in the inhibitory activity of these toxins.