Abstract

The effect of Pardaxin, a neurotoxin that induces neurotransmitter release from neurons, on the arachidonic acid (AA) cascade was studied in PC12 cells. Both native and the synthetic Pardaxin selectively stimulated phospholipase A₂ (PLA₂) activity (measured by [³H]AA release) in the presence as well as in the absence of extracellular calcium. Pardaxin-stimulated PLA₂ activity was also evident in the increased formation of lysophosphatidylcholine. Pardaxin analogs, lacking the α-helical structure that is essential for insertion into the plasma membrane, were ineffective in stimulating the AA cascade in PC12 cells. Pardaxin stimulation of PLA₂ was markedly inhibited by the nonselective PLA₂ inhibitors bromophenacyl bromide and mepacrine, by methyl arachidonyl fluorophosphonate, a dual inhibitor of calcium-dependent cytosolic PLA₂ and the calcium-independent PLA₂ and by bromoenol lactone[(E)-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl-2H-pyran-2-one], a highly specific inhibitor of calcium-independent PLA₂. After Pardaxin treatment, there was increased release of AA metabolites produced by the cyclooxygenase pathway as expressed in an 8-fold increase of PGE₂ release. The release of other eicosanoids, such as 6-keto-PGF_1α and thromboxane B₂, was also augmented. Pardaxin-induced PGE₂ release was observed in calcium-free medium and in the absence of any increase in cytosolic calcium. Dexamethasone partially inhibited Pardaxin-induced PGE₂ release. This effect was reversed by the type II corticosteroid receptor antagonist RU-38486. Our results indicate that Pardaxin stimulates release of AA and eicosanoids, independently of calcium, and suggest that calcium-independent PLA₂ plays an important role in Pardaxin stimulation of the AA cascade.