Venom phospholipase A2-induced impairment of glutamate uptake: An indirect and nonselective effect related to phospholipid hydrolysis

Abstract

In a nominally calcium-free medium, a toxic phospholipase A2, paradoxin, PDX (1-200nM) was able to significantly decrease glutamate uptake by rat hippocampal mini-slices. Under the same experimental conditions, PDX could also inhibit the reuptake of choline and dopamine, suggesting a nonselective action. Furthermore, we found no evidence of competition between PDX and [3H]L-Aspartate described as a marker of glutamate carrier proteins. A direct blockage of glutamate uptake by binding to the glutamate transporters is thus unlikely to occur. Implication of the free fatty acids (FFAs), or their metabolites, was clearly shown by the total suppression of PDX effect on reuptake in a medium inhibiting its catalytic activity (EGTA/Sr2+ buffer). Moreover, analysis of the FFAs liberated showed a significant increase in polyunsaturated fatty acid (PUFA) levels. Arachidonic acid (AA) concentration reached in the water phase, though in the low micromolar range, may be especially relevant in explaining this effect. Much higher concentrations are found in the membranes and may also participate in the action on reuptake. Evidence for the involvement of FFAs was also provided by the antagonistic, although partial, action of bovine serum albumine (BSA, 1%). Finally, free radicals or eicosanoids did not seem to play a significant role given the persistence of inhibition in the presence of NDGA (1 microM) or indomethacin (10 microM), inhibitors of the two major AA metabolic pathways. Altogether, PDX-induced uptake impairment may thus be related to the direct action of AA and other PUFAs on the glutamate transporter, as well as through less selective actions.