Involvement of group VICa2+-independent phospholipase A2 in protein kinase C-dependent arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells

We investigated the possible involvement of group VI Ca2+-independent phospholipase A(2) (iPLA(2)) in arachidonic acid (AA) liberation in zymosan-stimulated macrophage-like P388D(1) cells. Zymosan-induced AA liberation was markedly inhibited by methyl arachidonoyl fluorophosphonate, a dual inhibitor of group IV cytosolic phospholipase A, (cPLA(2)) and iPLA(2). We found that a relatively specific iPLA(2) inhibitor, bromoenol lactone, significantly decreased the zymosan-induced AA liberation in parallel with the decrease in iPLA(2) activity, without an effect on diacylglycerol formation. Consistent with this, attenuation of iPLA(2) activity by a group VI iPLA(2) antisense oligonucleotide resulted in a decrease in zymosan-induced prostaglandin D, generation. These findings suggest that zymosan-induced AA liberation may be, at least in part, mediated by iPLA(2). A protein kinase C (PKC) inhibitor diminished zymosan-induced AA liberation, while a PKC activator, phorbol la-myristate 13-acetate (PMA), enhanced the liberation. Bromoenol lactone suppressed the PMA-enhanced AA liberation without any effect on PIMA-induced PKC activation. Down-regulation of PKC alpha on prolonged exposure to PMA also decreased zymosan-induced AA liberation. Under these conditions, the remaining AA liberation was insensitive to bromoenol lactone, Furthermore, the PKC depletion suppressed increases in iPLA(2) proteins and the activity in the membrane fraction of zymosan-stimulated cells. In contrast, the zymosan-induced increases in iPLA(2) proteins and the activity in the fraction were facilitated by simultaneous addition of PMA Although intracellular Ca2+ depletion prevented zymosan-induced AA liberation, the translocation of PKCa to membranes was also inhibited. Taken together, we propose that zymosan may stimulate iPLA(2)-mediated AA liberation, probably through a PKC-dependent mechanism.