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Stimulation of macrophages with IgG2 immune complexes induced dose-dependently the O2- generation and the release of arachidonic acid and its metabolites. This Fc gamma R-mediated O2- generation was inhibited by a phospholipase A2 inhibitor, 4-p-bromophenacyl bromide (4-pBPB), in parallel to the dose-dependent inhibition of arachidonic acid release. The main arachidonic acid metabolites released were shown to be prostaglandin E2 and thromboxane B2 and blocking of the production of these metabolites by indomethacin did not inhibit the O2- generation. Inhibition of the Fc gamma R-mediated O2- generation and the arachidonic acid release by the C-kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), was less intense than by 4-pBPB. These results support the previously proposed hypothesis that arachidonic acid acts as an intracellular activator of the Fc gamma R-mediated O2- generation in macrophages. Although the C-kinase activation may also contribute to the activation of the O2--generating system, arachidonic acid release appears to play a major role in Fc gamma R-mediated O2- generation. In contrast, activation of C-kinase seems to be contributing mainly in the induction of both the arachidonic acid release and O2- generation by 12-o-tetradecanoylphorbol 13-acetate (TPA). Furthermore, suboptimal concentrations of TPA and arachidonate were found to act synergistically to stimulate O2- generation and the inhibition study suggested a positive synergism between C-kinase and arachidonic acid release to induce O2- generation. This synergistic action may have general importance in receptor-mediated O2- generation.

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A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), inhibited phorbol ester (12-o-tetradecanoylphorbol 13-acetate)-induced and Fc gamma receptor-mediated superoxide anion (O2-) generations in guinea pig macrophages, but the inhibitory effect on Fc gamma receptor-mediated O2- generation was only partial. Both O2- generations were inhibited extensively by a phospholipase A2 inhibitor, 4-p-bromophenacyl bromide (4-pBPB). It was confirmed in control experiments that H-7 and 4-pBPB had no direct inhibitory effect on NADPH-oxidase activity. Dose-dependent stimulation of O2- generation was induced by arachidonate in macrophages, and the arachidonate-induced O2- generation was not inhibited by H-7. Arachidonate could also induce NADPH-oxidase activation in a post-nuclear fraction obtained from unstimulated macrophages and this activation was not inhibited by H-7, indicating that protein kinase C activation was not involved in this cellfree system. These results support the hypothesis that the O2- generation induced by Fc gamma receptor stimulation is mainly mediated by arachidonic acid which is released by the action of phospholipase A2 activated by receptor stimulation. Arachidonic acid seems to be acting rather directly in activating the NADPH-oxidase system of macrophage membrane. Protein kinase C may have a significant role in Fc gamma receptor-mediated O2- generation but it is not obligatory, and protein kinase C seems to activate NADPH-oxidase rather indirectly, probably by inducing the arachidonic acid release.

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