Protein kinase C inhibition enhances platelet-activating factor-induced eicosanoid production in human eosinophils

Previous investigations have suggested that protein kinase C (PKC) may regulate guinea pig eosinophil responses through a suppressive "negative feedback" mechanism. Using the selective PI(C inhibitors bisindolylmaleimide I (Bis I, GF 109203X) and calphostin C, we examined the role of PKC in platelet activating factor (PAF)-induced respiratory burst and generation of arachidonic acid metabolites in human peripheral blood eosinophils, Ris I inhibited PAF-induced generation of superoxide anion with substantially lower potency (geometric mean IC50 = 1.41 mu M, 95% CI 0.94-2.11 mu M) than it exhibited against responses to the phorbol esters 4-beta-phorbol 12-myristate 13-acetate (PMA: IC50 = 0.25 mu M, 0.09-0.72 mu M; P < 0.01) and 4-beta-phorbol 12,13-dibutyrate (IC50 = 0.48 mu M, 0.20-1.14 mu M; P < 0.05). The production of thromboxane (measured as TxB(2)) induced by 1 mu M PAF was increased significantly by Bis Ist concentrations of 1 mu M (162 +/- 7.5% of control PAF response; P < 0.01) and 10 mu M (194 +/- 17%; P < 0.001): TxB(2) release induced by PMA was unaffected by concentrations of Bis I up to 1 mu M and inhibited by 10 mu M Bis I (48 +/- 11%; P < 0.05), Bis I (1 mu M) significantly increased both thromboxane and leukotriene C-4 (LTC4) production induced by 2 mu M (P < 0.01 and P < 0.05, respectively) or 20 mu M PAF (both P < 0.001). The actions of Bis I on PAF-stimulated thromboxane and leukotriene production were mimicked Dy a second PKC inhibitor, calphostin C, whereas the non-PKC-inhibitory analog, bisindolylmaleimide V, caused no enhancement of TxB(2) or LTC4 production, The increase in intracellular free calcium induced by 1 mu M PAF was heightened and prolonged in cells pre-treated with 1 mu M Bis I or 1 mu M calphostin C (peak increase, P < 0.05 for both drugs; level 60 s after addition of PAF, P < 0.001 and P < 0.05 for Bis I and calphostin C, respectively; time to return to 50% of peak, P < 0.05 for Bis I). We conclude that PI(C inhibition causes augmentation of thromboxane and LTC4 production in PAF-stimulated human eosinophils despite suppressing respiratory burst activity, indicating that different signaling pathways predominate hi these two responses and that PKC mediates a suppression of an early stage in an alternative pathway of activation.