Blockade of protein kinase C is involved in the inhibition by cycloheterophyllin of neutrophil superoxide anion generation

Cycloheterophyllin, a prenylflavone, inhibited the superoxide anion (O-2(.-)) generation from formyl-methionyl-leucyl-phenylalanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-stimulated rat neutrophils in a concentration-dependent manner with IC50 values of 47.0 +/- 5.0 and 1.7 +/- 0.4 mu M, respectively. Cycloheterophyllin had no effect on O-2(.-) generation in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. Cycloheterophyllin exerted a concentration-dependent inhibition of neutrophil cytosolic protein kinase C (PKC) and rat brain PKC, but had no effect on porcine heart protein kinase A (PKA). Unlike staurosporine, cycloheterophyllin did not affect the trypsin-treated rat brain PKC. [H-3]Phorbol 12,13-dibutyrate ([H-3]PDB) binding to neutrophil cytosolic PKC was significantly suppressed by cycloheterophyllin. However, cycloheterophyllin had negligible effect on the PMA-induced membrane translocation of PKC-beta and PKC-delta in neutrophils, Moreover, the fMLP-induced [Ca2+](i) elevation and inositol trisphosphate (IP3) formation of neutrophils were not affected by cycloheterophyllin at concentrations which significantly suppressed the O-2(.-) generation. In cell-free system, addition of arachidonate (AA) into the mixture of cytosol and membrane fractions of the resting neutrophils to make NADPH oxidase assembly and activation. Cycloheterophyllin had no effect on O-2(.-) generation in AA-activated cell-free system. These results suggest that the suppression of PKC activity through the interaction with the regulatory region of PKC is involved in the inhibition by cycloheterophyllin of the O-2(.-) generation in rat neutrophils.