The signaling mechanisms mediating the inhibitory effect of TCH-1116 on formyl peptide-stimulated superoxide anion generation in neutrophils

In fMLP (formyl-Met-Leu-Phe)-stimulated rat neutrophils, a mixture of regioisomers benzo[a]furo[2,3-c]phenazine-10-carboxylic acid and benzo[a]furo[2,3-c]phenazine-11-carboxylic acid (TCH-1116) inhibited O2- (superoxide anion) generation, which was not mediated by scavenging the generated O2- or by a cytotoxic effect on neutrophils. TCH-1116 had no effect on the arachidonic acid-induced NADPH oxidase activation in a cell-free system, whereas it effectively attenuated the phosphorylation of Ser residues in p47phox and the association between p47phox and p22phox in fMLP-stimulated neutrophils. The interaction of p47phox with PKC (protein kinase C) isoforms (α, βI, βII, δ and ζ) was attenuated by TCH-1116, whereas TCH-1116 did not affect the PKC isoforms membrane translocation, phosphorylation (Ser660) and kinase activity. TCH-1116 effectively attenuated the association between PKB/Akt (protein kinase B) and p47phox, Akt phosphorylation (Thr308/Ser473) and kinase activities of Akt and human recombinant PDK (3-phosphoinositide-dependent kinase) 1, whereas it had no effect on recruitment of Akt, phospho-PDK1 (Ser241) and p110γ to membrane. Moreover, the interaction of p21-activated kinase (PAK) 1 with p47phox and the phosphorylation of PAK1 (Thr423 but not Ser144) were inhibited by TCH-1116, but without affecting the membrane recruitment of PAK1. The cellular cyclic AMP level was not changed by TCH-1116. Taken together, these results suggest that TCH-1116 inhibits fMLP-stimulated O2- generation in rat neutrophils through the blockade of PKC, Akt and PAK signaling pathways.