A novel synthetic phenazine carboxylate derivative TCH inhibited formyl-Met-Leu-Phe (fMLP)-stimulated superoxide anion generation in rat neutrophils in a concentration- but not a time-dependent manner with IC50 value about 4.5 ± 0.8 μM. This inhibitory effect was not owing to the decrease in cell viability, scavenging of generated superoxide anion or direct blockade of NADPH oxidase activity. Under the same IC50 value, TCH inhibited the interaction of p47phox and Rac2 with p22phox and gp91phox, respectively, and the phosphorylation of p47phox, p21-activated kinase 1 (PAK1) and Vav in fMLP-stimulated cells. TCH had no effect on the phosphorylation of p38 mitogen-activated protein kinase and MAPK-activated protein kinase-2. Pretreatment of cells with TCH inhibited the interaction of Akt with p47phox, the phosphorylation of Akt and Akt activity, whereas, it did not affect the Akt activity in the cell-lysates of fMLP-stimulated cells. TCH inhibited the enzymatic activity of active human recombinant PDK1, whereas, it had no effect on the recruitment of Akt and PDK1 to membrane. TCH attenuated the interaction of PKC-α、-δ and -ζ with p47phox, whereas, it did not affect the association of PKC-β with p47phox. TCH decreased the membrane recruitment of PKC-α but not of PKC-β、-δ and -ζ. TCH had neither block the PKC activity nor increase the cellular cyclic AMP levels of fMLP-stimulated cells. Taken together, TCH probably attenuate PDK1/Akt, PKC and PAK1 signalings and Vav pathway leading to the inhibition of p47phox phosphorylation and Rac2 activation, respectively, which in turn blockade the assembly of active NADPH oxidase and then superoxide anion generation.
