Inhibition of superoxide anion generation by CHS-111 via blockade of the p21-activated kinase, protein kinase B/Akt and protein kinase C signaling pathways in rat neutrophils

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111) inhibited superoxide anion (O2−) generation, which was not mediated by scavenging the generated O2− or by a cytotoxic effect, and attenuated migration. CHS-111 had no effect on the arachidonic acid-induced NADPH oxidase activation or the GTPγS-stimulated Rac2 membrane translocation in cell-free systems, whereas it effectively attenuated the membrane recruitment of p40phox, p47phox and p67phox, phosphorylation of Ser residues in p47phox, association between p47phox and p22phox, and Rac activation in fMLP-stimulated neutrophils. Moreover, the phosphorylation and membrane recruitment of p21-activated kinase (PAK), PAK kinase activity and the interaction of PAK with p47phox were inhibited by CHS-111. CHS-111 effectively reduced Akt kinase activity and the association between Akt and p47phox, moderately inhibited the membrane recruitment of Akt and phospho-PDK1, and slightly attenuated Akt (Thr308) phosphorylation, whereas it had no effect on Akt (Ser473) phosphorylation or p110γ membrane translocation. The membrane recruitment of protein kinase C (PKC)-α, -βI, -βII, -δ and -ζ, PKC phosphorylation and PKC kinase activity was attenuated by CHS-111, whereas CHS-111 did not affect the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or downstream MAPK-activated protein kinase-2. Higher concentrations of CHS-111 were required to decrease fMLP-stimulated intracellular free Ca2+ concentration ([Ca2+]i) elevation in the presence but not in the absence of extracellular Ca2+, and to reduce cellular cyclic AMP but slightly increase cyclic GMP levels. Taken together, these results suggest that CHS-111 inhibits fMLP-stimulated O2− generation in rat neutrophils through the blockade of PAK, Akt and PKC signaling pathways.