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

Abstract

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111) inhibited superoxide anion (O 2 − ) generation, which was not mediated by scavenging the generated O 2 − 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 p40 phox, p47 phox and p67 phox, phosphorylation of Ser residues in p47 phox, association between p47 phox and p22 phox, 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 p47 phox were inhibited by CHS-111. CHS-111 effectively reduced Akt kinase activity and the association between Akt and p47 phox, 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 Ca 2+ concentration ([Ca 2+] i) elevation in the presence but not in the absence of extracellular Ca 2+, and to reduce cellular cyclic AMP but slightly increase cyclic GMP levels. Taken together, these results suggest that CHS-111 inhibits fMLP-stimulated O 2 − generation in rat neutrophils through the blockade of PAK, Akt and PKC signaling pathways.