title: 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 GTPgammaS-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 p110gamma membrane translocation. The membrane recruitment of protein kinase C (PKC)-alpha, -betaI, -betaII, -delta and -zeta, 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.
authors.affiliation: Institute of Medicine, Chung Shan Medical University, Taichung 403, Taiwan, ROC.
authors.forename: Ling-Chu
authors.initials: LC
authors.lastname: Chang
authors.forename: Ruey-Hseng
authors.initials: RH
authors.lastname: Lin
authors.forename: Li-Jiau
authors.initials: LJ
authors.lastname: Huang
authors.forename: Chi-Sen
authors.initials: CS
authors.lastname: Chang
authors.forename: Sheng-Chu
authors.initials: SC
authors.lastname: Kuo
authors.forename: Jih-Pyang
authors.initials: JP
authors.lastname: Wang
mesh_terms: D000818:Animals; D002474:Cell-Free System / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D007191:Indazoles / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: C544694:2-benzyl-3-(4-hydroxymethylphenyl)indazole; D007191:Indazoles; D013481:Superoxides; D000242:Cyclic AMP; D019255:NADPH Oxidases; D051057:Proto-Oncogene Proteins c-akt; D054462:p21-Activated Kinases; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D006152:Cyclic GMP