Inhibition of formyl peptide-stimulated superoxide anion generation by Fal-002-2 occurs mainly through the blockade of the p21-activated kinase and protein kinase C signaling pathways in ratneutrophils.
In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, a synthetic compound, 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), inhibited superoxide anion (O2(•-)) generation with an IC50 value of about 11μM, which was not mediated by scavenging the generated O2(•-) or by a cytotoxic effect on neutrophils. Fal-002-2 effectively attenuated the phosphorylation of Ser residues in p47(phox) and the association between p47(phox) and p22(phox) in fMLP-stimulated neutrophils. The interaction of p47(phox) with protein kinase C (PKC) isoforms (α, βI, βII, δ and ζ) was attenuated by Fal-002-2 with a similar IC50 value to that required for inhibition of O2(•-) generation, whereas Fal-002-2 had no prominent effect on PKC isoform membrane translocation and did not affect the kinase activity. Moreover, Fal-002-2 had no effect on the phosphorylation of Akt and downstream glycogen synthase kinase-3β, only slightly affected the intracellular free Ca(2+) concentration, phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase (MAPK), but effectively attenuated the downstream MAPK-activated protein kinase-2 phosphorylation. The interaction of p21-activated kinase (PAK) 1with p47(phox), phosphorylation of PAK1 (Thr423/Ser144) and the membrane recruitment of PAK1 were effectively inhibited by Fal-002-2. Fal-002-2 also blocked the activation of Rac1 and Cdc42 in a concentration range that effectively inhibited PAK activation. Taken together, these results suggest that Fal-002-2 inhibits fMLP-stimulated O2(•-) generation in neutrophils mainly through the blockade of PKC and PAK signaling pathways and partly through p38 MAPK signaling.
Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China.
Ya-Ru
YR
Tsai
Li-Jiau
LJ
Huang
Hui-Yi
HY
Lin
Yun-Jie
YJ
Hung
Miau-Rong
MR
Lee
Sheng-Chu
SC
Kuo
Mei-Feng
MF
Hsu
Jih-Pyang
JP
Wang
D000818:Animals; D002118:Calcium / Q000378:metabolism; D040502:Calgranulin B / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011804:Quinolines / Q000494:pharmacology*; D015363:Quinolones / Q000494:pharmacology*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000378:metabolism*
C582906:6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate; D040502:Calgranulin B; D011804:Quinolines; D015363:Quinolones; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000242:Cyclic AMP; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D051057:Proto-Oncogene Proteins c-akt; D054462:p21-Activated Kinases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium