| The chemotactic peptide N-formyl-met-leu-phe (fMLP) is known to induce various responses in neutrophils, including superoxide production catalyzed by NADPH oxidase, migration and actin polymerization. These responses are initiated by binding of fMLP with G protein-coupled receptor, which leads to the rapid activation of phospholipase C (PLC), a increase in the concentration of cytosolic free Ca2+, and the activation of protein kinase C (PKC). It is essential for the activation of NADPH oxidase that p47phox, a cytosolic component of NADPH oxidase, is phosphorylated by PKC and translocates to the plasma membrane to form an active complex with other cytosolic components and the membrane oxidase subunits. Parallel to the activation of PKC, the activation of phosphatidylinositide 3-kinase (PI3-K) also accurs after stimulation and induces the activation of protein kinase B (PKB, also called Akt) which was suggested to activate NADPH oxidase. It was also demonstrated that PI3-K participates in the regulation of neutrophil chemotaxis. Moreover, there is some evidence that mitogen-activated protein kinases (MAPK) such as p38 MAPK and extracellular response kinase (ERK) also regulate the activation of NADPH oxidase and the chemotaxis. However, the effects of intracellular Ca2+, PKC, PI3-K, p38 MAPK and ERK on the activation of NADPH oxidase and polymerization of actin are still unclear.In this study, the roles of these signaling moleculars in fMLP-induced the activation of NADPH oxidase and the polymerization of actin in neutrophilic HL-60 cells were investigated by using a chelator of intracellular Ca2+, and inhibitors of PKC, PI3-K, p38 MAPK and ERK. The effects of the chelator and inhibitors of PKC and PI3-K on fMLP-induced the activation of Akt were also examined.To evaluate the effects of intracellular Ca2+ and these kinases on the activation of NADPH oxidase, the influence of intracellular Ca2+ chelator and the kinase inhibitors on the activation of NADPH oxidase and the kinases were investigated in differentiated HL-60. 10 y mol/L BAPTA-AM, a chelator of intracellular Ca2+, significantly attenuated the fMLP-induced O2- generation; 8u mol/L GF109293x (PKC inhibitor) evidently inhibited O2- generation; 50 y mol/L SB203580 (p38 MAPK inhibitor), 50 y mol/L PD098059 (ERK inhibitor) and 0.1 y mol/L wortmannin (PI3-kinase inhibitor) respectively attenuated about 50%, 60% and 30% of fMLP-induced 62- generation. Pretreatment of cells with inhibitors of PKC, PI3-K, p38 MAPK and ERK did not inhibited the fMLP-induced response of intracellular Ca2+, though the pretreatment cells with 10 y mol/L BAPTA-AM markedly attenuated the fMLP-induced elevation of intracellular Ca2+. The activation of p38 MAPK could partially be inhibited in the cells pretreated with 10 y mol/L BAPTA-AM, 8u mol/L GF109293x or 0.1 y mol/L wortmannin. However, the activation of ERK and Akt was obviously inhibited only in the cells pretreated with 0.1 y mol/L wortmannin. Taken together, these results suggest that the Ca2+/PKC-dependent pathway is important in the fMLP-induced activation of NADPH oxidase in the differentiated HL-60 cells, while PI3-K-dependent pathway, including ERK, Akt, is also important but only partly participate in the activation of NADPH oxidase. The activation of these kinases was not essential to the fMLP-induced response of intracellular Ca2+. The activation of ERK and Akt is mainly regulated by PI3-K, while the activation of p38 MAPK may be regulated by intracellular Ca2+, PKC and PI3-K.In order to further understand the mechanism of activation of NADPH oxidase by p38 MAPK, the effects of p38 MAPK on the fMLP-induced phosphorylation of p47phox and translocation of p47phox to the cell membrane were studied with the inhibitor SB203580 in differentiated HL-60 cells. It was found that the kinetics of activation of p38 MAPK coincided with the kinetics of activation of NADPH oxidase, 50 y mol/L SB203580 could markedly inhibit the phosphorylation of p47phox in vitro and the translocation ofp47phox to the cell membrane. These...
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