Artocarpol A stimulation of superoxide anion generation in neutrophils involved the activation of PLC, PKC and p38 mitogen-activated PK signaling pathways

Artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, stimulated a slow onset and long-lasting superoxide anion generation in rat neutrophils, whereas only slightly activated the NADPH oxidase in a cell-free system. Pretreatment of neutrophils with pertussis toxin (1 μg ml -1), 50 μM 2′-amino-3′-methoxyflavone (PD 98059), or 1 μM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) had no effect on ART-stimulated superoxide anion generation. ART (30 μM) did not induce extracellular signal-regulated kinase (ERK) phosphorylation. 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) markedly attenuated the ART-stimulated superoxide anion generation (IC 50 value of 4.3 ± 0.3 μM). Moreover, ART induced p38 mitogen-activated PK (MAPK) phosphorylation and activation. The superoxide anion generation in response to ART was also substantially inhibited in a Ca 2+-free medium, and by pretreatment with 1 μM 1-[6-((17β-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)-hexyl]-1H-pyrrole-2,5-dione (U-73122) and 100 μM 2-aminoethyldiphenyl borate (2-APB). ART (30 μM) stimulated the [Ca 2+], elevation in the presence or absence of external Ca 2+, and also increased the D-myo-inositol 1,4,5-trisphosphate formation. 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)- maleimide (GF 109203X) greatly inhibited the ART-stimulated superoxide anion generation (IC 50 value of 7.8 ± 1.0 nM). ART increased the recruitment of PKC-α, -βI, and -βII to the plasma membrane of neutrophils, and stimulated Ca 2+-dependent PKC activation in the cytosol preparation. ART induced the phosphorylation of p47 phox, which was attenuated by GF 109203X. Moreover, ART evoked the membrane association of p47 phox, which was inhibited by GF 109203X and SB 203580. These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca 2+, and PKC signaling pathways in rat neutrophils.