Activation of phosphoinositide 3-kinase and Src family kinase is required for respiratory burst in rat neutrophils stimulated with artocarpol A

Artocarpol A (ART), a natural product isolated from Artocarpus rigida, stimulated superoxide anion (O2*-) generation, which was inhibited by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase (PI3K) inhibitor, in rat neutrophils. ART stimulated phosphorylation of protein kinase B (PKB/Akt) on both T308 and S473 residues, and LY 294002 inhibited these effects. Rat neutrophils expressed both class IA PI3K subunits (p85, p110alpha, p110beta, and p110delta) and a class IB PI3K subunit (p110gamma) as assessed by a combination of Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) approaches. Stimulation of neutrophils with ART evoked phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) formation, which reached a maximal level at 2 min and was attenuated by LY 294002, as evidenced by immunofluorescence microscopy and by flow cytometry. Detectable membrane-association of class IA PI3Ks, class IB PI3K and Ras was seen as early as 1.5, 0.5 and 1.5 min, respectively, after stimulation with ART. The kinetics of ART-induced Ras activation paralleled the kinetics of class IA PI3Ks recruitment to membrane caused by ART, and the p85 and p110gamma immunoprecipitates contain Ras. ART stimulated Src family kinase activation, which was detectable within 1.5 min of incubation with ART. Both Src kinase activity and PtdIns(3,4,5)P3 formation in ART-stimulated neutrophils were inhibited by 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine (PP1 analog). PP1 analog also attenuated the ART-stimulated O2*- generation in rat neutrophils. These results indicate that the stimulation of respiratory burst by ART in neutrophils implicates PI3K signaling.