Differential requirements of PI3K and Akt for PMA-induced O2− generation. (A) Neutrophils from WT, Akt1−/−, and Akt2−/− mice were stimulated with PMA (A), and O2− generation was recorded as counts per minute (CPS) on the basis of isoluminol-enhanced chemiluminescence. (B-C) WT mouse neutrophils were preincubated for 10 minutes with the PI3K inhibitor LY 294002 (B) or the Akt inhibitor SH X (C) at the indicated concentrations. The cells were then stimulated with PMA. O2− generation was recorded on the basis of isoluminol-enhanced chemiluminescence. The traces shown are representative of 2 independent experiments. (D) Neutrophils from WT mice were preincubated for 10 minutes with SB203580 (SB), GF109203x (GF), or vehicle (dimethyl sulfoxide, same concentration) and then stimulated with PMA. O2− generation was recorded on the basis of isoluminol-enhanced chemiluminescence. The traces shown are representative of 2 independent experiments. (E) The effect of SB203580 and GF109203x on PMA-induced Akt phosphorylation (top panel) was determined in neutrophils treated with the inhibitors described previously. PMA stimulation (200 ng/mL) was for 10 minutes. For comparison, neutrophils were also stimulated with C5a (100nM C5a). The relative level of Akt phosphorylation at Ser473 was determined on the basis of 3 experiments and are shown in the bar graph as mean ± SEM *P < .05 compared with the NS (no PMA or C5a stimulation) sample; #P < .05 compared with the PMA-stimulated sample. (F) Neutrophil from WT, Akt1−/−, and Akt2−/− mice were stimulated with different concentrations of PMA, and degranulation assay was performed as described in “Degranulation.” The percentage of released β-glucuronidase was determined and shown as mean ± SEM from 3 experiments, each in duplicate. *P < .05, compared to WT neutrophils.
