FIGURE 2.

Neutrophils cultured in hyperglycemic medium generate significantly increased superoxide, which is regulated by iPLA₂. A, HL-60 cells were differentiated into neutrophils by incubation with 1.25% DMSO for 6 d. The cells were then cultured under NG (5 mM), HG (25 mM), S100B (5 μg/ml), HG + S100B, and 25 mM mannose (MANN) as an osmotic control. After 24 h, the cells were washed and resuspended in PBS and were incubated with BEL 20 μM for 15 min at 37°C. The cells were stimulated with 1 μM fMLP, and superoxide generation was evaluated in nanomoles of O₂⁻. Results are expressed as mean ± SEM for n=4. B, Neutrophils (25 × 10⁶/ml) were incubated with either BEL (20 μM) or propranolol hydrochloride (150 μM) for 15 min at 37°C. Cells were washed once and resuspended in PBS and were stimulated with 1 μM fMLP. Superoxide release was evaluated by the reduction of cytochrome C read at 550 nM. Results are expressed as mean ± SEM for n= 3. *p < 0.05; **p < 0.01 (one-way ANOVA between groups NG, S100B, S100B+HG, and mannitol). #p<0.05; ##p<0.01 (one-way ANOVA between stimulated and BEL-treated groups).