Figure 4. β-NAD⁺_e-induced superoxide and nitrite generation in human granulocytes.

(A) Granulocytes were treated for 30 min at 37 °C with β-NAD⁺_e at the concentrations indicated, and O₂⁻ generation (white bars) was measured. For nitrite measurements, granulocytes were incubated for 60 min at 37 °C with β-NAD⁺_e at the concentrations indicated (grey bars), or pre-incubated for 90 min with 100 μM 8-Br-cADPR and then stimulated for 60 min at 37 °C with 100 μM β-NAD⁺_e (black bar). For both of these parameters, results are expressed as percentages of control values measured in unstimulated cells, and are the means±S.D. for four different experiments. The basal value of O₂⁻ production was 10.67±5.65 ΔmAU (n=15); the basal concentration of nitrite was 0.76±0.43 μM (n=14). (B) Before stimulation with β-NAD⁺_e, granulocytes were pre-incubated at 25 °C as follows: with 100 μM 8-Br-cADPR or with 50 μM ryanodine for 90 min; with 50 μM Cibacron Blue for 20 min; with 5 μM U73122 for 10 min; or with 20 mM nicotinamide for 1 min. Pre-treated and untreated (control) granulocytes were then incubated for 30 min at 37 °C with or without 100 μM β-NAD⁺_e, and the O₂⁻ production was measured. Results are expressed as the percentage of inhibition observed with each compound, and are the means±S.D. for at least three experiments.