Figure 4.

Influence of the redox state on the inhibition of 5-LOX induced by RF-Id. (A) Reduction of the DPPH radical (5 nmol) by RF-Id and RF-Ic, L-cysteine and ascorbic acid. Data are expressed as percentage of DPPH radical (100%); means ± SEM; n = 3. (B) Neutrophil homogenates (left panel) or partially purified human recombinant 5-LOX (right panel) were incubated with RF-Id (or DMSO as vehicle) for 15 min at 4°C. 5 min before stimulation, 1 mM DTT was added as indicated. After addition of 1 mM ATP, samples were warmed up for 30 s at 37°C and stimulated with 2 mM CaCl₂ and 20 μM AA for 10 min at 37°C. Data are expressed as percentage of vehicle control (0.1% DMSO); means ± SEM; n = 3. *P < 0.05, ***P <0.001 DTT treatment versus without, at indicated concentrations. (C) Partially purified 5-LOX was incubated with RED-RF-Id (or DMSO as vehicle) for 15 min at 4°C. 5 min before stimulation, 1 mM DTT was added as indicated. After addition of 1 mM ATP, samples were warmed up for 30 s at 37°C and stimulated with 2 mM CaCl₂ and 20 μM AA for 10 min at 37°C. Data are expressed as percentage of vehicle control; means ± SEM; n = 3. (D) To intact neutrophils, 500 μM diamide or vehicle (DMSO) was added 7.5 min before addition of RF-Id (left panel) or zileuton (right panel) at 37°C. After addition of compounds for another 7.5 min at 37°C, cells were stimulated with 2.5 μM A23187 plus 20 μM AA for 10 min at 37°C. Data are expressed as percentage of vehicle control (0.1% DMSO); means ± SEM; n = 3. *P <0.05, ***P <0.001 diamide treatment versus without, at indicated concentrations. (E) Neutrophils were pre-incubated with RF-Id (left panel) or zileuton (right panel) at 37°C. After 15 min, either 2.5 μM A23187 plus 40 μM AA or 0.3 M NaCl plus 40 μM AA was added, as indicated. After 10 min at 37°C, 5-LOX products were determined. Data are expressed as percentage of vehicle control (0.1% DMSO); means ± SEM; n = 3. ***P <0.001 A23187 versus NaCl at indicated concentrations.