Figure 2.

Effect of the test compounds on the biosynthesis of 5-LO products in transfected HEK293 cells (a) and thapsigargin-stimulated PMNL (b). (a) HEK293 cells expressing both 5-LO and FLAP were incubated in presence of the indicated compounds (1 μM) or their diluent (control, 0.5% DMSO) for 5 min, then biosynthesis of 5-LO products was initiated with the addition of 10 μM calcium ionophore A23187 and 10 μM arachidonic acid for 15 min. After stimulation, reactions were stopped and samples were processed for analysis of 5-LO products by RP-HPLC. Total 5-LO products measured represent the sum of LTB₄, its trans isomers, and 5-hydroxyeicosatetraenoic acid. (b) Freshly isolated human PMNL were incubated in presence of test compounds (or their diluent control, DMSO 0.5%) at the indicated concentrations for 5 min. Biosynthesis of the 5-LO products was initiated with the addition of 1 μM of thapsigargin, followed by a 15 min incubation period at 37°C. The stimulation was then stopped and samples were processed for RP-HPLC analysis and total 5-LO products quantification. Total 5-LO products measured represent the sum of LTB₄, its trans isomers, 20-COOH- and 20-OH-LTB₄ and 5-hydroxyeicosatetraenoic acid. Data are expressed as means ± SEM of at least 3 independent experiments. ^∗Different from control, as determined by one-way ANOVA with Dunnett's multiple comparison test (p < 0.05).