Dose response of PMA and mezerein for priming of 5-LO product formation and 5-LO translocation in PMNLs.
(A) To determine the effect on 5-LO product formation, PMNLs (5 × 106 cells in 1 mL PGC buffer) were primed with the indicated concentrations of PMA (●) or mezerein (○) for 5 minutes at 37°C before stimulation with 0.1 μM A23187 for an additional 10 minutes. 5-LO product formation was determined by HPLC. Results are given as mean ± SE; n = 3. (B) To determine the effect on 5-LO product formation in the presence of exogenous AA, PMNLs were primed for 5 minutes at 37°C with (■) or without PMA (▪) (100 nM) before stimulation with 0.1 or 2.5 μM A23187 in the presence of AA (40 μM) for 10 more minutes. Results are given as mean + SE; n = 3. (C) To determine the effect on 5-LO translocation, human PMNLs (3 × 107 in 1 mL PGC buffer) were primed with the indicated concentrations of PMA or mezerein for 5 minutes at 37°C and were subsequently stimulated with 0.1 μM ionophore for 10 minutes. Cells were fractionated, and soluble and nuclear fractions were analyzed for 5-LO by immunoblotting as described. Similar results were obtained in one additional experiment. Student t test; **P < .01.
