Calphostin C inhibits 5-LO product formation and 5-LO redistribution in MM6 cells and PMNLs.

To determine the effects on 5-LO product formation or translocation, the indicated cell numbers (in 1 mL PGC buffer) were incubated with the indicated concentrations of calphostin C for 30 minutes at 37°C under ordinary fluorescent light. Then PMA (100 nM) was added for the indicated times before stimulation with ionophore A23187. 5-LO product formation was determined by HPLC. Results are given as mean + SE; n = 3. Alternatively, cells were fractionated, and soluble and nuclear fractions were analyzed for 5-LO by immunoblotting as described. (A) Effect on 5-LO product formation in MM6 cells (3 × 10⁶ in 1 mL). Cells were primed with PMA for 10 minutes, before stimulation with 5 μM ionophore (●) or 5 μM ionophore plus 40 μM AA (○) for 10 minutes. (B) Effect on 5-LO product formation in PMNLs (5 × 10⁶ in 1 mL). Cells were primed with PMA for 5 minutes, before stimulation with 0.1 μM ionophore (○) for 10 minutes. Nonprimed cells were stimulated with 2.5 μM ionophore (●) or 2.5 μM ionophore plus 40 μM AA (▾) for 10 minutes. (C) Effect on translocation in MM6 cells (1 × 10⁷ in 1 mL). Cells were primed with PMA for 10 minutes, before stimulation with 5 μM ionophore for 5 minutes. Similar results were obtained in 2 additional experiments. (D) Effect on translocation in PMA-primed PMNLs (3 × 10⁷ in 1 mL). Cells were primed with PMA for 5 minutes, before stimulation with 0.1 μM ionophore for 5 minutes. Similar results were obtained in 2 additional experiments. (E) Effect on translocation in PMNLs stimulated only with ionophore (3 × 10⁷ cells in 1 mL). Nonprimed cells were stimulated with 2.5 μM ionophore for 5 minutes. Similar results were obtained in one additional experiment.