Figure 3.

Myrtucommulone (MC) inhibits microsomal prostaglandin E₂ synthase (mPGES)-1 in a reversible and substrate concentration-independent manner. (A) Microsomal preparations of interleukin-1β-stimulated A549 cells were pre-incubated with 3 µmol·L⁻¹ MK-886 or MC, or with vehicle (dimethyl sulphoxide, DMSO, w/o) for 15 min at 4°C, each. Then, one aliquot of the samples was diluted with assay buffer 10-fold, whereas the other one was not altered, and 20 µmol·L⁻¹ PGH₂ was added to start the reaction. For comparison, microsomal preparations were pre-incubated for 15 min at 4°C with 0.3 µmol·L⁻¹ MK-886 or MC, or with vehicle (DMSO), and then 20 µmol·L⁻¹ PGH₂ was added (no dilution). Then, all samples were incubated for 1 min on ice, and PGE₂ formation was analysed as described. Data are given as mean + SE, n = 3, *P < 0.05, anova + Tukey HSD post hoc tests. (B) The potency of MC for mPGES-1 inhibition was compared at 1 and 20 µmol·L⁻¹ PGH₂ as substrate. In a variation of the general procedure, the amount of PGE₂ was quantified for 1 µmol·L⁻¹ PGH₂ by use of a PGE₂ high sensitivity EIA Kit according to the manufacturer's protocol. Data are given as mean ± SE, n = 3–4. (C) The activity of mPGES-1 was determined at different PGH₂ and different MC concentrations, as indicated (left panel). In a variation of the general procedure, the amount of PGE₂ was quantified by use of a PGE₂ high sensitivity EIA Kit according to the manufacturer's protocol. Data are given as mean ± SE, n = 3–4. Eadie-Hofstee analysis (right panel) was performed by using the original data.