Figure 2.

Diflapolin inhibits epoxide hydrolase activity of sEH. (a) The epoxide hydrolase activity of human recombinant sEH was analyzed in fluorescence-based cell-free assay. Diflapolin, AUDA (300 nM), or DMSO (vehicle, 0.1%) was added to sEH and after 10 min at 4 °C, the reaction was started by addition of the substrate (50 µM PHOME) and stopped after 60 min before analyzing the fluorescent product. Data are expressed in percentage of control and are given as means ± S.E.M., n = 3–4, ***p < 0.001; vs vehicle (paired t-test). (b/c) sEH activity was analyzed in intact HepG2 cells and for recombinant sEH. Cells and enzyme were pre-incubated with DMSO (vehicle, 0.1%), diflapolin (indicated concentrations), SC57464A (0.3 µM), AUDA (5 µM) or MK886 (0.3 µM), and then incubated with 14,15-EET. Amounts of 14,15-EET and 14,15-DiHETrE were analyzed by UPLC-MS/MS. Data are expressed as percentage of control and are given as means ± S.E.M., n = 3, *p < 0.05; versus vehicle (paired t-test). (d) Phosphatase activity of sEH was analyzed in a fluorescence-based cell-free assay. Diflapolin or DMSO (vehicle, 0.1%) was added to human recombinant phosphatase domain of sEH for 10 min at 4 °C, the reaction was initiated by addition of DiFMUP (300 µM) and fluorescence was analyzed for 45 min. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3 *p < 0.05 vs. vehicle control (paired t-test). (e) Co-incubations of human recombinant 5-LOX and LTA₄-H in PBS plus 1 mM EDTA pre-incubated with diflapolin (10 µM), AUDA (10 µM), SC57461A (0.3 µM), zileuton (3 µM), or vehicle (0.1% DMSO) for 10 min on ice and subsequently stimulated by 20 µM AA and CaCl₂ for 10 min at 37 °C. LTB₄ isomers were analyzed by HPLC. Data are expressed as percentage of vehicle control (100%), are given as means ± S.E.M., n = 3–4. *p < 0.05 vs. vehicle control (paired t-test with logarithmized values).