SA induces 5-LO product formation and p38 MAPK activation.
(A) To determine 5-LO product formation, freshly isolated PMNLs (5 × 106 in 1 mL PGC buffer) were stimulated with SA for 3 minutes at 37°C. After addition of 40 μM AA the samples were incubated for another 5 minutes and 5-LO products were determined by HPLC. Results are given as mean + SE (n = 3-4). Student t test; *P < .05; **P < .01. To determine activation of p38 MAPK, freshly isolated PMNLs (5 × 106 in 100 μL PGC buffer) were stimulated with SA for 3 minutes at 37°C. Incubations were terminated by addition of the same volume of SDS-b. Samples were electrophoresed and analyzed for dually phosphorylated p38 MAPK by immunoblotting (upper panel); equal amounts of protein were evaluated with anti-p38 MAPK antibodies (lower panel). Results are representative of at least 3 separate experiments. (B) Typical HPLC chromatograms of 5-LO products extracted from PMNLs. Cells were stimulated for 5 minutes at 37°C with 40 μM AA alone (top), 10 μM SA for 3 minutes and subsequent addition of 40 μM AA (middle), or with 2.5 μM ionophore plus 40 μM AA (bottom); 1, prostaglandin B2; 2,3,trans-isomers of LTB4; and 4, LTB4were recorded at 280 nM, and 5, 5-H(p)ETE at 235 nM. (C) Activation of p38 MAPK-regulated 5-LO kinases. Samples prepared for determination of p38 MAPK activation from above were analyzed by in-gel kinase assay as described. Arrows indicate the positions of MK2 and MK3. Results are representative of at least 3 separate experiments.
