PGHS and LO expression and monohydroxy products analysis in A549 cells
(A) PGHS and LO expression in A549 cells. Cells were grown for 24 hr at 37°C in T-75 cm2 flasks in the presence or absence of IL-1β (1 ng/ml). Extracted total RNA (1 µg) was taken for RT and PCR using specific oligonucleotides for PGHS-1 and -2, 15-, 12-, and 5-LO, and GAPDH. Radioactive bands were quantified directly by phosphorimager analysis, normalized to the expression of GAPDH, and expressed as fold increase in mRNA levels after exposure to IL-1β. Inset: 15-LO mRNA expression in human lung tissue and peripheral blood monocytes (PBM). ND, not detected. (B) RP-HPLC profile of [3H]-labeled mono-HETEs from permeabilized IL-1β-treated A549 cells (1.5 µ 106 cells/ml) and exposed to [3H]-arachidonic acid (0.25 µCi/ml) plus unlabeled arachidonic acid (20 µM) for 20 min at 37°C. Products were extracted and chromatographed using a linear gradient of methanol:H2O:acetic acid (65:35:0.01; v/v/v) and methanol:acetic acid (99.99:0.01, v/v) at a flow rate of 1.0 ml/min. Arrows denote co-chromatography of synthetic standards.