Endothelial cells can synthesize leukotriene B4

Abstract

Leukotriene B4 (LTB4) from vascular endothelium may play a key role in the genesis of atherosclerotic lesions. However, the ability of this tissue to synthesize LTB4 is controversial. To resolve this issue arachidonic acid metabolism was characterized in cultures of confluent monolayers of a rabbit aortic endothelial cell line by use of both high-pressure liquid chromatography and radioimmunoassay. Cells were grown to confluence in Dulbecco's modified Eagle's medium/Ham's F12 with 5% fetal bovine serum. Lipoxygenase activity was studied by placing the cells in Hank's balanced salt solution with 2 μmol/L indomethacin. After 30 minutes preincubation with indomethacin cells were exposed to either arachidonic acid (10 μmol/L) or arachidonic acid labeled with radioactive carbon (14C) (1 μCi; SA 58 mCi/mmol) and then stimulated with 9.5 μmol/L calcium ionophore A23187 for 55 minutes. Studies of the cyclooxygenase activity were performed without preincubating with indomethacin. Samples were prepared for high-pressure liquid chromatography by evaporation to dryness under a vacuum and resuspending in 2 ml of 1:1 methanol/water. Tritium-labeled standards were added before loading the 14C-labeled samples on the column. Radiolabeled arachidonic acid metabolites were separated by high-pressure liquid chromatography and detected by means of a dual channel flow-through radiodetector that monitors both 14C and 3H. Based on coelution with authentic standards three lipoxygenase metabolites of arachidonic acid have been identified: LTB4, 12- and 5-hydroxyeicosatetraneoic acid. Leukotriene B4 was further characterized by ultraviolet spectral analysis and inhibition studies with use of nordihydroguaiaretic acid. Quantitation was facilitated by commercially available radioimmunoassay kits. An average of 600 pg LTB4/106 cells was measured from separate experiments. Negative controls produced no LTB4. These data demonstrate that rabbit aortic endothelial cells when stimulated with calcium ionophore in the presence of exogenous arachidonic acid synthesize LTB4, an inflammatory mediator that may play an important role in the pathogenesis of atheroslcerosis.