SYNTHESIS OF HYDROXYEICOSATETRAENOIC (HETES) AND EPOXYEICOSATRIENOIC ACIDS (EETS) BY CULTURED BOVINE CORONARY-ARTERY ENDOTHELIAL-CELLS

Endothelial cells release several factors which influence vascular ton e, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. Howeve r, many of the endothelial metabolites of arachidonic acid have not be en positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endoth elial cells. Cultured bovine coronary artery endothelial cells were in cubated with [C-14]arachidonic acid. The incubation media was extracte d and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). T he cells synthesized 6-keto prostaglandin (PG)F-1 alpha, PGE(2), 12-hy droxyheptadecatrienoic acid (HHT), 12-, 15-, and 11-hydroxyeicosatetra enoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrien oic acids (EET). Several of the HETEs were further analyzed by chiral- phase HPLC. The cells synthesized predominately 12(S)-, 15(S)-, and 11 (R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE su ggested that the 12- and 15-lipoxygenases were present in these cells. 11(R)-HETE is probably derived from cyclooxygenase. They also synthes ized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETE s and EETs were confirmed by mass spectrometry. The release of 6-keto PGF(1 alpha), and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all cond itions, the release of 6-keto PGF(1 alpha) exceed the release of 15-HE TE. Therefore, cultured bovine coronary artery endothelial cells synth esize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.