Cyclooxygenases-1 and -2 of endothelial cells utilize exogenous or endogenous arachidonic acid for transcellular production of thromboxane

The presence of prostaglandin (PG) H-2 in the supernatant of human umbilical vein endothelial cells (HUVEC) stimulated by thrombin restores the capacity of aspirin-treated platelets to generate thromboxane (TX) B-2. Induction of cyclooxygenase-2 (Cox-2) by interleukin (IL)-1 alpha or a phorbol ester increases this formation. HUVEC treated with aspirin lost their capacity to generate PGs but recovery occurred after 3 or 6-h induction of Cox-2 with phorbol ester or IL-1 alpha. Enzyme activity of the newly synthesized Cox-2 in aspirin-treated cells, evaluated after immunoprecipitation, was similar to untreated cells but after 18 h of cell stimulation only 50-60% recovery of Cox-1 was observed. The use of SC58125, a selective Cox-2 inhibitor, confirmed these findings in intact cells. Cyclooxygenase activity was related to the amount of Cox proteins present in the cells, but after induction of Cox-25, contribution of the latter to PG production was 6-8-fold that of Cox-1 . Aspirin-treated or untreated cells were incubated in the absence or presence of SC58125 and stimulated by thrombin, the ionophore A23187, or exogenous arachidonic acid. The production of endogenous (6-keto-PGF(1 alpha), PGE(2), PGF(2 alpha)) versus transcellular (TXB(2)) metabolites was independent of the inducer, the source of arachidonic acid and the Cox isozyme. However, in acetylsalicylic acid-treated cells, after 6-h stimulation with IL 1 alpha, newly synthesized Cox-2 produced less TXB(2) than 6-keto-PGF-(1 alpha) compared to untreated cells. At later times (>18 h), there was no metabolic difference between the cells. These studies suggest that in HUVEC, Cox compartmentalization occurring after short-term activation may selectively affect transcellular metabolism, but not constitutive production, of PGs.