Synthesis of 8-epi-prostaglandin F-2 alpha by human endothelial cells: role of prostaglandin H-2 synthase

The experiments described in this paper were designed to determine the mech anism underlying the increase in 8-isoprostaglandin F-2 alpha (8-epi-PGF(2 alpha)) production by cultured human endothelial cells during reoxygenation following hypoxia. Human umbilical artery endothelial cells were grown on microcarrier beads and exposed to sequential periods of normoxia, hypoxia, and reoxygenation. The amount of 8-epi-PGF(2 alpha) in the medium was deter mined by ELISA. The production of 8-epi-PGF(2 alpha) decreased by greater t han 90% during hypoxia. Upon reoxygenation 8-epi-PGF(2 alpha) production in creased linearly for 90 min reaching nearly 3 times normoxic levels. When a dded to the medium during reoxygenation, neither superoxide dismutase nor T iron, a cell-permeable superoxide scavenger, inhibited 8-epi-PGF(2 alpha) p roduction. However, 8-epi-PGF(2 alpha) production was inhibited by catalase . The production of 8-epi-PGF(2 alpha) was also inhibited by indomethacin a nd aspirin. Exogenous hydrogen peroxide stimulated 8-epi-PGF(2 alpha) produ ction by normoxic cells, and aspirin inhibited the hydrogen peroxide-mediat ed increase in 8-epi-PGF(2 alpha) production. These results indicate that t he reactive oxygen species responsible for 8-epi-PGF(2 alpha) synthesis dur ing reoxygenation is hydrogen peroxide and that in endothelial cells 8-epi- PGF(2 alpha) synthesis is mediated by prostaglandin H-2 synthase (PGHS). To verify the role of PGHS in 8-epi-PGF(2 alpha) synthesis, human PGHS-1 was expressed in COS-7 cells, a PGHS negative cell line that does not synthesiz e 8-epi-PGF(2 alpha). In the presence of exogenous arachidonic acid the COS -7 cells expressing human PGHS-1 produced substantial amounts of PGE(2) and 8-epi-PGF(2 alpha). These data indicate that human PGHS-1 can support the synthesis of 8-epi-PGF(2 alpha) and that 8-epi-PGF(2 alpha) synthesis by cu ltured human endothelial cells during reoxygenation is dependent on the act ivity of PGHS-1.