The experiments described in this paper were designed to determine the mechanism underlying the increase in 8-isoprostaglandin F (8-epi-PGF) 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 in the medium was determined by ELISA. The production of 8-epi-PGF decreased by greater than 90% during hypoxia. Upon reoxygenation 8-epi-PGF production increased linearly for 90 min reaching nearly 3 times normoxic levels. When added to the medium during reoxygenation, neither superoxide dismutase nor Tiron, a cell-permeable superoxide scavenger, inhibited 8-epi-PGF production. However, 8-epi-PGF production was inhibited by catalase. The production of 8-epi-PGF was also inhibited by indomethacin and aspirin. Exogenous hydrogen peroxide stimulated 8-epi-PGF production by normoxic cells, and aspirin inhibited the hydrogen peroxide-mediated increase in 8-epi-PGF production. These results indicate that the reactive oxygen species responsible for 8-epi-PGF synthesis during reoxygenation is hydrogen peroxide and that in endothelial cells 8-epi-PGF synthesis is mediated by prostaglandin H2 synthase (PGHS). To verify the role of PGHS in 8-epi-PGF synthesis, human PGHS-1 was expressed in COS-7 cells, a PGHS negative cell line that does not synthesize 8-epi-PGF. In the presence of exogenous arachidonic acid the COS-7 cells expressing human PGHS-1 produced substantial amounts of PGE2 and 8-epi-PGF. These data indicate that human PGHS-1 can support the synthesis of 8-epi-PGF and that 8-epi-PGF synthesis by cultured human endothelial cells during reoxygenation is dependent on the activity of PGHS-1.

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