Role of 8-epi PGF2α, 8-isoprostane, in H2O2-induced derangements of pulmonary artery endothelial cell barrier function

The non-enzymatic peroxidation product of arachidonic acid, 8-epi-PGF(2 alpha) or 8-isoprostane (8-IP) was measured in H2O2-exposed cultured pulmonary artery endothelial cell (PAEC) monolayers using a commercially-available enzyme immunoassay kit. H2O2 (50 mu M for 1-30 min) significantly increased 8-IP production in a time-dependent fashion. Treatment with higher H2O2 concentrations (100 or 250 mu M) failed to further increase 8-IP generation. Determinations of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH) were not sufficiently sensitive to detect lipid peroxidation in PAEC exposed to 50 mu M H2O2 for 15 min. 8-IP (100 pM-500 nM for 2 h) caused PAEC monolayer barrier dysfunction measured as the transmonolayer clearance of albumin without causing significant PAEC cytotoxicity (measured as intracellular lactate dehydrogenase release). This is the first report to provide evidence that 8-IP generated in H2O2-exposed PAEC contributes to oxidant-mediated alterations in monolayer barrier function at non-cytotoxic concentrations.