Abstract

F₂-Isoprostanes are isomers of the prostaglandin PGF_2α. At least one compound of this group, 8-epi-PGF_2α, exhibits biological activity, and therefore special interest is focused on the mechanism of isoprostane formation: enzyme catalyzed or radical mediated. We analyzed the formation of isoprostanes in vitro and in vivo. In both systems, purified cyclooxygenase isoenzymes and cell models specific for the cyclooxygenase isoenzymes, 8-epi-PGF_2α formation could be totally suppressed by cyclooxygenase inhibitors. Indomethacin inhibited concentration-dependent 8-epi-PGF_2α formation in platelets stimulated with calcium ionophore, arachidonic acid or thrombin. Nordihydroguaiaretic acid, an antioxidant, blocked isoprostane formation with a similar IC₅₀ value as thromboxane B₂ synthesis, pointing toward cyclooxygenase as the primary target of inhibition. Based on the turnover number, cyclooxygenase-2 formed higher levels of 8-epi-PGF_2α than cyclooxygenase-1. Endogenous 8-epi-PGF_2α production in rat mesangial cells correlated well with the mRNA and protein expression of cyclooxygenase-2 during interleukin-1 induction. However, in contrast to human platelets, which produced different forms of isoprostanes, rat mesangial cells appeared to form only 8-epi-PGF_2α. Further, this indicates that mesangial cells may represent a cellular origin for renal 8-epi-PGF_2αformation. Next, we analyzed the formation of isoprostanes in humans. A direct correlation was observed between indomethacin treatment and the decrease in 8-epi-PGF_2α and isoprostane levels, but compared with other prostanoids the inhibition was less pronounced. In summary, based on the in vitro studies, a clear cyclooxygenase-dependent formation of isoprostanes, especially 8-epi-PGF_2α, was observed. However, in vivoadditional formation via cyclooxygenase enzyme-independent mechanisms is likely.