Generation of the Isoprostane 8-Epi-prostaglandin F2a In Vitro and In Vivo via the Cyclooxygenases

F2-Isoprostanes are isomers of the prostaglandin PGF2a. At least one compound of this group, 8-epi-PGF2a, 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-PGF2a formation could be totally suppressed by cyclooxygenase inhibitors. Indomethacin inhibited concentration-dependent 8-epi-PGF2a 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-PGF2a than cyclooxygenase-1. Endogenous 8-epi-PGF2a 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-epiPGF2a. Further, this indicates that mesangial cells may represent a cellular origin for renal 8-epi-PGF2a formation. Next, we analyzed the formation of isoprostanes in humans. A direct correlation was observed between indomethacin treatment and the decrease in 8-epi-PGF2a 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-PGF2a, was observed. However, in vivo additional formation via cyclooxygenase enzyme-independent mechanisms is likely. Isoprostanes are recently discovered PG-like products formed from arachidonic acid (Morrow et al., 1990). The isoprostanes consist of stereoisomers and regioisomers of the common PGs and were first reported for PGF2. Later, isoprostanes of the E2 and D2 series were discovered (Morrow et al., 1994). The best characterized isoprostane is 8-epi-PGF2a, which has been found to modulate platelet aggregation (Morrow et al., 1992a); later potent vasoconstriction and bronchoconstriction were described in isolated lungs of rabbit (Banerjee et al., 1992) and rat (Kang et al., 1993). In rats, the intrarenal administration of 8-epi-PGF2a exerted reduction in the glomerular filtration rate and renal plasma flow (Takahashi et al., 1992). Interestingly, these effects can be fully prevented by antagonists of the thromboxane receptor (Banerjee et al., 1992; Takahashi et al., 1992), indicating an interaction with this receptor subtype. Recently, a separate receptor for 8-epi-PGF2a was postulated (Fukunaga et al., 1993). In explaining the formation of these compounds in vivo, two different mechanisms are discussed. On the one hand, a free radical-catalyzed peroxidation of arachidonic acid with endocyclization, leading to a PGG2-like compound, was suggested. Further reduction or rearrangement in the PGG2-like compound results in F2or E2/D2-isoprostane formation (Morrow et al., 1990, 1994). Regarding this mechanism, it has been proposed that detection of isoprostanes might offer a quantitative index for the generation of free radicals and lipid oxidation in vivo (Nourooz Zadeh et al., 1995; Roberts and Morrow, 1994). In accordance with this assumption, elevated levels of isoprostanes have been detected in situations associated with increased free radical generation: during coronary reperfusion and in chronic cigarette smokers (Bachi et al., 1996; Morrow et al., 1995; Takahashi et al., 1992). On the other hand, a COX-dependent isoprostane formation is discussed (Pratico et al., 1995; Pratico and FitzGerald 1996). The COXs are rate-limiting enzymes in the arachidonic acid cascade, providing the endoperoxide PGH2, which in turn is further metabolized by specific enzymes to PGs, Received for publication October 28, 1996. 1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (Nu 73/2–1, Se 263/11–1). ABBREVIATIONS: PG, prostaglandin; COX, cyclooxygenase; NSAID, nonsteroidal anti-inflammatory drug; GC/MS/MS, gas chromatographytandem mass spectrometry; RMC, rat mesangial cells; IL-1, interleukin-1b; TXB, thromboxane; HPS, hyperprostaglandin syndrome; RT, reverse transcription; PCR, polymerase chain reaction. 0022-3565/97/2823-1658$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 3 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:1658–1665, 1997 1658 at A PE T Jornals on A uust 9, 2017 jpet.asjournals.org D ow nladed from thromboxanes and prostacyclin. Several recent reports revealed the existence of two isoforms, COX-1 and COX-2 (for a review, see Otto and Smith, 1995). The former is thought to be constitutively expressed and involved in so-calling housekeeping functions, such as homeostasis, organ function or hormone modulation, whereas the latter is highly inducible and implicated in the signal cross-talk during pathological situations, such as inflammation. For further investigations into the mechanism of the formation of isoprostanes, especially 8-epi-PGF2a, we established a sensitive and specific assay based on GC/MS/MS for the detection of these compounds in diverse enzymatic and cellular systems as well as in humans and determined its formation under different conditions. In all systems used, in vitro and in vivo, we observed a COX-dependent formation of 8-epi-PGF2a. The fact that isoprostane formation in human samples could not be fully prevented by COX inhibitors suggests that further mechanisms are involved in isoprostane formation in vivo.