Induction of prostaglandin endoperoxide synthase‐2 in human monocytes associated with cyclo‐oxygenase‐dependent F₂‐isoprostane formation

Abstract

1 The isoprostane 8-epi-prostaglandin (PG)F2a is produced by free radical-catalyzed peroxidation of arachidonic acid. It may also be formed as a minor product of the cyclo-oxygenase activity of platelet PGH synthase (PGHS)-l. We investigated 8-epi-PGF2. production associated with induction of the human monocyte PGHS-2 and its pharmacological modulation. 2 Heparinized whole blood samples were drawn from healthy volunteers, 48 h following oral dosing with aspirin 300 mg to suppress platelet cyclo-oxygenase activity. One ml aliquots were incubated with lipopolysaccharide (LPS: 0.1-50 ug ml-') for 0-24 h at 37'C. PGE2 and 8-epi-PGF2a were measured in separated plasma by radioimmunoassay and enzyme immunoassay techniques. 3 Levels of both eicosanoids were undetectable (i.e. <60 pg ml-') at time 0. LPS induced the formation of PGE2 and 8-epi-PGF2, in a time-and concentration-dependent fashion, coincident with the induction of PGHS-2 detected by Western blot analysis of monocyte lysates. After 24 h at 10 mig ml-' LPS, immunoreactive PGE2 and 8-epi-PGF2, averaged 10,480+4,643 and 295+ 140 pg ml-' (mean + s.d., n = 6), respectively. 4 Dexamethasone-and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)-l-indanone (L-745,337), a selective inhibitor of the cyclo-oxygenase activity of PGHS-2, reduced PGE2 and 8-epi-PGF2. production in response to LPS.5 Isolated monocytes produced PGE2 and 8-epi-PGF2, in response to LPS (10 Mg ml-') in a timedependent fashion. Monocyte PGE2 and 8-epi-PGF2a production was largely prevented by dexamethasone (2 gM) and cycloheximide (10 Mg ml-') in association with suppression of PGHS-2 but not of PGHS-1 expression. 6 We conclude that the induction of PGHS-2 in human monocytes is associated with cyclo-oxygenasedependent generation of the vasoconstrictor and platelet-agonist 8-epi-PGF2,.

Pathophysiology of isoprostanes in the cardiovascular system: implications of isoprostane‐mediated thromboxane A2 receptor activation

“…In human monocytes, LPS induced the formation of PGE2 and the isoprostane 8-iso-PGF2α in a time-and dose-dependent manner, accompanied with the induction of COX-2 (PGHS-2). Incubation with the selective inhibitor of COX-2, L-745,337, decreased the production of PGE2 and 8-iso-PGF2α, indicating that the induction of COX-2 in monocytes is associated with an increased production of isoprostanes (Patrignani et al, 1996). The role of COX-2 in the formation of isoprostanes has been confirmed also by other groups.…”

Isoprostanes: Formation, Analysis and Use As Indices of Lipid Peroxidation in Vivo

“…An assay which coincidentally measured an F 2 -iP and a D 2 /E 2 -iP might allow correction for such differences in redox status if they occurred. A more immediate consideration is that iPF 2␣ -III can be formed by either COX isoform, in vitro and ex vivo (30,31,37,40), and COX activation and oxidant stress often coincide (41,42). The COX-dependent pathway does not appear to contribute to urinary iPF 2␣ -III, even in syndromes of COX activation (43,44).…”

Vascular Biology of the Isoprostanes

“…Isoprostaglandin F 2α type VI (IPF 2α -VI) has been clearly shown to be a free-radical product of arachidonic acid. However, several in vitro studies have described a cyclooxygenase-dependent formation of isoprostaglandin F 2α type III (iPF 2α -III) [11, 12, 13, 14, 15]. An efficient in vivo production of iPF 2α -III through the cyclooxygenase pathway would reduce the accuracy of iPF 2α -III as a valid marker of lipid peroxidation.…”