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. 1999 Dec 15;344(Pt 3):747–754.

Synthesis of 8-epi-prostaglandin F2alpha by human endothelial cells: role of prostaglandin H2 synthase.

M T Watkins 1, G M Patton 1, H M Soler 1, H Albadawi 1, D E Humphries 1, J E Evans 1, H Kadowaki 1
PMCID: PMC1220696  PMID: 10585861

Abstract

The experiments described in this paper were designed to determine the mechanism underlying the increase in 8-isoprostaglandin F(2alpha) (8-epi-PGF(2alpha)) 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(2alpha) in the medium was determined by ELISA. The production of 8-epi-PGF(2alpha) decreased by greater than 90% during hypoxia. Upon reoxygenation 8-epi-PGF(2alpha) 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(2alpha) production. However, 8-epi-PGF(2alpha) production was inhibited by catalase. The production of 8-epi-PGF(2alpha) was also inhibited by indomethacin and aspirin. Exogenous hydrogen peroxide stimulated 8-epi-PGF(2alpha) production by normoxic cells, and aspirin inhibited the hydrogen peroxide-mediated increase in 8-epi-PGF(2alpha) production. These results indicate that the reactive oxygen species responsible for 8-epi-PGF(2alpha) synthesis during reoxygenation is hydrogen peroxide and that in endothelial cells 8-epi-PGF(2alpha) synthesis is mediated by prostaglandin H(2) synthase (PGHS). To verify the role of PGHS in 8-epi-PGF(2alpha) synthesis, human PGHS-1 was expressed in COS-7 cells, a PGHS negative cell line that does not synthesize 8-epi-PGF(2alpha). In the presence of exogenous arachidonic acid the COS-7 cells expressing human PGHS-1 produced substantial amounts of PGE(2) and 8-epi-PGF(2alpha). These data indicate that human PGHS-1 can support the synthesis of 8-epi-PGF(2alpha) and that 8-epi-PGF(2alpha) synthesis by cultured human endothelial cells during reoxygenation is dependent on the activity of PGHS-1.

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Selected References

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  1. Awad J. A., Morrow J. D., Takahashi K., Roberts L. J., 2nd Identification of non-cyclooxygenase-derived prostanoid (F2-isoprostane) metabolites in human urine and plasma. J Biol Chem. 1993 Feb 25;268(6):4161–4169. [PubMed] [Google Scholar]
  2. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  3. Banerjee M., Kang K. H., Morrow J. D., Roberts L. J., Newman J. H. Effects of a novel prostaglandin, 8-epi-PGF2 alpha, in rabbit lung in situ. Am J Physiol. 1992 Sep;263(3 Pt 2):H660–H663. doi: 10.1152/ajpheart.1992.263.3.H660. [DOI] [PubMed] [Google Scholar]
  4. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745-2752.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davi G., Alessandrini P., Mezzetti A., Minotti G., Bucciarelli T., Costantini F., Cipollone F., Bon G. B., Ciabattoni G., Patrono C. In vivo formation of 8-Epi-prostaglandin F2 alpha is increased in hypercholesterolemia. Arterioscler Thromb Vasc Biol. 1997 Nov;17(11):3230–3235. doi: 10.1161/01.atv.17.11.3230. [DOI] [PubMed] [Google Scholar]
  6. Delanty N., Reilly M. P., Pratico D., Lawson J. A., McCarthy J. F., Wood A. E., Ohnishi S. T., Fitzgerald D. J., FitzGerald G. A. 8-epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo. Circulation. 1997 Jun 3;95(11):2492–2499. doi: 10.1161/01.cir.95.11.2492. [DOI] [PubMed] [Google Scholar]
  7. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  8. Fukunaga M., Makita N., Roberts L. J., 2nd, Morrow J. D., Takahashi K., Badr K. F. Evidence for the existence of F2-isoprostane receptors on rat vascular smooth muscle cells. Am J Physiol. 1993 Jun;264(6 Pt 1):C1619–C1624. doi: 10.1152/ajpcell.1993.264.6.C1619. [DOI] [PubMed] [Google Scholar]
  9. Fukunaga M., Takahashi K., Badr K. F. Vascular smooth muscle actions and receptor interactions of 8-iso-prostaglandin E2, an E2-isoprostane. Biochem Biophys Res Commun. 1993 Sep 15;195(2):507–515. doi: 10.1006/bbrc.1993.2075. [DOI] [PubMed] [Google Scholar]
  10. Fukunaga M., Yura T., Grygorczyk R., Badr K. F. Evidence for the distinct nature of F2-isoprostane receptors from those of thromboxane A2. Am J Physiol. 1997 Apr;272(4 Pt 2):F477–F483. doi: 10.1152/ajprenal.1997.272.4.F477. [DOI] [PubMed] [Google Scholar]
  11. Hecker M., Ullrich V., Fischer C., Meese C. O. Identification of novel arachidonic acid metabolites formed by prostaglandin H synthase. Eur J Biochem. 1987 Nov 16;169(1):113–123. doi: 10.1111/j.1432-1033.1987.tb13587.x. [DOI] [PubMed] [Google Scholar]
  12. Hemler M. E., Lands W. E. Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis. J Biol Chem. 1980 Jul 10;255(13):6253–6261. [PubMed] [Google Scholar]
  13. Hernandez L. A., Grisham M. B., Twohig B., Arfors K. E., Harlan J. M., Granger D. N. Role of neutrophils in ischemia-reperfusion-induced microvascular injury. Am J Physiol. 1987 Sep;253(3 Pt 2):H699–H703. doi: 10.1152/ajpheart.1987.253.3.H699. [DOI] [PubMed] [Google Scholar]
  14. Jourdan K. B., Mitchell J. A., Evans T. W. Release of isoprostanes by human pulmonary artery in organ culture: a cyclo-oxygenase and nitric oxide dependent pathway. Biochem Biophys Res Commun. 1997 Apr 28;233(3):668–672. doi: 10.1006/bbrc.1997.6523. [DOI] [PubMed] [Google Scholar]
  15. Klein T., Reutter F., Schweer H., Seyberth H. W., Nüsing R. M. Generation of the isoprostane 8-epi-prostaglandin F2alpha in vitro and in vivo via the cyclooxygenases. J Pharmacol Exp Ther. 1997 Sep;282(3):1658–1665. [PubMed] [Google Scholar]
  16. Krishna C. M., Liebmann J. E., Kaufman D., DeGraff W., Hahn S. M., McMurry T., Mitchell J. B., Russo A. The catecholic metal sequestering agent 1,2-dihydroxybenzene-3,5-disulfonate confers protection against oxidative cell damage. Arch Biochem Biophys. 1992 Apr;294(1):98–106. doi: 10.1016/0003-9861(92)90142-j. [DOI] [PubMed] [Google Scholar]
  17. Lynch S. M., Morrow J. D., Roberts L. J., 2nd, Frei B. Formation of non-cyclooxygenase-derived prostanoids (F2-isoprostanes) in plasma and low density lipoprotein exposed to oxidative stress in vitro. J Clin Invest. 1994 Mar;93(3):998–1004. doi: 10.1172/JCI117107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mallat Z., Philip I., Lebret M., Chatel D., Maclouf J., Tedgui A. Elevated levels of 8-iso-prostaglandin F2alpha in pericardial fluid of patients with heart failure: a potential role for in vivo oxidant stress in ventricular dilatation and progression to heart failure. Circulation. 1998 Apr 28;97(16):1536–1539. doi: 10.1161/01.cir.97.16.1536. [DOI] [PubMed] [Google Scholar]
  19. Mancini J. A., Riendeau D., Falgueyret J. P., Vickers P. J., O'Neill G. P. Arginine 120 of prostaglandin G/H synthase-1 is required for the inhibition by nonsteroidal anti-inflammatory drugs containing a carboxylic acid moiety. J Biol Chem. 1995 Dec 8;270(49):29372–29377. doi: 10.1074/jbc.270.49.29372. [DOI] [PubMed] [Google Scholar]
  20. Marshall P. J., Kulmacz R. J., Lands W. E. Constraints on prostaglandin biosynthesis in tissues. J Biol Chem. 1987 Mar 15;262(8):3510–3517. [PubMed] [Google Scholar]
  21. Minuz P., Andrioli G., Degan M., Gaino S., Ortolani R., Tommasoli R., Zuliani V., Lechi A., Lechi C. The F2-isoprostane 8-epiprostaglandin F2alpha increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO. Arterioscler Thromb Vasc Biol. 1998 Aug;18(8):1248–1256. doi: 10.1161/01.atv.18.8.1248. [DOI] [PubMed] [Google Scholar]
  22. Morrow J. D., Awad J. A., Boss H. J., Blair I. A., Roberts L. J., 2nd Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10721–10725. doi: 10.1073/pnas.89.22.10721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morrow J. D., Awad J. A., Kato T., Takahashi K., Badr K. F., Roberts L. J., 2nd, Burk R. F. Formation of novel non-cyclooxygenase-derived prostanoids (F2-isoprostanes) in carbon tetrachloride hepatotoxicity. An animal model of lipid peroxidation. J Clin Invest. 1992 Dec;90(6):2502–2507. doi: 10.1172/JCI116143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Morrow J. D., Frei B., Longmire A. W., Gaziano J. M., Lynch S. M., Shyr Y., Strauss W. E., Oates J. A., Roberts L. J., 2nd Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers. Smoking as a cause of oxidative damage. N Engl J Med. 1995 May 4;332(18):1198–1203. doi: 10.1056/NEJM199505043321804. [DOI] [PubMed] [Google Scholar]
  25. Morrow J. D., Hill K. E., Burk R. F., Nammour T. M., Badr K. F., Roberts L. J., 2nd A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9383–9387. doi: 10.1073/pnas.87.23.9383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Morrow J. D., Minton T. A., Roberts L. J., 2nd The F2-isoprostane, 8-epi-prostaglandin F2 alpha, a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist. Prostaglandins. 1992 Aug;44(2):155–163. doi: 10.1016/0090-6980(92)90077-7. [DOI] [PubMed] [Google Scholar]
  27. Morrow J. D., Roberts L. J., 2nd Quantification of noncyclooxygenase derived prostanoids as a marker of oxidative stress. Free Radic Biol Med. 1991;10(3-4):195–200. doi: 10.1016/0891-5849(91)90076-f. [DOI] [PubMed] [Google Scholar]
  28. Nanji A. A., Khwaja S., Tahan S. R., Sadrzadeh S. M. Plasma levels of a novel noncyclooxygenase-derived prostanoid (8-isoprostane) correlate with severity of liver injury in experimental alcoholic liver disease. J Pharmacol Exp Ther. 1994 Jun;269(3):1280–1285. [PubMed] [Google Scholar]
  29. O'Neill G. P., Mancini J. A., Kargman S., Yergey J., Kwan M. Y., Falgueyret J. P., Abramovitz M., Kennedy B. P., Ouellet M., Cromlish W. Overexpression of human prostaglandin G/H synthase-1 and -2 by recombinant vaccinia virus: inhibition by nonsteroidal anti-inflammatory drugs and biosynthesis of 15-hydroxyeicosatetraenoic acid. Mol Pharmacol. 1994 Feb;45(2):245–254. [PubMed] [Google Scholar]
  30. Paky A., Michael J. R., Burke-Wolin T. M., Wolin M. S., Gurtner G. H. Endogenous production of superoxide by rabbit lungs: effects of hypoxia or metabolic inhibitors. J Appl Physiol (1985) 1993 Jun;74(6):2868–2874. doi: 10.1152/jappl.1993.74.6.2868. [DOI] [PubMed] [Google Scholar]
  31. Palluy O., Bonne C., Modat G. Hypoxia/reoxygenation alters endothelial prostacyclin synthesis--protection by superoxide dismutase. Free Radic Biol Med. 1991;11(3):269–275. doi: 10.1016/0891-5849(91)90123-k. [DOI] [PubMed] [Google Scholar]
  32. Patrignani P., Santini G., Panara M. R., Sciulli M. G., Greco A., Rotondo M. T., di Giamberardino M., Maclouf J., Ciabattoni G., Patrono C. Induction of prostaglandin endoperoxide synthase-2 in human monocytes associated with cyclo-oxygenase-dependent F2-isoprostane formation. Br J Pharmacol. 1996 Jul;118(5):1285–1293. doi: 10.1111/j.1476-5381.1996.tb15535.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Patton G. M., Kadowaki H., Albadawi H., Soler H. M., Watkins M. T. Effect of hypoxia on steady-state arachidonic acid metabolism in bovine aortic endothelial cells. Am J Physiol. 1997 Mar;272(3 Pt 2):H1426–H1436. doi: 10.1152/ajpheart.1997.272.3.H1426. [DOI] [PubMed] [Google Scholar]
  34. Powell W. S. Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica. Prostaglandins. 1980 Nov;20(5):947–957. doi: 10.1016/0090-6980(80)90144-6. [DOI] [PubMed] [Google Scholar]
  35. Pratico D., Lawson J. A., FitzGerald G. A. Cyclooxygenase-dependent formation of the isoprostane, 8-epi prostaglandin F2 alpha. J Biol Chem. 1995 Apr 28;270(17):9800–9808. doi: 10.1074/jbc.270.17.9800. [DOI] [PubMed] [Google Scholar]
  36. Praticò D., Smyth E. M., Violi F., FitzGerald G. A. Local amplification of platelet function by 8-Epi prostaglandin F2alpha is not mediated by thromboxane receptor isoforms. J Biol Chem. 1996 Jun 21;271(25):14916–14924. doi: 10.1074/jbc.271.25.14916. [DOI] [PubMed] [Google Scholar]
  37. Praticó D., FitzGerald G. A. Generation of 8-epiprostaglandin F2alpha by human monocytes. Discriminate production by reactive oxygen species and prostaglandin endoperoxide synthase-2. J Biol Chem. 1996 Apr 12;271(15):8919–8924. doi: 10.1074/jbc.271.15.8919. [DOI] [PubMed] [Google Scholar]
  38. Proudfoot J. M., Beilin L. J., Croft K. D. PGF2-isoprostanes formed during copper-induced oxidation of low-density lipoproteins are the prostaglandins that cross-react with PGE2 antibodies. Biochem Biophys Res Commun. 1995 Jan 17;206(2):455–461. doi: 10.1006/bbrc.1995.1064. [DOI] [PubMed] [Google Scholar]
  39. Reilly M., Delanty N., Lawson J. A., FitzGerald G. A. Modulation of oxidant stress in vivo in chronic cigarette smokers. Circulation. 1996 Jul 1;94(1):19–25. doi: 10.1161/01.cir.94.1.19. [DOI] [PubMed] [Google Scholar]
  40. Schimke I., Griesmacher A., Weigel G., Holzhutter H. G., Muller M. M. Effects of reactive oxygen species on eicosanoid metabolism in human endothelial cells. Prostaglandins. 1992 Mar;43(3):281–292. doi: 10.1016/0090-6980(92)90096-c. [DOI] [PubMed] [Google Scholar]
  41. Soler H. M., Watkins M. T., Albadawi H., Kadowaki H., Patton G. M. Effects of oxygen tension and shear stress on human endothelial cell prostacyclin production. J Surg Res. 1997 Jan;67(1):46–53. doi: 10.1006/jsre.1996.4917. [DOI] [PubMed] [Google Scholar]
  42. Vacchiano C. A., Tempel G. E. Role of nonenzymatically generated prostanoid, 8-iso-PGF2 alpha, in pulmonary oxygen toxicity. J Appl Physiol (1985) 1994 Dec;77(6):2912–2917. doi: 10.1152/jappl.1994.77.6.2912. [DOI] [PubMed] [Google Scholar]
  43. Wang Z., Ciabattoni G., Créminon C., Lawson J., Fitzgerald G. A., Patrono C., Maclouf J. Immunological characterization of urinary 8-epi-prostaglandin F2 alpha excretion in man. J Pharmacol Exp Ther. 1995 Oct;275(1):94–100. [PubMed] [Google Scholar]
  44. Watkins M. T., Haudenschild C. C., al-Badawi H., Velazquez F. R., Larson D. M. Immediate responses of endothelial cells to hypoxia and reoxygenation: an in vitro model of cellular dysfunction. Am J Physiol. 1995 Feb;268(2 Pt 2):H749–H758. doi: 10.1152/ajpheart.1995.268.2.H749. [DOI] [PubMed] [Google Scholar]
  45. Watkins M. T., al-Badawi H., Cardenas R., Dubois E., Larson D. M. Endogenous reactive oxygen metabolites mediate sublethal endothelial cell dysfunction during reoxygenation. J Vasc Surg. 1996 Jan;23(1):95–103. doi: 10.1016/s0741-5214(05)80039-4. [DOI] [PubMed] [Google Scholar]
  46. Whorton A. R., Montgomery M. E., Kent R. S. Effect of hydrogen peroxide on prostaglandin production and cellular integrity in cultured porcine aortic endothelial cells. J Clin Invest. 1985 Jul;76(1):295–302. doi: 10.1172/JCI111960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yura T., Fukunaga M., Grygorczyk R., Makita N., Takahashi K., Badr K. F. Molecular and functional evidence for the distinct nature of F2-isoprostane receptors from those of thromboxane A2. Adv Prostaglandin Thromboxane Leukot Res. 1995;23:237–239. [PubMed] [Google Scholar]
  48. Zweier J. L., Kuppusamy P., Lutty G. A. Measurement of endothelial cell free radical generation: evidence for a central mechanism of free radical injury in postischemic tissues. Proc Natl Acad Sci U S A. 1988 Jun;85(11):4046–4050. doi: 10.1073/pnas.85.11.4046. [DOI] [PMC free article] [PubMed] [Google Scholar]

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