Arachidonic acid-induced carbon-centered radicals and phospholipid peroxidation in cyclo-oxygenase-2-transfected PC12 cells

Jianfei Jiang, Grigory G. Borisenko, Anatoly Osipov, Ian Martin, Renwu Chen, Anna A. Shvedova, Andrey Sorokin, Yulia Y. Tyurina, Alla Potapovich, Vladimir A. Tyurin, Steven H. Graham, Valerian E. Kagan

Research output: Contribution to journalArticle

54 Scopus citations

Abstract

Cyclo-oxygenase-2 (COX-2) is believed to induce neuronal oxidative stress via production of radicals. While oxygen radicals are not directly involved in COX-2-catalytic cycle, superoxide anion radicals have been repeatedly reported to play a critical role in COX-2-associated oxidative stress. To resolve the controversy, we characterized production of free radicals in PC12 cells in which COX-2 expression was manipulated either genetically or by direct protein transfection and compared them with those generated by a recombinant COX-2 in a cell-free system. Using spin-traps α-(4-pyridyl-1-oxide)-N-t-butylnitrone, 5,5-dimethyl-1-pyrroline-N-oxide and 4-((9-acridinecarbonyl) amino)-2,2,6,6-tetramethylpiperidine-1-oxyl (Ac-Tempo), we observed arachidonic acid (AA)-dependent production of carbon-centered radicals by heme-reconstituted recombinant COX-2. No oxygen radicals or thiyl radicals have been detected. COX-2 also catalyzed AA-dependent one-electron co-oxidation of ascorbate to ascorbate radicals. Next, we used two different approaches of COX-2 expression in cells, PCXII cells which express isopropyl-1-thio-β-D-galactopyranoside inducible COX-2, and PC12 cells transfected with COX-2 using a protein delivery reagent, Chariot. In both models, COX-2-dependent AA-induced generation of carbon-centered radicals was documented using spin-traps and Ac-Tempo. No oxygen radical formation was detected in COX-2-transfected cells by either spin-traps or fluorogenic probe, dihydroethidium. In the presence of ascorbate, AA-induced COX-2-dependent ascorbate radicals were detected. AA caused a significant and selective oxidation of one of the major phospholipids, phosphatidylserine (PS). PS was not a direct substrate for COX-2 but was co-oxidized in the presence of AA. The radical generation and PS oxidation were inhibited by COX-2 inhibitors, niflumic acid, nimesulide, or NS-398. Thus, COX-2 generated carbon-centered radicals but not oxygen radicals or thiyl radicals are responsible for oxidative stress in AA-challenged PC12 cells overexpressing COX-2.

Original languageEnglish (US)
Pages (from-to)1036-1049
Number of pages14
JournalJournal of Neurochemistry
Volume90
Issue number5
DOIs
StatePublished - Sep 1 2004

    Fingerprint

Keywords

  • Carbon-centered radical formation
  • Cyclo-oxygenase-2 inhibitor
  • Cyclo-oxygenase-2 transfection
  • PC12 cells
  • Phosphatidylserine oxidation
  • Radical spin-trapping

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Jiang, J., Borisenko, G. G., Osipov, A., Martin, I., Chen, R., Shvedova, A. A., Sorokin, A., Tyurina, Y. Y., Potapovich, A., Tyurin, V. A., Graham, S. H., & Kagan, V. E. (2004). Arachidonic acid-induced carbon-centered radicals and phospholipid peroxidation in cyclo-oxygenase-2-transfected PC12 cells. Journal of Neurochemistry, 90(5), 1036-1049. https://doi.org/10.1111/j.1471-4159.2004.02577.x