Arachidonic acid-induced carbon-centered radicals and phospholipid peroxidation in cyclo-oxygenase-2-transfected PC12 cells.
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 alpha-(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-beta-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.
Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Jianfei
J
Jiang
Grigory G
GG
Borisenko
Anatoly
A
Osipov
Ian
I
Martin
Renwu
R
Chen
Anna A
AA
Shvedova
Andrey
A
Sorokin
Yulia Y
YY
Tyurina
Alla
A
Potapovich
Vladimir A
VA
Tyurin
Steven H
SH
Graham
Valerian E
VE
Kagan
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D015153:Blotting, Western / Q000379:methods; D002244:Carbon / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003497:Cyclic N-Oxides; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D004347:Drug Interactions; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004996:Ethidium / Q000031:analogs & derivatives* / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism*; D005989:Glycerides / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D015227:Lipid Peroxidation / Q000187:drug effects*; D008054:Lipid Peroxides / Q000378:metabolism; D008565:Membrane Proteins; D009589:Nitrogen Oxides / Q000494:pharmacology; D010084:Oxidation-Reduction; D016716:PC12 Cells / Q000187:drug effects*; D010713:Phosphatidylcholines / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines; D051381:Rats; D014162:Transfection / Q000379:methods
C468114:1-palmitoyl-2-arachidonyl-3-phosphorylcholine; D003497:Cyclic N-Oxides; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D005609:Free Radicals; D005989:Glycerides; D007527:Isoenzymes; D008054:Lipid Peroxides; D008565:Membrane Proteins; D009589:Nitrogen Oxides; D010713:Phosphatidylcholines; D011725:Pyridines; C043438:alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone; C067883:dihydroethidium; D016718:Arachidonic Acid; D007544:Isopropyl Thiogalactoside; C037498:4-(9-acridinylamino)-2,2,6,6-tetramethyl-1-piperidinyloxy; D002244:Carbon; C068738:1-palmitoyl-2-parinaroylphosphatidylcholine; D006861:Hydrogen Peroxide; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D004996:Ethidium; C026433:1-arachidonyl monoglyceride; D015232:Dinoprostone