Faculty Collaboration Database

Arachidonic acid-induced carbon-centered radicals and phospholipid peroxidation in cyclo-oxygenase-2-transfected PC12 cells. J Neurochem 2004 Sep;90(5):1036-49

Date

08/18/2004

Pubmed ID

15312159

DOI

10.1111/j.1471-4159.2004.02577.x

Scopus ID

2-s2.0-4444356653 (requires institutional sign-in at Scopus site)   63 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 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.

Author List

Jiang J, Borisenko GG, Osipov A, Martin I, Chen R, Shvedova AA, Sorokin A, Tyurina YY, Potapovich A, Tyurin VA, Graham SH, Kagan VE

Author

Andrey Sorokin PhD Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Arachidonic Acid
Blotting, Western
Carbon
Chromatography, High Pressure Liquid
Cyclic N-Oxides
Cyclooxygenase 2
Cyclooxygenase 2 Inhibitors
Cyclooxygenase Inhibitors
Dinoprostone
Drug Interactions
Electron Spin Resonance Spectroscopy
Ethidium
Free Radicals
Glycerides
Humans
Hydrogen Peroxide
Isoenzymes
Isopropyl Thiogalactoside
Lipid Peroxidation
Lipid Peroxides
Membrane Proteins
Nitrogen Oxides
Oxidation-Reduction
PC12 Cells
Phosphatidylcholines
Prostaglandin-Endoperoxide Synthases
Pyridines
Rats
Transfection