EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines

• Published in: Free radical research 2011-04, Vol.45 (4), p.417-430
• Main Authors: Dikalov, Sergey I., Kirilyuk, Igor A., Voinov, Maxim, Grigor'ev, Igor A.
• Format: Article
• Language: English
• Subjects: Anions - metabolism; Cations - metabolism; cyclic hydroxylamine; Cyclic N-Oxides - metabolism; Electron Spin Resonance Spectroscopy; Endothelial Cells - drug effects; Endothelial Cells - metabolism; EPR; extracellular; Humans; Hydrocarbons, Cyclic - chemistry; Hydrocarbons, Cyclic - metabolism; Hydrophobic and Hydrophilic Interactions; Hydroxylamine - metabolism; Hydroxylamines - chemistry; Hydroxylamines - metabolism; intracellular; Mitochondria - drug effects; Mitochondria - metabolism; Neutrophils - drug effects; Neutrophils - metabolism; Organophosphates - metabolism; Permeability; Piperidines - metabolism; Pyrrolidines - metabolism; Rotenone - pharmacology; spin trap; Spin Trapping - methods; Static Electricity; Structure-Activity Relationship; Superoxide; Superoxides - analysis; Superoxides - metabolism; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 1071-5762; 1029-2470; 1029-2470
• DOI: 10.3109/10715762.2010.540242

Abstract Superoxide (O2*−) has been implicated in the pathogenesis of many human diseases, but detection of the O2*− radicals in biological systems is limited due to inefficiency of O2*− spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O2*− in neutrophils, cultured endothelial cells and isolated mitochondria using a new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O2*−, producing stable nitroxides and allowing site-specific O2*− detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O2*−. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O2*− both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected an increase in cytoplasm O2*− stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O2*−. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O2*− radical in extracellular or intracellular compartments, cytoplasm or mitochondria.