EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines.
Superoxide (O₂ⁱ⁻) has been implicated in the pathogenesis of many human diseases, but detection of the O(2)(•-) radicals in biological systems is limited due to inefficiency of O₂ⁱ⁻ spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O₂ⁱ⁻ 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 O₂ⁱ⁻, producing stable nitroxides and allowing site-specific cO₂ⁱ⁻ 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 O₂ⁱ⁻. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O₂ⁱ⁻ 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 O₂ⁱ⁻ stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O₂ⁱ⁻. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O₂ⁱ⁻ radical in extracellular or intracellular compartments, cytoplasm or mitochondria.
FRIMCORE, Emory University School of Medicine, Atlanta, GA, USA.
Sergey I
SI
Dikalov
Igor A
IA
Kirilyuk
Maxim
M
Voinov
Igor A
IA
Grigor'ev
D000838:Anions / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006844:Hydrocarbons, Cyclic / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D019811:Hydroxylamine / Q000378:metabolism; D006898:Hydroxylamines / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism; D010539:Permeability; D010880:Piperidines / Q000378:metabolism; D011759:Pyrrolidines / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D019097:Spin Trapping / Q000379:methods*; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013481:Superoxides / Q000032:analysis* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
C492269:1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine; C113868:1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine; D000838:Anions; D002412:Cations; D003497:Cyclic N-Oxides; D006844:Hydrocarbons, Cyclic; D006898:Hydroxylamines; D010755:Organophosphates; D010880:Piperidines; D011759:Pyrrolidines; D012402:Rotenone; D013481:Superoxides; D019811:Hydroxylamine; D013755:Tetradecanoylphorbol Acetate; C505333:TEMPOL-H