EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines

Superoxide (O???) 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 O??? spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O??? 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 O???, producing stable nitroxides and allowing site-specific cO??? 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 O???. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O??? 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 O??? stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O???. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O??? radical in extracellular or intracellular compartments, cytoplasm or mitochondria.