Enhanced mitochondrial superoxide in hyperglycemic endothelial cells: direct measurements and formation of hydrogen peroxide and peroxynitrite

Hyperglycemic challenge to bovine aortic endothelial cells (BAECs) increases oxidant formation and cell damage that are abolished by MnSOD overexpression, implying mitochondrial superoxide (O₂^•−) as a central mediator. However, mitochondrial O₂^•−and its steady-state concentrations have not been measured directly yet. Therefore, we aimed to detect and quantify O₂^•−through different techniques, along with the oxidants derived from it. Mitochondrial aconitase, a sensitive target of O₂^•−, was inactivated 60% in BAECs incubated in 30 mM glucose (hyperglycemic condition) with respect to cells incubated in 5 mM glucose (normoglycemic condition). Under hyperglycemic conditions, increased oxidation of the mitochondrially targeted hydroethidine derivative (MitoSOX) to hydroxyethidium, the product of the reaction with O₂^•−, could be specifically detected. An 8.8-fold increase in mitochondrial O₂^•−steady-state concentration (to 250 pM) and formation rate (to 6 μM/s) was estimated. Superoxide formation increased the intracellular concentration of both hydrogen peroxide, measured as 3-amino-2,4,5-triazole-mediated inactivation of catalase, and nitric oxide-derived oxidants (i.e., peroxynitrite), evidenced by immunochemical detection of 3-nitrotyrosine. Oxidant formation was further evaluated by chloromethyl dichlorodihydrofluorescein (CM-H₂DCF) oxidation. Exposure to hyperglycemic conditions triggered the oxidation of CM-H₂DCF and was significantly reduced by pharmacological agents that lower the mitochondrial membrane potential, inhibit electron transport (i.e., myxothiazol), and scavenge mitochondrial oxidants (i.e., MitoQ). In BAECs devoid of mitochondria (rho⁰cells), hyperglycemic conditions did not increase CM-H₂DCF oxidation. Mitochondrial O₂^•−formation in hyperglycemic conditions was associated with increased glucose metabolization in the Krebs cycle and hyperpolarization of the mitochondrial membrane.