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

Abstract

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.