Reactive Oxygen and Targeted Antioxidant Administration in Endothelial Cell Mitochondria

Abstract

We used fluorescent probes and EPR to study the mechanism(s) underlying reactive oxygen species (ROS) production by endothelial cell mitochondria and the action of mitoquinol, a mitochondria-targeted antioxidant. ROS measured by fluorescence resulted from complex I superoxide released to the matrix and converted to H 2 O 2 . In contrast, EPR largely detected superoxide generated at complex III and effluxed outward. ROS fluorescence by mitochondria fueled by the complex II substrate, succinate, was substantial but markedly inhibited by rotenone. Superoxide, detected by EPR, in succinate-fueled mitochondria was not inhibited by rotenone and likely derived from semiquinone formation at complex III. Mitoquinol decreased H 2 O 2 fluorescence by succinate-fueled mitochondria but had little effect on the EPR signal for superoxide. This was not associated with a detectable decrease in membrane potential. Mitoquinol markedly enhanced ROS fluorescence in mitochondria fueled by the complex I substrates, glutamate and malate. Inhibitor studies suggested that this occurred in complex I, at one or more Q binding pockets. The above effects of mitoquinol were determined in mitochondria isolated and subsequently exposed to the targeted antioxidant. However, similar effects were observed in mitochondria after antecedent exposure to mitoquinol/mitoquinone in culture, suggesting that the agent is retained after isolation of the organelles. In conclusion, ROS production in bovine aortic endothelial cell mitochondria results largely from reverse transport to complex I and through the Q cycle in complex III. Mitoquinol blocks ROS from reverse electron transport but increases superoxide production derived from forward transport. These effects likely occur at one or more Q binding sites in complex I. Control of reactive oxygen species (ROS)2 in endothelial cells is particularly important, because endothelial dysfunction, which may be triggered by ROS (1), is a major factor contributing to the development of atherosclerotic heart disease (2-4). Moreover, mitochondrial oxidative damage may underlie problems, including the vascular and neurologic complications of diabetes, cell damage in degenerative diseases, and aging (5-9). Reactive oxygen derived from endothelial cell mitochondria has also been implicated in the metabolic pathways leading to the microvascular complications of diabetes (6).Mitochondrial electron transport generates substantial amounts of superoxide derived from electron leaks as substrates are metabolized (10). The process may have adverse consequences as evidenced by observations in mice lacking the mitochondrial enzyme manganese superoxide dismutase. These mice develop dilated cardiomyopathy and live less than 2 weeks (11). The major sites of superoxide production have been somewhat controversial, but there is evidence that most derive from complexes I and III (12). There is also evidence that complex I superoxide is released exclusively to the matrix side of the inner membrane, whereas complex III likely generates su...

Mitochondria-Targeted Antioxidant MitoQ 10 Improves Endothelial Function and Attenuates Cardiac Hypertrophy

“…DecylTPP was used as a control compound because it is known to be taken up in a very similar way to MitoQ 10 in both cells in vitro 26 and into organs in vivo after IV injection (C. M. Porteous et al, personal communication, 2009). Therefore, our finding that decylTPP had no beneficial action is a strong indication that the effects of MitoQ 10 are attributable to the antioxidant action and not secondary to the accumulation of lipophilic cations within mitochondria.…”

Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes

“…Previous studies of bovine aortic endothelial cells using the potential-sensitive triphenylphosphonium ion yielded a ⌬ m of Ϫ220 mV (47). However, it is difficult to make a comparison, because the latter studies involved isolated mitochondria under hyperpolarizing conditions (e.g., in the presence of oligomycin) (47).…”

Reactive Oxygen Species in Inflammation and Tissue Injury

“…The main sites of O 2 2 production in the ETC are complex I and III, with complex I being more predominant in skeletal muscle and neural cells; whereas complex III is more predominant for endothelial cells (Fig. 3) (323,416). O 2 2 generated by mitochondria reacts with manganese SOD (MnSOD) in the mitochondrial matrix to generate H 2 O 2 , which can cross the mitochondrial outer membrane to access cytosolic targets.…”