Closure of VDAC causes oxidative stress and accelerates the Ca2+-induced mitochondrial permeability transition in rat liver mitochondria

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca2+-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O2−) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O2− from the IMS and sensitizing to the Ca2+-induced MPT. Treatment of isolated rat liver mitochondria with 5 μM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8 ± 1.4 min within a range of 100–250 μM Ca2+. G3139-mediated acceleration of the MPT was reversed by 20 μM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O2− in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O2− accumulation. Mathematical modeling of generation and turnover of O2− within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O2−. In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O2−, which causes an internal oxidative stress and sensitizes mitochondria toward the Ca2+-induced MPT.