VDAC isoforms affect NAD(P)H-dependent redox processes and ROS generation in mouse embryonic fibroblasts
Mitochondrial voltage dependent anion channels (VDAC) play an important role in the metabolite exchange between cytosol and mitochondria in cell. For recent years at least five research groups reported that VDAC1 exhibits the NADH-oxidoreductase activity. Other works demonstrated that blocking of VDAC causes the overproduction of ROS. However, the mechanisms of these effects are unclear, since VDAC1 is neither classical oxidoreductase nor antioxidant enzyme. We studied the impact of VDAC1/3 and VDAC2 isoforms on the redox processes in cells of mouse embryonic fibroblasts (MEF). It was demonstrated that total NADH-ferricyanide reductase activity in VDAC1/3-lacking cells (MEF VDAC1/3-) was almost identical, while in VDAC2-lacking cells (MEF VDAC2-) significantly increased in comparison with control (MEF wt). The suppression of known outer membrane NADH oxidoreductase, cytochrome b5 reductase (Cyb5R3), caused statistically significant inhibition of ferricyanide reduction. In the same time, intracellular and mitochondrial ROS production increased several fold in the range MEFwt < MEFVDAC1/3 < MEF VDAC2-. In the presense of redox-cycling compounds (menadione, lucigenin, nitrofurantoine), which are capable of receiving electrons from NAD(P)H dehydrogenases and transmit them to oxygen, the suppression of different VDAC isoforms also stimulated ROS production. This, at least in part, can be explained by the decrease of radical scavenging efficiency of cytochrome c/cytochrome c oxidase system. With use of original DBA-based fluorescent assay of NAD(P)H-oxidoreductase activity it was shown that VDAC1/3 and VDAC2 knockout lead to the decrease of activity of some non mitochondrial NADH-oxidoreductases, which, however, can be functionally associated with mitochondria.
Furthermore, increased expression of NADPH oxidases, including prosurvival NOX4 isoform D, was observed in MEF VDAC2- cells. Thus VDAC isoforms renders complex effect on the NAD(P)H oxidoreductase activity, ROS (and radical) production and utilization in cell. However, we were unable to reveal the own oxidoreductase activity of different VDAC isoforms.