Cellular Redox State Acts as Switch to Determine the Direction of NNT-Catalyzed Reaction in Cystic Fibrosis Cells

Abstract

The redox states of NAD and NADP are linked to each other in the mitochondria thanks to the enzyme nicotinamide nucleotide transhydrogenase (NNT) which, by utilizing the mitochondrial membrane potential (mΔΨ), catalyzes the transfer of redox potential between these two coenzymes, reducing one at the expense of the oxidation of the other. In order to define NNT reaction direction in CF cells, NNT activity under different redox states of cell has been investigated. Using spectrophotometric and western blotting techniques, the presence, abundance and activity level of NNT were determined. In parallel, the levels of NADPH and NADH as well as of mitochondrial and cellular ROS were also quantified. CF cells showed a 70% increase in protein expression compared to the Wt sample; however, regarding NNT activity, it was surprisingly lower in CF cells than healthy cells (about 30%). The cellular redox state, together with the low mΔΨ, pushes to drive NNT reverse reaction, at the expense of its antioxidant potential, thus consuming NADPH to support NADH production. At the same time, the reduced NNT activity prevents the NADH, produced by the reaction, from causing an explosion of ROS by the damaged respiratory chain, in accordance with the reduced level of mitochondrial ROS in NNT-loss cells. This new information on cellular bioenergetics represents an important building block for further understanding the molecular mechanisms responsible for cellular dysfunction in cystic fibrosis.

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“…To specifically detect O2 −• production of mitochondrial origin, use was made of MitoSOX Red, a specific mitochondrial dye which is highly and exclusively sensitive to superoxide [126,127] but not to other reactive oxygen/nitrogen species. MitoSOX Red is selectively targeted to mitochondria where it accumulates as a function of mitochondrial membrane potential and exhibits fluorescence upon oxidation by superoxide and subsequent binding to mitochondrial DNA.…”

“…(f) NADPH and NADH level measurements Nicotinamide adenine dinucleotide levels were performed by using the method involving 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), as terminal electron acceptor, with phenazine ethosulfate (PES), as an electron carrier, according to [127]. The reduction rate of the MTT is proportional to the concentration of the nicotinamide adenine dinucleotide and, thus, cycling time must be strictly kept the same for each assay.…”