Komlodi 2016 Abstract IOC116

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Komlodi T, Geibl F, Sassani M, Adam-Vizi V, Tretter L (2016) The effect of ΔΨmt and ΔpH on mitochondrial reactive oxygen species production - A comparative study using mitochondria isolated from different mammalian tissues. Mitochondr Physiol Network 21.11

Link: Mitochondr Physiol Network 21.11

Komlodi T, Geibl F, Sassani M, Adam-Vizi V, Tretter L (2016)

Event: IOC116

The generation of mitochondrial reactive oxygen species (ROS) has a great role in many neurodegenerative diseases (Alzheimer’s and Parkinson’s disease) and in myocardial ischemia-reperfusion injury. It is widely accepted that a high percentage of ROS production is dependent on the proton motive force (pmf), which is the electrochemical potential difference of protons across the inner mitochondrial membrane, composed of ΔΨmt and ΔpH. According to the literature, depolarization of the mitochondrial membrane and decline of the ΔpH result in decreased ROS generation. In this study we have compared the dependence of succinate supported ROS production on the ΔΨmt and ΔpH in mitochondria isolated from guinea-pig brain, heart and skeletal muscle. Consequently, we examined which one of the two components of pmf played a greater role in mitochondrial ROS generation.

Experiments were carried out on isolated brain, heart and skeletal muscle mitochondria prepared from guinea-pig. Mitochondria were energized with succinate, which supports the reverse electron flow, the major mitochondrial ROS source in vitro. Amplex Ultra Red fluorescent dye was used to measure mitochondrial ROS production. The changes of ΔΨmt were detected with safranine-O fluorescence, intramitochondrial pH was determined with BCECF fluorescence. Analysis of mitochondrial respiratory control was carried out with high-resolution respirometry. In order to separate the two components of the pmf (ΔΨmt and ΔpH) ionophores (valinomycin and nigericin) were used.

The effects of ionophores showed the same tendency in brain and heart mitochondria. In the presence of valinomycin, a K+ ionophore, at low [K+], we measured a drop in ΔΨmt, a consequent elevation of matrix pH and ΔpH with a corresponding decline of H2O2 production. The addition of nigericin, a K+/H+ exchanger, decreased mitochondrial ΔpH, this was followed by a compensatory increase of ΔΨmt resulted in an elevation of ROS generation.

Based on our measurements we can conclude that ΔΨmt has a greater role in modulation of mitochondrial ROS generation compared to ΔpH in brain, heart and skeletal muscle mitochondria; thus we have not seen tissue specificity regarding the effect of ionophores on ROS production.

O2k-Network Lab: HU Budapest Tretter L

Labels: MiParea: Respiration, mt-Membrane, mt-Medicine, Pharmacology;toxicology 

Stress:Oxidative stress;RONS  Organism: Guinea pig  Tissue;cell: Heart, Skeletal muscle, Nervous system  Preparation: Isolated mitochondria 

HRR: Oxygraph-2k, O2k-Fluorometer 

AmR, Safranin 

Affiliations and support

Dept Med Biochem, Semmelweis Univ Lab for Neurobiochem, Hungarian Acad Sc, Budapest, Hungary. - [email protected]

Supported by: OTKA (NK 81983), TAMOP (4.2.2./B-09/1), MTA (MTA TKI 2013), and Hungarian Brain Research Program (Grant No. KTIA_13_NAP-A-III/6)