Tag Archives: FCCP

mPTP opening differently affects electron transport chain and oxidative phosphorylation at succinate and NAD-dependent substrates oxidation in permeabilized rat hepatocytes

H. M. Mazur, V. M. Merlavsky, B. O. Manko, V. V. Manko*

Ivan Franko National University of Lviv, Ukraine;
*e-mail: volodymyr.manko@lnu.edu.ua

Received: 10 October 2019; Accepted: 15 May 2020

Mitochondrial Ca2+ overload may trigger the opening of mitochondrial permeability transition pore (mPTP) and its prolonged activation leads to cell death. ATP synthase is considered as a possible molecular component of the pore. The aim of this study was to investigate the state of oxidative phosphorylation at Ca2+-induced activation of mPTP in permeabilized hepatocytes. Hepatocytes were isolated by two-stage Seglen method. Permeabilization was performed using digitonin. Oxygen consumption rate was measured with Clark electrode. Oxidative phosphorylation was determined as the ratio of the ADP-stimulated respiration and substrate-stimulated respiration rates (ADP/S). It was established that increasing of Ca2+ concentration in the medium inhibited oligomycin effects and suppressed ADP- and FCCP-stimulated respiration upon succinate or glutamate, pyruvate and malate mixture oxidation. The mPTP inhibitor cyclosporin A did not directly affect respiration and oxidative phosphorylation after elevation of Ca2+ concentration and mPTP activation. When cyclosporine A was added before increasing Ca2+ concentration, the electron transport chain function (FCCP-stimulated respiration) was not impaired while the partial disruption of oxidative phosphorylation (ADP-stimulated respiration) was observed only upon succinate oxidation. The results obtained showed that inhibition of oxidative phosphorylation was the primary event in mPTP activation, possibly due to the involvement of ATP synthase in pore opening. In the case of NAD-dependent substrates oxidation that effect was stronger and faster than at succinate oxidation, due to the lower mitochondria energization.

Dependence of the mitochondrial adaptive capacity of hepatocytes on the oxidative substrates availability

H. M. Mazur, V. M. Merlavsky, B. O. Manko, V. V. Manko

Ivan Franko National University of Lviv, Ukraine;
e-mail: Volodymyr.Manko@lnu.edu.ua

Received: 11 February 2019; Accepted: 18 October 2019

The ability of the mitochondria to compensate for energy expenditure of cells largely depends on the availability of the oxidative substrates, transported across the intact plasma membrane with molecular carriers of limited affinity. The aim of this study was to investigate the dependence of adaptive respiratory responses of mitochondria of intact hepatocytes on the oxidative substrates. Basal and FCCP-stimulated respiration rates were determined with Clark electrode. After 15-minute incubation in the medium with the oxidative substrates or their combinations (glutamine, pyruvate, succinate, monomethyl succinate, α-ketoglutarate, dimethyl-α-ketoglutarate (2 mM) or glucose (10 mM)), isolated hepatocytes were added into the respiratory chamber. FCCP concentration was 0.25, 0.5 and 1 μM. The adaptive capacity of mitochondria was characterized by the maximal uncoupled respiration rate (the highest respiration rate among all tested FCCP concentrations), the optimal FCCP concentration (the concentration at which the maximal rate is achieved) and the area under the curve (AUC) of the dependence of the uncoupled respiration rate on FCCP concentration. The adaptive capacity of mitochondria, evaluated by AUC, increases in this order of substrates: glucose (0.063 r.u.), endogenous substrates (0.067 r.u.), glutamine (0.092 r.u.), pyruvate (0.113 r.u.), α-ketoglutarate (0.113 r.u.), succinate (0.152 r.u.), dimethyl-α -ketoglutarate (0.156 r.u.), and monomethyl succinate (0.172 r.u.). The adaptive capacity of mitochondria of hepatocytes seems to be partly dependent on plasma membrane transporters affinities (Km) to the oxidative substrates. The presence of glucose in the medium does not improve the adaptive capacity of hepatic mitochondria.