Ukr.Biochem.J. 2020; Volume 92, Issue 4, Jul-Aug, pp. 14-23


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;

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.

Keywords: , , , ,


  1. Gunter TE, Yule DI, Gunter KK, Eliseev RA, Salteret JD. Calcium and mitochondria. FEBS Lett. 2004;567(1):96-102. PubMed, CrossRef
  2. Territo PR, French SA, Dunleavy MC, Evans FJ, Balaban RS. Calcium activation of heart mitochondrial oxidative phosphorylation: rapid kinetics of mVO2, NADH, AND light scattering. J Biol Chem. 2001;276(4):2586-2599.  PubMed, CrossRef
  3. Merlavsky V, Ikkert O, Manko V. Са2+ influence on respiration processes upon streptozotocin-induced diabetes mellitus. Visnyk Lviv Univ. Biol Series. 2015;(70):294-304. (In Ukrainian).
  4. Kwong JQ, Molkentin JD. Physiological and pathological roles of the mitochondrial permeability transition pore in the heart. Cell Metab. 2015;21(2):206-214. PubMed, PubMedCentral, CrossRef
  5. Petronilli V, Penzo D, Scorrano L, Bernardi P, Di Lisa F. The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ. J Biol Chem. 2001;276(15):12030–12034. PubMed, CrossRef
  6. Bernardi P, Krauskopf A, Basso E, Petronilli V, Blachly-Dyson E, Di Lisa F, Forte MA. The mitochondrial permeability transition from in vitro artifact to disease target. FEBS J. 2006;273(10):2077-2099. PubMed, CrossRef
  7. Halestrap AP. What is the mitochondrial permeability transition pore? J Mol Cell Cardiol. 2009;46(6):821-831. PubMed, CrossRef
  8. Rasola A, Bernardi P. Mitochondrial permeability transition in Ca2+-dependent apoptosis and necrosis. Cell Calcium. 2011;50(3):222-233. PubMed, CrossRef
  9. Giorgio V, von Stockum S, Antoniel M, Fabbro A, Fogolari F, Forte M, Glick GD, Petronilli V, Zoratti M, Szabó I, Lippe G, Bernardi P. Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Natl Acad Sci USA. 2013;110(15):5887-5892. PubMed, PubMedCentral, CrossRef
  10. Chávez E, Rodríguez JS, García G, García N, Correa F. Oligomycin strengthens the effect of cyclosporin A on mitochondrial permeability transition by inducing phosphate uptake. Cell Biol Int. 2005;29(7):551-558. PubMed, CrossRef
  11. Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29-83. PubMed, CrossRef
  12. Crompton M, Ellinger H, Costi A. Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem J. 1988;255(1):357-360. PubMed, PubMedCentral
  13. Ponomarenko OV, Babich LG, Gorchev VF, Kosterin SO. Studies of Ca2+-dependent smooth muscle mitochondria swelling using flow cytometry and spermine effects on this process. Ukr Biokhim Zhurn. 2006;78(6):38-45. (In Ukrainian). PubMed
  14. Vergun O, Reynolds IJ. Distinct characteristics of Ca2+-induced depolarization of isolated brain and liver mitochondria. Biochim Biophys Acta. 2005; 1709(2):127-137. PubMed, CrossRef
  15. Sparagna GC, Gunter KK, Sheu SS, Gunter TE. Mitochondrial calcium uptake from physiological-type pulses of calcium. A description of the rapid uptake mode. J Biol Chem. 1995;270(46):27510-27515. PubMed, CrossRef
  16. Kröner H. The real kinetics of the mitochondrial calcium uniporter of the liver and its role in cell calcium regulation. Biol Chem Hoppe Seyler. 1988;369(3):149-155. PubMed, CrossRef
  17. Kupynyak NI, Ikkert OV, Shlykov SG, Babich LG, Manko VV. Mitochondrial ryanodine‐sensitive Ca2+ channels of rat liver. Cell Biochem Funct. 2017;35(1):42–49. PubMed, CrossRef
  18. McCormack JG. Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat-liver and within intact rat-liver mitochondria. Biochem J. 1985;231(3):581-595. PubMed, PubMedCentral, CrossRef
  19. Johnston JD, Brand MD. Stimulation of the respiration rate of rat liver mitochondria by sub-micromolar concentrations of extramitochondrial Ca2+. Biochem J. 1987;245(1):217-222. PubMed, PubMedCentral, CrossRef
  20. Nicholls DG, Budd SL. Mitochondrial and neuronal survival. Physiol Rev. 2000;80(1):315-360. PubMed, CrossRef
  21. Briston T, Roberts M, Lewis S, Powney B, Staddon JM, Szabadkai G, Duchen MR. Mitochondrial permeability transition pore: sensitivity to opening and mechanistic dependence on substrate availability. Sci Rep. 2017;7(1):10492. PubMed, PubMedCentral, CrossRef
  22. Kondrashova MN, Gogvadze VG, Medvedev BI, Babsky AM. Succinic acid oxidation as only energy support of intensive Ca2+ uptake by mitochondria. Biochem Biophys Res Commun. 1982;109(2):376-381. PubMed, CrossRef

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License.