Tag Archives: hepatocytes

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.

Fatty acids composition of inner mitochondrial membrane of rat cardiomyocytes and hepatocytes during hypoxia-hypercapnia

S. V. Khyzhnyak, S. V. Midyk, S. V. Sysoliatin, V. М. Voitsitsky

National University of Life and Environmental Sciences of Ukraine, Kyiv;
е-mail: khs2014@ukr.net

We studied the influence of hypoxic-hypercapnic environment under the effect of hypothermia (artificial hibernation) on fatty acids spectrum of inner mitochondrial membrane (IMM) lipids of rat cardiomyocytes and hepatocytes. Specific for cellular organelles redistribution of IMM fatty acids was determined. It led to the reduction of total amount of saturated fatty acids (SFAs) and increase of unsaturated fatty acids (UFAs) in cardiomyocytes and to the increase of SFAs and decrease of UFAs in hepatocytes. The decrease in the content of oleic acid and increased content of arachidonic and docosahexaenoic acids in IMM were shown. This may be due to their role in the regulatory systems during hibernation, as well as following exit therefrom. It is assumed that artificial hibernation state is characterized by the stress reaction leading to optimal readjustment of fatty acids composition of membrane lipids, which supports functional activity of mitochondria in hepatocytes and cardiomyocytes.

Cholecalciferol hydroxylation in rat hepatocytes under the influence of prednisolone

A. V. Khomenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: annavic@ukr.net

Glucocorticoid therapy is accompanied by development of processes typical of steroid osteoporosis. Indirect effects of glucocorticoids on the bone tissue are due to changes in mineral metabolism, which is regulated by vitamin D3. In this connection, we studied the influence of prednisolone on cholecalciferol metabolism. The study has shown that prednisolone action causes impairment of cholecalciferol metabolism in hepatocytes due to inhibiting vitamin D3 25-hydroxylase activity. Microsomal (CYP2R1) and mitochondrial (CYP27A1­) isoenzymes of vitamin D3 25-hydroxylase were found to function at different concentrations of the substrate. The relative protein contents of the isoenzymes greatly differed in the liver with the prevalence of CYP27A1 over CYP2R1. Prednisolone administration resulted in the lowering of both mitochondrial and microsomal isoenzymes of vitamin D3 25-hydroxylase. The inhibition of vitamin D3 25-hydroxylating system in hepatocytes contributed to a significant reduction in blood serum 25OHD3.

The sensitivity of cells with the various level of NAD(P)H:quinone oxidoreductase 1 to cytotoxic action of quinonimines and α-тоcopherol synthetic derivatives

G. V. Petrova1, А. V. Parshykov2

1Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv;
e-mail: petrova@biochem.kiev.ua
2Institute of Pharmacology and Toxocology, National Academy
of Medical Science of Ukraine, Kyiv

The effects of α-tocopherol with shortened to 6 carbon atoms side chain (α-Toc-C6), α-tocopherol succinate (α-TS) and quinonimine 2,6-dichlorophenolindophenol (DCPIP) on DT-diaphorase activity and viability of rat thymocytes, splenocytes and hepatocytes were investigated. It was shown that the lowest basal activity of the enzyme is inherent in splenocytes. In comparison to splenocytes, DT-diaphorase activity was 1.4 and 5 times higher in thymocytes and hepatocytes, respectively. It was found that the sensitivity of cells to the cytotoxic effect of DCPIP was inversely proportional to the basal level of DT-diaphorase activity and accompanied by its activation with subsequent inhibition at non-toxic and toxic concentrations, respectively. Hepatocytes were least sensitive to the cytotoxic effect of α-Toc-C6. In thymocytes and splenocytes α-Toc-C6 exerts inhibitory effects on DT-diaphorase, whereas in hepatocytes an increased activity of the enzyme was observed, which probably caused their high survival rate. Simultaneous induction of cytochrome P450 enzyme expression by α-Toc-C6 in hepatocytes is also possible. Cytotoxic effect of α-TS does not depend on the basal level of DT-diaphorase activity in cells, is not accompanied by its induction and it is most likely determined by the non-specific esterase activity.

NAADP-sensitive Сa(2+) stores in permeabilized rat hepatocytes

S. V. Bychkova1, T. I. Chorna2

1Ivan Franko National University of Lviv, Ukraine;
2National Centre for Biological Sciences, Tata Institute
of Fundamental Research, Bangalore, India;
e-mail: s.bychkova@gmail.com

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a nucleotide that is potent to release calcium from intracellular stores in different cell types. NAADP was shown to target specific type of intracellular store namely endolysosomal system or acidic store. Despite intense studies, its effect on endoplasmatic reticulum (ER) still remains to be elucidated. The main aim of our work was to investigate NAADP-sensitive store in permeabilized rat hepatocytes monitoring the level of Ca2+ inside intracellular organelles using chlorotetracycline (CTC). We have shown that NAADP triggered changes of stored Ca2+ in rat hepatocytes are dependent on concentration of EGTA-Ca2+-buffer in cell incubation medium, i.e. the higher is the EGTA concentration in incubation medium the smaller or absent is the effect of NAADP. Besides, the effect of NAADP was more pronounced upon cells pretreatment with the inhibitory concentration of ryanodine (100 µM). This might suggest that the effect of NAADP is dependent on ER luminal calcium. We have also found that NAADP-evoked Ca2+ release in permeabilized hepatocytes is sensitive to nigericin, bafilomycin A and thapsigargin. Additionally, NAADP triggered changes in stored Ca2+ were completely abolished by NED-19 as antagonist of NAADP.