Tag Archives: respiration

Experimental substantiation of permeabilized hepatocytes model for investigation of mitochondria in situ respiration

V. M. Merlavsky, B. O. Manko, O. V. Ikkert, V. V. Manko

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

TTo verify experimentally the model of permeabilized hepatocytes, the degree of cell permeability was assessed using trypan blue and polarographycally determined cell respiration rate upon succinate (0.35 mM) and α-ketoglutarate (1 mM) oxidation. Oxidative phosphorylation was stimulated by ADP (750 μM). Hepatocyte permeabilization depends on digitonin concentraion in medium and on the number of cells in suspension. Thus, the permeabilization of 0.9-1.7 million cells/ml was completed by 25 μg/ml of digitonin, permeabilization of 2.0-3.0 million cells/ml – by 50 μg/ml of digitonin and permeabilization of 4.0-5.6 million cells/ml – by 100 μg/ml. Thus, the higher is the suspension density, the higher digitonin concentration is required. Treatment of hepatocytes with digitonin resulted in a decrease of endogenous respiration rate to a minimum upon 20-22 μg of digitonin per 1 million cells. Supplementation of permeabilized hepatocytes with α-ketoglutarate maintained stable respiration rate on the level higher than endogenous respiration at the corresponding digitonin concentration, unlike the intact cells. Respiration rate of permeabilized hepatocytes at the simultaneous addition of α-ketoglutarate and ADP increased to the level of intact cell respiration, irrespective of digitonin concentration. Addition of solely succinate and especially succinate plus ADP markedly intensified the respiration of permeabilized hepatocytes to the level higher than that of intact cells. The dependence of succinate-stimulated respiration on digitonin concentration reached maximum at 20-22 μg of digitonin per 1 million cells. Optimal ratio of digitonin amount and the cell number in suspension is expected to be different in various tissues.

Influence of Са(2+) on kinetic parameters of pancreatic acinar mitochondria in situ respiration

B. O. Manko, V. V. Manko

Ivan Franko National University of Lviv, Ukraine;
e-mail: mankobo@gmail.com

The dependence of respiration rate of rat permeabilized acinar pancreacytes on oxidative substrates concentration was studied at various [Ca2+] – 10-8–10-6 M. Pancreacytes were permeabilized with 50 µg of digitonin per 1 million cells. Respiration rate was measured polarographically using the Clark electrode at oxidation of succinate or pyruvate either glutamate in the presence of malate. Parameters of Michaelis-Menten equation were calculated by the method of Cornish-Bowden or using Idi-Hofsti coordinates and parameters of Hill equation – using coordinates {v; v/[S]h}. In the studied range of [Ca2+] the kinetic dependence of respiration at pyruvate oxidation is described by the Michaelis-Menten equation, and at oxidation of succinate or glutamate – by Hill equation with h = 1.11–1.43 and 0.50–0.85, respectively. The apparent constant of respiration half-activation (K0.5) did not significantly change in the studied­ range of [Ca2+] while at 10-7 M Ca2+ it was 0.90 ± 0.06 mM for succinate, 0.096 ± 0.007 mM for pyruvate and 0.34 ± 0.03 mM for glutamate. Maximum respiration rate Vmax at pyruvate oxidation increased from 0.077 ± 0.002 to 0.119 ± 0.002 and 0.140 ± 0.002 nmol O2/(s·million cells) due to the increase of [Ca2+] from 10-7 to 5×10-7 or 10-6 M, respectively. At oxidation of succinate or glutamate Ca2+ did not significantly affect Vmax. Thus, the increase of [Ca2+] stimulates respiration of mitochondria in situ of acinar pancreacytes at oxidation of exogenous pyruvate (obviously due to pyruvate dehydrogenase activation), but not at succinate or glutamate oxidation.

Estimation of ATP-dependent K(+)-channel contribution to potential-dependent potassium uptake in the rat brain mitochondria

O. V. Akopova, V. I. Nosar, L. I. Kolchinskaya, I. N. Mankovska, V. F. Sagach

Bogomoletz Institute of  Physiology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: a-dubensky@mail.ru

The effect of potassium on state 4 respiration (substrate oxidation in the absence of ADP) was investigated. It was shown that potential-dependent potassium uptake in the brain mitochondria results in mitochondrial depolarization. Taking into account depolarization effect of potassium, the contribution of the endo­genous proton leak as well as K+-uptake to the re­spiration rate was calculated. It was shown that such estimation allows the share of ATP-dependent potassium channel contribution to potential-dependent potassium uptake to be determined by polarographic method.