Mazur G. Experimental substantiation of criteria of mitochondrial adaptive capacity of rat hepatocytes

The thesis is aimed at experimental substantiation of mitochondrial adaptive capacity criteria in rat hepatocytes and testing the feasibility of said criteria utilization in assessing the effects of regulatory or toxic agents such as Ca2+ or alcohol on mitochondrial oxidative function. It is suggested that mitochondrial adaptive capacity can be assessed by the following criteria: maximal uncoupled respiration rate, optimal protonophore (FCCP) concentration, acceleration of respiration upon stimulation with FCCP and the area under the curve of dependence of the uncoupled respiration rate on FCCP concentration. Oxidative substrates affect mitochondrial adaptive capacity of hepatocytes. The lowest maximal uncoupled respiration rate was upon glucose oxidation, and the highest upon α-ketoglutarate and succinate oxidation. Methyl esters of succinate and α-ketoglutarate maintain mitochondrial adaptive capacity at a higher level than free substrates. It was found that mitochondrial adaptive capacity of hepatocyte depends on the method of cell isolation, as indicated by a difference in maximal uncoupled respiration rate and optimal FCCP concentration. Upon in vitro liver perfusion the range of adaptive respiratory responses of mitochondria of hepatocytes is significantly narrower compared to cells isolated using in situ liver perfusion. It was shown that maximal rates of uncoupled respiration and the optimal concentrations of FCCP were higher upon in situ liver perfusion than upon in vitro liver perfusion. Irrespectively of the perfusion methods, the maximal rate of uncoupled respiration is highest with the use of monomethyl succinate and the optimal FCCP concentration is highest upon pyruvate oxidation. Thus, liver mitochondria are very sensitive to adequate perfusion during hepatocyte isolation procedure. The increase of Ca2+ concentration to 10 μM caused a marked diminishing of the adaptive capacity of mitochondria, as evidenced by a decrease of both ADP- stimulated and FCCP-stimulated respiration of permeabilized hepatocytes. Such changes are observed upon oxidation of either succinate or the mixture of malate, glutamate and pyruvate. It was established that ADP-stimulated respiration time-dependent decrease accelerated with increase of Ca2+ concentration. The addition of CsA after Ca2+ into the polarographic chamber did not change the respiration rate of hepatocytes both upon succinate or mixture of malate, glutamate and pyruvate oxidation. The addition of CsA into the chamber before Ca2+ prevented the Ca2+-induced decrease of ADP-stimulated and FCCP-stimulated respiration upon succinate oxidation. CsA also abolished the negative effect of high Ca2+ concentrations on the FCCP-stimulated respiration when mixture of malate, glutamate and pyruvate was present in the medium. However, CsA did not affect the negative effect of high Ca2+ concentrations on the ADP-stimulated respiration under these conditions. The effects of alcohol in vitro on the adaptive capacity of mitochondria were assessed. Alcohol in vitro did not affect the maximal FCCP-stimulated respiration, mitochondrial membrane potential and NAD(P)H autofluorescence of hepatocytes. CsA in vitro did not change the effect of alcohol on the respiration of hepatocytes. Unlike in vitro, alcohol in vivo increased maximal FCCP-stimulated respiration upon endogenous substrates, glucose or succinate oxidation, but did not affect the optimal FCCP concentration. Pyruvate in vivo did not affect maximal uncoupled respiration rate or optimal FCCP concentration and did not change the effect of alcohol on the respiration of hepatocytes. The obtained results prove that new criteria of adaptive capacity mitochondria are useful to study mitochondria of liver cells under physiological and pathological conditions.