Information on the thesis

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 Ca
2+
or alcohol on mito-
chondrial oxidative function.
It is suggested that mitochondrial adaptive capacity can be assessed by the fol-
lowing 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 concentra-
tion. Oxidative substrates affect mitochondrial adaptive capacity of hepatocytes. The
lowest maximal uncoupled respiration rate was upon glucose oxidation, and the high-
est 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 respira-
tion rate and optimal FCCP concentration. Upon in vitro liver perfusion the range of
adaptive respiratory responses of mitochondria of hepatocytes is significantly nar-
rower compared to cells isolated using in situ liver perfusion. It was shown that max-
imal 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 Ca
2+
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 ac-
celerated with increase of Ca
2+
concentration. The addition of CsA after Ca
2+
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 Ca
2+
prevented the Ca
2+
-induced
decrease of ADP-stimulated and FCCP-stimulated respiration upon succinate oxida-
tion. CsA also abolished the negative effect of high Ca
2+
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 Ca
2+
concentra-
tions 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.
Key words: adaptive capacity of mitochondria, oxidative substrates, hepato-
cytes, protonophores, mitochondrial permeability transition pore, cyclosporin A, al-
cohol.