Information on the thesis

The study focuses on the involvement of Ca 2+ channels of the plasma membrane,
endoplasmic reticulum and mitochondria in regulating energy processes in rat hepato-
cytes. In particular, liver perfusion with 20 μM eosin Y, incubation of the liver homog-
enate with it or adding it directly into the polarographic chamber were found to have
no effect on the oxygen consumption and oxidative phosphorylation. This suggests that
in the conditions of the experiment, the role of PMCA and SERCA in the regulatory
mechanisms of energy consumption in hepatocytes is insignificant. In contrast, the role
of IP 3 Rs in regulating oxygen consumption in hepatocytes is more significant, as add-
ing ІP 3 into the polarographic chamber resulted in an increased oxygen consumption
rate for the permeabilized hepatocytes in comparison with oxidation of the substrates
of Са 2+ -activated dehydrogenases (α ‐ ketoglutarate and pyruvate). It was found that
ryanodine has effect on Са 2+ concentration in the mitochondria matrix, membrane po-
tential of the mitochondria and oxygen consumption in them. 0.05–1 μM ryanodine
was shown to cause reduction of Са 2+ accumulation in the mitochondria matrix, which
appeared the most significant for pyruvate oxidation. This data is indicative of mRyRs
presence in rat hepatocytes, which, in contrast to RyRs of the endoplasmic reticulum,
are inhibited by ryanodine in the studied range. Due to this, the mitochondria mem-
brane potential drops for oxidation of pyruvate or α ‐ ketoglutarate, but not for succinate.
The inhibiting effect of mRyRs on oxygen consumption of isolated mitochondria de-
pends on Ca 2+ concentration in the medium, substrate of oxidation and duration of ex-
posure to ryanodine. For pyruvate at 0.1 μM Ca 2+ in the medium, the rate of ADP-
induced oxygen consumption by the mitochondria decreases considerably (and the
more so, the longer the exposure is); on the contrary, for 1 μM Ca 2+ it increases.
For α ‐ ketoglutarate oxidation, ADP-induced oxygen consumption by the mitochondria rises
following the addition of ryanodine into the polarographic cell with Ca 2+ in the con-
centration 0.1 μM. If succinate was the substrate of oxidation, adding ryanodine into
the polarographic cell results in the reduced rate of ADP-induced oxygen consumption
by the mitochondria for both Са 2+ concentrations, but only slightly. In order to establish
if the functional activity of RyRs of the endoplasmic reticulum influences the oxidation
processes in the mitochondria, we studied the effect of ryanodine (0.05, 0.1 and 1 μM)
on oxygen consumption in isolated intact hepatocytes. It appears that ryanodine effects
depend both on its concentration and on the duration of exposure to it. For instance,
oxygen consumption in the intact hepatocytes decreased after their prior incubation or
adding them to the media containing ryanodine in the concentrations 0.1 μM and 1 μM.
In case of adding ryanodine into the polarographic chamber after the hepatocytes, the
most significant drop was recorded for the ryanodine concentration 0.05 μM. At the
same time, adding ryanodine into the polarographic cell in the concentration of 1 μM
to the isolated hepatocytes did not result in statistically significant changes of their
oxygen consumption rate. Such relationship between the oxygen consumption rate of
the hepatocytes and duration of exposure to ryanodine and its concentration stems from
the fact that mRyRs and RyRs have different affinity to it. The inhibiting effects of
RyRs of the endoplasmic reticulum of the intact hepatocytes on the mitochondria oxy-
gen consumption are less marked and have a limited time span, which is why only in
case of a direct addition of ryanodine in the concentration of 1 μM into the polaro-
graphic cell they neutralize the mRyRs inhibiting effects. To verify the hypothesis that
suramin, which is RyRs agonist, can be also mRyRs agonist, we studied its effect on
the mitochondria membrane potential. Indeed, in the concentration of 1 μM, suramin
activates mRyRs. As a result, for oxidation of substrates of Са 2+ -activated dehydrogen-
ases (α ‐ ketoglutarate and pyruvate) and 0.1 μM Ca 2+ in the medium, the membrane
potential of the hepatocytes mitochondria rises, while for oxidation of succinate it
decreases. Therefore, different Са 2+ channels of the cells contribute differently to en-
ergy regulation in the liver mitochondria. A crucial role in Ca 2+ -activated regulation of
oxidation in the liver mitochondria is performed by mRyRs.
Key words: RyRs, mRyRs, ryanodine, cellular respiration, hepatocytes, suramin,
Δψ, Ca 2+ , substrates for oxidation.