The study focuses on the involvement of Ca2+ channels of the plasma membrane, endoplasmic reticulum and mitochondria in regulating energy processes in rat hepatocytes. In particular, liver perfusion with 20 μM eosin Y, incubation of the liver homogenate 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 IP3Rs in regulating oxygen consumption in hepatocytes is more significant, as adding ІP3 into the polarographic chamber resulted in an increased oxygen consumption rate for the permeabilized hepatocytes in comparison with oxidation of the substrates of Са2+-activated dehydrogenases (α‐ketoglutarate and pyruvate).
It was found that ryanodine has effect on Са2+ concentration in the mitochondria matrix, membrane potential of the mitochondria and oxygen consumption in them. 0.05–1 μM ryanodine was shown to cause reduction of Са2+ 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 membrane potential drops for oxidation of pyruvate or α‐ketoglutarate, but not for succinate. The inhibiting effect of mRyRs on oxygen consumption of isolated mitochondria depends on Ca2+ concentration in the medium, substrate of oxidation and duration of exposure to ryanodine. For pyruvate at 0.1 μM Ca2+ 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 Ca2+ it increases. For α‐ketoglutarate oxidation, ADP-induced oxygen consumption by the mitochondria rises following the addition of ryanodine into the polarographic cell with Ca2+ in the concentration 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 Са2+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.
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 Са2+-activated dehydrogenases (α‐ketoglutarate and pyruvate) and 0.1 μM Ca2+ in the medium, the membrane potential of the hepatocytes mitochondria rises, while for oxidation of succinate it decreases.
Therefore, different Са2+ channels of the cells contribute differently to energy regulation in the liver mitochondria. A crucial role in Ca2+-activated regulation of oxidation in the liver mitochondria is performed by mRyRs.
