Has been used homogenate from rat liver and isolated hepatocytes to assess the influence of ryanodine at concentrations of 0.01, 0.05, 0.1 and 1 mmol/l on energy processes in hepatocytes. The homogenate preincubated with ryanodine (0.01, 0.05 and 0.1 ?mol / L) for 5 minutes, after that of preincubated homogenate (100 ?l) was added to a polarographic chamber with a base extracellular solution and was recorded of oxygen consumption. Rate of oxygen consumption was determined using polarographic method using Clark’s electrode. Pyruvate, ?-ketoglutarate and succinate (5 mM) were used as the oxidation substrates. Respiration was stimulated by the addition of ADP (final concentration in the chamber was 200 ?M). We have registered a certain tendency to reduce oxygen consumption by liver homogenate after preincubation with ryanodine (0.01 and 0.05 ?mol / L), for the oxidation of ?-ketoglutarate, pyruvate and succinate. This may be due to the mediated effects of ryanodine through RyRs of the endoplasmic reticulum, which are localized near mitochondria, or maybe by direct action on mRyRs in the internal membrane of mitochondria. Given that the relationships between mitochondria and the endoplasmic reticulum are only partially preserved in the homogenate the other series of our experiments have been performed on isolated intact hepatocytes. In this series of experiments was used ryanodine at concentrations of 0.05, 0.1, and 1 ?mol/l, and carried out research in three variants. In the first variant, the cells were preincubated with ryanodine for 5 min and then inserted into a polarographic chamber where there was respiration medium. In the next variants of the experiment, ryanodine was added together with the respiration medium to the polarographic chamber, and then a suspension of hepatocytes or the first was added the suspension of hepatocytes and then ryanodine. After preincubation of isolated hepatocytes with ryanodine at concentrations of 0.1 and 1 ?mol/l, the oxygen consumption was reduced at 3 minutes of record and at 5 minutes (at concentrations of 1 ?mol/l). In the experiment, when hepatocytes after isolation were added to polarographic chamber, where before it was added to respiration medium the ryanodine, the oxygen consumption at 3 minutes of registration decreased under ryanodine at concentrations of 0.1 and 1 ?mol/l, and at 5 minutes at a concentration of 1 ?mol/l. Reducing the duration of exposure of ryanodine - adding it directly to the in the polarographic chamber to a suspension of hepatocytes the allowed to reveal its leveling effect on the concentration of 1 mmol/l. Such changes in oxygen consumption by hepatocytes, as well as rat homogenate liver, are related to the divergent action of riaodine on RyRs and mRyRs which apparently, have a different affinity for it. Consequently, RyRs and mRyRs play an important role in intracellular regulation of respiration of mitochondria in hepatocytes. The ryanodine at concentrations of 0.05-1 ?mol/l inhibits mRyRs, which leads to a decreasing accumulation of Ca2+ in the mitochondrial matrix and decreasing functional activity of Ca2+-dependent enzymes of the citric acid cycle and the function of the respiratory chain. At the same time, ryanodine activates the RyRs which causes an intensification of mitochondrial respiration in hepatocytes. However, this effect is limited in time, so it is recorded only with the direct addition of ryanodine at a concentration of 1 ?mol/l to cells to polarographic chamber.
Real Time Impact Factor:
Author Name: N.I. KUPYNYAK, O.V. IKKERT, V.V. MANKO
URL: View PDF
Keywords: RyRs, mRyRs, ryanodine, cellular respiration, hepatocytes