RESUMO
To examine ryanodine-sensitive Ca2+ channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α-ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage-sensitive fluorescent probe tetramethylrhodamine-methyl-ester (0.1µM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m-chlorophenyl hydrazone (CCCP, 10µM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo-4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05µM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1µM or 1µM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α-ketoglutarate in the presence of ryanodine (0.05µM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1µM or 1µM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05µM, 0.1µM, or 1µM) causes differential effects on Ca2+ concentration in the mitochondria matrix under oxidation of pyruvate or α-ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra-mitochondrial Ca2+ concentration and affecting the energy state of mictochondria in hepatocytes.