Merlavsky V. Ca2+-regulation of hepatocytes respiration upon various functional states of organism

Mechanisms of regulation of rat hepatocytes respiration by Ca2+ were investigated. A model of permeabilized hepatocytes for mitochondria in situ respiration studying was developed. It was revealed that hepatocytes permeabilization depends on digitonin concentraion in medium and on cell number in suspension. The highest respiration rate upon either succinate along or malate, glutamate and pyruvate mixture oxidation was in the solution with 1 mcM Ca2+. ADP-stimulated oxygen consumption was the most intensive within the medium containing 0.1 mcM Ca2+. Concentration of 10 mcM Ca2+ has been revealed to be toxic for hepatocytes mitochondria. The kinetic dependence of respiration upon either succinate or pyruvate oxidation was well described by Hill equation in the mediums with concentrations of Са2+ 0.1 and 1 mcM. Hill coefficient h and semi activation constant K0.5 for suc-cinate, at rotenone presence, were not changed after increase of Са2+ concentration from 0.1 to 1 mcM. And maximal velocity Vmax slightly increased. The kinetic dependence of respiration upon succinate and pyruvate oxidation was well described by Hill equation under taurine action, similar to control. Kinetic parameters of res-piration, upon succinate oxidation at both Са2+ concentrations, were not changed significantly under prolonged taurine injection. . Substrate inhibition, inherent to the dependence of respiration rate on pyruvate concentration within the medium with 1 mcM Са2+, started developing, under taurine effect, at higher concentration of this substrate in comparison with control. Short-term insulin action in vitro did not influence the examined processes. In vivo insulin effect was characterized by time dependence with activation of ADP-stimulated succinate and a-ketoglutarate oxidation in isolated hepatocytes in 4 hours after injection and absence of influence in case of prolonged action (6, 12 days). The greater intensification of respiration by mixture of malate, glutamate and pyruvate in diabetic animals was observed at 10 mcM Са2+. ADP-stimulated respiration rate in diabetic rats was maximal at 1 mcM Са2+.