Transient 45Ca Uptake and Release in Isolated Rat-Liver Cells during Recovery from Deenergized States

Abstract

1. Aerobic incubation of isolated rat liver cells--after dilution from the anaerobic stock suspension--transiently brings about a state, during which a reversible calcium uptake can be observed on addition of a respiratory substrate. Uptake varies greatly and can reach more than 50 nmol/mg protein, but declines to zero on prolonged preincubation, especially at higher temperature. Repeated additions of succinate or 3-hydroxybutyrate evoke new calcium transients. If ATP is simultaneously added, if greatly potentiates succinate-initiated reversible uptake. 2. If rotenone is present during the preincubation phase, calcium transients are strongly enhanced. Uptake is blocked by uncouplers and respiratory inhibitors, indicating the involvement of mitochondria. 3. Calcium uptake is not accompanied by increased oxygen consumption. The actual respiration cannot account sufficiently for the energy need of calcium uptake. Participation of cytoplasmic ATP is likely, as inhibitors of adenine nucleotide translocase affect uptake. 4. Lanthanum enhances calcium uptake in contrast to its action on mitochondria. 5. Pulse-labeling experiments indicate that the calcium taken up is removed from a rapidly exchangeable calcium pool by withdrawal into the mitochondria as a deep compartment. 6. Calcium uptake is accelerated either by increasing the phosphate level or by high temperature. It is prolonged by low temperature, high pH or high ATP concentration. Calcium release accelerates with increasing temperature, decreasing pH and a further rise in phosphate concentration. 7. The dependency on phosphate and temperature reveals a delicately poised equilibrium of uptake and release. At ambient temperature, phosphate increases uptake up to a concentration of 0.5 mM. Higher concentrations accelerate both uptake and release. At lower temperature, the accelerating effect on uptake predominates. A temperature shift during incubation results in adaptation of the calcium equilibrium to the new temperature, i.e. release of calcium at high temperature, uptake at low temperature. 8. Oxidizing metabolites inhibit succinate-stimulated calcium uptake and promote release of previously accumulated calcium. An increased sensitivity to phosphate is established. 9. With respect to isolated mitochondria, isolated liver cells appear to be a more realistic model for studying the physiological mechanism of mitochondrial calcium release, since compartmental constraints and regulations are maintained.