Regulation by calcium ions of pyruvate carboxylation, pyruvate transport, and adenine nucleotide transport in isolated rat liver mitochondria

The activity of pyruvate carboxylase in isolated, tightly coupled, rat liver mitochondria was estimated by measuring the incorporation of H¹⁴CO₃ into acid-stable metabolites in the presence of pyruvate and ATP. The addition of a given amount of Ca²⁺ (10 to 100 nmol/mg of protein) to isolated mitochondria catalyzing the incorporation of H¹⁴CO₃^- into acid-stable intermediates at a steady state rate caused a rapid decrease in the rate of H¹⁴CO₃^- incorporation, and the establishment of a new inhibited steady state rate. This effect corresponded with the uptake of Ca²⁺ (monitored with the use of ⁴⁵Ca) from the incubation medium by the mitochondria. When the steady state rate of H¹⁴CO₃^- incorporation was measured in the presence of various fixed concentrations of added Ca²⁺, it was found to decrease as the concentration of intramitochondrial Ca²⁺ increased from 21 (no added Ca²⁺) to 120 nmol/mg of protein (100 nmol/mg of added Ca²⁺). Inhibition by Ca²⁺ was more pronounced at low concentrations of pyruvate. Thus a 50% inhibition of the rate of H¹⁴CO₃^- incorporation was observed after the addition of either 25 or 10 nmol/mg of exogenous Ca²⁺ at 7.5 and 1 mM pyruvate, respectively. Inhibition of H¹⁴CO₃^- incorporation by Ca²⁺ could be completely reversed by the subsequent addition of Mn²⁺ (25 to 100 nmol/mg). An increase in intramitochondrial Ca²⁺ (from 20 to 50 nmol/mg of protein) was accompanied by 25% increase in the activity of active form of pyruvate dehydrogenase. Elevated concentrations of intramitochondrial Ca²⁺ (50 nmol/mg of exogenous Ca²⁺) were associated with (a) an 80% inhibition of the rate of mitochondrial ATP transport and a decrease of 50% in the pool of intramitochondrial adenine nucleotides available for exchange with exogenous [¹⁴C]ATP (measured at 0°); (b) a 40% inhibition of the rate of mitochondrial pyruvate transport measured at 6° in the presence of rotenone and antimycin A; (c) a 20% decrease in the intramitochondrial pyruvate concentration; (d) a small increase in the intramitochondrial concentration of acetyl-CoA; and (e) no significant change in the intramitochondrial concentrations of total ATP and ADP. A control experiment showed that an increase in intramitochondrial Ca²⁺ from 20 to 65 nmol/mg of protein caused a small stimulation of the activity of the citric acid cycle when this was estimated by measuring the release of ¹⁴CO₂ in the presence of [U-¹⁴C]malate. It is concluded that (a) inhibition by Ca²⁺ of H¹⁴CO₃^- incorporation is the result of inhibition of the pyruvate carboxylase enzyme by CaATP²⁺ and Ca²⁺ present in the mitochondrial matrix as well as a decrease in the concentration of intramitochondrial pyruvate; (b) the data are consistent with the proposal that changes in the intramitochondrial concentrations of Ca²⁺ can contribute to the regulation of the activity of pyruvate carboxylase in the liver; and (c) under some conditions, changes in the mitochondrial Ca²⁺ concentrations may regulate cellular metabolism through their effects on mitochondrial ATP and pyruvate transport.