Regulation of citric acid cycle by calcium

The relationship of extramitochondrial Ca²⁺ to intramitochondrial Ca²⁺ and the influence of intramitochondrial free Ca²⁺ concentrations on various steps of the citric acid cycle were evaluated. Ca²⁺ was measured using the Ca²⁺ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca²⁺ ranging from 0 to 1.2 μM. A change in matrix free Ca²⁺ from 0 to 0.3 μM caused a 135% increase in ADP stimulated oxidation of 0.6 mM α-ketoglutarate (K_0.5 = 0.15 μM). In the absence of ADP and the presence of 0.6 mM α-ketoglutarate, Ca²⁺ (0.3 μM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 μM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca²⁺ and isocitrate dehydrogenase was sensitive to Ca²⁺ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca²⁺, thus explaining the Ca²⁺ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess α-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca²⁺ and activity increased to 100% at 2 μM Ca²⁺. Activation by Ca²⁺ required more Ca²⁺ (K_0.5 = 1 μM) than for α-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria α-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca²⁺ action than pyruvate dehydrogenase.