Activation of pyruvate dehydrogenase during metabolism of ammonium ions in hemoglobin-free perfused rat liver.

Abstract

1 Ammonium chloride addition to perfused rat liver results in a shift of the steady state of the pyruvate dehydrogenase system towards the active (dephospho) form. This is evidenced by direct measurement of enzyme activity in freeze-stopped liver tissue, by net uptake of pyruvate from the perfusate, and also by an increased release of labeled CO2 from [1-14C]pyruvate. Half-maximal concentration of NH4Cl for extra pyruvate uptake is 0.5–0.7 mM. The effect is readily reversible. 2 Extra urea formation upon ammonia addition is matched by an extra O2 uptake as that required theoretically for the two ATP-consuming steps of urea synthesis. At 1.5 mM NH4Cl, there is no significant difference of ATP levels compared to the controls. 3 When extra urea formation and the concomitant O2 uptake are suppressed due to limitation at carbamoylphosphate synthesis in carbon-dioxide-free media, the activation of pyruvate dehydrogenase by NH4Cl is still observed. 4 When ammonia is generated intracellularly from glutamine, an extra uptake of pyruvate is not observed. 5 The NH4Cl-induced activation of pyruvate dehydrogenase is increased in presence of acetoacetate, and decreased in presence of 3-hydroxybutyrate. 6 Possible mechanisms are discussed. While some of the observations could also be explained by a direct interaction of ammonium ions with the pyruvate dehydrogenase interconversion system, the effect of ammonium ions on pyruvate dehydrogenase is interpreted to be mediated in an indirect manner by changes of the mitochondrial concentration of 2-oxoglutarate. As 2-oxoglutarate decreases during reductive amination to glutamate, a pool of high-energy compounds (eg. GTP or related substances) dependent upon 2-oxoglutarate oxidation is depleted, resulting in a de-inhibition of pyruvate dehydrogenase by the interconversion system. The detailed mechanism of the linkage between 2-oxoglutarate oxidation and pyruvate oxidation remains to be elucidated.