Influence of ammonium ions on hepatic de novo pyrimidine biosynthesis

Abstract

Carbamyl phosphate (CP) is synthesized in the liver by two separate enzymes, CPS I and CPS II. CPS I, an intramitochondrial enzyme involved in ureogenesis, has a relative activity of 500- to 1000-fold greater than CPS II, a cytoplasmic enzyme which initiates the sequence of reactions for pyrimidine biosynthesis. The contributions of NH 4Cl (substrate for CPS I) and glutamine (substrate for CPS II) as precursors for pyrimidine biosynthesis in isolated hepatocytes were compared by measuring their effect on uracil nucleotide pool size, the incorporation of NaH 14CO 3 into these pools, and the accumulation of orotic acid. Physiological concentrations of NH 4Cl caused a marked stimulation of incorporation of radioactivity into uracil nucleotides (6-fold increase at 0.5 m m NH 4Cl), and radioactive orotate appeared in both the cells and the medium. In contrast, glutamine (at concentrations up to 10 m m) had no effect on the incorporation of radioactivity into uracil nucleotides, and no orotic acid was detected. Uracil nucleotide pools were expanded up to 50% by low levels of NH 4Cl, but there was no expansion of this pool in the presence of added glutamine. NH 4Cl-driven pyrimidine de novo biosynthesis was insensitive to feedback inhibition by an expanded uracil nucleotide pool, to galactosamine treatment, and to acivicin treatment, indicating that NH 4 + stimulated pyrimidine biosynthesis as a result of CP synthesis by mitochondrial CPS I. The consequence of intramitochondrially produced CP being available for pyrimidine biosynthesis is that the controlling step of this pathway (CPS II) is bypassed. The appearance of orotic acid following NH 4Cl stimulation indicated that the rate-controlling step of hepatic de novo pyrimidine synthesis under these conditions was orotate phosphoribosyl transferase. These data indicate that, at physiological concentrations of NH 4 +, the majority of uracil nucleotides synthesized in isolated rat hepatocytes was derived from intramitochondrially generated CP. The effect of NH 4Cl on the output of uridine by the isolated perfused rat liver was examined. In the presence of a single addition of 20 m m NH 4Cl, the excretion of uridine was increased from 100–200 to 375 nmol h −1 g −1 liver and orotic acid was released into the circulating perfusate reaching a maximum of 2 μ m (in 220 ml of perfusate) after 2 h. With 40 m m NH 4Cl, uridine export was increased to 450 nmol h −1 g −1 and a maximum of 5 μ m orotic acid was released into the perfusate after 2 h. Thus, the feasibility of altering uridine excretion by the liver as a consequence of modulation of the urea cycle was demonstrated.