Effect of Ammonium Ion on Pyrimidine Synthesis <em>de novo</em> in Isolated Rat Hepatocytes.

Addition of ammonium ions to isolated rat hepatocytes stimulated the rate of synthesis of pyrimidines. Isolation and quantification of pyrimidine nucleotides, orotic acid an the acid-hydrolyzed product of carbamoylaspartic acid by non-exchange chromatography and high-pressure liquid chromatography show a marked simulation in the incorporation of [¹⁴C]bicarbonate in incubations with added ammonium ions. The incorporation into total uridine nucleotides (σUMP) was increased twofold in the presence of 5 mM ammonium ion, and approximately eightyfold into orotic acid. There was a parallel increase in labelling a carbamoylaspartic acid from undetectable to a level similar to that of orotic acid. The specific activity of urea formed during the incubations did not change during incubations or in the presence of ammonium ions confirming that the change in labelling of pyramidine was not due to a change in the specific activity of precursor. Despite the stimulation in incorporation of label into pyrimidines, there was no increase in the hepatcyte content of σUMP, which was 11.5 µmol/g dry weight, although the orotic acid content increased from 0.09 µmol/g dry weight in the absence of added ammonium ions (but in the presence of 2 mM L-glutamine) to 8.6 µmol/g dry weight with 5 mM ammonium ion. The stimulated incorporation of [¹⁴C]becarbonate in the presence of 5 mM and 10 mM ammonium ion was shown to be due to a stimulated synthesis of carbamoyl phosphate, since greater than 80% of label in the uracil ring was present at position C-2. Incubation of hepatocytes in basal medium (Eagles) containing 2.5% foetal calf serum and 20 mM bicarbonate showed that there was a significant stimulation of pyrimidine synthesis with 1 mM ammonium completely reversed by 5 mM L-ornithine and was partially reversed by 1 mM L-ornithine. Evidence for a contribution of the urea cycle carbamoyl phosphate synthetase to pyrimidine synthesis is discussed. [ABSTRACT FROM AUTHOR]