Stimulation of uracil nucleotide synthesis in mouse liver, intestine and kidney by ammonium chloride infusion

Abstract

De novo pyrimidine synthesis was studied in mouse liver, intestine, and kidney by intraperitoneal infusion of l5NH4C1 and analysis of "N incorporation into uracil nucleotide pools. When the dose of a 1-h infusion of "NH4C1 was increased from 50 pmol to 250 pmol the fraction of the total uracil nucleotide pool formed by de novo synthesis increased 4.0-fold in liver to 8.4% and 2.3-fold in intestine to 13.7%. The increase in intestine was independent of the increase in liver as evidenced by the lack of correlation between the increase observed in the intestine and liver of the same animal and the different distributions of label in the uracil ring nitrogens. A 2.4-fold increase in newly formed uracil nucleotides was observed in kidney when the infusion dose was raised from 150 pmol to 250 pmol. The increase in kidney was correlated with the increase in liver in the same animal and the distribution of label in the uracil ring nitrogens was similar to the distribution in liver. These results suggest that the increase in newly formed uracil nucleotides in intestine is due to increased de novo synthesis of pyrimidines in the intestine, while the increase in the kidney is due to increased salvage synthesis of uracil nucleotides from uridine synthesized in the liver and output to the circulation.In mammalian liver under normal physiological conditions the production of carbamoyl phosphate is thought to be rate-limiting for both the production of urea [1, 21 and the de novo synthesis of pyrimidines [3 -51. Carbamoyl phosphate for urea synthesis is formed by the mitochondrial enzyme carbamoyl phosphate synthase (CPS I). This enzyme uses ammonia for its substrate and is activated by N-acetyl-Lglutamate [6,7]. Carbamoyl phosphate for de novo pyrimidine synthesis is produced by the cytosolic enzyme, carbamoyl phosphate synthase I1 (CPS 11). CPS I1 is activated by 5'-phosphoribosyl diphosphate, inhibited by UTP and preferentially utilizes glutamine [3]. The intracellular separation of these enzymes serves to keep the two carbamoyl phosphate pools distinct and responsive to independent regulatory controls. However, in the presence of excess ammonium ions, the stimulation of CPS I increases mitochondrial carbamoyl phosphate levels to the point where mitochondrial carbamoyl phosphate can make substantial contributions to the cytosolic carbamoyl phosphate pool [8 -lo]. This provides a source

De novo synthesis of uracil nucleotides in mouse liver and intestine studied using [15N]alanine

“…Metabolism of L-alanine does generate ammonia [24] and at a high enough dose it would be expected that enough ammonia would be generated to stimulate de novo pyrimidine synthesis. It should be noted that for low doses of I5NH4CI, where stimulation of uracil were calculated from our previous work [23] nucleotide synthesis would be at a minimum, the calculated rate of uracil nucleotide synthesis in liver is virtually identical to the rate calculated after infusion of ~-["N]alanine. The rate in intestine, calculated after the lowest dose of I5NH4C1, is approximately 30% higher than the rate calculated after infusion of L -[~ 5N]alanine, but this difference is similar to differences in the rate calculated after infusion ~-['~N]alanine in different batches of animals.…”

“…Male CDFl mice were infused via an intraperitoneal catheter as previously described [23]. Tissue preparation, quantification of nucleotide pools by HPLC and measurement of isotopic enrichment in uracil were accomplished as previously described [23].…”

“…Tissue preparation, quantification of nucleotide pools by HPLC and measurement of isotopic enrichment in uracil were accomplished as previously described [23]. Calculation of the fraction of the uracil nucleotide pool synthesized by the de novo pathway and the enrichment in the uracil ring nitrogens of this newly synthesized fraction were also performed as before [23].…”

“…2 shows the effect of the rate of infusion of L-[15N]alanine on the measured rate of appearance of newly synthesized uracil nucleotides. For comparison, values from 15NH4CI infusions have been calculated from our previous work [23] and plotted. It can be seen that when the infusion rate of 15NH4CI increases from 50 pmol .…”

“…The method used most often to measure de novo pyrimidine synthesis in vitro, incorporation of radioactivity from [14C]bicarbonate, has met with limited success in vivo [19] and would be difficult to adapt to a clinical setting. Our laboratory has been investigating the use of stable isotope methodology to measure de novo pyrimidine synthesis [20 -231 and we have recently shown that the contribution of de novo synthesis to the uracil nucleotide pools of mouse tissues can be calculated from an analysis of the incorporation of 15N from 15NH4C1 into the uracil nucleotide pools [23] it unsuitable for the study of the unperturbed pathway, so we have concentrated on finding ways to supply 15N to pyrimidine precursor pools without stimulating de novo pyrimidine synthesis. In this paper we show that infusion of 15N-labelled amino acids meets this criterion and demonstrate the utility of 5N-labelled amino acid infusion for investigating de novo synthesis of uracil nucleotides in mouse liver and intestine in vivo.…”