Stimulation of uracil nucleotide synthesis in mouse liver, intestine and kidney by ammonium chloride infusion (original) (raw)
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De novo synthesis of uracil nucleotides in mouse liver and intestine studied using [15N]alanine
European Journal of Biochemistry, 1990
The amount of newly synthesized uracil nucleotides in mouse liver and intestine was determined by analysis of 15N incorporation into the uracil nucleotide pool of these tissues after intraperitoneal infusion of 15N-labelled amino acids. The appearance of newly synthesized uracil nucleotides was linear with time, and essentially independent of the rate of infusion of L-[15N]alanine. Varying the amino acid used in the infusion could affect the enrichment in the uracil ring nitrogens, but had no significant effect on the calculated amount of de novo synthesis. These results demonstrate the utility of this method in measuring de novo uracil nucleotide synthesis in mouse liver and intestine in vivo. The method should be a valuable tool in the effort to understand the regulation and pharmacological manipulation of de novo uracil nucleotide synthesis.
European Journal of Biochemistry, 1992
The relative contribution of de-novo and salvage synthesis to tissue pyrimidine nucleotide pools is an important parameter in the rational design of anti-pyrimidine therapies, but has not been measured in vivo. We have measured the contribution of de-novo synthesis to the total acid-soluble uracil nucleotide pool in mouse tissues by analysis of the incorporation of label after intra-peritoneal infusion of L-[15N]alanine. The contribution of salvage synthesis was measured by the incorporation of radiolabel after intravenous infusion of [14C]uridine. The results show that de-novo synthesis makes the larger contribution to the intestine uracil nucleotide pool, salvage synthesis makes the larger contribution to the kidney pool, and de-novo and salvage synthesis make roughly equal contributions to the liver pool. In tumors studied (L1210, P388, B16, Nettesheim), the contribution of de-novo synthesis was at least five times the contribution of salvage synthesis. The measurements were repeated 24 hours after a 400-mg/kg dose of N-phosphonacetyl-L-aspartic acid. De-novo synthesis was substantially inhibited in all tissues and tumors after this treatment, although significant residual activity was observed in the intestine and L1210 cells. Nettesheim carcinoma was the only tumor or tissue to show a significant increase in salvage synthesis after N-phosphonacetyl-L-aspartic acid treatment.
Life Sciences, 2002
Uric acid and allantoin are the key compounds of purine nucleotide catabolism formed in liver and many other organs of the rat. We observed that, after administration of 14 C-formate, incorporation of radioactivity into uric acid and allantoin is not similar, as one would expect. The phenomenon was demonstrated to be specific to liver and perfused liver, and not to other organs such as heart, jejunal mucosa, lung, spleen, and kidney. To interpret these results, the specific radioactivity of uric acid and allantoin in rat liver were analysed comparatively, after administration of the following labelled precursors: 14 C-glycine, 14 C-formate, 14 C-hypoxanthine, 14 C-uric acid and 14 C-adenine. After administration of 14 C-formate the specific radioactivity of allantoin was higher than that of uric acid and the same behavior was observed after 14 C-uric acid and 14 C-hypoxanthine, but not after 14 C-glycine and 14 C-adenine administration. The results indicate that the rate of their incorporation into uric acid and allantoin, and the subsequent export of these compounds into serum, can only partially explain the observed phenomenon, while the presence of different pools of uric acid and allantoin may give a complete explanation. D
Synthesis of pyrimidine nucleotide precursors in human and rat small intestinal mucosa
Biochimica et Biophysica Acta (BBA) - General Subjects, 1967
Methods of thin-layer chromatography have been devised to study the interconversions of the pyrimidine biosynthetic precursors aspartic acid, carbamoylaspartic acid, dihydroorotic acid, and orotic acid in preparations of rat and human small intestinal mucosa. Whole mucosai homogena~es, intact whole mucosal cells, crypt celt homogenates, and intact crypt cel!s obtained from the ra~ and human jejunal whole mucosal homogenates were utilized. Homogenate preparations were most active in utilizing aspartate and carbamoyiaspartate, whereas intact cells best formed orotic acid. Material obtained from the mucosal crypt areas, where cellular division occurs generally gave higher specific conversion rates than corresponding whole mueosal preparations. Human jejunai whole mucosal homogenates gave similar conversions to those of rat jejuno-ileai preparations. The methods have been scaled down to permit analysis of pyrimidine precursor metabolism in amounts of tissue obtainable by peroral biopsy.
Early induction of Na+-dependent uridine uptake in the regenerating rat liver
FEBS Letters, 1993
Na'-dependent uridine transport into liver plasma membrane vesicles from partially hepatectomized and sham-operated rats was studied. Preparations purified 6 h after 70% hepatectomy exhibited an increased V,,,,, of uridine uptake (3.7 vs. 1.4 pmollmg protl3 s) without any change in K,,, (6pM). Incubation of the vesicles in the presence of monensin decreased uridine uptake although the differences between both experimental groups remained identical. It is concluded that uridine transport is induced early after partial hepatectomy by a mechanism which does not involve changes in the transmembrane Na' gradient. This is the first evidence in favor of modulation of nucleoside transport into liver cells.
The relationship between urea and pyrimidine de novo synthesis in ruminant liver
Quarterly journal of experimental physiology (Cambridge, England), 1988
The influence of ammonia and inhibition of the urea cycle by norvaline on orotic acid synthesis in five Polish Merino ewes was investigated. The sheep were exposed to 60 min of successive infusions of 0.9% NaCl, NH4Cl (20 mumol kg-1 min-1), and NH4Cl with DL-norvaline (10 mumol kg-1 min-1) solutions administered to the mesenteric vein. The average rate of orotic acid output, urea output and ammonia uptake in the liver in control conditions (infusion of 0.9% NaCl) was: 1.09 nmol g-1 min-1, 0.97 mumol g-1 min-1 and 0.94 mumol g-1 min-1, respectively. Ammonia significantly stimulated the orotic acid output, urea output and ammonia uptake. At the same time liver uptake of glutamic acid, alanine and glycine increased. Norvaline as a competitive inhibitor of ornithine transcarbamylase depressed citrulline release and urea output in the liver, and elevated, although not significantly, the orotic acid output. It was concluded that ammonia stimulates the de novo synthesis of pyrimidine in th...
Pyrimidine nucleotide synthesis in the rat kidney in early diabetes
Biochemical Medicine and Metabolic Biology, 1991
Early renal hypertrophy of diabetes is associated with increases in the tissue content of RNA, DNA, and sugar nucleotides involved in the formation of carbohydrate-containing macromolecules. We have previously reported an increase in the activity of enzymes of the de nova and salvage pathways of purine synthesis in early diabetes; the present communication explores the changes in the pathways of pyrimidine synthesis. Measurements have been made of key enzymes of the de novo and salvage pathways at 3, 5, and 14 days after induction of diabetes with streptozotocin (STZ), phosphoribosyl pyrophosphate (PPRibP), and some purine and pyrimidine bases. Carbamoyl-phosphate synthetase II, the rate-limiting enzyme of the de novo route, did not increase in the first 5 days after STZ treatment, the period of most rapid renal growth; a significant rise was seen at 14 days (+38%). Dihydroorotate dehydrogenase, a mitochondrial enzyme, showed the most marked rise (+ 147%) at 14 days. The conversion of orotate to UMP, catalyzed by the enzymes of complex II, was increased at 3 days (+42%), a rise sustained to 14 days. The salvage route enzyme, uracil phosphoribosyltransferase (UPRTase), showed a pattern of change similar to complex II. The effect of the decreased concentration of PPRibP on the activities of CPSII, for which it is an allosteric activator, and on activities of OPRTase and UPRTase, for which it is an essential substrate, is discussed with respect to the relative
Distribution of Purine Nucleotides in Uremic Fluids and Tissues
Journal of Renal Nutrition, 2010
There are almost 100 different substances called uremic toxins. In this study, we analyze all findings concerning the new family of uremic compounds-nicotinamide end products: N-methyl-2-pyridone-5-carboxamide (Met2PY), N-methyl-4-pyridone-5-carboxamide, newly described 4-pyridone-3-carboxamide-1-b-D-ribonucleoside (4PYR) and 4-pyridone-3-carboxamide-1-b-D-ribonucleoside triphosphate (4PYTP). After few years of studies, we have found that these substances have higher plasma concentration in patients with chronic renal failure (CRF) in comparison with the healthy population. We noted a 40-fold increase in plasma 4PYR concentration in patients with CRF. This increment correlates significantly with the decline of kidney function measured as an increase of serum creatinine concentration and decrease of estimated glomerular filtration rate. Tested compounds are present and measurable in physiological fluids and tissues. We found higher saliva Met2PY concentration in patients with CRF in comparison with controls. Saliva Met2PY correlated negatively with estimated glomerular filtration rate and positively with serum creatinine concentration. One-third of studied group had higher concentration of Met2PY in the saliva than in plasma, and this segment of patients may be called as ''good excretors.'' In rats with experimental CRF, we found that both Met2PY and N-methyl-4-pyridone-5-carboxamide accumulated in selected tissues. We also demonstrated formation of 4PYTP in intact human erythrocytes during incubation with the precursor 4PYR. Incubation with 4PYR leads to lowering concentration of adenosine-5'-triphosphate. 4PYTP formation may be a way to remove 4PYR from the circulation and save adenosine-5'-triphosphate depletion. Summarizing, end products of the nicotinamide family are members of uremic toxins; however, exact pathophysiological role of these compounds in the development of uremic syndrome needs further studies.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1987
The anabolism of pyrimidine ribo-and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo-and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine-and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidinenucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.
Experimental Biology and Medicine, 1961
The mechanism involved in puromycin aminonucleoside (P-A) nephrosis is still not conclusively established. The available evidence favors the idea that the drug acts by competing with an essential precursor or inhibiting a key enzyme in the synthesis of nucleic acid.13 The striking specificity of chemical structure required for nephrotoxicity is unique, since even analogues with seemingly minor alterations of the molecule are ineffective in causing the disease.4 Similarly unique is the recent report by Hartman, Hartman and Baldridge 2 that in short-term experiments adenine partly protected animals from P-A toxicity while adenosine was totally ineffective. The present work extends these investigations, using adenine, adenosine and adenosine triphosphate (ATP) in doses relatively large compared to P-A. In addition, clinical, laboratory and pathologic observations have been extended over a period of several months following the course of drug administration. METHODS Female rats of the Holtzman strain, weighing approximately 200 (+20) gin., were used. Since aminonucleoside (P-A) causes nephrosis in ioo per cent of treated rats, relatively few animals were required for any experiments employing P-A animals as controls. The rats were housed in individual metabolism cages in a temperaturecontrolled environment and were fed a diet of Fox Chow and water ad libitum throughout the course of the experiment. Drugs that were given once daily were administered subcutaneously in the evening, and in some cases injections were given both moming and evening. When P-A (kindly furnished by Dr. Stanton Hardy of the Lederle Laboratories) was injected with another drug, the latter was always introduced a few minutes before the former and at a separate site. Except as indicated in the footnote of Table I, injections were given daily until the day of sacrifice. P-A was dissolved in distilled water, and o.6 ml. of an 0.5 per cent solution was injected daily. The other drugs (Nutritional Biochemicals Corporation) were dissolved or suspended in distilled water in the following concentrations: adenine, 2.5 to 5 per cent; adenosine, 2 per cent; and ATP, io per cent in the form of the tetrahydrate disodium salt. In order to insure and maintain stability, the ATP solution was adjusted to pH 7