UTP Content Research Articles

UMP Kinase Regulates Chloroplast Development and Cold Response in Rice.

Pyrimidine nucleotides are important metabolites that are building blocks of nucleic acids, which participate in various aspects of plant development. Only a few genes involved in pyrimidine metabolism have been identified in rice and the majority of their functions remain unclear. In this study, we used a map-based cloning strategy to isolate a UMPK gene in rice, encoding the UMP kinase that phosphorylates UMP to form UDP, from a recessive mutant with pale-green leaves. In the mutant, UDP content always decreased, while UTP content fluctuated with the development of leaves. Mutation of UMPK reduced chlorophyll contents and decreased photosynthetic capacity. In the mutant, transcription of plastid-encoded RNA polymerase-dependent genes, including psaA, psbB, psbC and petB, was significantly reduced, whereas transcription of nuclear-encoded RNA polymerase-dependent genes, including rpoA, rpoB, rpoC1, and rpl23, was elevated. The expression of UMPK was significantly induced by various stresses, including cold, heat, and drought. Increased sensitivity to cold stress was observed in the mutant, based on the survival rate and malondialdehyde content. High accumulation of hydrogen peroxide was found in the mutant, which was enhanced by cold treatment. Our results indicate that the UMP kinase gene plays important roles in regulating chloroplast development and stress response in rice.

Исследование содержания уридиловых нуклеотидов и активности аспартаткарбамоилтрансферазы в тканях облученных крыс при введении оротовой кислоты и перфторана

Purpose: Study of uridylic nucleotides content and aspartate carbamoyltransferase which was a key enzyme on the pathway for the synthesis of pyrimidine nucleotides in tissues of irradiated animals upon administration of orotic acid and perftorane was conducted.
 Material and methods: Studies were performed on random-bred albino rats subjected to a single γ-ray exposure at a total dose of 6 Gy. Orotic acid was injected as potassium salt in a dose of 60 mg / kg, perftoran salt in a dose of 1 ml / 100 g water.
 Results: A decrease in the content of UTP and UDP, as well as an increase in UMP after irradiation, especially on the 7th day, was established. The most pronounced changes in the studied biochemical parameters take place in the mucosa of the small intestine. The administration of orotic acid and perftorane to irradiated animals contributes to a significant correction of both the nucleotide content and the activity of aspartate carbamoyltransferase.
 Conclusion: The radiation leads to some decrease in the content of UDP and UTP. Changes in the content of nucleotides and activity of aspartate carbamoyltransferase in the mucosa of the small intestine are more pronounced in comparison with liver tissue. The combined administration of orotic acid and perftorane promotes the normalization of the content of nucleotides, and the activity of aspartate carbamoyltransferase in the liver and mucous membrane of the small intestine of irradiated animals.

The effects of galactosamine on UTP levels in the livers of young, adult and old rats.

Galactosamine (GalN), a well-known hepatotoxin that depletes the cellular pool of uracil nucleotides, was previously shown to have greater impact on the inhibition of protein synthesis in hepatocytes of old rats as compared with young animals (Kmieć 1994, Ann. N.Y. Ac. Sci. 717, 216-225). In the present study we compared the effects of GalN on the nucleotide content (measured by ion-exchange HPLC) in the livers of young (4 months), adult (12 months), and old (24-26 months old) rats two hours after its intraperitoneal administration. UTP content of the livers of old control rats was significantly lower (by 28%) than that of young animals. GalN administration decreased the UTP content in the livers of young, adult and old rats by, respectively, 55%, 65% and 89%, and increased the content of UDP-sugars by 189%, 175% and 305%. The hepatic content of ATP, ADP, AMP, NAD, GTP except CTP did not differ significantly among the age groups of rats studied, and was not changed by GalN treatment. The content of CTP was significantly higher in old rats (P < 0.03) upon GalN treatment. The lower hepatic content of UTP may partially explain the increased sensitivity of hepatocytes and livers of old rats to the action of galactosamine, and possibly to other hepatotoxic compounds that decrease transcription in the liver.

Elevated UTP and CTP Content in Cultured Neurons from HPRT-Deficient Transgenic Mice

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8.; HPRT) catalyzes the salvage synthesis of inosine-5'-monophosphate (IMP) and guanosine-5'-monophosphate (GMP) from the purine bases hypoxanthine and guanine, respectively. Complete deficiency of HPRT activity is associated with the Lesch-Nyhan syndrome (LNS), characterized by excessive purine production and severe neurological manifestations. The etiology of the metabolic consequences of HPRT deficiency is clarified, but that of the neurological manifestations is not yet understood. HPRT-deficient mice represent an experimental animal model of LNS. In search for a possible metabolic abnormality in LNS brains, connecting the neurological deficit to HPRT deficiency, the purine and pyrimidine nucleotide content of cultured neurons, prepared from HPRT-deficient transgenic mice, was now determined. The HPRT-deficient neuronal cultures exhibited a significantly elevated content of the pyrimidine nucleotides UTP (1.33-fold the normal level, p = 0.0002) and CTP (1.28-fold the normal level, p = 0.02), but normal content of the purine nucleotides ATP and GTP. This abnormality in neuronal pyrimidine nucleotide content may be associated with the pathophysiology of the neurological deficit in LNS.

Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury

ATP-dependent potassium (K ATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate K ATP channels. In addition, the effects of the K ATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N 2–5% CO 2 instead of 95% O 2–5% CO 2 for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%), probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (300 μM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 μM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 μM) antagonized the effects of diazoxide (300 μM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 μM). The ATP/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO 2. However, only the ATP/ADP ratio was decreased further by diazoxide (300 μM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of K ATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival via protein and DNA synthesis.

Effects of differentiation-inducing agents on purine nucleotide metabolism in an ovarian cancer cell line.

The effects of the differentiation-inducing agents sodium butyrate (NaOBt), dimethylsulfoxide (DMSO) and mycophenolic acid (MA), on purine nucleotide metabolism, was studied in an ovarian carcinoma cell line (GZL-8). Exposure to these agents inhibited cell proliferation, but did not affect cell viability. Three hours following exposure, NaOBt and DMSO moderately decelerated purine synthesis de novo, but MA accelerated it three-fold, this being associated with a two-fold increase in the excretion of hypoxanthine and xanthine into the incubation medium. NaOBt and DMSO did not affect the cellular nucleotide content, but MA caused a 73% decrease in GTP content and about a 50% increase in the cellular content of UTP. The following alterations in cellular enzyme activity were observed 72 h following exposure: NaOBt decreased the activity of hypoxanthine-guanine phosphoribosyltransferase and increased the activity of IMP and of AMP 5'-nucleotidases, DMSO increased the activity of IMP 5'-nucleotidase, and MA increased the activity of the two nucleotidases. The results suggest that, in the carcinoma cell line studied, the differentiation process induced by NaOBt and DMSO may be associated with a general shift in the direction of purine metabolism from anabolism to catabolism, whereas that induced by MA is associated with a specific decrease in the production of GTP.

Is cellular integrity responsible for the partial NMR invisibility of ATP in isolated ischemic rat liver?

The observability of nucleoside triphosphate (NTP) by 31P NMR spectroscopy was studied in the isolated rat liver during hypothermic perfusion and a subsequent 4-h cold ischemia. The influence of hypothermia (4 degrees C) was examined because of its delaying effects on cell injury induced by the ischemic conditions. The viability of the liver after hypothermic ischemia was assessed by measuring the recovery of the beta-NTP resonance after reperfusion. In 4-h cold ischemic liver, recovery was found to be in the range of 90-100% and consequently NTP visibility was studied under these conditions. Because the individual purine (or pyrimidine) NTPs are not distinguishable in the liver on the basis of their 31P NMR chemical shifts, the contributions of UTP and GTP were investigated by HPLC. The changes in liver NTP content measured either by NMR on isolated liver or by HPLC after perchloric acid extraction from the same organ are not significantly different. The total NTP level in normothermic perfused liver is 7.6 +/- 0.2 mumol NTP/g liver dry wt as determined by NMR. In such a liver, ATP + GTP + UTP and ATP contents measured by HPLC are, respectively, 7.9 +/- 1.0 and 6.3 +/- 0.9 mumol/g liver dry wt. This indicates that all NTP is detected by NMR and that a 20% contribution of the signal occurs from UTP + GTP. Under 4-h cold ischemic conditions, NTP visibility remains unchanged, furthermore the UTP + GTP contribution reaches 32% of the whole NTP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism and actions of 2‐deoxy‐2‐fluoro‐d‐galactose in vivo

The synthetic D-galactose analog 2-deoxy-2-fluoro-D-galactose (dGalF) offers unique advantages for studies of the D-galactose pathway by non-invasive techniques using 19F-NMR spectroscopy or positron emission from the 18F-labeled compound. The metabolism of 2-deoxy-2-fluoro-D-galactose was studied in rodents using the unlabeled, the 18F-labeled, and the 14C-labeled D-galactose analog. Analyses for the metabolites of 2-deoxy-2-fluoro-D-galactose were performed by HPLC, enzymatic methods, and 19F-NMR spectroscopy in vivo and in vitro. The metabolism of 2-deoxy-2-fluoro-D-galactose was most active in the liver which took up the major part of the administered dose of the 14C-labeled D-galactose analog, but renal excretion was also pronounced. This was confirmed by in vivo scanning of the rat using the 18F-labeled sugar (1.5 microCi/g; 25 nmol/g) and examination by positron-emission tomography and gamma camera. The dose dependence of the levels of the hepatic metabolites of 2-deoxy-2-fluoro-D-galactose was investigated for doses between 25 nmol/g body mass and 1 mumols/g body mass. After 1 h, the major part of the acid-soluble uracil nucleotides consisted of UDP-2-deoxy-2-fluoro-D-hexoses when the dose was at least 0.1 mumols/g. With higher doses, 2-deoxy-2-fluoro-D-galactose 1-phosphate became the predominant initial metabolite. After a dose of 1 mumols/g 2-deoxy-2-fluoro-D-galactose 1-phosphate accumulated rapidly (5.3 +/- 0.4 mumols/g liver after 30 min) followed by the formation of UDP-2-deoxy-2-fluoro-D-galactose and UDP-2-deoxy-2-fluoro-D-glucose (0.7 +/- 0.1 mumols/g and 1.8 +/- 0.1 mumols/g, respectively, after 5 h). The diversion of uridylate, due to the accumulation of UDP-2-deoxy-2-fluoro-D-hexoses, was associated with a rapid depletion of hepatic UTP, UDP-glucose, and UDP-galactose. The UTP content was decreased to 11 +/- 6% of normal within 15 min after administration of 2-deoxy-2-fluoro-D-galactose at a dose of 1 mumols/g. The UTP-depleting action was minimal, however, at a dose of 25 nmols/g or less, indicating that interference in uridylate metabolism would be negligible at the doses required for positron-emission tomography of the liver using the 18F-labeled compound. At higher doses, the UTP deficiency induced by 2-deoxy-2-fluoro-D-galactose could be useful in the chemotherapy of D-galactose-metabolizing tumors such as hepatocellular carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of purine and pyrimidine nucleosides and their catabolites from the perfused rat hindlimb in response to noradrenaline, vasopressin, angiotensin II and sciatic-nerve stimulation

Uric acid and uracil were released at constant rates (0.95 and 0.4 nmol/min per g respectively) by the perfused rat hindlimb. Noradrenaline, vasopressin or angiotensin II further increased the release of these substances 2-5-fold, coinciding with increases in both perfusion pressure (vasoconstriction) and O2 uptake. The hindlimb also released, but in lesser amounts, uridine, hypoxanthine, xanthine, inosine and guanosine, and all but hypoxanthine and guanosine were increased during intense vasoconstriction. Uric acid and uracil releases were increased by noradrenaline in a dose-dependent manner. However, the release of these substances did not fully correspond with the dose-dependent increase in O2 uptake and perfusion pressure, where changes in the latter occurred at lower doses of noradrenaline. Sciatic-nerve stimulation (skeletal-muscle contraction) did not increase the release of uracil, uric acid or uridine, but instead increased the release of inosine (7-fold) and hypoxanthine (2-fold). Since the UTP content as well as the UTP/ATP ratio are higher in smooth muscle than in skeletal muscle, it is proposed that release of uric acid and uracil arises from increased metabolism of the respective adenosine and uridine nucleotides during intense constriction of smooth muscle.

In vivo metabolism and UTP-depleting action of 2-deoxy-2-fluoro- d-galactose

The metabolism of 2-deoxy-2-fluoro- d-galactose (dGalF) was studied in rodents using HPLC, enzymatic methods, and 19F-NMR spectroscopy in vivo and in vitro. The liver took up the major part of the administered dose of the 14C-labeled d-galactose analog. This was confirmed in vivo by use of the 18F-labeled sugar (1.5 mCi/kg; 25 μmol/kg) and examination by positron emission tomography. After a dose of 1 mmol/kg, dGalF-1-phosphate accumulated rapidly (5.3 ± 0.4 mmol/kg after 30 min), followed by formation of UDP-dGalF and UDP-2-deoxy-2-fluoro- d-glucose (0.7 ± 0.1 and 1.8 ± 0.1 mmol/kg, respectively, after 5 hr). Good quantitative agreement was obtained between the measurements by HPLC and enzymatic analyses and by 19F-NMR. The noninvasive in vivo 19F-NMR technique is particularly advantageous, since it allows the simultaneous analysis of all dGalF metabolites. The diversion of uridylate, due to the accumulation of UDP-2-deoxy-2-fluoro- d-hexoses, was associated with a rapid depletion of hepatic UTP, UDP-glucose, and UDP-galactose. The UTP content was decreased to 11 ± 6% of normal within 15 min after administration of dGalF at a dose of 1 mmol/kg. The UTP-depleting action was minimal, however, at a dose of 25 μmol/kg or less, indicating that interference in uridylate metabolism will be negligible at the doses required for positron emission tomography of the liver using the 18F-labeled compound. At higher doses, the UTP deficiency induced by dGalF may be useful in the chemotherapy of d-galactose-metabolizing tumors such as hepatocellular carcinoma. At moderate doses of dGalF, 19F-NMR spectroscopy in vivo or in vitro could be used to pinpoint defects of the enzymes that cause galactosemia, i.e. of galactokinase, uridyltransferase, or 4-epimerase.

A comparison of rapid adenine nucleotide incorporation into phosphoglyceroyl-ATP and into RNA-like species in perfused rat heart

(1) Four main species of rapidly synthesised trichloroacetic acid-insoluble derivatives of adenylate can be separated in extracts from rat hearts. (2) The major species, accounting for more than 70% of the total, is phosphoglyceroyl-ATP; two others (16% of the total) are closely related to it. (3) Around 10% of incorporated radioactivity is in a high-molecular-weight form with a phosphate/purine ratio of 0.8; comparison of [ 14C]uridylate and [ 14C]adenylate incorporation supports the suggestion that this is a rapidly synthesised species of RNA which represents about 4% of total RNA in heart. (4) Values are given for the contents of UTP, UDP and UDPglucose in adult rat hearts.

Action de la D-galactosamine sur les nucléotides du cceur de rat. Effets de l'administration de cytidine et d'uridine

The treatment of rats by galactosamine (2 mmol/kg i.p.), which dramatically alters the metabolism of pyrimidine nucleotides in the liver, has been used to investigate the dynamics of pyrimidine nucleotides in the rat heart. Six hours after administration of the drug, the UTP and UDPG myocardial contents were decreased by respectively 40 and 52% while the sum of uracil nucleotides was increased by 66% and that of cytosine nucleotides by 15%. When administered 5 h after galactosamine treatment, cytidine (750 nmol/rat i.v.) induced a further increase in cytosine nucleotides (46% above control value 1 h later) without however effect on uracil nucleotides. On the other hand, the administration of uridine (250 nmol/rat, i.v. 5 h after galactosamine), while restoring UTP, UDPG and the pool of uracil nucleotides, provoked a decrease in cytosine nucleotide level (-17%). In the absence of galactosamine treatment, the administration of uridine and cytidine did not induce changes in nucleotide levels despite a rise in blood cytidine concentration. All these observations support the hypothesis that: 1. the pathway for cytosine nucleotide synthesis predominant in the heart is that utilizing preformed exogenous cytidine and 2. this pathway is mainly controlled by the intracellular concentration of UTP rather than that of CTP.

Mechanisms of action of pyrazolopyrimidines in Leishmania donovani.

We investigated the antileishmanial actions of the pyrazolopyrimidines allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine), thiopurinol (4-thiopyrazolo[3,4-d]pyrimidine), and aminopurinol (4-aminopyrazolo[3,4-d]pyrimidine). These compounds affect several metabolic processes. The first is the inhibition of GMP reductase by the IMP analogues allopurinol ribonucleoside monophosphate and thipurinol ribonucleoside monophosphate which reduces the organism's ability to synthesize ATP from guanine. Second, interconversion of adenine nucleotides to guanine nucleotides, is curtailed by the inhibition of IMP dehydrogenase by these same IMP analogues. Third, the IMP analogues reduce intracellular UTP content. The fourth affect is increased catabolism of RNA and consequent reduction of protein synthesis. This latter effect is due to the adenine nucleotide analogues aminopurinol ribonucleoside mono-, di-, and/or triphosphates, metabolic products of both allopurinol and aminopurinol.

Nucleotide Pools in the Eggs of Emma Field Cricket, Teleogryllus emma, and Two-Spotted Cricket, Gryllus bimaculatus(Orthoptera : Gryllidae)

Oxygen consumption in the eggs of two-spotted cricket, Gryllus bimaculatus (non-diapause species) increased gradually with embryonic development, but in the eggs of emma field cricket, Teleogryllus emma (diapause species) it decreased after diapause initiation. The contents of ATP, GTP, UTP and CTP in the eggs of two-spotted cricket increased throughout embryonic development. On the other hand, the contents of four triphosphates in the eggs of emma field cricket increased during the period of pre-diapause, but decreased after diapause initiation. In particular, the decrease of UTP and CTP was conspicuous at diapause initiation. The contents of UTP and CTP also increased at early stages of diapause development and embryonic development. UTP and CTP in the eggs of emma field cricket might play an important role in the regulation of early embryonic development, intervening diapause.

Metabolism of UDP-glucose by developing embryos of round and wrinkled varieties of Pisum sativum

Developing embryos from growing pods of round (Birte) and wrinkled (Greenshaft) varieties of Pisum sativum were studied during the period of rapid growth. No significant activity of UDP-glucose phosphorylase or of pyrophosphohydrolases for ATP or UTP was detected in extracts of embryos. Estimates of the maximum catalytic activities of UDP-glucose pyrophosphorylase and pyrophosphate: fructose 6-phosphate 1-phosphotransferase [PFK(PPi)] showed them to change during development roughly in parallel with that of sucrose synthase, and to exceed the maximum estimates of the rate of sucrose breakdown. Measurements of the embryo content of UTP, UDP-glucose, inorganic pyrophosphate, hexose monophosphates and fructose-1,6-bisphosphate suggest that the reactions catalysed by sucrose synthase, UDP-glucose pyrophosphorylase and PFK(PPi) approach equilibrium in vivo. Fructose-2,6-bisphosphate content showed a positive correlation with the rate at which the embryos converted sucrose to starch. Incubation of embryos in [U-14C]sucrose led to substantial labelling of UDP-glucose. It is suggested that sucrose synthase makes a major contribution to sucrose breakdown by forming UDP-glucose that is converted to glucose 1-phosphate by UDP-glucose pyrophosphorylase using pyrophosphate generated by PFK(PPi).

De novo pyrimidine biosynthesis in isolated rat hepatocytes. Quantitative aspects of the regulation by UTP.

Quantitative aspects of de novo pyrimidine biosynthesis in rat hepatocytes were monitored. A reduction of intracellular UTP contents by different concentrations of D-galactosamine led to a dose-dependent increase of 14CO2 incorporation into the sum of all acid-soluble uracil nucleotides. In controls the rate of de novo synthesis which was calculated from the incorporation rate of 14CO2 into the sum of all acid-soluble uracil nucleotides was 0.014 mumol X h-1 X g-1 compared to 0.056 mumol X h-1 X g-1 wet weight of liver in situations of a maximally stimulated de novo synthesis. Incubation of hepatocytes with uridine led to a dose-dependent reduction of 14CO2 incorporation to less than 25% of the amount incorporated in the controls. Alterations of the CTP content had no influence on the 14CO2 incorporation. In the presence of high D-galactosamine concentrations the increase of the total amount of acid-soluble uracil nucleotides exceeded the rate of the de novo synthesis derived from the incorporation of 14CO2 into the sum of the acid-soluble uracil nucleotide pool. It was also greater than the increase of the total amount of intra- and extracellular orotate after acidic hydrolysis--even in the presence of 6-azauridine, which stimulated de novo pyrimidine biosynthesis by itself.

Uridylate-trapping sugar analogs in combination with inhibitors of uridylate synthesis de novo and 5-fluorouridine

The combination of 3 types of antipyrimidines was studied in AS-30D hepatoma cells in suspension culture and in the rat in vivo. 1. 1.|Callulear UTP and CTP pools can be depleted most effectively by combining an inhibitor of de novo UMP synthesis with sugar analogs diverting UMP to UDP-sugar analogs. The following UMP-trapping sugar analogs were employed: d-galactosamine, d-galactosone, d-glucosome, and d-glucosamine. These d-galactose and d-glucose analogs intensified the depletion of UTP and CTP pools induced by the following inhibitors of de novo UMP synthesis: acivicin, PALA, palachol, pyrazofurin, and 6-azauridine. 2. 2.|The sugar analogs, in the absence of inhibitors of the de novo pathway, enhanced the rate of de novo UMP synthesis several-fold, as indicated by incorporation of 14CO 2 into intermediates and the products of the pathway and by the expansion of the acid-soluble uracil nucleotide pool. 3. 3.|Reduction of UTP and CTP contents to less than 5 and 15% of control, respectively, by d-galactosamine and PALA resulted in a decrease of the rate of RNA synthesis to 19% of control as calculated from the changes in specific activities of [ 14C]CTP and of [ 14C]cytidine in RNA after labeling with [ 14C]uridine. [cating that pyrimidine nucleoside excretion is regulated by pyrimidine nucleotide levels, possibly by UTP and CTP regulation of uridine kinase. 4. 5.|Determinaton of the rates of de novo pyrimidine synthesis, of the formation of RNA pyrimidines, and of pyrimidine nucleoside excreton indicates that de novo synthesis provides only about 67% of the pyrimidines required for the consuming processes. The difference, as well as the dilution of labeled pyrimidine nucleotide pools under conditions of a blocked de novo pathway, suggests a considerable salvage of pyrimidine nucleosides derived from RNA. This salvage of pyrimidines may be intracellular and/or by an excretion and re-uptake process. 5. 6.|Depletion of UTP and CTP pools, induced in hepatoma cells by d-galactosamine and 6-azauridine, leads to growth inhibition in suspension culture; this inhibition becomes irreversible in an increasing percentage of cells, killing all cells after 20 hr of UTP deficiency. 6. 7.|The enhanced uptake of 5-fluorouridine by UTP-deficient cells was associated with an increase of FUMP incorporation into RNA up to 4-fold and with stronger inhibition of cell growth. The amounts of FUDP-sugars formed from 5-fluorouridine in hepatoma cells were smaller than the amounts of UDP-sugars formed from uridine. All the FUDP-sugars served as substrates i vitro of the respective enzymes of UDP-sugar metablism. 7. 8.|Combination of UMP-trapping sugar analogs with an inhibitor of de novo UMP synthesis and followed by a pyrimidine ribonucleoside analog such as 5-fluorouridine may improve the tissue selectivity and the effectiveness of tumor chemotherapy with antipyrimidines.

Pyrimidine nucleotide metabolism in cardiac hypertrophy.

Cardiac hypertrophy was induced in rats either by isoprenaline treatment or by experimental abdominal aortic stenosis. In both models, the UTP content increased in the hypertrophying heart, while the ATP level remained depressed. The rate of net synthesis of uracil nucleotides, estimated by phosphate incorporation into alpha-phosphate groups, was largely increased during the first stage of hypertrophy (X 3.6 and 2.6, respectively). At identical stages of hypertrophy, the rate of synthesis of ribosomal RNA, measured after continuous intracardiac infusion of 3H-uridine, was increased by 1.6 and 2.5 times. A comparison of the changes in time course of the levels of UTP and RNA and their turnover suggests that the metabolism of uracil nucleotides and that of ribosomal RNA are closely interrelated. The increase in the rate of synthesis of pyrimidine is interpreted as an adaptational change in nucleotide metabolism in response to the increased requirement of precursors for RNA synthesis. The possible limiting role of pyrimidine nucleotides in the hypertrophic process is discussed.

Combined action of acivicin and d-galactosamine on pyrimidine nucleotide metabolism in hepatoma cells

The glutamine antagonist acivicin, l-(αS,5S)-α- amino-3- chloro-4,5- dihydro-5- isoxazoleacetic acid, strongly reduced CTP and GTP contents in AS-30D rat hepatoma cells in suspension. UTP only dropped to 63% of the respective control after 4hr; however, by combining acivicin with the uridylate-trapping sugar analogue d-galactosamine, a synergistic decrease in UTP contents to 7% of control was induced. Incorporation of 14CO 2 into purine and pyrimidine nucleotides followed by radio-high performance liquid chromatography showed marked inhibition of purine and pyrimidine biosynthesis de novo; the latter was reduced to 35% of control. The inhibitory potency of acivicin on glutamine-dependent carbamoyl-phosphate synthetase and consequently on de novo uracil nucleotide formation was also reflected by the complete suppression of the d-galactosamine-induced rise in total uridylate. Induction of UTP deficiency by interference with the first and rate-limiting step in pyrimidine biosynthesis de novo together with a trapping of uridylate by d-galactosamine may provide a promising approach to the chemotherapy of hepatocellular carcinoma.

Derangements in myocardial purine and pyrimidine nucleotide metabolism in patients with coronary artery disease and left ventricular hypertrophy.

Studies in animal models of myocardial ischemia and left ventricular hypertrophy have demonstrated a number of derangements in purine and pyrimidine nucleotide content of myocardium that are postulated to play a role in the pathogenesis of muscle dysfunction in these disorders. The present study examined myocardium of patients with coronary artery disease, left ventricular hypertrophy, or neither of these two abnormalities, to determine whether derangements in purine and pyrimidine nucleotide metabolism occur in humans. In patients with coronary artery disease, endocardial content of ATP, GTP, UTP, CTP, and creatine phosphate was reduced and ranged between 60% and 86% of the amount found in the epicardium. In patients without coronary artery disease or ventricular hypertrophy, endocardial content of these nucleotides was equal to or greater than that of epicardium. Endocardial and epicardial content of inosine was increased in patients with coronary artery disease, and after vein bypass grafting inosine content fell to the levels observed in myocardium of patients with normal coronary arteries. In patients with left ventricular hypertrophy, endocardial content of ATP, GTP, UTP, CTP, and creatine phosphate was also reduced and ranged between 64% and 88% of the epicardial content. In contrast to results obtained in patients without left ventricular hypertrophy, epicardial content of GTP, UTP, and CTP was increased by 131%, 123%, and 132% in hypertrophied myocardium. Thus the changes noted in myocardial nucleotide content in patients are similar to those noted in animal models of occlusive coronary disease and ventricular hypertrophy. These results suggest that the pathophysiological abnormalities in nucleotide metabolism noted in animal models also occur in human myocardium.