Uptake Of Orotic Acid Research Articles

OAT2 catalyses efflux of glutamate and uptake of orotic acid

OAT (organic anion transporter) 2 [human gene symbol SLC22A7 (SLC is solute carrier)] is a member of the SLC22 family of transport proteins. In the rat, the principal site of expression of OAT2 is the sinusoidal membrane domain of hepatocytes. The particular physiological function of OAT2 in liver has been unresolved so far. In the present paper, we have used the strategy of LC (liquid chromatography)-MS difference shading to search for specific and cross-species substrates of OAT2. Heterologous expression of human and rat OAT2 in HEK (human embryonic kidney)-293 cells stimulated accumulation of the zwitterion trigonelline; subsequently, orotic acid was identified as an excellent and specific substrate of OAT2 from the rat (clearance=106 μl·min⁻¹·mg of protein⁻¹) and human (46 μl·min⁻¹·mg of protein⁻¹). The force driving uptake of orotic acid was identified as glutamate antiport. Efficient transport of glutamate by OAT2 was directly demonstrated by uptake of [³H]glutamate. However, because of high intracellular glutamate, OAT2 operates as glutamate efflux transporter. Thus expression of OAT2 markedly increased the release of glutamate (measured by LC-MS) from cells, even without extracellular exchange substrate. Orotic acid strongly trans-stimulated efflux of glutamate. We thus propose that OAT2 physiologically functions as glutamate efflux transporter. OAT2 mRNA was detected, after laser capture microdissection of rat liver slices, equally in periportal and pericentral regions; previous reports of hepatic release of glutamate into blood can now be explained by OAT2 activity. A specific OAT2 inhibitor could, by lowering plasma glutamate and thus promoting brain-to-blood efflux of glutamate, alleviate glutamate exotoxicity in acute brain conditions.

Fluoroorotic acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines.

Resistance of hepatic nodules to orotic acid-induced accumulation of uridine nucleotides.

It has been hypothesized that orotic acid (OA) promotes rat liver carcinogenesis by a differential mitoinhibitory mode. Consistent with this hypothesis, hepatic nodules are relatively resistant to OA-induced mitoinhibition. OA-induced mitoinhibition is dependent on the metabolism of OA to uridine nucleotides. The present studies investigate the uptake and metabolic pathway of OA, both in vivo and in vitro, as a possible basis for the resistance of hepatic nodules to OA-induced mitoinhibition. Rats bearing hepatic nodules exposed to 1% dietary OA exhibited increased levels of uridine nucleotides in the surrounding non-nodular liver (from 0.44 to 0.70 mg/g liver) but not in the hepatic nodules. Further, following administration of [3H]OA i.p., nodules have significantly lower levels of acid-soluble radioactivity compared to the non-nodular surrounding tissue. Furthermore, most of the acid-soluble radioactivity was present as uridine nucleotides, suggesting that the OA taken up was converted to uridine nucleotides. Similarly, hepatocytes from nodules in primary culture incubated with radiolabeled OA, have significantly lower levels (46-60%) of acid-soluble radioactivity. These results suggest that the decreased uptake of OA by hepatic nodules may be a factor contributing to the observed resistance of hepatic nodules to the mitoinhibitory effects of OA.

Effects of ginsenoside‐Rb2 on RNA synthesis

AbstractThe effects of a single intraperitoneal injection of ginsenoside‐Rb2, 2.5 mg/100g body weight, on the synthesis of nuclear RNA in liver cells were determined serially after administration. Although the RNA content remained almost unchanged, the uptake of orotic acid by RNA reached a peak 6h after ginsenoside‐Rb2 administration. On the other hand, the total RNA polymerase activity increased after administration, reaching a peak at 4h; the RNA polymerase II activity also showed a pattern of variation similar to that of total RNA polymerase activity. In addition, the activity of RNA polymerase I also showed an increase suggesting that ginsenoside‐Rb2 promoted the synthesis of rRNA and mRNA.

Schistosoma mansoni: Analysis of cercarial transformation methods

Four methods of transforming cercariae to schistosomulae in vitro in ELAC buffer (pH 7.2, 37 C, 0–6 hr incubation) were compared in relation to biochemical and ultrastructural characteristics. The transformation methods used were chemical (3 m M linoleate), mechanical (centrifuge/vortex), mechanical/chemical, and heat (incubation at 37 C). Ultrastructural characteristics examined were based on the presence or absence of glycocalyx, heptalaminate membrane, cyton granules, and nuclear condition. Two EM fixation methods were used. Biochemical parameters assayed were loss of water tolerance (uptake of trypan blue dye), eicosanoid biosynthesis (PGE, LTB 4, and 5-HETE), protein synthesis (leucine uptake), RNA synthesis (uracil and orotic acid uptake), and DNA synthesis (thymidine uptake). EM characteristics were remarkably similar for all transformation methods except heat incubation, with transformed cercariae evidencing the characteristics of schistosomulae (cyton granule migration, absence of glycocalyx and heptalaminate membrane); however, euchromatic nuclei could not be demonstrated using in vivo or in vitro transformation methods. Despite the ultrastructural similarities between transformation methods, biochemical data demonstrated that the resultant organisms were quite different. The chemical transformation method gave the highest rate of loss of water tolerance and eicosanoid production. RNA and protein synthesis were not correlated to ultrastructural changes and were highest in those organisms undergoing mechanical transformation methods, significantly higher than in those cercariae transformed by the chemical method. DNA synthesis was not demonstrated using any transformation method, although thymidine uptake did occur. Our data indicate substantial biochemical differences exist between morphologically similar organisms. Thus, experiments using any type of artificially transformed schistosomule must be interpreted with caution until additional biochemical and physiological studies on cercarial transformation are undertaken.

Hormonal stimulation of 3H-orotic acid incorporation into RNA by serum-free cultured hepatocytes

Insulin and dexamethasone greatly stimulate the incorporation of 3H-orotic acid into RNA. Such a stimulation is associated to an increase in the uptake of the labelled precursor into the acid soluble fraction as well as in the specific radioactivity of the nucleoside plus nucleotide pool suggesting that hormone supplementation does not affect RNA synthesis by cultured cells. The lack of effect of insulin and dexamethasone on the level of total RNA polymerase activity in nuclei isolated from cultured hepatocytes is in line with this assumption. The hormone stimulated uptake of orotic acid is dependent on protein synthesis since it is completly abolished by cycloheximide.

RNA synthesis in primary cultures of adult rat hepatocytes.

The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver.

Characterization of hemadsorption-negative mutants of Mycoplasma pneumoniae.

Previously isolated mutants of Mycoplasma pneumoniae incapable of hemadsorption were characterized with respect to specific protein content, tracheal ring attachment capability, and virulence for both in vitro and in vivo model systems. Two-dimensional gel electrophoresis revealed both quantitative and qualitative differences between the protein complements of two different mutant strains and that of the virulent parent strain. Studies of mycoplasma attachment to hamster tracheal rings in vitro demonstrated that only one of these mutant strains still possessed the ability to attach to the respiratory epithelium via neuraminidase-sensitive receptors. Measurement of [3H]orotic acid uptake in mycoplasma-infected tracheal rings indicated that infection with the hemadsorption-negative mutants resulted in only slight reductions of ribonucleic acid synthesis, similar to levels observed for tracheal rings infected with an avirulent strain of M. pneumoniae. The virulence potential of the two mutant strains was further investigated by utilizing the hamster model system. Both mutant strains were rapidly cleared from the lungs of infected animals and produced little or no microscopic pneumonia.

Cumulative biochemical effects of repeated subclinical hydrogen sulfide intoxication in mouse brain.

Adult female NMRI mice exposed four times to 100 ppm hydrogen sulfide vapour for 2 h at 4-day intervals showed increasing inhibition of the cerebral cytochrome oxidase activity. Cerebral RNA decreased significantly after the fourth exposure. This change was accompanied by the reduced orotic acid uptake in the RNA fraction. At the same time, 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity used as a marker for glia increased. Acetylcholine esterase activity remained unchanged. The initial exposures also caused an increase in the superoxide dismutase activity and an increase in the glutathione concentration. The latter effects were abolished in the third and fourth exposures. The present data seem to indicate that the biochemical effects of repeated subclinical hydrogen sulfide intoxications are cumulative.

Effect of halothane inhalation on orotic acid uptake and uridine incorporation into mouse liver RNA

Repeated exposure of mice to an oxygen-halothane atmosphere (99.1, v/v) caused the liver wt and RNA content to increase about 30 per cent. When RNA was labeled with radioactive orotic acid during this time, the labeling of RNA decreased about 50 per cent. The RNA precursor pool was not affected by halothane treatment. It could be demonstrated that the application of the anesthetic hampered the uptake of orotic acid from the blood into liver cells. Using radioactive uridine, this effect could not be observed. The study demonstrates that unexpected side effects of drugs might complicate the interpretation of results.

Alterations in the metabolism of hamster tracheas in organ culture after infection by virulent Mycoplasma pneumoniae.

Exposure of hamster tracheal rings in organ culture to virulent Mycoplasma pneumoniae organisms leads to alterations in macromolecular biosynthesis and metabolic activity of the respiratory epithelial cells. Avirulent organisms derived from the same parent strain do not produce these effects. During the course of infection by virulent mycoplasmas, tracheal rings show an initial increase in [14C]galactose uptake followed by a significant decline as infection progresses which is also accompanied by abnormal processing of galactose as evidenced by amounts of 14CO2 released. Parallel decreases in the rate of [3H]orotic acid and [3H]amino acid uptake are observed. Within 24 h after infection of tracheal rings by virulent mycoplasmas, inhibition of host cell ribonucleic acid and protien synthesis is evident. Ribonucleic acid synthesis in infected cells, analyzed by gel electrophoresis, is reduced by 80% at 48 h and is negligible by 96 h. The course of mycoplasma infection can be interrupted or reversed by erythromycin after the initial mycoplasma-host cell interaction since addition of erythromycin 24 h or earlier after infection prevents the onset of abnormal orotic acid uptake. However, 48 h after infection, rescue of host cells by erythromycin cannot occur and cytopathology becomes evident. These data suggest that mediation of host cell injury requires continued protein synthesis by attached mycoplasmas, and the primary effect of mycoplasma infection on tracheal organ culture may be at a transcriptional or translational level.

Biochemical effects of miconazole on fungi—I: Effects on the uptake and/or utilization of purines, pyrimidines, nucleosides, amino acids and glucose by Candida albicans

The antifungal and antibacterial drug miconazole has been shown to inhibit, at concentrations lower than those affecting growth, the transport of adenine, guanine and hypoxanthine by Candida albicans in suspension culture. The decrease in the incorporation of purines into nucleic acids seems to be the consequence of an inhibitory effect on their uptake into the cells. When the purines were replaced by adenosine, deoxyadenosine and guanosine, miconazole increased the uptake and incorporation of the radioactivity derived from the nucleosides into macromolecules. The data suggest that the drug-induced increase of nucleoside incorporation into nucleic acids is secondary to enhanced nucleoside transport. Miconazole also slightly affected the uptake of orotic acid. The transport of glucose, glycine and leucine was not affected by miconazole whereas in some way the drug affected glutamine uptake. Studies on the distribution of miconazole and/or its metabolites in the Candida cell indicate that in log-phase cells most of the radioactivity was found in the fraction containing cell walls and plasmalemma. In stationary-phase cells the highest radioactivity was found in the fraction which contained the microsomes. Although more information will be needed, the data presented indicate that at low concentrations, miconazole acts primarily on the yeast cell membranes (cell wall and plasmalemma) resulting in a selective inhibition of the uptake of precursors of RNA and DNA (purines) and mucopolysaccharide (glutamine). Higher doses and longer incubation periods also alter the activities of microsomal membranes.

Evidence of changes in orotic acid uptake and/or metabolism during phaseollin production in the French bean Phaseolus vulgaris L.

Evidence of changes in orotic acid uptake and/or metabolism during phaseollin production in the French bean Phaseolus vulgaris L.

Metabolic alterations of liver regeneration. XI. Modulation of the uptake of orotic acid into ribonucleic acids in regenerating rat liver

Metabolic alterations of liver regeneration. XI. Modulation of the uptake of orotic acid into ribonucleic acids in regenerating rat liver

Differential sensitivity of rat liver and rat hepatoma cells to α-amanitin

α-Amanitin, an inhibitor of RNA polymerase derived from the mushroom Amanita phalloides, was shown to be about five times more potent than actinomycin-D in inhibiting rat liver tyrosine aminotransferase induction by cortisol. Rat hepatoma cells grown in culture, however, were resistant to these inhibitory effects of α-amanitin, while actinomycin-D effectively inhibited enzyme induction in these cells. The insensitivity of the hepatoma cells could not be accounted for by: (a) specific binding of α-amanitin to fetal calf serum protein present in the tissue culture medium; (b) metabolic conversion of this bicyclic octapeptide to an active compound in rat liver which does not occur in hepatoma cells; or (c) rapid inactivation of this substance by the hepatoma cells. RNA polymerase studies with isolated nuclei indicated that inhibition of cortisol induction of rat liver tyrosine aminotransferase by α-amanitin was accompanied by an inhibition of Mn 2+/(NH 4) 2SO 4-activated RNA polymerase. No effect on the Mg 2+-activated polymerase was noted. Similar experiments with hepatoma cells, in conjunction with [ 14C]-uracil and [ 3H 5]-orotic acid uptake studies, indicated that the RNA polymerase of these cells was sensitive to α-amanitin only when the cell membrane was broken. The two major conclusions from these data are: (1) hepatoma cells are insensitive to α-amanitin because they are impermeable to this substance, and (2) the correlation between inhibition of the Mn 2+/(NH 4) 2SO 4-activated RNA polymerase and inhibition of tyrosine aminotransferase induction by cortisol strongly suggests that modification of transcription by this hormone is the primary stimulus for increased synthesis of this enzyme.

The conversion of orotic acid into uridine 5'-monophosphate by isolated perfused normal and regenerating rat livers.

The release of (14)CO(2) from [7-(14)C]orotic acid was measured in isolated perfused normal and regenerating rat livers. With some limitations, the release of (14)CO(2) from [7-(14)C]orotic acid can be used to estimate UMP synthesis in perfused livers. Isolated perfused livers rapidly pick up labelled orotic acid added to perfusate and convert most of it into UMP. Perfused regenerating livers produce approx. 2.5 times as much UMP/g of liver as do perfused normal livers. However, the absolute amount of orotic acid converted into UMP is higher in perfused normal livers than in perfused regenerating livers. Perfused regenerating livers do not differ in their orotic acid uptake and UMP synthesis from livers of comparable size in which regeneration is not taking place. The total amount of orotic acid taken up by the liver (rather than the rate of uptake) and the size of the liver appear to be the determining factors in UMP production. The results suggest that the decrease in liver size caused by partial hepatectomy may be in itself sufficient to account for an increase in the flow of metabolites in the pyrimidine pathway at the early stages of liver regeneration.

Cytochalasin B: lack of effect on mucopolysaccharide synthesis and selective alterations in precursor uptake.

Synthesis and secretion of mucopolysaccharide in mouse 3T3 fibroblasts and in embryonic submandibular glands are unaffected by amounts of cytochalasin B that alter the morphology of these cells and tissues. The drug markedly and reversibly inhibits incorporation of [(3)H]glucosamine into mucopolysaccharide by preventing cellular uptake of the precursor, but does not affect incorporation of radiosulfate. Cytochalasin does not alter DNA, RNA, or protein synthesis, but stimulates the uptake of orotic acid and markedly inhibits the uptake of glucose. These selective effects on the transport of small molecules suggest that the primary action of the drug may be on cell membranes. Since processes unrelated to microfilament disruption may be altered by cytochalasin, great caution must be exercised in interpreting studies with the drug.

Recent advances in vitamin D metabolism: 1,25-Dihydroxycholecalciferol, a kidney hormone controlling calcium metabolism

The action of vitamin D in healing rickets is associated with an increased absorption of calcium from the intestine and with a direct effect on calcium metabolism in bone. Other likely target organs are the kidney and, possibly, striated muscle. The basic question which originally motivated the investigations of vitamin D metabolism was the occurrence of a lag period of at least 10 hours between administration of vitamin D and its physiological effects. It was agreed that the lag period might be due partly or entirely to the time required for the adequate formation of a protein carrier for calcium transport. There was, of course, the other possibility that a transformation of the vitamin into an active molecule might add to the time lag. As has been shown by now, both working hypotheses were correct: calcium-binding protein(s) have been found that are dependent on the vitamin D status (Wasserman and Corradino, 1971), and 1,25-dihydroxycholecalciferol (1,25-DHCC) has been isolated and identified as the active, hormone-like molecule which is formed from its precursor, 25-hydroxycholecalciferol (25-HCC) by a special hydroxylation reaction in the kidney. The latter can thus be considered an endocrine organ for the elaboration of 1,25-DHCC, a hormone which finds its way to the target organs. Eight years ago, Eisenstein and Passavoy (1964) reported that actinomycin D inhibited Ca2 + transport in everted intestinal sacs; this was followed and confirmed by observations from a number of laboratories (for literature, see Lawson, 1971) which showed among other things that if vitamin D was given early enough, the nuclear inhibitor had no effect. These findings focused the attention on nuclear events in the interpretation of vitamin D action. It could be shown that vitamin D, added in vivo, increased the uptake of orotic acid into nuclear and total RNA and increased the template activity of DNA (Norman, 1966; Stohs, Zull and DeLuca, 1967; Lawson et al 1969c; Hallick and DeLuca, 1969). Tables 1 and 2 show the effects of vitamin D administration on the orotic acid uptake observed by the Cambridge group.

Renal metabolic response to acid-base changes. II. The early effects of metabolic acidosis on renal metabolism in the rat.

The early renal metabolic response was studied in rats made acidotic by oral feeding of ammonium chloride. 2 hr after feeding of ammonium chloride there was already significant acidosis. Urinary ammonia also increased after ammonium chloride ingestion and at 1(1/2) hr was significantly elevated. In vitro gluconeogenesis by renal cortical slices was increased at 2 hr and thereafter increased steadily. Ammonia production by the same slices was also increased at 2 hr, but thereafter fell and at 6 hr had decreased to levels which, although higher than those of the control, were lower than those obtained from the rats acidotic for only 2 hr. There was no correlation between in vitro gluconeogenesis and ammonia production by kidney slices from rats during the first 6 hr of acidosis, but after 48 hr of ammonium chloride feeding, these two processes were significantly correlated. The early increase in renal gluconeogenesis was demonstrable with both glutamine and succinate as substrates. The activity of the enzyme phosphoenolpyruvate carboxykinase was increased after 4-6 hr of acidosis. During this time there was a decrease in renal RNA synthesis as shown by decreased uptake of orotic acid-(5)H into RNA. Metabolic intermediates were also measured in quick-frozen kidneys at varying times after induction of acidosis. There was an immediate rise in aspartate and a fall in alpha-ketoglutarate and malate levels. There was never any difference in pyruvate or lactate levels or lactate:pyruvate ratios between control and acidotic rats. Phosphoenolpyruvate rose significantly after 6 hr of acidosis. All the data indicate that increased gluconeogenesis is an early response to metabolic acidosis and will facilitate ammonia production by utilization of glutamate which inhibits the glutaminase I enzyme. The pattern of change in metabolic intermediates can also be interpreted as showing that there is not only enhanced gluconeogenesis, but also that there may be significant increase of activity of glutaminase II as part of the very early response to metabolic acidosis.

Absence of increased [ 14C]orotic acid uptake in perfused liver remnants

Absence of increased [ 14C]orotic acid uptake in perfused liver remnants