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Using rat striatal slices, we examined the effect of cytidine on the conversion of [3H]choline to [3H]-phosphatidylcholine ([3H]PC), and on net syntheses of PC, phosphatidylethanolamine (PE), and phosphatidylserine, when media did or did not also contain choline, ethanolamine, or serine. Incubation of striatal slices with cytidine (50-500 microM) caused dose-dependent increases in intracellular cytidine and cytidine triphosphate (CTP) levels and in the rate of incorporation of [3H] choline into membrane [3H]PC. In pulse-chase experiments, cytidine (200 microM) also increased significantly the conversion of [3H]choline to [3H]PC during the chase period. When slices were incubated with this concentration of cytidine for 1 h, small (7%) but significant elevations were observed in the absolute contents (nmol/mg of protein) of membrane PC and PE (p < 0.05), but not phosphatidylserine, the synthesis of which is independent of cytidine-containing CTP. Concurrent exposure to cytidine (200 microM) and choline (10 microM) caused an additional significant increase (p < 0.05) in tissue PC levels beyond that produced by cytidine alone. Exposure to choline alone at a higher concentration (40 microM) increased the levels of all three membrane phospholipids (p < 0.01); the addition of cytidine, however, did not cause further increases. Concurrent exposure to cytidine (200 microM) and ethanolamine (20 microM) also caused significantly greater elevations (p < 0.05) in tissue PE levels than those caused by cytidine alone. In contrast, the addition of serine (500 microM) did not enhance cytidine's effects on any membrane phospholipid. Exposure to serine alone, however, like exposure to sufficient choline, increased levels of all three membrane phospholipids significantly (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Cytidine, as cytidine 5'-diphosphate choline, is a major precursor in the synthesis of phosphatidylcholine in cell membranes. In the present study, we examined the relationships between extracellular levels of cytidine, the conversion of [14C]choline to [14C]phosphatidylcholine, and the net syntheses of phosphatidylcholine and phosphatidylethanolamine by PC12 cells. The rate at which cytidine (as [3H]cytidine) was incorporated into the PC12 cells followed normal Michaelis-Menten kinetics (Km = 5 microM; Vmax = 12 x 10(-3) mmol/mg of protein/min) when the cytidine concentrations in the medium were below 50 microM; at higher concentrations, intracellular [3H]cytidine nucleotide levels increased linearly. Once inside the cell, cytidine was converted mainly into cytidine triphosphate. In pulse-chase experiments, addition of cytidine to the medium caused a time- and dose-dependent increase (by up to 30%) in the incorporation of [14C]choline into membrane [14C]-phosphatidylcholine. When the PC12 cells were supplemented with both cytidine and choline for 14 h, small but significant elevations (p less than 0.05) were observed in their absolute contents of membrane phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, all increasing by 10-15% relative to their levels in cells incubated with choline alone. Exogenous cytidine, acting via cytidine triphosphate, can thus affect the synthesis and levels of cell membrane phospholipids.
4-N-hydroxy-cytidine was found to substitute for uridine as a pyrimidine supplement for the growth of Escherichia coli Bu(-). Measurement of the incorporation of 4-N-hydroxy-cytidine-2-(14)C into ribonucleic acid and deoxyribonucleic acid revealed that this compound was converted to cytidine or uridine before utilization. Two pathways for metabolism were considered: (i) the reduction of 4-N-hydroxy-cytidine to cytidine followed by deamination, (ii) the direct hydrolysis of hydroxylamine from 4-N-hydroxy-cytidine to yield uridine. A threefold increase in cytidine (deoxycytidine) deaminase (EC 3.5.4.5) activity, when the cells were grown on 4-N-hydroxy-cytidine, suggested the involvement of this enzyme. More direct proof was obtained by purifying the deaminase 185-fold and finding that it released hydroxylamine from 4-N-hydroxy-cytidine at one-fiftieth the rate at which ammonia was removed from cytidine. This result is consistent with the slower rate of growth of the Bu(-) cells on 4-N-hydroxy-cytidine than cytidine and suggests that the second pathway is the major route for utilization of this compound.
Pyrimidine nucleosides in blood plasma of rats were identified by different procedures, including chemical peak shift methods, before their quantification by reversed-phase high-performance liquid chromatography. The concentrations of uridine, cytidine, and deoxycytidine were 1.0 +/- 0.2, 10.6 +/- 1.9, and 33.4 +/- 5.4 mumol/l, respectively. Six hours after the administration of D-galactosamine, the level of circulating cytidine was severely depressed to 25% of control values; uridine decreased to 54% while deoxycytidine remained unchanged. 24 h after the dose of the amino sugar, the levels of cytidine and uridine returned to control values in blood plasma. Total acid-soluble uridine, cytidine, guanosine, and adenosine was determined by reversed-phase HPLC after treatment of the neutralized acid-soluble supernatant of freeze-clamped rat livers with phosphodiesterase and alkaline phosphatase. Six hours after its administration, D-galactosamine induced a 2.2-fold and a 1.6-fold rise in total acid-soluble uridine and cytidine, respectively. Co-administration of N-(phosphonoacetyl)-L-aspartate, an inhibitor of de novo pyrimidine synthesis, suppressed the increase in total acid-soluble uridine observed after D-galactosamine alone, but was without effect on the enhancement of total cytidine. Three hours after D-galactosamine and 15 min after [2-14C] cytidine, there was a rapid fall of the labeled nucleoside in blood plasma to 49% of control animals accompanied by a 2.8-fold rise in the total radioactivity of rat liver homogenates. From these results it can be concluded that the hepatocellular rise in total acid-soluble cytidine after D-galactosamine, in contrast to the increase in total acid-soluble uridine, originates from the phosphorylation of blood plasma cytidine via the salvage pathway. The depletion of circulating cytidine in the presence of hepatocellular UTP deficiency points to the importance of the liver and the hepatic UTP level for the clearance of blood plasma cytidine.
Cytidine deamination of nucleic acids underlies diversification of Ig genes and inhibition of retroviral infection, and thus, it would appear to be vital to host defense. The host defense properties of cytidine deamination require two distinct but homologous cytidine deaminases-activation-induced cytidine deaminase and apolipoprotein B-editing cytidine deaminase, subunit 3G. Although cytidine deamination has clear benefits, it might well have biological costs. Uncontrolled cytidine deamination might generate misfolded polypeptides, dominant-negative proteins, or mutations in tumor suppressor genes, and thus contribute to tumor formation. How cytidine deaminases target a given nucleic acid substrate at specific sequences is not understood, and what protects cells from uncontrolled mutagenesis is not known. In this paper, I shall review the functions and regulation of activation-induced cytidine deaminase and apolipoprotein B-editing cytidine deaminase, subunit 3G, and speculate about the basis for site specificity vis-à-vis generalized mutagenesis.
The metabolism of cytidine-2-(14)C and uridine-2-(14)C was studied in discs cut from leaflets of bean plants (Phaseolus vulgaris L.). Cytidine was degraded to carbon dioxide and incorporated into RNA at about the same rates as was uridine. Both nucleosides were converted into the same soluble nucleotides, principally uridine diphosphate glucose, suggesting that cytidine was rapidly deaminated to uridine and then metabolized along the same pathways. However, cytidine was converted to cytidine diphosphate and cytidine triphosphate more effectively than was uridine. Cytidine also was converted into cytidylic acid of RNA much more extensively and into RNA uridylic acid less extensively than was uridine. Azaserine, an antagonist of reactions involving glutamine (including the conversion of uridine triphosphate to cytidine triphosphate), inhibited the conversion of cytidine into RNA uridylic acid with less effect on its incorporation into cytidylic acid. On the other hand, it inhibited the conversion of orotic acid into RNA cytidylic acid much more than into uridylic acid. The results suggest that cytidine is in part metabolized by direct conversion to uridine and in part by conversion to cytidine triphosphate through reactions not involving uridine nucleotides.
Cytidine metabolism in the yeast Saccharomyces cerevisiae was analyzed by genetic and biochemical approaches. Disruption of a unique ORF (Genbank accession No. U 20865) bearing homology with eucaryotic or bacterial cytidine deaminases abolished cytidine deaminase activity and resulted in 5-fluorocytidine resistance. The gene encoding cytidine deaminase will be referred to as CDD1 (Genbank accession number AF080089). The ability to isolate mutants resistant to 5-fluorocytidine which mapped to five other loci demonstrated the existence of a complex cytidine metabolic network. Deciphering this network revealed several original features:(1) cytidine entry is mediated by the purine-cytosine transporter (Fcy2p),(2) cytidine is cleaved into cytosine by the uridine nucleosidase (Urh1p),(3) cytidine is phosphorylated into CMP by the uridine kinase (Urk1p),(4) a block in cytosine deaminase (Fcy1p), but not in cytidine deaminase (Cdd1p), constitutes a limiting step in cytidine utilisation as a UMP precursor.
Cytidine deaminase, an enzyme that catalyses the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara-C) and 5-azacytidine (5-azaC), has been partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70 degrees C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G-150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum activity over a broad pH range of 5-9.5. The enzyme is stabilized by the presence of the sulfhydryl reagent, dithiothreitol. Cytidine deaminase from normal human granulocytes has a greater affinity for its physiologic substrate cytidine (K(m) = 1.1 x 10(-5) M) than for ara-C (8.8 x 10(-5) M) or 5-azaC (4.3 x 10(-4) M). Halogenated analogues such as 5-fluorocytidine and 5-bromo-2'-deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a K(i) of 5.4 x 10(-8) M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, molecular weight, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52+/-1.86 x 10(3)/mg protein) than chronic myelocytic leukemia (CML) cells (1.40+/-0.70 x 10(3) U/mg protein) or acute myelocytic leukemia (AML) cells (0.19+/-0.17 x 10(3) U/mg protein). To explain these differences in enzyme levels in leukemic versus normal cells, the changes in cytidine deaminase levels associated with maturation of normal granulocytes were studied in normal human bone marrow. Myeloid precursors obtained from bone marrow aspirates were separated into mature and immature fractions by Ficoll density centrifugation. Deaminase activity in lysates of mature granulocytes was 3.55-14.2 times greater than the activity found in the lysates of immature cells. Decreased enzyme activity was also found in immature myeloid cells from a patient with CML as compared to mature granulocytes from the same patient. These observations support the conclusion that the greater specific activity of cytidine deaminase in normal mature granulocytes as compared to leukemic cells is related to the process of granulocyte maturation rather than a specific enzymatic defect in leukemic cells.
15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. The primary (15)(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H(2)O, pH 7.3), 1.0123 (in H(2)O, pH 4.2), and 1.0086 (in D(2)O, pD 7.3). Increasing solvent D(2)O content has no substantial effect on k(cat) but enhances k(cat)/K(m), with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction. Mutant cytidine deaminases with reduced catalytic activity show more pronounced (15)N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H(2)O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining. The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH(2) group, and represents the intrinsic isotope effect on C-N bond cleavage. This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme. The observed (15)N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate. These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step. The primary (15)N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 degrees C. These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C-N bond breaking is greater for the spontaneous reaction.
Based on its capacity to inhibit de novo pyrimidine biosynthesis by blocking dihydroorotate dehydrogenase activity, the antitumor agent brequinar sodium (BQR) has emerged as a new immunosuppressive agent. Since BQR is known to prevent the synthesis of nucleotides during cell proliferation, we hypothesized that it would be highly effective in controlling strong lymphocyte proliferative responses but might be less effective in controlling comparatively weak responses that do not necessarily involve new nucleotide synthesis. We addressed this question by culturing murine spleen cells with different types of stimuli, including Con A, phorbol myristate acetate +/- ionomycin, anti-CD3, and anti-Igs. Addition of BQR (0.001 microgram/ml to 10 micrograms/ml) at the start of a 72-hr culture period caused dose-dependent inhibition of strong proliferative responses, induced either by Con A (5 micrograms/ml) or PMA+ionomycin. A residual degree of proliferation persisted, however, even at the highest BQR concentrations. In contrast, no impairment of low-concentration Con A (0.5 or 0.1 microgram/ml), anti-CD3, or anti-Igs responses was observed. In order to ascertain its role in arresting nucleotide synthesis, we attempted to reverse the inhibitory effect of BQR by adding exogenous uridine or cytidine to lymphocyte cultures. BQR's inhibitory activity was reversed completely by adding uridine at 0.1 mM. In contrast, combination of BQR and cytidine (0.1 mM) potentiated BQR's activity and abrogated anti-CD3 or anti-Igs-induced lymphocyte proliferation in a dose-dependent manner. A synergistic inhibitory action between BQR and cytidine was observed when the BQR concentration was higher than 0.1 microgram/ml and with cytidine at 0.1 mM. Production of interleukin-2 and IL-4 was only slightly affected by BQR, but was significantly suppressed by coadministration of BQR and cytidine. Neither BQR (5 micrograms/ml) on its own, however, nor combination of BQR with cytidine affected production of mRNA for IL-2, IL-4, or interferon-gamma, as determined by reverse-transcription polymerase chain reaction. Our observations suggest that BQR may not only affect dihydroorotate dehydrogenase activity, but may also inhibit the enzyme cytidine deaminase, which converts cytidine to uridine. These antimetabolic effects of BQR complement the well-known cytokine synthesis inhibitory actions of FK506 or CsA. The combination of BQR and cytidine, however, offers a further possibility for inhibition of both cytokine production and T and B cell proliferation, and may have potential for the control of graft rejection.
Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.
The degradation of RNA in the cyclically perfused rat liver was determined from the release of labeled cytidine from RNA that had been previously labeled with [6-14C]orotic acid in vivo. Because cytidine is not appreciably degraded in rat liver (its deamination to uridine is virtually nil) or produced in significant amounts from free 5'-nucleotides, its release will directly reflect net RNA breakdown. This conclusion was substantiated by the fact that the specific radioactivity of released cytidine equaled that of CMP in RNA and remained unchanged for 180 min of perfusion. The initial rate of [14C]cytidine accumulation was slow, but after 10-20 min it increased abruptly by more than 4-fold and remained virtually constant. The addition of 0.5 mM unlabeled cytidine effectively prevented the reutilization of label and increased the rate of labeled cytidine release by an amount representing 13% of the maximal rate of cytidine accumulation. Rates of RNA degradation, measured between 20 and 60 min in the presence of 0.5 mM unlabeled cytidine, averaged 1.00 +/- 0.05 mg h-1 liver-1 (100-g rat), the equivalent of 65% of total RNA per day. This accelerated value, which was about 4-fold larger than the initial rate, is believed to be the direct consequence of amino acid deprivation since, in separate experiments, the increase was completely suppressed by the addition of plasma amino acids (Lardeux, B. R., and Mortimore, G. E. (1987) J. Biol. Chem. 262, 14514-14519). These findings demonstrate the potential value of cytidine as a marker for following moment-to-moment regulatory alterations in RNA degradation in the isolated liver or hepatocyte preparation.