Concentration of nucleotides and deoxynucleotides in peripheral and phytohemagglutinin-stimulated mammalian lymphocytes. Effects of adenosine and deoxyadenosine

Concentrations of purine and pyrimidine ribonucleotides were measured with HPLC in lymphocytes of man, horse, pig and sheep and in rat thymocytes. The ATP concentration was highest in lymphocytes of all species and about 850 pmol/10(6) cells in human and equine lymphocytes, higher in porcine and lower in ovine lymphocytes and rat thymocytes. The GTP concentration was comparable in human, equine and porcine lymphocytes, but lower in ovine lymphocytes. ATP concentration was also measured in lymphocytes of man, horse and pig with a luciferin-luciferase assay. During culturing with or without phytohemagglutinin the ATP concentrations decreased in these lymphocytes. The concentrations of TTP and dATP were measured with a DNA polymerase assay. Phytohemagglutinin-stimulation increased the TTP concentration in lymphocytes of all three species, the dATP concentration only in human lymphocytes. ATP, TTP and dATP concentrations and thymidine incorporation were measured in phytohemagglutinin-stimulated lymphocytes after 24 and 48 h culturing in the presence of adenosine or deoxyadenosine. Adenosine increased the ATP concentration in porcine and equine, but not in human lymphocytes. Deoxyadenosine and adenosine did not affect the TTP concentration. Deoxyadenosine decreased the ATP concentration only in the presence of EHNA in human lymphocytes, but increased it in other conditions and in equine and porcine lymphocytes. Deoxyadenosine in the presence of EHNA increased the dATP concentration in human, equine and porcine lymphocytes 3-, 10-, and 9-fold, respectively, and decreased considerably thymidine incorporation. Deoxyadenosine without EHNA increased the dATP concentration 2-5-fold, decreased the thymidine incorporation in lymphocytes of man and horse, but stimulated incorporation in porcine lymphocytes about 5-fold. The latter results indicate that accumulation of dATP is not always associated with inhibition of cell proliferation.     

Adenosine and deoxyadenosine metabolism in lymphocytes from horses,Equis cabalus

• 1.1. The toxic effects of adenosine and deoxyadenosine on lymphocytes from horses were evaluated.
• 2.2. Mitogen-stimulated peripheral blood lymphocytes (PBL) were found to be more sensitive to the inhibitory effects of adenosine than were lymphocytes from spleen, lymph node and thymus.
• 3.3. Adenosine deaminase activity was approximately 10 times lower in horse lymphoid tissue in comparison to that found in human lymphoid tissue. In horse, the highest activity was in spleen while the lowest activity was in thymus.
• 4.4. Adenosine inhibition of mitogenesis in PBL was prevented by uridine or cytidine, suggesting pyrimidine starvation as the major mechanism for adenosine toxicity.
• 5.5. Deoxyadenosine inhibition of mitogenesis in PBL was not prevented by the addition of various ribo- or deoxyribonucleosides. This and the finding that treated cells show no increase in deoxyATP suggest that some other mechanism for deoxyadenosine toxicity other than deoxyATP inhibition of ribonucleotide reductase operates in horse PBL.

     

Adenosine inhibits cell division and promotes neurite extension in PC12 cells

Low concentrations (10-50 microM) of adenosine (EC50 = 17 microM) or chloroadenosine (EC50 = 23 microM) prevent the division of PC12 cells. This inhibition is not mimicked by guanosine, inosine, 3',5' dideoxyadenosine, phenylisopropyladenosine, or adenylylimidodiphosphate. The growth inhibition is not relieved by addition of uridine or deoxycytidine, nor is it potentiated by homocysteine thiolactone. Inhibition of adenosine uptake does not inhibit adenosine-dependent growth arrest. PC12 variants that are deficient in adenosine kinase are as sensitive as wild-type cells to the growth-inhibitory effects of adenosine. These experiments suggest that adenosine prevents cell division at an adenosine receptor rather than acting after being metabolically altered. The adenosine receptor that inhibits cell division does not appear to be the adenosine receptor that stimulates adenylate cyclase for these reasons: (1) phenylisopropyladenosine, which is a potent agonist of this receptor, does not inhibit cell division; (2) 3',5' dideoxyadenosine does not antagonize the effect of adenosine on cell division; and (3) theophylline does not affect growth inhibition by adenosine. Thus, these experiments suggest the existence of a second adenosine receptor that can inhibit cell division. Adenosine also promotes the morphological differentiation of PC12 cells. In the presence of the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenosine (EHNA), adenosine causes the formation of short neurites (one-half to one and one-half cell diameters in length). Adenosine also increases the rate of neurite formation of both long and short neurites in response to NGF.     

Determination of ribonucleosides,deoxyribonucleosides, and purine and pyrimidine bases in adult rabbit cerebrospinal fluid and plasma

Purine and pyrimidine base and nucleoside levels were measured in adult rabbit cisternal CSF and plasma by reversed-phase high-performance liquid chromatography. The concentrations of bases, nucleosides, and nucleoside phosphates were similar in plasma and CSF except for the adenosine phosphates and uracil which were higher in the plasma. In plasma and CSF, adenosine levels were low (0.12 microM) and guanosine, deoxyadenosine, deoxyguanosine, and deoxyinosine were not detectable (less than 0.1 microM); inosine and xanthine concentrations were 1-2 microM and hypoxanthine concentrations were approximately 5 microM; uridine (approximately 8 microM), cytidine (2-3 microM), and thymidine, deoxyuridine, and deoxycytidine (0.5-1.4 microM) were easily detectable. In both plasma and CSF, guanine, and thymine were undetectable (less than 0.1 microM), adenine and cytosine were less than 0.2 microM, but uracil was present (greater than 1 microM). Adenosine, inosine, and guanosine phosphates were also detectable at low concentrations in CSF and plasma. These results are consistent with the hypothesis that purine deoxyribonucleosides are synthesized in situ in the adult rabbit brain. In contrast, pyrimidine deoxyribonucleosides and ribonucleosides, and purine and pyrimidine bases are available in the CSF for use by the brain.     

Thymidine and Thymine Incorporation into Deoxyribonucleic Acid: Inhibition and Repression by Uridine of Thymidine Phosphorylase of Escherichia coli

Thymidine is poorly incorporated into deoxyribonucleic acid (DNA) of Escherichia coli. Its incorporation is greatly increased by uridine, which acts in two ways. Primarily, uridine competitively inhibits thymidine phosphorylase (E.C.2.4.4), and thereby prevents the degradation of thymidine to thymine which is not incorporated into normally growing E. coli. Uridine also inhibits induction of the enzyme by thymidine. It prevents the actual inducer, probably a deoxyribose phosphate, from being formed rather than competing for a site on the repressor. The inhibition of thymidine phosphorylase by uridine also accounts for inhibition by uracil compounds of thymine incorporation into thymine-requiring mutants. Deoxyadenosine also increases the incorporation of thymidine, by competitively inhibiting thymidine phosphorylase. Deoxyadenosine induces the enzyme, in contrast to uridine. But this is offset by a transfer of deoxyribose from deoxyadenosine to thymine. Thus, deoxyadenosine permits incorporation of thymine into DNA, even in cells induced for thymidine phosphorylase. This incorporation of thymine in the presence of deoxyadenosine did not occur in a thymidine phosphorylase-negative mutant; thus, the utilization of thymine seems to proceed by way of thymidine phosphorylase, followed by thymidine kinase. These results are consistent with the data of others in suggesting that wild-type E. coli cells fail to utilize thymine because they lack a pool of deoxyribose phosphates, the latter being necessary for conversion of thymine to thymidine by thymidine phosphorylase.     

Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.     

Thymidine incorporation into Rhizobium meliloti.

Thymidine is rapidly catabolized to thymine, beta-aminoisobutyric acid, and carbon dioxide by Rhizobium meliloti cells. The incorporation of labelled thymidine into the DNA of R. meliloti cells can be enhanced by the addition of low concentrations (10-20 micrograms/mL) of deoxyadenosine or other nucleosides (adenosine, uridine, guanosine). However, at high concentrations ( greater than 50 micrograms/mL) these compounds inhibit thymidine incorporation. Conditions to obtain highly radioactive DNA of Rhizobium are described.     

EHNA is a poor inhibitor of deoxyadenosine catabolism in cultured human lymphocytes

Erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) has been used by many workers as enzyme inhibitor in vitro to simulate the in vivo situation in inherited adenosine deaminase (ADA) deficiency. In this study the metabolism of 8-14C deoxyadenosine (dAR) has been followed in cultured lymphocytes from patients deficient in enzymes associated with the catabolism and salvage of dAR, in the absence and presence of 10 microM EHNA. The results show that EHNA, at these concentrations, does not prevent the catabolism of dAR and thus does not provide a valid model for investigating the toxicity to the immune system in inherited ADA deficiency.     

The effect of nucleosides and deoxycoformycin on adenosine and deoxyadenosine inhibition of human lymphocyte activation.

Micromolar deoxyadenosine inhibits leucine uptake during the 1st day of proliferation in mitogen-stimulated lymphocytes if adenosine deaminase is inhibited. This inhibition occurs before DNA synthesis begins, suggesting that deoxyadenosine can affect mitogenesis by mechanisms that do not involve ribonucleotide reductase inhibition. If deoxyadenosine addition to mitogen-stimulated lymphocytes is delayed to the 2nd or 3rd day post-stimulation, inhibition of proliferation is markedly reduced. Although the time dependence of deoxyadenosine toxicity resembles that of adenosine, these compounds appear to inhibit early protein synthesis by different mechanisms: 1) deoxycoformycin markedly potentiates deoxyadenosine but not adenosine; 2) deoxycytidine and thymidine reverse deoxyadenosine toxicity but do not alter adenosine toxicity.     

Deoxyadenosine blockade of G0 to G1 transition in lymphocytes: Possible involvement of protein kinases

Immunodeficiency in adenosine deaminase deficiency has been attributed to the lymphotoxicity of deoxyadenosine that accumulates to high levels in patients. To gain insight into the mechanism of deoxyadenosine toxicity, we investigated the dose-response and time course of its toxic effects on concanavalin A-stimulated mouse splenic lymphocytes by thymidine incorporation and flow cytometry. Deoxyadenosine at a level as low as 0.3 μM inhibited the progression of G₀. In contrast, higher concentrations of the nucleoside, i.e., in the range of 1 to 3 μM, were needed to block transition of the stimulated lymphocytes from G₀ to G₁. The inhibition of their S entry and progression required even higher concentrations. Furthermore, staurosporine, a potent inhibitor of protein kinases, was found to potentiate the toxicity of deoxyadenosine in mitogen-stimulated lymphocytes. Calcium mobilization in mitogen-activated lymphocytes was inhibited by deoxyadenosine. Our data suggest that, while ribonucleotide reductase inhibition by dATP could explain the blockade of S entry and progression by deoxyadenosine in cycling lymphocytes or leukemic cells, more important effects of this compound on antigen-activated lymphocytes occur at the early G₀ phase. A possible mechanism of deoxyadenosine lethality is its inhibition of protein phosphorylation. © 1996 Wiley-Liss, Inc.     

Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.     

Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor-positive and -negative human breast cancer cells via the intrinsic pathway

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.     

Adenosine deaminase 1 and concentrative nucleoside transporters 2 and 3 regulate adenosine on the apical surface of human airway epithelia: implications for inflammatory lung diseases

Adenosine is a multifaceted signaling molecule mediating key aspects of innate and immune lung defenses. However, abnormally high airway adenosine levels exacerbate inflammatory lung diseases. This study identifies the mechanisms regulating adenosine elimination from the apical surface of human airway epithelia. Experiments conducted on polarized primary cultures of nasal and bronchial epithelial cells showed that extracellular adenosine is eliminated by surface metabolism and cellular uptake. The conversion of adenosine to inosine was completely inhibited by the adenosine deaminase 1 (ADA1) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The reaction exhibited Km and Vmax values of 24 microM and 0.14 nmol x min(-1) x cm(-2). ADA1 (not ADA2) mRNA was detected in human airway epithelia. The adenosine/mannitol permeability coefficient ratio (18/1) indicated a minor contribution of paracellular absorption. Adenosine uptake was Na+-dependent and was inhibited by the concentrative nucleoside transporter (CNT) blocker phloridzin but not by the equilibrative nucleoside transporter (ENT) blocker dipyridamole. Apparent Km and Vmax values were 17 microM and 7.2 nmol x min(-1) x cm(-2), and transport selectivity was adenosine = inosine = uridine > guanosine = cytidine > thymidine. CNT3 mRNA was detected throughout the airways, while CNT2 was restricted to nasal epithelia. Inhibition of adenosine elimination by EHNA or phloridzin raised apical adenosine levels by >3-fold and stimulated IL-13 and MCP-1 secretion by 6-fold. These responses were reproduced by the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and blocked by the adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline (8-SPT). This study shows that adenosine elimination on human airway epithelia is mediated by ADA1, CNT2, and CNT3, which constitute important regulators of adenosine-mediated inflammation.     

Adenosine deaminase and immunodeficiency: An in vitro model

We have examined the effect of adenosine and EHNA, a competitive inhibitor of adenosine deaminase (ADA), upon the ability of human peripheral blood lymphocytes to respond to mitogen. Addition of adenosine at concentrations greater than 10 μm (10^?5m) resulted in inhibition of lymphocyte proliferation at 48 hr of culture, provided that the culture medium was relatively free of ADA activity. The actual concentrations of adenosine remaining in inhibited cultures at the time of harvest were considerably lower than those added initially. EHNA alone also inhibited PHA response (and to a lesser extent PWM and Con A responses), but only at high concentrations. Noninhibitory concentrations of EHNA and adenosine together acted synergistically to produce profound inhibition of lymphocyte proliferation. This may provide an in vitro model to explore further the mechanism of the immunodeficiency associated with deficiency of ADA. Adenosine deaminase activity in stimulated cultures did not differ significantly from that found in unstimulated cultures, and the activity per protein or per DNA actually decreased in stimulated versus unstimulated cultures.     

Regulation of pyrimidine biosynthesis by purine and pyrimidine nucleosides in slices of rat tissue

Evidence of the primary sites for the regulation of de novo pyrimidine biosynthesis by purine and pyrimidine nucleosides has been obtained in tissue slices through measurements of the incorporation of radiolabeled precursors into an intermediate and end product of the pathway. Both purine and pyrimidine nucleosides inhibited the incorporation of [¹⁴C]-NaHCO₃ into orotic acid and uridine nucleotides, and the inhibition was found to be reversible upon transferring the tissue slices to a medium lacking nucleoside. The ammonia-stimulated incorporation of [¹⁴C]NaHCO₃ into orotic acid, which is unique to liver slices, was sensitive to inhibition by pyrimidine nucleosides at physiological levels of ammonia, but this regulatory mechanism was lost at toxic levels of ammonia. Adenosine, but not uridine, was found to have the additional effects of inhibiting the conversion of [¹⁴C]orotic acid to UMP and depleting the tissue slices of PRPP. Since PRPP is required as an activator of the first enzyme of the de novo pathway, CPSase II, and a substrate of the fifth enzyme, OPRTase, these results indicate that adenosine inhibits the incorporation of [¹⁴C]NaHCO₃ into orotic acid and the incorporation of [¹⁴C]orotic acid into UMP by depriving CPSase II and OPRTase, respectively, of PRPP. Uridine or its metabolites, on the other hand, appear to control the de novo biosynthesis of pyrimidines through end product inhibition of an early enzyme, most likely CPSase II. We found no evidence of end product inhibition of the conversion of orotic acid to UMP in tissue slices.     

Sodium-dependent nucleoside transport in mouse lymphocytes, human monocytes, and hamster macrophages and peritoneal exudate cells.

Mouse splenocytes and hamster peritoneal exudate cells (PEC), including macrophages, were shown to contain a predominantly Na(+)-dependent and inhibitor (6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside, NBMPR)-resistant transport system for adenosine and other nucleosides. Adenosine (1 microM) was transported about equally in mouse thymocytes and human monocytes from peripheral blood by a Na(+)-dependent system and the NBMPR-sensitive facilitated diffusion system. Hamster PEC also transported inosine, tubercidin, formycin B, uridine, and thymidine in a NBMPR-insensitive manner. With the exception of formycin B, all nucleosides were phosphorylated intracellularly to varying degree, adenosine being almost fully phosphorylated. During the time course of routine experiments (30 s) formycin B was concentrated twofold over external medium levels (1 microM) without any drop-off in the transport rate. On the basis of metabolic studies it was estimated that uridine and tubercidin were also transported against a concentration gradient. Inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, and the pyrimidines uridine, thymidine, and cytidine (all 100 microM) inhibited transport of adenosine and inosine about 50-100%, while 3'-deoxyinosine showed weak inhibitory action. Transport of thymidine was strongly inhibited by nucleosides except by 3'-deoxyinosine. The Na(+)-dependent, active, and concentration transport system appears to be a feature of many immune-type cells, and its presence offers particular conceptual possibilities for the therapy of infections located in these cells.     

An analysis of multiple mechanisms of adenosine toxicity in baby hamster kidney cells

Analysis of the response of baby hamster kidney cells to adenosine in the presence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine has revealed two distinct mechanisms of toxicity. The first is apparent at low concentrations of adenosine (less than 5 microM) and is dependent upon the presence of a functional adenosine kinase. The initial toxicity is abolished by uridine, is unrelated to the inhibition of ribonucleotide reductase, and is accompanied by a decrease in the size of the pyrimidine nucleotide pool. Toxicity at higher concentrations of adenosine is adenosine kinase independent and is potentiated by homocysteine thiolactone. An elevation in the intracellular level of S-adenosylhomocysteine, which was observed following treatment with higher concentrations of adenosine (greater than 10 microM), is believed to mediate toxicity at these levels. Interestingly, BHK cells were resistant to intermediate levels of adenosine. The mechanism of resistance is currently unknown, but appears unrelated to a lack of inhibition of adenosine deaminase. It is proposed that substrate inhibition of adenosine kinase may be a determinant of this property.     

Metabolism of purine nucleosides and phosphoribosylpyrophosphate in thymocytes and splenocytes of various mammalian species

1. Activities of ADA, PNP and AK were measured in splenocytes and thymocytes of newborn children, young horses, pigs, sheep, rats and mice and compared with the activities previously found in peripheral lymphocytes. 2. With all species, except horse, the activity of ADA (per 10(6) cells) was higher in thymocytes than in lymphocytes. Activity of ADA was highest in splenocytes of pig and sheep. Activity of ADA was lowest in all lymphoid cells of the horse and only about 10% of the activity in human splenocytes and lymphocytes. 3. With all species, except horse, the activity of PNP was lower in thymocytes than in lymphocytes. Activity of PNP was highest in human lymphocytes and lowest in ovine thymocytes. 4. Activity of AK is comparable in thymocytes of all species and always lower than the ADA activity. In splenocytes of man, horse and pig the activity of AK is comparable to that in thymocytes. 5. Activity of deoxyguanosine kinase was lowest in rat thymocytes and highest in those of man. 6. When enzyme activities are expressed per milligram of protein, the differences between thymocytes and lymphocytes are less pronounced. 7. Activity of PRPP synthetase per 10(6) cells was comparable in thymocytes, splenocytes and lymphocytes of the same species and between the various species. 8. The concentration of PRPP was lowest in ovine thymocytes and higher in splenocytes than in thymocytes of the same species, except man.     

Effects of Hydroxyurea and Deoxyadenosine on the Synthesis of Deoxycytidine Phosphate and on the Uptake of Nucleosides in Excised Root Tips of Vicia faba

The effects of hydroxyurea and deoxyadenosine on the synthesis of deoxycytidine phosphate was studied by measuring the incorporation of [¹⁴C]-cytidine into acid soluble deoxycytidine phosphate in root tips of Vicia faba. Hydroxyurea and deoxyadenosine both markedly depressed the incorporation of [¹⁴C]-cytidine. Deoxyadenosine had the additional effect of inhibiting the uptake of [¹⁴C]-cytidine. Furthermore, millimolar concentrations of deoxyadenosine inhibited the uptake of micromolar concentrations of adenosine, thymidine, and deoxycytidine. The incorporation of [¹⁴C]-cytidine into RNA was only slightly affected by hydroxyurea. Deoxyadenosine inhibited the incorporation into RNA to about the same extent as the uptake of [¹⁴C]-cytidine. It is suggested that hydroxyurea reduced the incorporation of radioactive cytidine into deoxycytidine phosphate mainly by interfering with ribonucleotide reduction. The depression of [¹⁴C]-cytidine incorporation into deoxycytidine phosphate in the presence of deoxyadenosine is believed to be the result of an inhibition of both ribonucleotide reduction and [¹⁴C]-cytidine uptake.