Evidence against functional adenosine receptors on murine lymphocytes

The effects of adenosine, 2-chloroadenosine (2Cl Ado) and N6-phenylisopropyladenosine (PIA) were examined on peripheral blood and splenic lymphocytes from mice. Lectin-stimulated DNA synthesis was antagonized by adenosine and 2Cl Ado at high concentrations. Lower concentrations of all three nucleosides produced an enhancement of lectin-stimulated thymidine uptake in splenic lymphocytes. Peripheral blood lymphocytes were found to exhibit only inhibitions of mitogenic stimulation suggesting a difference in response to nucleoside exposure between spleen and peripherally circulating cells. The synthesis of antibody to sheep red blood cells was inhibited in a non-cytotoxic manner by 2Cl Ado and PIA while adenosine was without effects. The receptor antagonist 8-phenyltheophylline was found to block nucleoside increases in thymidine uptake and low concentrations of 2Cl Ado with regard to antibody production. The effects of high concentrations of 2Cl Ado or PIA on humoral responses were not antagonized by receptor blockade. The data suggest that functional alterations of lymphocyte responses to nucleoside exposure are not a consequence of surface receptors for adenosine nucleosides in murine cells.     

Characterization of nucleoside uptake and transport in Entamoeba histolytica

The uptake and transportation of purine and pyrimidine based nucleosides by trophozoites of axenically grown Entamoeba histolytica (HMI-IMSS) were studied. The trophozoites transported adenosine and its analog tubercidin (1 μM) at a significant rate but poor transportation was observed in case of uridine (about 10% relative rate), inosine (3%?), thymidine (2%?) and formycin B (1%?). The Km for adenosine was 160?±?42 μM. Unlabeled nucleosides (100 μM) inhibited adenosine and tubercidin transport. Adenosine related compounds 5′-deoxyadenosine and nebularin inhibited adenosine and tubercidine transport by 50%? or more. However, inosine related compounds guanosine, 3′-deoxyinosine and formycin B were less inhibitory. The pyrimidine nucleosides uridine, thymidine and cytidine were poorly inhibitory. 6-[(4 nitrobenzyl)-mercapto] purine ribonucleoside, an inhibitor of mammalian nucleoside transporter, inhibited adenosine or tubercidin transport in E. histolytica variably between 0–30%?at 10?μM, but dilazep, a known inhibitor, was inactive upto 10 μM. Increase in temperature from 22°C to 33°C enhanced the rate of transport of adenosine 4.5 fold, tubercidin 7.3 fold and of inosine 4 fold. These findings along with the structure activity figures suggested that transport was mediated and not passive.     

Uptake and release of adenosine in isolated rat fat cells

Radioactively labelled adenosine and adenine were rapidly taken up by isolated rat fat cells, and incorporated into nucleotides, of which ATP dominated. The overall process had an apparent K_m of 1–5 μM. During incubation, especially in the presence of lipolytic agents, there was a reduction in labelled ATP with a compensatory increase in ADP, AMP, cAMP and nucleosides. The build-up of adenosine during incubation was inhibited by theophylline, which inhibits 5′-nucleotidase. Radioactivity released from perifused fat cells consisted mainly of nucleoside material, of which adenosine predominated. Lipolytic stimulation caused no significant increase in nucleoside outflow from perifused cells, whereas oxygenation was capable of reducing this outflow. It is concluded that adenosine is formed by fat cells as a consequence of ATP breakdown. Stimulation of lipolysis during activation of the sympathetic nerves leads to reversible ATP breakdown and adenosine release. Adenosine might therefore act as a modulator of lipolysis in vivo under these conditions, even though it does not serve as a feed back regulator in the proper sense.     

Two high affinity nucleoside transporters in Leishmania donovani

A rapid sampling kinetic technique has been used to evaluate the nucleoside transport functions of Leishmania donovani. The results indicated that L. donovani promastigotes possessed two independent purine nucleoside transporters with nonoverlapping substrate specificity. The first transported inosine, guanosine, and their analogs, while the second carried adenosine, analogs of adenosine, and the pyrimidine nucleosides, uridine, cytidine, and thymidine. The apparent Km values of the two nucleoside permeases for their purine nucleoside substrates were extraordinarily low, in the micromolar range. The organisms were capable of concentrating purine nucleosides from the medium and converting them to the nucleotide level with great efficiency and rapidity. Inosine and adenosine transport could be distinguished by different sensitivities to sulfhydryl reagents, suggesting structural differences between the two transporters. Finally, the two nucleoside transport systems of L. donovani were virtually refractory to inhibition by 4-nitrobenzylthioinosine and dipyridamole, two potent inhibitors of nucleoside entry into mammalian cells.     

Molecular biology studies of tubercidin resistance inTrypanosoma cruzi

Trypanosomatids are incapable of de novo purine synthesis; purines are obtained through the scavenging of exogenous nucleosides. To advance our understanding of purine utilization, we mutagenized aTrypanosoma cruzi stock and selected for resistance to high levels of tubercidin (7-deazaadanosine, TUB), a purine analog. The TUB-resistant stocks were>100 times more resistant to TUB than was the parental stock. TUB and uridine transport in the TUB-resistant stocks decreased by 50%–90%, whereas thymidine and adenosine transport were unaffected. These data imply that TUB-resistant stocks have defects in the pathways involved in the transport of TUB and uridine but not in the thymidine and adenosine transport pathways. Karyotype analyses using specific probes showed that the deletion of a 950-kb chromosome-size DNA occurred in both of the TUB-resistant stocks. These data suggest that genes involved in nucleoside transport are located in this DNA region. This study will facilitate the identification and characterization of the specific genes involved in nucleoside transport and aid in the elucidation and development of new chemotherapeutics for Chagas' disease.Abbreviations TUB tubercidin (7-deazaadenosine) - TUBr tubercidin-resistant - PFG pulse field gradient     

N-ethylmaleimide differentially affects adenosine and thymidine uptake by rat thymocytes

The involvement of membrane sulfhydryl groups in the uptake of adenosine and thymidine was examined in rat thymocytes pretreated with 6,6'-dithiodinicotinic acid (CPDS) and N-ethylmaleimide (NEM). CPDS, which is known to react uniquely with external membrane sulfhydryls, under short incubation conditions, did not significantly affect the uptake of adenosine and thymidine. Formation of cAMP in nonstimulated and adenosine-stimulated cells was also unaltered by CPDS. However, inhibition of adenosine uptake by competitive inhibitor, dipyridamole, was significantly stronger when the cells were pretreated with CPDS. Preincubation of cells with NEM showed differential sensitivity of adenosine and thymidine uptake, depending on concentration of this sulfhydryl alkylating agent. The results suggest the involvement of NEM-accessible sulfhydryls in membrane transport of adenosine and thymidine. Dual effect of NEM on nucleoside transport may be related to the complexity of nucleoside carrier(s) or to the existence of different nucleoside carriers within thymocyte membranes. On the other hand, the easily accessible, external membrane -SH groups which can be blocked with CPDS, are not essential in thymocyte nucleoside transport but they appear to be situated at a site which interacts with the membrane transport system of nucleoside.     

Stage-specific alteration of nucleoside membrane permeability and nitrobenzylthioinosine insensitivity in Plasmodium falciparum infected erythrocytes

In human erythrocytes, the intracellular presence of malarial parasites (Plasmodium falciparum) markedly changed the permeation characteristics of the nucleosides, adenosine and tubercidin, an adenosine analogue. We report parasite-induced changes in the kinetics of cellular uptake of the nucleosides and in the appearance in infected cells of a nucleoside permeation route of low sensitivity to the classical inhibitor of erythrocytic nucleoside transport, nitrobenzylthioinosine (NBMPR). These changes and a diminution in NBMPR effectiveness during parasite maturation to the trophozoite or schizont stage, suggest the presence in the infected cells of an altered or new nucleoside permeation mechanism of low sensitivity to NBMPR. The incorporation of adenosine into polynucleotides was also of low sensitivity to 10 microM NBMPR. Binding studies of [3H]NBMPR with both normal erythrocytes and those harbouring parasites at each morphological stage indicated that fewer high affinity NBMPR binding sites were present on cells containing mature parasites than on the uninfected cells. The apparent low sensitivity to NBMPR of nucleoside permeation in erythrocytes containing P. falciparum forms may enable therapeutic measures with cytotoxic nucleosides to be directed with selectivity toward parasite-containing cells.     

Single-step selection of mouse FM3A cell mutants defective in thymidylate synthetase

A tritium-suicide method for isolating thymidine auxotrophic mutants is described. Mutagenized mouse FM3A cells were cultured in medium containing [³H] deoxyuridine. Most of the surviving clones examined showed a phenotype of absolute thymidine auxotrophy. This phenotype is very stable and was found to be genetically recessive in cell-cell hybridization experiments. The growth of these variant clones was not supported by various pyrimidine nucleosides other than thymidine. The activity of thymidylate synthetase in crude extracts of these clones was less than 1% of that of FM3A cells. These results strongly indicate that the thymidine auxotrophic phenotype resulted from a genetic defect in thymidylate synthetase.     

The accumulation of [3H]phenylisopropyl adenosine ([3H]PIA) and [3H]adenosine into rabbit retinal neurons is inhibited by nitrobenzylthioinosine (NBI)

Uptake of [3H]adenosine and [3H]R-phenylisopropyladenosine (R-PIA) into retinal cells was assessed autoradiographically, in the presence and absence of the purine nucleoside transport inhibitor, nitrobenzylthioinosine (NBI). Under control conditions, both purine nucleosides were accumulated in cell bodies localized to the ganglion cell layer, and the inner nuclear layer. In the presence of NBI, significantly less accumulation of nucleosides within cell bodies was observed, particularly within the inner nuclear layer, suggesting that most of the uptake occurred via the transport of both substrates. The stereoisomer of adenosine, L-[3H]adenosine, was not accumulated into retinal cells consistent with the view that the accumulation of both adenosine and R-PIA occurs via the purine nucleoside transporter.     

Isolation and characterization of S49 mouse lymphoma cell mutants deficient in adenosine deaminase

Adenosine deaminase-deficient mutants of a mouse lymphoma cell line S49 have been isolated by a two-step selection process. In the first step, we derived mutant lines containing haploid levels of adenosine deaminase activity from wild-type cells. The selective medium contained tritiated deoxyadenosine, deoxycytidine, and deoxycoformycin. Wild-type cells were killed, presumably because of suicidal incorporation of tritiated deoxy adenosine via the adenosine deaminase pathway. The second step was to derive, from the partially deficient mutants, sublines that were virtually lacking adenosine deaminase, using tritiated deoxyadenosine and deoxycytidine. Four mutant clones were found to contain less than 5% of the enzyme activity of wild-type cells and virtually no immunoreactive adenosine deaminase protein. Northern blot analysis showed that the levels of adenosine deaminase mRNA were drastically reduced. Back-selection for adenosine deaminase-positive revertants can be accomplished by using a medium containing deoxyadenosine (as a sole source of purine), aminopterin, and thymidine or, alternatively, by using deoxyadenosine alone in a serum-free medium.     

Purine salvage and metabolism inBabesia bovis

Studies of the incorporation of radio-labelled purine precursors into the erythrocytic forms ofBabesia bovis under tissue-culture conditions have confirmed the presence in the parasite of enzymatic activities responsible for the salvage of preformed purines. The results also revealed that the parasite was capable of a variety of nucleotide interconversions, such that exogenous hypoxanthine and adenosine were incorporated into both adenine and guanine nucleotides followed by the incorporation of these nucleotides into the adenine and guanine moieties of RNA and DNA. No evidence was found for salvage of preformed pyrimidines. Evidence was also obtained for the insertion of a parasite-specific nucleoside/nucleobase transporter into the membrane of the bovine (host) red cell. Thus, whereas normal (non-parasitised) bovine red cells are essentially incapable of transporting nucleosides across their membranes, the invasion of these cells byB. bovis introduces a transporter that can be inhibited by classic nucleoside transport inhibitors.     

Uptake and release of adenosine in isolated rat fat cells.

Radioactively labelled adenosine and adenine were rapidly taken up by isolated rat fat cells, and incorporated into nucleotides, of which ATP dominated. The overall process had an apparent Km of 1--5 micrometers. During incubation, especially in the presence of lipolytic agents, there was a reduction in labelled ATP with a compensatory increase in ADP, AMP, cAMP and nucleosides. The build-up of adenosine during incubation was inhibited by theophylline, which inhibits 5'-nucleotidase. Radioactivity released from perifused fat cells consisted mainly of nucleoside material, of which adenosine predominated. Lipolytic stimulation caused no significant increase in nucleoside outflow from perifused cells, whereas oxygenation was capable of reducing this outflow. It is concluded that adenosine is formed by fat cells as a consequence of ATP breakdown. Stimulation of lipolysis during activation of the sympathetic nerves leads to reversible ATP breakdown and adenosine release. Adenosine might therefore act as a modulator of lipolysis in vivo under these conditions, even though it does not serve as a feed back regulator in the proper sense.     

Evidence for indirect involvement of thymidine kinase in excision repair processes in mouse cell lines

Wild-type cells and thymidine kinase-deficient clones from two mouse lymphoma cell lines, P388 and L5178Y, were compared for sensitivity to killing by the mutagens, ultraviolet irradiation (UV), ethyl methane sulfonate (EMS), and N-methyl-N-nitro-N-nitrosoguanidine (MNNG). Two out of three thymidine kinase-deficient P388 clones showed significantly enhanced sensitivity to killing by all three mutagens. This increased sensitivity to killing was also reflected in increased mutagenesis by the three mutagens. In the L5178Y cell line, wild-type cells showed little difference to two thymidine kinase-deficient clones in terms of mutagen sensitivity. This indicates that thymidine kinase may be significant for DNA repair processes in P388 but not in L5178Y cells. Unscheduled DNA synthesis (UDS) experiments were carried out on P388 and L5178Y wild-type cells and wild-type Friend leukemia cells (which are mutagen-sensitive when deficient in thymidine kinase). The UDS experiments showed the L5178Y cells were low in excision repair abilities relative to the P388 cells and the Friend cell clone. This indicates that the increased mutagen sensitivity in thymidine kinase-deficient P388 and clone 707 Friend cells may be due to thymidine kinase playing an indirect role in DNA excision repair, a process which is of little significance in the L5178Y cell line.     

Reduced purine accumulation is encoded on an amplified DNA in Leishmania mexicana amazonensis resistant to toxic nucleosides

Nucleoside analogs are potential anti-Leishmania agents. To better understand how these compounds might lose their effectiveness, Leishmania were independently selected for resistance to inosine dialdehyde or tubercidin. Each of the resistant cells exhibited resistance to inosine dialdehyde and tubercidin as well as to formycin B and allopurinol ribonucleoside. Resistant cells had a greatly reduced capability of accumulating exogenous adenosine, guanosine, thymidine and guanine. This decreased ability to accumulate nucleosides and at least one nucleobase appeared to be due to reduced activity of a number of distinct purine transporters, as the differences between purine metabolizing enzymes were not sufficiently different to account for the decreased accumulation capability. The resistance to toxic nucleosides and the decreased ability to accumulate purines were due to the presence in the resistant cells of an extrachromosomal DNA approximately 55 kb in size. The extrachromosomal DNA was not detected in wild-type cells or revertants which have lost resistance to toxic nucleosides. Except for a 1.2-kb difference, the extrachromosomal DNA from both independently selected resistant cells appeared to be identical. The resistant cells contained 2–4 times as much DNA homologous to the extrachromosomal DNA as compared to wild type cells. When cloned into an E. coli/Leishmania shuttle vector, a portion of the amplified DNA had the ability to confer upon wild-type cells resistance to the toxic purine nucleoside analogs tubercidin and inosine dialdehyde. These transformed cells also exhibited a decreased ability to accumulate non-toxic purine nucleosides.     

Purine metabolism in the intact sporozoites and merozoites of Eimeria tenella

Intact Eimeria tenella sporozoites and merozoites did not incorporate radiolabeled formate or glycine into their purine nucleotides suggesting a lack of de novo purine synthesis. However, [U-14C]glucose was incorporated into the cellular purine and pyrimidine nucleotide pools of both forms probably via conversion to radiolabeled ribose-1-phosphate and/or 5'-phosphoribosyl-1-alpha-pyrophosphate and the resulting action of various purine and pyrimidine salvage enzymes. Both forms of the parasite salvaged radiolabeled purine bases and nucleosides in a similar fashion. These purines were incorporated into ribonucleotides and into RNA and DNA. Adenine and inosine were transformed to hypoxanthine. Adenosine was converted to both inosine and hypoxanthine. Hypoxanthine and xanthine were not oxidized to uric acid but were metabolized to nucleotides. Guanosine was cleaved to guanine; guanine was deaminated to xanthine. The results demonstrate the presence of several purine salvage pathways. Purine phosphoribosylating and nucleoside phosphorylating activities as well as purine nucleoside cleaving and adenosine, adenine and guanine deaminating activities were evident. The metabolic evidence suggests the enzymes required to convert the newly formed nucleoside monophosphates to ATP and GTP were present also.     

Specificity of anti-nucleoside antibodies in systemic lupus erythematosus

The titer of IgG antinucleoside antibodies in the sera of 162 individuals was determined by an enzyme-linked immunosorbent assay. The nucleosides used in the assay were adenosine, cytidine, guanosine, and thymine-riboside conjugated to human serum albumin. The specificity of IgG antinucleoside antibodies was indicated by appropriate reduction in antibody binding after solid-phase adsorptions of antibody with specific immobilized nucleoside conjugates. Disease-associated increases in serum IgG antibodies to cytidine and guanosine but not to adenosine or thymine-riboside occurred in patients with systemic lupus erythematosus (SLE). The epitope density of nucleosides in the conjugates and differences in the sensitivity of each nucleoside assay were not responsible for disease-associated IgG antinucleoside antibody responses. These findings support a possible pathogenic role for cytidine and guanosine as antigens or crossreactive antigenic determinants in some patients with SLE.     

Complementation mapping in microcell hybrids: Localization ofFpgs andAk-1 onmus musculus chromosome 2

The gene encoding folylpolyglutamyl synthetase (FPGS) was assigned to mouse chromosome 2 by complementation mapping. Chinese hamster ovary cells (AuxBl) deficient in FPGS, and consequently auxotrophic for glycine, adenosine, and thymidine (gat^?), were employed as recipients in microcell-mediated chromosome transfer experiments. Mouse chromosomes derived from diploid embryo fibroblasts were introduced into hamster AuxBl cells, and gat⁺ microcell hybrids were selected in medium lacking adenosine and thymidine. Mouse chromosome 2 was the only donor chromosome whose presence correlated with expression of FPGS activity. Furthermore, every gat⁺ hybrid clone expressed murine AK-1, a marker previously assigned to chromosome 2. Eight of 20 clones analyzed retained deletion chromosomes derived from mouse chromosome 2. These clones were used to localize murine Fpgs and Ak-1 to a region of this chromosome, namely 2(cen→C1).     

Uphill transport of pyrimidine nucleosides in renal brush border vesicles

The uptake of cytidine, of thymidine and of uridine in brush border vesicles prepared from the cortex of rat kidney has been studied by the technique of rapid filtration. The nucleosides were not metabolized in the vesicles. The time-courses of uptake in the presence of inwardly directed gradients of Na⁺ and of K⁺ showed an overshoot, indicating uphill transport. The overshoot was much more pronounced with Na⁺ than with K⁺; it was not observed when Na⁺ was at equilibrium across the membrane. The uptake of the nucleosides was stimulated by an inside negative potential in the presence of Na⁺. These results provide evidence for a cotransport of pyrimidine nucleosides with Na⁺. The apparentK _m's for the uptake of cytidine, of thymidine and of uridine were 3.76 mol · l^–1, 4.18 mol · l^–1 and 7.21 mol · l^–1 respectively. The uptake of the pyrimidine nucleosides was insensitive to 6-nitrobenzylthioinosine. This insensitivity as well as the high affinity for the nucleosides and the capacity for uphill transport indicate that the nucleoside carrier(s) is renal brush border is (are) different from the carriers found in most other cell types.Abbreviations HEPES N-2-hydroxymethylpiperazine-N-2-ethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - NBMPR 6-nitrobenzylthioinosine - Tris tris(hydroxymethyl)-aminomethane