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PubMed1980

Passive diffusion of nucleosides into Micrococcus sodonensis membrane vesicles.

Nucleoside entry into isolated membrane vesicles of Micrococcus sodonensis (luteus) was studied using 14C-labelled nucleosides: adenosine, inosine, cytidine, uridine, guanosine, and thymidine. All nucleosides were recovered unmetabolized from the vesicles except adenosine and cytidine which were partly deaminated by membrane-bound enzymes. Vesicle preparations actively transported proline but no energy source was found capable of supporting concentrative nucleoside uptake. The entry of nucleosides into M. sodonensis vesicles was not saturable, nor was there competition between the nucleosides studied for entry. It was concluded that nucleoside entry into M. sodonensis vesicles occurs by passive diffusion.

Adenosine↗
PubMed1997

Sodium-dependent nucleoside transport in the human intestinal brush-border membrane.

The objective of the study was to determine the identity and kinetic characteristics of nucleoside transporters present in the brush-border membrane of the human jejunum. With use of brush-border membrane vesicles, uptake of [3H]uridine was stimulated two- to threefold by an inwardly directed Na+ gradient and was inhibited by both 100 microM thymidine and 100 microM guanosine nucleosides, which serve as model substrates for purine (N1, cif) and pyrimidine (N2, cit) transporters, respectively. [3H]thymidine and [3H]guanosine transport exhibited an overshoot phenomenon only in the presence of a Na+ gradient. Na(+)-thymidine uptake was inhibited by 100 microM cytidine or thymidine but not by guanosine, inosine, formycin B, or hypoxanthine. [3H]guanosine uptake was inhibited by 100 microM inosine, guanosine, or formycin B but not by thymidine or cytidine. Both adenosine and uridine inhibited uptake of [3H]thymidine and [3H]guanosine to a similar extent, indicating that both N1, cif and N2, cit Na(+)-nucleoside transporters are expressed in human jejunum. Enhanced uptake of Na(+)-thymidine by an inside-negative potential difference generated by K+ and valinomycin provides evidence that nucleoside transport is rheogenic, involving net transfer of a positive charge. The Hill coefficient was unity for all three substrates, indicating a Na(+)-nucleoside coupling stoichiometry of 1:1. At saturating Na+ concentration (150 mM) the kinetic parameters (n = 3-4) Michaelis-Menten constant and maximum velocity for uridine, thymidine, and guanosine uptake were 4.15 +/- 1.79, 2.74 +/- 0.58, 12.02 +/- 1.34 microM and 25.93 +/- 7.38, 16.10 +/- 3.64, 63.92 +/- 10.23 pmol.mg-1.10 s-1, respectively. These results suggest that, in contrast to the human kidney that expresses the N4 nucleoside transporter, the human jejunum expresses both N1 and N2 Na(+)-nucleoside transporters.

Adult↗
PubMed2000

The enantioselectivity of enzymes involved in current antiviral therapy using nucleoside analogues: a new strategy?

This review is primarily intended for synthetic bio-organic chemists and enzymologists who are interested in new strategies in the design of virus inhibitors. It is an attempt to assess the importance of the enzymatic properties of L-nucleosides and their analogues, particularly those that are active against viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), herpes simplex virus (HSV), etc. Only data obtained with purified enzymes have been considered and discussed. The examined enzymes include nucleoside- or nucleotide-phosphorylating enzymes, catabolic enzymes, viral target enzymes and cellular polymerases. The enantioselectivities of these enzymes were determined from existing data and are significant only when a sufficient number of enantiomeric pairs of substrates could be examined. The reported data emphasize the weak enantioselectivities of cellular or viral nucleoside kinases and some viral DNA polymerases. Thus, cellular deoxycytidine kinase has a considerably relaxed enantioselectivity with respect to a large number of nucleosides or their analogues, and it occupies a strategic position in the intracellular activation of the compounds. Similarly, HIV-1 reverse transcriptase often has a relatively weak enantioselectivity and can be inhibited by the 5-triphosphates of a large series of L-nucleosides and analogues. In contrast, degradation enzymes, such as adenosine or cytidine deaminases, generally demonstrate strict enantioselectivities favouring D-enantiomers and are used by chemists in asymmetric syntheses. The weak enantioselectivities of some enzymes involved in nucleoside metabolism are more or less pronounced, and one enantiomer or the other is favoured depending on the substrate. This suggests that the low enantioselectivity is fortuitous and does not result from evolutionary pressure, since these enzymes do not create or modify asymmetric centres in substrates. The combined enantioselectivities of the enzymes examined in this review strongly suggest that the field of L-nucleosides and their analogues should be systematically explored in the search for new virus inhibitors.

Antiviral Agents↗
PubMed2003

Cellular pharmacology of D-d4FC, a nucleoside analogue active against drug-resistant HIV.

The backbone of effective highly active antiretroviral therapy regimens for the treatment of HIV infections currently contains at least two nucleosides. Among the features that influence the potency of each component of a regimen and the overall efficacy of the combination are the cellular uptake and bioconversion of nucleoside analogues to their active triphosphate form, and the extent of possible interactions in these steps that might occur when more than one nucleoside is used in a regimen. D-d4FC (Reverset), a new cytidine analogue with the ability to inhibit many nucleoside-resistant viral variants, was examined for these parameters. In phytohemaglutinin-stimulated human peripheral blood mononuclear cells, D-d4FC was taken up in a rapid (8 h to 50% maximal value), saturable (plateau above 10 microM parent nucleoside concentration) process, resulting in levels of D-d4FC triphosphate that should provide potent antiviral activity against a variety of virus genotypes. Based on measurement of antiviral effects in cell culture, additive and in some cases, synergistic interactions were observed with protease inhibitors, non-nucleoside reverse transcriptase inhibitors or other nucleosides, including cytidine analogues.

Anti-HIV Agents↗
PubMed1989

Nucleosides. CXXXV. Synthesis of some 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-9H-purines and their biological activities.

Seven purine nucleosides containing the 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl moiety were synthesized and tested for their antitumor activity. Direct condensation of 3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranosyl bromide (1) with N6-benzoyladenine in CH2Cl2 followed by saponification of the product afforded the adenine nucleoside (I, 2'-F-ara-A). Deamination of I with NaNO2 in HOAc gave the hypoxanthine analogue (II, 2'-F-ara-H). The 6-thiopurine nucleoside (III, 2'-F-ara-6MP) was prepared by condensation of 1 with 6-chloropurine by the mercury procedure followed by thiourea treatment and saponification of the product. Methylation of III gave the 6-SCH3 analogue (IV). Raney Ni desulfurization of III afforded the unsubstituted purine nucleoside (V, 2'-F-ara-P). Condensation of 1 with 2-acetamido-6-chloropurine by the silyl procedure afforded the protected 2-acetamido-6-chloropurine nucleoside which served as the precursor for both the guanine and 6-thioguanine nucleosides (VI, 2'-F-ara-G and VII, 2'-F-ara-TG, respectively). Thus, alkaline hydrolysis of the precursor gave VI. Thiourea treatment prior to alkaline hydrolysis gave VII. The new nucleoside, 2'-F-ara-G (VI) is found to be selectively toxic to human T-cell leukemia CCRF-CEM.

Animals↗
PubMed2004

Characterization of novel nucleoside analogs by electrospray ionization mass spectra.

In this paper, we synthesized a novel nucleoside analog by coupling thymine with dimethyl dicarboxylate biphenyl (DDB). The structure of the target compound was determined using 1H nuclear magnetic resonance (NMR) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). The fragmentation pathways were studied in details through ESI-MS/MS. By comparing with unsubstituted nucleosides, such as AZT, MCI, d4T and DDI, it was found that the nucleoside analog coupled with DDB would not yield the daughter ions corresponding to the fragments of the nucleoside base and arabinofuranose analogs, but would lose a neutral molecule HF and DDB easily. However, the unsubstituted nucleosides could lightly yield the fragment ions of the nucleoside base and sugar ring. Hence, electrospray ionization mass spectrometry combined with tandem mass spectrometry (MS/MS) provides a convenient method to recognize the substituted and unsubstituted nucleosides.

Dioxoles↗
PubMed2003

Pyrimidine nucleoside analogs in cancer treatment.

Pyrimidine nucleoside analogs are essential components of hematological malignancy therapy and are also used in the treatment of solid tumors. These agents act as antimetabolites, compete with physiologic pyrimidine nucleosides and, consequently, interact with a large number of intracellular targets to induce cytotoxicity. Pyrimidine nucleoside analogs share some common characteristics, such as requiring both transport by specific membrane transporters and intracellular metabolism. However, these compounds differ in regards to their preferential interaction with certain targets, which may explain why some compounds are more effective against rapidly proliferating tumors and others against neoplasias with a more protracted evolution. Recent progress in the identification and characterization of nucleoside transporters and the enzymes of nucleoside metabolism, as well as an understanding of the molecular mechanisms of anticancer nucleoside activity, provides opportunities for the development of new pyrimidine nucleoside analogs. Strategies to optimize intracellular analog accumulation and to enhance cancer cell selectivity are proving beneficial in clinical trials.

Animals↗
PubMed2006

Base release in nucleosides induced by low-energy electrons: a DFT study.

Low-energy electrons are known to induce strand breaks and base damage in DNA and RNA through fragmentation of molecular bonding. Recently the glycosidic bond cleavage of nucleosides by low-energy electrons has been reported. These experimental results call for a theoretical investigation of the strength of the C(1)'-N link in nucleosides (dA, dC and dT) between the base and deoxyribose before and after electron attachment. Through density functional theory (DFT) calculations, we compare the C(1)'-N bond strength, i.e., the bond dissociation energy of the neutral and its anionic radical, and find that an excess electron effectively weakens the C(1)'- N bond strength in nucleosides by 61-75 kcal/mol in the gas phase and 76-83 kcal/mol in the solvated environment. As a result, electron-induced fragmentation of the C(1)'-N bond in the gas phase is exergonic for dA (DeltaG=-14 kcal/mol) and for dT (DeltaG=-6 kcal/mol) and is endergonic (DeltaG=+1 kcal/ mol) only for dC. In the gas phase all the anionic nucleosides are found to be in valence states. Solvation is found to increase the exergonic nature by an additional 20 kcal, making the fragmentation both exothermic and exergonic for all nucleoside anion radicals. Thus C(1)'-N bond breaking in nucleoside anion radicals is found to be thermodynamically favorable both in the gas phase and under solvation. The activation barrier for the C(1)'-N bond breaking process was found to be about 20 kcal/mol in every case examined, suggesting that a 1 eV electron would induce spontaneous cleavage of the bond and that stabilized anion radicals on the DNA strand would undergo base release at only a modest rate at room temperature. These results suggest that base release from nucleosides and DNA is an expected consequence of low-energy electron-induced damage but that the high barrier would inhibit this process in the stable anion radicals.

Base Pairing↗
PubMed2001

Active intestinal absorption of nucleosides by Na+-dependent transport across the brush border membrane in cows.

Transport of 3H-labeled nucleosides across the bovine intestinal brush border membrane (BBM) was characterized with BBM vesicles (BBMV) isolated from mid-jejunum of cows because large amounts of nucleic acids are digested in the small intestine of ruminants. Two Na+-dependent electrogenic nucleoside transporters with overlapping substrate specificity were shown to be present in the jejunal BBM, one for pyrimidine nucleosides and one for purine nucleosides. As indicated by inhibitory studies, thymidine seemed to be a specific substrate for the pyrimidine nucleoside transporter, while this applied to guanosine and deoxyguanosine for the purine nucleoside transporter. Uridine and adenosine appear to have an affinity to both transporters. This also applies to deoxyadenosine and deoxyuridine. Nucleobases (uracil, hypoxanthine) did not affect transport of nucleosides. The kinetic constants (Km and Vmax) for Na-dependent thymidine and guanosine transport were 29 and 24 micromol/L and 78 and 51 pmol (mg protein)(-1) s(-1), respectively. These values are much higher than those reported for monogastric species.

Animals↗
PubMed1995

Profile of nucleotides and nucleosides of human milk.

The content of nucleotides and nucleosides of human milk was analyzed using a newly developed method for high performance liquid chromatography. By this method it is possible to analyze nucleotides and nucleosides simultaneously. Human milk was pooled according to season, lactation period, and geographical area. Three kinds of nucleosides--cytidine, uridine, and adenosine--and 6 kinds of nucleotides--5'-CMP, 5'-UMP, 5'-AMP, 5'-GMP, 5'-IMP, and 5'-CDP--were detected. Cytidine, 5'-CMP, and 5'-CDP predominated throughout lactation. Also there seemed to be geographical differences in nucleoside composition. The overall amounts of nucleotides and nucleosides were higher in winter than in summer. No nucleosides were detected in bovine milk, nor in bovine milk-based infant formula, and bovine milk contained much less nucleotides than human milk. These results suggest that nucleosides and nucleotides found in human milk may play some important roles in the development of infants.

Adenosine↗
PubMed1975

Characterization of nucleoside transport in hymenolepidid cestodes.

Three species of cestodes, Hymenolepis diminuta, H. citelli, and H. microstoma, were shown to transport nucleosides by a mediated process. In H. diminuta the Ki values of various competitive inhibitors led to the conclusion that at least 2 different loci are involved in the transport of nucleosides. One of these loci has greater specificity for the purine nucleosides, the other for pyrimidine nucleosides. No significant difference was observed in their affinity for ribo- or deoxyribonucleosides, although thymidine was slightly less effective as an inhibitor of ribonucleosides than was uridine. Transport by the pyrimidine nucleoside locus was stimulated by thymine, but not by hypoxanthine, whereas transport by the purine nucleoside locus was stimulated by hypoxanthine but not by thymine. Preloading the worms with thymine gave less stimulation of transport than did the presence of eoxgenous modulator. Efflux of previously accumulated nucleoside was not blocked by the presence of exogenous modulator. The presence of exgenous thymine enhanced the incorporation of uridine into the nonextractable pool. Thymine also stimulated uracil transport in H. citelli, but not in H. microstoma, and had no effect on uridine transport in either of these species. The results of transport and modulator studies on H. diminuta grown in hamsters were not different from the results with worms grown in rats indicating that the regulatory effects observed were an inherent function of the parasite and not the host.

Adenine↗
PubMed2001

Mechanisms of uptake and resistance to troxacitabine, a novel deoxycytidine nucleoside analogue, in human leukemic and solid tumor cell lines.

Troxacitabine (Troxatyl; BCH-4556; (-)-2'-deoxy-3'-oxacytidine), a deoxycytidine analogue with an unusual dioxolane structure and nonnatural L-configuration, has potent antitumor activity in animal models and is in clinical trials against human malignancies. The current work was undertaken to identify potential biochemical mechanisms of resistance to troxacitabine and to determine whether there are differences in resistance mechanisms between troxacitabine, gemcitabine, and cytarabine in human leukemic and solid tumor cell lines. The CCRF-CEM leukemia cell line was highly sensitive to the antiproliferative effects of troxacitabine, gemcitabine, and cytarabine with inhibition of proliferation by 50% observed at 160, 20, and 10 nM, respectively, whereas a deoxycytidine kinase (dCK)-deficient variant (CEM/dCK(-)) was resistant to all three drugs. In contrast, a nucleoside transport-deficient variant (CEM/ARAC8C) exhibited high levels of resistance to cytarabine (1150-fold) and gemcitabine (432-fold) but only minimal resistance to troxacitabine (7-fold). Analysis of troxacitabine transportability by the five molecularly characterized human nucleoside transporters [human equilibrative nucleoside transporters 1 and 2, human concentrative nucleoside transporter (hCNT) 1, hCNT2, and hCNT3] revealed that short- and long-term uptake of 10-30 microM [(3)H]troxacitabine was low and unaffected by the presence of either nucleoside transport inhibitors or high concentrations of nonradioactive troxacitabine. These results, which suggested that the major route of cellular uptake of troxacitabine was passive diffusion, demonstrated that deficiencies in nucleoside transport were unlikely to impart resistance to troxacitabine. A troxacitabine-resistant prostate cancer subline (DU145(R); 6300-fold) that exhibited reduced uptake of troxacitabine was cross-resistant to both gemcitabine (350-fold) and cytarabine (300-fold). dCK activity toward deoxycytidine in DU145(R) cell lysates was <20% of that in DU145 cell lysates, and no activity was detected toward troxacitabine. Sequence analysis of cDNAs encoding dCK revealed a mutation of a highly conserved amino acid (Trp(92)-->Leu) in DU145(R) dCK, providing a possible explanation for the reduced phosphorylation of troxacitabine in DU145(R) lysates. Reduced deamination of deoxycytidine was also observed in DU145(R) relative to DU145 cells, and this may have contributed to the overall resistance phenotype. These results, which demonstrated a different resistance profile for troxacitabine, gemcitabine, and cytarabine, suggest that troxacitabine may have an advantage over gemcitabine and cytarabine in human malignancies that lack or have low nucleoside transport activities.

Amino Acid Sequence↗
PubMed1988

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.

Animals↗
PubMed1983

Heterogeneity of nucleoside transport in mammalian cells. Two types of transport activity in L1210 and other cultured neoplastic cells.

The characteristics of nucleoside transport were examined in L1210 murine leukemia cells and five other cultured neoplastic cells. The initial rates of uridine, adenosine, and thymidine transport in L1210 cells were only partially inhibited by 1 microM nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport in other cells. The IC50 for NBMPR inhibition of uridine transport was 5 nM, but 20% of the activity remained insensitive to concentrations as high as 3 microM. Uridine uptake in the presence of 1 microM NBMPR was saturable and was inhibited by other nucleosides, suggesting the participation of an NBMPR-insensitive transport mechanism. There appeared to be little difference in the specificity of NBMPR-sensitive and -insensitive transport for the physiological nucleosides. Uridine, adenosine, and thymidine were all substrates for both mechanisms, and the Km values for total and NBMPR-insensitive uridine transport were the same (250 microM). Furthermore, little difference was found in the ability of several other nucleosides to inhibit total or NBMPR-insensitive uridine transport. In both cases, adenosine was the most effective inhibitor while cytidine and deoxycytidine were the least effective. The two transport processes did, however, differ from each other in their sensitivity to p-mercuribenzenesulfonate (pMBS). NBMPR-insensitive uridine transport was inhibited by pMBS with an IC50 less than 25 microM, while the IC50 for NBMPR-sensitive transport was greater than 400 microM. Cloning of the parent L1210 cell line indicated that both NBMPR-sensitive and -insensitive transport occurred in the same cell. Both types of uridine transport activity were also observed in three other cell lines (RPMI 6410, L5178Y, and P388), while two lines, S49 and Walker 256, exhibited only NBMPR-sensitive and -insensitive transport, respectively. The level of NBMPR-insensitive transport was an important determinant in the ability of NBMPR to inhibit uridine uptake over prolonged periods (10 min), with as little as 20% NBMPR-insensitive transport sufficient to render uptake over 10 min virtually insensitive to NBMPR. The existence of these two types of nucleoside transport activity in mammalian cells may have important implications in the chemotherapeutic use of transport inhibitors in combination with cytotoxic nucleosides or with inhibitors of pyrimidine and purine biosynthesis.

4-Chloromercuribenzenesulfonate↗
PubMed1983

Urinary excretion of modified nucleosides in chronic myelogenous leukemia.

Urinary excretion of modified nucleosides was measured in 15 patients with Philadelphia chromosome-positive CML to determine the correlation with activity of this disease. Resolution and quantitation of seven nucleosides were accomplished with reversed-phase HPLC. Patients in the stable phase of CML had excretion levels one to two times normal, whereas patients in the blastic phase showed elevations up to 12 times normal. The nucleosides showing the most significant differences in excretion between stable phase and blastic phase were 1-methylinosine, pseudouridine, and N2,N2-dimethylguanosine (p less than 0.01, p less than 0.001, and p less than 0.01, respectively). Nucleoside excretion was also determined in patients with bacterial pneumonia and urinary tract infection for comparison. Serial nucleoside determinations were made in two patients with CML and found to correlate closely with disease activity. The degree of elevation and the correlation with disease activity suggest the potential value of urinary nucleoside quantitation in monitoring patients with CML; in particular, nucleoside excretion may be useful in detecting early blastic transformation.

Adult↗
PubMed1997

Uptake and release of [3H]formycin B via sodium-dependent nucleoside transporters in mouse leukemic L1210/MA27.1 cells.

At least seven functionally distinct nucleoside transport processes exist; however, mouse leukemic L1210/MA27.1 cells possess only one subtype, a Na+-dependent transporter termed N1/cif. The capacity of this transporter subtype to release nucleosides from L1210/MA27.1 cells was investigated with the poorly metabolized inosine analog [3H]formycin B. Uptake of [3H]formycin B into these cells was inhibited by replacement of Na+ in the buffer with choline, or by blocking Na+/K+ ATPase with 2 mM ouabain, inhibiting glycolysis with 5 mM iodoacetic acid or inhibiting nucleoside transport with 1 mM phloridzin. Sodium stimulated uptake with an EC50 value of 12 mM. To measure release of [3H]formycin B, cells were loaded with [3H]formycin B (10 microM) then washed and resuspended in buffer. Replacement of Na+ in the buffer with choline enhanced [3H]formycin B release by 20 to 47%, and significant stimulation of release was observed with Na+ concentrations of 30 mM or less. Resuspending loaded cells into Na+ buffer containing 2 mM ouabain or 10 microM monensin, a Na+ ionophore, significantly enhanced [3H]formycin B release during 20 min by 39% or 29%, respectively. Release of [3H]formycin B into choline buffer was inhibited 26.5% by 10 mM phloridzin and 39.6% by 10 mM propentofylline, compounds known to inhibit various transporters including Na+-dependent nucleoside transporters. Release was also inhibited significantly by 100 microM concentrations of dilazep, dipyridamole and nitrobenzylthioinosine, inhibitors with selectivity for Na+-independent nucleoside transporters. In the absence of Na+, the permeants adenosine and uridine enhanced [3H]formycin B release by up to 40.9% and 21.4%, respectively. These data indicate that in the absence of an inwardly directed Na+ gradient, Na+-dependent nucleoside transporters can function in the release of nucleosides.

Adenosine Triphosphate↗
PubMed1998

Differential expression and regulation of nucleoside transport systems in rat liver parenchymal and hepatoma cells.

Primary cultures of rat-liver parenchymal cells show carrier-mediated nucleoside uptake by a mechanism that mainly involves concentrative, Na+-dependent transport activity. In contrast, the hepatoma cell line FAO shows high nucleoside transport activity, although it is mostly accounted for by Na+-independent transport processes. This is associated with a low amount of sodium purine nucleoside transporter (SPNT) mRNA. SPNT encodes a purine-preferring transporter expressed in liver parenchymal cells. To analyze whether SPNT expression is modulated during cell proliferation, SPNT mRNA levels were determined in the early phase of liver growth after partial hepatectomy and in synchronized FAO cells that had been induced to proliferate. SPNT mRNA amounts increased as early as 2 hours after partial hepatectomy. FAO cells induced to proliferate after serum refeeding show an increase in SPNT mRNA levels, which is followed by an increase in Na+-dependent nucleoside uptake and occurs before the peak of 3H-thymidine incorporation into DNA. FAO cells also express significant equilibrative nucleoside transport activity, which may be accounted for by the expression of the nitrobenzylthioinosine (NBTI)-sensitive and -insensitive isoforms, rat equilibrative nucleoside transporter 1 (rENT1) and rENT2, respectively. Interestingly, rENT2 mRNA levels follow a similar pattern to that described for SPNT when FAO cells are induced to proliferate, whereas rENT1 appears to be constitutively expressed. Liver parenchymal cells show low and negligible mRNA levels for rENT1 and rENT2 transporters, respectively, although most of the equilibrative transport activity found in hepatocytes is NBTI-resistant. It is concluded that: 1) SPNT expression is regulated both in vivo and in vitro in a way that appears to be dependent on cell cycle progression; 2) SPNT expression may be a feature of differentiated hepatocytes; and 3) equilibrative transporters are differentially regulated, rENT2 expression being cell cycle-dependent. This is consistent with its putative role as a growth factor-induced delayed early response gene.

Animals↗
PubMed2003

Genomic organization and functional characterization of the human concentrative nucleoside transporter-3 isoform (hCNT3) expressed in mammalian cells.

Human CNT3 encodes the concentrative nucleoside transport N3 system. Previous expression studies in oocytes showed that the Km values for nucleosides of the cloned hCNT3 were 7- to 25-fold lower than the endogenous N3 transporter in HL60 cells. Therefore, in the present study we re-examined the kinetic properties of the cloned hCNT3 using mammalian cell expression systems by transient expression in Cos7L cells and stably expression in nucleoside transporter deficient PK15NTD cells. We demonstrated that hCNT3 is a Na-dependent, broadly-selective nucleoside transporter with affinities (<11 microM) for nucleosides closely resembling the endogenous N3 transporter. Pharmacological studies showed that phloridzin is a mixed-type inhibitor of hCNT3 (Ki=15 microM), and the dideoxyuridine analogs are poor substrates. By epitope-tagging, we further demonstrated that hCNT3 is N-glycosylated as PNGase F and Endo H deglycosylated hCNT3 from 67 kDa to 58 kDa. Searching the human genome database, we identified the genomic organization of hCNT3. This gene contains 19 exons and its exon-intron boundaries within the coding sequence exactly match with those of hCNT1 and hCNT2 with one additional exon in the N-terminus. Our data suggest that hCNT3 gene is evolutionarily conserved with hCNT1 and hCNT2. Physiologically, hCNT3 is a glycoprotein, which transports purine and pyrimidine nucleosides in a Na-dependent manner with high affinities.

Animals↗