Nucleoside Transporter Proteins Research Articles

Differential Expression of Human Equilibrative Nucleoside Transporter 1 (hENT1) Protein in the Reed-Sternberg Cells of Hodgkin's Disease

Gemcitabine is a cytotoxic nucleoside analog with activity in relapsing/refractory Hodgkin's disease (HD). Because gemcitabine is hydrophilic, it requires plasma membrane nucleoside transporter proteins to access intracellular targets. The most abundant and widely distributed transporter in human cells is human equilibrative nucleoside transporter 1 (hENT1). Because our prior studies showed that a deficiency in hENT1 confers high-level resistance to gemcitabine toxicity in vitro, we developed an immunohistochemical method to assess the hENT1 abundance of cells in tumor tissue. We now report the application of this method for visualizing the hENT1 protein abundance in the plasma membranes of Reed-Sternberg cells in lymph nodes of HD patients. Frozen sections of 30 lymph nodes were stained with monoclonal antibodies (mAb 10D7G2) raised against a synthetic peptide comprised of residues 254-271 from the large intracellular loop of hENT1 and staining intensity was scored on a 0-4+ scale. hENT1-staining intensity varied among HD lymph node samples (score/ n ; 0/8; 1/10; 2/9; 3/3; 4/0) and suggested that at least 60% of the tumors appeared hENT1 deficient. Because Epstein-Barr virus (EBV) is often associated with HD, staining for Epstein-Barr early RNA was also examined. Although 9/30 patients tested positive for EBV, there was no correlation with hENT1 staining. hENT1-staining intensities were positively correlated with age of the patient but were independent of other clinical, laboratory or pathology features (tumor stage, histologic subtype, presence of B symptoms, staining for CD15 or CD30, serum biochemistry, disease free survival, and overall survival). We conclude that, because hENT1 deficiency has been previously related to nucleoside-drug resistance, immunohistochemical staining for hENT1 warrants evaluation as a predictive tool for guiding the appropriate use of gemcitabine in the treatment of HD.

Characterisation of a concentrative type of adenosine transporter from Arabidopsis thaliana (ENT1,At)

Here we report on the isolation of an Arabidopsis thaliana cDNA that is able to complement a Saccharomyces cerevisiae mutant unable to synthesise adenine. This cDNA encodes a highly hydrophobic protein (ENT1,At) of 428 amino acids, showing high similarity to the human nucleoside transporter hENT1. Yeast cells expressing ENT1,At are able to grow on adenosine-containing media, adenosine import exhibited an apparent affinity (K(M)) of 3.6 microM, and led to accumulation of this nucleoside within the yeast cell. Transport is inhibited by various nucleosides. Typical inhibitors of ENT-type nucleoside transporters do not inhibit (3)H-adenosine import. The presence of protonophores abolished adenosine import, indicating that ENT1,At catalyse a proton-dependent adenosine transport. This is the first functional characterisation of a plant nucleoside transport protein.

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes.

In the present study, one has determined the relative role of plasma membrane equilibrative (Na+-independent) ENT nucleoside transport proteins (particularly ENT2) in the uptake of antiviral nucleoside analogues for comparison with the previously reported drug transport properties of concentrative (Na+-dependent) CNT nucleoside transport proteins. The human and rat nucleoside transport proteins hENT1, rENT1, hENT2 and rENT2 were produced in Xenopus oocytes and investigated for their ability to transport three 3'-deoxy-nucleoside analogues, ddC (2'3'-dideoxycytidine), AZT (3'-azido-3'-deoxythymidine) and ddI (2'3'-dideoxyinosine), used in human immunodeficiency virus (HIV) therapy. The results show, for the first time, that the ENT2 transporter isoform represents a mechanism for cellular uptake of these clinically important nucleoside drugs. Recombinant h/rENT2 transported ddC, ddI and AZT, whilst h/rENT1 transported only ddC and ddI. Relative to uridine, h/rENT2 mediated substantially larger fluxes of ddC and ddI than h/rENT1. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 transport-positive for AZT and enhanced the uptake of both ddC and ddI, identifying this region as a major site of 3'-deoxy-nucleoside drug interaction.

Distinct regional distribution of human equilibrative nucleoside transporter proteins 1 and 2 (hENT1 and hENT2) in the central nervous system

Nucleoside transport processes play an important role in human cells in salvage of nucleosides used in the biosynthesis of nucleic acids and in regulating endogenous adenosine concentrations in the human central nervous system (CNS). By altering the levels of adenosine available to interact with cell–surface receptors, nucleoside transporters have profound effects on the ability of adenosine to modulate neurotransmission, vascular tone and other physiological events. Although the human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) are believed to play a crucial role in modulating brain function, their distribution within the major divisions of the human CNS is not known. In this work, antibodies specific for hENT1 and hENT2 were produced against fragments of the transporter proteins and used for immunoblot analysis of enriched membrane fractions prepared from several regions of the human brain. While hENT1 was most prevalent in the frontal and parietal lobes of the cerebral cortex, thalamus, midbrain and basal ganglia, hENT2 was concentrated in the cerebellum and brainstem regions, particularly the pons. The apparent reciprocal distribution of hENT1 and hENT2 in human brain suggests that these nucleoside transporter proteins are produced in distinct regions of the CNS where they function in nucleoside salvage and/or regulation of exogenous adenosine. Within the brain regions that were investigated, the pattern of hENT1 distribution correlated well with adenosine A 1 receptor abundance. The regional co-localization of hENT1 and A 1 receptor protein suggests an important role of hENT1-mediated transport process in the control of neuromodulatory actions mediated by adenosine A 1 receptors in human brain.

Identification of Cys140 in helix 4 as an exofacial cysteine residue within the substrate-translocation channel of rat equilibrative nitrobenzylthioinosine (NBMPR)-insensitive nucleoside transporter rENT2

The human and rat equilibrative nucleoside transporter proteins hENT1, rENT1, hENT2 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs), and are distinguished functionally by differences in transport of nucleobases and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs. In the present study, we have produced recombinant hENT1, rENT1, hENT2 and rENT2 in Xenopus oocytes and investigated uridine transport following exposure to the impermeant thiol-reactive reagent p-chloromercuriphenyl sulphonate (PCMBS). PCMBS caused reversible inhibition of uridine influx by rENT2, but had no effect on hENT1, hENT2 or rENT1. This difference correlated with the presence in rENT2 of a unique Cys residue (Cys140) in the outer half of TM4 that was absent from the other ENTs. Mutation of Cys140 to Ser produced a functional protein (rENT2/C140S) that was insensitive to inhibition by PCMBS, identifying Cys140 as the exofacial Cys residue in rENT2 responsible for PCMBS inhibition. Uridine protected wild-type rENT2 against PCMBS inhibition, suggesting that Cys140 in TM4 lies within or is closely adjacent to the substrate-translocation channel of the transporter. TM4has been shown previously to be within a structural domain (TMs 3Ő6) responsible for interactions with NBMPR, vasoactive drugs and nucleobases.

Functional production of mammalian concentrative nucleoside transporters inSaccharomyces cerevisiae

The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described. A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins. The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S. cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids. Transporter-mediated rescue of S. cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine. This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins.

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced inXenopusoocytes

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in<i>Xenopus</i>oocytes

Nucleoside transporter proteins: emerging targets for drug discovery

Nucleosides are central salvage metabolites and, as precursors of nucleotides, play an essential role in intermediary metabolism and biosynthesis, as well as signal transduction through interaction with purinergic receptors. Nucleoside analogues are used to treat haematological malignancies, certain solid tumours and many viral diseases. Because most nucleosides are relatively hydrophilic molecules, the presence of specialised transporter proteins in plasma membranes is essential for cellular uptake. Nucleoside transport is mediated by either bi-directional equilibrative processes, driven by chemical gradients, or inwardly directed concentrative processes, driven by sodium gradients. Recent advances in the identification of nucleoside transporter proteins from humans and lower organisms, using molecular cloning and functional expression of cDNAs, have transformed our understanding of nucleoside transport processes. Most of the nucleoside transporters identified thus far are members of two previously unrec...

A Solid State NMR Approach to the Structure-Activity Relationship of the Nucleoside Transport Protein NupC of Escherichia coli

A Solid State NMR Approach to the Structure-Activity Relationship of the Nucleoside Transport Protein NupC of <i>Escherichia coli</i>

Differential Transport of Cytosine-Containing Nucleosides by Recombinant Human Concentrative Nucleoside Transporter Protein hCNT1

The transportability of cytosine-containing nucleosides by recombinant hCNT1 was investigated in transfected mammalian cells. Apparent Km values for hCNT1-mediated transport of uridine, cytidine and deoxycytidine were, respectively, 59, 140 and 150 μM. Uridine transport was inhibited 89, 32 and 11%, respectively, by 500 μM gemcitabine, cytarabine and lamivudine, demonstrating that, unlike gemcitabine (a high-affinity hCNT1 permeant), cytarabine and lamivudine are poor hCNT1 permeants.

Gemcitabine transport in xenopus oocytes expressing recombinant plasma membrane mammalian nucleoside transporters.

Gemcitabine, a pyrimidine analogue of deoxycytidine, is an anticancer nucleoside drug that requires functional plasma membrane nucleoside transporter proteins to reach its intracellular targets and cause cytotoxicity. Because of technical difficulties inherent in studying nucleoside transport in human cells, we rigorously defined gemcitabine membrane transportability by producing each of the available human (h) and rat (r) recombinant nucleoside transporters (NTs) individually in Xenopus laevis oocytes. Oocytes were microinjected with in vitro-transcribed RNAs derived from complementary DNAs encoding (C = concentrative) rCNT1, rCNT2, hCNT1, hCNT2, (E = equilibrative) rENT1, rENT2, hENT1, and hENT2. Uptake of [(3)H]gemcitabine and [(14)C] uridine was measured 3 days after microinjection to determine kinetic constants. We also used the two-electrode, voltage-clamp technique to investigate the electrophysiology of hCNT1-mediated gemcitabine transport. Gemcitabine was transported by most of the tested proteins (the exceptions being the purine-selective rCNT2 and hCNT2), with the greatest uptake occurring in oocytes producing recombinant rCNT1 and hCNT1. Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent K(m) values of 24, 160, and 740 microM, respectively. Gemcitabine had a limited ability to cross the lipid bilayer of oocyte membranes by simple diffusion. External application of gemcitabine to oocytes producing recombinant hCNT1 induced an inward current, which demonstrated that hCNT1 functions as a Na(+)/nucleoside co-transport protein and confirmed the transporter's ability to transport gemcitabine. Mammalian nucleoside transporters vary widely in their affinity and capacity to transport gemcitabine. Variation in the tumor and tissue distribution of plasma membrane nucleoside transporter proteins may contribute to the solid tumor activities and schedule-dependent toxic effects of gemcitabine.

Stimulation of long-chain fatty acid uptake by dipyridamole in isolated myocytes.

This study was designed to investigate the effects of the cardiovascular drug dipyridamole on fatty acid metabolism in isolated cardiac myocytes. Effects of dipyridamole on the oxidation of long-chain (palmitate) fatty acid, medium-chain (octanoate) fatty acid, and the carbohydrate intermediate (pyruvate) were determined by using isolated cardiac myocytes from both normal and diabetic rats. Dipyridamole increased palmitate oxidation in a concentration-dependent manner in both normal and diabetic myocytes. Maximal stimulation of palmitate oxidation (175% of control) was observed with 100 microM dipyridamole. In contrast, oxidation of octanoate and pyruvate was not affected. The stimulation of palmitate oxidation by dipyridamole persisted despite its removal from the incubation medium. In contrast to the effect in myocytes, palmitate oxidation was not affected by dipyridamole in isolated rat heart mitochondria. Palmitate uptake was increased by 2.5- and 1.6-fold when palmitate concentration was adjusted to 0.05 and 0.2 mM, respectively. Dipyridamole did not affect lipolysis in isolated myocytes. When dipyridamole (100 microM) and L-carnitine (5 mM) were added together to the incubation medium, palmitate oxidation was further increased to 223% of the control. The nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) failed to increase palmitate oxidation in isolated myocytes. Although palmitate oxidation in diabetic cells is much higher than that in normal myocytes, dipyridamole increased palmitate oxidation by 243% in diabetic myocytes over its baseline oxidation rate in normal cells. These results suggest that increased palmitate oxidation in isolated cardiac myocytes after dipyridamole administration occurs independent of effects on either the phosphodiesterase enzyme or nucleoside transport protein, but it may result from increased palmitate transport across the plasma membrane.

Adenosine transport: Recent advances in the molecular biology of nucleoside transporter proteins

Adenosine is a hydrophilic molecule that requires specialized transport proteins for permeation of cell membranes. Functional studies have identified two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients and inwardly directed concentrative processes driven by the sodium electrochemical gradient. The equilibrative nucleoside transport processes (es, ei) are widely distributed among various cell types, whereas the concentrative nucleoside transport processes (cit, cif, cib, csg, cs) are present primarily in specialized epithelial Using molecular cloning techniques and functional expression of cDNAs in oocytes of Xenopus laevis, we have isolated and characterized cDNAs encoding integral membrane proteins of the four major nucleoside transport processes of rat and human cells (es, ei, cit, cif). The equilibrative and concentrative nucleoside transport processes are mediated by members of two previously unrecognized groups of integral membrane proteins, which we have designated the Equilibrative Nucleoside Transporter (ENT) and the Concentrative Nucleoside Transporter (CNT) protein families. This review summarizes the current state of knowledge of the molecular biology of the ENT and CNT protein families, focusing on the role of these proteins (r/hENT1, r/hENT2, r/hCNT1, r/ hCNT2) in the transport of adenosine.

Recent advances in the molecular biology of nucleoside transporters of mammalian cells

Nucleosides are hydrophilic molecules and require specialized transport proteins for permeation of cell membranes. There are two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients and inwardly directed concentrative processes driven by the sodium electrochemical gradient. The equilibrative nucleoside transport processes (es, ei) are found in most mammalian cell types, whereas the concentrative nucleoside transport processes (cit, cif, cib, csg, cs) are present primarily in specialized epithelia. Using a variety of cloning strategies and functional expression in oocytes of Xenopus laevis, we have isolated and characterized cDNAs encoding the rat and human nucleoside transporter proteins of the four major nucleoside transport processes of mammalian cells (es, ei, cit, cif). From the sequence relationships of these proteins with each other and with sequences in the public data bases, we have concluded that the equilibrative and concentrative nucleoside transport processes are mediated by members of two previously unrecognized groups of integral membrane proteins, which we have designated the equilibrative nucleoside transporter (ENT) and the concentrative nucleoside transporter (CNT) protein families. This review summarizes the current state of knowledge in the molecular biology of the ENT and CNT protein families, focusing on the characteristics of the four human (h) and rat (r) nucleoside transport proteins (r/hENT1, r/hENT2, r/hCNT1, r/hCNT2).Key words: nucleoside transporter, equilibrative, concentrative, ENT, CNT.

Recent advances in the molecular biology of nucleoside transporters of mammalian cells.

Nucleosides are hydrophilic molecules and require specialized transport proteins for permeation of cell membranes. There are two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients and inwardly directed concentrative processes driven by the sodium electrochemical gradient. The equilibrative nucleoside transport processes (es, ei) are found in most mammalian cell types, whereas the concentrative nucleoside transport processes (cit, cif, cib, csg, cs) are present primarily in specialized epithelia. Using a variety of cloning strategies and functional expression in oocytes of Xenopus laevis, we have isolated and characterized cDNAs encoding the rat and human nucleoside transporter proteins of the four major nucleoside transport processes of mammalian cells (es, ei, cit, cif). From the sequence relationships of these proteins with each other and with sequences in the public data bases, we have concluded that the equilibrative and concentrative nucleoside transport processes are mediated by members of two previously unrecognized groups of integral membrane proteins, which we have designated the equilibrative nucleoside transporter (ENT) and the concentrative nucleoside transporter (CNT) protein families. This review summarizes the current state of knowledge in the molecular biology of the ENT and CNT protein families, focusing on the characteristics of the four human (h) and rat (r) nucleoside transport proteins (r/hENT1, r/hENT2, r/hCNT1, r/hCNT2).

Nucleoside transport and its significance for anticancer drug resistance

This article discusses the role of nucleoside transport processes in the cytotoxicity of clinically important anticancer nucleosides. This article summarizes recent advances in the molecular biology of nucleoside transport proteins, review the current state of knowledge of the transportability of therapeutically useful anticancer nucleosides, and provide an overview of the role of nucleoside transport deficiency as a mechanism of resistance to nucleoside cytotoxicity are summarized. Several strategies for utilization of nucleoside transport processes to improve the therapeutic index of anticancer therapies, including the use of nucleoside-transport inhibitors to modulate toxicity of both nucleoside and non-nucleoside antimetabolite drugs are also presented.

Molecular Cloning and Functional Characterization of Nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) Equilibrative Nucleoside Transporter Proteins (rENT1 and rENT2) from Rat Tissues

Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we isolated a cDNA from human placenta encoding the 456-residue glycoprotein hENT1. When expressed in Xenopus oocytes, hENT1 mediated es-type transport activity and was inhibited by coronary vasoactive drugs (dipyridamole and dilazep) that may compete with nucleosides and NBMPR for binding to the substrate binding site. We now report the molecular cloning and functional expression of es and ei homologs of hENT1 from rat tissues; rENT1 (457 residues) was 78% identical to hENT1 in amino acid sequence, and rENT2 (456 residues) was 49-50% identical to rENT1/hENT1 and corresponded to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in truncated form. rENT1 was inhibited by NBMPR (IC50 = 4.6 nM at 10 microM uridine), whereas rENT2 was NBMPR-insensitive (IC50 > 1 microM). Both proteins mediated saturable uridine influx (Km = 0.15 and 0.30 mM, respectively), were broadly selective for purine and pyrimidine nucleosides, including adenosine, and were relatively insensitive to inhibition by dipyridamole and dilazep (IC50 > 1 microM). These observations demonstrate that es and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.

Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1).

We report identification of a new human nucleoside transporter protein by molecular cloning and functional expression of its cDNA. Previously, we used expression selection in Xenopus oocytes to isolate a cDNA from rat jejunal epithelium encoding the pyrimidine-selective Na+-dependent nucleoside transporter rCNT1 (Q.-Q. Huang, S. Y. M. Yao, M. W. L. Ritzel, A. R. P. Paterson, C. E. Cass, and J. D. Young. J. Biol. Chem. 269: 17757-17760, 1994). cDNAs for a human homologue of rCNT1, designated hCNT1, have been isolated from human kidney by hybridization cloning and reverse transcriptase polymerase chain reaction amplification strategies. hCNT1 was 83% identical to rCNT1 in amino acid sequence and exhibited the transport characteristics of an Na+-dependent nucleoside transporter with selectivity for pyrimidine nucleosides and adenosine when expressed in Xenopus oocytes. Deoxyadenosine, which undergoes net renal secretion, and guanosine were poor permeants. hCNT1 did, however, transport 3'-azido-3'-deoxythymidine. This is the first demonstration that members of the CNT family exist in human cells and provides evidence of their involvement in the renal transport of physiological nucleosides and nucleoside drugs. The hCNT1 gene was mapped to chromosome 15q25-26.

Effect of Detergents on Ligand Binding and Translocation Activities of Solubilized/Reconstituted Nucleoside Transporters

Purification of equilibrative nucleoside transporters has been hampered by the functional instability of the detergent-solubilized proteins. A variety of nonionic detergents were compared with octylglucoside (the most commonly used detergent in this regard) for their abilities to solubilize functionally stable nucleoside transporter proteins from Ehrlich cell plasma membranes. Transporter stability was assessed through the binding of the specific probe [3H]nitrobenzylthi- oinosine to freshly solubilized and stored (48 h/6°C) preparations. The most promising detergents were decylmaltoside and cyclohexylbutylmaltoside, both of which, like octylglucoside, solubilized over 70% of the transporters from the membrane. Decylmaltoside- and cyclohexylbutylmaltoside-solubilized transport proteins retained 61 and 83%, respectively, of their [3H]nitrobenzylthioinosine binding activity upon storage, compared to about 30% using octylglucoside. Decylmaltoside was also superior to octylglucoside in its capacity to solubilize the transporter in a state that retained its high affinity for the transport inhibitors dilazep (Ki= 11 nM, vs 75 nMin octylglucoside) and dipyridamole (Ki= 260 nM, vs 12 μMin octylglucoside). Reconstitution studies indicated that both the de- cylmaltoside- and cyclohexylbutylmaltoside-solubilized transporters were capable of mediating the uptake of [3H]uridine. Decylmaltoside was superior to cyclohexylbutylmaltoside, however, in both the enhanced transport activity of the resulting proteoliposomes (Vi= 21 pmol/mg/s vs 13 pmol/mg/s, respectively) and the lower nonmediated uptake observed in the decylmaltoside-derived vesicles (27% of total uptake at 4 min incubation). Nevertheless, cyclohexylbutylmaltoside may be useful in initial solubilization procedures due to its ability to selectively solubilize the nucleoside transporter from the plasma membrane. The rational use of these detergents will enable a more extensive purification of functional nucleoside transporters.

Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells.

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the 'equilibrative sensitive' or 'es' transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximately Km values of 21 microM, 12.5 microM and 15 microM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.