Vol. 52 No. 4/2005, 749–758 Review

Nucleoside transporters (NT) facilitate the movement of nucleosides and nucleobases across cell membranes. NT-mediated transport is vital for the synthesis of nucleic acids in cells that lack de novo purine synthesis. Some nucleosides display biological activity and act as signalling molecules. For example, adenosine exerts a potent action on many physiological processes including vasodilatation, hormone and neurotransmitter release, platelet aggregation, and lipolysis. Therefore, carrier-mediated transport of this nucleoside plays an important role in modulating cell function, because the efficiency of the transport processes determines adenosine availability to its receptors or to metabolizing enzymes. Nucleoside transporters are also key elements in anticancer and antiviral therapy with the use of nucleoside analogues. Mammalian cells possess two major nucleoside transporter families: equilibrative (ENT) and concentrative (CNT) Na(+)-dependent ones. This review characterizes gene loci, substrate specificity, tissue distribution, membrane topology and structure of ENT and CNT proteins. Regulation of nucleoside transporters by various factors is also presented.

The effect of adenosine is mediated by specific cell-surface receptors A1, A2a, A2b and A3 (Olah & Stiles, 2000).The affinity of these receptors for adenosine varies.Thus their activation depends on adenosine concentration.Metabolism and transport across the plasma membrane are the main factors influencing the adenosine level.Most nucleosides and their analogues such as anticancer and antiviral nucleoside drugs are hydrophilic molecules and require specific transport proteins to be transported in and out of the cell.Two major classes of nucleoside transport systems have been described in mammalian cells, the equilibrative transport system and the concentrative one (Baldwin et al., 2004;Gray et al., 2004;Kong et al., 2004).Nucleosides, nucleobases and their analogues are also substrates for other car-750 2005 M. Podgorska and others rier proteins such as organic anion and cation transporters and ABC transporter proteins that mostly function as efflux transporters (Pastor-Anglada et al., 2005).In our review we focus on equilibrative (ENT) and concentrative (CNT) nucleoside transporters.
The equilibrative transport system mediates nucleoside transport in both directions depending on the nucleoside concentration gradient across the plasma membrane (Fig. 1).This class of nucleoside transport system is subdivided in two types (es and ei) based on their sensitivity to nitrobenzylthioinosine (NBTI).The es transport system is highly sensitive to inhibition by NBTI (K i = 0.1 to 10 nM).In contrast, the ei transport system is resistant to NBTI up to 1 µM (Griffith & Jarvis, 1996;Kiss et al., 2000;Leung et al., 2001).Currently four transporters (ENT1, ENT2, ENT3 and ENT4) belonging to the equilibrative transport system have been identified and cloned (Table 1).ENT1 and ENT3 are susceptible to NBTI, whereas this compound does not inhibit ENT2.ENT4 has been identified only recently and its transporting characteristics have not been fully determined.These four members of the equilibrative transport system are widely distributed among various cell types, but the number of molecules of each nucleoside transporter depends on the cell and tissue type.All transporters are able to transport adenosine, but they have different capacities to transport other nucleosides and nucleobases (Baldwin et al., 2004;Kong et al., 2004).
The concentrative transport system is Na +dependent and the movement of the nucleoside regardless of its concentration gradient is coupled to that of the sodium ion (Fig. 1).Six functionally different concentrative nucleoside transport activities have been described in human cells and they have been named in two different ways (Table 1).The first nomenclature is numerical and follows the order of discovery (N1-N6), and the second one is based on the substrate specificity (cit, cif, cib, cit-like, cs, csg).The N1-cif activity is purine-selective, the N2-cit transport system accepts pyrimidine nucleosides, the N3-cib system transports both purine and pyrimidine nucleosides, the N4-cit-like system is selective for pyrimidine nucleosides but also accepts adenosine and guanosine, the N5-cs system transports adenosine and its analogues, and the N6-csg activity is guanosine-selective (Thorn & Jarvis, 1996;Flanagan & Meckling-Gill, 1997;Cass et al., 1998;Baldwin et al., 1999;Cabrita et al., 2002).To date three different proteins responsible for the cit (CNT1), cif (CNT2), and cib (CNT3) activities have been identified (Gray et al., 2004;Kong et al., 2004).Proteins that demonstrate the N4-cit-like, N5-cs or N6-csg activities have not been identified yet.
Human ENT1 protein consists of 456 amino acids and its sequence displays about 78% identity to the rat (rENT1) and mouse (mENT1) homologues.Splice variants of ENT1 have been found only in the mouse.The mRNA for the shorter form of mENT1 termed mENT1.2(458 aa) is produced by alternative splicing at the end of exon 7. Mouse ENT1.2 protein was shown to be commonly co-expressed with mENT1.1 (460 aa) and the highest level was found in the liver, heart and testis (Handa et al., 2001).The two forms of mENT1 protein appear to be functionally identical, although mENT1.2lacks the potential casein kinase II phosphorylation site.
Both rat and human ENT1 proteins display broad substrate specificity for pyrimidine and purine nucleosides, but are unable to transport the pyrimidine base uracil (Yao et al., 1997).Human ENT1 has been reported to transport the nucleoside analogues used in the treatment of cancers, including CdA and ara-C.On the other hand, hENT1 only weakly transports the antiviral nucleoside analogues such as ddC and does not accept AZT (Griffiths et al., 1997;Mackey et al., 1998).Among the ENT1 proteins expressed in various species there are some differences in susceptibility to inhibition by specific compounds.Human ENT1, which is sensitive to NBTI, is also in- Properties of nucleoside transporters hibited by the coronary vasodilators dipyridamole, dilazep, and draflazine.In contrast, rat ENT1 that is also inhibited by NBTI appears to be resistant to those compounds (Yao et al., 1997;Sundaram et al., 1998;Kiss et al., 2000;Ward et al., 2000;Baldwin et al., 2004).

ENT2
Human ENT2 (hENT2) protein that is responsible for the ei type nucleoside transport is encoded by a gene localized at position 13q on chromosome 11 (Table 2).The mRNA for ENT2 was reported to be present in several tissues including heart, kidney, brain, placenta, thymus, pancreas, intestine, and prostate, but the highest expression level was observed in skeletal muscle (Griffiths et al., 1997;Crawford et al., 1998;Lum et al., 2000;Pennycooke et al., 2001).
Human ENT2 consists of 456 amino acids and their sequence displays 88% identity to mouse (mENT2) and rat (rENT2) homologues.In human, beside the 456-amino acid ENT2 protein, there exist at least two shorter forms of ENT2 generated from mRNA splice variants.The 326 aa protein termed hHNP36 lacks the first three transmembrane domains and is inactive as a nucleoside transporter.Inactive is also the second splice variant, a 301-amino acid protein named hENT2A that lacks the C-terminal domain (Crawford et al., 1998).
The ENT2 protein accepts a broad range of substrates, including purine and pyrimidine nucleosides and nucleobases.It has been postulated that human ENT2 plays a role in the efflux and reuptake of inosine and hypoxanthine generated from adenosine during and a�er strenuous physical exercise (Griffiths et al., 1997).ENT2 (both rat and human) is much less susceptible to inhibition by NBTI and the coronary vasodilators dipyridamole and draflazine than ENT1 (Yao et al., 1997;Sundaram et al., 1998;Ward et al., 2000;Baldwin et al., 2004).In contrast to hENT1 protein, hENT2 can transport the antiviral nucleoside analogue AZT and exhibits a greater affinity for ddC (Griffiths et al., 1997;Crawford et al., 1998;Yao et al., 2001).

ENT3
The gene encoding the human ENT3 (hENT3) protein is located at position q22.1 on chromosome  et al., 2001;Baldwin et al., 2004;2005).Human ENT3 is a 475-residue protein displaying 73% identity to the mouse homologue (mENT3) (Baldwin et al., 2004;2005;Kong et al., 2004).ENT3 has a characteristic, long (51 aa) hydrophilic N-terminal region preceding the first transmembrane (TM1) domain.The N-terminal region of ENT3 consists of two dileucine motifs characteristic for endosomal, lysosomal targeting motifs.This architectural design distinguishes the ENT3 protein from other members of the equilibrative transporters family.Indeed, it was demonstrated that hENT3 protein is predominantly localized intracellularly and that mutation of the dileucine motif to alanine triggers the relocation of ENT3 protein to the cell surface (Baldwin et al., 2004;2005).
In comparison with ENT1, the ENT3 protein is much less susceptible to inhibition by NBTI and coronary vasodilatory drugs like dipyridamole and dilazep.Human ENT3 demonstrates a broad selectivity for nucleosides, but does not transport hypoxanthine.The hENT3 protein facilitates transport of several adenosine analogues like CdA and cordycepin (3'-deoxyadenosine).The antiviral purine and pyrimidine nucleoside analogues ddI, ddC and in particular AZT are efficiently transported by this protein (Baldwin et al., 2004;2005).

ENT4
The gene encoding the human ENT4 protein is located on chromosome 7 at position p22.1.The mRNA for hENT4 was detected in several human tissues (Table 2).Human ENT4 is a 530-residue protein displaying 86% identity to its 528-residue mouse homologue (Baldwin et al., 2004).The substrate specificity of hENT4 has not been determined in detail, but among the ENT proteins hENT4 has the lowest affinity for adenosine (Kong et al., 2004).

CNT1
The mRNA for rCNT1 was detected in several tissues including jejunum, kidney, liver, small intestine, and in many regions of the brain (Huang et al., 1993;Anderson et al., 1996;Pennycooke et al., 2001).The gene encoding human CNT1 protein is located at position q25-26 on chromosome 15 (Table 2).Human CNT1 is a 650-amino acid protein displaying 83% identity to its rat 648-residue homologue (Ritzel et al., 1997).Rat CNT1 protein appears to be a typical cit type transporter (N2 subtype) dependent upon sodium and preferentially transporting pyrimidine nucleosides (Huang et al., 1994;Ritzel et al., 1997;Wang et al., 1997).This protein also binds adenosine, but this purine is weakly transported.Rat CNT1 transports several antiviral nucleoside analogues, including AZT and ddC.Nucleoside analogues such as ara-C and dFdC that are used in anticancer therapy are also substrates for hCNT1 (Yao et al., 1997).

CNT2
The protein responsible for the cif type process (N1) that was identified by molecular cloning in the rat liver was named sodium-dependent purine nucleoside transporter (SPNT).The concentrative transporter cloned later from rat intestine was called rCNT2 (Huang et al., 1993;Wang et al., 1997).These two transporters are almost identical proteins that differ in a conservative substitution of glycine for alanine and valine for isoleucine at positions 419 and 522, respectively (Che et al., 1995;Yao et al., 1997;Cass et al., 1998).

CNT3
The gene for human CNT3 protein is located on chromosome 9 at position q22.The mRNA for hCNT3 has been detected in pancreas, trachea, bone marrow, and mammary gland (Table 2).This protein exhibits a cib activity (N3 subtype) and transports both purine and pyrimidine nucleosides in a sodium Properties of nucleoside transporters dependent manner (Ritzel et al., 2001).The hCNT3 protein exhibits a 2:1 stoichiometry for cotransport of Na + and nucleoside (Ritzel et al., 2001) in contrast to the 1:1 ratio employed by CNT1 and CNT2 (Plagemann & Aran, 1990).The CNT3 protein facilitates transport of several purine and pyrimidine analogues such as CdA, dFdC and FdU that are used in anticancer therapy (Ritzel et al., 2001;Zhang et al., 2003;Badagnani et al., 2005).

TOPOLOGY AND FUNCTION OF THE ENT AND CNT PROTEINS
Each member of the ENT family consists of 11 hydrophobic transmembrane (TM) α-helices arranged in the plasma membrane by such way that the N-terminus is cytoplasmic while the C-terminus is located in the extracellular space (Sundaram et al., 2001a).These transporters are pos�ranslationally modified by glycosylation.The sites of glycosylation in hENT1 and hENT2 are located in the large extracellular loop between TM helices 1 and 2 (Fig. 2).Human ENT1 contains one such site, whereas hENT2 has two (Griffiths et al., 1997;Vickers et al., 1999;Sundaram et al., 2001a).Experiments with cells grown in a medium containing a drug that prevents glycosylation (tunicamycin) showed that glycosylation of the ENT1 protein is not essential for its es activity (Hogue et al., 1990).However, a study on a glycosylation-defective mutant of hENT1 has demonstrated that glycosylation of hENT1 modulates its sensitivity to inhibition by NBTI and coronary vasodilators dipyridamole and dilazep (Vickers et al., 1999).In contrast, N-glycosylation of hENT2 has no effect on its nucleoside transport kinetics, but is required for efficient targeting of this carrier protein to the plasma membrane (Ward et al., 2003).Human and mouse ENT3 are also glycosylated within the 1-2 TM loop, whereas the exact location of the glyco-sylation site in the C-terminal tail of ENT4 remains unknown.Another feature of the mammalian ENT proteins is a large hydrophilic loop between helices 6 and 7 at the cytoplasmic side (Baldwin et al., 2004).
The structural determinants for substrate recognition and discrimination by ENT have been recently uncovered based on experiments with recombinant proteins and site-directed mutagenesis.The results of these experiments indicated that the transmembrane region of ENT2 encompassing helices 1-6 is responsible for transport of 3'-deoxynucleosides (Yao et al., 2001), while the TM helices 5-6 of this transporter appear to recognize nucleobases (Yao et al., 2002).Mutation of glycine to serine at position 154 in hENT1 resulted in a loss of its ability to bind NBTI and decreased the affinity for cytidine and adenosine (Hyde et al., 2001;SenGupta & Unadkat, 2004).Another glycine residue at position 184 in hENT1 was shown to be important for targeting of the transporter to the plasma membrane or/and for the correct folding (SenGupta et al., 2002).
Work on chimeric rat and human ENT proteins indicated that TM domains 3-6 contain residues responsible for the sensitivity to coronary vasodilators and to NBTI (Sundaram et al., 1998;2001b).The amino acid at position 33 in hENT1 (Met) and hENT2 (Ile) was shown to be responsible for the sensitivity of these transporters to coronary vasodilators such as dilazep, draflazine, and dipyridamole (Visser et al., 2002).
The topological model of hCNT proteins consists of 13 TM helices with the N-terminal region located at the cytoplasmic side (Wang & Giacomini, 1997;Ritzel et al., 1998;Hamilton et al., 2001).In hCNT the C-terminal hydrophilic part positioned in the extracellular space is glycosylated (Fig. 2) (Hamilton et al., 2001).Experiments on chimeras constructed from rat CNT1 and CNT2 proteins indicated that region encompassing TM 7  et al., 1999) and hCNT1 (Loewen et al., 1999).and 9 is responsible for substrate selectivity (Wang & Giacomini, 1997;Hamilton et al., 2001).Those studies showed that replacing four residues (Ser 319 , Gln 320 , Ser 353 , Leu 354 ) in the TM 7-9 region of hCNT1 to the corresponding residues in hCNT2 (Gly 313 , Met 314 , Thr 347 , Val 348 ) resulted in a change of the substrate specificity of the transporter from the cit (pyrimidine specific) to the cif (purine specific) type.Further work indicated that even a single mutation of any of these residues changed the transporter specificity and/or affinity (Loewen et al., 1999).Mutation of serine 319 to glycine in TM7 of hCNT1 produced a transporter able to transport both purines and pyrimidines (cib type), whereas additional mutation of glutamine 320 to methionine increased the activity of the transporter (Loewen et al., 1999).Mutational studies performed on rat CNT1 yielded similar results (Wang & Giacomini, 1999).Those studies indicate that serine 319/318 (human/rat) is responsible for pyrimidine selectivity of CNT1, whereas the adjacent glutamine (320 and 319 in human and rat, respectively) is important for the modulation of the apparent affinity for nucleosides.

REGULATION OF NUCLEOSIDE TRANSPORTERS
Our understanding of the mechanisms that regulate the nucleoside transporters is relatively weak comparing to knowledge on their structure and kinetics.Data gathered so far indicate that the expression level of nucleoside transporters depends on the kind of tissue and its physiological state.Down-regulation and recycling is a relatively well-described phenomenon among the regulatory mechanisms of nucleoside transporters.A study performed on adrenal chromaffin cells, a cellular model with a high similarity to neuronal cells, showed that NBTI binding caused quick internalization of the transporter protein.As a result, 50-60% of the transporter protein was destroyed, but the remaining 40-50% returned to the plasma membrane (Torres et al., 1992).Other studies demonstrated the presence of a cytoplasmic pool of nucleoside transporters in mammalian reticulocytes (Blostein & Grafova, 1987;Liang & Johnstone, 1992).
A study performed on starved rats has demonstrated that food supply modulates the expression level of CNT1 in rat small intestine.It was showed that nucleotide-deficient diets increased the amount of CNT1 protein in jejunum brush border membranes.On the other hand, nucleotide-supplemented diet increased the CNT1 level in the liver but decreased it in the jejunum.These findings suggest that the supply of nucleotides modulates CNT1 expression in a tissue-specific manner (Lopez-Navarro et al., 1996;Mercader et al., 1996;Patil & Unadkat, 1997;Valdes et al., 2000).
Hormonal regulation of nucleoside transport activity has been reported in several tissues and cell types.Exposition of cultured chromaffin cells to T3 resulted in an increase in the number of nucleoside transporters and stimulation of adenosine transport (Fideu & Miras-Portugal, 1992).In thyroidectomized rats the NBTI-sensitive adenosine transport was shown to be reduced by 45% in brain stem synaptosomal preparations (Fideu et al., 1994).A study on rat liver documented up-regulation of nucleoside transport by glucagon through a mechanism involving membrane hyperpolarization (Gomez-Angelats et al., 1996).Insulin was also able to stimulate Na + -dependent uridine uptake by a process consistent with de novo synthesis of the carrier protein (Gomez-Angelats et al., 1996;Pastor-Anglada et al., 1998).The important role of insulin in the regulation of nucleoside transporters supports the multiple observations from experimental and human diabetes.Decreased nucleoside uptake was reported in hippocampal slices from diabetic rats (Morrison et al., 1992;Cassar et al., 1998).It was also reported that the NBTI-sensitive adenosine transport is reduced in endothelial cells isolated from human diabetic umbilical vein (Sobrevia et al., 1994;Vasquez et al., 2004).On the other hand, in human cells isolated from diabetic umbilical artery smooth muscle adenosine transport was significantly raised (Aguayo et al., 2001).A study performed on rats showed that the mRNA levels of rENT1, rENT2, rCNT1, and CNT2 were significantly altered in diabetic heart, liver, and kidney (Pawelczyk et al., 2003).Experiments performed on cultured rat T and B lymphocytes demonstrated that nucleoside transporters expression levels were independently and differentially regulated by glucose and insulin (Sakowicz et al., 2004;2005).It appears that in rat lymphocytes the expression level of rENT2 and rCNT2 highly depends on insulin but is not affected by changes in extracellular glucose level, whereas the expression level of rENT1 is sensitive to extracellular glucose level but not to insulin.Experiments conducted on rat B lymphocytes have showed that insulin utilizes different signalling pathways to regulate expression of CNT2 and ENT2 transporters (Sakowicz et al., 2005).It was demonstrated that insulin up-regulated the level of ENT2 by signalling through PI3K with no involvement of MAK kinases.On the other hand, insulininduced suppression of CNT2 expression was totally blocked by an inhibitor of MEK-1 (PD98059), indicating that insulin controls CNT2 level by signalling through the MAPK pathway.An elevated level of glucose suppressed expression of ENT1 transporter in rat B lymphocytes by signalling through the MAP kinase pathway (Sakowicz et al., 2005).The ability of elevated glucose to regulate nucleoside transporters by signalling through the MAPK pathway was also reported for human umbilical vein endothelial cells Properties of nucleoside transporters and human aortic smooth muscle cells (Montecinos et al., 2000;Leung et al., 2001).In cultured human aortic smooth muscle cells, an increase of extracellular glucose concentration induced expression of ENT1 (Leung et al., 2001;2005).On the other hand, in human umbilical vein endothelium (HUVEC) the expression level of ENT1 and its transport activity were shown to be reduced by elevated level of glucose (Aguayo et al., 2005).In HUVEC, elevated glucose level affected adenosine transport by a mechanism that involved endothelial NO synthase, PKC and MAP kinases (Montecinos et al., 2000;Aguayo et al., 2005).An involvement of P 2Y2 purinoceptors in the effect of elevated glucose on the NBTI-sensitive adenosine transport in HUVEC was also reported (Parodi et al., 2002).A modulatory role of ATP on the NBTI-sensitive nucleoside transporter in bovine chromaffin cells was also described (Casillas et al., 1993;Delicado et al., 1994).
The data gathered so far indicate that the functioning of nucleoside transporters depends also on cell cycle progression and cell differentiation.Studies performed on LA-N-2 human neuroblastoma cells documented an increased expression of NBTI-insensitive equilibrative nucleoside transporter and enhanced uptake of formycin B during cell differentiation (Jones et al., 1994).Studies conducted on hepatocytes have indicated that hepatocarcinogenesis leads to a selective loss of the concentrative nucleoside transporter CNT2 and an increased expression of nucleoside transporter ENT2 (Plagemann & Wohlhueter, 1985;del Santo et al., 1998;Dragan et al., 2000;Pastor-Anglada et al., 2001).On the other hand, synchronized hepatoma cell line FAO that had been induced to proliferate displayed an increase in ENT2, CNT1 and CNT2 mRNA levels just before the peak of thymidine incorporation into DNA.In those cells the mRNA level of ENT1 did not change during proliferation.This suggests that ENT2, CNT1 and CNT2 expression appears to be cell cycle-dependent, but ENT1 is constitutively expressed (del Santo et al., 1998;Valdes et al., 2002).It has been demonstrated that in rat during liver regeneration a�er partial hepatectomy, the activity of sodium-dependent transporters increase concurrently with elevated mRNA and protein levels of CNT1 and CNT2 (Ruiz-Montasell et al., 1993;Felipe et al., 1997;Pastor-Anglada et al., 1998;del Santo et al., 1998).An involvement of growth factors in the regulation of expression of nucleoside transporters was demonstrated in murine bone marrow-derived macrophages.These cells express both the equilibrative (ENT1, ENT2) and concentrative (CNT1, CNT2) nucleoside transporters.A study with the use of isoform-specific probes and antibodies showed that M-CSF-induction of cultured macrophages to proliferate specifically up-regulated only ENT1 at both the mRNA and protein levels.In contrast, exposure of these cells to INF-γ increased the expression of CNT1 and CNT2 by a STAT1-independent pathway.INF-γ also down-regulated the M-CSF-induced expression of ENT1 (Soler et al., 2001a;2003).Exposition of rat foetal hepatocytes to growth hormones such as dexamethasone or T3 induced selective upregulation of CNT2 protein and mRNA level and suppressed ENT1 and ENT2 expression (del Santo et al., 2001).Studies performed on human B cell lines have shown that differentiation factors such as phorbol esters (PMA) and LPS up-regulate concentrative transport systems, but down-regulate the equilibrative transporters in a PKC-dependent manner (Soler et al., 1998).PMA and LPS induce also expression of TNF-α, but TNF-α alone alters the expression level of equilibrative nucleoside transporters in a PKC-independent manner.On the other hand, it has been demonstrated that activation of concentrative transporters by TNF-α is a process dependent on PKC (Soler et al., 1998;2001b).Another player involved in PMA-induced alteration of nucleoside transporters is NO, which is required in the regulation of the concentrative transporters but not of the equilibrative ones (Soler et al., 2000).

CONCLUSIONS
Over the past two decades important advances in the understanding of nucleoside transporter functioning have been achieved.Identification and molecular cloning of the ENT and CNT families from mammals and protozoan parasites have provided detailed information about the structure, function, regulation, and tissue and cellular localization.Structure-function analyses of various nucleoside transporter chimeras and mutants revealed important elements involved in substrate and inhibitor recognition and binding.However, the mechanisms that regulate nucleoside transporters in various tissues and cell types are just beginning to be understood.Because of the ability of these transporters to handle nucleoside analogues used in treatment of patients with cancer and viral diseases, the ongoing research allow the designing of more specifically targeted new compounds or improving the existing drugs.New nucleoside-based drugs are welcome not only in the treatment of cancer and viral diseases, but also in cardiovascular disorders and parasitic infections.