A nucleoside transporter is functionally linked to ectonucleotidases in rat liver canalicular membrane.

Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.     

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.     

The broadly selective human Na+/nucleoside cotransporter (hCNT3) exhibits novel cation-coupled nucleoside transport characteristics

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. hCNT3, a Na+/nucleoside symporter, transports a broad range of physiological purine and pyrimidine nucleosides as well as anticancer and antiviral nucleoside drugs, and belongs to a different CNT subfamily than hCNT1/2. H+-dependent Escherichia coli NupC and Candida albicans CaCNT are also CNT family members. The present study utilized heterologous expression in Xenopus oocytes to investigate the specificity, mechanism, energetics, and structural basis of hCNT3 cation coupling. hCNT3 exhibited uniquely broad cation interactions with Na+, H+, and Li+ not shared by Na+-coupled hCNT1/2 or H+-coupled NupC/CaCNT. Na+ and H+ activated hCNT3 through mechanisms to increase nucleoside apparent binding affinity. Direct and indirect methods demonstrated cation/nucleoside coupling stoichiometries of 2:1 in the presence of Na+ and both Na+ plus H+, but only 1:1 in the presence of H+ alone, suggesting that hCNT3 possesses two Na+-binding sites, only one of which is shared by H+. The H+-coupled hCNT3 did not transport guanosine or 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, demonstrating that Na+- and H+-bound versions of hCNT3 have significantly different conformations of the nucleoside binding pocket and/or translocation channel. Chimeric studies between hCNT1 and hCNT3 located hCNT3-specific cation interactions to the C-terminal half of hCNT3, setting the stage for site-directed mutagenesis experiments to identify the residues involved.     

Complex regulation of nucleoside transporter expression in epithelial and immune system cells

Nucleoside transporters have a variety of functions in the cell, such as the provision of substrates for nucleic acid synthesis and the modulation of purine receptors by determining agonist availability. They also transport a wide range of nucleoside-derived antiviral and anticancer drugs. Most mammalian cells co-express several nucleoside transporter isoforms at the plasma membrane, which are differentially regulated. This paper reviews studies on nucleoside transporter regulation, which has been extensively characterized in the laboratory in several model systems: the hepatocyte, an epithelial cell type, and immune system cells, in particular B cells, which are non-polarized and highly specialized. The hepatocyte co-expresses at least two Na+-dependent nucleoside transporters, CNT1 and CNT2, which are up-regulated during cell proliferation but may undergo selective loss in certain experimental models of hepatocarcinomas. This feature is consistent with evidence that CNT expression also depends on the differentiation status of the hepatocyte. Moreover, substrate availability also modulates CNT expression in epithelial cells, as reported for hepatocytes and jejunum epithelia from rats fed nucleotide-deprived diets. In human B cell lines, CNT and ENT transporters are co-expressed but differentially regulated after B cell activation triggered by cytokines or phorbol esters, as described for murine bone marrow macrophages induced either to activate or to proliferate. The complex regulation of the expression and activity of nucleoside transporters hints at their relevance in cell physiology.     

Complex regulation of nucleoside transporter expression in epithelial and immune system cells

Nucleoside transporters have a variety of functions in the cell, such as the provision of substrates for nucleic acid synthesis and the modulation of purine receptors by determining agonist availability. They also transport a wide range of nucleoside-derived antiviral and anticancer drugs. Most mammalian cells coexpress several nucleoside transporter isoforms at the plasma membrane, which are differentially regulated. This paper reviews studies on nucleoside transporter regulation, which has been extensively characterized in the laboratory in several model systems: the hepatocyte, an epithelial cell type, and immune system cells, in particular B cells, which are non-polarized and highly specialized. The hepatocyte co-expresses at least two Na+-dependent nucleoside transporters, CNT1 and CNT2, which are up-regulated during cell proliferation but may undergo selective loss in certain experimental models of hepatocarcinomas. This feature is consistent with evidence that CNT expression also depends on the differentiation status of the hepatocyte. Moreover, substrate availability also modulates CNT expression in epithelial cells, as reported for hepatocytes and jejunum epithelia from rats fed nucleotide-deprived diets. In human B cell lines, CNT and ENT transporters are co-expressed but differentially regulated after B cell activation triggered by cytokines or phorbol esters, as described for murine bone marrow macrophages induced either to activate or to proliferate. The complex regulation of the expression and activity of nucleoside transporters hints at their relevance in cell physiology.     

Nucleoside transport in human colonic epithelial cell lines: evidence for two Na+-independent transport systems in T84 and Caco-2 cells.

RT-PCR of RNA isolated from monolayers of the human colonic epithelial cell lines T84 and Caco-2 demonstrated the presence of mRNA for the two cloned Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2, but not for the cloned Na+-dependent concentrative nucleoside transporters, CNT1 and CNT2. Uptake of [3H]uridine by cell monolayers in balanced Na+-containing and Na+-free media confirmed the presence of only Na+-independent nucleoside transport mechanisms. This uptake was decreased by 70-75% in the presence of 1 microM nitrobenzylthioinosine, a concentration that completely inhibits ENT1, and was completely blocked by the addition of 10 microM dipyridamole, a concentration that inhibits both ENT1 and ENT2. These findings indicate the presence in T84 and Caco-2 cells of two functional Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2.     

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

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

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.     

Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2

Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.     

Na(+)-dependent, active and Na(+)-independent, facilitated transport of formycin B in mouse spleen lymphocytes

Na(+)-dependent, active and Na(+)-independent facilitated nucleoside transport were characterized in mouse spleen cells using rapid kinetic techniques and formycin B, a metabolically inert analog of inosine, as substrate. The Michaelis-Menten constants for formycin B transport by the two transporters were about 30 and 400 microM, respectively. The first-order rate constant for Na(+)-dependent transport was about 4-times higher than that for facilitated formycin B transport. The Na(+)-dependent carrier is specific for uridine and purine nucleosides and accumulates formycin B concentratively in an unmodified form. Concentrative accumulation was inhibited by ATP depletion and gramicidin and ouabain treatment of the cells. Our data indicate a single Na(+)-binding site on the Na(+)-dependent nucleoside carrier and a Michaelis-Menten constant for Na+ of about 10 mM. This transporter was not significantly inhibited by dipyridamole and nitrobenzylthioinosine, inhibitors of the facilitated transporter. The Na(+)-independent, facilitated nucleoside transporter of spleen cells exhibits properties comparable to those of the carriers present in mammalian cells in general. The B lymphocytes remaining after depletion of spleen cell populations of T lymphocytes by incubation with a combination of T-cell specific monoclonal antibodies plus complement exhibited about the same activities of active and facilitated nucleoside transport as the original suspension.     

Na+-dependent and -independent transport of uridine and its phosphorylation in mouse spleen cells

Rapid kinetic techniques were used to study the transport and salvage of uridine and other nucleosides in mouse spleen cells. Spleen cells express two nucleoside transport systems: (1) the non-concentrative, symmetrical, Na+-independent transporter with broad substrate specificity, which has been found in all mammalian cells and is sensitive to inhibition by dipyridamole and nitrobenzylthioinosine; and (2) a Na+-dependent nucleoside transport, which is specific for uridine and purine nucleosides and resistant to inhibition by dipyridamole and nitrobenzylthioinosine. The kinetic properties of the two transporters were determined by measuring uridine influx in ATP-depleted cells and dipyridamole-treated cells, respectively. The Michaelis-Menten constants for Na+-independent and -dependent transport were about 40 and 200 microM, respectively, but the first-order rate constants were about the same for both transport systems. Nitrobenzylthioinosine-sensitivity of the facilitated nucleoside transporter correlated with the presence of about 10,000 high-affinity (Kd = 0.6 nM) nitrobenzylthioinosine-binding sites per cell. The turnover number of the nitrobenzylthioinosine-sensitive nucleoside transporter was comparable to that of mouse P388 leukemia cells. The activation energy of this transporter was 20 kcal/mol. Entry of uridine via either of the transport routes was rapidly followed by its phosphorylation and conversion to UTP. The Michaelis-Menten constant for the in situ phosphorylation of uridine was about 50 microM and the first-order rate constants for phosphorylation and transport were about the same. The spleen cells also efficiently salvaged adenosine, adenine, and hypoxanthine, but not thymidine.     

Recent advances in studies on biochemical and structural properties of equilibrative and concentrative nucleoside transporters

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.     

Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket

SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.     

A two sodium ion/D-glucose symport mechanism: membrane potential effects on phlorizin binding

Apical membrane vesicles isolated from a continuous renal cell line, LLC-PK1, catalyze electrogenic Na+-stimulated hexose transport and Na+-dependent binding of 3H-labeled 1-[2-(beta-D-glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone [( 3H]phlorizin), a competitive ligand of this transport system. Phlorizin was not itself transported across the membrane and thus can serve as a probe of the binding step. The stoichiometry of Na+-dependent phlorizin binding in vesicles was 1:1, whereas Na+/hexose cotransport in vesicles exhibited a 2:1 stoichiometry. Na+ increased the affinity of phlorizin binding without affecting the total number of binding sites. An increased number of Na+-dependent phlorizin binding sites was observed under conditions of interior-negative membrane potential. These results are consistent with a model of the Na+/glucose cotransport cycle in which the unloaded transporter is negatively charged and its orientation influenced by membrane potential. Glucose and one sodium ion interact with the transporter, resulting in an uncharged complex. Binding of a second sodium ion triggers translocation of glucose and both sodium ions via formation of a loaded carrier complex bearing a single positive charge.     

Arabidopsis ANTR1 is a thylakoid Na+-dependent phosphate transporter: functional characterization in Escherichia coli

In this study, the putative anion transporter 1 (ANTR1) from Arabidopsis thaliana was shown to be localized to the chloroplast thylakoid membrane by Western blotting with two different peptide-specific antibodies. ANTR1 is homologous to the type I of mammalian Na+-dependent inorganic phosphate (Pi) transporters. The function of ANTR1 as a Na+-dependent Pi transporter was demonstrated by heterologous expression and uptake of radioactive Pi into Escherichia coli cells. The expression of ANTR1 conferred increased growth rates to the transformed cells and stimulated Pi uptake in a pH- and Na+-dependent manner as compared with the control cells. Among various tested effectors, Pi was the preferred substrate. Although it competed with the uptake of Pi, glutamate was not transported by ANTR1 into E. coli. In relation to its function as a Pi transporter, several physiological roles for ANTR1 in the thylakoid membrane are proposed, such as export of Pi produced during nucleotide metabolism in the thylakoid lumen back to the chloroplast stroma and balance of the trans-thylakoid H+ electrochemical gradient storage.