Current Progress on Equilibrative Nucleoside Transporter Function and Inhibitor Design

Equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs) mediate the cellular uptake of nucleosides and nucleobases across the plasma membrane and play important roles in the salvage pathways of nucleotide synthesis. However, information about nucleoside transport systems in the lung alveolar epithelial cells is limited. Therefore, in the present study, we examined the function and expression of nucleoside transporters using primary cultured alveolar type II cells and transdifferentiated type I-like cells. The uptake of uridine, a substrate for ENTs and CNTs, in type II and type I-like cells was time, temperature, and concentration dependent, and was inhibited by other nucleoside transporter substrates such as adenosine. Uridine uptake in both cells was insensitive to nanomolar concentrations of NBMPR, a potent ENT1 inhibitor, while it was inhibited by higher concentrations of NBMPR, suggesting that ENT2, but not ENT1, is involved in uridine uptake in these cells. Additionally, uridine uptake was higher in the presence of Na+ than in the absence of Na + and was partially inhibited by a CNT inhibitor phloridzin in these cells, suggesting that CNT is also involved in uridine uptake. In both cells, the mRNA expression of ENT1, ENT2, CNT2, and CNT3 was observed. Finally, the activity of uridine uptake was considerably higher in type II cells than in type I-like cells. In addition, the mRNA expression of ENT2, CNT2, and CNT3, but not ENT1, was lower in type I-like cells than in type II cells. These findings would help understand the functional roles of equilibrative and concentrative nucleoside transporters in alveolar epithelial cells.

Editorial FocusDueling nucleosides: cross-regulation of extracellular adenosine by guanosine. Focus on "Extracellular guanosine regulates extracellular adenosine levels"George R. DubyakGeorge R. DubyakDepartment of Physiology and Biophysics, Case Western Reserve University, Cleveland, OhioPublished Online:01 Mar 2013https://doi.org/10.1152/ajpcell.00012.2013This is the final version - click for previous versionMoreSectionsPDF (139 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations in addition to serving as components of nucleic acids and central players in intracellular energy metabolism, adenosine- and guanosine-based metabolites play critical but distinct roles in signal transduction processes. ATP has two major functions in intracellular signaling. It serves as the principal nucleotide phosphate donor for the myriad protein kinases that act as molecular switches for most critical intracellular functions. In addition, ATP is the substrate for adenylyl cyclases and inositol lipid kinases, serving, respectively, as the source of the ubiquitous cyclic AMP second messenger and the various inositol lipid second messengers, e.g., 3,4,5-phosphatidylinositol-tris phosphate or PIP3. Similarly, GTP has two major functions in intracellular signaling. It is the allosteric ligand for the activation of GTP-binding regulatory proteins that comprise the other major group of molecular switches for tuning cell function. Like ATP, GTP also serves as a source of second messengers by providing the substrate for the guanylyl cyclases that catalyze formation of the second messenger cyclic GMP.Notably, nature has selected adenosine-containing metabolites for a third discrete role in cellular signal transduction: as extracellular agonists for purinergic receptors (8). Purinergic receptors include the P2X-family of ATP-gated ion channel receptors (seven subtypes), the P2Y-family of G protein-coupled nucleotide receptors (eight subtypes), and the P1-family of G protein-coupled adenosine receptors (four subtypes). Some P2Y receptor subtypes additionally (or exclusively) recognize nonpurinergic uridine nucleotides as agonists. In contrast, despite the "purinergic" sobriquet, no members of these three receptor groups recognize the guanosine-containing compounds (the "other" purines) as agonistic or antagonistic ligands. Thus, despite their critical roles in intracellular signal transduction, guanosine-containing compounds have historically been considered as nonentities in terms of extracellular signaling by nucleotides and nucleosides. However, a new study by Jackson et al. (1) in this issue of American Journal of Physiology-Cell Physiology reveals a hitherto unappreciated role for extracellular guanosine (and guanosine nucleotides) in the regulation of extracellular adenosine clearance/metabolism. Thus, these "poor relative" purines may indeed be involved in the complex world of extracellular purinergic signaling.As for any molecules (e.g., neurotransmitters, hormones, cytokines) that function as agonists for cell surface receptors, the extracellular levels of adenosine nucleotides (ATP, ADP, AMP) and adenosine per se are regulated at the levels of production and clearance (2). Cells release intracellular stores of ATP either by traumatic cell lysis or by highly regulated, nonlytic processes that include exocytosis of ATP-containing vesicles and gating of nucleotide-permeable channels (Fig. 1). Released extracellular ATP is efficiently catabolized to ADP and, ultimately, AMP via CD39-family ecto-nucleoside 5' triphosphate diphosphohydrolylases (eNTPDases) or ecto-nucleotide pyrophosphatases/phosphodiesterases (eNPPs) (10). In turn, extracellular AMP is dephosphorylated to adenosine via the actions of the CD73 ecto-5'nucleotidase and/or GPI-anchored ecto-alkaline phosphatases (APs). Extracellular adenosine has several possible fates: 1) metabolism via adenosine deaminase to inosine; 2) active reaccumulation into cells via Na+-coupled concentrative nucleoside transporters (CNTs); or 3) passive reuptake into cells via equilibrative nucleoside transporters (ENTs) (4). Reuptake via CNTs or ENTs links the disposition of extracellular adenosine to the network of intracellular adenosine metabolism that includes (among multiple other reactions) phosphorylation of adenosine to AMP via adenosine kinase or the conjugation of adenosine with homocysteine via S-adenosylhomocysteine hydrolase (SAH).Fig. 1.Purinergic signaling pathways: modulation of extracellular adenosine clearance by extracellular guanosine. The pathways for release, extracellular metabolism, and downstream receptor-mediated signaling of ATP and its purine metabolites are indicated. ATP can be serially hydrolyzed to ADP and then AMP via the CD39-family nucleoside triphosphate diphosphohydrolases (NTPDs). Alternatively, ATP can be directly metabolized to AMP via ecto-nucleotide pyrophosphatases/phosphodiesterases (ENPPs). AMP hydrolysis by either the CD73 ecto-5'-nucleotidases or ecto-alkaline phosphatases (APs) generates extracellular adenosine. It is important to note that extracellular GTP, GDP, and GMP are also substrates for these various ecto-nucleotidases. Extracellular adenosine (or guanosine) can be directly released from metabolically stressed cells via efflux through equilibrative nucleoside transporters (ENTs). ENTs can also function as reuptake pathways for adenosine, guanosine, and the pyrimidine nucleosides. Alternatively, these nucleosides can be actively accumulated via the concentrative nucleoside transporters (CNTs) that function as Na+/nucleoside cotransporters. In some tissues, extracellular adenosine is readily converted to inosine via extracellular versions of the adenosine deaminase enzymes. Accumulation of extracellular guanosine acts to sustain higher extracellular levels of adenosine by competition for these or other mechanisms for adenosine clearance. Thus, guanosine may potentiate protective purinergic receptor signaling via adenosine receptors despite its lack of direct agonistic on these receptors. GPCR, G protein-coupled receptor.Download figureDownload PowerPointJackson et al. tested whether the levels of extracellular adenosine (added exogenously or generated endogenously) in cultures of rat preglomerular vascular smooth muscle cells (VSMCs) are modulated by the additional presence of either guanosine or guanosine nucleotides. Although high micromolar (10–30 μM) guanosine did not increase accumulation of extracellular adenosine generated from endogenous VSMC sources under basal conditions, the presence of guanosine markedly suppressed the rate at which exogenously added adenosine (in the micromolar range) was cleared. The result was that the extracellular adenosine was sustained at concentrations sufficient to activate adenosine receptors for considerably longer durations in the presence than in the absence of guanosine. When iodoacetate (IAA) treatment was used to induce metabolic stress and injury, the VSMCs generated increased extracellular levels of both adenosine and guanosine from endogenous sources, presumably by ecto-nucleotidases acting on released ATP and GTP. Notably, when the clearance of endogenously generated extracellular guanosine was accelerated by supplementing the culture medium with purine nucleoside phosphorylase (which metabolizes guanosine but not adenosine), the clearance of endogenously generated extracellular adenosine was also accelerated. Exogenous extracellular uridine, thymidine, or cytidine also retarded the rate of adenosine clearance but with reduced efficacies relative to that of guanosine. Moreover, only minor extracellular accumulation of these alternative "competing" nucleosides from endogenous pools was observed in the conditioned media from IAA-treated cells. Thus, based on these observations and data from other experiments, Jackson et al. propose that extracellular guanosine—perhaps uniquely among nonadenosine nucleosides—may indirectly potentiate purinergic signaling via adenosine receptors in metabolically stressed or injured tissues by attenuating the rate and extent of extracellular adenosine clearance (1). Although most experiments utilized the rat preglomerular VSMC model, similar effects of guanosine on extracellular adenosine handling were observed in other rodent and human cell types.What are the likely mechanisms that underlie this heretofore unappreciated action of extracellular guanosine? Jackson et al. used pharmacological approaches to test whether guanosine might compete with adenosine for either the metabolic enzymes (adenosine deaminase, adenosine kinase, SAH) or the nucleoside reuptake transporters (CNTs and ENTs) that participate (directly or indirectly) in the regulation of extracellular adenosine homeostasis (1). However, none of these "players" appeared to be the target of guanosine. As noted by the authors, the ability of the pyrimidine nucleosides to mimic this action of guanosine (albeit with reduced efficacy) suggests that a nucleoside transporter rather than an adenosine-metabolizing enzyme is the more likely mediator. The inability of Na+-deficient medium to alter the action of guanosine effectively ruled out participation of the three CNT-family members. Jackson et al. argue against likely roles for one of more of the four ENT-family members based on pharmacology (insensitivity of the guanosine action to drugs that potently block ENT 1 or ENT2 transport function), subcellular localization (ENT3 predominantly traffics to lysosomes rather than the plasma membrane), or specific allosteric requirements (ENT4 function is highly dependent on an acidic extracellular pH) (1, 4). Unequivocal assessment of these ENTs will require genetic approaches and more extensive pharmacological/cell biological characterizations. However, if more stringent experiments also negate roles for the molecularly identified ENTs, this would suggest the existence of a novel nucleoside transporter(s). This is a highly salient issue given the critical roles for such transporters in the uptake of not only physiological nucleosides but also therapeutically relevant nucleoside analogs.Microdialysis analyses in several types of metabolically stressed tissues have indicated that interstitial concentrations of both guanosine- and adenosine-containing metabolites can rise to micromolar levels (5–7, 9). Localized adenosine receptor stimulation can drive both antiinflammatory signaling and protective ischemic preconditioning responses (3). Thus, this newly identified competitive "duel" between extracellular guanosine and adenosine for nucleoside clearance mechanisms may be highly relevant to understanding and manipulating these protective actions of purinergic signaling in ischemic diseases and tissue trauma.GRANTSThis work was supported in part by National Institutes of Health Grant R01-GM36387.DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the author.AUTHOR CONTRIBUTIONSG.R.D. drafted the manuscript; edited and revised the manuscript; approved the final version of the manuscript.REFERENCES1. Jackson EK , Cheng D , Jackson TC , Verrier JD , Gillespie DG. Extracellular guanosine regulates extracellular adenosine levels. Am J Physiol Cell Physiol (December 12, 2012). doi:10.1152/ajpcell.00212.2012.ISI | Google Scholar2. Lazarowski ER , Boucher RC , Harden TK. Mechanisms of release of nucleotides and integration of their action as P2X- and P2Y-receptor activating molecules. Mol Pharmacol 64: 785–795, 2003.Crossref | PubMed | ISI | Google Scholar3. Linden J. Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection. Annu Rev Pharmacol Toxicol 41: 775–787, 2001.Crossref | PubMed | ISI | Google Scholar4. Loffler M , Morote-Garcia JC , Eltzschig SA , Coe IR , Eltzschig HK. Physiological roles of vascular nucleoside transporters. Arterioscler Thromb Vasc Biol 27: 1004–1013, 2007.Crossref | PubMed | ISI | Google Scholar5. Melani A , Turchi D , Vannucchi MG , Cipriani S , Gianfriddo M , Pedata F. ATP extracellular concentrations are increased in the rat striatum during in vivo ischemia. Neurochem Int 47: 442–448, 2005.Crossref | PubMed | ISI | Google Scholar6. Ninomiya H , Otani H , Lu K , Uchiyama T , Kido M , Imamura H. Complementary role of extracellular ATP and adenosine in ischemic preconditioning in the rat heart. Am J Physiol Heart Circ Physiol 282: H1810–H1820, 2002.Link | ISI | Google Scholar7. Nishiyama A , Majid DS , Taher KA , Miyatake A , Navar LG. Relation between renal interstitial ATP concentrations and autoregulation-mediated changes in renal vascular resistance. Circ Res 86: 656–662, 2000.Crossref | PubMed | ISI | Google Scholar8. Ralevic V , Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413–492, 1998.PubMed | ISI | Google Scholar9. Valen G , Owall A , Takeshima S , Goiny M , Ungerstedt U , Vaage J. Metabolic changes induced by ischemia and cardioplegia: a study employing cardiac microdialysis in pigs. Eur J Cardiothorac Surg 25: 69–75, 2004.Crossref | PubMed | ISI | Google Scholar10. Zimmermann H. Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs Arch Pharmacol 362: 299–309, 2000.Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: G. R. Dubyak, Dept. of Physiology and Biophysics, Case Western Reserve Univ., School of Medicine, 1900 Euclid Ave., Cleveland, OH 44106 (e-mail: george.[email protected]edu). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Cited ByCytokine-Induced Guanylate Binding Protein 1 (GBP1) Release from Human Ovarian Cancer Cells19 February 2020 | Cancers, Vol. 12, No. 2Synthesis and Evaluation of the Antitumor Properties of a Small Collection of Pt II Complexes with 7-Deazaadenosine as Scaffold6 September 2017 | European Journal of Organic Chemistry, Vol. 2017, No. 33Extracellular guanosine regulates extracellular adenosine levelsEdwin K. Jackson, Dongmei Cheng, Travis C. Jackson, Jonathan D. Verrier, and Delbert G. Gillespie1 March 2013 | American Journal of Physiology-Cell Physiology, Vol. 304, No. 5 More from this issue > Volume 304Issue 5March 2013Pages C403-C405 Copyright & PermissionsCopyright © 2013 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.00012.2013PubMed23325409History Published online 1 March 2013 Published in print 1 March 2013 Metrics

Concentrative nucleoside transporters (CNTs) and equilibrative nucleoside transporters (ENTs) are important in physiological and pharmacological activity and disposition of nucleosides and nucleoside drugs. A better understanding of the structural requirements of inhibitors for these transporters will aid in designing therapeutic agents. To define the relative and unified structural requirements of nucleoside analogs for interaction with hCNT1, hCNT2, and hENT1, we applied an array of structure-activity techniques. Unique pharmacophore models for each respective nucleoside transporter were generated. These models reveal that hCNT2 affinity is dominated by hydrogen bonding features, whereas hCNT1 and hENT1 displayed mainly electrostatic and steric features. Hydrogen bond formation over 3'-OH is essential for all nucleoside transporters. Inhibition of nucleoside transporters by a series of uridine and adenosine analogs and a variety of drugs was analyzed by comparative molecular field analysis. Cross-validated r2 (q2) values were 0.65, 0.52, and 0.74 for hCNT1, hCNT2, and hENT1, respectively. The predictive quality of the models was further validated by successful prediction of the inhibition of a set of test compounds. Addition of a hydroxyl group around the 2-position of purine (or 3-position of pyrimidine) may increase inhibition to hCNT2 transporter; addition of hydroxyl group around the 2,7-position of purine (or the 3,5-position of pyrimidine) would increase the inhibition to hENT1 transporter. Utilization of these models should assist the design of high-affinity nucleoside transporter inhibitors and substrates for both anticancer and antiviral therapy.

Permeases of the equilibrative nucleoside transporter family mediate the uptake of nucleosides and/or nucleobases in a diverse array of eukaryotes and transport a host of drugs used for treatment of cancer, heart disease, AIDS, and parasitic infections. To identify residues that play central roles in transport function, we have systematically substituted by site-directed mutagenesis all the charged residues located within predicted transmembrane domains of the Leishmania donovani nucleoside transporter 1.1, LdNT1.1, which transports adenosine and the pyrimidine nucleosides. Substitution of three of these ten residues by uncharged amino acids resulted in loss of >95% transport activity, and we hence designated them "key" residues. These amino acids were Glu94, Lys153, and Arg404 located in transmembrane domains 2, 4, and 9, respectively. In addition, previous studies on the related LdNT2 inosine/guanosine transporter identified the highly conserved Asp389 and Arg393 (equivalent to Asp374 and Arg378 in LdNT1.1) in transmembrane domain 8 as key residues. Among these residues, the mutants in Arg393 (LdNT2) and Arg404 were strongly impaired in trafficking to the plasma membrane, but the other mutants were expressed with high to moderate efficiency at the cell surface, indicating that their mutation impaired transport activity per se. A conservative K153R substitution exhibited a change in substrate specificity, acquiring the ability to transport inosine, a nucleoside that is not a substrate for the wild-type LdNT1.1 permease. These results imply that the Glu94, Lys153, and Asp374 residues may play central roles in the mechanism of substrate translocation in LdNT1.1.

Under several adverse conditions, such as hypoxia or ischaemia, extracellular levels of adenosine are elevated because of increased energy demands and ATP metabolism. Because extracellular adenosine affects metabolism through G-protein-coupled receptors, its regulation is of high adaptive importance. CNT2 (concentrative nucleoside transporter 2) may play physiological roles beyond nucleoside salvage in brain as it does in other tissues. Even though nucleoside transport in brain has mostly been seen as being of equilibrative-type, in the present study, we prove that the rat phaeochromocytoma cell line PC12 shows a concentrative adenosine transport of CNT2-type when cells are differentiated to a neuronal phenotype by treatment with NGF (nerve growth factor). Differentiation of PC12 cells was also associated with the up-regulation of adenosine A1 receptors. Addition of adenosine receptor agonists to cell cultures increased CNT2-related activity by a mechanism consistent with A₁ and A2A receptor activation. The addition of adenosine to the culture medium also induced the phosphorylation of the intracellular regulatory kinase AMPK (AMP-activated protein kinase), with this effect being dependent upon adenosine transport. CNT2-related activity of differentiated PC12 cells was also dramatically down-regulated under hypoxic conditions. Interestingly, the analysis of nucleoside transporter expression after experimental focal ischaemia in rat brain showed that CNT2 expression was down-regulated in the infarcted tissue, with this effect somehow being restricted to other adenosine transporter proteins such as CNT3 and ENT1 (equilibrative nucleoside transporter 1). In summary, CNT2 is likely to modulate extracellular adenosine and cell energy balance in neuronal tissue.

Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides, nucleobases, and a variety of therapeutic drugs in eukaryotes from protozoa to mammals. Despite its importance, the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers. Previous genetic studies on the LdNT1.1 nucleoside transporter from Leishmania donovani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway. The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method. A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional, cysteine-less, parental LdNT1.1. Cysteine replacement at six positions (M(176)C, T(186)C, S(187)C, Q(190)C, V(193)C, and K(194)C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents, denoting their solvent accessibility to the translocation pathway. Adenosine was able to block this thiol modification, implying that access to the domain becomes restricted as a consequence of the substrate binding. Strikingly, the Q(190)C substitution interacted differentially with the substrates adenosine and uridine, suggesting that binding of adenosine but not uridine might directly occlude this position. When superimposed on a helical model, all six mutants clustered along one face of the amphipathic alpha-helix predicted for TMD5, strongly suggesting its involvement in the translocation pathway through LdNT1.1.

Uptake of purine and pyrimidine nucleosides in astrocytes is important for several reasons: (1) uptake of nucleosides contributes to nucleic acid synthesis; (2) astrocytes synthesize AMP, ADP, and ATP from adenosine and GTP from guanosine; and (3) adenosine and guanosine function as neuromodulators, whose effects are partly terminated by cellular uptake. It has previously been shown that adenosine is rapidly accumulated by active uptake in astrocytes (Hertz and Matz, Neurochem Res 14:755-760, 1989), but the ratio between active uptake and metabolism-driven uptake of adenosine is unknown, as are uptake characteristics for guanosine. The present study therefore aims at providing detailed information of nucleoside transport and transporters in primary cultures of mouse astrocytes. Reverse transcription-polymerase chain reaction identified the two equilibrative nucleoside transporters, ENT1 and ENT2, together with the concentrative nucleoside transporter CNT2, whereas CNT3 was absent, and CNT1 expression could not be investigated. Uptake studies of tritiated thymidine, formycin B, guanosine, and adenosine (3-s uptakes at 1-4 degrees C to study diffusional uptake and 1-60-min uptakes at 37 degrees C to study concentrative uptake) demonstrated a fast diffusional uptake of all four nucleosides, a small, Na(+)-independent and probably metabolism-driven uptake of thymidine (consistent with DNA synthesis), larger metabolism-driven uptakes of guanosine (consistent with synthesis of DNA, RNA, and GTP) and especially of adenosine (consistent with rapid nucleotide synthesis), and Na(+)-dependent uptakes of adenosine (consistent with its concentrative uptake) and guanosine, rendering neuromodulator uptake independent of nucleoside metabolism. Astrocytes are accordingly well suited for both intense nucleoside metabolism and metabolism-independent uptake to terminate neuromodulator effects of adenosine and guanosine.

In this study, we have established new syncytiotrophoblast cell lines (TR-TBTs) from the recently developed transgenic rat harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg-rat). Four conditionally immortalized syncytiotrophoblast cell lines (TR-TBT 18d-1 approximately 4) were obtained from pregnant Tg-rats at gestational day 18. These cell lines had a syncytium-like morphology, could be prepared as monolayers, expressed cytokeratins and rat syncytiotrophoblast markers, and exhibited apical or basal GLUT1 localizations and apical GLUT3 localizations. TR-TBTs express large T-antigen and grow well at 33 degrees C with a doubling time of about 30 h. TR-TBTs have processes for the uptake of dehydroepiandrosteron-3-sulfate (DHEAS) and these are predominantly located on the basal side, and this is the first report of an in vitro model of blood placental barrier (BPB) able to incorporate DHEAS. Therefore, TR-TBTs are an appropriate in vitro model for investigating carrier-mediated transport functions at the BPB. Moreover, TR-TBTs express betaine/GABA transporter (GAT-2/BGT-1), concentrative nucleoside transporter 2 (CNT2), equilibrative nucleoside transporter 1 (ENT1), and ENT2 and the expression of these transporters has been reported in blood-brain barrier (BBB). Thus, the expression patterns of nucleoside and neurotransmitter transporters examined are quite similar in both the BPB and BBB.

Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2')- and C(5')-positions on the ribose sugar and is not stimulated by a membrane pH differential. [(3)H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [(3)H]adenosine or [(3)H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.

The gastrointestinal tract is the absorptive organ for nutrients found in foods after digestion. Nucleosides and, to a lesser extent nucleobases, are the late products of nucleoprotein digestion. These metabolites are absorbed by nucleoside (and nucleobase) transporter (NT) proteins. NTs are differentially distributed along the gastrointestinal tract showing also polarized expression in epithelial cells. Concentrative nucleoside transporters (CNTs) are mainly located at the apical side of enterocytes, whereas equilibrative nucleoside transporters (ENTs) facilitate the basolateral efflux of nucleosides and nucleobases to the bloodstream. Moreover, selected nucleotides and the bioactive nucleoside adenosine act directly on intestinal cells modulating purinergic signaling. NT-polarized insertion is tightly regulated. However, not much is known about the modulation of intestinal NT function in humans, probably due to the lack of appropriate cell models retaining CNT functional expression. Thus, the possibility of nutritional regulation of intestinal NTs has been addressed using animal models. Besides the nutrition-related role of NT proteins, orally administered drugs also need to cross the intestinal barrier, this event being a major determinant of drug bioavailability. In this regard, NT proteins might also play a role in pharmacology, thereby allowing the absorption of nucleoside- and nucleobase-derived drugs. The relative broad selectivity of these membrane transporters also suggests clinically relevant drug-drug interactions when using combined therapies. This review focuses on all these physiological and pharmacological aspects of NT protein biology. © 2017 American Physiological Society. Compr Physiol 8:1003-1017, 2018.

Nucleoside transporters in the disposition and targeting of nucleoside analogs in the kidney

Glycine 154 of the equilibrative nucleoside transporter, hENT1, is important for nucleoside transport and for conferring sensitivity to the inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.

Brief exposure of the heart to hypoxia results in less cellular damage after subsequent hypoxia, an effect known as preconditioning (PC). PC has been widely studied but is still not fully understood. Adenosine (Ado), adenosine receptors, and protein kinase C (PKC) have been implicated as integral components of PC. Adenosine (nucleoside) transporters (NTs) facilitate flux of Ado across cell membranes, but their role in PC is unknown. Therefore, we used the murine cardiomyocyte cell line, HL-1, and asked if there was feedback regulation of NTs by Ado, Ado receptors, and PKC following either hypoxic or pharmacological PC. Activation (by specific agonists) of A1 or A3 Ado receptors or PKC resulted in PC in HL-1. The A1 (but not A3) receptor is coupled to PKCepsilon, and activation of PKCepsilon (by specific peptide agonist) resulted in PC. Moreover, PKCepsilon stimulates Ado uptake via the predominant NT in HL-1, mouse equilibrative nucleoside transporter 1 (mENT1). Studies in primary neonatal mouse cardiomyocytes confirmed our observations in HL-1 cells. Hypoxic challenge led to a rapid increase in, and efflux of, intracellular Ado from cells, which was blocked by NT inhibitors (dipyridamole/nitrobenzylthioinosine). Moreover, NT inhibition during hypoxia or PC was highly protective, suggesting that Ado loss contributes to decreased cell viability. Our data suggest that hypoxic challenge causes an efflux of Ado via ENTs, activation of A1 and/or A3 receptors, signaling through PKCepsilon, and activation of ENT1. Since Ado is required for ATP synthesis on reperfusion, this feedback regulation of mENT1 would promote reuptake of Ado.

Functional and molecular characterization of adenosine transport at the rat inner blood–retinal barrier