Nucleoside transporter expression and function in cultured mouse astrocytes

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.

Uptake, Transport and Metabolism of Exogenous Nucleosides in Intestinal Epithelial Cell Cultures

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.

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

Na+-dependent nucleoside transporters mediate the intracellular uptake of purine and pyrimidine nucleosides. The N1, N2, and N3 Na+-nucleoside transporters differ in substrate selectivity. N1 is purine-selective, N2 is pyrimidine-selective, and N3 is broadly selective. Recently, we created a chimeric transporter, T8, from the cloned rat N1 and N2 transporters. Whereas most chimeric proteins exhibit the characteristics of one of the two parent proteins, limited studies suggested that T8 possesses either a combined substrate selectivity of N1 and N2 or the selectivity of N3. The purpose of this study was to determine the substrate profile, transport mechanisms, and Na+-coupling stoichiometry of T8 and to compare these measurements with those of wild-type N1, N2, and N3. In Xenopus laevis oocytes expressing T8, Na+-dependent uptake of 3H-labeled purine (adenosine, inosine, and guanosine) and pyrimidine nucleosides (uridine, thymidine, and cytidine) was significantly enhanced (3.5-18.6-fold), which suggests that T8 accepts both purine and pyrimidine nucleosides as permeants. T8-mediated uptake of [3H]thymidine was competitively inhibited by inosine, and T8-mediated uptake of [3H]inosine was competitively inhibited by thymidine, which suggests that purine and pyrimidine nucleosides share a common binding site. Base-modified ribo- and 2'-deoxyribonucleosides were potent inhibitors of T8. In contrast, 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and 3'-azidothymidine, which are known inhibitors of N1 or N2, did not inhibit T8-mediated uptake. These data suggest that the substrate profile of T8 is not a combination of those of N1 and N2; rather, it is similar to that of N3. However, the Na+/nucleoside stoichiometric ratio of T8 was determined to be 1, consistent with both N1 and N2 but different from N3.

Several mammalian nucleoside transporters have been identified at the molecular level. Human and rat equilibrative nucleoside transporter 2 (hENT2 and rENT2, respectively) was previously reported to have the dual ability of transporting both nucleosides and nucleobases. In the present study, we characterized the transport of a variety of nucleosides and nucleobases via recombinant mouse ENT2 (mENT2). Cloned mENT2 mediated the uptake of nucleosides and purine nucleobases, but not pyrimidine nucleobases. The mENT2-mediated uptake of adenosine was significantly inhibited by nucleosides and nucleobases, irrespective of purine and pyrimidine. The K(m) values for the uptake of nucleosides and purine nucleobases mediated by mENT2 varied between 1.24 and 16.3 microM, and the transport clearances of adenosine and hypoxanthine via the transporter were greater than those of other substrates. Therefore, we concluded that mENT2 is nucleoside and purine nucleobase transporter, and pyrimidine nucleobases are blockers for the transporter, differing from hENT2 and rENT2 that were reported to also transport pyrimidine nucleobases.

The metabolism of nitrogen-rich nucleosides in Arabidopsis seedlings was investigated at the level of import and subsequent salvage or degradation. Uptake and fate of nucleosides imported by equilibrative nucleoside transporter 3 (ENT3) was analysed and, furthermore, a comprehensive analysis of the effect of exogenously fed nucleosides at the level of metabolic as well as transcriptomic alterations was performed. Expression of nucleoside transporters ENT1 and ENT3, together with nucleoside import, was increased upon nitrogen limitation. Thereby a role for ENT3, which is expressed mainly in the vasculature of roots and leaves, as a major import route for nucleosides was supported. Exogenously fed nucleosides were able to attenuate nitrogen starvation effects such as chlorophyll breakdown, anthocyanin accumulation, RNA breakdown and reduced levels of amino acids. In response to nucleoside supply, up-regulation of genes involved in nitrogen distribution in plants was observed. In addition, genes involved in nucleoside metabolism were identified as regulated upon nitrogen limitation. In summary, an overall beneficial effect of nucleoside supply to Arabidopsis seedlings, especially under limiting nitrogen conditions, was observed.

Spodoptera frugiperda (Sf9) cells express novel purine- and pyrimidine-selective nucleoside transporters

Current Progress on Equilibrative Nucleoside Transporter Function and Inhibitor Design

Facilitated diffusion and sodium-dependent transport of purine and pyrimidine nucleosides in rat liver

Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild-type promastigotes that were transferred to medium deficient in purines. Pulse labelling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations.

This study investigated mRNA expression and protein localization of equilibrative and concentrative nucleoside transporters (ENTs, CNTs) in primary cultures of rat brain endothelial cells (RBEC) and rat choroid plexus epithelial cells (RCPEC). Reverse transcriptase PCR analysis revealed that RBEC and RCPEC contained mRNA for rENT1, rENT2 and rCNT2 and for rENT1, rENT2, rCNT2 and rCNT3, respectively. Immunoblotting of membrane fractions of RBEC, fresh RCPEC and primary cultures of RCPEC revealed the presence of rENT1, rENT2 and rCNT2 proteins in all samples. Measurement of [14C]adenosine uptake into cells grown as monolayers on permeable plastic supports revealed a polarized distribution of Na+-dependent adenosine uptake in that CNT activity was associated exclusively in membranes of RBEC facing the lower chamber (which corresponds to the surface facing the interstitial fluid in situ) and in membranes of RCPEC facing the upper chamber (which corresponds to the surface facing the cerebrospinal fluid in situ). In both RBEC and RCPEC, adenosine uptake from the opposite chambers was Na+-independent and partially inhibited by nitrobenzylthioinosine, indicating the presence of the equilibrative sensitive transporter rENT1.

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.

Immunosuppression by adenosine is an important cancer immune checkpoint. Extracellular adenosine signals through specific receptors and can be transported across the cell membrane through nucleoside transporters. Although adenosine receptors are well-known to regulate tumor immunity, the impact of adenosine transporters remains unexplored. In this study, we investigated the effect on tumor immunity of equilibrative nucleoside transporter-1 (ENT1), the major regulator of extracellular adenosine concentrations. Blocking or deleting host ENT1 significantly enhanced CD8+ T-cell-dependent antitumor responses. Tumors inoculated into ENT1-deficient mice showed increased infiltration of effector CD8+ T cells with an enhanced cytotoxic transcriptomic profile and significant upregulation of granzyme B, IFNγ, IL2, TNFα, and CXCL10. ENT1 deficiency was further associated with decreased tumor-infiltrating T regulatory cells and CD206high macrophages and suppressed CCL17 production. ENT1 deficiency notably potentiated the therapeutic activity of PD-1 blockade. T cells upregulated ENT1 upon activation, and blocking ENT1 enhanced their function when cocultured with cognate antigen/HLA-matched melanoma cells. Mechanistically, ENT1-mediated adenosine uptake inhibited the activity of phosphoribosyl pyrophosphate synthetase in activated T cells, thereby suppressing production of uridine 5'-monophosphate and its derivatives required for DNA and RNA synthesis. In summary, this study identified ENT1-mediated adenosine uptake as an important mechanism of adenosine-mediated immunosuppression and pyrimidine starvation that can be targeted to enhance antitumor T-cell responses. Significance: ENT1 is a potential therapeutic target to overcome immunosuppression induced by extracellular adenosine and to increase the activity of PD-1 blockade.

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.