Nucleoside transport in rat cerebral-cortical synaptosomes. Evidence for two types of nucleoside transporters.

The transport of [U-14C]uridine was investigated in rat cerebral-cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions the rapid efflux of uridine from the synaptosomes is prevented and uridine is not significantly metabolized in the synaptosome during the first 1 min of uptake. The dose-response curve for the inhibition of uridine transport by nitrobenzylthioinosine (NBMPR) was biphasic: approx. 40% of the transport activity was inhibited with an IC50 (concentration causing half-maximal inhibition) value of 0.5 nM, but the remaining activity was insensitive to concentrations as high as 1 microM. Similar biphasic dose-response curves were observed for dilazep inhibition, but both transport components were equally sensitive to dipyridamole inhibition. Uridine influx by both components was saturable (Km 300 +/- 51 and 214 +/- 23 microM, and Vmax. 12 +/- 3 and 16 +/- 3 pmol/s per mg of protein, for NBMPR-sensitive and NBMPR-insensitive components respectively), and inhibited by other nucleosides such as 2-chloroadenosine, adenosine, inosine, thymidine and guanosine with similar IC50 values for the two components. Inhibition of uridine transport by NBMPR was associated with high-affinity binding of NBMPR to the synaptosome membrane (Kd 58 +/- 15 pM). Binding of NBMPR to these sites was competitively blocked by uridine and adenosine and inhibited by dilazep and dipyridamole, with Ki values similar to those measured for inhibiting NBMPR-sensitive uridine influx. These results demonstrate that there are two components of nucleoside transport in our rat synaptosomal preparation that differ in their sensitivity to inhibition by NBMPR. Thus conclusions regarding nucleoside transport in rat brain based only on NBMPR-binding activity must be viewed with caution.     

A single glycine mutation in the equilibrative nucleoside transporter gene, hENT1, alters nucleoside transport activity and sensitivity to nitrobenzylthioinosine

The human equilibrative nucleoside transporter, hENT1, which is sensitive to inhibition by nitrobenzylthioinosine (NBMPR), is expressed in a wide variety of tissues. hENT1 is involved in the uptake of natural nucleosides, including regulation of the physiological effects of extracellular adenosine, and transports nucleoside drugs used in the treatment of cancer and viral diseases. Structure-function studies have revealed that transmembrane domains (TMD) 3 through 6 of hENT1 may be involved in binding of nucleosides. We have hypothesized that amino acid residues within TMD 3-6, which are conserved across equilibrative transporter sequences from several species, may have a critical role in the binding and transport of nucleosides. Therefore, we explored the role of point mutations of two conserved glycine residues, at positions 179 and 184 located in transmembrane domain 5 (TMD 5), using a GFP-tagged hENT1 in a yeast nucleoside transporter assay system. Mutations of glycine 179 to leucine, cysteine, or valine abolished transporter activity without affecting the targeting of the transporter to the plasma membrane, whereas more conservative mutations such as glycine to alanine or serine preserved both targeting to the plasma membrane and transport activity. Similar point mutations at glycine 184 resulted in poor targeting of hENT1 to the plasma membrane and little or no detectable functional activity. Uridine transport by G179A mutant was significantly lower (p < 0.05) and less sensitive (p < 0.05) to inhibition by NBMPR when compared to the wild-type transporter (IC(50) 7.7 +/- 0.8 nM versus 46 +/- 14.6 nM). Based on these data, we conclude that when hENT1 is expressed in yeast, glycine 179 is critical not only to the ability of hENT1 to transport uridine but also as a determinant of hENT1 sensitivity to NBMPR. In contrast, glycine 184 is likely important in targeting the transporter to the plasma membrane. This is the first identification and characterization of a critical amino acid residue of hENT1 that is important in both nucleoside transporter function and sensitivity to inhibition by NBMPR.     

Purine uptake and release in rat C6 glioma cells: nucleoside transport and purine metabolism under ATP-depleting conditions

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.     

Identification and characterisation of high affinity nucleoside and nucleobase transporters in Toxoplasma gondii

The protozoan parasite Toxoplasma gondii depends upon salvaging the purines that it requires. We have re-analysed purine transport in T. gondii and identified novel nucleoside and nucleobase transporters. The latter transports hypoxanthine (TgNBT1; K(m)=0.91+/-0.19 microM) and is inhibited by guanine and xanthine: it is the first high affinity nucleobase transporter to be identified in an apicomplexan parasite. The previously reported nucleoside transporter, TgAT1, is low affinity with K(m) values of 105 and 134 microM for adenosine and inosine, respectively. We have now identified a second nucleoside transporter, TgAT2, which is high affinity and inhibited by adenosine, inosine, guanosine, uridine and thymidine (K(m) values 0.28-1.5 microM) as well as cytidine (K(i)=32 microM). TgAT2 also recognises several nucleoside analogues with therapeutic potential. We have investigated the basis for the broad specificity of TgAT2 and found that hydrogen bonds are formed with the 3' and 5' hydroxyl groups and that the base groups are bound through H-bonds with either N3 of the purine ring or N(3)H of the pyrimidine ring, and most probably pi-pi-stacking as well. The identification of these high affinity purine nucleobase and nucleoside transporters reconciles for the first time the low abundance of free nucleosides and nucleobases in the intracellular environment with the efficient purine salvage carried out by T. gondii.     

Complex effects of sulfhydryl reagents on ligand interactions with nucleoside transporters: evidence for multiple populations of ENT1 transporters with differential sensitivities to N-ethylmaleimide

Functional studies have implicated cysteines in the interaction of ligands with the ENT1 nucleoside transporter. To better define these interactions, N-ethylmaleimide (NEM) and p-chloromercuribenzylsulfonate (pCMBS) were tested for their effects on ligand interactions with the [(3)H] nitrobenzylthioinosine (NBMPR) binding site of the ENT1 transporters of mouse Ehrlich ascites cells and human erythrocytes. NEM had biphasic, concentration-dependent effects on NBMPR binding to intact Ehrlich cells, plasma membranes, and detergent-solubilized membranes, with about 35% of the binding activity being relatively insensitive to NEM inhibition. NBMPR binding to human erythrocyte membranes also displayed heterogeneity in that about 33% of the NBMPR binding sites remained, albeit with lower affinity for NBMPR, even after treatment with NEM at concentrations in excess of 1 mM. However, unlike that seen for Ehrlich cells, no "reversal" in NBMPR binding to human erythrocyte membranes was observed at the higher concentrations of NEM. pCMBS inhibited 100% of the NBMPR binding to both Ehrlich cell and human erythrocyte membranes, but had no effect on the binding of NBMPR to intact cells. The effects of NEM on NBMPR binding could be prevented by coincubation of membranes with nonradiolabeled NBMPR, adenosine, or uridine. Treatment with NEM and pCMBS also decreased the affinity of other nucleoside transport inhibitors for the NBMPR binding site, but enhanced the affinities of nucleoside substrates. These data support the existence of at least two populations of ENT1 in both erythrocyte and Ehrlich cell membranes with differential sensitivities to NEM. The interaction of NEM with the mouse ENT1 protein may also involve additional sulphydryl groups not present in the human ENT1.     

Nucleobase transport by human equilibrative nucleoside transporter 1 (hENT1)

The human equilibrative nucleoside transporters hENT1 and hENT2 (each with 456 residues) are 40% identical in amino acid sequence and contain 11 putative transmembrane helices. Both transport purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inhibition by nanomolar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR), hENT1 being NBMPR-sensitive. Previously, we used heterologous expression in Xenopus oocytes to demonstrate that recombinant hENT2 and its rat ortholog rENT2 also transport purine and pyrimidine bases, h/rENT2 representing the first identified mammalian nucleobase transporter proteins (Yao, S. Y., Ng, A. M., Vickers, M. F., Sundaram, M., Cass, C. E., Baldwin, S. A., and Young, J. D. (2002) J. Biol. Chem. 277, 24938-24948). The same study also revealed lower, but significant, transport of hypoxanthine by h/rENT1. In the present investigation, we have used the enhanced Xenopus oocyte expression vector pGEMHE to demonstrate that hENT1 additionally transports thymine and adenine and, to a lesser extent, uracil and guanine. Fluxes of hypoxanthine, thymine, and adenine by hENT1 were saturable and inhibited by NBMPR. Ratios of V(max) (pmol/oocyte · min(-1)):K(m) (mm), a measure of transport efficiency, were 86, 177, and 120 for hypoxantine, thymine, and adenine, respectively, compared with 265 for uridine. Hypoxanthine influx was competitively inhibited by uridine, indicating common or overlapping nucleobase and nucleoside permeant binding pockets, and the anticancer nucleobase drugs 5-fluorouracil and 6-mercaptopurine were also transported. Nucleobase transport activity was absent from an engineered cysteine-less version hENT1 (hENT1C-) in which all 10 endogenous cysteine residues were mutated to serine. Site-directed mutagenesis identified Cys-414 in transmembrane helix 10 of hENT1 as the residue conferring nucleobase transport activity to the wild-type transporter.     

Mutation of residue 33 of human equilibrative nucleoside transporters 1 and 2 alters sensitivity to inhibition of transport by dilazep and dipyridamole.

Human equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met33 to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevis oocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity. To determine whether the reciprocal mutation in hENT2 (Ile33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis. Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR. These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified.     

Functional analysis of site-directed glycosylation mutants of the human equilibrative nucleoside transporter-2

Protein glycosylation is important for nucleoside transport, and this has been demonstrated for the human equilibrative nucleoside transporter-1 (hENT1). It is not known whether glycosylation affects the functions of hENT2 or where hENT2 is glycosylated. We address these questions using N-glycosylation mutants (N48D, N57D, and N48/57D) and demonstrate that hENT2 is glycosylated at Asn(48) and Asn(57). Our results show that although the apparent affinities for [3H]uridine and [3H]cytidine of the mutants were indistinguishable from those of the wild-type protein, N-glycosylation was required for efficient targeting of hENT2 to the plasma membrane. All mutants had a two- to threefold increase in IC(50) for dipyridamole. N57D and N48/57D, but not N48D, also had a twofold increase in IC(50) for NBMPR. We conclude that the relative insensitivity of hENT2 to inhibitors is primarily due to its primary structure and not to glycosylation. Glycosylation modulates hENT1 function, but is not required for hENT2.     

Mutation of Trp29 of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity

hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved. Mutations at Trp29 were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp29 mutations significantly reduced the apparent V(max) and/or increased the apparent K(m) values for adenosine transport. Trp29 mutations increased the IC50 values for hENT1 inhibition by dipyridamole, dilazep, NBMPR, soluflazine and draflazine. NBMPR and soluflazine displayed remarkably similar trends, with large aromatic substitutions at residue 29 resulting in the lowest IC50 values, suggesting that both drugs could interact via ring-stacking interactions with Trp29. The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L442I mutant that displayed a selective loss of purine nucleoside transport. W29T, L442I and the double mutant W29T/L442I were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp29 is positioned close to Met33, implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp29 forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine.     

Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.     

Dipyridamole-insensitive nucleoside transport in mutant murine T lymphoma cells

From a mutagenized population of S49 murine T lymphoma cells, a mutant cell line, JPA4, was selected that expressed an altered nucleoside transport capability. JPA4 cells transported low concentrations of purine nucleosides and uridine more rapidly than the parental S49 cell line. The transport of these nucleosides by mutant cells was insensitive to inhibition by either dipyridamole (DPA) or 4-nitrobenzylthioinosine (NBMPR), two potent inhibitors of nucleoside transport in mammalian cells. Kinetic analyses revealed that the apparent Km values for the transport of uridine, adenosine, and inosine were 3-4-fold lower in JPA4 cells compared to wild type cells. In contrast, the transport of both thymidine and cytidine by JPA4 cells was similar to that of parental cells, and transport of these pyrimidine nucleosides remained sensitive to inhibition by both NBMPR and DPA. Furthermore, thymidine was a 10-12-fold weaker inhibitor of inosine transport in JPA4 cells than in wild type cells. Thus, JPA4 cells appeared to express two types of nucleoside transport activities; a novel (mutant) type that was insensitive to inhibition by DPA and NBMPR and transported purine nucleosides and uridine, and a parental type that retained sensitivity to inhibitors and transported cytidine and thymidine. The phenotype of the JPA4 cell line suggests that the sensitivity of mammalian nucleoside transporters to both NBMPR and DPA can be genetically uncoupled from its ability to transport certain nucleoside substrates and that the determinants on the nucleoside transporter that interact with each nucleoside are not necessarily identical.     

Functional and molecular characterization of nucleobase transport by recombinant human and rat equilibrative nucleoside transporters 1 and 2. Chimeric constructs reveal a role for the ENT2 helix 5-6 region in nucleobase translocation

The human (h) and rat (r) equilibrative (Na(+)-independent) nucleoside transporters (ENTs) hENT1, rENT1, hENT2, and rENT2 belong to a family of integral membrane proteins with 11 transmembrane domains (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine and coronary vasoactive drugs. Structurally, the proteins have a large glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, hENT1, rENT1, hENT2, and rENT2 were produced in Xenopus laevis oocytes and investigated for their ability to transport pyrimidine and purine nucleobases. hENT2 and rENT2 efficiently transported radiolabeled hypoxanthine, adenine, guanine, uracil, and thymine (apparent K(m) values 0.7-2.6 mm), and hENT2, but not rENT2, also transported cytosine. These findings were independently confirmed by hypoxanthine transport experiments with recombinant hENT2 produced in purine-cytosine permease (FCY2)-deficient Saccharomyces cerevisiae and provide the first direct demonstration that the ENT2 isoform is a dual mechanism for the cellular uptake of nucleosides and nucleobases, both of which are physiologically important salvage metabolites. In contrast, recombinant hENT1 and rENT1 mediated negligible oocyte fluxes of hypoxanthine relative to hENT2 and rENT2. Chimeric experiments between rENT1 and rENT2 using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) identified TMs 5-6 of rENT2 (amino acid residues 172-231) as a determinant of nucleobase transport activity, suggesting that this domain forms part(s) of the ENT2 substrate translocation channel.     

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

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

[3H]dipyridamole binding to nucleoside transporters from guinea-pig and rat lung.

Membranes from guinea-pig lung exhibited high-affinity binding of [3H]dipyridamole, a potent inhibitor of nucleoside transport. Binding (apparent KD 2 nM) was inhibited by the nucleoside-transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and lidoflazine and by the transported nucleosides uridine and adenosine. In contrast, there was no detectable high-affinity binding of [3H]dipyridamole to lung membranes from the rat, a species whose nucleoside transporters exhibit a low sensitivity to dipyridamole inhibition. Bmax. values for high-affinity binding of [3H]dipyridamole and [3H]NBMPR to guinea-pig membranes were similar, suggesting that these structurally unrelated ligands bind to the NBMPR-sensitive nucleoside transporter with the same stoichiometry.     

Inosine and equilibrative nucleoside transporter 2 contribute to hypoxic preconditioning in the murine cardiomyocyte HL-1 cell line

The purine nucleoside adenosine is a physiologically important molecule in the heart. Brief exposure of cardiomyocytes to hypoxic challenge results in the production of extracellular adenosine, which then interacts with adenosine receptors to activate compensatory signaling pathways that lead to cellular resistance to subsequence hypoxic challenge. This phenomenon is known as preconditioning (PC), and, while adenosine is clearly involved, other components of the response are less well understood. Flux of nucleosides, such as adenosine and inosine, across cardiomyocyte membranes is dependent on equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2). We have previously shown in the murine cardiomyocyte HL-1 cell line that hypoxic challenge leads to an increase in intracellular adenosine, which exits the cell via ENT1 and preconditions via A1 and A3 adenosine receptor-dependent mechanisms. However, the role and contribution of inosine and ENT2 are unclear. In this study, we confirmed that ENT1 and ENT2 are both capable of transporting inosine. Moreover, we found that hypoxic challenge leads to a significant increase in levels of intracellular inosine, which exits the cell via both ENT1 and ENT2. Exogenously added inosine (5 microM) preconditions cardiomyocytes in an A1 adenosine receptor-dependent manner since preconditioning can be blocked by the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM) but not the A3 adenosine receptor antagonist MRS-1220 (200 nM). These data suggest that cardiomyocyte responses to hypoxic PC are more complex than previously thought, involving both adenosine and inosine and differing, but overlapping, contributions of the two ENT isoforms.     

Coupling of CFTR-mediated anion secretion to nucleoside transporters and adenosine homeostasis in calu-3 cells

The purpose of this study was to characterize the role of adenosine-dependent regulation of anion secretion in Calu-3 cells. RT-PCR studies showed that Calu-3 cells expressed mRNA for A2A and A2B but not A1 or A3 receptors, and for hENT1, hENT2 and hCNT3 but not hCNT1 or hCNT2 nucleoside transporters. Short-circuit current measurements indicated that A2B receptors were present in both apical and basolateral membranes, whereas A2A receptors were detected only in basolateral membranes. Uptake studies demonstrated that the majority of adenosine transport was mediated by hENT1, which was localized to both apical and basolateral membranes, with a smaller hENT2-mediated component in basolateral membranes. Whole-cell current measurements showed that application of extracellular nitrobenzylmercaptopurine ribonucleoside (NBMPR), a selective inhibitor of hENT1-mediated transport, had similar effects on whole-cell currents as the application of exogenous adenosine. Inhibitors of adenosine kinase and 5'-nucleotidase increased and decreased, respectively, whole-cell currents, whereas inhibition of adenosine deaminase had no effect. Single-channel studies showed that NBMPR and adenosine kinase inhibitors activated CFTR Cl- channels. These results suggested that the equilibrative nucleoside transporters (hENT1, hENT2) together with adenosine kinase and 5'-nucleotidase play a crucial role in the regulation of CFTR through an adenosine-dependent pathway in human airway epithelia.     

Design, synthesis, and evaluation of 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate (8MDP-fluor), as a novel equilibrative nucleoside transporter probe

Nucleoside transporters are integral membrane glycoproteins that play critical roles in physiological nucleoside and nucleobase fluxes, and influence the efficacy of many nucleoside chemotherapy drugs. Fluorescent reporter ligands/substrates have been shown to be useful in the analysis of nucleoside transporter (NT) protein expression and discovery of new NT inhibitors. In this study, we have developed a novel dipyridamole (DP)-based equilibrative nucleoside transporter 1 (ENT1) fluorescent probe. The potent ENT1 and ENT2 inhibitor analogue of dipyridamole, 2,6-bis(diethanolamino)-4,8-diheptamethyleneiminopyrimido[5,4-d]pyrimidine (4, 8MDP), was modified to replace one β-hydroxyethyl group of the amino substituent at the 2-position with a β-aminoethyl group and then conjugated through the amino group to 6-(fluorescein-5-carboxamido)hexanoyl moiety to obtain a new fluorescent molecule, 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate, designated 8MDP-fluorescein (8MDP-fluor, 6). The binding affinities of 8MDP-fluor at ENT1 and ENT2 are reflected by the uridine uptake inhibitory K(i) values of 52.1 nM and 285 nM, respectively. 8MDP-fluor was successfully demonstrated to be a flow cytometric probe for ENT1 comparable to the nitrobenzylmercaptopurine riboside (NBMPR) analogue ENT1 fluorescent probe SAENTA-X8-fluorescein (SAENTA-fluor, 1). This is the first reported dipyridamole-based ENT1 fluorescent probe, which adds a novel tool for probing ENT1, and possibly ENT2.     

Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V(max) of 3.4 +/- 1 pmol.microl(-1).s(-1), with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (K(m) = 96 +/- 37 microM; V(max) = 1.6 +/- 0.3 pmol.microl(-1).s(-1)) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (K(i) = 19 +/- 7 microM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na(+) or H(+). PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.     

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.     

Novel C2-purine position analogs of nitrobenzylmercaptopurine riboside as human equilibrative nucleoside transporter 1 inhibitors

Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. S⁶-(4-nitrobenzyl)mercaptopurine riboside (NBMPR) is a prototype inhibitor of the human equilibrative nucleoside transporter (hENT1), and is a high affinity ligand with a K_d of 0.1–1.0 nM. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel C²-purine position substituted analogs of NBMPR at the hENT1. The aim of this research was to understand the substituent requirements at the C²-purine position of NBMPR. Structure–activity relationships (SAR) indicate that increasing the steric bulk at the C²-purine position of NBMPR led to a decrease in binding affinity of these ligands at the hENT1. New high affinity inhibitors were identified, with the best compound, 2-fluoro-4-nitrobenzyl mercaptopurine riboside (7), exhibiting a K_i of 2.1 nM. This information, when coupled with the information obtained from other structure–activity relationship studies should prove useful in efforts aimed at modeling the NMBPR and analogs pharmacophore of hENT1 inhibitors.