scholarly journals Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells

1996 ◽  
Vol 317 (2) ◽  
pp. 457-465 ◽  
Author(s):  
Xiao FANG ◽  
Fiona E. PARKINSON ◽  
Delores A. MOWLES ◽  
James D. YOUNG ◽  
Carol E. CASS
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
Functional Characterization ◽  
Transport Processes ◽  
Nucleoside Transport ◽  
Single Type ◽  
Nucleoside Transporter ◽  
Pyrimidine Nucleosides ◽  
Transporter Protein ◽  
Sodium Dependent

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the ‘equilibrative sensitive’ or ‘es’ transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximate Km values of 21 μM, 12.5 μM and 15 μM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.

Recent advances in the molecular biology of nucleoside transporters of mammalian cells

1998 ◽  
Vol 76 (5) ◽  
pp. 761-770 ◽  
Author(s):  
Carol E Cass ◽  
James D Young ◽  
Stephen A Baldwin
Keyword(s):  
Molecular Biology ◽  
Mammalian Cells ◽  
Cell Types ◽  
Functional Expression ◽  
Transport Proteins ◽  
Transport Processes ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Protein Families ◽  
Public Data

Nucleosides are hydrophilic molecules and require specialized transport proteins for permeation of cell membranes. There are two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients and inwardly directed concentrative processes driven by the sodium electrochemical gradient. The equilibrative nucleoside transport processes (es, ei) are found in most mammalian cell types, whereas the concentrative nucleoside transport processes (cit, cif, cib, csg, cs) are present primarily in specialized epithelia. Using a variety of cloning strategies and functional expression in oocytes of Xenopus laevis, we have isolated and characterized cDNAs encoding the rat and human nucleoside transporter proteins of the four major nucleoside transport processes of mammalian cells (es, ei, cit, cif). From the sequence relationships of these proteins with each other and with sequences in the public data bases, we have concluded that the equilibrative and concentrative nucleoside transport processes are mediated by members of two previously unrecognized groups of integral membrane proteins, which we have designated the equilibrative nucleoside transporter (ENT) and the concentrative nucleoside transporter (CNT) protein families. This review summarizes the current state of knowledge in the molecular biology of the ENT and CNT protein families, focusing on the characteristics of the four human (h) and rat (r) nucleoside transport proteins (r/hENT1, r/hENT2, r/hCNT1, r/hCNT2).Key words: nucleoside transporter, equilibrative, concentrative, ENT, CNT.

A purine-selective nucleobase/nucleoside transporter in PK15NTD cells

2008 ◽  
Vol 294 (6) ◽  
pp. R1988-R1995 ◽  
Author(s):  
Kazi Mirajul Hoque ◽  
Linxi Chen ◽  
George P. H. Leung ◽  
Chung-Ming Tse
Keyword(s):  
Transport System ◽  
Mammalian Cells ◽  
Organic Cation ◽  
Organic Cation Transporter ◽  
Nucleoside Transport ◽  
Acidic Ph ◽  
Nucleoside Transporter ◽  
High Affinity ◽  
Naturally Occurring ◽  
Purine Analog

Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified an Na-independent purine-selective nucleobase/nucleoside transport system in the nucleoside transporter-deficient PK15NTD cells. This transport system has 1,000-fold higher affinity for nucleobases than nucleosides with Kmvalues of 2.5 ± 0.7 μM for [3H]adenine, 6.4 ± 0.5 μM for [3H]guanine, 1.1 ± 0.1 mM for [3H]guanosine, and 4.2 ± 0.5 mM [3H]adenosine. The uptake of [3H]guanine (0.05 μM) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5–1 mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine >>> thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2′ and/or 5 positions (all at 1 mM) were more potent than adenosine in competing the uptake of [3H]guanine: 2-chloro-2′-deoxyadenosine > 2-chloroadenosine > 2′3′-dideoxyadenosine = 2′-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-Chloro-2′-deoxyadenosine and 2-chloroadenosine inhibited [3H]guanine uptake with IC50values of 68 ± 5 and 99 ± 10 μM, respectively. The nucleobase/nucleoside transporter was resistant to nitrobenzylthioinosine {6-[(4-nitrobenzyl) thiol]-9-β-d-ribofuranosylpurine}, dipyridamole, and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor decynium-22 (IC50of ∼1 μM), and by acidic pH (pH = 5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells.

scholarly journals Differential uptake of [3H]guanosine by nucleoside transporter subtypes in Ehrlich ascites tumour cells

1992 ◽  
Vol 287 (2) ◽  
pp. 431-436 ◽  
Author(s):  
J R Hammond
Keyword(s):  
Mammalian Cells ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Transporter ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Tumour ◽  
Transport Inhibitors ◽  
Influx Kinetics ◽  
Ascites Tumour Cells ◽  
Ehrlich Ascites Tumour Cells

Intracellular metabolism of [3H]guanosine was minimal (< 15%) during the first 22 s of incubation, and hence reasonable estimates of initial-rate influx kinetics could be derived by using metabolically active cells. Na(+)-dependent concentrative [3H]guanosine uptake was not observed. Data suggest that [3H]guanosine was accumulated primarily via the nitrobenzylthioguanosine (NBTGR)-sensitive subtype of facilitated nucleoside transporter. Incubation of cells with 100 nM-NBTGR significantly decreased the potency of guanosine as an inhibitor of [3H]uridine influx. The Vmax. for [3H]guanosine influx (9.2 pmol/s per microliters) was significantly lower than that for [3H]uridine influx (16 pmol/s per microliters). The Km for transporter-mediated [3H]guanosine influx determined in the presence of 100 nM-NBTGR was 16-fold higher (1780 microM) than that determined in its absence, whereas the Km for [3H]uridine influx was shifted by only 2-fold. In other respects, the cellular accumulations of [3H]guanosine and [3H]uridine were similar; both had Km values of approx. 140 microM for total mediated influx, and both were inhibited similarly by other nucleosides and transport inhibitors. These characteristics, and the fact that guanosine is an endogenous nucleoside, suggest that [3H]guanosine may prove useful as a poorly metabolized, relatively selective, substrate for study of the NBTGR-sensitive nucleoside transport systems of mammalian cells.

scholarly journals Cysteine-accessibility analysis of transmembrane domains 11–13 of human concentrative nucleoside transporter 3

2006 ◽  
Vol 394 (2) ◽  
pp. 389-398 ◽  
Author(s):  
Jing Zhang ◽  
Tracey Tackaberry ◽  
Mabel W. L. Ritzel ◽  
Taylor Raborn ◽  
Gerry Barron ◽  
...  
Keyword(s):  
Expression System ◽  
Transmembrane Domains ◽  
Nucleoside Transport ◽  
Cysteine Residues ◽  
Nucleoside Transporter ◽  
Yeast Expression System ◽  
Cysteine Substitution ◽  
Concentrative Nucleoside Transporter ◽  
Translocation Pathway ◽  
Nucleoside Drugs

hCNT3 (human concentrative nucleoside transporter 3) is a nucleoside–sodium symporter that transports a broad range of naturally occurring purine and pyrimidine nucleosides as well as anticancer nucleoside drugs. To understand its uridine binding and translocation mechanisms, a cysteine-less version of hCNT3 was constructed and used for cysteine-accessibility and permeant-protection assays. Cysteine-less hCNT3, with 14 endogenous cysteine residues changed to serine, displayed wild-type properties in a yeast expression system, indicating that endogenous cysteine residues are not essential for hCNT3-mediated nucleoside transport. A series of cysteine-substitution mutants spanning predicted TMs (transmembrane domains) 11–13 was constructed and tested for accessibility to thiol-specific reagents. Mutants M496C, G498C, F563C, A594C, G598C and A606C had no detectable transport activity, indicating that a cysteine substitution at each of these positions was not tolerated. Two functional mutants in putative TM 11 (L480C and S487C) and four in putative TM 12 (N565C, T557C, G567C and I571C) were partially inhibited by MTS (methanethiosulphonate) reagent and high concentrations of uridine protected against inhibition, indicating that TMs 11 and 12 may form part of the nucleoside translocation pathway. The lack of accessibility of MTS reagents to TM 13 mutants suggests that TM 13 is not exposed to the nucleoside translocation pathway. Furthermore, G567C, N565C and I571C mutants were only sensitive to MTSEA (MTS-ethylammonium), a membranepermeant thiol reagent, indicating that these residues may be accessible from the cytoplasmic side of the membrane, providing evidence in support of the predicted orientation of TM 12 in the current putative topology model of hCNT3.

Genomic analysis of nucleoside transporters in Diptera and functional characterization of DmENT2, a Drosophila equilibrative nucleoside transporter

2007 ◽  
Vol 28 (3) ◽  
pp. 337-347 ◽  
Author(s):  
Jerry Machado ◽  
Parween Abdulla ◽  
W. J. Brad Hanna ◽  
Arthur J. Hilliker ◽  
Imogen R. Coe
Keyword(s):  
Sequence Similarity ◽  
Functional Characterization ◽  
Genomic Analysis ◽  
Structure And Function ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Nucleoside Transport Inhibitors ◽  
And Function

The recent completion of genome sequencing projects in a number of eukaryotes allows comparative analysis of orthologs, which can aid in identifying evolutionary constraints on protein structure and function. Nucleoside transporters (NTs) are present in a diverse array of organisms and previous studies have suggested that there is low protein sequence similarity but conserved structure in invertebrate and vertebrate NT orthologs. In addition, most taxa possess multiple NT isoforms but their respective roles in the physiology of the organism are not clear. To investigate the evolution of the structure and function of NTs, we have extended our previous studies by identifying NT orthologs in the Dipteran Anopheles gambiae and comparing these proteins to human and Drosophila melanogaster (Dm) NTs. In addition, we have functionally characterized DmENT2, one of three putative D. melanogaster ENTs that we have previously described. DmENT2 has broad substrate specificity, is insensitive to standard nucleoside transport inhibitors and is expressed in the digestive tract of late stage embryos based on in situ hybridization. DmENT1 and DmENT2 are expressed in most stages during development with the exception of early embryogenesis suggesting specific physiological roles for each isoform. These data represent the first complete genomic analysis of Dipteran NTs and the first report of the functional characterization of any Dipteran NT.

scholarly journals Uptake of Nitrobenzylthioinosine and Purine β-l-Nucleosides by Intracellular Toxoplasma gondii

2003 ◽  
Vol 47 (10) ◽  
pp. 3247-3251 ◽  
Author(s):  
Omar N. Al Safarjalani ◽  
Fardos N. M. Naguib ◽  
Mahmoud H. el Kouni
Keyword(s):  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
Nucleoside Transporters ◽  
Host Cells ◽  
Purine Nucleoside ◽  
Nucleoside Transport ◽  
Fibroblast Cells ◽  
Nucleoside Transporter ◽  
Intracellular Parasites ◽  
Infected Cells

ABSTRACT Intracellular Toxoplasma gondii grown in human foreskin fibroblast cells transported nitrobenzylthioinosine {NBMPR; 6-[(4-nitrobenzyl)mercapto]-9-β-d-ribofuranosylpurine}, an inhibitor of nucleoside transport in mammalian cells, as well as the nonphysiological β-l-enantiomers of purine nucleosides, β-l-adenosine, β-l-deoxyadenosine, and β-l-guanosine. The β-l-pyrimidine nucleosides, β-l-uridine, β-l-cytidine, and β-l-thymidine, were not transported. The uptake of NBMPR and the nonphysiological purine nucleoside β-l-enantiomers by the intracellular parasites also implies that Toxoplasma-infected cells can transport these nucleosides. In sharp contrast, under the same conditions, uninfected fibroblast cells did not transport NBMPR or any of the unnatural β-l-nucleosides. β-d-Adenosine and dipyridamole, another inhibitor of nucleoside transport, inhibited the uptake of NBMPR and β-l-stereoisomers of the purine nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. Furthermore, infection with a Toxoplasma mutant deficient in parasite adenosine/purine nucleoside transport reduced or abolished the uptake of β-d-adenosine, NBMPR, and purine β-l-nucleosides. Hence, the presence of the Toxoplasma adenosine/purine nucleoside transporters is apparently essential for the uptake of NBMPR and purine β-l-nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. These results also demonstrate that, in contrast to the mammalian nucleoside transporters, the Toxoplasma adenosine/purine nucleoside transporter(s) lacks stereospecificity and substrate specificity in the transport of purine nucleosides. In addition, infection with T. gondii confers the properties of the parasite's purine nucleoside transport on the parasitized host cells and enables the infected cells to transport purine nucleosides that were not transported by uninfected cells. These unique characteristics of purine nucleoside transport in T. gondii may aid in the identification of new promising antitoxoplasmic drugs.

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

2005 ◽  
Vol 52 (4) ◽  
pp. 749-758 ◽  
Author(s):  
Marzena Podgorska ◽  
Katarzyna Kocbuch ◽  
Tadeusz Pawelczyk
Keyword(s):  
Mammalian Cells ◽  
Cell Function ◽  
De Novo ◽  
Transport Processes ◽  
Nucleoside Transporters ◽  
Membrane Topology ◽  
Nucleoside Transporter ◽  
Signalling Molecules ◽  
Physiological Processes ◽  
Carrier Mediated Transport

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

scholarly journals Functional expression of the nitrobenzylthioinosine-sensitive nucleoside transporter of human choriocarcinoma (BeWo) cells in isolated oocytes of Xenopus laevis

1994 ◽  
Vol 299 (3) ◽  
pp. 769-773 ◽  
Author(s):  
C E Boumah ◽  
C M Harvey ◽  
A R P Paterson ◽  
S A Baldwin ◽  
J D Young ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Oocytes ◽  
Functional Expression ◽  
Transport Processes ◽  
Nucleoside Transport ◽  
Xenopus Laevis Oocytes ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Bewo Cells ◽  
Ic50 Value

Cultured human choriocarcinoma (BeWo) cells have previously been shown to exhibit, in comparison with other cultured cell types, elevated nitrobenzylthioinosine (NBMPR)-sensitive transport activity and large numbers (> 10(7)/cell) of high-affinity NBMPR-binding sites [Boumah, Hogue and Cass (1992) Biochem. J. 288, 987-996]. The present study investigates whether NBMPR-sensitive nucleoside transport activity could be induced in Xenopus laevis oocytes by microinjection of poly(A)+ RNA isolated from proliferating cultures of BeWo cells. Expression of uridine transport activity was assayed by comparing rates of uptake (22 degrees C) of 100 microM [3H]uridine by RNA-injected oocytes with uptake by water-injected or uninjected oocytes. A 4-fold stimulation of uridine uptake (2.0 versus 0.5 pmol/90 min per oocyte) was seen when oocytes were injected with 50 ng of BeWo poly(A)+ RNA, and this stimulation was abolished when the RNA-injected oocytes were assayed in the presence of 10 microM NBMPR. The expressed uridine transport activity in oocytes was highly sensitive to NBMPR, with a 50% reduction seen at 1.1 nM NBMPR (IC50 value). The IC50 value for NBMPR inhibition of uptake of 100 microM [3H]uridine by intact BeWo cells was 1.4 nM. Inward fluxes of [3H]uridine in the RNA-injected oocytes were greatly reduced in the presence of high concentrations (2 mM) of non-radioactive nucleosides (adenosine, thymidine, inosine) that are known permeants of NBMPR-sensitive nucleoside transport processes. These results establish that the abundance of NBMPR-sensitive nucleoside transporter mRNA in poly(A)+ RNA preparations from BeWo cells is sufficient to achieve production of functionally active transporter protein in Xenopus oocytes and that, when expressed in Xenopus oocytes, the transporters exhibit NBMPR sensitivity and permeant selectively similar to that of the native transporters.

[125I]Iodohydroxynitrobenzylthioinosine: a new high-affinity nucleoside transporter probe

1987 ◽  
Vol 65 (5) ◽  
pp. 467-473 ◽  
Author(s):  
Wendy P. Gati ◽  
Leonard I. Wiebe ◽  
Edward E. Knaus ◽  
Alan R. P. Paterson
Keyword(s):  
Mammalian Cells ◽  
Specific Binding ◽  
Sodium Dodecyl ◽  
Nucleoside Transport ◽  
Relative Molecular Mass ◽  
Scatchard Analysis ◽  
Nucleoside Transporter ◽  
High Affinity ◽  
Transport Inhibitor ◽  
Nucleoside Transport Inhibitor

[125l]Iodohydroxynitrobenzylthioinosine ([125I]IH-NBMPR), a new γ-labeled nucleoside transport inhibitor, has been prepared at a theoretical specific activity of 2000 Ci/mmol (1 Ci = 37 GBq). IH-NBMPR was more acidic than hydroxynitrobenzylthioinosine (H-NBMPR), having a pKa of 4.6. Site-specific binding of [125I]IH-NBMPR to membrane-enriched fractions (MEF) from S49 mouse lymphoma cells was pH dependent, increasing with the fraction of undissociated molecules present; it was maximal at pH 4.5 and negligible at pH 7.0. Scatchard analysis of specific binding to MEF from S49 cells under equilibrium conditions at pH 5.0 yielded a Kd of 15 nM (equivalent to 4.0 nM for the undissociated fraction of inhibitor molecules) and maximum number of binding sites (Bmax) of 4.9 pmol/mg protein. Specific binding of IH-NBMPR could not be demonstrated in MEF from AE1 cells, a nucleoside transport-deficient mutant of S49 cells. Influx of uridine into mouse erythrocytes at pH 5.0 in the presence of 5 μM IH-NBMPR (1.4 μM undissociated IH-NBMPR) was reduced to about 7% of the control value, indicating that this compound is an effective nucleoside transport inhibitor. Photoactivation of site-bound [125I]IH-NBMPR, following equilibration of the ligand with MEF from S49 cells at pH 5.0, resulted in specific covalent labeling of a polypeptide with a relative molecular mass of 52 000 – 63 000, identified on sodium dodecyl sulfate – polyacrylamide gels. These results indicate that the new, iodinated ligand is an inhibitor of nucleoside transport and that it binds specifically and with high affinity to nucleoside transporter polypeptides in mammalian cells.

Sign in / Sign up

Export Citation Format

Share Document