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.

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.

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.

scholarly journals Sensitivity of Acute Leukemia Cells to Cytarabine Is a Correlate of Cellular es Nucleoside Transporter Site Content Measured by Flow Cytometry With SAENTA-Fluorescein

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 346-353 ◽  
Author(s):  
Wendy P. Gati ◽  
Alan R.P. Paterson ◽  
Loree M. Larratt ◽  
A. Robert Turner ◽  
Andrew R. Belch
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Cell Types ◽  
Transport Processes ◽  
Nucleoside Transport ◽  
Leukemia Cells ◽  
Nucleoside Transporter ◽  
Flow Cytometric Method ◽  
Ic50 Values

Abstract Cytarabine (araC) is converted to araC 5′-triphosphate after entering leukemia cells as a substrate for nucleoside transport processes. This study tested the relationship between araC cytotoxicity, measured in an in vitro tetrazolium dye reduction assay of cell viability, and the cellular abundance of es nucleoside transport elements, assayed by a flow cytometric method that used the es-specific stain, 5-(SAENTA-x8)-fluorescein (5-(Sx8)-F), in cultured leukemia cells and in myeloblasts and lymphoblasts (blasts) from leukemia patients. Cellular es site abundance (Bmax value for 5-(Sx8)-F binding) varied sixfold among nine leukemic myeloblast samples from patients. In cultured OCI/AML-2 myeloblasts and CCRF-CEM T-lymphoblasts, and in fresh leukemic blasts, es sites were fractionally blocked by treatment with graded concentrations of nitrobenzylthioinosine (NBMPR), an inhibitory es site ligand, to simulate the variation in es expression found in leukemic blasts from patients with acute myeloid leukemia. When the cytotoxicity of a single concentration of araC was determined in NBMPR-treated leukemia cells, cell kill correlated closely with the intensity of 5-(Sx8)-F fluorescence (r = .92 to .99), a measure of the cell surface abundance of functional es nucleoside transporter sites. Concentrations of NBMPR that achieved half-maximal reduction (4.3 to 12 nmol/L) of cellular 5-(Sx8)-F fluorescence (measured by flow cytometry) approximated IC50 values (1 to 10 nmol/L) previously found for inhibition by NBMPR of es-mediated nucleoside fluxes in several cell types, supporting the view that 5-(Sx8)-F interacted with the es transporter. The correlation of araC cytotoxicity and the Bmax for 5-(Sx8)-F binding to es sites in cultured leukemia cells and in leukemic blasts from acute leukemia patients (r = .95) suggests that the flow cytometry assay of es capacity may be useful in predicting clinical response to araC.

scholarly journals Sensitivity of Acute Leukemia Cells to Cytarabine Is a Correlate of Cellular es Nucleoside Transporter Site Content Measured by Flow Cytometry With SAENTA-Fluorescein

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 346-353 ◽  
Author(s):  
Wendy P. Gati ◽  
Alan R.P. Paterson ◽  
Loree M. Larratt ◽  
A. Robert Turner ◽  
Andrew R. Belch
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Cell Types ◽  
Transport Processes ◽  
Nucleoside Transport ◽  
Leukemia Cells ◽  
Nucleoside Transporter ◽  
Flow Cytometric Method ◽  
Ic50 Values

Cytarabine (araC) is converted to araC 5′-triphosphate after entering leukemia cells as a substrate for nucleoside transport processes. This study tested the relationship between araC cytotoxicity, measured in an in vitro tetrazolium dye reduction assay of cell viability, and the cellular abundance of es nucleoside transport elements, assayed by a flow cytometric method that used the es-specific stain, 5-(SAENTA-x8)-fluorescein (5-(Sx8)-F), in cultured leukemia cells and in myeloblasts and lymphoblasts (blasts) from leukemia patients. Cellular es site abundance (Bmax value for 5-(Sx8)-F binding) varied sixfold among nine leukemic myeloblast samples from patients. In cultured OCI/AML-2 myeloblasts and CCRF-CEM T-lymphoblasts, and in fresh leukemic blasts, es sites were fractionally blocked by treatment with graded concentrations of nitrobenzylthioinosine (NBMPR), an inhibitory es site ligand, to simulate the variation in es expression found in leukemic blasts from patients with acute myeloid leukemia. When the cytotoxicity of a single concentration of araC was determined in NBMPR-treated leukemia cells, cell kill correlated closely with the intensity of 5-(Sx8)-F fluorescence (r = .92 to .99), a measure of the cell surface abundance of functional es nucleoside transporter sites. Concentrations of NBMPR that achieved half-maximal reduction (4.3 to 12 nmol/L) of cellular 5-(Sx8)-F fluorescence (measured by flow cytometry) approximated IC50 values (1 to 10 nmol/L) previously found for inhibition by NBMPR of es-mediated nucleoside fluxes in several cell types, supporting the view that 5-(Sx8)-F interacted with the es transporter. The correlation of araC cytotoxicity and the Bmax for 5-(Sx8)-F binding to es sites in cultured leukemia cells and in leukemic blasts from acute leukemia patients (r = .95) suggests that the flow cytometry assay of es capacity may be useful in predicting clinical response to araC.

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 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 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 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 Characterization of African bat henipavirus GH-M74a glycoproteins

2014 ◽  
Vol 95 (3) ◽  
pp. 539-548 ◽  
Author(s):  
Michael Weis ◽  
Laura Behner ◽  
Markus Hoffmann ◽  
Nadine Krüger ◽  
Georg Herrler ◽  
...  
Keyword(s):  
G Protein ◽  
Mammalian Cells ◽  
Nipah Virus ◽  
Syncytium Formation ◽  
Cell Types ◽  
Functional Expression ◽  
Surface Expression ◽  
Hendra Virus ◽  
Proteolytic Activation ◽  
F Protein

In recent years, novel henipavirus-related sequences have been identified in bats in Africa. To evaluate the potential of African bat henipaviruses to spread in non-bat mammalian cells, we compared the biological functions of the surface glycoproteins G and F of the prototype African henipavirus GH-M74a with those of the glycoproteins of Nipah virus (NiV), a well-characterized pathogenic member of the henipavirus genus. Glycoproteins are central determinants for virus tropism, as efficient binding of henipavirus G proteins to cellular ephrin receptors and functional expression of fusion-competent F proteins are indispensable prerequisites for virus entry and cell-to-cell spread. In this study, we analysed the ability of the GH-M74a G and F proteins to cause cell-to-cell fusion in mammalian cell types readily permissive to NiV or Hendra virus infections. Except for limited syncytium formation in a bat cell line derived from Hypsignathus monstrosus, HypNi/1.1 cells, we did not observe any fusion. The highly restricted fusion activity was predominantly due to the F protein. Whilst GH-M74a G protein was found to interact with the main henipavirus receptor ephrin-B2 and induced syncytia upon co-expression with heterotypic NiV F protein, GH-M74a F protein did not cause evident fusion in the presence of heterotypic NiV G protein. Pulse–chase and surface biotinylation analyses revealed delayed F cleavage kinetics with a reduced expression of cleaved and fusion-active GH-M74a F protein on the cell surface. Thus, the F protein of GH-M74a showed a functional defect that is most likely caused by impaired trafficking leading to less efficient proteolytic activation and surface expression.

The SLC28 (CNT) and SLC29 (ENT) nucleoside transporter families: a 30-year collaborative odyssey

2016 ◽  
Vol 44 (3) ◽  
pp. 869-876 ◽  
Author(s):  
James D. Young
Keyword(s):  
Cell Signalling ◽  
Cell Types ◽  
Chemotherapeutic Agents ◽  
Nucleoside Analogues ◽  
Nucleoside Transporter ◽  
Protein Families ◽  
Lower Vertebrate ◽  
Nucleotide Biosynthesis ◽  
Cellular Events ◽  
Concentrative Nucleoside Transporter

Specialized nucleoside transporter (NT) proteins are required for passage of nucleosides and hydrophilic nucleoside analogues across biological membranes. Physiologic nucleosides serve as central salvage metabolites in nucleotide biosynthesis, and nucleoside analogues are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases. The nucleoside adenosine modulates numerous cellular events via purino-receptor cell signalling pathways. Human NTs are divided into two structurally unrelated protein families: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family. Human CNTs are inwardly directed Na+-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types. Human ENTs mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types. Both protein families are evolutionarily old: CNTs are present in both eukaryotes and prokaryotes; ENTs are widely distributed in mammalian, lower vertebrate and other eukaryote species. This mini-review describes a 30-year collaboration with Professor Stephen Baldwin to identify and understand the structures and functions of these physiologically and clinically important transport proteins.

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