scholarly journals Reduction of equilibrative nitrobenzylthioinosine-sensitive nucleoside transporter in tamoxifen-treated MCF-7 cells: an oestrogen-reversible phenomenon

1997 ◽  
Vol 327 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Lay-Beng GOH ◽  
Chee-Wee LEE
Keyword(s):  
Cell Growth ◽  
Growth Retardation ◽  
Binding Sites ◽  
Kinetic Studies ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Binding Affinities ◽  
Side Group ◽  
Mcf 7

MCF-7 cells display both nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative, but not the Na+-dependent, nucleoside transport. Transport of uridine by es is more sensitive to inhibition by purine nucleosides, whereas the ei component is more sensitive to nucleosides without an amino side group, such as inosine and thymidine. When exposed to 10 μM tamoxifen for 5 days, MCF-7 cells displayed a 44% decrease in the total number of NBMPR-binding sites [Bmax from 245000±18000 to 136000±25000 sites per cell (mean±S.E.M.; n = 5; P< 0.05)], and a 57% decrease in cell growth with no significant change in binding affinities [Kd from 0.37±0.05 to 0.45±0.08 nM (n = 5; P> 0.05)]. Kinetic studies of [3H]uridine transport showed a decrease in the Vmax of the es component from 21.7±0.3 (n = 8) to 8.4±2.2 μM/s (n = 4; P<0.05), whereas the Vmax of the ei component [from 4.7±0.5 (n =8) to 5.8±1.6 μM/s (n = 4; P> 0.05)] and Km values for both components [es from 460±80 to 630±280 μM (n ⩾ 3; P> 0.05) and ei from 355±115 to 440±220 μM (n ⩾ 4; P> 0.05)] did not change significantly. Oestradiol at 100 nM reversed almost completely the NBMPR-binding site decrease and growth retardation in tamoxifen-treated cells. Thus tamoxifen is shown to cause an oestrogen-reversible decrease of es nucleoside transporters in MCF-7 cells.

scholarly journals Nucleoside transport and proliferative rate in human thymocytes and lymphocytes

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2038-2042 ◽  
Author(s):  
CL Smith ◽  
LM Pilarski ◽  
ML Egerton ◽  
JS Wiley
Keyword(s):  
Cytosine Arabinoside ◽  
Binding Sites ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
S Phase ◽  
Nucleoside Transporters ◽  
Labeling Index ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

scholarly journals Nucleoside transport and proliferative rate in human thymocytes and lymphocytes

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2038-2042 ◽  
Author(s):  
CL Smith ◽  
LM Pilarski ◽  
ML Egerton ◽  
JS Wiley
Keyword(s):  
Cytosine Arabinoside ◽  
Binding Sites ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
S Phase ◽  
Nucleoside Transporters ◽  
Labeling Index ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter

Abstract The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

Characteristics of membrane transport losses during reticulocyte maturation

1987 ◽  
Vol 65 (10) ◽  
pp. 869-875 ◽  
Author(s):  
Rhoda Blostein ◽  
Eva Grafova
Keyword(s):  
Membrane Transport ◽  
Binding Sites ◽  
Sodium Pump ◽  
Proteolytic Processing ◽  
Atp Depletion ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Transporter ◽  
Transport Losses

The decline in activity of distinct membrane transport systems was followed during in vitro maturation of sheep reticulocytes, namely the sodium pump (measured as specific ouabain binding sites), Na+–glycine cotransport, and the nucleoside transporter (measured as specific nitrobenzylthioinosine binding sites). Certain features of this maturation-associated decline in membrane transport are clarified. Thus, the apparent retardation of loss by metabolic (ATP) depletion, reported previously for the sodium pump and Na+–glycine cotransport, is applicable also to the decline in nucleoside transport. The absolute losses, as well as relative effects of ATP depletion, are different for the three distinct systems. Inhibitors of membrane recycling and (or) intracellular processing, such as chloroquine, as well as ATP depletion, prevent not only the loss but also cause a transient increase in nucleoside transport sites apparent at the surface. Proteolytic processing, at least in the case of the nucleoside transporter, is probably also involved since leupeptin retards the loss in binding sites. Protection against the decline in transporters can also be affected by specific ligands as evidenced in ouabain protection of sodium pump sites. The results provide evidence that membrane transporter recycling is a fundamental process underlying the energy-dependent, maturation-associated loss in membrane transport functions.

Nucleoside transporters and liver cell growth

1998 ◽  
Vol 76 (5) ◽  
pp. 771-777 ◽  
Author(s):  
Marçal Pastor-Anglada ◽  
Antonio Felipe ◽  
F Javier Casado ◽  
Belén Del Santo ◽  
João F Mata ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Growth ◽  
Liver Cell ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Liver Growth ◽  
Liver Parenchymal ◽  
Hepatocyte Regeneration ◽  
Parenchymal Cells

Liver parenchymal cells show a wide variety of plasma membrane transporters that are tightly regulated by endocrine and nutritional factors. This review summarizes work performed in our laboratory on these transport systems, particularly nucleoside transporters, which are up-regulated in physiological situations associated with liver cell growth. Rat hepatocytes show a Na+-dependent nucleoside transport activity that is stimulated by pancreatic hormones. Indeed, this biological activity appears to be the result of the co-expression of at least two isoforms of nucleoside carriers, CNT1 and CNT2 (also called SPNT). These two transporters are up-regulated during the early phase of liver growth after partial hepatectomy, although to different extents, suggesting differential regulation of the two isoforms. The recent generation of isoform-specific antibodies allowed us to demonstrate that carrier expression may also have complex post-transcriptional regulation on the basis of the lack of correspondence between mRNA and protein levels. The analysis of nucleoside transport systems in hepatoma cells and the comparison with those in hepatocytes has also provided evidence that the differentiation status of liver parenchymal cells may determine the pattern of nucleoside transporters expressed.Key words: liver, hepatocyte, regeneration, cell cycle, nucleoside, plasma membrane, transport systems.

scholarly journals Subtype-specific regulation of equilibrative nucleoside transporters by protein kinase CK2

2005 ◽  
Vol 386 (2) ◽  
pp. 281-289 ◽  
Author(s):  
Meaghan STOLK ◽  
Elizabeth COOPER ◽  
Greg VILK ◽  
David W. LITCHFIELD ◽  
James R. HAMMOND
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Interacting Protein ◽  
Human Osteosarcoma Cells ◽  
Specific Regulation ◽  
Membrane Lifetime ◽  
Kinase Ck2

Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive α′ and α subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2α′ (decreased CK2α′ activity) increased the number of binding sites (∼1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (∼1.8-fold) the Vmax for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the Vmax of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2α′ activity had no effect, however, on either the KD of [3H]NBMPR binding or the Km of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4–12 h of induction of the inactive CK2α′ subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2α′ reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2α′-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.

scholarly journals Interferon-γ regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways

2003 ◽  
Vol 375 (3) ◽  
pp. 777-783 ◽  
Author(s):  
Concepció SOLER ◽  
Antonio FELIPE ◽  
José GARCÍA-MANTEIGA ◽  
Maria SERRA ◽  
Elena GUILLÉN-GÓMEZ ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Signal Transduction ◽  
Interferon Γ ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Transcriptional Level ◽  
Nucleoside Transporter ◽  
Protein Levels ◽  
Ifn Γ

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-γ (interferon-γ). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-γ was studied using macrophages from STAT1 knockout mice. IFN-γ triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-γ is required for DNA synthesis. Interestingly, IFN-γ led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-γ up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-γ is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-γ in macrophages.

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

1986 ◽  
Vol 240 (3) ◽  
pp. 879-883 ◽  
Author(s):  
M M Shi ◽  
J D Young
Keyword(s):  
Guinea Pig ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Affinity Binding ◽  
Nucleoside Transporter ◽  
High Affinity Binding ◽  
High Affinity ◽  
Nucleoside Transport Inhibitors ◽  
Transport Inhibitors ◽  
Low Sensitivity

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.

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.

Regulation of K+ current in human airway epithelial cells by exogenous and autocrine adenosine

2001 ◽  
Vol 281 (6) ◽  
pp. C1991-C2002 ◽  
Author(s):  
Artur J. Szkotak ◽  
Amy M. L. Ng ◽  
Jolanta Sawicka ◽  
Stephen A. Baldwin ◽  
S. F. Paul Man ◽  
...  
Keyword(s):  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Nucleoside Transporters ◽  
Membrane Receptors ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Airway Epithelial ◽  
Equilibrative Nucleoside Transporter ◽  
K Current ◽  
Uptake Studies

The regulatory actions of adenosine on ion channel function are mediated by four distinct membrane receptors. The concentration of adenosine in the vicinity of these receptors is controlled, in part, by inwardly directed nucleoside transport. The purpose of this study was to characterize the effects of adenosine on ion channels in A549 cells and the role of nucleoside transporters in this regulation. Ion replacement and pharmacological studies showed that adenosine and an inhibitor of human equilibrative nucleoside transporter (hENT)-1, nitrobenzylthioinosine, activated K+ channels, most likely Ca2+-dependent intermediate-conductance K+ ( I K) channels. A1 but not A2 receptor antagonists blocked the effects of adenosine. RT-PCR studies showed that A549 cells expressed mRNA for I K-1 channels as well as A1, A2A, and A2B but not A3 receptors. Similarly, mRNA for equilibrative (hENT1 and hENT2) but not concentrative (hCNT1, hCNT2, and hCNT3) nucleoside transporters was detected, a result confirmed in functional uptake studies. These studies showed that adenosine controls the function of K+ channels in A549 cells and that hENTs play a crucial role in this process.

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