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 Nucleoside uptake in rat liver parenchymal cells

1996 ◽  
Vol 317 (3) ◽  
pp. 835-842 ◽  
Author(s):  
Joan MERCADER ◽  
Mireia GOMEZ-ANGELATS ◽  
Belén del SANTO ◽  
Javier CASADO ◽  
Antonio F. FELIPE ◽  
...  
Keyword(s):  
Rat Liver ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Dependent Manner ◽  
Transport Activity ◽  
Liver Parenchymal ◽  
Uridine Uptake ◽  
High Concentrations ◽  
Parenchymal Cells ◽  
Dose Dependent

Rat liver parenchymal cells express Na+-dependent and Na+-independent nucleoside transport activity. The Na+-dependent component shows kinetic properties and substrate specificity similar to those reported for plasma membrane vesicles [Ruiz-Montasell, Casado, Felipe and Pastor-Anglada (1992) J. Membr. Biol. 128, 227–233]. This transport activity shows apparent Km values for uridine in the range 8–13 μM and a Vmax of 246 pmol of uridine per 3 min per 106 cells. Most nucleosides, including the analogue formycin B, cis-inhibit Na+-dependent uridine transport, although thymidine and cytidine are poor inhibitors. Inosine and adenosine inhibit Na+-dependent uridine uptake in a dose-dependent manner, reaching total inhibition. Guanosine also inhibits Na+-dependent uridine uptake, although there is some residual transport activity (35% of the control values) that is resistant to high concentrations of guanosine but may be inhibited by low concentrations of adenosine. The transport activity that is inhibited by high concentrations of thymidine is similar to the guanosine-resistant fraction. These observations are consistent with the presence of at least two Na+-dependent transport systems. Na+-dependent uridine uptake is sensitive to N-ethylmaleimide treatment, but Na+-independent transport is not. Nitrobenzylthioinosine (NBTI) stimulates Na+-dependent uridine uptake. The NBTI effect involves a change in Vmax, it is rapid, dose-dependent, does not need preincubation and can be abolished by depleting the Na+ transmembrane electrochemical gradient. Na+-independent uridine transport seems to be insensitive to NBTI. Under the same experimental conditions, NBTI effectively blocks most of the Na+-independent uridine uptake in hepatoma cells. Thus the stimulatory effect of NBTI on the concentrative nucleoside transporter of liver parenchymal cells cannot be explained by inhibition of nucleoside efflux.

scholarly journals Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells

1998 ◽  
Vol 330 (2) ◽  
pp. 997-1001 ◽  
Author(s):  
Antonio FELIPE ◽  
Raquel VALDES ◽  
Belén del SANTO ◽  
Jorge LLOBERAS ◽  
Javier CASADO ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Polyclonal Antibodies ◽  
High Specificity ◽  
Liver Cells ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Plasma Membrane Vesicles

Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the ‘epithelial-specific isoform’ called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization.

Quantitative Estimation of the Loss of Membrane Images Resulting From Oblique Sectioning of Biological Membranes

1967 ◽  
Vol 25 ◽  
pp. 144-145
Author(s):  
Alden V. Loud
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Liver Cell ◽  
Quantitative Estimation ◽  
Simple Method ◽  
Thin Sections ◽  
Liver Parenchymal ◽  
Qualitative Interpretation ◽  
Considerable Length ◽  
Parenchymal Cells

Williams and Kallman pointed out one of the major artifacts in the electron microscopy of biological thin sections, namely, the failure to form images of membranes which are inclined at large angles within the section. This loss is a significant consideration in the qualitative interpretation of electron micrographs and especially in the quantitative assay of endoplasmic reticulum and mitochondrial cristae membranes. In order to estimate the effective loss of membrane images it would be desirable to use a specimen which provides a considerable length of membrane tilted at a known angle and a simple method of measuring its “visibility”. The spherical nuclear envelopes of rat liver parenchymal cells satisfy these conditions. Figure 1 shows part of a binucleate liver cell in which the nuclear membrane is clearly visible around the larger section but blurred by oblique orientation in the smaller section.

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 The role of the plasma membrane in fatty acid uptake by rat liver parenchymal cells

1971 ◽  
Vol 123 (5) ◽  
pp. 837-844 ◽  
Author(s):  
J. D. Wright ◽  
C. Green
Keyword(s):  
Plasma Membrane ◽  
Palmitic Acid ◽  
Membrane Fraction ◽  
Specific Radioactivity ◽  
Fatty Acid Uptake ◽  
Acid Uptake ◽  
Liver Parenchymal ◽  
Plasma Membrane Fraction ◽  
Parenchymal Cells

1. Suspensions of isolated rat liver parenchymal cells incorporate [14C]palmitic acid into glycerides at about 40% of the rate obtained with liver slices. 2. At short time-intervals most of the incorporation is into phosphatidylcholine and this is recovered mainly in the plasma-membrane fraction. 3. At later times (5min to 2h) the [14C]palmitic acid is mainly found in triglyceride, but this is not recovered in the plasma-membrane fraction. 4. Addition of lysophosphatidylcholine increases incorporation of palmitic acid into both phosphatidylcholine and triglyceride, with maximum effect at about 0.1mm. 5. In vivo, 1min after injection of [14C]palmitic acid, radioactive phosphatidylcholine is concentrated in the plasma-membrane fraction, but the proportion present in this fraction declines rapidly. 6. The phosphatidylcholine of the plasma-membrane fraction has, at 1min after injection, a specific radioactivity 30-fold greater than that of the whole tissue. 7. This phosphatidylcholine reaches its maximum specific radioactivity before the tissue phosphatidic acid or diglyceride. 8. The phosphatidylcholine of the plasma-membrane fraction has a very rapid turnover. 9. It is proposed that the rapid formation of phospholipids in the plasma membrane is by acylation of their lyso-derivatives and the role of this process in fatty acid uptake is discussed.

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.

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