The interaction of ionophore A23187 with the uridine uptake system of quiescent and serum-activated hamster fibroblasts (original) (raw)
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Nucleoside uptake in rat liver parenchymal cells
Biochemical Journal, 1996
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 ...
Cancer Research
Ar-Trifluoroacetyladriamycin-14-0-heiniadipate (AD 143), a DNA nonbinding derivative of Adriamycin, was studied for its effect on the uptake of labeled nucleosides into human leukemia (Ml.-l) and lymphoma (P3HR-1) cells in culture. After preincubation with AD 143 at concentrations as low as 5.2^M (Ml.-l) or 13 ¿IM (P3HR-1), the ability of the cells to take up extracellular labeled nucleosides was decreased by more than 50%. Similar experiments with Escherichia coli cells showed that AD 143 at the same concentrations did not have any effect. Influx of [3H]thymidine or |3H]uridine was studied by centrifugation of the cells through phthalate oil mixture, and it was found that the influx of the labeled nucleosides was decreased after treatment of the cells with AD 143. An increase in the membrane fluidity was observed after treatment of the cells with AD 143, as revealed by electron paramagnetic resonance spectroscopy studies. These observations suggest that the decreased incorporation of [3H]thymidine and |3H)uridine into acid-precipitable material that we observed earlier in the AD 143-treated cells may in part be the result of the AD 143-induced alteration of cell membrane activities, which in turn causes an inhibition of nucleoside uptake.
Biochemical Journal, 1982
A kinetic study of the inward transport of uridine in erythrocytes of rabbit, human, mouse, rat and guinea-pig demonstrated that the apparent Km of this process was similar (about 0.2mM) in these cell types, but Vmax. values differed markedly. In this array of cell types, Vmax. values were proportional to the number of transport-inhibitory, high-affinity binding sites present per cell of each type. Transport of uridine or adenosine was not detected in dog erythrocytes, nor was saturable, high-affinity binding of nitrobenzylthioinosine demonstrable. These findings demonstrate that species differences in nucleoside transport capacity are attributable to differences in the cell-surface content of functional nucleoside transport sites, rather than to differences in the kinetic properties of these sites.
European Journal of Biochemistry, 1994
Uridine transport was investigated in cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue. In intact cells, the kinetic parameters for uridine uptake were K, 150 2 45 pM, and V,,, 414 2 17 pmol . lo6 cells-' . min-I. This low affinity for uridine and its inhibition by low concentrations of nitrobenzylthioinosine (K, 3 nM) and dipyridamole (K, 54 nM) are consistent with a facilitated diffusion nucleoside transport system. The IC,* value for the adenosine transport inhibition by uridine was very high (240 pM), agreeing with the relative affinities of these nucleosides in the chromaffin cell nucleoside transport system, which was 150-fold higher for adenosine than for uridine. Uridine was significantly metabolized in chromaffin cells but not in plasma membrane vesicles. The affinity of uridine transport measured in these membrane vesicles was reproducible and similar to the affinity found for intact cells with a K,,, value of 185 % 11 pM and a V,, value of 4.2420.10 pmol . mg protein-' . s-I. These membrane preparations were employed to investigate the regulatory action of ATP and other nucleotide analogues on nucleoside transport. ATP increased the V,,, value but the K, value was not significantly modified. Adenosine 5'-[p,y-imino]triphosphate, 1 ,W-ethenoadenosine 5'-triphosphate, and adenosine(5')-tetraphospho(5')adenosine (Ap,A) at 100 pM were able to mimic the ATP effect. These results agree with a regulatory role of ATP, and the uridine transport on chromaffin plasma membrane vesicles is a good model for analyzing the nucleoside-transporter function and regulation.
Early induction of Na+-dependent uridine uptake in the regenerating rat liver
FEBS Letters, 1993
Na'-dependent uridine transport into liver plasma membrane vesicles from partially hepatectomized and sham-operated rats was studied. Preparations purified 6 h after 70% hepatectomy exhibited an increased V,,,,, of uridine uptake (3.7 vs. 1.4 pmollmg protl3 s) without any change in K,,, (6pM). Incubation of the vesicles in the presence of monensin decreased uridine uptake although the differences between both experimental groups remained identical. It is concluded that uridine transport is induced early after partial hepatectomy by a mechanism which does not involve changes in the transmembrane Na' gradient. This is the first evidence in favor of modulation of nucleoside transport into liver cells.
Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells
Biochemical Journal, 1989
Uptake of [3H]uridine by Ehrlich cells was mediated by both nitrobenzylthioinosine (NBMPR)-sensitive (75%) and NBMPR-insensitive (25%) mechanisms. Each cell contained approx. 26,000 high-affinity (KD = 0.19 nM) recognition sites for [3H]NBMPR, and binding was inhibited by dipyridamole and adenosine at concentrations similar to those required for inhibition of [3H]uridine uptake. Calculations show that each cell contains a total of about 35,000 nucleoside transporters. Photoaffinity labelling of a partially purified preparation of plasma membranes with [3H]NBMPR resulted in a single broad 3H-labelled band on SDS/polyacrylamide gels, with an apparent molecular-mass peak of 42 kDa. This is in contrast with human erythrocyte membranes, where [3H]NBMPR photolabelled two broad bands with peaks at 55 and 80 kDa. Treatment of photoaffinity-labelled membranes with endoglycosidase F decreased the apparent molecular masses of both the Ehrlich-cell and erythrocyte [3H]NBMPR-labelled proteins to...
Biochemical Pharmacology, 1973
The effects of chloropromazine, dipyridamole and 2-phenylethanol on the permeability of the cell membrane and on the metabolism of exogenous uridine by Ehrlich ascites cells were compared, by following the rate of fluorescein efflux from the cells, the incorporation of the labelled nucleoside into RNA and by determining the distribution of the label between the various metabolites. The main effect of chloropromaxine was on the passive permeability of the cell membrane, decreasing it at low concentration and increasing it at higher ones; the latter effect was enhanced by irradiation with visible light. Dipyridamole did not modify the passive permeability, but blocked the entry of the nucleoside into the cell. Phenylethanol inhibited the entry of the nucleoside and increased the cell permeability. MATERIALS AND METHODS Ehrlich ascites cells, Lipschtitz IV tetraploid strain, obtained from Arsal, Pomezia, Italy, were grown in male Swiss mice and harvested 6-8 days after i.p. transplantation of 4-7 million cells. Cell viability was checked by the dye exclusion method using trypan blue, 1 mg/ml, in 0.9% NaCl solution, according to Tennant.' Only nonhemorrhagic cell samples containing more than 90 per cent viable cells were used.