Nucleoside uptake in rat liver parenchymal cells

Abstract

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.

Differential expression and regulation of nucleoside transport systems in rat liver parenchymal and hepatoma cells

“…Moreover, a high nucleoside uptake through Na ϩ -independent transport systems should not be considered as a determinant feature of highly proliferating liver cells. Indeed, the Na ϩ -dependent transport activity, which has been characterized in detail both in liver plasma membrane vesicles and in hepatocytes, [30][31][32]35,43 is significantly induced (nearly a threefold increase in V max ) in the prereplicative phase of liver growth after partial hepatectomy. 33 Moreover, pathophysiological conditions associated with liver hypertrophia, like genetic obesity, are also characterized by a selective induction of the Na ϩ -dependent transport activity for nucleoside uptake.…”

Enhancement of fludarabine sensitivity by all-trans-retinoic acid in chronic lymphocytic leukemia cells

“…Uptake of H]-fludarabine (1 μM) was measured using a rapid filtration method adapted from a previously described technique. 38 Cells were resuspended in either a sodium chloride buffer or a choline chloride buffer. Each uptake assay started by mixing a cell suspension with an equal volume of buffer, supplemented with 1 μM [8- ]-mannitol (specific activity 4,000 dpm/pmol) was included in the incubation medium to assess the amount of extracellular medium that became trapped in the bottom acidic layer.…”

Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia

“…26 Cells were washed and resuspended in either a sodium or a choline chloride buffer, as previously reported. Uptake assays were started by mixing cell suspensions with an equal volume of buffer, supplemented with a radionucleoside at a specific activity of 4000 dpm/pmol for JVM-2 cells and 17 500 dpm/pmol for CLL cells.…”