Nucleoside transporter subtype expression: effects on potency of adenosine kinase inhibitors.

1. Adenosine kinase (AK) inhibitors can enhance adenosine levels and potentiate adenosine receptor activation. As the AK inhibitors 5' iodotubercidin (ITU) and 5-amino-5'-deoxyadenosine (NH(2)dAdo) are nucleoside analogues, we hypothesized that nucleoside transporter subtype expression can affect the potency of these inhibitors in intact cells. 3. Three nucleoside transporter subtypes that mediate adenosine permeation of rat cells have been characterized and cloned: equilibrative transporters rENT1 and rENT2 and concentrative transporter rCNT2. We stably transfected rat C6 glioma cells, which express rENT2 nucleoside transporters, with rENT1 (rENT1-C6 cells) or rCNT2 (rCNT2-C6 cells) nucleoside transporters. 3. We tested the effects of ITU and NH(2)dAdo on [(3)H]-adenosine uptake and conversion to [(3)H]-adenine nucleotides in the three cell types. NH(2)dAdo did not show any cell type selectivity. In contrast, ITU showed significant inhibition of [(3)H]-adenosine uptake and [(3)H]-adenine nucleotide formation at concentrations < or =100 nM in rENT1-C6 cells, while concentrations > or =3 microM were required for C6 or rCNT2-C6 cells. 4. Nitrobenzylthioinosine (NBMPR; 100 nM), a selective inhibitor of rENT1, abolished the effects of nanomolar concentrations of ITU in rENT1-C6 cells. 5. This study demonstrates that the effects of ITU, but not NH(2)dAdo, in whole cell assays are dependent upon nucleoside transporter subtype expression. Thus, cellular and tissue differences in expression of nucleoside transporter subtypes may affect the pharmacological actions of some AK inhibitors.

Purine uptake and release in rat C6 glioma cells: nucleoside transport and purine metabolism under ATP-depleting conditions.

Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, (3)H-nucleoside uptake experiments, and [(3)H]nitrobenzylthioinosine ([(3)H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [(3)H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [(3)H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [(3)H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP --> IMP --> inosine --> hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.