Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells

Abstract

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the ‘equilibrative sensitive’ or ‘es’ transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1_rat) of rat intestine in COS-1 cells. The production of recombinant cNT1_rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of ³H-labelled nucleosides. Recombinant cNT1_rat was sodium-dependent and selective for pyrimidine nucleosides, with approximate K_m values of 21 μM, 12.5 μM and 15 μM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its V_max value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1_rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1_rat is a representative, may play a role in cellular uptake of these drugs. The cNT1_rat/COS-1 expression system is a useful tool for analysis of cNT1_rat-mediated transport processes.