Nucleosides need to be transported into cells either to provide precursors for purine nucleotide biosynthesis by salvage pathways or to regulate the extracellular concentration of adenosine, which in turn modulates purinergic receptors implicated in a variety of physiological processes. High-affinity Na⁺-dependent concentrative nucleoside transport was initially thought to be restricted to (re)absorptive epithelia, thus mediating vectorial transport of nucleosides. We kinetically characterized and later cloned a hepatic pyrimidine-preferring nucleoside transporter, CNT1, that appeared to be up-regulated during liver regeneration. CNT1 is targeted to the apical membrane through the hepatic transcytotic pathway, whereas the purine-preferring high affinity nucleoside transporter CNT2 is mostly located at the basolateral membrane of the hepatocyte (Duflot et al. 2002), in which it seems to modulate extracellular adenosine availability to A1 type receptors. CNT expression is up-regulated by hepatomitogens and multifunctional cytokines such as IL-6 and TNF-α, both in cultured cells and in vivo. Although CNT (particularly CNT1) protein amounts change when progressing into the cell cycle, this may be the result of adaptive responses to alterations in nucleotide metabolism, as recently shown in hepatoma cells synchronized by inhibiting ribonucleotide reductase with hydroxyurea (Valdés et al. 2002). Differentiation is indeed the major determinant of nucleoside transporter expression in hepatocytes, as deduced from the analysis of CNT expression in cultured rat fetal hepatocytes induced to differentiate (del Santo et al. 2001) and in hepatocarcinogenesis (del Santo et al. 1998; Dragan et al. 2000). Non-epithelial cells, such as macrophages, also express CNT1, CNT2 and the equilibrative nucleoside transporters ENT1 and ENT2. We recently took advantage of this feature to demonstrate that ENT1 appears to be responsible for nucleoside channelling into DNA and thus required for proliferation, whereas CNT1 and CNT2 are up-regulated after macrophage activation (Soler et al. 2001a,b). Thus, CNT and ENT proteins are differentially regulated and may play different roles in cell physiology. High-affinity uptake of nucleoside-derived anticancer drugs is also mediated by CNT-type proteins, as recently described using the two electrode voltage clamp technique on Xenopus laevis oocytes expressing selected CNT isoforms (Lostao et al. 2000; Mata et al. 2001). Heterologous expression of CNT1 confers sensitivity to CNT1 substrates even in cells overexpressing the ENT1 protein (Mata et al. 2001). Although ENT1 activity has recently been correlated with ex vivo sensitivity of chronic lymphocytic leukaemia (CLL) cells to fludarabine (Molina-Arcas et al. 2002), retention of high affinity CNT1-mediated transport may confer increased sensitivity to nucleoside-derived drugs. We are currently addressing this issue by analyzing CNT1 expression in human tumors using a tissue array approach.

The Regulation of Transport Systems Research Group is currently funded by Ministerio de Ciencia y Tecnolog’a (Spain), Ministerio de Sanidad y Consumo (Spain), Generalitat de Catalunya (Catalonia, Spain), Fundación Ramón Areces and Universitat de Barcelona.