Primary cultures of rat-liver parenchymal cells show carrier-mediated nucleoside uptake by a mechanism that mainly involves concentrative, Na ؉ -dependent transport activity. In contrast, the hepatoma cell line FAO shows high nucleoside transport activity, although it is mostly accounted for by Na ؉ -independent transport processes. This is associated with a low amount of sodium purine nucleoside transporter (SPNT) mRNA. SPNT encodes a purinepreferring transporter expressed in liver parenchymal cells. To analyze whether SPNT expression is modulated during cell proliferation, SPNT mRNA levels were determined in the early phase of liver growth after partial hepatectomy and in synchronized FAO cells that had been induced to proliferate. SPNT mRNA amounts increased as early as 2 hours after partial hepatectomy. FAO cells induced to proliferate after serum refeeding show an increase in SPNT mRNA levels, which is followed by an increase in Na ؉ -dependent nucleoside uptake and occurs before the peak of 3 H-thymidine incorporation into DNA. FAO cells also express significant equilibrative nucleoside transport activity, which may be accounted for by the expression of the nitrobenzylthioinosine (NBTI)-sensitive and -insensitive isoforms, rat equilibrative nucleoside transporter 1 (rENT1) and rENT2, respectively. Interestingly, rENT2 mRNA levels follow a similar pattern to that described for SPNT when FAO cells are induced to proliferate, whereas rENT1 appears to be constitutively expressed. Liver parenchymal cells show low and negligible mRNA levels for rENT1 and rENT2 transporters, respectively, although most of the equilibrative transport activity found in hepatocytes is NBTI-resistant. It is concluded that: 1) SPNT expression is regulated both in vivo and in vitro in a way that appears to be dependent on cell cycle progression; 2) SPNT expression may be a feature of differentiated hepatocytes; and 3) equilibrative transporters are differentially regulated, rENT2 expression being cell cycle-dependent. This is consistent with its putative role as a growth factor-induced delayed early response gene. (HEPATOLOGY 1998;28:1504-1511.)Nucleosides and nucleoside analogues have a wide range of potent physiological and pharmacological properties. Purines, essentially adenosine, play a multifactorial role in liver physiology by modulating key metabolic pathways of the hepatocyte 1-5 and influencing, among other functions, hepatic arterial pressure-flow autoregulation, 6 vasodilation, 7 and superoxide anion generation. 8

Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the 'epithelial-specific isoform' called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization.

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Na(+)-dependent uridine uptake is stimulated in isolated rat liver parenchymal cells by glucagon. This effect is transient, reaches maximum levels of stimulation 10 min after hormone addition, and is dose-dependent. Glucagon action can be mimicked by agents that are able to hyperpolarize the plasma membrane (e.g. monensin) and by dibutyryl cyclic AMP. The effects triggered by glucagon, monensin and dibutyryl cyclic AMP are not additive, suggesting a common mechanism of action. 8-(4-Chloro-phenylthio)adenosine 3':5'-cyclic monophosphate (PCT), a cyclic AMP analogue but also a nucleoside analogue, markedly stimulates Na(+)-dependent uridine uptake in an additive manner to that triggered by monensin, similarly to the effect described for nitrobenzylthioinosine. Considering the roles reported for nucleosides in liver metabolism, the use of PCT as a cyclic AMP analogue should be precluded. Insulin is also about to up-regulate Na(+)-dependent uridine uptake by a mechanism which involves a stable induction of this transport activity at the plasma-membrane level. This is consistent with a mechanism involving synthesis and insertion of more carriers into the plasma membrane. It is concluded that the recently characterized hepatic concentrative nucleoside transporter is under short-term hormonal regulation by glucagon, through mechanisms which involve membrane hyperpolarization, and under long-term control by insulin. This is the first report showing hormonal modulation of the hepatic concentrative nucleoside transporter.

Liver parenchymal cells show a wide variety of plasma membrane transporters that are tightly regulated by endocrine and nutritional factors. This review summarizes work performed in our laboratory on these transport systems, particularly nucleoside transporters, which are up-regulated in physiological situations associated with liver cell growth. Rat hepatocytes show a Na+-dependent nucleoside transport activity that is stimulated by pancreatic hormones. Indeed, this biological activity appears to be the result of the co-expression of at least two isoforms of nucleoside carriers, CNT1 and CNT2 (also called SPNT). These two transporters are up-regulated during the early phase of liver growth after partial hepatectomy, although to different extents, suggesting differential regulation of the two isoforms. The recent generation of isoform-specific antibodies allowed us to demonstrate that carrier expression may also have complex post-transcriptional regulation on the basis of the lack of correspondence between mRNA and protein levels. The analysis of nucleoside transport systems in hepatoma cells and the comparison with those in hepatocytes has also provided evidence that the differentiation status of liver parenchymal cells may determine the pattern of nucleoside transporters expressed.