Developmental regulation of the concentrative nucleoside transporters CNT1 and CNT2 in rat liver

Abstract

Background/Aims: The pattern of nucleoside transporter expression in hepatocytes was studied in the developing rat liver.

Methods: Hepatocytes isolated from fetuses, neonates and adult rats were used for uridine uptake measurements and concentrative nucleoside transporter (CNT) expression.

Results: Adult hepatocytes showed the highest Na⁺-dependent uridine uptake, but fetal hepatocytes exhibited a significant NBTI-sensitive component of equilibrative Na⁺-independent transport, which was either negligible or absent in neonatal and adult rat hepatocytes. Low Na⁺-dependent uridine uptake was associated with low amounts of CNT1 and CNT2 transporter proteins, both with apparent K_m values in the low micromolar range. Hepatocyte primary cultures from 20-day-old fetuses showed very low amounts of CNT2 mRNA, and expressed both carrier proteins. Incubation of fetal hepatocytes with dexamethasone and T₃ resulted in a significant increase in Na⁺-dependent uridine uptake and an accumulation of the CNT2 protein and mRNA.

Conclusions: The expression of concentrative nucleoside carriers in hepatocytes from developing rat liver is developmentally regulated. Addition of endocrine factors known to induce differentiation of fetal hepatocytes results in selective up-regulation of CNT2 expression.

Introduction

Plasma membrane transporters selectively up-regulate their activity during liver cell proliferation, probably in response to either increased metabolic needs or osmotic challenges during cell cycle progression. One of these transport systems, system A for neutral amino acid transport, is up-regulated soon after partial hepatectomy in rat liver plasma membrane vesicles in a manner that is consistent with de novo synthesis of carrier proteins [1]. Selective activation of Na⁺-dependent concentrative transporters is counterbalanced by the early increase in Na,K-ATPase activity that follows the accumulation of both α1 and β1 pump subunit protein and mRNA[2].

In an attempt to determine whether other plasma membrane carriers show this type of regulatory adaptation during liver cell growth, we initially characterized nucleoside transport activity in rat liver plasma membrane vesicles [3], showed that this activity was regulated by the endocrine status of the animal, both in vivo [4], [5] and in isolated hepatocytes [5], and found that it was up-regulated simultaneously to system A during the early phase of liver regeneration after partial hepatectomy [4], [6]. More recently, it has been established that the transport of nucleosides into liver parenchymal cells is mediated by at least two Na⁺-dependent transport systems [7], which are likely to be CNT2 (SPNT), initially cloned as a ‘liver’ specific nucleoside transporter [8], and CNT1 [9], previously characterized as a specific transporter from absorptive epithelia, such as small intestine and kidney [10] CNT2 mRNA levels increase only 2 h after partial hepatectomy in the rat [4] and it has recently been shown that CNT2 may be a cell-cycle-dependent regulated gene [11]. Polyclonal isoform-specific antibodies against CNT1 and CNT2 proteins [9] have allowed us to show that they are differentially regulated during liver regeneration [4], whereas a selective loss of nucleoside carrier expression has been reported in hepatoma cell lines [11].

The possibility that the expression of the concentrative nucleoside carrier isoforms CNT1 and CNT2 may be dependent on the differentiation and proliferation status of the hepatocyte prompted us to analyze a physiological model of liver growth and differentiation: fetal and postnatal development in the rat. We also tried to determine whether differentiation of fetal parenchymal cells in culture is associated with nucleoside carrier up-regulation.