Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells

Abstract

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.

Regulation of Nucleoside Transport by Lipopolysaccharide, Phorbol Esters, and Tumor Necrosis Factor-α in Human B-lymphocytes

“…The ultimate physiological role of this response requires further analysis, although a possible relationship between cell growth and up-regulation of concentrative nucleoside transporters has recently been shown in other cell types (19,46,47). These actions appear to be dependent on protein kinase C activation, which is consistent with previous reports based on HL-60 cells induced to differentiate (20 -22).…”

“…CNT1 and CNT2 are coexpressed in absorptive epithelia, liver, and, probably, brain (14 -18). Coexpression of two isoforms in a single cell type may also involve isoform-specific regulation, as recently shown in liver parenchymal cells for the CNT1-and CNT2-related carrier proteins (19).…”

Lipopolysaccharide-induced Apoptosis of Macrophages Determines the Up-regulation of Concentrative Nucleoside Transporters Cnt1 and Cnt2 through Tumor Necrosis Factor-α-dependent and -independent Mechanisms

“…Proteins were transferred onto filters (Immobilon-P, Millipore, New Bedford, MA), which were then blocked in a 5% dry milk-supplemented 0.2% Tween 20, phosphate-buffered saline prior to immunoreaction. To monitor CNT1 expression, a monospecific rabbit polyclonal antibody raised against an N terminus fragment (residues 45-67) of the corresponding rat horthologue was used (37,38). This antibody specifically cross-reacted with the murine transporter as described previously (50).…”

“…After high stringency washing (31,36,37), filters were exposed, and films were processed for densitometric analysis.…”

Nitric oxide regulates nucleoside transport in activated B lymphocytes

“…N1 is involved in the Na ϩ -dependent uptake of purine nucleosides, its activity is associated with the protein product of the concentrative nucleoside transporter 2 (CNT2) sodium purine nucleoside transporter (SPNT) cDNA [6,7], and it is mostly expressed in epithelial tissues such as small intestine, kidney, and liver [6][7][8]. In liver cells, N1 activity and CNT2 mRNA levels appear to be dependent on cell cycle progression [9], and they are modulated by the endocrine and nutritional status of the cell [10,11].…”