Abstract
The HepaRG cell line is a highly differentiated human hepatoma cell line, displaying the expression of various drug transporters. However, functional expression of nucleoside transporters remains poorly characterized in HepaRG cells, although these transporters play a key role in hepatic uptake of antiviral and anticancer drugs. The present study was, therefore, designed to characterize the expression, activity and regulation of equilibrative (ENT) and concentrative (CNT) nucleoside transporter isoforms in differentiated HepaRG cells. These cells were found to exhibit a profile of nucleoside transporter mRNAs similar to that found in human hepatocytes, i.e., notable expression of ENT1, ENT2 and CNT1, with very low or no expression of CNT2 and CNT3. ENT1 activity was, next, demonstrated to be the main uridine transport activity present in HepaRG cells, like in cultured human hepatocytes. Various physiological factors, such as protein kinase C (PKC) activation or treatment by inflammatory cytokines or hepatocyte growth factor (HGF), were additionally found to regulate expression of ENT1, ENT2 and CNT1; PKC activation and HGF notably concomitantly induced mRNA expression and activity of ENT1 in HepaRG cells. Overall, these data suggest that HepaRG cells may be useful for analyzing cellular pharmacokinetics of nucleoside-like drugs in human hepatic cells, especially of those handled by ENT1.
Highlights
The hepatic cell line HepaRG is an original human hepatoma cell line, which expresses various liver-specific functions when cultured in appropriate conditions, i.e., in the presence of 2% dimethyl sulfoxide (DMSO) [1]
ENT1 and ENT2 mediate passive bidirectional transport of purine and pyrimidine nucleosides down a concentration gradient across the plasma membrane; ENT3 transports nucleosides across intracellular membranes, notably those of lysosomes [12], whereas ENT4, known as plasma membrane monoamine transporter (PMAT), mainly functions as a polyspecific organic cation transporter [13], handling only adenosine among nucleosides [14]
Expression of nucleoside transporter mRNAs was determined by RT-Quantitative polymerase chain reaction (qPCR) in proliferating HepaRG cells and in differentiated HepaRG cells, as well as in freshly isolated hepatocytes and primary cultured human hepatocytes
Summary
The hepatic cell line HepaRG is an original human hepatoma cell line, which expresses various liver-specific functions when cultured in appropriate conditions, i.e., in the presence of 2% (vol/vol) dimethyl sulfoxide (DMSO) [1] These hepatoma cells have, been proposed as surrogates for the use of primary human hepatocytes, especially for xenobiotic metabolism and toxicity studies [2,3]. Whether HepaRG cells exhibit functional expression and regulation of nucleoside transporters remains unknown This point is worthy of interest, owing to (i) the established role played by nucleoside transporters in pharmacokinetics and toxicity of various drugs and xenobiotics; and (ii) the growing use of HepaRG cells as surrogates for human hepatocytes in pharmacological and toxicological studies [2,3]. The present study was designed to characterize the expression of plasma membrane nucleoside transporters, i.e., ENT1, ENT2, CNT1, CNT2 and CNT3, in HepaRG cells, as well as the putative regulation of some of them by factors known to modulate drug transporter expression in human hepatic cells, like protein kinase C (PKC) activation [23] or inflammatory cytokine treatment [24]
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