Characterization of ribavirin uptake systems in human hepatocytes.
OBJECTIVE
The purpose of this study was to identify the major ribavirin uptake transporter(s) in human hepatocytes and to determine if these previously unidentified transporters are involved in hepatic ribavirin uptake. Furthermore, we aimed to address what causes the difference in uptake levels among human hepatocytes.


METHODS
Profiles of ribavirin uptake and nucleoside transporter mRNA expression in Caucasian hepatocytes (HH268, HH283 and HH291) were characterized by transport assay and reverse transcription-polymerase chain reaction (RT-PCR). The 5'-side of the SLC29A1 gene structure was characterized by determination of transcription start sites and by RT-PCR.


RESULTS
Equilibrative nucleoside transporter 1 (ENT1)-mediated uptake was exclusively involved in ribavirin uptake in HH268 and HH283 and was responsible for the largest ribavirin uptake fraction in HH291. The level of ENT1-mediated uptake in HH291 was higher than that in HH268 and HH283. Characterization of the SLC29A1 gene structure revealed the existence of several ENT1 mRNA isoforms in the human liver, and the levels of four ENT1 mRNA isoforms in HH291 were higher than those in HH268 or HH283. No ENT2-mediated uptake was observed in any hepatocyte lines. Na(+)-dependent uptake was detected only in HH291; however, mRNA levels of concentrative nucleoside transporters (CNTs) were at trace levels in all hepatocyte lines.


CONCLUSIONS
ENT1, but not ENT2 or CNTs, is a major ribavirin uptake transporter in human hepatocytes. The different ENT1-mediated ribavirin uptake levels in different hepatocyte lines are associated with different expression levels of specific isoforms of ENT1 mRNAs. Furthermore, an unidentified Na(+)-dependent ribavirin transport system might exist in human hepatocytes.
Fukuchi
Yukina
Y
Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba-shi, Chiba, Japan.
Furihata
Tomomi
T
Hashizume
Misato
M
Iikura
Minami
M
Chiba
Kan
K
D020121:5' Untranslated Regions / Q000235:genetics; D000998:Antiviral Agents / Q000493:pharmacokinetics*; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics* / Q000378:metabolism; D033722:Equilibrative-Nucleoside Transporter 2 / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012254:Ribavirin / Q000493:pharmacokinetics*; D012964:Sodium / Q000378:metabolism; D014162:Transfection
D020121:5' Untranslated Regions; D000998:Antiviral Agents; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C438649:SLC29A1 protein, human; C438654:SLC29A2 protein, human; C065171:cif nucleoside transporter; D012254:Ribavirin; D012964:Sodium