Molecular Cloning and Functional Characterization of Nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) Equilibrative Nucleoside Transporter Proteins (rENT1 and rENT2) from Rat Tissues

Abstract

Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we isolated a cDNA from human placenta encoding the 456-residue glycoprotein hENT1. When expressed in Xenopus oocytes, hENT1 mediated es-type transport activity and was inhibited by coronary vasoactive drugs (dipyridamole and dilazep) that may compete with nucleosides and NBMPR for binding to the substrate binding site. We now report the molecular cloning and functional expression of es and ei homologs of hENT1 from rat tissues; rENT1 (457 residues) was 78% identical to hENT1 in amino acid sequence, and rENT2 (456 residues) was 49 -50% identical to rENT1/hENT1 and corresponded to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in truncated form. rENT1 was inhibited by NBMPR (IC 50 ‫؍‬ 4.6 nM at 10 M uridine), whereas rENT2 was NBMPR-insensitive (IC 50 > 1 M). Both proteins mediated saturable uridine influx (K m ‫؍‬ 0.15 and 0.30 mM, respectively), were broadly selective for purine and pyrimidine nucleosides, including adenosine, and were relatively insensitive to inhibition by dipyridamole and dilazep (IC 50 > 1 M). These observations demonstrate that es and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.

Host-Based Ribavirin Resistance Influences Hepatitis C Virus Replication and Treatment Response

“…9). Treatment with 100 M NBMPR, which inhibits ENT1 and ENT2 (34,52), yielded similar results. Therefore, ENT1 is a major RBV transporter in primary human PBMCs.…”

“…1D). No significant reduction in RBV uptake was observed with a higher concentration of NBMPR (100 M), which inhibits both ENT1 and ENT2 (34,52). While the literature indicates that NBMPR treatment specifically inhibits ENT-mediated transport, we cannot exclude the remote possibility that another NBMPR-sensitive RBV transporter exists (34,52).…”

“…No significant reduction in RBV uptake was observed with a higher concentration of NBMPR (100 M), which inhibits both ENT1 and ENT2 (34,52). While the literature indicates that NBMPR treatment specifically inhibits ENT-mediated transport, we cannot exclude the remote possibility that another NBMPR-sensitive RBV transporter exists (34,52). Inhibition of concentrative transport through phloridzin treatment, an inhibitor of sodium-dependent nucleoside transport (25,47), did not reduce RBV uptake in cells, unless they expressed CNT3 via transfection ( Fig.…”

Adenosine Signaling in Striatal Circuits and Alcohol Use Disorders

“…These types include ENT1, ENT2, ENT3, ENT4, and CNT1, CNT2, CNT3. ENT1 is sensitive to nanomolar concentrations of nitrobenzylthioinosine (nitrobenzylmercaptopurine riboside; NBMPR), whereas ENT2 is resistant to NBMPR up to 1 mM (Baldwin et al, 1999;Yao et al, 1997). Furthermore, ENT1 and ENT2 are widely expressed throughout the central nervous system (Jennings et al, 1998;, while ENT3 appears to be expressed outside the CNS (Baldwin et al, 2005).…”

Functional characterization of a H+/nucleoside co‐transporter (CaCNT) from Candida albicans, a fungal member of the concentrative nucleoside transporter (CNT) family of membrane proteins

“…Two ENT and three CNT functional isoforms have been identified. Human (h) and rat (r) ENT1 and ENT2 transport pyrimidine and purine nucleosides and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs, and by the ability of hENT2 and rENT2 to also transport nucleobases (Griffiths et al, 1997a(Griffiths et al, , 1997bYao et al, 1997;Crawford et al, 1998;Yao et al, 2002a). CNT1 and CNT2 both transport uridine and adenosine, but are otherwise selective for pyrimidine (hCNT1 and rCNT1) and purine (hCNT2 and rCNT2) nucleosides (Huang et al, 1994;Che et al, 1995, Yao et al, 1996aWang et al, 1997;Ritzel et al, 1997Ritzel et al, , 1998.…”