Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta

Abstract

Mammalian equilibrative nucleoside transporters are typically divided into two classes, es and ei, based on their sensitivity or resistance respectively to inhibition by nitrobenzylthioinosine (NBMPR). Previously, we have reported the isolation of a cDNA clone encoding a prototypic es-type transporter, hENT1 (human equilibrative nucleoside transporter 1), from human placenta. We now report the molecular cloning and functional expression in Xenopus oocytes of a cDNA from the same tissue encoding a homologous ei-type transporter, which we designate hENT2. This 456-residue protein is 46% identical in amino acid sequence with hENT1 and corresponds to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in a form bearing a sequence deletion. In addition to placenta, hENT2 is found in brain, heart and ovarian tissue. Like hENT1, hENT2 mediates saturable transport of the pyrimidine nucleoside uridine (Km 0.2±0.03 mM) and also transports the purine nucleoside adenosine. However, in contrast with hENT1, which is potently inhibited by NBMPR (Ki 2 nM), hENT2 is NBMPR-insensitive (IC50 < 1 μM). It is also much less sensitive to inhibition by the coronary vasoactive drugs dipyridamole and dilazep and to the lidoflazine analogue draflazine, properties that closely resemble those reported for classical ei-type transport in studies with intact cells.

Excretion of biliary compounds during intrauterine life

“…There is evidence of the presence of two ENT isoforms in the human placenta: ENT1 (SLC29A1) and ENT2 (SLC29A2) [116,117] . Moreover, concentrative nucleoside transporters (CNTs), CNT2 (SLC28A2) and CNT3 (SLC28A3) are also expressed in human placenta.…”

“…Moreover, concentrative nucleoside transporters (CNTs), CNT2 (SLC28A2) and CNT3 (SLC28A3) are also expressed in human placenta. Both ENT1 and ENT2 are able to transport a wide variety of therapeutic agents such as the anticancer drugs cytarabine and gemcitabine and the antiviral drugs zalcitabine (ddC) and zidovudine [117,118] , and they therefore probably play a role in fetal exposure to these types of drugs.…”

Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy

“…The isolation of cDNAs encoding hENT1 (Griffiths et al, 1997a) and hENT2 (Griffiths et al, 1997b;Crawford et al, 1998) permitted detailed molecular and functional characterization of these transporters. The hENT1 cDNA encodes a protein with 11 transmembrane domains and a single extracellular site for glycosylation (Griffiths et al, 1997a, b;Yao et al, 1997;Kiss et al, 2000;Handa et al, 2001).…”

Nucleoside analogues: mechanisms of drug resistance and reversal strategies

“…The two cloned hENTs differ in their sensitivity to inhibition by nanomolar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR): hENT1 has es activity (equilibrative and NBMPR-sensitive) while hENT2 possesses ei (equilibrative and NBMPR-insensitive) nucleoside transport activity. [12][13][14] The human concentrative nucleoside transporters (hCNT) are inwardly-directed transporters that are capable of transporting nucleosides against a concentration gradient by utilizing the transmembrane sodium concentration gradient. [15][16][17] The hCNT1 protein has greater affinity for pyrimidine nucleosides but also transports adenosine, while the hCNT2 protein transports purine nucleosides and uridine.…”