Mouse Equilibrative Nucleoside Transporter 2 (mENT2) Transports Nucleosides and Purine Nucleobases Differing from Human and Rat ENT2

Abstract

Nucleosides and nucleobases are important precursors for nucleotide synthesis.1) Individual nucleosides and nucleobases also serve a variety of specialized functions. [2][3][4][5] Various structural analogues of nucleoside and nucleobase are cytotoxic and have found expanding therapeutic use as antineoplastic agents.6) Nucleosides and nucleobases are hydrophilic and require specific transporters for permeation through the cell membrane. [7][8][9] Several mammalian nucleoside transporters, which are expressed in the plasma membrane of cells, have been cloned.9) The detailed transport characteristics of human and rat equilibrative nucleoside transporter 2 (hENT2 and rENT2, respectively) have been examined, and they transported a broad range of natural nucleosides and their analogues in Na ϩ -independent and nitrobenzylmercaptopurine riboside (NBMPR)-resistant fashions. [10][11][12] Furthermore, hENT2 and rENT2 were previously reported to transport not only nucleosides but also purine and pyrimidine nucleobases.13) However, the transport of nucleosides and nucleobases via mouse ENT2 (mENT2) was not characterized in detail, except for the sensitivity of natural nucleosides and cardioprotective agents to uridine uptake mediated by the transporter.14) The transport mechanisms of various nucleosides and nucleobases in mouse primary-cultured cells have been investigated.15) Furthermore, tumor cells-bearing mouse has been used for the developments of improved chemotherapeutic strategies.16) Therefore, the functional characterization of mENT2 using a variety of nucleosides and nucleobases is physiologically and pharmacologically important. Recently, we clarified that the uracil uptake by mENT2-overexpressing Cos-7 cells (Cos-7/ENT2) for 2 min was similar to that by mock cells (Cos-7/pCI-neo), 17) although uracil was reported to be a substrate for hENT2 and rENT2.13) These findings hypothesized that there are species differences in transport characteristics of ENT2.In this study, therefore, we evaluated the transport of nucleosides and nucleobases mediated by recombinant mENT2. Cell Culture Cos-7 cells were maintained in Dulbecco's modified Eagle's MEM (Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (MP Biochemicals, LLC., CA, U.S.A.) at 37°C under a humidified atmosphere of 5% CO 2 in air. MATERIALS AND METHODS ChemicalsGeneration of mENT2-Transfectants As described previously, a cDNA of mENT2 subcloned into pCI-neo expression vector (Promega Co., WI, U.S.A.) was introduced into Cos-7 cells using LipofectAMINE2000 (Invitrogen, CA, U.S.A.).17) The peptide encoded by cloned mENT2 was identical to that previously reported. 14)Uptake Assay The uptake assaying was performed by the modified method of Nagasawa et al.18) The uptake reaction was initiated by adding 3 H-labeled nucleoside or nucleobase (1 mCi/ml), or 14 C-labeled hypoxanthine (0.1 mCi/ml) to the cells. In the case of nucleoside uptake, the cells were treated with 100 nM NBMPR to completely block the uptake mediated by ENT1 in Cos-...

Characterization of Nucleobase Transport by Mouse Sertoli Cell Line TM4

“…It is possible that these systems may have the dual capability of transporting both nucleobases and corresponding nucleosides, as observed in ENT2. Recently, it was reported that purine-selective nucleoside transporter mouse ENT2 transported purine nucleobase as well as purine nucleosides, 43) which is consistent with the observation in TM4 cells. Although, we have reported that primary-cultured rat Sertoli cells contain similar purine-or pyrimidine-selective transport systems, 29) the characteristics such as K m values and sodium-dependence in guanine uptake were different from those of TM4 cells.…”

“…However, guanine transport in TM4 cells is inhibited only by purine nucleobases, which is different from mouse ENT2 that is inhibited by both of purine-and pyrimidine-nucleobases. 43) From these results, it appears that nucleobase transport in TM4 cells is similar but molecularly different from mouse ENT2.…”

“…Recently, it was reported that mouse ENT2 transports only purine nucleobases, 43) which is similar to guanine transport in TM4 cells. Furthermore, K m value of guanine transport by mouse ENT2 was close to that of TM4 cells.…”

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

“…Ent/ENT transport is sodium-independent and, like Cnt/CNT transporters, endogenous nucleosides as well as cancer and antiviral nucleoside analogs are common substrates (Table 3) Mackey et al, 1999;Kiss et al, 2000;Baldwin et al, 2005;Damaraju et al, 2005;Nagai et al, 2007;Govindarajan et al, 2008). It is noteworthy that ENT1 is also expressed in the mitochondria, where it may be involved in the cellular toxicity of antiviral nucleoside drugs (Lai et al, 2004;Govindarajan et al, 2009).…”

Expression of Nucleoside Transporters in the Mouse Testis and Ovary

“…Five major isoforms of the mammalian nucleoside transporter have been reported (Yao et al, 1997;Kong et al, 2004;Nagai et al, 2007b). Two genetically distinct Equilibrative Nucleoside Transporters (ENT) were identified, termed ENT1 and ENT2.…”