Humans possess three members of the cation-coupled concentrative nucleoside transporter CNT (SLC 28) family, hCNT1-3: hCNT1 is selective for pyrimidine nucleosides but also transports adenosine, hCNT2 transports purine nucleosides and uridine, and hCNT3 transports both pyrimidine and purine nucleosides. hCNT1/2 transport nucleosides using the transmembrane Na+ electrochemical gradient, while hCNT3 is both Na+- and H+-coupled. By producing recombinant hCNT3 in Xenopus laevis oocytes, we have used radiochemical high performance liquid chromatography (HPLC) analysis to investigate the metabolic fate of transported [3H] or [14C] pyrimidine and purine nucleosides once inside cells. With the exception of adenosine, transported nucleosides were generally subject to minimal intracellular metabolism. We also used radiochemical HPLC analysis to study the mechanism by which adenosine functions as a low Km, low Vmax permeant of hCNT1. hCNT1-producing oocytes were pre-loaded with [3H] uridine, after which efflux of accumulated radioactivity was measured in transport medium alone, or in the presence of extracellular non-radiolabelled adenosine or uridine. hCNT1-mediated [3H]-efflux was stimulated by extracellular uridine, but inhibited by extracellular adenosine, with >95% of the radioactivity exiting cells being unmetabolized uridine, consistent with a low transmembrane mobility of the hCNT1/adenosine complex. Humans also possess four members of the equilibrative nucleoside transporter ENT (SLC 29) family, hENT1-4. Of these, hENT1 and hENT2 transport both nucleosides and nucleobases into and out of cells, but their relative contributions to nucleoside and nucleobase homeostasis and, in particular, to adenosine signaling via purinoreceptors, are not known. We therefore used HPLC to determine plasma nucleoside and nucleobase concentrations in wild-type, mENT1-, mENT2- and mENT1/mENT2-knockout (KO) mice, and to compare the findings with knockout of mCNT3. Results demonstrated that ENT1 was more important than ENT2 or CNT3 in determining plasma adenosine concentrations, indicated modest roles of ENT1 in the homeostasis of other nucleosides, and suggested that none of the transporters is a major participant in handling of nucleobases.

中文翻译：

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HPLC揭示了核苷和核苷碱基稳态，核苷代谢和核苷转运的新特征。

人类拥有阳离子耦合的浓缩核苷转运蛋白CNT（SLC 28）系列的三个成员，hCNT1-3：hCNT1对嘧啶核苷具有选择性，但也可以转运腺苷，hCNT2转运嘌呤核苷和尿苷，hCNT3转运嘧啶和嘌呤核苷。hCNT1 / 2使用跨膜Na +电化学梯度转运核苷，而hCNT3既与Na +偶联，又与H +偶联。通过在非洲爪蟾卵母细胞中产生重组hCNT3，我们已使用放射化学高效液相色谱（HPLC）分析了一旦进入细胞后转运的[3H]或[14C]嘧啶和嘌呤核苷的代谢命运。除腺苷外，转运的核苷通常仅需极少的细胞内代谢。我们还使用放射化学HPLC分析来研究腺苷作为hCNT1的低Km，低Vmax渗透物起作用的机理。在产生hCNT1的卵母细胞中预装[3H]尿苷，然后在单独的运输介质中或存在细胞外非放射性标记腺苷或尿苷的情况下测量累积的放射性外排。hCNT1介导的[3H]外排受细胞外尿苷的刺激，但被细胞外腺苷抑制，大于95％的放射活性细胞未代谢尿苷，这与hCNT1 /腺苷复合物的跨膜迁移率低相符。人类还拥有hENT1-4平衡核苷转运蛋白ENT（SLC 29）家族的四个成员。其中，hENT1和hENT2将核苷和核碱基均运入和运出细胞，但是它们对核苷和核苷碱基稳态，尤其是通过嘌呤受体对腺苷信号传导的相对贡献尚不清楚。因此，我们使用HPLC测定了野生型mENT1，mENT2-和mENT1 / mENT2-敲除（KO）小鼠的血浆核苷和核碱基浓度，并将其与敲除mCNT3的结果进行了比较。结果表明，ENT1在确定血浆腺苷浓度方面比ENT2或CNT3更重要，表明ENT1在其他核苷的稳态中具有适度的作用，并且表明这些转运蛋白均不是处理核碱基的主要参与者。mENT2-和mENT1 / mENT2-基因敲除（KO）小鼠，并将其与mCNT3基因敲除的结果进行比较。结果表明，ENT1在确定血浆腺苷浓度方面比ENT2或CNT3更重要，表明ENT1在其他核苷的稳态中具有适度的作用，并且表明这些转运蛋白均不是处理核碱基的主要参与者。mENT2-和mENT1 / mENT2-基因敲除（KO）小鼠，并将其与mCNT3基因敲除的结果进行比较。结果表明，ENT1在确定血浆腺苷浓度方面比ENT2或CNT3更重要，表明ENT1在其他核苷的稳态中具有适度的作用，并且表明这些转运蛋白均不是处理核碱基的主要参与者。