Equilibrative nucleoside transporter 2 is expressed in human umbilical vein endothelium, but is not involved in the inhibition of adenosine transport induced by hyperglycaemia
Equilibrative nucleoside transporter 2 is expressed in human umbilical vein endothelium, but is not involved in the inhibition of adenosine transport induced by hyperglycaemia
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DOI：
10.1016/j.placenta.2004.10.006
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发表时间：
2005-09-01
期刊：
影响因子：
3.8
通讯作者：
Sobrevia, L
中科院分区：
文献类型：
--
作者：
Aguayo, C;Casado, J;Sobrevia, L
人体平衡、不依赖 Na+ 的核苷转运由对硝基苄基硫代肌苷 (NBMPR) 敏感（系统 es、hENT1）或不敏感（系统 ei、hENT2）的膜蛋白介导。妊娠糖尿病和细胞外 D-葡萄糖浓度升高会减少人脐静脉内皮 (HUVEC) 中的腺苷转运。我们研究了 HUVEC 中 hENT2 和 hENT1 的表达，以及 D-葡萄糖对其在与 25 MM D-葡萄糖预孵育（24 小时）的 HUVEC 中的活性和表达的影响。通过实时逆转录聚合酶链式反应对hENT2和hENT1 mRNA进行定量，并通过Western blotting检测其蛋白。 hENT2和hENT1蛋白在HUVEC中共表达，并且位于质膜上，然而，hENT2主要位于细胞质和核周。 D-葡萄糖降低 hENT1 和 hENT2 mRNA 表达，但仅降低质膜上 hENT1 蛋白的丰度。完整细胞和膜囊泡中的腺苷转运被 D-葡萄糖和 NMBPR (1 μM) 抑制。在存在或不存在 1 μM NBMPR 的情况下，次黄嘌呤均抑制腺苷转运。 D-葡萄糖降低了完整细胞、膜囊泡和质膜组分中 NBMPR 的最大结合。总之，本研究表明 hENT2 和 hENT1 在 HUVEC 中共表达，即使腺苷转运也由 hENT2 介导，hENT2 介导的转运活性也不参与 D-葡萄糖诱导的总腺苷转运的下调。
Human equilibrative, Na+-independent nucleoside transport is mediated by membrane proteins sensitive (system es, hENT1) or insensitive (system ei, hENT2) to nitrobenzylthioinosine (NBMPR). Gestational diabetes and elevated extracellular concentrations of D-glucose reduce adenosine transport in human umbilical vein endothelium (HUVEC). We studied hENT2 and hENT1 expression in HUVEC, and the effect of D-glucose on their activity and expression in HUVEC preincubated with 25 MM D-glucose (24 h). hENT2 and hENT1 mRNA were quantified by real-time reverse transcription polymerase chain reaction, and their proteins were detected by Western blotting. hENT2 and hENT1 proteins are co-expressed in HUVEC and are located at the plasma membrane, however, hENT2 was mainly cytoplasmatic and perinuclear in location. D-Glucose reduced hENT1 and hENT2 mRNA expression, but only hENT1 protein abundance at the plasma membrane. Adenosine transport was inhibited by D-glucose and NMBPR (1 mu M) in intact cells and membrane vesicles. Hypoxanthine inhibited adenosine transport in the absence or in the presence of 1 mu M NBMPR. D-Glucose reduced NBMPR maximal binding in intact cells, membrane vesicles, and plasma membrane fractions. In conclusion, the present study demonstrates that hENT2 and hENT1 are co-expressed in HUVEC, and even when adenosine transport is also mediated by hENT2, the hENT2-mediated transport activity is not involved in the D-glucose-induced down-regulation of total adenosine transport.