Hypoxia increases equilibrative nucleoside transporter 2 activity by a transcriptional independent mechanism in human umbilical vein endothelium
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Date
2006
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Abstract
Low oxygen tension (hypoxia) reduces adenosine transport in several types of mammalian cells. Adenosine transport is mediated by human equilibrative nucleoside transporter 1 (hENT1) and hENT2 in human umbilical vein endothelium (HUVEC), a fetal cell type that grows under 5% O2 (ie. normoxia for this cell type). We studied whether hypoxia alters hENT2 expression and activity in HUVEC. Methods: Cells were cultured (0-24 h) in 5% or 2% O2 (hypoxia), and [3H]adenosine uptake (125 and 500 μM, 4 μCi/ml, 20 s, 37°C) was measured in absence or presence of 100 nM nitrobenzylthioinosine (NBMPR, hENT1 inhibitor). hENT2 mRNA was quantified by real time RT-PCR, and protein abundance was determined by Western blot. SLC29A2 (for hENT2) promoter activity was measured following transfection (electroporation, 320 V, 30 ms) with pGL3 basic plasmid (firefly/renilla luciferase reporter gene) carrying -1477 bp and -587 bp of the promoter sequence. Results: Hypoxia reduced hENT2 mRNA expression (~55%), and promoter activity (~50%), but did not alter hENT2 protein abundance. Adenosine uptake via hENT2 was increased (2-fold) in hypoxia. Conclusions: Adenosine uptake via hENT2 may be modulated by post-translational mechanisms in hypoxia in HUVEC. Supported by FONDECYT 1030781/1030607/7050030. A Torres holds a School of Medicine research fellowship, and M Farías holds a CONICYT-PhD fellowship.