Equilibrative, Na+-independent nucleoside transport in mammalian cells is mediated by specific membrane proteins sensitive (system es, hENT1) or insensitive (system ei, hENT2) to inhibition by nitrobenzyl-thioinosine, NBMPR (Griffith & Jarvis, 1996). We have reported that adenosine transport mediated by hENT1 (Sobrevia et al. 1994; Montecinos et al. 2000) and hENT1 mRNA levels are reduced in human umbilical vein endothelial cells (HUVECs) exposed to elevated D-glucose (25 mM, Aguayo et al. 2001). The aim of this study was to determine the effect of elevated D-glucose on the protein levels and localization of hENT1 and hENT2 in cultures of human umbilical vein endothelium.
Endothelial cells were isolated (0.2 mg ml-1 collagenase) from umbilical veins of normal pregnancies (Ethics Committee approval and informed patient consent were obtained) and cultured in medium 199 (M199), supplemented with 15 % fetal calf serum, 3.2 mM L-glutamine, and 5 mM D-glucose, 100 i.u. ml-1 penicillin-streptomycin. Transport of adenosine (10 µM, 20 s, 4 µCi ml-1, 22 °C) was determined in Hepes-buffered Krebs solution in cells cultured in 5 mM or 25 mM D-glucose (24 h) in the presence or absence of 10 µM NBMPR (30 min). Subconfluent endothelial cells (~70 % confluence) cultured on glass slides were fixed in paraformaldehyde (2 %) and permeabilized with saponin (0.05 %), incubated with specific primary rabbit antibody anti-hENT1 or anti-hENT2 (dilutions 1:40) and immunofluorescence detected by Leika Laser confocal microscopy. Western blot for hENT1 and hENT2 (antibody dilution 1:2000) was performed in plasma membrane and cytosolic fractions obtained by centrifugation (100 000 g, 60 min, 4 oC) from endothelial cells harvested in a lysis buffer (20 mM Tris-HCl, 2 mM EDTA, 0.5 mM EGTA, 5 mM β-mercapto-ethanol, 250 mM sucrose) as previously described (Montecinos et al. 2000). Immunohistochemistry of a cross-section (7 µm) of umbilical cord was performed in paraformaldehyde (4 %)-fixed tissue and processed using standard protocols. The antigen-antibody complexes were visualized by the biotin-streptavidin-peroxidase method with aminoethyl carbazole as the chromogen.
Transport of 10 µM adenosine (22 ± 3 pmol (106 cells)-1 s-1, mean ± S.E.M., n = 16) was significantly (Student’s unpaired t test) inhibited by 1 nM NBMPR (4 ± 2 pmol (106 cells)-1 s-1, n = 16, P < 0.05) or 10 µM NMBPR (3 ± 1 pmol (106 cells)-1 s-1, n = 22, P < 0.04). Adenosine transport was also inhibited by incubation of cells with 25 mM D-glucose (8 ± 2 pmol (106 cells)-1 s-1, n = 12, P < 0.05). Cross-sections of umbilical cords show positive immunoreactivity for hENT1 and hENT2. Laser confocal microscopy shows hENT1 and hENT2 immuno-fluorescence, with hENT1 expressed both in the cytosol and plasma membrane. However, hENT2 was detected only in the cytosol. Incubation of cells with 25 mM D-glucose did not change hENT1 and hENT2 cellular distribution. By Western blot analysis, hENT1 was detected both at the cytosolic and plasma membrane fractions, but hENT2 was detected only in the cytosolic fraction. High D-glucose decreased hENT1 protein levels in the membrane fraction (25 mM D-glucose/5 mM D-glucose = 0.5 arbitrary units, P < 0.05), without significant changes in hENT2 protein levels (25 mM D-glucose/5 mM D-glucose = 0.9 a.u., P > 0.05).
In summary, these results show a differential localization of hENT1 and hENT2 in human umbilical endothelial cells, with hENT1 being preferentially localized at the plasma membrane and hENT2 at the cytosol. Thus adenosine transport could be mediated by hENT1 and D-glucose-induced inhibition of adenosine transport could be due to recycling of hENT1 from the plasma membrane to subcellular fractions.This work was supported by FONDECYT 1000354 and 7000354, DIUC Iniciativa Grupo de Investigaciùn de Avanzada (201034006-1.4, University of Concepciùn)-Chile, and The Wellcome Trust (UK) to L.S., and grants SAF99-0115 and 2F097-1268 to M.P.-A. C.A. holds a CONICYT (Chile) PhD fellowship.
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Montecinos, V.P., Aguayo, C., Flores, C., Wyatt, A.W., Pearson, J.D., Mann, G.E. & Sobrevia, L. (2000). J. Physiol. 529, 777-790. abstract
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