Equilibrative nucleoside transporter 2 is expressed in human umbilical vein endothelium, but is not involved in the inhibition of adenosine transport induced by hyperglycaemia.
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 microM) in intact cells and membrane vesicles. Hypoxanthine inhibited adenosine transport in the absence or in the presence of 1 microM 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.
Aguayo
C
C
Cellular and Molecular Physiology Laboratory (CMPL), Department of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago.
Casado
J
J
González
M
M
Pearson
J D
JD
Martín
R San
RS
Casanello
P
P
Pastor-Anglada
M
M
Sobrevia
L
L
D000241:Adenosine / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics / Q000378:metabolism*; D033722:Equilibrative-Nucleoside Transporter 2 / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007003:Hypoglycemia / Q000378:metabolism*; D019271:Hypoxanthine / Q000494:pharmacology; D033703:Nucleoside Transport Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014471:Umbilical Veins
D004338:Drug Combinations; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D033703:Nucleoside Transport Proteins; D012333:RNA, Messenger; C438649:SLC29A1 protein, human; C438654:SLC29A2 protein, human; C444719:adenosine transporter; D019271:Hypoxanthine; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D005947:Glucose; D000241:Adenosine