Insulin increases L-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelium (HUVEC)(Gonzalez et al. 2004). The nucleoside adenosine also activates L-arginine transport and NO synthesis, an effect that depends on its extracellular concentration which is maintained by an efficient uptake by the endothelium (Vasquez et al. 2004). Adenosine transport is mediated by equilibrative nucleoside transporters 1 (hENT1, sensible to ?1 μM nitrobenzylthioinosine, NBMPR) and hENT2 (sensible to >1 μM NBMPR) in HUVEC (Aguayo et al. 2004). However it is unknown whether insulin alters adenosine transport in this cell type. We studied the effect of insulin on activity and expression of hENT1 and hENT2 in HUVEC. Cells (Ethics committee approval and informed patient consent were obtained) were cultured in medium 199 containing 20% bovine sera, 3.2 mM L-glutamine, 100 iu/ml penicillin-streptomycin (37C, 5% CO2). Passage 2 cells were exposed (0-24 h) to insulin (0.001-100 nM) and adenosine transport ([³H]adenosine, 5-500 μM, 4 μCi/ml, 37C, 20 s) was measured in absence or presence of nitrobenzylthioinosine (NBMPR, 0.001-100 μM) or hypoxanthine (2 mM). Specific [³H]NBMPR equilibrium binding (0.01-5 nM, 30 min) was measured. hENT1 and hENT2 mRNA were quantified by real-time PCR and proteins were detected by Western blot. Total 10 μM adenosine uptake (0.22 ± 0.01 pmol/μg protein/s, means ± S.E.M, n=8-16) was significantly (P1 μM NBMPR (Ki = 3.3 ± 0.6 μM) and by 2 mM hypoxanthine. Insulin increased [half-maximal (K1/2) effect = 6.5 ± 1.2 h] the Vmax for hENT2 (2.3 ± 0.2 vs 0.9 ± 0.1 pmol/μg protein/s), with no significant changes in the apparent Km (91 ± 12 vs 101 ± 15 μM). Insulin also increased the hENT1 (3.1-fold, K1/2 = 4.5 ± 0.5 h) and hENT2 (3.1-fold, K1/2 = 4.1 ± 0.6 h) mRNA copies, and hENT1 (1.9-fold, K1/2 = 6.8 ± 0.9 h) and hENT2 (2.5-fold, K1/2 = 5.9 ± 1.1 h) protein level. Maximal binding (Bmax) was increased by insulin (0.79 ± 0.02 vs 0.51 ± 0.02 pmol/μg protein). In conclusion, hENT1 and hENT2 expression are modulated by insulin, and only hENT2 activity is increased by this hormone in HUVEC.