Insulin and hyperglycaemia induced vasodilatation is explained by activation of endothelial L-arginine transport and nitric oxide (NO) synthesis (L-arginine/NO pathway)(Gonzalez et al. 2004). Hyperglycaemia reduces uptake of the endogenous nucleoside adenosine in human umbilical vein endothelium (HUVEC). Since biological effects of adenosine depend on an efficient uptake via human equilibrative nucleoside transporters 1 (hENT1) and hENT2 in HUVEC (Vasquez et al. 2004), changes in hENT1 and/or hENT2 expression and activity are crucial to keep physiological levels of extracellular adenosine. We studied whether insulin alters adenosine transporters in HUVEC. Cells were cultured in medium 199 containing 20% bovine serum, 3.2 mM L-glutamine, 100 iu/ml penicillin-streptomycin (37°C, 5% CO₂). Passage 2 cells were exposed (0-24 h) to medium containing 5 (normal) or 25 mM (high) D-glucose and insulin (0.001-100 nM). Adenosine transport ([³H]adenosine, 5-500 μM, 4 μCi/ml, 37°C, 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. Overall adenosine transport (hENT1- and hENT2-mediated) was significantly (P<0.05, unpaired Student's t test, values are means ± S.E.M., n=4-12) increased (2.1-fold) by insulin, associated with increased V_max for hENT2-, but reduced V_max for hENT1-mediated transport in normal D-glucose. Insulin also increased hENT2 (1.7-fold), but reduced (45±12%) hENT1 protein abundance and mRNA number of copies (66±10%), and returned high D-glucose induced reduction of hENT1 and hENT2 mRNA expression, and hENT2 protein abundance to values in cells in normal D-glucose. However, insulin did not alter (P>0.05) hENT1 protein abundance. Insulin and high D-glucose increased eNOS expression (protein and mRNA) and activity (1.2±0.2, 3.1±0.4 and 2.6±0.1 pmol/μg protein/30 min for control, insulin and high D-glucose, respectively). Insulin effect on hENT1 was blocked by N^G-nitro-L-arginine methyl ester (L-NAME, eNOS inhibitor), and only hENT1-mediated transport was reduced by S-nitroso-N-acetylpenicillamine (SNAP, NO donor). Thus, insulin increases overall adenosine transport by increasing hENT2 expression and activity, a phenomenon that does not involve NO. However, NO synthesis is required for insulin-inhibition of hENT1 transport, but not for its expression.