Expression of equilibrative nucleoside transporters 1 (ENT1, inhibited by nanomolar nitrobenzylthioinosine, NBMPR) is reduced by D-glucose, an effect associated with activation of the L-arginine/NO pathway (Parodi et al. 2002). We investigated whether the inhibitory effect of elevated D-glucose on adenosine transport depends on expression of cationic amino acid transporters 2 (CAT2) and the inducible NO synthase (iNOS).

Mouse embryonic fibroblasts (MEFs) from humanely killed wild-type, Cat2– and iNOS-deficient mouse (Ethics Committee approval was obtained) were cultured (24 h) in medium 199 (M199), containing 10 % newborn calf serum, 3.2 mM L-glutamine, and 5 or 25 mM D-glucose. Adenosine transport ([2,8,5-³H]adenosine, 60 Ci mmol^-1, 0-500 µM, 2 µCi ml^-1, 22°C, 20 s) was measured in cells incubated (0-24 h) with M199 in the absence or presence of cytokines (10 ng ml^-1 IL1-β + 10 ng ml^-1 TNF-α + 20 I.U. ml^-1 INF-λ), N^G-nitro-L-arginine methyl ester (L-NAME, 100 µM), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, 10 µM), or NBMPR (1-1000 nM). CAT2 and iNOS mRNA was amplified by reverse transcriptase-polymerase chain reactions. iNOS activity was determined by L-[³H]citrulline formation from L-[³H]arginine (4 µCi ml^-1, 30 min)(Casanello & Sobrevia, 2002).

Adenosine transport in wild-type, Cat2-/- and iNOS-/- MEFs (1.2 ± 0.12, 1.1 ± 0.06 and 1.2 ± 0.2 pmol (mg protein)^-1 min^-1, respectively; means ± S.E.M., n = 8-16) was inhibited (P < 0.05, Student’s unpaired t test) by 1 nM NBMPR (0.1 ± 0.02, 0.2 ± 0.01 and 0.2 ± 0.02 pmol (mg protein)^-1 min^-1, respectively). Elevated D-glucose reduced the V_max (12 ± 2 vs 43 ± 5 pmol (mg protein)^-1 min^-1, P < 0.05, n = 12) with non- significant changes in the apparent K_m (84 ± 12 vs 101 ± 24 µM) for NBMPR-sensitive adenosine transport in wild-type MEFs. Adenosine transport in Cat2-/- or iNOS-/- MEFs was unaltered (P > 0.05). Cytokine-activated wild-type MEFs cultured in 5 mM D-glucose reduced adenosine transport to similar values determined in 25 mM D-glucose. ENT1 mRNA level was reduced (47 ± 4 %) by 25 mM D-glucose only in wild-type MEFs. D-Glucose or cytokine effects on adenosine transport activity and expression were blocked by L-NAME, and mimicked by SNAP. These results suggest that adenosine transport via ENT1 depends on expression of CAT2 and iNOS in MEFs.

This work was supported by FONDECYT (1030781, 1030607, 1000354, 7000354) and DIUC-University of Concepción (201.084.003-1.0)-Chile, The Welcome Trust (UK) and Fulbright Commission (USA).