Hyperglycaemia is associated with endothelial damage, a low cellular level of L-ascorbic acid, and increased L-arginine transport and eNOS expression and activity in human umbilical vein endothelial cells (HUVECs) (Beckman et al. 2001; Flores et al. 2003). Endothelial nitric oxide synthase (eNOS) activity is highly dependent on its redox state and is altered by reactive oxygen species (ROS) (Wang et al. 2001). L-Ascorbic acid has long been proposed as a major antioxidant and strong reducing agent, and a recent study suggests its role as an enhancer of NO bioactivity (Price et al. 2001). This study intended to determine the effect of L-ascorbic acid on L-arginine transport and eNOS protein expression and activity in HUVECs exposed to high D-glucose.

Cells isolated from normal pregnancies (Ethics Committee approval and informed patient consent were obtained) were cultured in medium 199 (containing 20 % bovine serum, 3.2 mM L-glutamine), and exposed (1 -24 h) to 5 or 25 mM D-glucose, in the absence or presence of L-ascorbic acid (0.1 -100 µM). L-Arginine transport (100 µM, L-[³H]arginine, 2 µCi ml^-1, 37°C, 1 min) was determined in the absence or presence of L-ascorbic acid (100 µM). eNOS activity was determined by L-[³H]citrulline formation from L-[³H]arginine (4 µCi ml^-1, 30 min), and eNOS protein was detected by Western blot. mRNA for hCAT-1, hCAT-2B and eNOS was quantified by real-time PCR. ROS formation was characterized by measuring the fluorescence intensity of the ROS indicator 2Ô,7Ô-dichlorofluorescein (DCF).

High D-glucose increased the L-arginine transport (1.1 ± 0.02 vs. 0.4 ± 0.1 pmol (µg protein)^-1 min^-1, n = 15, P < 0.05, means ± S.E.M., Student’s unpaired t test). L-Ascorbic acid blocked the effect of D-glucose on L-arginine transport (0.3 ± 0.1 pmol (µg protein)^-1 min^-1, n = 4, P < 0.05), with half-maximal effect at 0.3 ± 0.02 µM L-ascorbic acid, and at 3.5 ± 0.3 h incubation). In addition, D-glucose also increased (P < 0.05) L-citrulline synthesis (1.7-fold), eNOS protein (2.5-fold) and mRNA number of copies (2.4-fold), and hCAT-1 mRNA (2.3-fold) and hCAT-2B (2.6-fold) number of copies. The effects of D-glucose were blocked (P < 0.05) by L-ascorbic acid. Incubation of cells with 25 mM D-glucose increased ROS levels (1.5-fold) in HUVECs, but not in cells incubated with L-ascorbic acid.

In summary, D-glucose-induced alterations in the L-arginine-NO pathway in human umbilical vein endothelium could be due to higher levels of ROS, and L-ascorbic acid could act as a protective factor in hyperglycaemia.

This work was supported by FONDECYT (1030781, 1030607, 7030004, 7030109) and DIUC-University of Concepción (201.084.003-1.0)-Chile, and The Welcome Trust (UK).