The atherogenic effect of oxLDL has been reported to be mediated by eNOS transcriptional inhibition and post-transcriptional eNOS mRNA destabilization (Liao et al. 1995). Inhibition of eNOS activity by oxLDL is antagonized by pretreatment of endothelial cells with L-arginine (Vergnani et al. 2000). Transport of L-arginine has been shown to be unaltered by oxLDL in human endothelium (Jay et al. 1997), and inhibited by the major lipid constituent of oxLDL, lysophosphatidylcholine (LPC), in bovine aortic endothelium (Kikuta et al. 1998). We have investigated the effect of oxLDL on the L-arginine/NO pathway in human endothelium.

Endothelial cells were isolated (0.2 µg ml^-1 collagenase) from human umbilical veins (Ethics Committee approval and informed patient consent was obtained) and cultured in medium 199, supplemented with 10 % fetal and 10 % newborn calf serum, 3.2 mM L-glutamine, and 5 mM D-glucose. Cells were exposed for 24 h to serum-free culture medium followed by a further 16 h incubation period at 37 °C in serum-free medium containing native LDL (nLDL, 50 µg ml^-1) or oxLDL (50 µg ml^-1), in the absence or presence of N ^G-nitro-L-arginine methylester (L-NAME, 100 µM, 16 h, eNOS inhibitor), PD-98059 (10 µM, 16 h, MAP kinase kinase inhibitor), or calphostin C (100 nM, 16 h, PKC inhibitor). L-[³H] Arginine transport (1-1000 µM, 2 µCi ml^-1, 37 °C, 1 min) was determined in phosphate buffer saline solution for the last minute of the 16 h incubation period with LDLs. Protein levels for eNOS and phosphorylated p42/44^mapk were determined by Western blotting and immunocytochemistry. Endothelial NO synthase activity was estimated by conversion of L-[³H] arginine into L-[³H] citrulline (L-[³H] arginine, 100 µM, 4 µCi ml^-1, 37 °C) for the last 30 min of the 16 h incubation period with LDLs. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to determine mRNA levels for hCAT-1 (human Cationic Amino acid Transporters) and hCAT-2B using β-actin as housekeeper.

Incubation of endothelial cells with oxLDL resulted in an inhibition (P < 0.05, unpaired Student’s t test) of maximal transport capacity for L-arginine (V_max/K_m = 0.052 ± 0.01 vs. 0.011 ± 0.007 pmol (µg protein)^-1 min^-1 µM^-1, mean ± S.E.M., n = 3, for control and oxLDL, respectively) due to a reduced maximal velocity (V_max = 5.6 ± 0.6 vs. 1.7 ± 0.2 pmol (µg protein)^-1 min^-1) with no changes (P > 0.05) in the apparent K_m (108 ± 10 vs. 151 ± 15 µM). However, nLDL did not change significantly (P > 0.05) the kinetic parameters for L-arginine transport in endothelial cells (V_max = 4.8 ± 0.6 pmol (µg protein)^-1 min^-1, K_m = 135 ± 14 µM, n = 3). The inhibitory effect of oxLDL on 100 µM L-arginine transport was unaltered by PD-98059 (0.7 ± 0.3 pmol (µg protein)^-1 min^-1, n = 4) or calphostin C (0.8 ± 0.7 pmol (µg protein)^-1 min^-1, n = 4). Basal NO synthesis (17 200 ± 1600 d.p.m. (mg protein)^-1 30 min^-1) was significantly inhibited by oxLDL (9700 ± 300 d.p.m. (mg protein)^-1 30 min^-1, P < 0.05) and L-NAME (10 100 ± 125 d.p.m. (mg protein)^-1 30 min^-1, P < 0.05). As for L-arginine transport, inhibition of NO synthesis by oxLDL was unaltered by PD-98059 (9600 ± 300 d.p.m. (mg protein)^-1 30 min^-1, P > 0.05) or calphostin C (9650 ± 165 d.p.m. (mg protein)^-1 30 min^-1, P > 0.05). Inhibition of L-arginine transport and NO synthesis by oxLDL was associated with increased phosphorylation of p42/44^mapk, inhibitable by PD-98059, and increased eNOS protein levels. Oxidized LDL induced a reduction (P < 0.05, n = 3) of the mRNA ratios for hCAT-1/β-actin (control 0.088 ± 0.009 arbitrary units (a.u.), oxLDL 0.039 ± 0.004 a.u.) and hCAT-2B/β-actin (control 0.223 ± 0.02 a.u., oxLDL 0.036 ± 0.004 a.u.).

In conclusion, inhibition of L-arginine transport by oxLDL could result from a reduced mRNA level for hCAT-1 and/or hCAT-2B transporters in human umbilical vein endothelial cells in vitro. oxLDL-induced reduction in NO synthesis is associated with a reduced eNOS activity and eNOS mRNA level in this cell type. The finding that the inhibitory effects of oxLDL are paralleled by increased phosphorylation of p42/44^mapk suggests a NO-independent alternative pathway for activation of p42/44^mapk in response to oxLDL, in addition to a NO-dependent activation of these MAP kinases as reported for hyperglycaemia (Montecinos et al. 2000).

This work was supported by FONDECYT (1000354 and 7000354) and DIUC-Iniciativa de Grupos de Investigaciùn de Avanzada (University of Concepciùn)-Chile, and The Wellcome Trust (UK). L.L. holds a University of Concepciùn-PhD fellowship. R.G. is the recipient of a University of Concepciùn-MINEDUC (Chile) fellowship for undergraduate biochemistry studies.

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