Transport of the cationic amino acid L-arginine is mediated by system y⁺/CATs (cationic amino acid transporters), and nitric oxide (NO) is synthesised by the endothelium NO synthase (eNOS) in human umbilical vein endothelial cells, HUVECs (Sobrevia & Mann, 1997). It has been reported that HUVECs isolated from pregnancies with intra-uterine growth retardation (IUGR) exhibit a reduced accumulation of cGMP (Parra et al. 2001). Other studies show that maternal plasma L-arginine concentration is increased in IUGR pregnancies (Ceti et al. 1990), which could result from a reduced removal of this amino acid by vascular endothelium. Studies performed in IUGR rat models show that L-arginine supplementation significantly prevents fetal growth restriction (Vosatka et al. 1998). The aim of this study was to characterize the effect of IUGR on the expression and activity of L-arginine/NO signalling pathway in cultures of human umbilical vein endothelium.

Endothelial cells were isolated (0.2 mg ml^-1 collagenase) from umbilical veins of normal pregnancies or pregnancies diagnosed with IUGR (ethics committee approval and informed patient consent were obtained), and cultured in medium 199 (M199), supplemented with 10 % fetal and 10 % newborn calf serum, 3.2 mM L-glutamine, and 5 mM D-glucose. Kinetics of L-arginine transport (7.5-1000 µM, 2 µCi ml^-1, 37 °C, 1 min) and initial rates of influx of the membrane potential sensitive probe, tetra-[³H]phenyl-phosphonium ([³H] TPP⁺, 1 µCi ml^-1, 45 nM, 0-120 s, 37 °C) were determined in endothelial cell monolayers. Activity of eNOS was estimated by conversion of L-[³H]arginine into L-[³H]citrulline (100 µM L-[³H]arginine, 4 µCi ml^-1, 37°C, 30 min) in the presence or absence of N ^G-nitro-L-arginine methylester (L-NAME, 100 µM, 30 min, eNOS inhibitor). Reverse transcriptase-polymerase chain reactions (RT-PCR) were performed to determine mRNA levels for human CAT-1, CAT-2B and eNOS.

Overall rates of L-arginine transport were fitted best by a Michaelis-Menten equation plus a linear non-saturable component in normal and IUGR endothelial cells. IUGR has no effect on the non-saturable component (K_D) of L-arginine transport (control = 0.03 ± 0.01 vs. IUGR = 0.025 ± 0.009 pmol (µg protein)^-1 min^-1 µM^-1, means ± S.E.M., n = 4, P > 0.05, Student’s unpaired t test), but reduced the V_max for saturable transport (control = 5.0 ± 0.3 vs. IUGR = 3.6 ± 0.4 pmol (µg protein)^-1 min^-1, n = 3-5, P < 0.05), with negligible changes in the apparent K_m (control = 167 ± 29 vs. IUGR = 141 ± 41 µM, P > 0.05). Initial rates of [³H] TPP⁺ influx in IUGR cells (0.3 ± 0.03 pmol (mg protein)^-1 (15 s)^-1, n = 4) was lower (P < 0.05) than values determined in control cells (0.6 ± 0.1 pmol (mg protein)^-1 (15 s)^-1, n = 5). IUGR was also associated with a reduction (P < 0.05) of mRNA levels for hCAT-1 (IUGR/normal = 0.31 arbitrary units (a.u.)) and hCAT-2B (IUGR/normal = 0.29 a.u.) compared with normal cells (hCAT-1 or hCAT-2B = 1 a.u.). However, IUGR decreased (33 %) eNOS mRNA levels compared with values determined in normal cells. Preliminary experiments show that synthesis of L-citrulline from L-arginine is reduced (P < 0.05) in IUGR cells (0.8 ± 0.1 pmol (10⁶ cells)^-1 (30 min)^-1, n = 6) compared with normal cells (1.0 ± 0.2 pmol (10⁶ cells)^-1 (30 min)^-1, n = 4-15).

In summary, IUGR is associated with a reduced activity of L-arginine/NO signalling pathway that could be due to a reduced expression of hCAT-1 and hCAT-2B transporters in human umbilical vein endothelial cells. IUGR-induced decrease of NO synthesis and L-arginine transport could be a stimulatory factor to increase eNOS mRNA expression in this cell type in IUGR pregnancies.This work was supported by FONDECYT 1000354 and 7000354, DIUC-2001 (P.C. and L.S.), DIUC Iniciativa Grupo de Investigaciùn de Avanzada (201034006-1.4, University of Concepciùn)-Chile, and The Wellcome Trust (UK). P.C. holds University of Concepciùn-MSc fellowship.

Ceti, I., Corbetta, C., Sereni, L., Marconi, A., Bozzetti, P., Pardi, G. & Battaglia, F.C. (1990). Am. J. Obstet. Gynecol. 162, 253-261.

Parra, M.C., Lees, C., Mann, G.E., Pearson, J.D. & Nicolaides, K.H. (2001). Am. J. Obstet. Gynecol. 184, 497-502.

Sobrevia, L. & Mann, G.E. (1997). Exp. Physiol. 82, 423-452.

Vosatka, R.J., Hassoun, P.M. & Harvey-Wilkes, K.B. (1998). Am. J. Obstet. Gynecol. 178, 242-246.