Experimental Physiology : Translation and Integration Endothelial cells are crucial in modulating vascular tone in the human feto-placental circulation which lacks autonomic innervation (Fox & Khong, 1990), thus circulating and locally released vasoactive molecules, such as nitric oxide (NO), are likely to be involved in the control of feto- placental haemodynamics. The potent vasodilator NO is a byproduct of the conversion of L-arginine into L-citrulline via the Ca 2+ –calmodulin-dependent endothelial NO synthase (eNOS)(Moncada et al. 1991; Alderton et al. 2001). In several cell types, eNOS activity seems to depend on the availability of extracellular L-arginine, and L-arginine transport has been considered to be a rate- limiting factor for NO synthesis (Poston & Taylor, 1995; Sobrevia & Mann, 1997). It has been shown that L-arginine transport is mediated by the cationic amino acid transporter (CAT) isoforms y + /CAT-1 (K m , 100–200 mM; Sobrevia et al. 1995; Casanello & Sobrevia, 2002; Flores et al. 2003), y + /CAT-2B (K m , ~200–400 mM; Casanello & Sobrevia, 2002; Flores et al. 2003), and systems b 0,+ (K m , ~3–4 mM; Bussolati et al. 1993) and y + L (K m , ~1–40 mM; Arancibia & Sobrevia, 1999; Sala et al. 2002) in human umbilical vein endothelial cells (HUVECs). System y + L is a heterodimer formed by the interaction of the heavy chain of the cell surface antigen Nitric oxide synthesis requires activity of the cationic and neutral amino acid transport system y + L in human umbilical vein endothelium Yerko Arancibia-Garavilla *†, Fernando Toledo ‡, Paola Casanello *§ and Luis Sobrevia *¶ * Cellular and Molecular Physiology Laboratory (CMPL), Department of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, PO Box 114-D, Santiago, † Faculty of Sciences, Universidad de Chile, Santiago, ‡ Department of Basic Sciences, Faculty of Sciences, Universidad del Bio-Bio, Chillán and § Department of Obstetrics and Gynaecology, Faculty of Medicine, Universidad de Concepción, Concepción, Chile (Manuscript resubmitted 28 August 2003; accepted 17 September 2003) L-Arginine transport is mediated by the cationic/neutral amino acid transport system y + L and cationic amino acid transporters y + /CATs in human umbilical vein endothelial cells (HUVECs). System y + /CATs activity may be rate-limiting for nitric oxide (NO) synthesis, but no reports have demonstrated system y + L involvement in NO synthesis in endothelium. We investigated the role of system y + L in NO synthesis in HUVECs. Transport of 1.5 mML-arginine was inhibited (P < 0.05) by L-lysine (K i , 1.4 mM), L-leucine (K i , 1.8 mM) and L-phenylalanine (K i , 4.1 mM), but was unaltered (P > 0.05) by L-alanine or L-cysteine. The system y + /CATs inhibitor, N-ethylmaleimide (NEM), did not alter 1.5 mML-arginine transport, but inhibited (92 ± 3 %) 100 mML-arginine transport. L-Arginine transport in the presence of NEM was saturable (V max , 0.37 ± 0.02 pmol (mg protein) _1 min _1 ; K m , 1.5 ± 0.3 mM) and competitively inhibited by L-leucine in the presence of Na + (V max , 0.49 ± 0.06 pmol (mg protein) _1 min _1 ; K m , 6.5 ± 0.9 mM). HUVECs express SLC3A2/4F2hc, SLC7A7/4F2-lc2 and SLC7A6/4F2-lc3 genes encoding for the high-affinity transport system y + L. N G -Nitro-L-arginine methyl ester and L-leucine, but not NEM, inhibited NO synthesis in medium containing 1.5 mML-arginine. Cells exposed to 25 mM D-glucose (24 h) exhibited reduced system y + L activity (V max , 0.15 ± 0.008 pmol (mg protein) _1 min _1 ; K m , 1.4 ± 0.3 mM) and NO synthesis. However, 25 mMD-glucose increased NO synthesis and L-arginine transport via system y + . Thus, L-arginine transport through system y + L plays a role in NO synthesis, which could be a determining factor in pathological conditions where the endothelial L-arginine–NO pathway is altered, such as in diabetes mellitus. Experimental Physiology (2003) 88.6, 699–710. 2647 Publication of The Physiological Society ¶ Corresponding author: sobrevia@med.puc.cl ) by guest on September 28, 2011 ep.physoc.org Downloaded from Exp Physiol (