Nitric oxide synthesis requires activity of the cationic and neutral amino acid transport system y+L in human umbilical vein endothelium.
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 microM L-arginine was inhibited (P < 0.05) by L-lysine (K(i), 1.4 micro M), L-leucine (K(i), 1.8 micro M) and L-phenylalanine (K(i), 4.1 microM), 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 microM L-arginine transport, but inhibited (92 +/- 3 %) 100 microM L-arginine transport. L-arginine transport in the presence of NEM was saturable (V(max), 0.37 +/- 0.02 pmol (microg protein)(-1) min(-1); K(m), 1.5 +/- 0.3 microM) and competitively inhibited by L-leucine in the presence of Na+ (V(max), 0.49 +/- 0.06 pmol (microg protein)(-1) min(-1); K(m), 6.5 +/- 0.9 microM). 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 microM L-arginine. Cells exposed to 25 mM D-glucose (24 h) exhibited reduced system y+L activity (V(max), 0.15 +/- 0.008 pmol (microg protein)(-1) min(-1); K(m), 1.4 +/- 0.3 microM) and NO synthesis. However, 25 mM D-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.
Arancibia-Garavilla
Yerko
Y
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, Chile.
Toledo
Fernando
F
Casanello
Paola
P
Sobrevia
Luis
L
D027241:Amino Acid Transport System y+L / Q000378:metabolism*; D026923:Amino Acid Transport Systems, Basic / Q000378:metabolism; D026921:Amino Acid Transport Systems, Neutral / Q000378:metabolism; D001120:Arginine / Q000493:pharmacokinetics*; D001693:Biological Transport, Active / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009569:Nitric Oxide / Q000096:biosynthesis*; D014471:Umbilical Veins / Q000378:metabolism*
D027241:Amino Acid Transport System y+L; D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D009569:Nitric Oxide; D001120:Arginine