Nitric Oxide Synthesis Requires Activity of the Cationic and Neutral Amino Acid Transport System y⁺L in Human Umbilical vein Endothelium

Abstract

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 μM L‐arginine was inhibited (P < 0.05) by L‐lysine (Ki, 1.4 μM), L‐leucine (Ki, 1.8 μM) and L‐phenylalanine (Ki, 4.1 μM), 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 μM L‐arginine transport, but inhibited (92 ± 3%) 100 μM L‐arginine transport. L‐Arginine transport in the presence of NEM was saturable (Vmax, 0.37 ± 0.02 pmol (μg protein)−1 min−1; Km, 1.5 ± 0.3 μM) and competitively inhibited by L‐leucine in the presence of Na+ (Vmax, 0.49 ± 0.06 pmol (μg protein)−1 min−1; Km, 6.5 ± 0.9 μM). HUVECs express SLC3A2/4F2hc, SLC7A7/4F2‐lc2 and SLC7A6/4F2‐lc3 genes encoding for the high‐affinity transport system y+L. NG‐Nitro‐L‐arginine methyl ester and L‐leucine, but not NEM, inhibited NO synthesis in medium containing 1.5 μM L‐arginine. Cells exposed to 25 mM D‐glucose (24 h) exhibited reduced system y+L activity (Vmax, 0.15 ± 0.008 pmol (μg protein)−1 min−1; Km, 1.4 ± 0.3 μM) 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.

Modulation of the Cationic Amino Acid Transport System y⁺L by Surface Potential, Ouabain and Thrombin in Human Platelets: Effects of Uremia

“…To our knowledge, there are only three studies investigating the modulation of L -arginine transport via system y + L in leukocytes, erythrocytes and endothelium [7, 24, 34]. Experiments with human umbilical vein endothelium reported that system y + L activity was reduced by elevated D -glucose, but the exact mechanisms involved were not described [34]. In contrast, we did not observe any effect of elevated D -glucose on system y + L activity in human platelets, suggesting that glucose may not hyperpolarize platelets and/or affect system y + L activity.…”

Cell signalling-mediating insulin increase of mRNA expression for cationic amino acid transporters-1 and -2 and membrane hyperpolarization in human umbilical vein endothelial cells

“…In HUVEC, L-arginine is taken up primarily by the Na + -independent, high-affinity (K m 100-400 µM) systems y + / hCAT-1 and y + /hCAT-2B (human cationic amino acid transporter) [3,10] and, to a lesser extent, via system y + L (K m 1-40 µM) [2,38,42]. Insulin increases the activity of system y + /CATs in HUVEC [45], rat pancreas [29], gastric mucosa [6] and CAT-1 expression in rat liver cells [49] and coronary myocytes [43].…”

Loss of Collectrin, an Angiotensin-Converting Enzyme 2 Homolog, Uncouples Endothelial Nitric Oxide Synthase and Causes Hypertension and Vascular Dysfunction

“…In several cell types, including the ECs and renal epithelium, the synthesis of NO requires the de novo import of extracellular l-arginine. [26][27][28] l-Arginine transport into cells is mediated by different classes of amino acid transporters that are defined by their ion dependency, substrate specificity, and relative affinity. 29,30 In ECs, system y + , a sodium-independent system, accounts for ≈60% of l-arginine transport, and system y + L, a sodium-dependent system, accounts for ≈40% of l-arginine transport.…”

“…29,30 In ECs, system y + , a sodium-independent system, accounts for ≈60% of l-arginine transport, and system y + L, a sodium-dependent system, accounts for ≈40% of l-arginine transport. 26,31,32 System y + selectively mediates the cellular transport and exchange of cationic amino acids, including l-arginine, whereas system y + L transports and mediates the exchange of both cationic and neutral amino acids. 26,28 Endothelium-dependent relaxation has been shown to be impaired in a patient with l-arginine deficiency who was shown to be a compound heterozygote for 2 mutations in the SLC7A7 gene that encodes for the y + LAT1 amino acid transporter.…”

“…26,31,32 System y + selectively mediates the cellular transport and exchange of cationic amino acids, including l-arginine, whereas system y + L transports and mediates the exchange of both cationic and neutral amino acids. 26,28 Endothelium-dependent relaxation has been shown to be impaired in a patient with l-arginine deficiency who was shown to be a compound heterozygote for 2 mutations in the SLC7A7 gene that encodes for the y + LAT1 amino acid transporter. The patient's endothelium-dependent relaxation was corrected with l-arginine infusion.…”