s-nitrosocysteine and 2-aminobicyclo(2-2-1)heptane-2-carboxylic-acid

The system L neutral amino acid transporter (LAT; LAT1, LAT2, LAT3, or LAT4) has multiple functions in human biology, including the cellular import of S-nitrosothiols (SNOs), biologically active derivatives of nitric oxide (NO). SNO formation by haemoglobin within red blood cells (RBC) has been studied, but the conduit whereby a SNO leaves the RBC remains unidentified. Here we hypothesised that SNO export by RBCs may also depend on LAT activity, and investigated the role of RBC LAT in modulating SNO-sensitive RBC-endothelial cell (EC) adhesion. We used multiple pharmacologic inhibitors of LAT in vitro and in vivo to test the role of LAT in SNO export from RBCs and in thereby modulating RBC-EC adhesion. Inhibition of human RBC LAT by type-1-specific or nonspecific LAT antagonists increased RBC-endothelial adhesivity in vitro, and LAT inhibitors tended to increase post-transfusion RBC sequestration in the lung and decreased oxygenation in vivo. A LAT1-specific inhibitor attenuated SNO export from RBCs, and we demonstrated LAT1 in RBC membranes and LAT1 mRNA in reticulocytes. The proadhesive effects of inhibiting LAT1 could be overcome by supplemental L-CSNO (S-nitroso-L-cysteine), but not D-CSNO or L-Cys, and suggest a basal anti-adhesive role for stereospecific intercellular SNO transport. This study reveals for the first time a novel role of LAT1 in the export of SNOs from RBCs to prevent their adhesion to ECs. The findings have implications for the mechanisms of intercellular SNO signalling, and for thrombosis, sickle cell disease, and post-storage RBC transfusion, when RBC adhesivity is increased.

Topics: Amino Acid Transport System L; Amino Acids, Cyclic; Animals; Benzoxazoles; Cell Adhesion; Cysteine; Endothelial Cells; Erythrocyte Deformability; Erythrocytes; Human Umbilical Vein Endothelial Cells; Humans; In Vitro Techniques; Leucine; Mice; Mice, Nude; Reticulocytes; RNA, Messenger; S-Nitrosothiols; Tyrosine

System L amino acid transporters have been shown to be responsible for cellular uptake of S-nitroso-L-cysteine (l-CSNO). In this study, we examined the characteristics of L-CSNO uptake in Xenopus laevis oocytes expressing system L transporters and found that uptake increased only when both 4F2 heavy chain (4F2HC) and either L-type amino acid transporter 1 (LAT1) or LAT2 light chain were coexpressed. The K(m) for transport was 57 +/- 8 microM for 4F2HC-LAT1 and 520 +/- 52 microM for 4F2HC-LAT2. Vascular endothelial and smooth muscle cells were shown to express transcripts for 4F2HC and for both LAT1 and LAT2. Transport of L-CSNO into red blood cells, endothelial cells, and smooth muscle cells was inhibited by 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) and by large neutral amino acids demonstrating functional system L transporters in each cell type. Uptake of L-CSNO led to accumulation of cellular S-nitrosothiols and inhibition of both growth factor-induced ERK phosphorylation and TNF-alpha-mediated IkappaB degradation. Similar effects were seen when cells were incubated simultaneously with S-nitrosoalbumin and L-cysteine but not with d-cysteine or with S-nitrosoalbumin alone. In each case, nitrosylation of proteins and cellular responses were blocked by BCH. Together, these data suggest that transmembrane movement of nitric oxide (NO) equivalents from the plasma albumin NO reservoir is mediated by cysteine, which serves as a carrier. The mechanism requires transnitrosylation from S-nitrosoalbumin to free cysteine and activity of system L transporters, thereby providing a unique pathway for cellular responses to S-nitrosoalbumin.

Topics: Amino Acid Transport System L; Amino Acids, Cyclic; Amino Acids, Neutral; Animals; Biological Transport, Active; Cells, Cultured; Cysteine; Endothelium, Vascular; Erythrocytes; Extracellular Signal-Regulated MAP Kinases; Female; Fusion Regulatory Protein 1, Heavy Chain; Humans; I-kappa B Proteins; In Vitro Techniques; Large Neutral Amino Acid-Transporter 1; Muscle, Smooth, Vascular; Nitric Oxide; Oocytes; Phosphorylation; S-Nitrosothiols; Tumor Necrosis Factor-alpha; Xenopus laevis

Previously, we proposed that S-nitroso-L-cysteine, an endogenous S-nitrosothiol, was incorporated via the system L-like amino acid transporter(s) in rat brain slices. In this study, we investigated the effect of S-nitroso-L-cysteine on L-[3H]leucine uptake in PC12 cells (a neuronal cell line). L-[3H]Leucine uptake in PC12 cells was Na(+) independent and significantly inhibited by an inhibitor of system L and by L-phenylalanine, L-cysteine, L-methionine and L-leucine at 1 mM. The effects of L-alanine, L-serine and L-threonine were limited. S-Nitroso-L-cysteine, but not other nitric oxide compounds, inhibited L-[3H]leucine uptake, and this inhibitory effect was eliminated by washing with buffer. System L is composed of the 4F2 light chains (LAT1 or LAT2) and the heavy chain, and the transcripts of these components were detected in RNA from PC12 cells. These findings suggest that S-nitroso-L-cysteine is incorporated via the system L amino acid transporter and thus regulates cell responses in PC12 cells.

Topics: Alanine; Amino Acid Transport Systems; Amino Acids, Cyclic; Animals; beta-Alanine; Biological Transport; Calcium; Cysteine; Dose-Response Relationship, Drug; Leucine; Nitric Oxide Donors; PC12 Cells; Phenylalanine; Rats; RNA, Messenger; S-Nitrosothiols; Stereoisomerism; Tritium