Functional characterization of two S-nitroso-L-cysteine transporters, which mediate movement of NO equivalents into vascular cells.
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.
Li
Sheng
S
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Whorton
A Richard
AR
D027062:Amino Acid Transport System L / Q000378:metabolism*; D000598:Amino Acids, Cyclic / Q000494:pharmacology; D021542:Amino Acids, Neutral / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D005260:Female; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D027283:Large Neutral Amino Acid-Transporter 1 / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D026403:S-Nitrosothiols / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014982:Xenopus laevis
D027062:Amino Acid Transport System L; D000598:Amino Acids, Cyclic; D021542:Amino Acids, Neutral; D027282:Fusion Regulatory Protein 1, Heavy Chain; D020672:I-kappa B Proteins; D027283:Large Neutral Amino Acid-Transporter 1; D026403:S-Nitrosothiols; D014409:Tumor Necrosis Factor-alpha; C006504:2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; D009569:Nitric Oxide; C021315:S-nitrosocysteine; D048049:Extracellular Signal-Regulated MAP Kinases; D003545:Cysteine