Identification of stereoselective transporters for S-nitroso-L-cysteine: role of LAT1 and LAT2 in biological activity of S-nitrosothiols.
Many of the biological effects of nitric oxide are mediated by S-nitrosothiols. However, the mechanisms by which S-nitrosothiols transduce their activity across cell membranes are unclear. We show that the pathway responsible for the cellular effects of S-nitrosothiols is specific for S-nitrosocysteine (CSNO), is stereoselective, and requires direct uptake of intact L-CSNO. Transport is independent of extracellular sodium, competitively inhibited by leucine, and blocked by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, a specific inhibitor of the system L amino acid transporter family. Other nitrosothiols such as S-nitrosoglutathione are not substrates for transport and require reaction with L-cysteine for activity. To show that system L family members mediate uptake, we expressed two members, LAT1 and LAT2, in Xenopus oocytes. Both LAT1 and LAT2, when co-expressed with 4F2 heavy chain, were found to efficiently transport L-CSNO. Mammalian cells were shown to express LAT1 and LAT2. A431 cells express both proteins, whereas T24 cells express only LAT1. Overexpression of LAT1 in T24 cells using recombinant adenoviruses led to increased uptake of L-CSNO, whereas knockdown using a specific small interfering RNA led to decreased uptake. These data definitively identify LAT1 and LAT2 as members of system L that mediate transmembrane movement of l-CSNO and suggest that system L family members are involved in the cellular activity of small molecular weight nitrosothiols.
Li
Sheng
S
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Whorton
A Richard
AR
D027182:Amino Acid Transport System y+ / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D005260:Female; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000235:genetics / Q000378:metabolism; D027301:Fusion Regulatory Protein 1, Light Chains / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D027283:Large Neutral Amino Acid-Transporter 1 / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D026403:S-Nitrosothiols / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D014982:Xenopus laevis
D027182:Amino Acid Transport System y+; D027282:Fusion Regulatory Protein 1, Heavy Chain; D027301:Fusion Regulatory Protein 1, Light Chains; D027283:Large Neutral Amino Acid-Transporter 1; D011994:Recombinant Proteins; D026403:S-Nitrosothiols; C420646:SLC7A8 protein, human; D004247:DNA; C021315:S-nitrosocysteine; D003545:Cysteine