Identification of Stereoselective Transporters for S-Nitroso-L-cysteine (2005)

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. 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. Nitrosothiols including the low molecular weight nitrosothiols, S-nitrosoglutathione (GSNO) 1The abbreviations used are: GSNO, S-nitrosoglutathione; CSNO, S-nitrosocysteine; SNAP, S-nitroso-N-acetylpenicillamine; MTSES, sodium 2-sulfonatoethyl methanethiosulfonate; MPB, N-(3-maleimidylpropionyl)biocytin; LAT1, system L amino acid transporter 1; LAT2, system L amino acid transporter 2; PTP1B, protein-tyrosine phosphatase 1B; NO, nitric oxide; BCH, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid; RIPA, radioimmune precipitation buffer; HRP, horseradish peroxidase; RT, reverse transcriptase. 1The abbreviations used are: GSNO, S-nitrosoglutathione; CSNO, S-nitrosocysteine; SNAP, S-nitroso-N-acetylpenicillamine; MTSES, sodium 2-sulfonatoethyl methanethiosulfonate; MPB, N-(3-maleimidylpropionyl)biocytin; LAT1, system L amino acid transporter 1; LAT2, system L amino acid transporter 2; PTP1B, protein-tyrosine phosphatase 1B; NO, nitric oxide; BCH, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid; RIPA, radioimmune precipitation buffer; HRP, horseradish peroxidase; RT, reverse transcriptase. and S-nitrosocysteine (CSNO), have been proposed to be important mediators of nitric oxide signaling (1Foster M.W. McMahon T.J. Stamler J.S. Trends Mol. Med. 2003; 9: 160-168Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 2Gow A.J. Farkouh C.R. Munson D.A. Posencheg M.A. Ischiropoulos H. Am. J. Physiol. 2004; 287: L262-L268Crossref PubMed Scopus (334) Google Scholar, 3Hogg N. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 585-600Crossref PubMed Scopus (352) Google Scholar). The biological activity of these compounds resides in their ability to reversibly modify critical proteins in cell signaling pathways through targeted oxidation of cysteine thiols (1Foster M.W. McMahon T.J. Stamler J.S. Trends Mol. Med. 2003; 9: 160-168Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 2Gow A.J. Farkouh C.R. Munson D.A. Posencheg M.A. Ischiropoulos H. Am. J. Physiol. 2004; 287: L262-L268Crossref PubMed Scopus (334) Google Scholar, 3Hogg N. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 585-600Crossref PubMed Scopus (352) Google Scholar, 4Hess D.T. Matsumoto A. Nudelman R. Stamler J.S. Nat. Cell Biol. 2001; 3: E46-E49Crossref PubMed Scopus (216) Google Scholar). Although their biological activity is well established, the mechanisms by which they transduce their activity from the extracellular space to intracellular targets are largely unknown. Insight into pathways that may participate in the transfer of NO equivalents across cell membranes comes from experiments that show that nitrosothiols are not equivalent in their ability to alter signaling pathways. For example, CSNO but not GSNO or S-nitroso-N-acetylpenacillamine (SNAP) has been shown to modify protein-tyrosine phosphatase 1B (PTP1B) (5Li S. Whorton A.R. Arch. Biochem. Biophys. 2003; 410: 269-279Crossref PubMed Scopus (96) Google Scholar) and glyceraldehyde-3-phosphate dehydrogenase (6Padgett C.M. Whorton A.R. Am. J. Physiol. 1995; 269: C739-C749Crossref PubMed Google Scholar) in intact cells. Similarly, CSNO but not GSNO is able to transfer NO equivalents across red cell membranes, leading to the formation of intracellular GSNO and S-nitrosohemoglobin (7Tsikas D. Sandmann J. Rossa S. Gutzki F.M. Frolich J.C. Anal. Biochem. 1999; 270: 231-241Crossref PubMed Scopus (62) Google Scholar, 8Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1461) Google Scholar). The transfer of NO equivalents does not appear to involve NO release through the breakdown of nitrosothiols (1Foster M.W. McMahon T.J. Stamler J.S. Trends Mol. Med. 2003; 9: 160-168Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 9Mathews W.R. Kerr S.W. J. Pharmacol. Exp. Ther. 1993; 267: 1529-1537PubMed Google Scholar, 10Stamler J.S. Toone E.J. Curr. Opin. Chem. Biol. 2002; 6: 779-785Crossref PubMed Scopus (107) Google Scholar, 11McMahon T.J. Pawloski J.R. Hess D.T. Piantadosi C.A. Luchsinger B.P. Singel D.J. Stamler J.S. Blood. 2003; 102: 410-411Crossref PubMed Scopus (8) Google Scholar). Rather it seems that transfer requires specific interactions with proteins since the bioactivity of low molecular weight thiols is known to be stereoselective and unrelated to chemical half-life (1Foster M.W. McMahon T.J. Stamler J.S. Trends Mol. Med. 2003; 9: 160-168Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, 9Mathews W.R. Kerr S.W. J. Pharmacol. Exp. Ther. 1993; 267: 1529-1537PubMed Google Scholar, 10Stamler J.S. Toone E.J. Curr. Opin. Chem. Biol. 2002; 6: 779-785Crossref PubMed Scopus (107) Google Scholar, 11McMahon T.J. Pawloski J.R. Hess D.T. Piantadosi C.A. Luchsinger B.P. Singel D.J. Stamler J.S. Blood. 2003; 102: 410-411Crossref PubMed Scopus (8) Google Scholar, 12Kwon J. Lee S.R. Yang K.S. Ahn Y. Kim Y.J. Stadtman E.R. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16419-16424Crossref PubMed Scopus (511) Google Scholar, 13Lipton A.J. Johnson M.A. Macdonald T. Lieberman M.W. Gozal D. Gaston B. Nature. 2001; 413: 171-174Crossref PubMed Scopus (285) Google Scholar). For example, in the circulation, l-CSNO is a potent vasodilator, but d-CSNO is not (14Davisson R.L. Travis M.D. Bates J.N. Lewis S.J. Circ. Res. 1996; 79: 256-262Crossref PubMed Scopus (86) Google Scholar), and in the central nervous system, l-CSNO rather than d-CSNO is important in regulating ventilatory signaling (13Lipton A.J. Johnson M.A. Macdonald T. Lieberman M.W. Gozal D. Gaston B. Nature. 2001; 413: 171-174Crossref PubMed Scopus (285) Google Scholar). Furthermore, in cells that possess γ-glutamyl transpeptidase, GSNO is only active following cleavage to S-nitrosocysteinylglycine (15Gaston B. Reilly J. Drazen J.M. Fackler J. Ramdev P. Arnelle D. Mullins M.E. Sugarbaker D.J. Chee C. Singel D.J. Loscalzo J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10957-10961Crossref PubMed Scopus (576) Google Scholar). Together, these data strongly suggest the existence of a stereoselective mechanism that is characterized either by a receptor-mediated process selective for l-CSNO or by selective transport of intact CSNO from outside to inside the cell. The involvement of a transporter is supported by data from our group and from others demonstrating that amino acids that competitively inhibit members of the system L amino acid transporter family block the biological activity of CSNO in intact cells (5Li S. Whorton A.R. Arch. Biochem. Biophys. 2003; 410: 269-279Crossref PubMed Scopus (96) Google Scholar, 16Nemoto T. Shimma N. Horie S. Saito T. Okuma Y. Nomura Y. Murayama T. Eur. J. Pharmacol. 2003; 458: 17-24Crossref PubMed Scopus (18) Google Scholar, 17Zhang Y. Hogg N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7891-7896Crossref PubMed Scopus (156) Google Scholar). These transporters exist as heterodimers between a common 80-kDa heavy chain (4F2HC) and a variable 40-kDa light chain (4F2LC) that confers transport activity (18Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar, 19Yanagida O. Kanai Y. Chairoungdua A. Kim D.K. Segawa H. Nii T. Cha S.H. Matsuo H. Fukushima J. Fukasawa Y. Tani Y. Taketani Y. Uchino H. Kim J.Y. Inatomi J. Okayasu I. Miyamoto K. Takeda E. Goya T. Endou H. Biochim. Biophys. Acta. 2001; 1514: 291-302Crossref PubMed Scopus (572) Google Scholar, 20Nakamura E. Sato M. Yang H. Miyagawa F. Harasaki M. Tomita K. Matsuolka S. Noma A. Iwai K. Minato N. J. Biol. Chem. 1999; 274: 3009-3016Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 21Uchino H. Kanai Y. Kim D.K. Wempe M.F. Chairoungdua A. Morimoto E. Anders M.W. Endou H. Mol. Pharmacol. 2002; 61: 729-737Crossref PubMed Scopus (340) Google Scholar, 22Rossier G. Meier C. Bauch C. Summa V. Sordat B. Verrey F. Kuhn L.C. J. Biol. Chem. 1999; 274: 34948-34954Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 23Segawa H. Fukasawa Y. Miyamoto K. Takeda E. Endou H. Kanai Y. J. Biol. Chem. 1999; 274: 19745-19751Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 24Pineda M. Fernandez E. Torrents D. Estevez R. Lopez C. Camps M. Lloberas J. Zorzano A. Palacin M. J. Biol. Chem. 1999; 274: 19738-19744Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). System L transporter activity is derived from two characterized light chains, LAT1 and LAT2. LAT1 is relatively specific for large neutral amino acids, whereas LAT2 has a broader specificity and transports both large and small neutral amino acids (18Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar, 22Rossier G. Meier C. Bauch C. Summa V. Sordat B. Verrey F. Kuhn L.C. J. Biol. Chem. 1999; 274: 34948-34954Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 23Segawa H. Fukasawa Y. Miyamoto K. Takeda E. Endou H. Kanai Y. J. Biol. Chem. 1999; 274: 19745-19751Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 24Pineda M. Fernandez E. Torrents D. Estevez R. Lopez C. Camps M. Lloberas J. Zorzano A. Palacin M. J. Biol. Chem. 1999; 274: 19738-19744Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Both transporters have wide tissue distribution and are critical for amino acid uptake and transfer across endothelial and epithelial barriers, especially in the brain, kidney, and intestine (21Uchino H. Kanai Y. Kim D.K. Wempe M.F. Chairoungdua A. Morimoto E. Anders M.W. Endou H. Mol. Pharmacol. 2002; 61: 729-737Crossref PubMed Scopus (340) Google Scholar, 22Rossier G. Meier C. Bauch C. Summa V. Sordat B. Verrey F. Kuhn L.C. J. Biol. Chem. 1999; 274: 34948-34954Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 23Segawa H. Fukasawa Y. Miyamoto K. Takeda E. Endou H. Kanai Y. J. Biol. Chem. 1999; 274: 19745-19751Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 24Pineda M. Fernandez E. Torrents D. Estevez R. Lopez C. Camps M. Lloberas J. Zorzano A. Palacin M. J. Biol. Chem. 1999; 274: 19738-19744Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 25Verrey F. Pfluegers Arch. Eur. J. Physiol. 2003; 445: 529-533Crossref PubMed Scopus (230) Google Scholar). Both are sodium-independent exchangers with symmetrical extracellular and intracellular substrate specificities but greatly different extracellular (high) and intracellular (low) affinities (25Verrey F. Pfluegers Arch. Eur. J. Physiol. 2003; 445: 529-533Crossref PubMed Scopus (230) Google Scholar). Importantly, both have been reported to transport mercurial derivatives of cysteine (26Simmons-Willis T.A. Koh A.S. Clarkson T.W. Ballatori N. Biochem. J. 2002; 367: 239-246Crossref PubMed Scopus (188) Google Scholar). In the present study, we have investigated pathways used by exogenous nitrosothiols to modify cellular proteins. We report the existence of a transport mechanism that stereoselectively transports l-CSNO into cells and show that uptake activity is mediated by proteins that constitute system L amino acid transport. In addition, we provide definitive data that show that l-CSNO is a substrate for LAT1 and LAT2. These studies are important since they show that specific mechanisms exist for cellular responses to nitrosothiols and identify a novel pathway for movement of nitrosothiols into cells, where they can efficiently exert their biological effects. Materials—GSNO was synthesized as described previously (5Li S. Whorton A.R. Arch. Biochem. Biophys. 2003; 410: 269-279Crossref PubMed Scopus (96) Google Scholar). CSNO was prepared by mixing cysteine hydrochloride (pH 2.5) and sodium nitrite at a molar ratio of 2:3 in water. Complete reaction was verified by UV absorption at 335 nm. SNAP was purchased form Alexis Biochemicals. [3H]leucine was from Amersham Biosciences, l-[35S]cysteine was from PerkinElmer Life Sciences, sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) was from Toronto Research Chemicals, N-(3-maleimidylpropionyl)biocytin (MPB) was from Molecular Probes, and N-ethyl maleimide, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), α-(methylamino)isobutyrate, l-cysteine, d-cysteine, and sodium nitrite were purchased from Sigma. Cell Culture—A431 or T24 were obtained from American Type Culture Collection. Cells were grown in Dulbecco's modified Eagle's medium with high glucose (A431) or McCoy's 5a medium (T24) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. To begin an experiment, confluent cells were rinsed twice with Hanks' balanced salt solution (Invitrogen) buffered with 25 mm Hepes (HBSH, pH 7.4) and incubated at 37 °C with various agents in HBSH for the indicated times. Determination of PTP1B Oxidation—PTP1B oxidation was determined using MPB as described previously (5Li S. Whorton A.R. Arch. Biochem. Biophys. 2003; 410: 269-279Crossref PubMed Scopus (96) Google Scholar). Briefly, after treatment, cells were washed three times with ice-cold HBSH and then scraped into RIPA buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mm phenylmethylsulfonyl fluoride) containing 20 mm N-ethyl maleimide. After incubation on ice for 30 min, samples were centrifuged at 14,000 rpm for 10 min at 4 °C, and the supernatant was applied to a PD-10 desalting column (Amersham Biosciences) to remove excess N-ethyl maleimide. The eluate that contained cellular protein was treated with 20 mm dithiothreitol for 30 min to reduce all oxidized thiols. Samples were reapplied to a PD-10 column to remove excess dithiothreitol, and the eluates were treated with 50 μm MPB. Excess MPB was removed by elution from a PD-10 column, and eluates were treated with 50 μm glutathione. Aliquots containing equivalent amounts of protein were immunoprecipitated using 1 μg of PTP1B monoclonal antibody (Oncogene) by incubation at 4 °C overnight. Immune complexes were precipitated by incubation for 2 h with 20 μl of protein A-agarose (Santa Cruz Biotechnology) and collected by centrifugation. Immunoprecipitates were washed five times with RIPA buffer, dissolved in 20 μl of electrophoresis sample buffer (50 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerin, and 0.01% bromphenol blue), separated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. After blocking with 10% nonfat dried milk in TBST buffer (20 mm Tris-HCl, pH 7.6, 137 mm sodium chloride, 0.1% Tween 20), MPB-labeled PTP1B was visualized by incubation with 1:4000 dilution of NeutrAvidin-linked horseradish peroxidase (avidin-HRP, Molecular Probes) followed by incubation with the ECL Plus detection system (Amersham Biosciences). Blots were stripped using 100 mm β-mercaptoethanol, 2% SDS, 62.5 mm Tris-HCl, pH 6.7, at 50 °C for 30 min, reprobed using rabbit polyclonal PTP1B antibody (Santa Cruz Biotechnology), and visualized using anti-rabbit IgG coupled to horseradish peroxidase followed by visualization with ECL Plus. Direct Measurement of Amino Acid Uptake—l-[35S]CSNO was prepared by mixing l-cysteine (30 μCi/μmol [35S]cysteine, pH 2.5) and sodium nitrite at a molar ratio of 2:3 in water. The resulting CSNO was neutralized by diluting the reaction mixture in HBSH. Cells were incubated with either l-[35S]CSNO or [3H]leucine (25 μCi/μmol) in HBSH at 37 °C, washed five times with ice-cold HBSH to remove extracellular radioactivity, and lysed into RIPA buffer with 1% SDS. Extracts collected after trichloroacetic acid precipitation produced identical results. Protein concentration was quantified by BCA assay (Pierce), and radioactivity was determined by liquid scintillation spectrometry. Uptake was linear for at least 15 min. RT-PCR—Total RNA was isolated from cells using the RNeasy Mini kit (Qiagen) and reverse-transcribed by oligo(dT) using an Omniscript RT kit (Qiagen) at 37 °C for 60 min. A fragment of LAT1 (554 bp) was amplified by PCR using primers corresponding to the partial sequence from nucleotides 418 to 971 of human LAT1 (forward, 5′-ATC CGG CCT TCA TCG CAG TAC ATC-3′, and reverse, 5′-GAC ATG ACG CCC AGG TGA TAG TTC-3′). A fragment of LAT2 (706 bp) was amplified using primers corresponding to the partial sequence from nucleotides 4 to 710 of human LAT2 (forward, 5′-GAA GAA GGA GCC AGG CAC CGA AAC-3′, and reverse, 5′-CCA GTG CGA CGA GGC CGA TGT CAG-3′). PCR products were separated by electrophoresis in a 2% agarose gel and visualized under UV light in the presence of ethidium bromide. Expression of System L in Xenopus Oocytes and Measurement of l-CSNO Uptake—Xenopus laevis ovarian lobes containing stage V or VI oocytes were purchased from Nasco. Oocytes were treated with collagenase A (2 mg/ml) for 30–50 min at room temperature in Ca2+-free medium (82.5 mm NaCl, 2 mm KCl, 1 mm MgCl2, and 5 mm HEPES, pH 7.5) to remove the follicular layer and then maintained in buffer containing 100 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, and 100 μg/ml gentamicin, pH 7.6 at 18 °C. 4F2HC cDNA was obtained from human aortic smooth muscle cells by RT-PCR using the following primer pair: forward, 5′-TAA GGA TCC ATG AGC CAG GAC ACC GAG G-3′, and reverse, 5′-TAA GAA TTC TCA GGC CGC GTA GGG GAA-3′. Human LAT2 cDNA was obtained from A431 cells by RT-PCR using the following primer pair: forward, 5′-TA GGA TCC ATG GAA GAA GGA GCC AGG CAC-3′, and reverse, 5′-TA GAA TTC GCC AGG GAA TGG TGG TCC TCA-3′. LAT1 cDNA was obtained as described below. The 4F2HC, LAT1, and LAT2 cDNAs were subcloned into WCV vector (a kind gift of Dr. Antonius M. J. VanDongen) under the control of the T7 promoter. Capped cRNAs were synthesized in vitro using T7 RNA polymerase, purified by RNeasy Mini kit (Qiagen), and injected (73.6 nl of cRNA, 200 ng/μl) into the defolliculated oocytes under a dissecting microscope with a micrometer-driven micropipetter. Pure water was injected as following oocytes were washed and then incubated in μl of uptake buffer mm chloride, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, and 5 mm pH 7.4) containing 25 μm l-[35S]CSNO (30 μCi/μmol) with 1 mm for 15 min at room The oocytes were then washed five times with ice-cold uptake buffer, and radioactivity was determined by liquid scintillation spectrometry. of LAT1 cDNA was obtained from A431 cells by RT-PCR using the following primer pair: forward, TGG GTG CGG GCC CGA and reverse, TCC TCC TGG GGG ACC cDNA was subcloned into vector and then into cells by The were by and to cells using (Qiagen) following the adenoviruses were and as described S. J. B. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). of RNA to RNA small RNA was used to the of LAT1. containing and corresponding to nucleotides of the LAT1 were synthesized as forward, TTC CGG TCA TCA AGC ATG ACG GAG ATG TCC and reverse, GGA TGT TGA TGA CGG TGT The were and into the vector with the sequence under the control of the promoter. The was amplified from cells by RT-PCR using the following primer pair: forward, 5′-TA GAC CCC CAG TGG GAC CAG and reverse, TCG TGT CCT TTC CAC The RT-PCR was into vector by and the were with and for with the described The recombinant was then with and and the fragment containing the and the sequence was subcloned into an vector The were prepared as the were by of CSNO have previously shown that l-CSNO PTP1B in intact cells, leading to increased of others have found that the activity of nitrosothiols is it to that PTP1B oxidation is mediated by l-CSNO but not by shown in when A431 cells were treated with prepared l-CSNO or with an mixture of SNAP and l-cysteine, intracellular PTP1B was oxidized as indicated by increased with MPB d-CSNO or the of SNAP were largely with studies (5Li S. Whorton A.R. Arch. Biochem. Biophys. 2003; 410: 269-279Crossref PubMed Scopus (96) Google Scholar), l-cysteine SNAP when used In addition, or were (5Li S. Whorton A.R. Arch. Biochem. Biophys. 2003; 410: 269-279Crossref PubMed Scopus (96) Google Scholar). be that and d-CSNO have leading to the formation of and and d-CSNO have such different to modify PTP1B, these breakdown products are These that the mechanism is stereoselective and strongly our that a specific transporter or protein that is specific for l-CSNO is involved (5Li S. Whorton A.R. Arch. Biochem. Biophys. 2003; 410: 269-279Crossref PubMed Scopus (96) Google Scholar). CSNO by the mechanism involved in movement of NO equivalents from the extracellular to intracellular we the of on PTP1B is well known to thiols but does not the cell. thiols in the medium or on the extracellular are for intracellular thiols The of on PTP1B oxidation was investigated by 10 mm to the medium cells on the ability of l-CSNO to PTP1B, demonstrating that cell thiols are not involved in the bioactivity of l-CSNO and that does not cells and modify The for l-CSNO was by the of GSNO in We previously shown that the incubation of cells with GSNO l-cysteine PTP1B (5Li S. Whorton A.R. Arch. Biochem. Biophys. 2003; 410: 269-279Crossref PubMed Scopus (96) Google Scholar). However, when we mm l-cysteine to medium containing 10 mm followed by the of mm GSNO, we PTP1B that thiols and the formation of is in with the PTP1B oxidation l-cysteine and GSNO were for 5 min at room temperature to since cell it is that thiols participate in a mechanism and that movement of NO equivalents into cells transport of intact l-CSNO. In the following we investigated direct cellular uptake of l-CSNO. A431 cells were treated with 25 μm uptake was with 1 mm to thiols blocked uptake of cysteine by The is These show that uptake of cysteine and show that cysteine is not In we prepared l-[35S]CSNO by mixing l-[35S]cysteine with at pH followed by dilution into HBSH to the acid and a concentration of 25 μm CSNO on cells. Uptake of l-CSNO was not different from uptake of l-[35S]cysteine under these Importantly, when with MTSES, uptake of l-[35S]CSNO was largely uptake of uptake when cells are incubated with l-[35S]CSNO in the presence of transport of intact the of not alter [3H]leucine uptake, demonstrating that system L transport is not by These suggest that CSNO is intact by cells. Direct uptake of intact CSNO was shown in the following experiments in which we investigated the pH of both CSNO formation and uptake. In these l-cysteine and were at the pH and CSNO formation was determined at 335 nm. To uptake, l-[35S]cysteine and were under the into HBSH pH and to cells incubated in HBSH containing 1 mm CSNO formation in solution when l-cysteine and are is L. J. Chem. 2002; PubMed Scopus Google Scholar), uptake using the pH CSNO formation was reaction at pH uptake pH In the between CSNO formation and uptake was linear the of pH which that CSNO is the responsible for the uptake uptake is in and is by not is an important to uptake of since was prepared and to cells. of System L in of acid transporters were characterized on their for a of amino In the of system transport resides in two proteins, LAT1 neutral amino acid and LAT2 large and small neutral amino acid (21Uchino H. Kanai Y. Kim D.K. Wempe M.F. Chairoungdua A. Morimoto E. Anders M.W. Endou H. Mol. Pharmacol. 2002; 61: 729-737Crossref PubMed Scopus (340) Google