Pulmonary alveolar epithelial uptake of S-nitrosothiols is regulated by L-type amino acid transporter.
Nitric oxide (NO) effects are often mediated via S-nitrosothiol (SNO) formation; SNO uptake has recently been shown to be mediated in some cell types via system L-type amino acid transporters (LAT-1, 2). Inhaled NO therapy may exert some biological effects via SNO formation. We therefore sought to determine if pulmonary epithelial SNO uptake depended on LAT or peptide transporter 2 (PEPT2). Both LAT-1 and PEPT2 proteins were detected by immunoblot and immunocytochemistry in L2 cells and rat lung. We tested SNO uptake through the transporters by exposing rat alveolar epithelial cells (L2 and type II) to RSNOs: S-nitrosoglutathione, S-nitrosocysteinylglycine (SNO-Cys-Gly), S-nitrosocysteine (CSNO), and to NO donor diethylamine NONOate (DEA-NONOate). SNO was detected in cell lysates by ozone chemiluminescence. NO uptake was detected by fluorescence in alveolar epithelial cells loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate cultured in submersion and exposed to RSNOs and DEA NONOate. Addition of L-Cys but not D-Cys to RSNOs or DEA NONOate increased SNO and DAF-FM signal that was inhibited by coincubation with LAT competitors. Incubation of cells with PEPT2 substrate SNO-Cys-Gly showed no increase in SNO or DAF-FM signal unless incubated with L-Cys. This was unaffected by PEPT2 inhibition. We conclude that RSNOs (thionitrites, S-nitrosothiols) and NO enter alveolar epithelial cells predominantly by S-nitrosation of L-Cys, which is then imported through LAT.
Granillo
Olivia M
OM
School of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Brahmajothi
Mulugu V
MV
Li
Sheng
S
Whorton
A Richard
AR
Mason
S Nicholas
SN
McMahon
Timothy J
TJ
Auten
Richard L
RL
D027182:Amino Acid Transport System y+ / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism* / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005452:Fluoresceins / Q000494:pharmacology; D027301:Fusion Regulatory Protein 1, Light Chains / Q000378:metabolism*; D006834:Hydrazines / Q000494:pharmacology; D027283:Large Neutral Amino Acid-Transporter 1 / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000378:metabolism* / Q000494:pharmacology; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D027981:Symporters / Q000378:metabolism
C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; D027182:Amino Acid Transport System y+; D004151:Dipeptides; D005452:Fluoresceins; D027301:Fusion Regulatory Protein 1, Light Chains; D006834:Hydrazines; D027283:Large Neutral Amino Acid-Transporter 1; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; C486582:Slc7a8 protein, rat; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; D009569:Nitric Oxide; C028505:cysteinylglycine; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D003545:Cysteine