Pulmonary alveolar epithelial uptake of S-nitrosothiols is regulated by L-type amino acid transporter

Abstract

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.

Fig. 1.

L-type amino acid transporter 1 (LAT-1) and peptide transporter 2 (PEPT2) expression. A: LAT-1 expression, whole rat lung immunoblot, 50 μg protein/lane. B, left: LAT-1 expression in L2 cell lysates, 50 μg protein/lane. B, right: PEPT2 expression in L2 cell lysates, 40 μg protein/lane. C: LAT-1 (left) and PEPT2 (right) expression in rat lung.

Fig. 2.

S-nitrosothiol (SNO)/nitric oxide (NO) uptake by L2 cells treated with RSNO ± LAT inhibition. A: SNO detected by triiodide reduction of NO in cell lysates via ozone chemiluminescence after incubation with 200 μM GSNO ± 50 μMd-Cys, 200 μM GSNO + 50 μMl-Cys, 200 μM GSNO + 50 μMl-Cys + 10 mMl-Leu, 200 μM GSNO + 50 μMl-Cys + 10 mM BCH (competitive inhibitors of LAT-1). Data are means ± SE representative of 3 experiments with samples analyzed in duplicate. B: DAF-FM fluorescence signal in L2 cells treated with RSNO ± LAT inhibition. Effect of 100 μMl-Cys on 200 μM GSNO induced intracellular DAF-FM signal ± 10 mM l-Leu or 10 mM BCH (competitive inhibitors of LAT-1). Values were normalized to the maximum signal. Data are means ± SE, n = 8/condition, *P < 0.001 vs. maximum.

Fig. 3.

NO uptake in isolated rat type II cells treated with RSNO ± LAT inhibition. Effect of 100 μM l- and d-Cys on 200 μM GSNO induced intracellular DAF-FM signal. Conditions include 200 μM GSNO + 100 μM l-Cys + 10 mM l-Leu or 10 mM BCH (competitive inhibitors of LAT-1). Values normalized to maximum signal. Data are means ± SE, n = 8/condition, *P < 0.001 vs. maximum.

Fig. 4.

SNO/NO accumulation in L2 cells treated with DEA NONOate. A: SNO detected by ozone chemiluminescence after incubating with 100 μM GSNO + 100 μM l-Cys, 100 μM DEA NONOate ± 100 μM d- or l-Cys, and 100 μM DEA NONOate + 100 μM l-Cys ± LAT-1 competitors l-Leu and BCH (10 mM). Data are means ± SE, n = 6. B: DAF-FM signal in L2 cells treated with NONOate. L2 cells incubated with 100 μM GSNO + 100 μM l-Cys, 100 μM DEA NONOate ± 100 μM d-Cys, 100 μM DEA NONOate + 100 μM l-Cys ± LAT-1 competitors l-Leu and BCH (10 mM). Data are normalized to maximum signal. Data are means ± SE, n = 8/condition, *P < 0.001 vs. maximum.

Fig. 5.

PEPT2 mediated uptake of SNO/NO in alveolar epithelium. A: SNO in lysates from L2 cells treated with 200 μM SNO-Cys-Gly ± 100 μM l-Cys or 10 mM Gly-Sar. B: DAF-FM fluorescence in L2 cells treated with SNO-Cys-Gly ± Gly-Sar, with CSNO ± l-Leu for comparison. C: DAF-FM fluorescence in type II cells treated with SNO-Cys-Gly + Gly-Sar or l-Cys for comparison. Data are means ± SE, n = 3/condition, representative of 3 replicate experiments, *P < 0.001 vs. maximum.