The mechanism of transmembrane S-nitrosothiol transport

Abstract

S-nitrosothiols have been suggested to play an important role in nitric oxide (NO)-mediated biological events. However, the mechanisms by which an S-nitrosothiol (or the S-nitroso functional group) is transferred across cell membrane are still poorly understood. We have demonstrated previously that the degradation of S-nitrosoglutathione (GSNO) by cells absolutely required the presence of cystine in the extracellular medium and proposed a mechanism that involved the reduction of cystine to cysteine, followed by the reaction of cysteine with GSNO to form S-nitrosocysteine (CysNO), mixed disulfides, and nitrosyl anion. In the present study we have assessed the effect of cystine on the transfer of the S-nitroso functional group from the extracellular to the intracellular space. Using RAW 264.7 cells, we found that the presence of L-cystine enhanced GSNO-dependent S-nitrosothiol uptake, increasing the intracellular S-nitrosothiol level from approximately 60 pmol/mg of protein to approximately 3 nmol/mg of protein. The uptake seems to depend on the reduction of L-cystine to L-cysteine, which involves the xc- amino acid transport system, the transnitrosation between GSNO and L-cysteine to form L-CysNO, and uptake of L-CysNO via amino acid transport system L. Compared with GSNO, (Z)-1-[N-(3-ammoniopropyl)-N-[4-(3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate, an NO donor, is much less effective at intracellular S-nitrosothiol formation in the presence of L-cystine or L-cysteine, suggesting that the biochemical changes that occur after exposure of cells to S-nitrosothiol, with respect to thiol chemistry, are distinctly different from those observed with NO.

Fig. 1.

The effect of l-cystine on intracellular S-nitrosothiol formation. RAW 264.7 cells were treated with GSNO (500 μM) for 60 min in HBSS, FBS-free medium, or HBSS supplemented with l-cystine (200 μM). Representative chemiluminescence traces of sulfanilamide-treated (A) or HgCl₂/sulfanilamide-treated (B) samples are shown. (C) Quantification of S-nitrosothiol content in the cell lysate. Data represent mean ± SEM. (D) Time course of the formation of intracellular S-nitrosothiol in the presence (○) or absence (•) of l-cystine as well as the decay of extracellular S-nitrosothiol level in the presence of l-cystine (▴). Data represent mean ± SEM (n = 3).

Fig. 2.

Concentration response of l-cystine- or l-cysteine-mediated intracellular S-nitrosothiol formation and extracellular S-nitrosothiol decay. RAW 264.7 cells were treated with GSNO (500 μM) in HBSS for 60 min in the presence of various concentrations of l-cystine (filled symbols) or l-cysteine (open symbols). The S-nitrosothiol content in the cell lysate (• and ○) was measured by chemiluminescence. The S-nitrosothiol level in HBSS (▴ and ▵) was detected by UV-visible spectroscopy. Data represent mean ± SEM (n = 3).

Fig. 3.

The effect of l-glutamate on the formation of intracellular S-nitrosothiol and extracellular thiol. S-nitrosothiol formation (gray bars): RAW 264.7 cells were treated with GSNO (500 μM) for 60 min in the absence or presence of l-cystine (200 μM) and l-glutamate (3 mM). The S-nitrosothiol content in the cell lysate was measured by chemiluminescence. Thiol formation (black bars): RAW 264.7 cells were incubated in HBSS for 60 min in the absence and presence of l-cystine and l-glutamate. The thiol level in the HBSS was detected by a DTNB assay. Data represent mean ± SEM (n = 3).

Scheme 1.

A model for l-cystine-mediated S-nitrosothiol uptake.