The mechanism of transmembraneS-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 Ϸ60 pmol͞mg of protein to Ϸ3 nmol͞mg of protein. A lthough S-nitrosothiols have been thought to play an important role in many nitric oxide (NO)-mediated biological events, the processes by which S-nitrosothiol or their bound S-nitroso groups are transferred between the extracellular and intracellular spaces are still poorly understood. Transport mechanisms involving cell-surface protein disulfide isomerase (1, 2), ␥-glutamyl transpeptidase (3, 4), or anion exchanger (5) have been proposed.Previous studies have shown that some cellular effects of either S-nitrosoglutathione (GSNO) or S-nitroso-N-acetylpenicillamine (SNAP) depend on the presence of L-cysteine (6-9). It was suggested that cysteine can increase the efficiency of NO release from GSNO by the formation of unstable Snitrosocysteine (CysNO) through a transnitrosation reaction (9). Increases in cytosolic GSNO and cysteine levels were observed during the incubation of CysNO with human red blood cells (RBCs), implying the transport of CysNO into RBCs and the transnitrosation between CysNO and cytosolic glutathione (GSH) (10). Mallis and Thomas (8) indicated that a stereospecific transporter is not involved in the direct transfer of CysNO across the cell membrane, because the combination of GSNO with L-cysteine or D-cysteine gave a similar CysNO-like effect. However, other studies indicate that amino acid transporter system L (L-AT) is involved in the uptake of CysNO by cells (7,(11)(12)(13). 2-Aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), a specific L-AT inhibitor, and L-leucine, a substrate for L-AT, were shown to inhibit CysNO-stimulated cellular effects. L-leucine uptake in PC12 cells was inhibited by CysNO, not by SNAP, sodium nitroprusside, 3-morpholinosydnonimine (SIN-1)⅐HCl, (ϩ or Ϫ)-N-{(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl}-3-pyridinecarboximide (NOR-4), or sodium nitrite (12). Although these studies suggest the involvement of specific transport mechanisms for S-nitrosothiols, the change of intracellular S-nitrosothiol level was not examined. Consequently, the direct involvement of these pathways...

Identification of S-nitrosylation motifs by site-specific mapping of the S -nitrosocysteine proteome in human vascular smooth muscle cells

“…These two conditions were used to explore the S-nitrosoproteome of HASMC. The difference in the yield of protein S-nitrosocysteine between CysNO and the nitric oxide donor treatment may reflect the higher efficiency of S-nitrosylation by CysNO consistent with previous results (23). Cell culture studies have shown that exogenous CysNO is effectively transported intracellularly via the amino acid transporter system transporter system (23).…”

“…The difference in the yield of protein S-nitrosocysteine between CysNO and the nitric oxide donor treatment may reflect the higher efficiency of S-nitrosylation by CysNO consistent with previous results (23). Cell culture studies have shown that exogenous CysNO is effectively transported intracellularly via the amino acid transporter system transporter system (23). Consequently, intracellular CysNO may facilitate the formation of protein Snitrosocysteine adducts primarily by replenishment of endogenous S-nitrosoglutathione or by direct transnitrosation.…”

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

“…Both NOͲdonors are cell permeable: SNOC can be transported into RBCs by the amino acid transporters LATͲ1 and 2 [42,43]. However, as a nitrosothiol, SNOC not only releases NO but is also an efficient nitrosating agent.…”

Bioanalytical profile of the l-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis

“…It can be regulated via intracellular traffic and active plasma membrane transport of S-nitroso-L-cysteine by an L-type amino acid transport system (LATs of the SLC7 family) [158][159][160][161]. S-Nitrosothiols may be important component of the NO pathway, but this issue is problematic and controversially discussed with regard both to concentration and biological roles.…”