Long-distance transport of S-nitrosothiols is possible via the blood ow along the vascular tree, but the kinetics of transport on a cellular scale in tissues is still being investigated. The transport of S-nitrosothiols across cell membranes was particularly controversial [50,80,81]. Experimental evidence suggests that molecules like GSNO or GSH cannot cross cellular membranes, neither by passive diffusion nor by an active transport mechanism. The carboxyl groups of glutathione are only weakly acid, so that GSNO and GSH should be a dynamic mixture of neutral, anionic and dianionic forms. But even the neutrals are not capable of crossing into the intracellular compartment. Studies  on NIH/3T3 broblasts showed that exposure to extracellular GSNO or SNAP failed to increase intracellular S-nitrosation. Low levels of extracellular CysNO (<0.1 mM) enhanced intracellular cysteine levels but failed to raise S-nitrosation. Signi cant S-nitrosation was only observed at high levels (1 mM) of extracellular CysNO. S-nitrosation of the intracellular compartment took place on a timescale of about 10 min . NMR studies  con rmed that no signi cant exchange of GSH takes place over the membrane of human erythrocytes. Moreover, the extracellular glu-tathione affected neither the intracellular GSH/GSSG ratio nor the glucose metabolism of the erythrocytes. This indicates that extracellular GSH did not provoke intracellular release of thiols from cleavage of disul de bonds in the interior compartment. Taken together, this study showed that intracellular GSH cannot be raised by suppletion of extracellular GSH.
The cellular transport of small S-nitrosothiols was extensively studied by Hogg et al. [84,85]. They showed that extracellular GSNO and SNAP did not signi cantly increase the pool of intracellular S-nitrosothiols in RAW 264.7 macrophages. However, the coincubation of GSNO with L-cysteine or its disul de (L-cystine) greatly enhanced the intracellular S-nitrosation. The S-nitroso moieties were predominantly located on the cysteine residues of proteins. Signi cant intracellular S-nitrosation was also observed after incubation with only extracellular L-CysNO. The data suggested that L-CysNO was transported intact across the membrane, and initiated subsequent transnitrosation towards the intracellular proteins. The effect of L-cysteine on extracellular GSNO was plausibly explained by the following sequence of events: First, transnitrosation from GSNO to L-CysNO. Second, active transport of L-CysNO across the membrane into the interior compartment. Third, transnitrosation from L-CysNO to the sulfhydryl groups of the proteins. The transport of L-CysNO was stereospe-ci c for the L-isomer and could be blocked by inhibitors of the cellular transport system of amino acids. The uptake of L-CysNO was speci cally attributed to the amino acid transporter system L (L-AT) [81,85].
The amino acid transporter system of the cellular membrane is not the only factor determining entry of S-nitrosothiols into the interior compartment and the timescale of intracellular transnitrosation. The cell membrane itself is rich in thiol-containing proteins, and each one of these is a potential carrier for cellular entry of NO. Protein disul de isomerase (PDI) is a membrane enzyme that normally catalyzes thiol disul de exchange reaction. This protein was found to raise the level of S-nitrosothiols inside human erythroleukemia cells . Additional pathways for the entry of S-nitrosothiols will undoubtedly be recognized in the future.
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