Requirement of Transmembrane Transport for S-Nitrosocysteine-dependent Modification of Intracellular Thiols

Abstract

S-Nitrosothiols have been implicated as intermediary transducers of nitric oxide bioactivity; however, the mechanisms by which these compounds affect cellular functions have not been fully established. In this study, we have examined the effect of S-nitrosothiol transport on intracellular thiol status and upon the activity of a target protein (caspase-3), in bovine aortic endothelial cells. We have previously demonstrated that the specific transport of amino acid-based S-nitrosothiols (S-nitroso-L-cysteine and S-nitrosohomocysteine) occurs via amino acid transport system L to generate high levels of intracellular protein S-nitrosothiols (Zhang, Y., and Hogg, N. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 7891-7896). In this study, we demonstrate that the transport of S-nitrosothiols is essential for these compounds to affect intracellular thiol levels and to modify intracellular protein activity. Importantly, the ability of these compounds to affect intracellular processes occurs independently of nitric oxide formation. These findings suggest that the major action of these compounds is not to liberate nitric oxide in the extracellular space but to be specifically transported into cells where they are able to modify cellular functions through nitric oxide-independent mechanisms. S-Nitrosothiols (RSNO),2 thioesters of nitrite, have been implicated to play a role in the complex biological responses of nitric oxide (NO). S-Nitrosation has been observed in whole animals and in the cell systems under basal (1-3) and inflammatory conditions (4, 5). It has been suggested that S-nitrosation of enzyme cysteine residues represents a post-translation modification involved in signal transduction and the alteration of enzyme function in a similar way to phosphorylation (6). Indeed, there are numbers of protein targets that have been reported to be nitrosated on cysteine residues including p21ras (7), L-type Ca 2ϩ channels (8), transcription factor NF-B (9), inhibitory B kinase (10), and caspase-3 (11, 12).Although the evidence is strong, in test systems, that thiol nitrosation can affect many cellular processes and enzyme activities, it is less clear that this represents a physiological or a pathophysiological effect of NO formation. NO per se is a poor and inefficient nitrosating agent (13), and intracellular systems that catalyze nitrosation from NO have not yet been defined. Although the plasma copper-protein ceruloplasmin has been shown to catalyze RSNO formation (14), this has not been demonstrated in whole blood containing high concentrations of hemoglobin, a potent NO scavenger (15). From a pharmacological perspective, however, it has been demonstrated in many studies that the exposure of cells to low molecular weight RSNOs, such as S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitroso-L-cysteine (L-CysNO), can affect cellular processes (16). Although it is often assumed that these compounds spontaneously release NO in cell culture and that the effects of these compounds can be attributed t...

Differential mechanisms of inhibition of glyceraldehyde-3-phosphate dehydrogenase byS-nitrosothiols and NO in cellular and cell-free conditions

“…Rigorous comparisons between the effects of NO and the effects of S-nitrosation on a specific cellular activity have rarely been accomplished, and NOdependent effects are often ascribed to S-nitrosation with little to no quantitative confirmatory data. We have previously demonstrated clear differences between NO and CysNO in their ability to inhibit caspase 3-like activity in doxorubicintreated bovine aortic endothelial cells (BEACs) (1). Whereas CysNO appeared to inhibit this enzyme through S-nitrosation, as has been previously proposed, we observed no inhibition upon the exposure of cells to NO.…”

“…Previously, we reported that CysNO could rapidly deplete GSH levels in BAECs (1). Figure 3B shows that immediately after CysNO treatment (0 h recovery), the GSH levels were increased at low micromolar levels of CysNO, but above 40 M CysNO, GSH was substantially depleted in a concentration-dependent manner.…”

Redox Regulation in the Extracellular Environment

“…29 Extracellular levels of RSNO can also influence intracellular thiol levels by the transport of a low-molecular-weight RSNO, thus representing a different method of RSNO influx independent of NO generation. 30 …”

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…High levels of NO donor will not only limit selectively, but also have the potential to compromise the cellular reducing systems thus stabilising S-nitrosothiols, allowing them to accumulate at a higher concentration. S-nitrosocysteine is known to deplete GSH levels in a concentration-dependent manner in multiple cell types [131,132]. Severe depletion of the reduced thiol pool was shown to generate non-physiological levels of intracellular S-nitrosothiols.…”