Requirement of transmembrane transport for S-nitrosocysteine-dependent modification of intracellular thiols

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.     

Biological chemistry and clinical potential of S-nitrosothiols

S-Nitrosothiols are endogenous metabolites of nitric oxide that have been detected in extra- and intracellular spaces. Many biological functions of S-nitrosothiols have been described that can be categorized as being due to one or more of the following: (i) nitric oxide release, (ii) transnitrosation, (iii) S-thiolation, and (iv) direct action. This emphasizes the fact that S-nitrosothiols are more than simply nitric oxide donors. Many of the biological functions that have been described for S-nitrosothiols have clinical correlates. This review describes the biological chemistry, biological actions, and clinical potential of these compounds.     

Formation and stability of S-nitrosothiols in RAW 264.7 cells

S-Nitrosothiols have been suggested to be mediators of many nitric oxide-dependent processes, including apoptosis and vascular relaxation. Thiol nitrosation is a poorly understood process in vivo, and the mechanisms by which nitric oxide can be converted into a nitrosating agent have not been established. There is a discrepancy between the suggested biological roles of nitric oxide and its known chemical and physical properties. In this study, we have examined the formation of S-nitrosothiols in lipopolysaccharide-treated RAW 264.7 cells. This treatment generated 17.4 +/- 1.0 pmol/mg of protein (means +/- SE, n =27) of intracellular S-nitrosothiol that slowly decayed over several hours. S-Nitrosothiol formation depended on the formation of nitric oxide and not on the presence of nitrite. Extracellular thiols were nitrosated by cell-generated nitric oxide. Oxygenated ferrous hemoglobin inhibited the formation of S-nitrosothiol, indicating the nitrosation occurred more slowly than diffusion. We discuss several mechanisms for S-nitrosothiol formation and conclude that the nitrosation propensity of nitric oxide is a freely diffusible element that is not constrained within an individual cell and that both nitric oxide per se and nitric oxide-derived nitrosating agents are able to diffuse across cell membranes. To achieve intracellular localization of the nitrosation reaction, mechanisms must be invoked that do not involve the formation of nitric oxide as an intermediate.     

Characterization and application of the biotin-switch assay for the identification of S-nitrosated proteins

S-Nitrosation of protein cysteinyl residues has been suggested to be an important nitric oxide-dependent posttranslational modification. The so-called biotin-switch method has been developed to identify S-nitrosated proteins. This method relies on the selective reduction of S-nitrosothiols by ascorbate. In this study we have assessed the ability of ascorbate to reduce S-nitrosothiols and show that ascorbate is a very inefficient reducing agent. We show that higher concentrations of ascorbate and longer incubation times can significantly improve immunological detection of S-nitrosothiols. We have compared immunological detection of S-nitrosothiols with the level of intracellular S-nitrosothiols measured by tri-iodide chemiluminescence and show that the biotin-switch method is capable of detecting only high (nmol/mg protein) levels of intracellular S-nitrosothiols obtained after exposing cells to S-nitrosocysteine, but not the low levels observed during physiological nitric oxide formation. Preliminary proteomic analysis of protein S-nitrosothiols has identified elongation factor 2, heat shock protein 90 beta, and a 65-kDa macrophage protein homologous to human L-plastin as major nitrosation targets at high intracellular nitrosation levels in the murine macrophage-derived RAW 264.7 cell line. While the biotin-switch method may be a useful tool to aid in the positive identification of protein S-nitrosothiols, it cannot match the sensitivity of chemiluminescence-based methods and its use in proteomic studies likely suffers from selective detection of more easily reducible S-nitrosothiols.     

S-Nitrosylation signaling regulates cellular protein interactions

BACKGROUND: S-Nitrosothiols are made by nitric oxide synthases and other metalloproteins. Unlike nitric oxide, S-nitrosothiols are involved in localized, covalent signaling reactions in specific cellular compartments. These reactions are enzymatically regulated. SCOPE: S-Nitrosylation affects interactions involved in virtually every aspect of normal cell biology. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation. MAJOR CONCLUSIONS AND SIGNIFICANCE: S-Nitrosylation is a regulated signaling reaction.     

Formation of nanomolar concentrations of S-nitroso-albumin in human plasma by nitric oxide

S-Nitrosothiols are potentially important mediators of biological processes including vascular function, apoptosis, and thrombosis. Recent studies indicate that the concentrations of S-nitrosothiols in the plasma from healthy individuals are lower than previously reported and in the range of 30-120 nM. The mechanisms of formation and metabolism of these low nM concentrations, capable of exerting biological effects, remain unknown. An important issue that remains unresolved is the significance of the reactions of low fluxes of nitric oxide (NO) with oxygen to form S-nitrosothiols in a complex biological medium such as plasma, and the impact of red blood cells on the formation of S-nitrosothiols in blood. These issues were addressed by exposing plasma to varying fluxes of NO and measuring the net formation of S-nitrosothiols. In the presence of oxygen and physiological fluxes of NO, the predominant S-nitrosothiol formed is S-nitroso-albumin at concentrations in the high nM range (approximately 400-1000 nM). Although the formation of S-nitrosothiols by NO was attenuated in whole blood, presumably by erythrocytic hemoglobin, significant amounts of S-nitrosothiols within the physiological range of S-nitrosothiol concentrations (approximately 80 nM) were still formed at physiological fluxes of NO. Little is known about the stability of S-nitroso-albumin in plasma, and this is central to our understanding of the biological effectiveness of S-nitrosothiols. Low molecular weight thiols decreased the half-life of S-nitroso-albumin in plasma, and the stability of S-nitroso-albumin is enhanced by the alkylation of free thiols. Our data suggests that physiologically relevant concentrations of S-nitrosothiols can be formed in blood through the reaction of NO with oxygen and proteins, despite the low rates of reaction of oxygen with NO and the presence of erythrocytes.     

Nitric oxide stimulates the erythrocyte for ascorbate recycling.

S-Nitrosothiols act as carrier and reservoir of nitric oxide (NO), and release NO under stimulation of ascorbate (Asc). Erythrocyte can regenerate Asc from its oxidised products, thus saving this powerful antioxidant. In this paper the effect of donors of NO, superoxide, and peroxynitrite (SpNONOate, KO(2), and SIN-1, respectively) on the erythrocyte production of Asc was investigated. We report here that NO stimulated, while superoxide and peroxynitrite decreased, the Asc recycling. The NO-stimulating effect on the erythrocyte production of Asc was confirmed by using GSNO, a natural occurring S-nitrosothiol, as NO donor. These data highlight a new property of NO, that is the stimulation of erythrocytes for their Asc recycling. Such a property might contribute to regenerate Asc from its oxidised forms, thus preventing its depletion in the circulation. Temperature and pH significantly affected, both in absence and presence of NO, the recycling of Asc by erythrocytes. We propose that a positive feedback, involving the reciprocal stimulation between Asc and S-nitrosothiols, might enhance productions of Asc by erythrocytes and NO release by circulating S-nitrosothiols.     

Autowave mode of functioning of the system nitric oxide + free iron + thiols may ensure the regulation of biological action of nitric oxide and its endogenic compounds

It has been shown earlier that, in a system NO + Fe2+ + thiols in aqueous solution, an oscillatory mode of changes with time in the concentration of paramagnetic dinitrosyl iron complexes with thiol-containing legends and S-nitrosothiols formed in this system and in the concentration of free iron (not included into dinitrosyl iron complexes) can be realized. It is assumed that, in this system, autowaves can arise, which ensure periodic changes with time and space in the concentration of the system constituents. These changes may underlie the regulation of the physiologic effect of nitric oxide, dinitrosyl iron complexes, and S-nitrosothiols as agents affecting various intracellular and tissue targets.     

Role of thiamine thiol form in nitric oxide metabolism

In alkaline media the thiamine cyclic form is converted into a thiol form (pK(a) 9.2) with an opened thiazole ring. The thiamine thiol form releases nitric oxide from S-nitrosoglutathione (GSNO). Thiamine disulfide, mixed thiamine disulfide with glutathione, and nitric oxide are produced in the reaction. Free glutathione was recorded in small amounts. The concentration of formed nitric oxide agreed well with the concentration of degraded GSNO. The concentration of released nitric oxide was determined under anaerobic conditions spectrophotometrically by production of nitrosohemoglobin. In air, the release of nitric oxide was recorded by the production of nitrite or the oxidation of oxyhemoglobin to methemoglobin. The concentration of the thiol form in the body under physiological pH values (7.2-7.4) did not exceed 1.5-2.0%. We believe that due to the exchange reactions between the thiamine thiol form and S-nitrosocysteine protein residues, nitric oxide can be released and mixed thiamine-protein disulfides are formed. The mixed thiamine disulfides (including thiamine ester disulfides) as well as the thiamine disulfide form are quite easily reduced by low molecular weight thiols to form the thiamine cyclic form with a closed thiazole ring. A possible role of the thiamine thiol form in releasing deposited nitric oxide from low-molecular-weight S-nitrosothiols and protein S-nitrosothiols and in regulation of blood flow in the vascular bed is discussed.     

Increased oxidative stress in the RAW 264.7 macrophage cell line is partially mediated via the S-nitrosothiol-induced inhibition of glutathione reductase

We investigated whether endogenously or exogenously produced nitric oxide (NO) can inhibit cellular glutathione reductase (GR) via the formation of S-nitrosothiols to decrease cellular glutathione (GSH) and increase oxidative stress in RAW 264.7 cells. The specificity of this inhibition was demonstrated by addition of a NO-synthase inhibitor, and met- or oxyhemoglobin. Using isolated GR we found that only certain NO donors inhibit this enzyme via S-nitrosothiol. Furthermore, we found that cellular GSH decrease is paralleled by an increase of superoxide anion production. Our results show that the GR enzyme is a potential target of S-nitrosothiols to decrease cellular GSH levels and to induce oxidative stress in macrophages.     

Endothelial heme oxygenase-1 induction by hypoxia. Modulation by inducible nitric-oxide synthase and S-nitrosothiols

The stress protein heme oxygenase-1 (HO-1) is induced in endothelial cells exposed to nitric oxide (NO)-releasing agents, and this process is finely modulated by thiols (Foresti, R., Clark, J. E., Green, C. J., and Motterlini R. (1997) J. Biol. Chem. 272, 18411-18417). Here, we report that up-regulation of HO-1 in aortic endothelial cells by severe hypoxic conditions (pO(2) 相似文献   

Cytochrome c-mediated formation of S-nitrosothiol in cells

S-nitrosothiols are products of nitric oxide (NO) metabolism that have been implicated in a plethora of signalling processes. However, mechanisms of S-nitrosothiol formation in biological systems are uncertain, and no efficient protein-mediated process has been identified. Recently, we observed that ferric cytochrome c can promote S-nitrosoglutathione formation from NO and glutathione by acting as an electron acceptor under anaerobic conditions. In the present study, we show that this mechanism is also robust under oxygenated conditions, that cytochrome c can promote protein S-nitrosation via a transnitrosation reaction and that cell lysate depleted of cytochrome c exhibits a lower capacity to synthesize S-nitrosothiols. Importantly, we also demonstrate that this mechanism is functional in living cells. Lower S-nitrosothiol synthesis activity, from donor and nitric oxide synthase-generated NO, was found in cytochrome c-deficient mouse embryonic cells as compared with wild-type controls. Taken together, these data point to cytochrome c as a biological mediator of protein S-nitrosation in cells. This is the most efficient and concerted mechanism of S-nitrosothiol formation reported so far.     

Identification of stereoselective transporters for S-nitroso-L-cysteine: role of LAT1 and LAT2 in biological activity of S-nitrosothiols

Many of the biological effects of nitric oxide are mediated by S-nitrosothiols. However, the mechanisms by which S-nitrosothiols transduce their activity across cell membranes are unclear. We show that the pathway responsible for the cellular effects of S-nitrosothiols is specific for S-nitrosocysteine (CSNO), is stereoselective, and requires direct uptake of intact L-CSNO. Transport is independent of extracellular sodium, competitively inhibited by leucine, and blocked by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, a specific inhibitor of the system L amino acid transporter family. Other nitrosothiols such as S-nitrosoglutathione are not substrates for transport and require reaction with L-cysteine for activity. To show that system L family members mediate uptake, we expressed two members, LAT1 and LAT2, in Xenopus oocytes. Both LAT1 and LAT2, when co-expressed with 4F2 heavy chain, were found to efficiently transport L-CSNO. Mammalian cells were shown to express LAT1 and LAT2. A431 cells express both proteins, whereas T24 cells express only LAT1. Overexpression of LAT1 in T24 cells using recombinant adenoviruses led to increased uptake of L-CSNO, whereas knockdown using a specific small interfering RNA led to decreased uptake. These data definitively identify LAT1 and LAT2 as members of system L that mediate transmembrane movement of l-CSNO and suggest that system L family members are involved in the cellular activity of small molecular weight nitrosothiols.     

Ceramide in nitric oxide inhibition of glioma cell growth. Evidence for the involvement of ceramide traffic

The treatment of C6 glioma cells with the nitric oxide donor, PAPANONOate ((Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate), resulted in a dose-dependent inhibition of cell proliferation. This was associated to a rapid and significant increase of ceramide levels and was mimicked by treatments that augment cellular ceramide. Metabolic experiments with radioactive sphingosine, serine, and choline showed that nitric oxide strongly reduced the utilization of ceramide for the biosynthesis of both sphingomyelin and glucosylceramide. Nevertheless, nitric oxide did not modify the activity of different enzymes of ceramide metabolism. The possibility that nitric oxide impairs the availability of ceramide for sphingolipid biosynthesis was then investigated. The metabolism of N-hexanoyl-[(3)H]sphingosine demonstrated that nitric oxide did not affect the biosynthesis of N-hexanoyl-[(3)H]sphingolipids but inhibited the metabolic utilization of long chain [(3)H]ceramide, synthesized in the endoplasmic reticulum (ER) from the recycled [(3)H]sphingosine. Moreover, results obtained with fluorescent ceramides, brefeldin A, ATP depletion, as well as in a ceramide transport assay indicate that nitric oxide impairs the traffic of ceramide from ER to Golgi apparatus. All this supports that, in glioma cells, the modulation of ceramide traffic can contribute to the regulation of its intracellular levels and participate in the nitric oxide-activated signaling pathway involved in the control of cell proliferation.     

Interaction between endothelium-derived relaxing factors, S-nitrosothiols, and endothelin-1 on Ca2+ mobilization in rat vascular smooth muscle cells.

S-Nitrosothiols (S-nitrosocysteine, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine), which belong to the group of endothelium-derived relaxing factors (EDRFs), caused decreases of cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMCs). The endothelin-1 (ET-1)-induced sustained increase of [Ca2+]i in rat VSMCs was completely abolished by preaddition of at least an equal molar quantity of S-nitrosocysteine (Cys-SNO). Also exposure of VSMCs to a mixture of Cys-SNO and ET-1 at the same time resulted in the transient increase only. These results suggest that S-nitrosothiols may have no significant effect on ET-1-induced Ca2+ release from intracellular stores via inositol 1,4,5-triphosphate production but do affect Ca2+ influx through Ca2+ channels in the plasma membrane.     

Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase.

Although normal intracellular levels of arginine are well above the K(m), and should be sufficient to saturate nitric oxide synthase in vascular endothelial cells, nitric oxide production can, nonetheless, be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox," has suggested the existence of a separate pool of arginine directed to nitric oxide synthesis. In this study, we demonstrate that exogenous citrulline was as effective as exogenous arginine in stimulating nitric oxide production and that citrulline in the presence of excess intracellular and extracellular arginine further enhanced bradykinin stimulated endothelial nitric oxide production. The enhancement of nitric oxide production by exogenous citrulline could therefore be attributed to the capacity of vascular endothelial cells to efficiently regenerate arginine from citrulline. However, the regeneration of arginine did not affect the bulk intracellular arginine levels. This finding not only supports the proposal for a unique pool of arginine, but also suggested channeling of substrates that would require a functional association between nitric oxide production and arginine regeneration. To support this proposal, we showed that nitric oxide synthase, and the enzymes involved in arginine regeneration, argininosuccinate synthase and argininosuccinate lyase, cofractionated with plasmalemmal caveolae, a subcompartment of the plasma membrane. Overall, the results from this study strongly support the proposal for a separate pool of arginine for nitric oxide production that is defined by the cellular colocalization of enzymes involved in nitric oxide production and the regeneration of arginine.     

Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cells

S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a critical link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, respectively. Although low molecular weight S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothiols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and determined, via confocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temperature sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low molecular weight S-nitroso-l-cysteine ethyl ester (l-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter molecule. Transnitrosation of FRET-MT by SNO-albumin, but not l-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide isomerase (csPDI). These results extend the original observations of Ramachandran et al. (Ramachandran N, Root P, Jiang XM, Hogg PJ, Mutus B. Proc Natl Acad Sci U S A 98: 9539-9544, 2001) and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell molecular targets (e.g. MT).     

S-nitrosoglutathione reductase affords protection against pathogens in Arabidopsis, both locally and systemically

Nitric oxide and S-nitrosothiols (SNOs) are widespread signaling molecules that regulate immunity in animals and plants. Levels of SNOs in vivo are controlled by nitric oxide synthesis (which in plants is achieved by different routes) and by S-nitrosoglutathione turnover, which is mainly performed by the S-nitrosoglutathione reductase (GSNOR). GSNOR is encoded by a single-copy gene in Arabidopsis (Arabidopsis thaliana; Martínez et al., 1996; Sakamoto et al., 2002). We report here that transgenic plants with decreased amounts of GSNOR (using antisense strategy) show enhanced basal resistance against Peronospora parasitica Noco2 (oomycete), which correlates with higher levels of intracellular SNOs and constitutive activation of the pathogenesis-related gene, PR-1. Moreover, systemic acquired resistance is impaired in plants overexpressing GSNOR and enhanced in the antisense plants, and this correlates with changes in the SNO content both in local and systemic leaves. We also show that GSNOR is localized in the phloem and, thus, could regulate systemic acquired resistance signal transport through the vascular system. Our data corroborate the data from other authors that GSNOR controls SNO in vivo levels, and shows that SNO content positively influences plant basal resistance and resistance-gene-mediated resistance as well. These data highlight GSNOR as an important and widely utilized component of resistance protein signaling networks conserved in animals and plants.     

Thiyl radicals react with nitric oxide to form S-nitrosothiols with rate constants near the diffusion-controlled limit

A possible route to S-nitrosothiols in biology is the reaction between thiyl radicals and nitric oxide. D. Hofstetter et al. (Biochem. Biophys. Res. Commun.360:146-148; 2007) claimed an upper limit of (2.8+/-0.6)x10(7) M(-1)s(-1) for the rate constant between thiyl radicals derived from glutathione and nitric oxide, and it was suggested that under physiological conditions S-nitrosation via this route is negligible. In the present study, thiyl radicals were generated by pulse radiolysis, and the rate constants of their reactions with nitric oxide were determined by kinetic competition with the oxidizable dyes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and a phenothiazine. The rate constants for the reaction of nitric oxide with thiyl radicals derived from glutathione, cysteine, and penicillamine were all in the range (2-3) x10(9) M(-1)s(-1), two orders of magnitude higher than the previously reported estimate in the case of glutathione. Absorbance changes on reaction of thiyl radicals with nitric oxide were consistent with such high reactivity and showed the formation of S-nitrosothiols, which was also confirmed in the case of glutathione by HPLC/MS. These rate constants imply that formation of S-nitrosothiols in biological systems from the combination of thiyl radicals with nitric oxide is much more likely than claimed by Hofstetter et al.