Oxidation and nitrosation of thiols at low micromolar exposure to nitric oxide. Evidence for a free radical mechanism

Although the nitric oxide (.NO)-mediated nitrosation of thiol-containing molecules is increasingly recognized as an important post-translational modification in cell signaling and pathology, little is known about the factors that govern this process in vivo. In the present study, we examined the chemical pathways of nitrosothiol (RSNO) production at low micromolar concentrations of .NO. Our results indicate that, in addition to nitrosation by the .NO derivative dinitrogen trioxide (N2O3), RSNOs may be formed via intermediate one-electron oxidation of thiols, possibly mediated by nitrogen dioxide (.NO2), and the subsequent reaction of thiyl radicals with .NO. In vitro, the formation of S-nitrosoglutathione (GSNO) from .NO and excess glutathione (GSH) was accompanied by the formation of glutathione disulfide, which could not be ascribed to the secondary reaction of GSH with GSNO. Superoxide dismutase significantly increased GSNO yields and the thiyl radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), inhibited by 45 and 98% the formation of GSNO and GSSG, respectively. Maximum nitrosation yields were obtained at an oxygen concentration of 3%, whereas higher oxygen tensions decreased GSNO and increased GSSG formation. When murine fibroblasts were exposed to exogenous .NO, RSNO formation was sensitive to DMPO and oxygen tension in a manner similar to that observed with GSH alone. Our data indicate that RSNO formation is favored at oxygen concentrations that typically occur in tissues. Nitrosothiol formation in vivo depends not only on the availability of .NO and O2 but also on the degree of oxidative stress by affecting the steady-state concentration of thiyl radicals.     

A nanoparticle delivery vehicle for S-nitroso-N-acetyl cysteine: Sustained vascular response

Interest in the development of nitric oxide (NO) based therapeutics has grown exponentially due to its well elucidated and established biological functions. In line with this surge, S-nitroso thiol (RSNO) therapeutics are also receiving more attention in recent years both as potential stable sources of NO as well as for their ability to serve as S-nitrosating agents; S-nitrosation of protein thiols is implicated in many physiological processes. We describe two hydrogel based RSNO containing nanoparticle platforms. In one platform the SNO groups are covalently attached to the particles (SNO-np) and the other contains S-nitroso-N-acetyl cysteine encapsulated within the particles (NAC-SNO-np). Both platforms function as vehicles for sustained activity as trans-S-nitrosating agents. NAC-SNO-np exhibited higher efficiency for generating GSNO from GSH and maintained higher levels of GSNO concentration for longer time (24h) as compared to SNO-np as well as a previously characterized nitric oxide releasing platform, NO-np (nitric oxide releasing nanoparticles). In vivo, intravenous infusion of the NAC-SNO-np and NO-np resulted in sustained decreases in mean arterial pressure, though NAC-SNO-np induced longer vasodilatory effects as compared to the NO-np. Serum chemistries following infusion demonstrated no toxicity in both treatment groups. Together, these data suggest that the NAC-SNO-np represents a novel means to both study the biologic effects of nitrosothiols and effectively capitalize on its therapeutic potential.     

Exogenous vs. endogenous γ-glutamyltransferase activity: Implications for the specific determination of S-nitrosoglutathione in biological samples

The determination of S-nitrosoglutathione (GSNO) levels in biological fluids is controversial, partly due to the laborious sample handling and multiple pretreatment steps required by current techniques. GSNO decomposition can be effected by the enzyme gamma-glutamyltransferase (GGT), whose involvement in GSNO metabolism has been suggested. We have set up a novel analytical method for the selective determination and speciation of GSNO and its metabolite S-nitrosocysteinylglycine, based on liquid chromatography separation coupled to on-line enzymatic hydrolysis of GSNO by commercial GGT. In a post-column reaction coil, GGT allows the specific hydrolysis of the γ-glutamyl moiety of GSNO, and the S-nitrosocysteinylglycine (GCNO) thus formed is decomposed by copper ions originating oxidized cysteinylglycine and nitric oxide (NO). NO immediately reacts with 4,5-diaminofluorescein (DAF-2) forming a triazole derivative, which is detected fluorimetrically. The limit of quantitation (LOQc) for GSNO and GCNO in plasma ultrafiltrate was 5 nM, with a precision (CV) of 1-6% within the 5-1500 nM dynamic linear range.The method was applied to evaluate the recovery of exogenous GSNO after addition of aliquots to human plasma samples presenting with different total GGT activities. By inhibiting GGT activity in a time dependent manner, it was thus observed that the recovery of GSNO is inversely correlated with plasmatic levels of endogenous GGT, which indicates the need for adequate inhibition of endogenous GGT activity for the reliable determination of endogenous GSNO.     

A role of iron ions in the SOS DNA repair response induced by nitric oxide in Escherichia coli

An induction of the SOS DNA repair response by physiological nitric oxide donors (dinitrosyl iron complexes (DNIC) with thiols and S-nitrosothiols (RSNO)) was studied in E. coli cells. DNIC with thiols were the most effective SOS-inducers. Being more toxic, RSNO mediated a similar response at 10-100 microM, but they were inactive at concentrations above 0.5 mM. Pretreatment of the cells with chelating agents, o-phenanthroline and picolinic acid, prevented induction of the SOS response by all NO-donors used and led to a decrease in the DNIC-type EPR signal that appeared after incubation of the cells with DNIC or S-nitrosoglutathione (GSNO). Analysis of these effects revealed a dual role of iron ions in reactivity and toxicity of the NO-donating agents. On one hand, they could stabilize GSNO in the form of less toxic DNIC, and, on the other hand, they took part in the formation of the SOS-inducing signal by NO-donating agents.     

The ectoenzyme gamma-glutamyl transpeptidase regulates antiproliferative effects of S-nitrosoglutathione on human T and B lymphocytes.

Expression of the ectoenzyme gamma-glutamyl transpeptidase (GGT) is regulated on T lymphocytes. It is present at a low level on naive T cells, at a high level on activated T cells, and at an intermediate level on resting memory T cells. GGT cleaves the glutamyl group from glutathione, which is the first step in the uptake of extracellular glutathione. In vitro, purified GGT also metabolizes the naturally occurring nitrosothiol, S-nitrosoglutathione (GSNO). Because of this relationship, the effects of cellular GGT on the metabolism of and cellular response to GSNO were tested. The GGT-negative lymphoblasts Ramos and SupT1 were transfected with cDNA for human GGT. In the presence of cells lacking GGT, GSNO is extremely stable. In contrast, GGT-expressing cells rapidly metabolize GSNO leading to nitric oxide release. The nitric oxide causes a rapid (<2-h) inhibition of DNA synthesis. There is a concomitant decrease in the concentration of intracellular deoxyribonucleotides, suggesting that one effect of the nitric oxide generated from GSNO is the previously described inactivation of the enzyme ribonucleotide reductase. GSNO also caused a rapid, GGT-dependent cytostatic effect in Hut-78, a human T cell lymphoma, as well as in activated peripheral blood T cells. Although DNA synthesis was decreased to 16% of control values in anti-CD3-stimulated Hut-78, the production of IL-2 was unchanged by GSNO. These data show that GGT, a regulated ectoenzyme on T cells, controls the rate of nitric oxide production from GSNO and thus markedly affects the physiological response to this biologically active nitrosothiol.     

γ-Glutamyltransferase catabolism of S-nitrosoglutathione modulates IL-8 expression in cystic fibrosis bronchial epithelial cells

S-nitrosoglutathione (GSNO) is an endogenous nitrosothiol involved in several pathophysiological processes. A role for GSNO has been envisaged in the expression of inflammatory cytokines such as IL-8; however, conflicting results have been reported. γ-Glutamyltransferase (GGT) enzyme activity can hydrolyze the γ-glutamyl bond present in the GSNO molecule thus greatly accelerating the release of bioactive nitric oxide. Expression of GGT is induced by oxidative stress, and activated neutrophils contribute to GGT increase in cystic fibrosis (CF) lung exudates by releasing GGT-containing microvesicles. This study was aimed at evaluating the effect of GSNO catabolism mediated by GGT on production of IL-8 in CF transmembrane regulation protein-mutated IB3-1 bronchial cells. The rapid, GGT-catalyzed catabolism of GSNO caused a decrease in both basal and lipopolysaccharide-stimulated IL-8 production in IB3-1 cells, by modulating both NF-κB and ERK1/2 pathways, along with a decrease in cell proliferation. In contrast, a slow decomposition of GSNO produced a significant increase in both cell proliferation and expression of IL-8, the latter possibly through p38-mediated stabilization of IL-8 mRNA. Our data suggest that the differential GSNO catabolism mediated by GGT enzyme activity can downregulate the production of IL-8 in CF cells. Hence, the role of GGT activity should be considered when evaluating GSNO for both in vitro and in vivo studies, the more so in the case of GSNO-based therapies for cystic fibrosis.     

Investigations of S-transnitrosylation reactions between low- and high-molecular-weight S-nitroso compounds and their thiols by high-performance liquid chromatography and gas chromatography-mass spectrometry.

S-Transnitrosylation reactions are supposed to be the basic principle by which nitric oxide-related biological activities are regulated in vivo. Mechanisms of S-transnitrosylation reactions are poorly understood and equilibria constants for physiological S-nitroso compounds and thiols are rare. In the present study we investigated S-transnitrosylation reactions of the thiols homocysteine, cysteine, glutathione, N-acetylcysteine, N-acetylpenicillamine, and human plasma albumin and their corresponding S-nitroso compounds SNhC, SNC, GSNO, SNAC, SNAP, and SNALB utilizing high-performance liquid chromatographic and gas chromatographic-mass spectrometric techniques. These methods allowed to study S-transnitrosylation reactions in mixtures of several S-nitroso compound/thiol pairs, to determine equilibria constants, and to elucidate the mechanism of S-transnitrosylation reactions. We obtained the following order for the equilibria constants in aqueous buffered solution at pH 7.4: SNhC approximately SNAC > GSNO approximately SNALB > SNAP > SNC. Our results suggest that the mechanism of S-transnitrosylation reactions of these S-nitroso compounds and their thiols involve heterolytic cleavage of the S&sbond;N bond. Incubation of SNC with human red blood cells resulted in a dose-dependent formation of GSNO in the cytosol through S-transnitrosylation of intracellular GSH by the SNC transported into the cells. This reaction was accompanied with an almost complete disappearance of the SNC fraction transported into the cells. This finding is in full agreement with the equilibrium constant Keq of 1.9 for the reaction SNC + GSH <--> Cys + GSNO in aqueous buffer.     

Endothelial ��-Glutamyltransferase Contributes to the Vasorelaxant Effect of S-Nitrosoglutathione in Rat Aorta

S-nitrosoglutathione (GSNO) involved in storage and transport of nitric oxide (^•NO) plays an important role in vascular homeostasis. Breakdown of GSNO can be catalyzed by γ-glutamyltransferase (GGT). We investigated whether vascular GGT influences the vasorelaxant effect of GSNO in isolated rat aorta. Histochemical localization of GGT and measurement of its activity were performed by using chromogenic substrates in sections and in aorta homogenates, respectively. The role of GGT in GSNO metabolism was evaluated by measuring GSNO consumption rate (absorbance decay at 334 nm), ^•NO release was visualized and quantified with the fluorescent probe 4,5-diaminofluorescein diacetate. The vasorelaxant effect of GSNO was assayed using isolated rat aortic rings (in the presence or absence of endothelium). The role of GGT was assessed by stimulating enzyme activity with cosubstrate glycylglycine, as well as using two independent inhibitors, competitive serine borate complex and non-competitive acivicin. Specific GGT activity was histochemically localized in the endothelium. Consumption of GSNO and release of free ^•NO decreased and increased in presence of serine borate complex and glycylglycine, respectively. In vasorelaxation experiments with endothelium-intact aorta, the half maximal effective concentration of GSNO (EC50 = 3.2±0.5.10⁻⁷ M) increased in the presence of the two distinct GGT inhibitors, serine borate complex (1.6±0.2.10⁻⁶ M) and acivicin (8.3±0.6.10⁻⁷ M), while it decreased with glycylglycine (4.7±0.9.10⁻⁸ M). In endothelium-denuded aorta, EC₅₀ for GSNO alone increased to 2.3±0.3.10⁻⁶ M, with no change in the presence of serine borate complex. These data demonstrate the important role of endothelial GGT activity in mediating the vasorelaxant effect of GSNO in rat aorta under physiological conditions. Because therapeutic treatments based on GSNO are presently under development, this endothelium-dependent mechanism involved in the vascular effects of GSNO should be taken into account in a pharmacological perspective.     

A novel mechanism underlying the susceptibility of neuronal cells to nitric oxide: the occurrence and regulation of protein S-nitrosylation is the checkpoint

The susceptibility of neuronal cells to nitric oxide (NO) is a key issue in NO-mediated neurotoxicity. However, the underlying mechanism remains unclear. As a cyclic guanosine monophosphate (cGMP)-independent NO signaling pathway, S -nitrosylation (or S -nitrosation) has been suggested to occur as a post-translational modification in parallel with O-phosphorylation. The underlying mechanism of the involvement of protein S -nitrosylation in the susceptibility of neuronal cells to NO has been little investigated. In this study, we focused on the role of S -nitrosothiols (RSNO) in the susceptibility of a cerebellar cell line R2 to NO. Our results showed the following: (i) S -nitrosoglutathione (GSNO) induced a burst of RSNO in GSH-depleted R2 cells, the majority of which were primarily contributed by the S -nitrosylation of proteins (Pro-SNOs), and was followed by severe neuronal necrosis; (ii) the elevation in the level of Pro-SNOs resulted from a dysfunction of S -nitroglutathione reductase (GSNOR) as a result of its substrate, GSNO, being unavailable in GSH-depleted cells. In the meantime, the suppression of GSNOR increased NO-mediated neurotoxicity in R2 cells, as well as in cerebellar granule neurons; (iii) Our results also demonstrate that the burst of RSNO is the "checkpoint" of cell fate: if RSNO can be reduced to free thiol proteins, cells will survive; if they are further oxidized, cells will die; and (iv) GSH-ethyl ester and Vitamin C protected R2 cells against GSNO neurotoxicity through two distinct mechanisms: by inhibiting the elevation of Pro-SNOs and by reducing Pro-SNOs to free thiol proteins, respectively. A novel mechanism underlying the susceptibility of neuronal cells to NO is proposed and some potential strategies to prevent the NO-mediated neurotoxicity are discussed.     

A kinetic study of gamma-glutamyltransferase (GGT)-mediated S-nitrosoglutathione catabolism

S-Nitrosoglutathione (GSNO) is a nitric oxide (NO) donor compound which has been postulated to be involved in transport of NO in vivo. It is known that γ-glutamyl transpeptidase (GGT) is one of the enzymes involved in the enzyme-mediated decomposition of GSNO, but no kinetics studies of the reaction GSNO-GGT are reported in literature.In this study we directly investigated the kinetics of GGT with respect to GSNO as a substrate and glycyl-glycine (GG) as acceptor co-substrate by spectrophotometry at 334 nm. GGT hydrolyses the γ-glutamyl moiety of GSNO to give S-nitroso-cysteinylglycine (CGNO) and γ-glutamyl-GG. However, as both the substrate GSNO and the first product CGNO absorb at 334 nm, we optimized an ancillary reaction coupled to the enzymatic reaction, based on the copper-mediated decomposition of CGNO yielding oxidized cysteinyl-glycine and NO. The ancillary reaction allowed us to study directly the GSNO/GGT kinetics by following the decrease of the characteristic absorbance of nitrosothiols at 334 nm. A K_m of GGT for GSNO of 0.398 ± 31 mM was thus found, comparable with K_m values reported for other γ-glutamyl substrates of GGT.     

The aqueous garlic, onion and leek extracts release nitric oxide from S-nitrosoglutathione and prolong relaxation of aortic rings

Garlic, onion and leek have beneficial effects in treatment of numerous health disorders. The aim of the present study was to investigate underlying molecular mechanisms. To test the potency of the aqueous garlic, onion and leek extracts to release NO from GSNO we have measured NO oxidation product, NO(2)-, by the Griess reagent method. Further, we studied the ability of garlic extract to relax noradrenaline-precontracted rat aortic rings in the presence of GSNO and effects of garlic extract on electrical properties of rat heart intracellular chloride channels. We have observed that: i) garlic, onion and leek extracts released NO from GSNO in the order: garlic > onion > leek; ii) the ability of garlic extract to release NO was pH-dependent (8.0 > 7.4 > 6.0) and potentiated by thiols (Cys > GSH = N-acetyl-cysteine > oxidized glutathione) at concentration 100 μmol/l; iii) the garlic extract (0.045 mg/ml) prolonged relaxation time of aortic rings induced by GSNO (50 nmol/l) and inhibited intracellular chloride channels. We suggest that NO-releasing properties of the garlic, onion and leek extracts and their interaction with Cys and GSH are involved in NO-signalling pathway which contributes to some of its numerous beneficial biological effects.     

Increased stability of S-nitrosothiol solutions via pH modulations

S-nitrosothiol (RSNO) solutions represent a valuable source of nitric oxide and could be used as topical vasodilators, but their fast decomposition rate poses a serious obstacle to their potentially widespread therapeutic use. Our aim was to characterize and quantify the effect of pH on S-nitrosothiol formation and decomposition in simple aqueous solutions of S-nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC) and S-nitroso-3-mercaptopropionic acid (SN3MPA). Furthermore, we investigated the effect of storage pH on the stability of GSNO incorporated in poly(ethylene glycol)/ poly(vinyl alcohol) matrices. S-nitrosothiol concentrations were measured spectrophotometrically and laser Doppler scanning method was used to assess dermal blood flow. GSH and NAC solutions reached a complete transformation to nitrosothiols when synthesized using acidic NaNO(2) solution. The initial concentration of all investigated RSNOs decreased more slowly with pH adjusted to mildly basic values (8.4-8.8) for the storage period. Polymer gels of PVA/PEG compositions at mildly basic storage pH further reduced the decomposition rate succeeding to contain 46.8% of the initial GSNO concentration for 25 days. This amount of topically administered GSNO was still capable of increasing the dermal blood flow over 200% in human subjects.     

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.     

Dynamic state of S-nitrosothiols in human plasma and whole blood

In the vasculature, nitrosothiols derived from the nitric oxide (NO)-mediated S-nitrosation of thiols play an important role in the transport, storage, and metabolism of NO. The present study was designed to examine the reactions that promote the decomposition, formation, and distribution of extracellular nitrosothiols in the circulation. The disappearance of these species in plasma and whole blood was examined using a high-performance liquid chromatography method to separate low- and high-molecular weight nitrosothiols. We found that incubation of S-nitrosocysteine (CySNO) or S-nitrosoglutathione (GSNO) with human plasma resulted in a rapid decomposition of these nitrosothiols such that <10% of the initial concentration was recovered after 10-15 min. Neither metal chelators (DTPA, neocuproine), nor zinc chloride (glutathione peroxidase inhibitor), acivicin (gamma-glutamyl transpeptidase inhibitor), or allopurinol (xanthine oxidase inhibitor) inhibited the decomposition of GSNO. With both CySNO and GSNO virtually all NO was recovered as S-nitrosoalbumin (AlbSNO), suggesting the involvement of a direct transnitrosation reaction. Electrophilic attack of the albumin-associated thiols by reactive nitrogen oxides formed from the interaction of NO with O(2) was ruled out because one would have expected 50% yield of AlbSNO. Similar results were obtained in whole blood. The amount of S-nitrosohemoglobin recovered in the presence of 10 microM GSNO or CySNO was less than 100 nM taking into consideration the detection limit of the assay used. Our results suggest that serum albumin may act as a sink for low-molecular-weight nitrosothiols and as a modulator of NO(+) transfer between the vascular wall and intraerythrocytic hemoglobin.     

Increased stability of S-nitrosothiol solutions via pH modulations

S-nitrosothiol (RSNO) solutions represent a valuable source of nitric oxide and could be used as topical vasodilators, but their fast decomposition rate poses a serious obstacle to their potentially widespread therapeutic use. Our aim was to characterize and quantify the effect of pH on S-nitrosothiol formation and decomposition in simple aqueous solutions of S-nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC) and S-nitroso-3-mercaptopropionic acid (SN3MPA). Furthermore, we investigated the effect of storage pH on the stability of GSNO incorporated in poly(ethylene glycol)/ poly(vinyl alcohol) matrices. S-nitrosothiol concentrations were measured spectrophotometrically and laser Doppler scanning method was used to assess dermal blood flow. GSH and NAC solutions reached a complete transformation to nitrosothiols when synthesized using acidic NaNO₂ solution. The initial concentration of all investigated RSNOs decreased more slowly with pH adjusted to mildly basic values (8.4–8.8) for the storage period. Polymer gels of PVA/PEG compositions at mildly basic storage pH further reduced the decomposition rate succeeding to contain 46.8% of the initial GSNO concentration for 25 days. This amount of topically administered GSNO was still capable of increasing the dermal blood flow over 200% in human subjects.     

A new assay for the determination of low-molecular-weight nitrosothiols (nitrosoglutathione), NO, and nitrites by using a specific and sensitive solid-state amperometric gas sensor.

Nitric oxide (NO) is generated in biological systems and plays an important role as a bioregulatory molecule. Its ability to bind hemoglobin and myoglobin is well known. Moreover, it may lose an electron forming the nitrosyl group involved in the formation of S-nitrosothiols. The main problem in analyzing NO is its extreme reactivity. We have tackled this task by using an amperometric sensor to determine free NO, S-nitrosothiols (such as S-nitrosoglutathione), and nitrite in cell-free systems and murine microglial cell cultures. The determination of nitrosothiols is of biochemical relevance and a difficult task particularly at low concentration values. In this article we describe a new method based on the reductive cleavage of the S-NO bond by cuprous ions followed by a solid-state amperometric determination. The system described by us is sensitive, rapid, does not require previous purification steps, is easy to perform, and is inexpensive. For this reason, we think that it may represent an important analytical improvement. It has been suggested that nitrosothiols may exert biological activity by acting as a reservoir of NO. We tested the production of nitrite and of RSNO in stimulated, cultured murine microglial cells and we have shown that nitrite accumulates in these conditions. GSNO also accumulates, provided that GSH is present in the medium.     

Direct observation of trapping and release of nitric oxide by glutathione and cysteine with electron paramagnetic resonance spectroscopy

While the biosynthesis of nitric oxide (NO) is well established, one of the key issues that remains to be solved is whether NO participates in the biological responses right after generation through biosynthesis or there is a "secret passage" via which NO itself is trapped, transported, and released to exert its functions. It has been shown that NO reacts with thiol-containing biomolecules (RSH), like cysteine (Cys), glutathione (GSH), etc., to form S-nitrosothiols (RSNOs), which then release nitrogen compounds, including NO. The direct observation of trapping of NO and its release by RSNO has not been well documented, as most of the detection techniques measure the content of NO as well as nitrite and nitrate. Here we use spin-trapping electron paramagnetic resonance (EPR) technique to measure NO content directly in the reaction time course of samples of GSH and Cys ( approximately mM) mixed with NO ( approximately microM) in the presence of metal ion chelator, which pertains to physiological conditions. We demonstrate that NO is readily trapped by these thiols in less than 10 min and approximately 70-90% is released afterward. These data imply that approximately 10-30% of the reaction product of NO does not exist in the free radical form. The NO release versus time curves are slightly pH dependent in the presence of metal ion chelator. Because GSH and Cys exist in high molar concentrations in blood and in mammalian cells, the trapping and release passage of NO by these thiols may provide a mechanism for temporal and spatial sequestration of NO to overcome its concentration gradient-dependent diffusion, so as to exert its multiple biological effects by reacting with various targets through regeneration.     

Effect of S-nitrosothiols on cellular glutathione and reactive protein sulfhydryls

S-Nitrosothiols may cause many of the biological effects of NO and cellular effects have been attributed to S-nitrosylation of reactive protein sulfhydryls. This report examines the effect of S-nitrosothiols on the low-molecular-weight thiols and protein thiols in NIH/3T3 cells. A low concentration of S-nitrosocysteine increased the cysteine content of the cells, with no evidence of either low-molecular-weight thiol or protein S-nitrosylation. Millimolar amounts of S-nitrosocysteine produced S-nitrosoglutathione (GSNO), cysteinyl glutathione, cysteine, and glutathione disulfide. Large amounts of protein S-nitrosylation and lesser amounts of protein S-glutathiolation and S-cysteylation were also observed. GSNO and S-nitroso-N-acetylpenicillamine (SNAP) were much less effective than S-nitrosocysteine, but a combination of cysteine and GSNO produced S-nitrosocysteine-like effects. In cultured hepatocytes, millimolar S-nitrosocysteine was significantly less effective since the cells contained three times more glutathione than NIH/3T3 cells. Results suggest that S-nitrosocysteine enters cells intact, and low concentrations do not significantly increase cellular pools of S-nitrosothiol or S-nitrosylated protein. Millimolar concentrations of S-nitrosocysteine generate S-nitrosylated, S-glutathiolated, and S-cysteylated proteins, as well as a variety of low-molecular-weight disulfides and S-nitrosothiols.     

Iron catalyzed conversion of NO into nitrosonium (NO+) and nitroxyl (HNO/NO-) species.

The conversion of NO into its congeners, nitrosonium (NO+) and nitroxyl (HNO/NO-) species, has important consequences in NO metabolism. Dinitrosyl iron complex (DNIC) combined with thiol ligands was shown to catalyze the conversion of NO into NO+, resulting in the synthesis of S-nitrosothiols (RSNO) both in vitro and in vivo. The formation mechanism of DNIC was proposed to involve the intermediate release of nitroxyl. Since the detection of hydroxylamine (as the product of a rapid reaction of HNO/NO- with thiols) is taken as the evidence for nitroxyl generation, we examined the formation of hydroxylamine, RSNO, and nitrite (the product of a rapid reaction of NO+ with water) in neutral solutions containing iron ions and thiols exposed to NO under anaerobic conditions. Hydroxylamine was detected in NO treated solutions of iron ions in the presence of cysteine, but not glutathione (GSH). The addition of urate, a major "free" iron-binding agent in humans, to solutions of GSH and iron ions, and the subsequent treatment of these solutions with NO increased the synthesis of GSNO and resulted in the formation of hydroxylamine. This caused a loss of urate and yielded a novel nitrosative/nitration product. GSH attenuated the urate decomposition to such a degree that it could be reflected as the function of GSH:urate. Results described here contribute to the understanding of the role of iron ions in catalyzing the conversion of NO into HNO/NO- and point to the role of uric acid not previously described.     

Investigations of S-Transnitrosylation Reactions between Low- and High-Molecular-WeightS-Nitroso Compounds and Their Thiols by High-Performance Liquid Chromatography and Gas Chromatography–Mass Spectrometry

S-Transnitrosylation reactions are supposed to be the basic principle by which nitric oxide-related biological activities are regulatedin vivo.Mechanisms of S-transnitrosylation reactions are poorly understood and equilibria constants for physiologicalS-nitroso compounds and thiols are rare. In the present study we investigated S-transnitrosylation reactions of the thiols homocysteine, cysteine, glutathione,N-acetylcysteine,N-acetylpenicillamine, and human plasma albumin and their correspondingS-nitroso compounds SNhC, SNC, GSNO, SNAC, SNAP, and SNALB utilizing high-performance liquid chromatographic and gas chromatographic–mass spectrometric techniques. These methods allowed to study S-transnitrosylation reactions in mixtures of severalS-nitroso compound/thiol pairs, to determine equilibria constants, and to elucidate the mechanism of S-transnitrosylation reactions. We obtained the following order for the equilibria constants in aqueous buffered solution at pH 7.4: SNhC ≈ SNAC > GSNO ≈ SNALB > SNAP > SNC. Our results suggest that the mechanism of S-transnitrosylation reactions of theseS-nitroso compounds and their thiols involve heterolytic cleavage of the S---N bond. Incubation of SNC with human red blood cells resulted in a dose-dependent formation of GSNO in the cytosol through S-transnitrosylation of intracellular GSH by the SNC transported into the cells. This reaction was accompanied with an almost complete disappearance of the SNC fraction transported into the cells. This finding is in full agreement with the equilibrium constantK_eqof 1.9 for the reaction SNC + GSH ↔ Cys + GSNO in aqueous buffer.