Formation Of GSNO Research Articles

Biotransformation of organic nitrates by glutathione S-transferases and other enzymes: An appraisal of the pioneering work by William B. Jakoby

Organic nitrates (R–ONO2; R, organic residue) such as nitroglycerin are used as drugs in part for more than a century. Their pharmacological use is associated with clinically relevant tolerance which is reportedly known since 1888. The underlying mechanisms of both, the mechanisms of action and the main pharmacological effect, which is vasodilatation and reduction of blood pressure, and the development of tolerance, which means increasing need of drug amount in sustained long-term therapy, are still incompletely understood. William B. Jakoby and associates were the first to report the biotransformation of organic nitrates, notably including nitroglycerin (i.e., glycerol trinitrate; GTN), by glutathione S-transferase (GST)-catalyzed conjugation of glutathione (GSH) to the nitrogen atom of one of the three nitrate groups of GTN to generate glutathione sulfenyl nitrite (glutathione thionitrate, S-nitroglutathione; GSNO2). Jakoby's group was also the first to suggest that GSNO2 reacts with a second GSH molecule to produce inorganic nitrite (ONO−) and glutathione disulfide (GSSG) without the catalytic involvement of GST. This mechanism has been adopted by others to the biotransformation of GTN by mitochondrial aldehyde dehydrogenase (mtALDH-(CysSH)2) which does not require GSH as a substrate. The main difference between these reactions is that mtALDH forms an internal thionitrate (mtALDH-(CysSH)-CysSNO2) which releases inorganic nitrite upon intra-molecular reaction to form mtALDH disulfide (mtALDH-(CysS)2). Subsequently, ONO− and GSNO2 are reduced by several proteins and enzymes to nitric oxide (NO) which is a very potent activator of soluble guanylyl cyclase to finally relax the smooth muscles thus dilating the vasculature. GSNO2 is considered to rearrange to GSONO which undergoes further reactions including GSNO and GSSG formation. The present article is an appraisal of the pioneering work of William B. Jakoby in the area of the biotransformation of organic nitrates by GST. The two above mentioned enzymatic reactions are discussed in the context of tolerance development to organic nitrates, still a clinically relevant pharmacological concern.

S-Nitrosoglutathione formation at gastric pH is augmented by ascorbic acid and by the antioxidant vitamin complex, Resiston

Context: Exogenous nitrogen oxides must be made bioavailable to sustain normal physiology because nitric oxide synthase (NOS) deficient mice are viable. In the stomach, S-nitrosoglutathione (GSNO) is formed from ingested nitrite and high levels of airway glutathione (GSH) that are cleared and swallowed. However, gastric GSNO may be broken down by nutrients like ascorbic acid (AA) before it is absorbed.Objective: To study the effect of AA on GSNO formation and stability.Materials and methods: GSH and nitrite were reacted with or without 5 mM AA or Resiston (5 mM AA with retinoic acid and α-tocopherol). GSNO was measured by reduction/chemiluminescence and HPLC. AA and reduced thiols were measured colorimetrically. O-Nitrosoascorbate and AA were measured by gas chromatography–mass spectrometry (GC–MS).Results: GSNO was formed in saline and gastric samples (pH ∼4.5) from physiological levels of GSH and nitrite. Neither AA nor Resiston decreased [GSNO] at pH >3; rather, they increased [GSNO] (0.12 ± 0.19 μM without AA; 0.42 ± 0.35 μM with AA; and 0.43 ± 0.23 μM with Resiston; n = 4 each; p ≤ 0.05). However, AA compounds decreased [GSNO] at lower pH and with incubation >1 h. Mechanistically, AA, but not dehydroascorbate, increased GSNO formation; and the O-nitrosoascorbate intermediate was formed.Conclusions: AA, with or without other antioxidants, did not deplete GSNO formed from physiological levels of GSH and nitrite at pH >3. In fact, it favoured GSNO formation, likely through O-nitrosoascorbate. Gastric GSNO could be a NOS-independent source of bioavailable nitrogen oxides.

Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets

Nitric oxide (NO), S-nitrosoglutathione (GSNO) and S-nitrosocysteine are highly potent signaling molecules, acting both by cGMP-dependent and cGMP-independent mechanisms. The NO metabolite nitrite (NO2 (-)) is a major NO reservoir. Hemoglobin, xanthine oxidoreductase and carbonic anhydrase (CA) have been reported to reduce/convert nitrite to NO. We evaluated the role and the physiological importance of CA for an extra-platelet CA/nitrite/NO/cGMP pathway in human platelets. Authentic NO was analyzed by an NO-sensitive electrode. GSNO and GS(15)NO were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). cGMP was determined by LC-MS/MS or RIA. In reduced glutathione (GSH) containing aqueous buffer (pH 7.4), human and bovine erythrocytic CAII-mediated formation of GSNO from nitrite and GS(15)NO from (15)N-nitrite. In the presence of L-cysteine and GSH, this reaction was accompanied by NO release. Incubation of nitrite with bovine erythrocytic CAII and recombinant soluble guanylyl cyclase resulted in cGMP formation. Upon incubation of nitrite with bovine erythrocytic CAII and washed human platelets, cGMP and P-VASP(S239) were formed in the platelets. This study provides the first evidence that extra-platelet nitrite and erythrocytic CAII may modulate platelet function in a cGMP-dependent manner. The new nitrite-dependent CA activity may be a general principle and explain the cardioprotective effects of inorganic nitrite in the vasculature. We propose that nitrous acid (ONOH) is the primary CA-catalyzed reaction productof nitrite.

Effect of alkyl group on transnitrosation of N-nitrosothiazolidine thiocarboxamides

S-Nitrosoglutathione (GSNO) relaxes vascular smooth muscles, prevents platelet aggregation, and acts as a potential in vivo nitric oxide donor. 3-Nitroso-1,3-thiazolidine-4-thiocarboxamide (1), a N-nitrosothio-proline analogue, exhibited a high GSNO formation activity. In this study, two compounds (2 and 3) based on compound 1 were newly synthesized by introducing either one or two methyl groups onto a nitrogen atom on the thioamide substituent in 1. The pseudo-first-order rate constants (kobs) for the GSNO formation for the reaction between the compound and glutathione followed the order 1>2≒3. Thus, the introduction of a methyl group(s) onto the thioamide group led to a decrease in the transnitrosation activity. On the basis of density functional theoretical calculations, the transnitrosation for the N-nitrosothiazolidine thiocarboxamides was proposed to proceed via a bridged intermediate pathway. Specifically, the protonated compound 1 forms a bridged structure between the nitrogen atom in the nitroso group and two sulfur atoms—one in the ring and the other in the substituent. The bridged intermediate gives rise to a second intermediate in which the nitroso group is bonded to the sulfur atom in the thioamide group. Finally, the nitroso group is transferred to GSH to form GSNO.

Transnitrosation of alicyclic N-nitrosamines containing a sulfur atom

Aromatic and aliphatic nitrosamines are known to transfer a nitrosonium ion to another amine. The transnitrosation of alicyclic N-nitroso compounds generates S-nitrosothiols, which are potential nitric oxide donors in vivo. In this study, certain alicyclic N-nitroso compounds based on non-mutagenic N-nitrosoproline or N-nitrosothioproline were synthesised, and the formation of S-nitrosoglutathione (GSNO) was quantified under acidic conditions. We then investigated the effect of a sulfur atom as the substituent and as a ring component on the GSNO formation. In the presence of thiourea under acidic conditions, GSNO was formed from N-nitrosoproline and glutathione, and an N-nitroso compound containing a sulfur atom and glutathione produced GSNO without thiourea. The quantity of GSNO derived from the reaction of the N-nitrosamines containing a sulfur atom and glutathione was higher than that from the N-nitrosoproline and glutathione plus thiourea. Among the analogues that contained a sulfur atom either in the ring or as a substituent, the thiazolidines produced a slightly higher quantity of GSNO than the analogue with a thioamide group. A compound containing sulfur atoms both in the ring and as a substituent exhibited the highest activity for GSNO formation among the alicyclic N-nitrosamines tested. The results indicate that the intramolecular sulfur atom plays an important role in the transnitrosation via alicyclic N-nitroso compounds to form GSNO.

Glutathiyl radical as an intermediate in glutathione nitrosation

Nitrosation of thiols is thought to be mediated by dinitrogen trioxide (N2O3) or by nitrogen dioxide radical (NO2). A kinetic study of glutathione (GSH) nitrosation by NO donors in aerated buffered solutions was undertaken. S-nitrosoglutathione (GSNO) formation was assessed spectrophotometrically and by chemiluminescence. The results suggest an increase in the rate of GSNO formation with an increase in GSH with a half-maximum constant EC50 that depends on NO concentration. Our observed increase in EC50 with NO concentration suggests a significant contribution of NO2-mediated nitrosation with the glutathiyl radical as an intermediate in the production of GSNO.

Role of nitric oxide-mediated glutathionylation in neuronal function: potential regulation of energy utilization

Excessive generation of nitric oxide radical (NO*) in neuroinflammation, excitotoxicity and during age-related neurodegenerative disorders entails the localized and concerted increase in nitric oxide synthase(s) expression in glial cells and neurons. The aim of the present study was to assess the biological significance of the impact of NO* on the cell's thiol status with emphasis on S-glutathionylation of targeted proteins. Exposure of primary cortical neurons or astrocytes to increasing flow rates of NO* (0.061-0.25 microM/s) resulted in the following. (i) A decrease in GSH (glutathione) in neurons accompanied by formation of GSNO (S-nitrosoglutathione) and GSSG (glutathione disulfide); neurons were far more sensitive to NO* exposure than astrocytes. (ii) A dose-dependent oxidation of the cellular redox status: the neuron's redox potential increased approximately 42 mV and that of astrocytes approximately 23 mV. A good correlation was observed between cell viability and the cellular redox potential. The higher susceptibility of neurons to NO* can be partly explained by a reduced capacity to recover GSH through lower activities of GSNO and GSSG reductases. (iii) S-glutathionylation of a small subset of proteins, among them GAPDH (glyceraldehyde-3-phosphate dehydrogenase), the S-glutathionylation of which resulted in inhibition of enzyme activity. The quantitative analyses of changes in the cell's thiol potential upon NO* exposure and their consequences for S-glutathionylation are discussed in terms of the distinct redox environment of astrocytes and neurons.

Kinetics of nitrosation of GSH using NO donors

Modification of critical thiols by reactive nitrogen species (RNS) act as regulators of protein mediated signaling. Nitrosothiols are also believed to act as vasodilators which can release NO and activate the heme protein guanylate cyclase, thus initiating the mechanism for vasodilation. Nitrosation is thought to be mediated by dinitrogen trioxide (N2O3). Combination of NO with thiyl radical (GS.) following oxidation of the thiyl anion by NO2 has also been suggested as a mechanism for nitrosothiol formation. We investigate the kinetic mechanism of GSH (0–20mM) nitrosation in vitro by NO donor PAPA/NO (0.5 – 10 mM). GSNO formation was assessed spectrophotometrically at 338 nm. Results suggest a Michaelis‐Menten dependence of the rate on GSH concentration with a half‐maximum constant (Km,GSH) that depends on NO concentration. If there were solely one mechanism of nitrosation, one would expect a constant Km GSH value. Increasing [NO] up to 10 fold showed a proportional increase in Km,GSH. Pseudo steady state approximation yields the following kinetic rate for GS mediated nitrosation: urn:x-wiley:08926638:media:fsb2fasebj241supplement98422:fsb2fasebj241supplement98422-math-0001The reported increase in Km,GSH with NO concentration suggest a significant contribution of GS.mediated nitrosation in the formation of GSNO.(Supported by NIH SC1HL95101)

Fibrinogen-Dependent Signaling in Microvascular Erythrocyte Function: Implications on Nitric Oxide Efflux

Experimental evidence has shown that plasma fibrinogen plays a key role as a major cardiovascular risk factor, acting directly to trigger erythrocyte aggregation in occlusive vascular disease. However, due to the complex and hitherto unclear interaction between fibrinogen and the erythrocyte membrane, no study has yet evaluated the effects of fibrinogen, under physiological range values, on the erythrocyte nitric oxide (NO) mobilization. Taking into consideration the potential NO-derived molecules, we have raised the hypothesis that fibrinogen, under physiological conditions, may act to influence blood flow via erythrocyte NO modulation. In this in vitro study whole-blood samples were harvested from healthy subjects, erythrocyte suspensions were incubated in the absence (control aliquots) and presence of different fibrinogen concentrations and levels of NO, nitrite, nitrate and S-nitroglutathione (GSNO) were determined. Our results showed, when compared with control aliquots, that the presence of fibrinogen modulates the NO mobilization in erythrocytes by (1) decreasing erythrocyte NO efflux levels (P < 0.001); (2) increasing levels of intraerythrocytic NO oxidative metabolites, namely, nitrite (P < 0.0001) and nitrate (P < 0.0001); and (3) enhancing the formation of GSNO (P < 0.001). In conclusion, this study provides new insights into an unknown mechanism by which fibrinogen modulates the erythrocyte capacity to supply NO, the effects of which on inflammation profiles (generally associated with blood hyperviscosity and hyperaggregation) still need to be elucidated. Also, increased erythrocyte GSNO levels may be associated with platelet NO metabolism, its activation status and hypotension, which may be extremely relevant in the clinical setting as biomarkers.

S‐Nitrosoglutathione and Hypoxia‐Inducible Factor‐1 Confer Chemoresistance against Carbamoylating Cytotoxicity of BCNU in Rat C6 Glioma Cells

BCNU (1,3-bis[2-chloroethyl]-1-nitrosourea) is the mainstay in glioblastoma multiform chemotherapy with only minimal effects. BCNU may kill tumor cells via carbamoylating cytotoxicity, which irreversibly inhibits glutathione reductase with resultant accumulation of oxidized form of glutathione causing oxidative stress. S-nitrosoglutathione (GSNO) is a product of glutathione and nitric oxide interaction. We report that GSNO formation may underlie carbamoylating chemoresistance mediated by activation of inducible nitric oxide synthase. Transactivation of hypoxia-inducible factor-1 (HIF-1)-responsive genes reduces oxidative stress caused by glutathione depletion. We also noted that preconditioning of C6 glioma cells to induce HIF-1 and its downstream genes confers chemoresistance against carbamoylating cytotoxicity of BCNU.

Production of nitric oxide elevates nitrosothiol formation resulting in decreased glutathione in macrophages exposed to asbestos or asbestos substitutes

The purpose of this study was to investigate the effects of pneumoconiogenic particles, such as asbestos, on nitrosothiol formation in macrophages. In addition, the effects of man-made mineral fibers (MMMFs) were also evaluated, because they have come into heavy use as substitutes for asbestos. RAW264.7 cells and J774 cells of murine macrophage cell lines were cultured with chrysotile B (CH) asbestos, crocidolite (CR) asbestos, or MMMFs comprised of glass wool (GW), rock wool (RW), or ceramic (RF1). All of these fibers significantly increased nitric oxide (NO) production in the culture with macrophages. Chrysotile B, CR, and GW significantly decreased the level of reduced glutathione (GSH) in RAW264.7 cells. S-nitrosothiol (RS-NO) formation was increased by both types of cells on exposure to every fiber. A large portion of this increased RS-NO may be in the form of S-nitrosoglutathione (GS-NO), because GSH is the most abundant thiol substance in the cell. Both CH and GW significantly increased superoxide anion in the media cultured of RAW264.7 cells. These results indicate that macrophages exposed to asbestos or MMMFs are subject to oxidative stress, not only through the generation of reactive oxygen and nitrogen species, but also through decreases in the level of the cellular antioxidant, GSH, by GS-NO formation. The increase of RS-NO in macrophages exposed to asbestos or MMMFs may deserve more attention as the indicator of continuous oxidative stress by NO on cells and tissues, which causes inflammation and involves the development of asbestos-induced diseases.

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.

S-nitrosation of glutathione by nitric oxide, peroxynitrite, and (*)NO/O(2)(*-).

To elucidate potential mechanisms of S-nitrosothiol formation in vivo, we studied nitrosation of GSH and albumin by nitric oxide ((*)NO), peroxynitrite, and (*)NO/O(2)(*)(-). In the presence of O(2), (*)NO yielded 20% of S-nitrosoglutathione (GSNO) at pH 7.5. Ascorbate and the spin trap 4-hydroxy-[2,2,4,4-tetramethyl-piperidine-1-oxyl] (TEMPOL) inhibited GSNO formation by 67%. Electron paramagnetic resonance spectroscopy with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) demonstrated intermediate formation of glutathionyl radicals, suggesting that GSNO formation by (*)NO/O(2) is predominantly mediated by (*)NO(2). Peroxynitrite-triggered GSNO formation (0.06% yield) was stimulated 10- and 2-fold by ascorbate and TEMPOL, respectively. Co-generation of (*)NO and O(2)(*)(-) at equal fluxes yielded less GSNO than (*)NO alone, but was 100-fold more efficient (8% yield) than peroxynitrite. Moreover, in contrast to the reaction of peroxynitrite, GSNO formation by (*)NO/O(2)(*)(-) was inhibited by ascorbate. Similar results were obtained with albumin instead of GSH. We propose that sulfhydryl compounds react with O(2)(*)(-) to initiate a chain reaction that forms radical intermediates which combine with (*)NO to yield GSNO. In RAW 264.7 macrophages, S-nitrosothiol formation by (*)NO/O(2) and (*)NO/O(2)(*)(-) occurred with relative efficiencies comparable to those in solution. Our results indicate that concerted generation of (*)NO and O(2)(*)(-) may essentially contribute to nitrosative stress in inflammatory diseases.

Metabolism of S-nitrosoglutathione in intact mitochondria

S-nitrosation of protein thiol groups by nitric oxide (NO•) is a widely recognized protein modification. Only few intracellular S-nitrosated proteins have been identified and it has been reported that S-nitrosation/denitrosation can serve as a regulatory process in signal-transduction pathways. Given the potential physiological importance of S-nitrosothiols, and considering that mitochondria are endowed with high levels of thiols and the biochemical requisites for synthesizing NO•, we examined the occurrence of S-nitrosoglutathione (GSNO) in intact, coupled rat liver mitochondria. These organelles contained 0.34nmol of GSNO/mg of protein, detected by HPLC with UV–visible and electrochemical detections. This concentration was dynamically modulated by the availability of NO•; its decay was affected mainly by GSH and superoxide dismutase in a reaction that entailed the generation of GSSG. On the basis of the relatively long half-life of GSNO and the negligible recovery of NO• during its decay, roles for GSNO as a storage and transport molecule for NO• are discussed. Moreover, the formation of GSNO and its reaction with GSH can be considered to be partly responsible for the catabolism of NO• via a complex mechanism that might result in the formation of hydroxylamine, nitrite or nitrous oxide depending upon the availability of oxygen, superoxide dismutase and glutathione. Finally, the high concentrations of GSH in the cytosol and mitochondria might favour the formation of GSNO by reacting with NO• ‘in excess’, thereby avoiding damaging side reactions (such as peroxynitrite formation), and facilitate the inactivation of NO• by generating other nitrogen-related species without the chemical properties characteristic of NO•.

Metabolism of S-nitrosoglutathione in intact mitochondria.

S-nitrosation of protein thiol groups by nitric oxide (NO*) is a widely recognized protein modification. Only few intracellular S-nitrosated proteins have been identified and it has been reported that S-nitrosation/denitrosation can serve as a regulatory process in signal-transduction pathways. Given the potential physiological importance of S-nitrosothiols, and considering that mitochondria are endowed with high levels of thiols and the biochemical requisites for synthesizing NO*, we examined the occurrence of S-nitrosoglutathione (GSNO) in intact, coupled rat liver mitochondria. These organelles contained 0.34 nmol of GSNO/mg of protein, detected by HPLC with UV-visible and electrochemical detections. This concentration was dynamically modulated by the availability of NO*; its decay was affected mainly by GSH and superoxide dismutase in a reaction that entailed the generation of GSSG. On the basis of the relatively long half-life of GSNO and the negligible recovery of NO* during its decay, roles for GSNO as a storage and transport molecule for NO* are discussed. Moreover, the formation of GSNO and its reaction with GSH can be considered to be partly responsible for the catabolism of NO* via a complex mechanism that might result in the formation of hydroxylamine, nitrite or nitrous oxide depending upon the availability of oxygen, superoxide dismutase and glutathione. Finally, the high concentrations of GSH in the cytosol and mitochondria might favour the formation of GSNO by reacting with NO* 'in excess', thereby avoiding damaging side reactions (such as peroxynitrite formation), and facilitate the inactivation of NO* by generating other nitrogen-related species without the chemical properties characteristic of NO*.

Artifactual-free analysis of S-nitrosoglutathione and S-nitroglutathione by neutral-pH, anion-pairing, high-performance liquid chromatography : Study on peroxynitrite-mediated S-nitration of glutathione to S-nitroglutathione under physiological conditions

The endogenous potent vasodilators and inhibitors of platelet aggregation S-nitrosoglutathione (GSNO) and S-nitroglutathione (GSNO 2) are frequently analyzed by high-performance liquid chromatography (HPLC) using mobile phases of acidic pH. These systems are associated with problems stemming from rapid and considerable artifactual formation of GSNO from glutathione (GSH) and ubiquitous nitrite. We describe a novel ion-pairing HPLC method with UV absorbance detection at 334 nm for the highly specific and interference-free analysis of GSNO and GSNO 2 in the presence of high GSH and nitrite concentrations. Complete avoidance of artifactual formation of GSNO was accomplished by using the anion-pairing agent tetrabutylammoniumhydrogen sulphate in the mobile phase that enables analysis of GSNO at neutral pH, at which GSH and nitrite do not react to form GSNO. This HPLC system was used to study formation of GSNO 2 from GSH and peroxynitrite under physiological conditions. We found by this HPLC system that peroxynitrite (0–300 μ M) reacts with GSH (0–5 m M) to form GSNO 2 at a mean yield of 2%. Analysis of the same samples by a cation-pairing HPLC system with acidic mobile phase (pH 2.0) revealed, however, GSNO plus GSNO 2 formation of the order of 20% due to on column reaction of GSH with peroxynitrite-derived nitrite to form GSNO. Ammonium sulfamate is frequently used to remove nitrite from thiol-containing solutions under acidic conditions. By means of the anion-pairing HPLC system it is demonstrated that nitrite removal by this method is incomplete even when ammonium sulfamate is used at high concentrations. These findings underscore the absolute requirement of neutral pH conditions for the analysis of GSNO. The novel anion-pairing HPLC method should be useful to provide reliable data on formation, reaction and metabolism of GSNO and GSNO 2 in biological fluids using various detectors including mass spectrometers.

Nitric oxide inactivates glyoxalase I in cooperation with glutathione.

We previously found that glyoxalase I (Glo I) is inactivated upon exposure of human endothelial cells to extracellular nitric oxide (NO), and this event correlates with an increase in its pI on two-dimensional gels. In this study, we demonstrate that NO can modulate Glo I activity in cooperation with cellular glutathione (GSH). Severe depletion of intracellular GSH prevents the inactivation of Glo I in response to NO, although such depletion enhances the inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a well-known enzyme susceptible to NO-induced oxidation. S-Nitrosoglutathione (GSNO), an adduct of GSH and NO, lowers the activity of purified human Glo I, while S-nitrosocysteine (CysNO) inactivates the enzyme only in the presence of GSH. This indicates that a dysfunction in Glo I would require the formation of GSNO in situ. Competitive inhibitors of Glo I, S-(4-bromobenzyl)glutathione and its membrane-permeating form, completely abolish the NO action in vitro and inside cells, respectively. Taken together, these results reveal that Glo I can interact directly with GSNO, and that the interaction converts Glo I into an inactive form. Moreover, the data suggest that the substrate recognition site of Glo I might be involved in the interaction with GSNO.

Determination of S-Nitrosoglutathione in Human and Rat Plasma by High-Performance Liquid Chromatography with Fluorescence and Ultraviolet Absorbance Detection after Precolumn Derivatization with o-Phthalaldehyde

An analytical method is described for the quantification of S-nitrosoglutathione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmaleimide, the conversion of GSNO by 2-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricyclic isoindole derivative, and subsequent high-performance liquid chromatographic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to be identical by mass spectrometry. GSH (up to 50 μM) did not interfere with the analysis of GSNO (up to 1000 nM). The limits of detection of the method for buffered aqueous solutions of GSNO were determined as 3 nM using fluorescence and 70 nM using UV absorbance detection. Isolation of GSNO by HPLC analysis (pH 7.0) of plasma ultrafiltrate samples (200 μl) prior to derivatization allows specific and artifact-free quantification of GSNO in human and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LOQ) of the combined method was determined as 100 nM of GSNO in human plasma ultrafiltrate using fluorescence detection. No endogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 healthy humans at concentrations exceeding the LOQ of the method. After iv infusion of GSNO (125 μmol/kg body wt) in a rat for 20 min GSNO and GSH were detected in rat plasma at 60 and 130 μM, respectively. The method should be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations.

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 SN 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 constantKeqof 1.9 for the reaction SNC + GSH ↔ Cys + GSNO in aqueous buffer.

Formation of S-Nitrosothiols via Direct Nucleophilic Nitrosation of Thiols by Peroxynitrite with Elimination of Hydrogen Peroxide

Peroxynitrite (ONOO-), a potent oxidant formed by reaction of nitric oxide (NO.) with superoxide anion, can activate guanylyl cyclase and is able to induce vasodilation or inhibit platelet aggregation and leukocyte adhesion, via thiol-dependent formation of NO. Reaction of ONOO- with thiols is thought to proceed through formation of a S-nitrothiol (thionitrate; RSNO2) intermediate and yields low levels of S-nitrosothiols (thionitrites; RSNO), both of which are theoretical sources of NO. Kinetic analysis of NO. production after reaction of ONOO- with GSH established that NO. originates exclusively from the thionitrite GSNO. Further mechanistic investigations indicated that GSNO formation by ONOO- does not occur via one-electron oxidation mechanisms. Nitrosation of GSH could theoretically proceed via intermediate formation of the thionitrate GSNO2, which, after rearrangement to the corresponding sulfenyl nitrite (GSONO), can react with GSH to form GSNO and GSOH. However, no evidence for such a mechanism was found in experiments with NO2. or with the stable nitrothiol tert-butylthionitrate. Using high performance liquid chromatography with chemiluminescence detection, formation of H2O2 was observed after reaction of ONOO- with GSH under both aerobic and anaerobic conditions, at levels similar to the yield of GSNO, indicative of a direct nucleophilic nitrosation mechanism with elimination of HOO-. Our results indicate that ONOO- may contribute to S-nitrosation in vivo and that direct nitrosation of thiols or other nucleophilic substrates by ONOO- may represent an important and often overlooked component of NO. biochemistry.