Mechanism of superoxide dismutase/H(2)O(2)-mediated nitric oxide release from S-nitrosoglutathione--role of glutamate

S-Nitrosoglutathione (GSNO), a physiologically relevant nitric oxide ((*)NO) donor, exhibits antioxidant, anti-ischemic, and antiplatelet properties. The exact mechanism of (*)NO release from GSNO in biological systems has not been determined. Both copper ions and copper-containing enzymes have been shown to catalyze (*)NO release from GSNO. In this study we observed that copper-zinc superoxide dismutase (Cu,ZnSOD) in the presence of H(2)O(2) caused a rapid decomposition of GSNO, forming oxidized glutathione (GSSG) and (*)NO. The cupric ions (Cu(2+)) released from Cu,ZnSOD were bound to the glutamate moiety of GSNO, yielding a 2:1 (GSNO)(2)Cu(2+) complex. Strong chelators of cupric ions, such as histidine and diethylenetriaminepentaacetic acid, inhibited the formation of (GSNO)(2)Cu(2+) complex, GSSG, and (*)NO. GSSG alone inhibited Cu(2+)-induced decomposition of GSNO. This effect is attributed to complexation of copper by GSSG. We conclude that binding of copper to GSNO is obligatory for (*)NO release from GSNO; however, the rate of this reaction was considerably slowed due to binding of Cu(2+) by GSSG. The glutamate moiety in GSNO and GSSG controls copper-catalyzed (*)NO release from GSNO. Cu,ZnSOD and H(2)O(2) enhanced peroxidation of unsaturated lipid that was inhibited by GSNO. The antioxidant function of GSNO is related to the sequestering of copper by GSNO and its ability to slowly release (*)NO. Implications of these findings are discussed in relation to GSNO-induced cardioprotection and to neuropathological processes.     

Optimization of Cardiovascular Stent against Restenosis: Factorial Design-Based Statistical Analysis of Polymer Coating Conditions

The objective of this study was to optimize the physicodynamic conditions of polymeric system as a coating substrate for drug eluting stents against restenosis. As Nitric Oxide (NO) has multifunctional activities, such as regulating blood flow and pressure, and influencing thrombus formation, a continuous and spatiotemporal delivery of NO loaded in the polymer based nanoparticles could be a viable option to reduce and prevent restenosis. To identify the most suitable carrier for S-Nitrosoglutathione (GSNO), a NO prodrug, stents were coated with various polymers, such as poly (lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG) and polycaprolactone (PCL), using solvent evaporation technique. Full factorial design was used to evaluate the effects of the formulation variables in polymer-based stent coatings on the GSNO release rate and weight loss rate. The least square regression model was used for data analysis in the optimization process. The polymer-coated stents were further assessed with Differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy analysis (FTIR), Scanning electron microscopy (SEM) images and platelet adhesion studies. Stents coated with PCL matrix displayed more sustained and controlled drug release profiles than those coated with PLGA and PEG. Stents coated with PCL matrix showed the least platelet adhesion rate. Subsequently, stents coated with PCL matrix were subjected to the further optimization processes for improvement of surface morphology and enhancement of the drug release duration. The results of this study demonstrated that PCL matrix containing GSNO is a promising system for stent surface coating against restenosis.     

trans-[Ru(NO)(NH3)4(py)](BF4)3.H2O encapsulated in PLGA microparticles for delivery of nitric oxide to B16-F10 cells: Cytotoxicity and phototoxicity

The NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (py=pyridine) was loaded into poly-lactic-co-glycolic acid (PLGA) microparticles using the double emulsification technique. Scanning electron microscopy (SEM) and dynamic light scattering revealed that the particles are spherical in shape, have a diameter of 1600nm, and have low tendency to aggregate. The entrapment efficiency was 25%. SEM analysis of the melanoma cell B16-F10 in the presence of the microparticles containing the complex trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (pyMP) showed that the microparticles were adhered to the cell surface after 2h of incubation. The complex with concentrations lower than 1x10(-4)M did not show toxicity in B16-F10 murine cells. The complex in solution is toxic at higher concentrations (>1x10(-3)M), with cell death attributed to NO release following the reduction of the complex. pyMP is not cytotoxic due to the lower bioavailability and availability of the entrapped complex to the medium and its reducing agents. However, pyMP is phototoxic upon light irradiation. The phototoxicity strongly suggests that cell death is due to NO release from trans-[Ru(NO)(NH(3))(4)(py)](3+). This work shows that pyMP can serve as a model for a drug delivery system carrying the NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O, which can release NO locally at the tumor cell by irradiation with light only.     

S-nitrosophytochelatins: investigation of the bioactivity of an oligopeptide nitric oxide delivery system

This study investigates the in vitro bioactivity of S-nitrosophytochelatins (SNOPCs), oligopeptide analogues of S-nitrosoglutathione (GSNO), and their mechanisms of nitric oxide (NO) delivery. SNOPCs were more potent than GSNO in inhibiting platelet aggregation and stimulating vasorelaxation. Their potency was related to the number of S-nitrosated moieties per mole compound. Transnitrosation reactions with cell membrane surface components were shown to be the primary mode of NO delivery to intracellular targets for SNOPCs, while delivery via γ-glutamyl transpeptidase was unique to GSNO. Due to rapid NO release, larger SNOPCs elicited a more transitory effect compared to smaller compounds. The duration of effect was influenced by compound molecular weight, NO release kinetics, ability to undergo transnitrosation, and incubation time with tissues. In summary, a new oligopeptide NO delivery system based on SNOPCs was shown to be biologically active and can be used to investigate the mechanisms of NO delivery to intracellular targets.     

Do nitric oxide donors mimic endogenous NO-related response in plants?

Huge advances achieved recently in elucidating the role of NO in plants have been made possible by the application of NO donors. However, the application of NO to plants in various forms and doses should be subjected to detailed verification criteria. Not all metabolic responses induced by NO donors are reliable and reproducible in other experimental designs. The aim of the presented studies was to investigate the half-life of the most frequently applied donors (SNP, SNAP and GSNO), the rate of NO release under the influence of light and reducing agents. At a comparable donor concentration (500 μM) and under light conditions the highest rate of NO generation was found for SNAP, followed by GSNO and SNP. The measured half-life of the donor in the solution was 3 h for SNAP, 7 h for GSNO and 12 h for SNP. A temporary lack of light inhibited NO release from SNP, both in the solution and SNP-treated leaf tissue, which was measured by the electrochemical method. Also a NO, selective fluorescence indicator DAF-2DA in leaves supplied with different donors showed green fluorescence spots in the epidermal cells mainly in the light. SNP as a NO donor was the most photosensitive. The activity of PAL, which plays an important role in plant defence, was also activated by SNP in the light, not in the dark. S-nitrosothiols (SNAP and GSNO) also underwent photodegradation, although to a lesser degree than SNP. Additionally, NO generation capacity from S-nitrosothiols was shown in the presence of reducing agents, i.e. ascorbic acid and GSH, and the absence of light. The authors of this paper would like to polemicize with the commonly cited statement that “donors are compounds that spontaneously break down to release NO” and wish to point out the fact that the process of donor decomposition depends on the numerous external factors. It may be additionally stimulated or inhibited by live plant tissue, thus it is necessary to take into consideration these aspects and monitor the amount of NO released by the donor.     

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 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.     

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 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.     

Nitric oxide induces apoptosis via hydrogen peroxide, but necrosis via energy and thiol depletion

We investigated the mechanisms by which two nitric oxide (NO) donors, diethylenetriamine/NO adduct (DETA/NO) and S-nitrosoglutathione (GSNO), induced cell death in a J774 macrophage cell line. Both NO donors induced caspase activation within 6 h, but only DETA/NO-induced caspase activation was sensitive to inhibition of p38 and was completely prevented by antioxidants catalase, ascorbate, dehydroascorbate, or N-acetylcysteine, suggesting that DETA/NO-induced apoptosis may be mediated by H(2)O(2). Consistent with this, DETA/NO acutely stimulated reactive oxygen species (ROS) production by mitochondria and cells, and inhibited catalase-mediated H(2)O(2) breakdown in cells. After prolonged, 24 h exposure of cells to DETA/NO, inactivation of caspases occurred, which was accompanied by an increase in necrosis. DETA/NO-induced necrosis was insensitive to caspase inhibitors, but was partially prevented by catalase or N-acetylcysteine, and was preceded by inhibition of glyceraldehyde-3-phosphate dehydrogenase and a decrease in cellular adenosine triphosphate (ATP). GSNO was even more potent in inhibiting glycolysis and switching apoptosis to necrosis. In cells depleted of glutathione, GSNO and DETA/NO induced rapid necrosis, which resulted from rapid depletion of ATP due to inhibition of glycolysis. Glycolytic intermediate 3-phosphoglycerate decreased DETA/NO-induced necrosis and increased apoptosis. We conclude that: (i). NO-induced apoptosis is mediated by H(2)O(2); (ii). NO-induced necrosis is mediated by energy failure speeded by thiol depletion.     

Kinetics and mechanism of the decomposition of S-nitrosoglutathione by l-ascorbic acid and copper ions in aqueous solution to produce nitric oxide.

S-Nitrosothiols serve as a good source of nitric oxide ((*)NO) mainly due to the ease of cleavage of the S-N bond which consequently produces (*)NO. The reductive decomposition of S-nitrosoglutathione (GSNO) by l-ascorbic acid (vitamin C) yields (*)NO which was monitored both electrochemically (using NO-probe) and spectrophotometrically. The rate of reaction and (*)NO release was found to be pH dependent in a manner which drastically increases with pH demonstrating that the l-ascorbic acid dianion (A(2-)) is by far the most reactive species of l-ascorbic acid (H(2)A). The derived rate expression (measuring the disappearance of the absorption at ca. 336 nm due to GSNO) was established as rate = -d[GSNO](t)/dt = ((k(a)[H(+)](2) + k(b)[H(+)]K(1) + k(c)K(1)K(2))/([H(+)](2) + K(1)[H(+)] + K(1)K(2)))[GSNO](t)[H(2)A](t). k(a), k(b), and k(c) are second-order rate constants via the H(2)A, HA(-), and A(2-) pathways, respectively, while K(1) and K(2) represent the first and second equilibrium dissociation constants of l-ascorbic acid. There is little or no reaction at low pH (below 5.5), where H(2)A is a predominant species, and as a result the rate constant (k(a)) via this route was found to be negligible. At 25 degrees C, k(b) = 5.23 +/- 1.47 x 10(-3) dm(3) mol(-1) s(-1) and k(c) = 1.22 +/- 0.04 x 10(3) dm(3) mol(-1) s(-1), activation parameters DeltaH(double dagger)(b) = 54.4 +/- 4.3 kJ mol(-1), DeltaS(double dagger)(b) = -106 +/- 16 J K(-1) mol(-1), DeltaH(double dagger)(c) = 80.5 +/- 7.5 kJ mol(-1), DeltaS(double dagger)(c) = 84 +/- 7 kJ mol(-1). The experimental rate and activation parameters suggest that this redox process follows an outer-sphere electron transfer mechanism. GSNO is relatively stable in the dark, aqueous medium and even in the presence of trace quantities of Cu(2+). Induced catalytic decomposition of GSNO only becomes significant above ca. 10 microM Cu(2+), but after this it shows linear dependency. To nullify any catalysis by Cu(2+) or any other transition metal ions, EDTA was added to all experimental reactions except those where catalysis by Cu(2+) was studied.     

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.     

S-Nitrosoglutathione in Rat Cerebellum: Identification and Quantification by Liquid Chromatography-Mass Spectrometry

Abstract: Given the extreme lability and the facile inactivation of the messenger nitric oxide (NO) by many reactive biochemical species, it has been suggested that some intermediate compounds, for example, S -nitrosothiols, may act to stabilize NO and at the same time to preserve its biological activity. To test this hypothesis, we investigated if the S -nitrosothiol of glutathione, which is the predominant low molecular weight thiol in CNS, is present in the rat brain. The HPLC analysis of cerebellar extract from [³⁵S]cysteine-prelabeled slices suggested that S -nitrosoglutathione (GSNO) was indeed present in rat brain. To detect endogenous GSNO, a methodology based on liquid chromatography-mass spectrometry was developed. Besides an unequivocal identification of the endogenous GSNO, this method also permitted its precise quantification using ¹⁵N-labeled GSNO ([¹⁵N]-GSNO) as internal standard. GSNO level in adult cerebellum amounts to 15.4 ± 1.4 pmol/mg of protein. This is the first direct demonstration of the presence of endogenous GSNO in CNS. The packaging of NO in the form of GSNO might serve to facilitate its transport, prolong its life, and target its delivery to specific effectors.     

Effect of nitric oxide donors on oxygen-dependent cytotoxic responses mediated by neutrophils

We analyzed the effect of nitric oxide (NO) on oxygen-dependent cytotoxic responses mediated by neutrophils against unopsonized erythrocytes using three NO donors: S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and sodium nitroprusside (SNP). Neutrophils were treated with these compounds for 1-2 min at 37 degrees C and cytotoxicity was then triggered in the presence of NO donors by precipitating immune complexes, aggregated IgG, the chemotactic peptide FMLP, or opsonized zymosan. GSNO induced, in all cases, a marked increase in cytotoxic responses, while SNAP moderately increased cytotoxicity triggered by immune complexes, aggregated IgG, or Z, opsonized zymosen, without modifying those responses induced by FMLP. By contrast, SNP dramatically suppressed cytotoxicity triggered by all of the stimuli assessed. The enhancing effects mediated by GSNO and SNAP did not depend on the stimulation of guanylyl cyclase and were prevented by the NO scavengers hemoglobin and PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide). The inhibitory activity of SNP, on the other hand, was not prevented by NO scavengers, suggesting that it cannot be ascribed to the release of NO. In another set of experiments, neutrophils were pretreated with GSNO or SNAP for different times. Then cells were washed to remove NO donors from the culture medium, and cytotoxicity was triggered by different stimuli. It was found that neutrophils must be pretreated with NO donors for at least 4 h to increase cytotoxic responses, and pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity. Not only cytotoxic responses, but also the production of O2- and H2O2, and the release of myeloperoxidase were increased under these conditions.     

RuII(NO+)]3+-core complexes with 4-methyl-pyrimidine and ethyl-isonicotinate: synthesis, X-ray structure, spectroscopy, and computational and NO-release studies upon UVA irradiation

The reaction of RuCl(3)(NO).H(2)O with 4-methylpyrimidine (MePYM) and ethylisonicotinate (EINT), in absolute ethanol at 40-55 degrees C afforded crystalline trans-[RuCl(3)(NO)L(2)] complexes. Structural studies via X-ray diffraction, and spectroscopic methods (NMR, IR, UV-visible (UV-Vis)) revealed that the molecular structures have the two Ls in trans positions (axial) and the chloride anions and the NO(+) cation as equatorial ligands; pyrimidine...pyrimidine pairing pattern via two weak C-H...N interactions occur. The molecular structures for the EINT derivative was inferred from spectroscopy and computations. Under irradiation at 366 nm several solutions of the title compounds deliver NO via first order processes. Visible light (420-700 nm) does not produce significant NO release from CH(2)Cl(2) and CH(3)CN solutions within 24h.     

Polynitrosated polyesters: preparation, characterization, and potential use for topical nitric oxide release

New nitric oxide (NO) donor macromolecules, containing multiple S-nitrosothiol (S-NO) groups covalently attached to the polymer backbone, were prepared through the polycondensation reaction of diols (ethylene glycol and poly(ethylene glycol)) with mercaptosuccinic acid, followed by the S-nitrosation of the SH groups by a gaseous NO/O2 mixture. The polynitrosated polyesters (PNPEs) obtained were characterized by IR spectroscopy and gel permeation chromatography and displayed biological activity as vasodilators, leading to local hyperaemia when applied topically on healthy skin. Kinetic measurements in either dry or aqueous conditions have shown that PNPEs can provide sustained NO release for more than 20 h at physiological temperature. Their increased viscosity at low temperatures greatly reduces the rate of NO release, allowing for their storage for more than 90 days at -20 degrees C without decomposition. These results indicate that PNPEs have potential for topical delivery of NO in biomedical applications.     

Protein S-glutathiolation triggered by decomposed S-nitrosoglutathione

Recombinant human brain calbindin D(28K) (rHCaBP), human Cu,Zn-superoxide dismutase (HCuZnSOD), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) were found to be S-glutathiolated in decomposed S-nitrosoglutathione (GSNO) solutions. Tryptic or Glu-C digestion and MALDI-TOF MS analyses of the digests are consistent with S-thiolation of Cys111 and Cys187 of HCuZnSOD and rHCaBP, respectively, upon exposure to decomposed GSNO. GAPDH activity analysis reveals that S-glutathiolation most likely occurs on the active site Cys149, and the single free Cys34 is assumed to be the site of S-glutathiolation in BSA. The yields of S-glutathiolation of rHCaBP, GAPDH, and BSA were much higher than those of HCuZnSOD. The latter is limited by the accessibility of Cys111 to the glutathiolating reagent in the HCuZnSOD dimer. Unlike decomposed GSNO, fresh GSNO, reduced glutathione (GSH), and oxidized glutathione (GSSG) are not efficient S-glutathiolating agents for the proteins examined here. On the basis of analysis by mass spectrometry and UV-visible absorption, GSNO decomposition in the dark at room temperature yields glutathione disulfide S-oxide [GS(O)SG], glutathione disulfide S-dioxide (GSO(2)SG), and GSSG as products. GS(O)SG is the efficient protein S-glutathiolating agent in GSNO solutions, not GSNO, which does not carry out efficient S-glutathiolation of rHCaBP, HCuZnSOD, or GAPDH in vitro. A hydrolysis pathway yielding GSOH and nitroxyl (HNO/NO(-)) as intermediates is proposed for GSNO decomposition in the dark. This is based on inhibition of GSNO breakdown by dimedone, a reagent specific for sulfenic acids, and on nitroxyl scavenging by metmyoglobin. The results presented here are contrary to numerous reports of protein S-thiolation by low-molecular weight S-nitrosothiols.     

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.     

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.