Decomposition Of S-nitrosothiols Research Articles

Copper-Cystine Biohybrid-Embedded Nanofiber Aerogels Show Antibacterial and Angiogenic Properties.

Copper-cystine-based high aspect ratio structures (CuHARS) possess exceptional physical and chemical properties and exhibit remarkable biodegradability in human physiological conditions. Extensive testing has confirmed the biocompatibility and biodegradability of CuHARS under diverse biological conditions, making them a viable source of essential Cu2+. These ions are vital for catalyzing the production of nitric oxide (NO) from the decomposition of S-nitrosothiols (RSNOs) found in human blood. The ability of CuHARS to act as a Cu2+ donor under specific concentrations has been demonstrated in this study, resulting in the generation of elevated levels of NO. Consequently, this dual function makes CuHARS effective as both a bactericidal agent and a promoter of angiogenesis. In vitro experiments have shown that CuHARS actively promotes the migration and formation of complete lumens by redirecting microvascular endothelial cells. To maximize the benefits of CuHARS, they have been incorporated into biomimetic electrospun poly(ε-caprolactone)/gelatin nanofiber aerogels. Through the regulated release of Cu2+ and NO production, these channeled aerogels not only provide antibacterial support but also promote angiogenesis. Taken together, the inclusion of CuHARS in biomimetic scaffolds could hold great promise in revolutionizing tissue regeneration and wound healing.

Development of Poly(1,8-octanediol-co-citrate-co-ascorbate) Elastomers with Enhanced Ascorbate Performance for Use as a Graft Coating to Prevent Neointimal Hyperplasia.

Small-diameter expanded polytetrafluoroethylene (ePTFE) graft surfaces have poor long-term patency due to limited endothelial cell (EC) coverage and anastomotic intimal hyperplasia. Multifunctional elastomers that coat the ePTFE graft surface to promote EC adhesion while simultaneously inhibiting intimal hyperplasia are highly desirable. Poly(diol-co-citrate) (PDC), a thermoset elastomer, is biodegradable, biocompatible, and mimics vascular mechanical properties. Engineering antioxidant components into PDC polymeric structures improves biocompatibility by attenuating oxidative stress yet is limited by bioavailability. Herein, we develop a new ascorbate protection and deprotection strategy (APDS) for loading bioactive ascorbic acid into the structure of PDC elastomers to improve poly(1,8-octanediol-co-citrate-co-ascorbate) (POCA) prepolymer ascorbate activity. Elastomers cured from APDS POCA prepolymers provide twice the active ascorbate sites on the elastomer surface (35.19 ± 1.64 ng mg-1 cm-2) versus unprotected POCA (Un.POCA, 18.31 ± 0.97 ng mg-1 cm-2). APDS POCA elastomers displayed suitable mechanical properties for vascular graft coating [Young's modulus (2.15-2.61 MPa), elongation (189.5-214.6%) and ultimate tensile strength (2.73-3.61 MPa)], and superior surface antioxidant performance through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging and lipid peroxidation inhibition as compared to poly(1,8-octanediol-co-citrate) (POC) and Un.POCA. Hydrolytic degradation of APDS POCA occurred within 12 weeks under physiological conditions with a mass loss of 25.8 ± 3.4% and the degradation product retaining ascorbate activity. APDS POCA elastomer surfaces supported human aortic endothelial cell proliferation while inhibiting human aortic smooth muscle cell proliferation in vitro. APDS POCA elastomer surfaces displayed superior decomposition of S-nitrosothiols compared to POC and Un.POCA. Taken together, these findings indicate the potential of APDS POCA elastomers to serve as bioactive, therapeutic coatings that enhance the long-term patency of small diameter ePTFE grafts.

The mechanism of nitric oxide release through gold nanoparticles-mediated catalytic decomposition of S-nitrosothiols by density functional theory

First principles calculations were performed to study the mechanism of nitric oxide release through the catalytic decomposition of S-nitrosothiols mediated by gold nanoparticles within the density functional theory. The gold nanoparticles and S-nitrosothiols were modeled by Au38 cluster and SPEN compound, respectively. The results reveals a two-step reaction process. The primary step is SPEN adsorbs on Au38 cluster surface by S atom to form the intermediate. In the next step, NO dissociates from SPEN under the catalysis of Au38 cluster and thereafter a specie of Au-thiolate complex was produced as the product. Furthermore, we found that the overall catalytic reaction is exothermic with a small energy barrier, which confirms the reaction process is kinetically feasible.

Colorimetric copper ion sensing in solution phase and on paper substrate based on catalytic decomposition of S-nitrosothiol

A S-nitrosothiol (RSNO) is used for highly selective and sensitive copper ion sensing for the first time. Cu(II) in the sample is reduced to Cu(I) by a low amount of thiols generated from hydrolysis of RSNO molecules or added thiols such as l-glutathione or l-cysteine. Cu(I) is able to trigger cleavage of the SNO bond, which converts colored RSNOs to colorless products. The dark green S-nitroso-N-acetylpenicillamine is used as an exemplary RSNO in this report. In the spectrophotometric test, the detection limit toward Cu(II) is 0.23 μM without added thiol reductants, and 0.08 and 0.06 μM in the presence of l-glutathione and l-cysteine, respectively. Furthermore, we prepared fully inkjet printed paper-based sensors by deposition of all reaction reagents and buffers on the same piece of cellulose paper. A smartphone equipped with a color analysis app enables quantification of the color change of the paper-based Cu(II) sensors. In this method, a detection limit of 1.2 μM and a linear range of 0–10 μM were obtained. Finally, we successfully applied this instrumentation-free and reagent-free senor for Cu(II) analysis in real drinking water and river water samples.

Flush with a flash: natural three-component antimicrobial combinations based on S-nitrosothiols, controlled superoxide formation and "domino" reactions leading to peroxynitrite.

S-Nitrosothiols are ˙NO releasing agents renowned for vasodilatory and antioxidant properties. O2˙- promotes their decomposition, forming highly aggressive peroxynitrite ions (ONOO-). Since the production of O2˙- can be controlled by enzymes or by visible light, such otherwise harmless components can be turned into effective antimicrobial and nematicidal combinations with numerous potential applications in medicine.

Colorimetric analysis of the decomposition of S-nitrosothiols on paper-based microfluidic devices.

A disposable microfluidic paper-based analytical device (μPAD) was developed to easily analyse different S-nitrosothiols (RSNOs) through colorimetric measurements. RSNOs are carriers of nitric oxide (NO) that play several physiological and physiopathological roles. The quantification of RSNOs relies on their decomposition using several protocols and the colorimetric detection of the final product, NO or nitrite. μPADs were fabricated by wax printing technology in a geometry containing one central zone for the sample inlet and eight circular detection zones interconnected by microfluidic channels for decomposition and posterior detection of decayed products. Different decomposition protocols including mercuric ions and light (UV, visible, and infrared) were tested on μPADs. For this purpose, a 3D printed holder was coupled with μPADs to easily design a simultaneous decomposition procedure using different light sources. The Griess reagent was added to detect NO and nitrite produced by the different decomposition methods. μPADs were then scanned using a flat board scanner and calibration curves based on color intensity were plotted. The limit of detection (LOD) values achieved for nitrite (used as a reference compound) and S-nitrosoglutathione (GSNO) using mercuric decomposition were 3 and 4 μM, respectively. The LOD reported herein for nitrite is considered among the lowest LODs already reported for this compound using μPADs. The results also show that low-molecular-weight RSNO, namely S-nitrosocysteine, decomposes more easily than high-molecular-weight RSNOs with light. As a proof of concept, RSNOs in human plasma were successfully detected on μPADs. For this purpose, a preliminary treatment step was optimized and the presence of high-molecular-weight (HMW) RSNOs was evidenced in the available plasma samples. The concentrations of HMW-RSNOs and nitrite in the various samples ranged from 5 to 16 μM and from 37 to 58 μM, respectively.

Hyperbaric oxygen treatment reduces neutrophil‐endothelial adhesion in chronic wound conditions through S‐nitrosation

Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and tumor necrosis factor-alpha, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced nitrous oxide-related protein modifications. The transnitrosating agent S-nitroso-L-cysteine ethyl ester (600 μM) also reduced neutrophil adhesion to human umbilical vein endothelial cell monolayers, and the iNOS inhibitor 1400 W (10 μM) and HgCl2, which promotes the decomposition of S-nitrosothiols (1 mM), reversed the effect of HBO, suggesting that S-nitrosation may inhibit neutrophil-endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S-nitrosation.

Preparation and characterization of an improved Cu2+–cyclen polyurethane material that catalyzes generation of nitric oxide from S-nitrosothiols

A new, stable and highly efficient Cu(2+)-cyclen-polyurethane material is described and shown to exhibit improved performance compared to prior materials for the catalytic decomposition of S-nitrosothiols to physiologically active nitric oxide.

The hemocompatibility of a nitric oxide generating polymer that catalyzes S-nitrosothiol decomposition in an extracorporeal circulation model

Nitric oxide (NO) generating (NOGen) materials have been shown previously to create localized increases in NO concentration by the catalytic decomposition of blood S-nitrosothiols (RSNO) via copper (Cu)-containing polymer coatings and may improve extracorporeal circulation (ECC) hemocompatibility. In this work, a NOGen polymeric coating composed of a Cu o-nanoparticle (80 nm)-containing hydrophilic polyurethane (SP-60D-60) combined with the intravenous infusion of an RSNO, S- nitroso- N-acetylpenicillamine (SNAP), is evaluated in a 4 h rabbit thrombogenicity model and the anti-thrombotic mechanism is investigated. Polymer films containing 10 wt.% Cu o-nanoparticles coated on the inner walls of ECC circuits are employed concomitantly with systemic SNAP administration (0.1182 μmol/kg/min) to yield significantly reduced ECC thrombus formation compared to polymer control + systemic SNAP or 10 wt.% Cu NOGen + systemic saline after 4 h blood exposure (0.4 ± 0.2 NOGen/SNAP vs 4.9 ± 0.5 control/SNAP or 3.2 ± 0.2 pixels/cm 2 NOGen/saline). Platelet count (3.9 ± 0.7 NOGen/SNAP vs 1.8 ± 0.1 control/SNAP or 3.0 ± 0.2 × 10 8/ml NOGen/saline) and plasma fibrinogen levels were preserved after 4 h blood exposure with the NOGen/SNAP combination vs either the control/SNAP or the NOGen/saline groups. Platelet function as measured by aggregometry (51 ± 9 NOGen/SNAP vs 49 ± 3% NOGen/saline) significantly decreased in both the NOGen/SNAP and NOGen/saline groups while platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry was not decreased after 4 h on ECC to ex vivo collagen stimulation (26 ± 2 NOGen/SNAP vs 29 ± 1 MFI baseline). Western blotting showed that fibrinogen activation as assessed by Aγ dimer expression was reduced after 4 h on ECC with NOGen/SNAP (68 ± 7 vs 83 ± 3% control/SNAP). These results suggest that the NOGen polymer coating combined with SNAP infusion preserves platelets in blood exposure to ECCs by attenuating activated fibrinogen and preventing platelet aggregation. These NO-mediated platelet changes were shown to improve thromboresistance of the NOGen polymer-coated ECCs when adequate levels of RSNOs are present.

S-nitrosothiol-modified dendrimers as nitric oxide delivery vehicles.

The synthesis and characterization of two generation-4 polyamidoamine (PAMAM) dendrimers with S-nitrosothiol exteriors are reported. The hyperbranched macromolecules were modified with either N-acetyl-D, L-penicillamine (NAP) or N-acetyl-L-cysteine (NACys) and analyzed via 1H and 13C NMR, UV absorption spectroscopy, MALDI-TOF mass spectrometry, and size exclusion chromatography. Treatment of the dendritic thiols with nitrite solutions yielded the corresponding S-nitrosothiol nitric oxide (NO) donors (G4-SNAP, G4-NACysNO). Chemiluminescent NO detection demonstrated that the dendrimers were capable of storing approximately 2 micromol NO x mg (-1) when exposed to triggers of S-nitrosothiol decomposition (e.g., light and copper). The kinetics of NO release were found to be highly dependent on the structure of the nitrosothiol (i.e., tertiary vs primary) and exhibited similar NO release characteristics to classical small molecule nitrosothiols reported in the literature. As a demonstration of utility, the ability of G4-SNAP to inhibit thrombin-mediated platelet aggregation was assayed. At equivalent nitrosothiol concentrations (25 microM), the G4-SNAP dendrimer resulted in a 62% inhibition of platelet aggregation, compared to only 17% for the small molecule NO donor. The multivalent NO storage, the dendritic effects exerted on nitrosothiol stability and reactivity, and the utility of dendrimers as drug delivery vehicles highlight the potential of these constructs as clinically useful S-nitrosothiol-based therapeutics.

Effect of pH and Metal Ions on the Decomposition Rate of S-nitrosocysteine

S-nitrosothiols (RSNOs) have many biological functions including platelet deactivation, immunosupression, neurotransmission, and host defense. Most of the functions are attributed to nitric oxide (NO) release during S-nitrosothiol decomposition. As the simplest biologically occurring S-nitrosothiol, S-nitrosocysteine (CySNO) has been widely used as an NO donor and has also been incorporated into biomedical polymers. Knowledge of the CySNO decomposition rate is important for assessing the impact of CySNO on various bioengineering applications or biological systems. In this work, spectrophotometer measurements of CySNO decomposition in the presence of metal ions showed that the decomposition rate is highly susceptible to the pH. The maximum decomposition occurs near physiological pH (near 7.4) while in the acidic (pH < 6) and alkaline (pH > 9) condition CySNO is very stable. This demonstrates that blood provides an optimized environment for the decomposition of CySNO leading to the release of NO. The CySNO decomposition rate can also be affected by buffers with different purity levels in the presence and absence of metal ion chelators-although all buffers show the same pH phenomenon of maximizing near physiological pH. An equilibrium model of metal ions as a function of pH provides a plausible explanation for the pH dependence on the experimental decomposition rate.

Effects of S-Nitrosation on Hemoglobin-Induced Microvascular Damage

Blood substitutes, such as diaspirin cross-linked hemoglobin (Hb), cause microvascular leakiness to macromolecules. Because of the potentially stabilizing effects of nitric acid (NO) on endothelium, experiments were performed to determine whether S-nitrosohemoglobin (SNO-Hb), a potential NO-donor Hb-based blood substitute, would not cause microvascular damage. Release of NO, or its metabolites, from the SNO-Hb was facilitated by addition of glutathione, which aids in the decomposition of S-nitrosothiols. In anesthetized rats, the mesenteric microvasculature was perfused with SNO-Hb with glutathione (six rats), SNO-Hb alone (six rats), or saline (eight rats) for 10 min, followed by fluorescein isothiocyanate (FITC)-albumin for 1 min, and finally fixed for epifluorescence microscopic examination. When comparing the SNO-Hb group with saline, both the numbers and areas of leaks were significantly increased [0.019 +/- 0.003 (SEM) microm vs. 0.0030 +/- 0.0004 and 7.36 +/- 1.50 vs. 0.156 +/- 0.035 (p < 0.005)]. With the addition of glutathione, leakage was still high (0.005 +/- 0.00005 microm and 5.086 +/- 0.064 microm) but decreased compared with SNO-Hb alone (p < 0.005). In conclusion, NO, or a related vasodilator, when released from SNO-Hb, significantly reduces but does not eliminate microvascular damage. Further improvements may result by S-nitrosating a more stable form of modified hemoglobin.

Metalloprotein-dependent decomposition of S-nitrosothiols: Studies on the stabilization and measurement of S-nitrosothiols in tissues

The stabilization of S-nitrosothiols is critical for the development of assays to measure their concentration in tissues. Low-molecular-weight S-nitrosothiols are unstable in tissue homogenates, even in the presence of thiol blockers or metal-ion chelators. The aim of this study was to try and stabilize low-molecular-weight S-nitrosothiols in tissue and gain insight into the mechanisms leading to their decomposition. Rat tissues (liver, kidney, heart, and brain) were perfused and homogenized in the presence of a thiol-blocking agent ( N-ethylmaleimide) and a metal-ion chelator (DTPA). Incubation of liver homogenate with low-molecular-weight S-nitrosothiols ( l-CysNO, d-CysNO, and GSNO) resulted in their rapid decomposition in a temperature-dependent manner as measured by chemiluminescence. The decomposition of l-CysNO requires a cytoplasmic factor, with activity greatest in liver > kidney > heart > brain > plasma, and is inhibitable by enzymatic proteolysis or heating to 80°C, suggesting that a protein catalyzes the decomposition of S-nitrosothiols. The ability of liver homogenate to catalyze the decomposition of l-CysNO is up-regulated during endotoxemia and is dependent on oxygen, with the major product being nitrate. Multiple agents were tested for their ability to block the decomposition of l-CysNO without success, with the exception of potassium ferricyanide, which completely blocked CysNO decomposition in liver homogenates. This suggests that a ferrous protein (or group of ferrous proteins) may be involved. We also show that homogenization of tissues in ferricyanide-containing buffers in the presence of N-ethylmaleimide and DTPA can stabilize both low- and high-molecular-weight S-nitrosothiols in tissues before the measurement of their concentration.

Discriminating formation of HNO from other reactive nitrogen oxide species

Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GS(O)NH 2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GS(O)NH 2 was not observed with other nitrogen oxides (NO, N 2O 3, NO 2, or ONOO −), indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GS(O)NH 2. Detection of GS(O)NH 2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and N G-hydroxy- l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.

Nitrosonium-Catalyzed Decomposition of S-Nitrosothiols in Solution: A Theoretical and Experimental Study

The decomposition of S-nitrosothiols (RSNO) in solution under oxidative conditions is significantly faster than can be accounted for by homolysis of the S-N bond. Here we propose a cationic chain mechanism in which nitrosation of nitrosothiol produces a nitrosated cation that, in turn, reacts with a second nitrosothiol to produce nitrosated disulfide and the NO dimer. The nitrosated disulfide acts as a source of nitrosonium for nitrosothiol nitrosation, completing the catalytic cycle. The mechanism accounts for several unexplained facets of nitrosothiol chemistry in solution, including the observation that the decomposition of an RSNO is accelerated by O(2), mixtures of O(2) and NO, and other oxidants, that decomposition is inhibited by thiols and other antioxidants, that decomposition is dependent on sulfur substitution, and that decomposition often shows nonintegral kinetic orders.

Direct Detection of S-Nitrosothiols Using Planar Amperometric Nitric Oxide Sensor Modified with Polymeric Films Containing Catalytic Copper Species

The direct amperometric detection of S-nitrosothiol species (RSNOs) is realized by modifying a previously reported amperometric nitric oxide gas sensor with thin hydrophilic polyurethane films containing catalytic Cu(II)/(I) sites. Catalytic Cu(II)/(I)-mediated decomposition of S-nitrosothiols generates NO(g) in the thin polymeric film at the distal tip of the NO sensor. Three different species are examined to create the catalytic layer: (1) a lipophilic Cu(II)-ligand complex; (2) Cu(II)-phosphate salt; and (3) small (3-microm) metallic Cu(0) particles. All three catalytic layers yield reversible amperometric response in proportion to the concentration of S-nitrosothiols (e.g., nitrosocysteine, nitrosoglutathione, S-nitroso-N-acetylcysteine, S-nitrosoalbumin) present in the aqueous test solution. Sensitivity toward the different RSNO species is dependent on the respective catalytic rates of decomposition of the RSNO species by reactive Cu(I), accessibility of the species into the polyurethane layer containing the catalyst, the level of reducing agents (ascorbate) used in solution to help generate reactive Cu(I) species, and the concentration of metal ion complexing agents present in the test solution (e.g., EDTA). Under optimized conditions, all RSNO species can be detected at < or =1 microM levels, with sensor lifetimes of at least 10 days for the sensors based on Cu(II)-phosphate and Cu0 particles. It is further shown that the new RSNO sensors can be used to assess the "NO-generating" ability of fresh blood samples by effectively detecting the total level of reactive RSNO species present in such samples.

The mechanisms of S-nitrosothiol decomposition catalyzed by iron

The mechanisms of S-nitrosothiol transformation into paramagnetic dinitrosyl iron complexes (DNICs) with thiol- or non-thiol ligands or mononitrosyl iron complex (MNICs) with N-methyl- d-glucamine dithiocarbamate catalyzed by iron(II) ions under anaerobic conditions were studied by monitoring EPR or optical features of the complexes and S-nitrosothiols. The kinetic investigations demonstrated the appearance of short-living paramagnetic mononitrosyl–iron complex with l-cysteine prior to the formation of stable dinitrosyl–iron complex with cysteine in the solution of iron(II)–citrate complex (50–100 μM), S-nitrosocysteine (400 μM), and l-cysteine (20 mM) in 100 mM Hepes buffer (pH 7.4). The addition of deoxyhemoglobin (100 μM) did not influence the process, which points to a direct interaction between S-nitrosocysteine and iron(II) ions to yield DNIC. The reaction of DNIC–cysteine formation is first- and second-order in iron and S-nitrosocysteine, respectively. The third-order rate constant is (1.0 ± 0.2) × 10 5 M −2 s −1 (estimated from EPR results) or (2.0 ± 0.1) × 10 4 M −2 s −1 (estimated by optical method). A similar process of DNIC–cysteine formation was observed in a solution of iron(II)–citrate complex, l-cysteine, and NO-proline (200 μM) as a NO donor. The appearance of a less stable dinitrosyl–iron complex with phosphate was detected when solutions of iron(II)–citrate containing 100 mM phosphate buffer (pH 7.4) were mixed with S-nitrosocysteine or NO-proline. The rapid formation of DNIC with phosphate was followed by its decay. When the concentration of l-cysteine in solutions was reduced from 20 to 1 mM, the life-time of the DNIC–cysteine diminished notably; this was caused by consumption of l-cysteine in the process of DNIC–cysteine formation from S-nitrosocysteine and iron. Thus, l-cysteine is consumed. Formation of DNIC with glutathione was also observed in a solution of glutathione (20 mM), S-nitrosoglutathione (400 μM), and iron(II) complex (800 μM) in 100 mM Hepes buffer (pH 7.4), but the rate of formation was about 10 times slower than the formation of the DNIC–cysteine. The rate of MNIC–MGD formation from iron(II)–MGD complexes and S-nitrosocysteine was first-order in both reactants. The second-order rate constant for this reaction, estimated from EPR measurements, was 30 ± 5 M −1 s −1. Rate constants of MNIC–MGD formation from iron(II)–MGD and the more stable S-nitrosoglutathione and S-nitroso- d, l-penicillamine were equal to 3.0 ± 0.3 and 0.3 ± 0.05 M −1 s −1, respectively. Thus, the concerted mechanism of DNIC and MNIC formation from S-nitrosothiols and iron(II) ions can be suggested to be predominant.

30] Quantitation of S-nitrosothiols in cells and biological fluids

The chapter presents a study related to the quantitation of s-nitrosothiols (RSNO) in cells and biological fluids. RSNO are a class of chemical compounds that are widespread in vivo, thus providing a reliable source of nitric oxide (NO). Major RSNO compounds in blood differ with respect to their circulating concentrations and half-life. Plasma concentrations of s-nitrosoalbumin exceed that of low molecular weight nitrosothiols by at least an order of magnitude. Reported levels of s-nitrosothiols in biological fluids and tissues vary from low nanomolor to micromolar concentrations. The chapter discusses the formation and decomposition of s-nitrosothiols, stating that several biochemical pathways may yield s-nitrosothiols. The chapter mentions the major pathways of s-nitrosothiol decomposition that are important in the quantification of these compounds based on NO release. The techniques for the assessment of s-nitrosothiols are described. DAF-2 has been introduced as a highly sensitive and specific fluorogenic scavenger of NO in the presence of dioxygen, yielding the fluorescent product DAF-2 triazole (DAF-2T) with a sensitivity of 5 nM for NO. The chapter discusses analysis of s-nitrosothiols in plasma samples using 4,5-diaminofluorescein, analysis of s-nitrosothiols in pregnancy plasma fractions, and S-nitrosylation of metallothioneins.

The reaction mechanism of nitrosothiols with copper(I).

Copper and other transition metal ions and their complexes are catalysts for the decomposition of nitrosothiols. In this way they catalyze the biological functions of nitrosothiols. The kinetics and mechanism of the reaction of two nitrosothiols, S-nitrosothiolactic acid and S-nitrosoglutathione (GSNO), with copper(I) are reported. The kinetics of the reaction of Cu(MeCN)+(n) (n=0-3) with the nitrosothiols were studied. The results indicate that Cu+(aq) is the active species in the GSNO system, with k(Cu+(aq)+GSNO)=(9.4 +/- 2.0) x 10(7) dm3 mol(-1) s(-1). The results also indicate that the Cu(MeCN)+(n) (n=0-3) complexes react with S-nitrosothiolactic acid. Transient species are formed in these processes. The results suggest that these species contain copper(I) and thiol. The results shed light on the catalytic role of copper complexes in the decomposition of S-nitrosothiols.

A redox-based mechanism for nitric oxide-induced inhibition of DNA synthesis in human vascular smooth muscle cells.

1. The current study explored potential redox mechanisms of nitric oxide (NO)-induced inhibition of DNA synthesis in cultured human and rat aortic smooth muscle cells. 2. Exposure to S-nitrosothiols, DETA-NONOate and NO itself inhibited ongoing DNA synthesis and S phase progression in a concentration-dependent manner, as measured by thymidine incorporation and flow cytometry. Inhibition by NO donors occurred by release of NO, as detected by chemiluminescence and judged by the effects of NO scavengers, haemoglobin and cPTIO. 3. Co-incubation with redox compounds, N-acetyl-L-cysteine, glutathione and L-ascorbic acid prevented NO inhibition of DNA synthesis. These observations suggest that redox agents may alternatively attenuate NO bioactivity extracellularly, interfere with intracellular actions of NO on the DNA synthesis machinery or restore DNA synthesis after established inhibition by NO. 4. Recovery of DNA synthesis after inhibition by NO was similar with and without redox agents suggesting that augmented restoration of DNA synthesis is an unlikely mechanism to explain redox regulation. 5. Study of extracellula interactions revealed that all redox agents potentiated S-nitrosothiol decomposition and NO release. 6. Examination of intracellular NO bioactivity showed that as opposed to attenuation of NO inhibition of DNA synthesis by redox agents, there was no inhibition (potentiation in the presence of ascorbic acid) of soluble guanylate cyclase (sGC) activation judged by cyclic GMP accumulation in rat cells. 7. These data provide evidence that NO-induced inhibition of ongoing DNA synthesis is sensitive to redox environment. Redox processes might protect the DNA synthesis machinery from inhibition by NO, in the setting of augmented liberation of biologically active NO from NO donors.