Decomposition Of S-nitrosothiols Research Articles

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.

Superoxide-mediated Decomposition of BiologicalS-Nitrosothiols

Incubation of S-nitrosocysteine or S-nitrosoglutathione (5-100 M) in the presence of a generator of superoxide (xanthine/xanthine oxidase) resulted in a time-dependent decomposition of S-nitrosothiols and accumulation of nitrite/nitrate in reaction mixtures. Quantitatively, the amounts of nitrite/nitrate represented >90% of nitrosonium equivalent of S-nitrosothiols degraded during the incubation. The reaction rates were unaffected by the presence catalase (1 unit/ml). Kinetic analysis showed that the degradation of S-nitrosothiols in the presence of superoxide proceeded at second order rate constants of 76,900 M-1 s-1 (S-nitrosocysteine) and 12,800 M-1 s-1 (S-nitrosoglutathione), respectively, with a stoichiometric ratio of 1 mol of S-nitrosothiol per 2 mol of superoxide. The findings provide the evidence for the involvement of superoxide in the metabolism of S-nitrosothiols. Furthermore, substantially slower reaction rates of superoxide with S-nitrosothiols relative to the reaction rate with NO are consistent with the contention that the transient formation of S-nitrosothiols in biological systems may protect NO from its rapid destruction by superoxide, thus enabling these compounds to serve as carriers or buffers of NO.

The Reaction of S-Nitrosoglutathione with Superoxide

The nitric oxide (NO)-dependent S-nitrosation of thiols to generate S-nitrosothiols has been proposed as an important pathway for the metabolism of NO in vivo. Although it has been suggested that these S-nitrosated compounds are resistant to decomposition by reactive oxygen metabolites (ROMs), very little information is available regarding the interaction between S-nitrosothiols and ROMs. We found that S-nitrosoglutathione (GSNO) rapidly reacted with O−2to generate glutathione disulfide and equimolar quantities of nitrite and nitrate. The reaction was second order with respect of GSNO and first order with respect of O−2with a rate equation of −d[GSNO]/dt = 2k3[GSNO]2[O−2], where k3= 3 − 6×108M−2s−1. In addition, the reaction of GSNO with O−2generated a strong oxidant as an intermediate capable of oxidizing dihydrorhodamine in the absence of the apparent generation of NO. We conclude that O−2may act as a physiological modulator of S-nitrosation reactions by directly promoting the decomposition of S-nitrosothiols.

Decomposition of S-nitrosothiols: the effects of added thiols

The Cu+ (added as Cu2+) mediated decomposition of the five S-nitrosothiols, derived from penicillamine, cysteamine, thiomalic acid, N-acetylpenicillamine and cysteine have been examined kinetically in the presence of varying amounts of the corresponding thiols. Large differences in behaviour were found. In some cases, reactions were catalysed by added thiols, whereas in others, stability was conferred, often resulting in the appearance of quite large induction effects. The results are explained in terms of the dual function of the thiol (as thiolate), (a) as a reducing agent generating Cu+, and (b) as a complexing agent for Cu2+, when it is then less available for reduction. The balance of these two effects depends on the structure and concentration of the added thiol. The findings were supported by examining the two effects separately, using ascorbate as a reducing agent, and ethylenediaminetetraacetic acid as the complexing agent. For penicillamine, cysteine and thiomalic acid, the Cu2+ complexes were identified from their UV spectra, and their decomposition was followed kinetically.

Decomposition of S-nitrosothiols by mercury(II) and silver salts

Rate measurements have been obtained for the reaction of a number of S-nitrosothiols (RSNO) in water with mercury(II) salts. Reaction is first order in both reactants, and the products are nitrous acid and the corresponding thiol–Hg2+ complex. Decomposition is ca. 103 faster when mercury(II) nitrate, rather than mercury(II) chloride is used as the source of mercury(II). This is consistent with the fact that whereas the nitrate salt is fully ionised in water, the chloride exists almost entirely in the undissociated molecular form. There is very little variation in the rate constant with RSNO structure. Silver ion reacts similarly, although less rapidly, and the kinetic order with respect to [Ag+] is ca. 2. The results are discussed in terms of a mechanism involving rate-limiting attack by water, at the nitrogen atom in the mercury(II) (or silver) ion complex of the S-nitrosothiol, and are contrasted with the corresponding reactions of S-nitrosothiols with Cu+, which generate nitric oxide.

Seleno Compounds and Glutathione Peroxidase Catalyzed Decomposition ofS-Nitrosothiols

Seleno compounds such as selenocystamine and seleno-D, L-cystine were found to catalyze the decomposition ofS-nitrosothiols (e.g.S-nitroso-glutathione andS-nitroso-N-acetyl-D, L-penicillamine) in the presence of different thiols (e.g. glutathione,N-acetyl-D-penicillamine and 2-mercaptoethanol), and liberate nitric oxide. It was also found that glutathione peroxidase itself can catalyze the decomposition ofS-nitroso-glutathione without the presence of any thiol or H2O2.

Mechanism of Nitric Oxide Release from S-Nitrosothiols

S-Nitrosothiols have many biological activities and have been suggested to be intermediates in signal transduction. The mechanism and products of S-nitrosothiol decomposition are of great significance to the understanding of nitric oxide (.NO) biochemistry. S-Nitrosothiols are stable compounds at 37 degrees C and pH 7.4 in the presence of transition metal ion chelators. The presence of trace transition metal ions (present in all buffers) stimulates the catalytic breakdown of S-nitrosothiols to .NO and disulfide. Thiyl radicals are not formed as intermediates in this process. Photolysis of S-nitrosothiols results in the formation of .NO and disulfide via the intermediacy of thiyl radicals. Reduced metal ion (e.g. Cu+) decomposes S-nitrosothiols more rapidly than oxidized metal ion (e.g. Cu2+) indicating that reducing agents such as glutathione and ascorbate can stimulate decomposition of S-nitrosothiol by chemical reduction of contaminating transition metal ions. Transnitrosation can also stimulate S-nitrosothiol decomposition if the product S-nitrosothiol is more susceptible to transition metal ion-catalyzed decomposition than the parent S-nitrosothiol. Equilibrium constants for the transnitrosation reactions of reduced glutathione, either with S-nitroso-N-acetyl-dl-penicillamine or with S-nitroso-L-cysteine indicate that S-nitrosoglutathione formation is favored. The biological relevance of S-nitrosothiol decomposition is discussed.

Identification of Cu+ as the effective reagent in nitric oxide formation from S-nitrosothiols (RSNO)

Decomposition of S-nitrosothiols (RSNO) in aqueous solution at pH 7.4 is brought about by copper ions, either present as an impurity or specifically added. The primary products are nitric oxide and the disulfide. In the presence of the specific Cu+ chelator, neocuproine, reaction is progressively inhibited as the [neocuproine] is increased, the reaction eventually stopping completely. The characteristic UV–VIS spectrum of the Cu+ adduct can be obtained from the reaction solutions. This shows clearly that Cu+ and not Cu2+ is the effective catalyst. Two limiting kinetic conditions can be identified for a range of S-nitrosothiols at specific copper ion concentrations (a) a first-order dependence and (b) a zero-order dependence upon [RSNO]. Normally both situations also have a short induction period. This induction period can be removed by the addition of the corresponding thiol RSH. A mechanism is proposed in which Cu+ is formed by reduction of Cu2+ by thiolate anion via an intermediate, possibly RSCu+. Loss of nitric oxide from RSNO is then brought about by Cu+, probably via another intermediate in which Cu+ is bound to the nitrogen atom of the NO group and another electron-rich atom (such as nitrogen from an amino group, or oxygen from a carboxylate group) involving a six-membered ring. As well as NO this produces both RS– and Cu2+ which then are part of the cycle regenerating Cu+. Thiolate ion is oxidised to RS˙ which dimerizes to give the disulfide. Depending on the structure (and hence reactivity) of RSNO either Cu+ formation or its reaction with RSNO can be rate-limiting. Computer modelling of the reaction scheme allows the generation of absorbance time plots of the same forms as those generated experimentally, i.e. first- or zero-order, both with or without induction periods. We suggest that the thiolate ion necessary to bring about Cu2+ reduction is either present as a thiol impurity or is generated in small quantities by partial hydrolysis of the nitrosothiol, which results in an induction period. Addition of small quantities of thiol removes the induction period and leads to catalysis but larger quantities bring about a rate reduction by, it is suggested, complexation of the Cu2+. For two very unreactive substrates, S-nitrosoglutathione and S-nitroso-N-acetylcysteine very large induction periods were observed, typically three hours. This results, we suggest, from competitive re-oxidation of Cu+ to Cu2+ by the dissolved oxygen. Experiments carried out anaerobically confirm this, since there is then no induction period. Addition of hydrogen peroxide extends the induction period ever further. The results are discussed in terms of the biological properties of S-nitrosothiols which are related to nitric oxide release.

Photosensitized decomposition of S-nitrosothiols and 2-methyl-2-nitrosopropane Possible use for site-directed nitric oxide production

Irradiation of S-nitrosoglutathione (GSNO) with light ( λ = 550 nm) resulted in the homolytic decomposition of GSNO to generate glutathionyl radical (GS ·) and nitric oxide (·NO), which were monitored by ESR spectrometry. Inclusion of Rose Bengal (RB) resulted in a 9-fold increase in the quantum yield for ·NO production and also an increase in the rate of thiyl radical formation. The bimolecular rate constant for the interaction of triplet RB with GSNO has been estimated to be approximately 1.2 × 10 9 M −1s −1 by competition with oxygen. Hematoporphyrin (HP) also enhanced the rate of ·NO and tert-butyl radical. Aluminum 2-Methyl-2-nitrosopropane (MNP) decomposed on irradiation ( λ = 660 nm) to form ·NO and tert-butyl radical. Aluminum phthalocyanine tetrasulphonate enhanced the rate of decomposition of MNP by 10-fold. These studies show that photosensitizers enhance the release of ·NO from donor compounds.

Strand Scission in DNA Induced by S-Nitrosothiol with Hydrogen Peroxide

Strand breaks can be produced in pBluescript plasmid DNA by S-nitrosothiol and H 2O 2 in the presence of a metal chelator, diethylenetriaminepentaacetic acid. S-Nitrosothiols with a wide range of stability were found to be active as a DNA cleaver in the presence of H 2O 2. Strand breaks were temperature dependent, occurring more rapidly at higher temperature. Sodium azide and mannitol inhibited S-nitrosothiol/H 2O 2-induced strand breaks in DNA. Catalase inhibited damage to DNA in a concentration-dependent manner whereas both superoxide dismutase and a yeast protector protein did not prevent damage to DNA. It is suggested that observed strand breaks in DNA are mediated by hydroxyl radicals arising from the reaction between H 2O 2 and thiyl radicals generated by homolytic decomposition of S-nitrosothiol.

The reactions of 3,5-dibromo-4-nitrosobenzenesulfonate and its biological applications.

Recently, 5,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) has been applied to detect biological free radicals. However, DBNBS has various non-specific reactions which lead to preplexing results. Thus, we investigated some basic reactions of DBNBS in combination of other nitroso spin traps to assign DBNBS spin adducts derived from human platelets which presumably related to the endothelium-derived relaxing factor (EDRF). The collagen activated platelets yielded four spin adducts (ST, LT, SS, and LS) in the presence of DBNBS (40 mM). The broad triplet due to ST was also observed by bubbling NO gas into a DBNBS solution. To identify ST, nitrosobenzene (NB) in dry dioxane was mixed with NO-saturated dioxane. The NB-NO spin adduct was observed but decomposed into diphenyl aminoxyl by the addition of H2O indicating that the primary adduct formed by the reaction of NO and DBNBS is unstable and turns into a dimerization product. Although ST could be eliminated by the inhibitor of EDRF, ST was shown to be produced by non-specific reactions. Another triplet was assigned to an S-centered radical because thiyl radicals which were generated from either the decomposition of S-nitrosothiol, or glutathione oxidation exhibited almost identical triplet signals. The other two sextets were assigned to C-centered radical adducts. Thus, DBNBS detected NQ-related, S-centered, and two C-centered radicals derived from human platelets. Special cautions are necessary for the identification of DBNBS spin adducts in a biological system to exclude artifactual radicals.