RSNOs

Some acyclic sulfur-nitrogen compounds also exhibit intense colours. For example, S-nitrosothiols RSNO are either green, red or pink (Section 9.7). Their UV-visible spectra show an intense band in the 330-350 nm region (no it ) and a weaker band in the visible region at 550-600 nm... 

In solution the cis and trans isomers may co-exist, as demonstrated by N NMR and UV-visible spectra. The N NMR chemical shift of the trans isomer is shifted ca. 60 ppm downfield relative to the cis isomer." The visible absorption band of S-nitrosothiols corresponds to a weak n K transition in the 520-590 nm region. The absorption maxima of trans conformers are red-shifted by ca. 30 nm relative to those of the cis isomer. Two absorptions are observed in the 520-590 nm region in the experimental spectra of RSNO derivatives." ... 

Methylanaline could be transnitrosated with nitrite and S-nitrosocysteine and also by a simulated protein bound nitrite. In the latter case, an important factor was the local concentration of nitrosothiol groups on the matrix. The effects of S-nitrosocysteine as an inhibitor of lipid oxidation, as a color developer, and as an anticlostridial, have been reported recently in a turkey product (31). The Molar concentration of RSNO equating to 25 ppm nitrite gave similar results for color and inhibition of lipid oxidation but had less anti-clostridial activity. Transnitrosation between RSNO and heme protein was demonstrated. 

S-nitrosothiols (RSNO) have emerged as important species in the storage and transport of nitric oxide. As NO donors these S-N compounds have potential medical applications in the treatment of blood circulation problems. 

S -nitrosothiols, several of which occur naturally, e.g., iS -nitrosocysteine and S-nitrosoglutathione, have an important role in NO transport and regulation in biological systems. Potential applications of RSNO compounds include their use as vasodilators in the treatment of angina and in the search for a cure for male impotence.11 The most convenient route to S-nitrosothiol formation is the nitrosation of thiols. 

The ability of S -nitrosothiols to mimic many of the biological properties of NO itself may emanate from in vivo decomposition to generate NO. This decomposition is catalysed by Cu2+,n and may be important in the development of thrombo-resistant devices used in kidney dialysis or coronary by-pass surgery.196 It is also possible that direct transfer of NO from RSNO occurs in biological systems.197... 

Reaction (52) occurs at the gradient interface of the bolus addition until local Hb(02) concentrations have been reduced, at which point additional NO reduces the iron(III) to iron(II) which can further react with free NO to form Hb(NO). The validity of this mechanism was verified by the observation that addition of CN- ion, which binds irreversibly to metHb to form metHb(CN), significantly attenuated the formation of Hb(NO) in both cell-free Hb and RBC. Mathematical models used to simulate bolus addition of NO to cell-free Hb and RBC were compatible with the experimental results (147). In the above experiments, SNO-Hb was a minor reaction product and was formed even in the presence of 10 mM CN, suggesting that RSNO formation does not occur as a result of (hydrolyzed) NO+ formation during metHb reduction. However, formation of SNO-Hb was not detectable when NO was added as a bolus injection to RBC or through thermal decomposition of DEA/NO in cell free Hb (DEA/NO = 2-(A/ A/ diethylamino)diazenolate). SNO-Hb was observed... 

Recently, it has been found that NO donors inhibit HIV-1 replication in acutely infected human peripheral blood mononuclear cells (PBMCs), and have an additive inhibitory effect on HIV-1 replication in combination with 3 -azido-3 -deoxythymisylate (AZT) [139, 140]. S-nitrosothiols (RSNOs) inhibit HIV-1 replication at a step in the viral replicative cycle after reverse transcription, but before or during viral protein expression through a cGMP-independent mechanism. In the latently infected U1 cell line, NO donors and intracellular NO production stimulate HIV-1 reactivation. These studies suggest that NO both inhibits HIV-1 replication in acutely infected cells and stimulates HIV-1 reactivation in chronically infected cells. Thus, NO donors may be useful in the treatment of HIV-1 disease by inhibiting acute infection, or reactivating a latent virus. 

This chapter summarizes the current state of knowledge on the role of S-nitroso-thiols in mammalian systems under the following headings Structure and cellular reactivity of RSNOs formation of RSNOs in the biological milieu and physiological role of RSNOs. 

The principal in vitro route to the formation of RSNOs is through the reaction of nitrous acid or protonated nitrite (HN02) with thiols (Eq. (1)). Since the pKa of HN02 is 3.37, this reaction is unlikely to occur in cells and tissues where the pH is maintained at 7.4. 

However, this reaction can and has led to errors in the measurement of RSNOs in biological fluids when the samples are improperly buffered to avoid the HN02 -route to S-nitrosation (Tsikas, 2003). 

Enzymatic conversion of nitrite to RSNOs has been reported. Glutathione-S-transferase catalyzed generation of RSNOs from organic nitrites was initially demonstrated in rat liver microsomes (Ji et ah, 1996). Subsequently, this activity has been identified in the rat heart and lung GSTs (Akerboom et al., 1997). 

NO reacts directly with thiols in vitro to yield RSNOs only in the absence of oxygen (Gow et al, 1997). Therefore, this reaction, Scheme 4.1, is unlikely to occur in biological systems. 

N203-dependent S-nitrosation has been demonstrated in the case of semm albumin (Nedospasov et al, 2000 Rafikova et al., 2002), in membranes (Liu et al., 1998) and in the protein-disulfide isomerase dependent transfer of NO-equivalents from extracellular RSNOs to the cytosol (Ramachandran et al., 2001). 

Other postulated routes (Jourd heuil et al., 2003) to RSNO formation include the reaction between NO and 02 to yield N02 via a second-order reaction. NO and thiolate anion, RS, react giving rise to thiyl radical, (RS ) [e]. RS then reacts with NO to yield RSNO [f]. The reaction between RS and RS- can also be the source of non-enzymatic generation of superoxide anion (02 ) [g], [h]. 02 reacts with NO to produce peroxynitrite (ONOO ) [i] (Szabo, 2003). Thiols react with ONOOH to form RSNOs [k] (van der Vliet et al.,1998). 

Intracellular and extracellular RSNO metabolism has been studied in LPS activated macrophages (Zhang and Hogg, 2003). This study showed that 0.02% of the NO produced in response to LPS, (detected as N02 ) was converted to cytosolic RSNOs and that all of the RSNOs detected were in the large molecular weight (>3K) protein fraction and were very stable to denitrosation (t1(f2 3h). These authors also showed that the molecular specie(s) responsible for S-nitrosation is freely diffusible and has to be transported to the cell surface before internal S-nitrosation could take place. 

In a recent comprehensive study, Feelish and coworkers (Bryan et al, 2004) determined the concentrations of RSNOs, N-nitrosamines (RNNOs) N02 , N03, heme nitrosyl (NO-heme) in plasma, RBCs, as well as brain, heart, liver, kidney, lung, and aortic tissues of rats. Furthermore, the levels of these analytes were monitored under conditions of eNOS inhibition, hypoxia and redox state. The emerging picture was that RSNOs were detected in all of the tissues examined in comparable levels to NO-... 

The multi-copper carrying enzyme ceruloplasmin (CP), found in large amounts in liver and nervous tissues, has been shown to convert NO to RSNOs. The proposed mechanism involves the binding of NO to the CP type I Cu-sites. The NO is then oxidized to NO+ and transferred to RS giving rise to RSNO (Innoue et al., 1999). 

Hbp C93 S-nitrosation could also be accomplished by exposure to RSNOs (GSNO or CysNO). The rates RSNO-dependent Hb-S-nitrosation was 10-fold larger in oxy-Hb than in deoxy-Hb. Conversely, the rate of spontaneous decay of deoxy-Hb-SNO was -20-fold larger than oxy-Hb-SNO. An explanation for this differential reactivity was presented in a subsequent study (Stamler et al, 1997) where protein modeling data based on the X-ray structures of Hb in T and R states indicated that in OxyHb the SNO of Cys (1 93 is protected from solvent. In contrast, in deoxyHb the SNO is highly exposed to solvent. The implication was that the NO+ on Cys (193-S-NO could be transferred to thiols in RBC and eventually effluxed to induce vasodilation under conditions of low 02 saturation. 

Gow and Stamler (Gow and Stamler, 1998) looked more carefully at Hb-SNO chemistry under more physiological conditions i.e. at NO to Hb ratios ranging from 1 100 to 10 1. They reported that at NO Hb ratios of -1 20 NO is bound to the hemes. As 02 is slowly introduced the NO is transferred from the Heme to the )G93 thiol and 02Hb-SNO is converted to the R state. They also presented spectral evidence that in the absence of oxygen, at low NO Hb ratios (< 1 20) the major product is nitrosyl Hb, whereas metHb is produced at intermediate NO Hb ratios (1 20 to 1 2) and nitrosylH b is once again produced at ratios of > 1 2. The authors proposed that when Heme-Fe(II)-NO is in close proximity to [5G93-S11, NO is directly transferred to yield SNO H. They further proposed that 02 would then act as an electron acceptor to yield RSNO plus superoxide. 

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