Stability Of Rsno In Vivo

In biological systems, additional pathways exist for removal of RSNO. The consumption of S-nitrosogroups (S-denitrosation) by endogenous enzymes has not been often reported in the literature. Mammalian physiology does not seem to require a dedicated system to remove excess quantitites of RSNO. A modest capacity for S-denitrosation has been recently reported for the protein disul de isomerase enzyme [62] and for anaerobic xanthine oxidase [63]. Cu Zn superoxide dismutase was capable to denitrosate low concentrations of GSNO in vitro, but the signi cance of this nding is questionable since the reaction was inhibited by in-vivo levels of GSH (Table 1). Thioredoxin reductase (TR) [64] and glutathione peroxidase [65] will denitrosate GSNO to GSH under release of free NO. Finally, the y-glutamyl transferase enzyme was implicated in the stereoselective effect of L-CysNO on posthypoxic ventilation of mice [66]. The preceding examples do not consume large quantities of LMW nitrosothiols and are not expected to have signi cance for GSNO levels in vivo.

In contrast, S-nitrosoglutathione reductase (GSNOR) was found to have an impact on GSNO metabolism in vivo. In the literature, this enzyme is also referred to as alcohol dehydrogenase-3 (ADH3) or as GSH-dependent formaldehyde dehydrogenase. It can consume large quantities of GSNO [4,67 70]. GSNOR activity is found in many different tissues and found highest in liver [71]. The physiological signi cance of the GSNOR pathway was impressively demonstrated by experiments with GSNOR knockout mutant mice [4], which showed greatly enhanced levels of RSNO and became hypotensive under anesthesia. Human asthmatics show the combination of enhanced GSNOR levels and depressed GSNO in the bronchial uids. It suggests that chemically incorporated NO stores are depleted in asthmatic patients. Since the remainder of NO is exhaled, the unbalance between NO and S-nitrosocompounds may be the reason for the observed increase of exhaled NO in asthma [70,72]. Recent studies on the decomposition of low-molecular-weight S-nitrosothiols in tissue homogenates of rats have provided more evidence that enzymatic pathways contribute signi cantly to the process [73]. The above publications leave no doubt that signi cant enzymatic pathways for S-denitrosation of LMW nitrosothiols operate in mammals. Their role in the regulation of endogenous pool of S-nitrosothiols remains the subject of intense research for the moment being.

In addition to this enzymatic pathway, GSNO may be depleted by superoxide according to the third order reaction [74]

dt 2

The third order reaction rate in vitro was reported as k = 6 x 108 M-2 s . With GSNO concentrations in tissue below ca 50 nM (cf Table 1) and superoxide levels below micromolar range, the loss of GSNO via this pathway is insigni cant in comparison to loss by catalytic action of spurious metal ions. This observation supports the hypothesis that the transient formation of RSNO may help to increase the effective lifetime of NO in biological systems by protection against the reaction with superoxide.

The hydroxyl radical OH is a very reactive oxygen species that can be generated in solutions when hydrogen peroxide undergoes a Fenton reaction with spurious ferrous iron. Using pulse radiolysis for generation of the hydroxyl radicals at pH = 7, the main reaction products were found to be disul des at nitrite. The reaction was second order with diffusion limited reaction rates as usually found for hydroxyl. The reaction rates for CysNO, GSNO and ACysNO were 2.27, 1.46 and 1.94 x 1010 (Ms)-1, respectively [75].

Finally, the reaction with oxy-heme proteins, in particular oxy-hemoglobin (oxyHb) and oxy-myoglobin (oxyMb) should be considered. Oxy-hemoglobin is known as the major sink for free NO radicals in the vascular system. The reaction products are methemoglobin and nitrate oxyHb + NO ^ metHb + NO-

The reaction is very fast with second order reaction rates of 8.9 x 107 (Ms)-1 for oxyHb and 4.4 x 107 (Vs)-1 for oxyMb (pH = 7, 20°C) [76]. Therefore, one might expect a signi cant oxidation of S-nitrosothiols by oxyHb as well. However, in-vitro experiments show that the NO moiety of S-nitrosothiols is well protected against oxidation to nitrate by oxy-heme. Exposure of a small quantity of CysNO to oxyHb leads to S-nitrosation of the Cys|393 residue of the protein but not to the formation of nitrate [77,78]. In fact, the rates and extent of transnitrosation from CysNO were very similar for oxyHb and metHb.

This transnitrosation reaction to oxyHb was completely inhibited by the addition of the metal chelators neocuproine and DTPA [78]. Therefore, the transnitrosation of oxyHb seems to require the presence of catalytic traces of copper, either in free form or in the form of Cu Zn dismutase.

Clearly, reaction (11) is highly signi cant for free NO radicals but does not apply to S-nitrosothiols. Phrased otherwise, a small quantity of low-molecular-weight S-nitrosothiols can survive for a very long time in the presence of oxy-heme. This property is a very signi cant distinction with free NO. It explains why signi cant quantities of S-nitrosothiols can coexist with oxygenated erythrocytes and blood (Table 1), in clear contrast to free NO. Oxidation to nitrate becomes feasible only if GSNO is supplied in excess over Cys|393 residues [78]. This situation is very unlikely to happen in vivo.

Under anaerobic conditions, GSNO can be reduced by deoxyhemoglobin in a slow and irreversible reaction [79]. In presence of excess deoxyHb, the half life of GSNO is about an hour with concomitant release of equimolar quantities of GSH and nitrosylated ferric hemoglobin. The reaction rate was dependent on the conformer state (R or T) of the Hb tetramer. Interestingly, transnitrosation from GSNO to the Cys^93 residue of Hb did not occur in this assay. As with oxyHb, the reaction pathway with deoxyHb seems not relevant for the lifetime of GSNO in vivo.

Recapitulating these various reactions, we recall that in vivo RSNO concentrations remain below ca 100 nM (Table 1). At such low levels, the thermolytic and photolytic pathways are negligible in comparison with the effect of trace metal ions, superoxide and the chemical equilibria with other stores of NO. Signi cantly, S-nitrosothiols are essentially stable against oxidation by oxyHb, whereas free NO is rapidly oxidized to nitrate.

Dealing With Asthma Naturally

Dealing With Asthma Naturally

Do You Suffer From ASTHMA Chronic asthma is a paralyzing, suffocating and socially isolating condition that can cause anxiety that can trigger even more attacks. Before you know it you are caught in a vicious cycle Put an end to the dependence on inhalers, buying expensive prescription drugs and avoidance of allergenic situations and animals. Get control of your life again and Deal With Asthma Naturally

Get My Free Ebook


Post a comment