Manganese-salen complexes (Mn-Salen), including EUK-8 [manganese N,N'-bis(salicylidene)ethylenediamine chloride] and EUK-134 [manganese 3-methoxy N,N'-bis(salicylidene)ethylenediamine chloride], have been reported to possess combined superoxide dismutase (SOD) and catalase mimetic functions. Because of this SOD/catalase mimicry, EUK-8 and EUK-134 have been investigated as possible therapeutic agents in neurological disorders resulting from oxidative stress, including Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis. These actions have been explained by the ability of the Mn-Salen to remove deleterious superoxide (O(2)(-)) and H(2)O(2). However, in addition to oxidative stress, cells in models for neurodegenerative diseases may also be subjected to damage from reactive nitrogen oxides (nitrosative stress), resulting from elevated levels of NO and sister compounds, including peroxynitrite (ONOO(-)). We have been examining the interaction of EUK-8 and EUK-134 with NO and ONOO(-). We find that in the presence of a per-species (H(2)O(2), ONOO(-), peracetate and persulphate), the Mn-Salen complexes are oxidized to the corresponding oxo-species (oxoMn-Salen). OxoMn-Salens are potent oxidants, and we demonstrate that they can rapidly oxidize NO to NO(2) and also oxidize nitrite (NO(2)(-) to nitrate (NO(2)(-)). Thus these Mn-Salens have the potential to ameliorate cellular damage caused by both oxidative and nitrosative stresses, by the catalytic breakdown of O(2)(-), H(2)O(2), ONOO(-) and NO to benign species: O(2), H(2)O, NO(2)(-) and NO(3)(-).

Reactive nitrogen species, such as peroxynitrite, can nitrate tyrosine in proteins to form nitrotyrosine. Nitrotyrosine is metabolized to 3-nitro-4-hydroxyphenylacetic acid (NHPA), which is excreted in the urine. This has led to the notion that measurement of urinary NHPA may provide a time-integrated index of nitrotyrosine formation in vivo. However, it is not known whether NHPA is derived exclusively from metabolism of nitrotyrosine, or whether it can be formed by nitration of circulating para -hydroxyphenylacetic acid (PHPA), a metabolite of tyrosine. In the present study, we have developed a gas chromatography MS assay for NHPA and PHPA to determine whether or not NHPA can be formed directly by nitration of PHPA. Following the injection of nitrotyrosine, 0.5+/-0.16% of injected dose was recovered unchanged as nitrotyrosine, and 4.3+/-0.2% as NHPA in the urine. To determine whether or not NHPA could be formed by the nitration of PHPA, deuterium-labelled PHPA ([(2)H(6)]PHPA) was injected, and the formation of deuterated NHPA ([(2)H(5)]NHPA) was measured. Of the infused [(2)H(6)]PHPA, 78+/-2% was recovered in the urine unchanged, and approx. 0.23% was recovered as [(2)H(5)]NHPA. Since the plasma concentration of PHPA is markedly higher than free nitrotyrosine (approx. 400-fold), the nitration of high-circulating endogenous PHPA to form NHPA becomes very significant and accounts for the majority of NHPA excreted in urine. This is the first study to demonstrate that NHPA can be formed by nitration of PHPA in vivo, and that this is the major route for its formation.

Acceleration of the heart rate in response to catecholamines is impaired in cirrhosis. In this study, we tested the hypothesis that increased formation of reactive nitrogen species in biliary cirrhosis causes nitration of cardiac proteins and leads to impaired chronotropic function. Bile duct-ligated (rats with cirrhosis) or sham-operated rats were injected daily with either saline, N G -L-nitro-arginine methyl ester (L-NAME), or N-acetylcysteine for 7 days from week 3 to week 4 after surgery. Cardiac chronotropic responsiveness to ␤-adrenergic stimulation was assessed in vitro using spontaneous beating isolated atria. Nitration of cardiac proteins was measured by mass spectrometry and located by immunogold electron microscopy. Marked impairment of chronotropic responses of isolated atria to isoproterenol was seen in rats with cirrhosis, which normalized after the administration of N-acetylcysteine or L-NAME. The levels of protein-bound nitrotyrosine in atrial tissue increased from 16 ؎ 1 to 23 ؎ 3 pg/g tyrosine in rats with cirrhosis, and decreased to 15 ؎ 1 and 17 ؎ 1 pg/g after treatment with L-NAME and N-acetylcysteine, respectively (P < .05). Immunogold electron microscopy demonstrated increased nitration of mitochondrial proteins in the atria of rats with cirrhosis. The plasma nitrite/nitrate levels were elevated in rats with biliary cirrhosis, and decreased after administration of L-NAME but were unchanged by N-acetylcysteine. In conclusion, abnormal cardiac chronotropic function in cirrhosis is associated with increased nitration of cardiac proteins. Two independent treatments (N-acetylcysteine and L-NAME) that decrease nitration of cardiac proteins led to normalization of cardiac responses. Nitration of critical proteins in cardiac tissue may lead to abnormal cardiac function. (HEPATOLOGY 2006;43:847-856.)

Nitric oxide (NO) is a powerful antiplatelet agent, but its notoriously short biological half-life limits its potential to prevent the activation of circulating platelets. Here we used diethylamine diazeniumdiolate (DEA/NO) as an NO generator to determine whether the antiplatelet effects of NO are prolonged by the formation of a durable, plasma-borne S-nitrosothiol reservoir. Preincubation of both platelet rich plasma (PRP) and washed platelets (WP) with DEA/NO (2 M) for 1 min inhibited collagen-induced platelet aggregation by 82 ؎ 5 and 91 ؎ 2%, respectively. After 30 min preincubation with DEA/ NO, NO was no longer detectable in either preparation, but aggregation remained markedly inhibited (72 ؎ 7%) in PRP. In contrast, the inhibitory effect in WP was almost completely lost at this time (5 ؎ 3%) but was partially restored (39 ؎ 10%) in WP containing human serum albumin (1%) and fully restored by co-incubation with albumin and the low molecular weight (LMW) thiols, glutathione, (5 M), cysteinyl-glycine (10 M), or cysteine (10 M). This NO-mediated effect was not seen with LMW thiols in the absence of albumin and was associated with S-nitrosothiol formation. Our results demonstrate that LMW thiols play an important role in both the formation and activation of an S-nitrosoalbumin reservoir that significantly prolongs the duration of action of NO. Nitric oxide (NO)1 is a crucial free radical messenger with potent antiplatelet activity (1-5). NO synthesized in vascular endothelial cells and platelets is recognized to be a key mediator that protects against both atherogenesis and thrombosis (6). In platelets, NO primarily acts to stimulate soluble guanylate cyclase, ultimately resulting in a cyclic guanosine monophosphate (cGMP) and G kinase-mediated reduction in calcium mobilization (7,8), although cGMP-independent inhibitory effects have also been identified (9). Under physiological conditions, the half-life of NO is short (ϳ3-10 s) (10, 11), suggesting that NO bioactivity should rapidly dissipate and only impact on cells within close diffusible range of the site of production (12, 13). However, a number of studies suggest that NO can be incorporated into relatively stable endogenous reservoirs that modify its biological activity (14 -19). S-Nitrosothiols rank high among the likely candidates for such a reservoir because of the relative abundance of suitable thiols in the biological environment (20). A physiological role for S-nitrosothiols has been implicated following identification of endogenous S-nitrosothiols at relevant concentrations (14, 21-24), together with plausible pathways that could result in their formation (25-28). In plasma, it has been shown that the vast majority of the Snitrosothiol pool exists in the form of the high molecular weight species S-nitrosoalbumin (14, 29, 30). However, low molecular weight (LMW) thiols such as glutathione are also present in plasma in the low micromolar range and have previously been shown to potentiate the antiplatelet action of S-nitrosoalbumin (31). Given the c...

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