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The redox siblings nitroxyl (HNO) and nitric oxide (NO) have often been assumed to undergo casual redox reactions in biological systems. However, several recent studies have demonstrated distinct pharmacological effects for donors of these two species. Here, infusion of the HNO donor Angeli's salt into normal dogs resulted in elevated plasma levels of calcitonin gene-related peptide, whereas neither the NO donor diethylamine/NONOate nor the nitrovasodilator nitroglycerin had an appreciable effect on basal levels. Conversely, plasma cGMP was increased by infusion of diethylamine/NONOate or nitroglycerin but was unaffected by Angeli's salt. These results suggest the existence of two mutually exclusive response pathways that involve stimulated release of discrete signaling agents from HNO and NO. In light of both the observed dichotomy of HNO and NO and the recent determination that, in contrast to the [O.sub.2]/[O.sup.-.sub.2] couple, HNO is a weak reductant, the relative reactivity of HNO with common biomolecules was determined. This analysis suggests that under biological conditions, the lifetime of HNO with respect to oxidation to NO, dimerization, or reaction with [O.sub.2] is much longer than previously assumed. Rather, HNO is predicted to principally undergo addition reactions with thiols and ferric proteins. Calcitonin gene-related peptide release is suggested to occur via altered calcium channel function through binding of HNO to a ferric or thiol site. The orthogonality of HNO and NO may be due to differential reactivity toward metals and thiols and in the cardiovascular system, may ultimately be driven by respective alteration of cAMP and cGMP levels. Angeli's salt | superoxide dismutase | heme protein | cGMP | calcitonin gene-related peptide

Nitroxyl anion (HNO/N[O.sup.-]), the one-electron reduced form of nitric oxide (NO), induces positive cardiac inotropy and selective venodilation in the normal in vivo circulation. Here we tested whether HNO/N[O.sup.-] augments systolic and diastolic function of failing hearts, and whether contrary to NO/nitrates such modulation enhances rather than blunts [beta]-adrenergic stimulation and is accompanied by increased plasma calcitonin gene-related peptide (CGRP). HNO/N[O.sup.-] generated by Angelis' salt (AS) was infused (10 [micro]g/kg per min, i.v.) to conscious dogs with cardiac failure induced by chronic tachycardia pacing. AS nearly doubled contractility, enhanced relaxation, and lowered cardiac preload and afterload (all P < 0.001) without altering plasma cGMP. This contrasted to modest systolic depression induced by an NO donor diethylamine(DEA)/NO or nitroglycerin (NTG). Cardiotropic changes from AS were similar in failing hearts as in controls despite depressed [beta]-adrenergic and calcium signaling in the former. Inotropic effects of AS were additive to dobutamine, whereas DEA/NO blunted [beta]-stimulation and NTG was neutral. Administration of propranolol to nonfailing hearts fully blocked isoproterenol stimulation but had minimal effect on AS inotropy and enhanced lusitropy. Arterial plasma CGRP rose [beta]-fold with AS but was unaltered by DEA/NO or NTG, supporting a proposed role of this peptide to HNO/N[O.sup.-] cardiotropic action. Thus, HNO/N[O.sup.-] has positive inotropic and lusitropic action, which unlike NO/nitrates is independent and additive to [beta]-adrenergic stimulation and stimulates CGRP release. This suggests potential of HNO/N[O.sup.-] donors for the treatment of heart failure. nitroxyl | contractility | heart failure | nitric oxide | CGRP

Free radical-induced cellular stress contributes to cancer during chronic inflammation. Here, we investigated mechanisms of p53 activation by the free radical, NO. NO from donor drugs induced both ataxia-telangiectasia mutated (ATM)- and ataxiatelangiectasia mutated and Rad3-related-dependent p53 posttranslational modifications, leading to an increase in p53 transcriptional targets and a [G.sub.2]/M cell cycle checkpoint. Such modifications were also identified in cells cocultured with NO-releasing macrophages. In noncancerous colon tissues from patients with ulcerative colitis (a cancer-prone chronic inflammatory disease), inducible NO synthase protein levels were positively correlated with p53 serine 15 phosphorylation levels. Immunostaining of HDM-2 and [p21.sup.WAF1] was consistent with transcriptionally active p53. Our study highlights a pivotal role of NO in the induction of cellular stress and the activation of a p53 response pathway during chronic inflammation. posttranslational modification | phosphorylation

The chemical origins of nitrated tyrosine residues (NT) formed in proteins during a variety of pathophysiological conditions remain controversial. Although numerous studies have concluded that NT is a signature for peroxynitrite (ONO[O.sup.-]) formation, other works suggest the primary involvement of peroxidases. Because metal homeostasis is often disrupted in conditions bearing NT, the role of metals as catalysts for protein nitration was examined. Cogeneration of nitric oxide (NO) and superoxide ([O.sup.-.sub.2]), from spermine/NO (2.7 [micro]M/min) and xanthine oxidase (1-28 [micro]M [O.sup.-.sub.2]/min), respectively, resulted in protein nitration only when these species were produced at approximately equivalent rates. Addition of ferriprotoporphyrin IX (hemin) to this system increased nitration over a broad range of 02 concentrations with respect to NO. Nitration in the presence of superoxide dismutase but not catalase suggested that ONO[O.sup.-] might not be obligatory to this process. Hemin-mediated NT formation required only the presence of N[O.sup.-.sub.2] and [H.sub.2][O.sub.2], which are stable end-products of NO and [O.sup.-.sub.2] degradation. Ferrous, ferric, and cupric ions were also effective catalysts, indicating that nitration is mediated by species capable of Fenton-type chemistry. Although ONO[O.sup.-] can nitrate proteins, there are severe spatial and temporal constraints on this reaction. In contrast, accumulation of metals and N[O.sup.-.sub.2] subsequent to NO synthase activity can result in far less discriminate nitration in the presence of an [H.sub.2][O.sub.2] source. Metal catalyzed nitration may account for the observed specificity of protein nitration seen under pathological conditions, suggesting a major role for translocated metals and the labilization of heme in NT formation. nitrotyrosine | nitric oxide | peroxynitrite | oxidation | hemin

A potential of about -0.8 ([+ or -] 0.2) V (at 1 M versus normal hydrogen electrode) for the reduction of nitric oxide (NO) to its one-electron reduced species, nitroxyl anion ([sup.3]N[O.sup.-]) has been determined by a combination of quantum mechanical calculations, cyclic voltammetry measurements, and chemical reduction experiments. This value is in accord with some, but not the most commonly accepted, previous electrochemical measurements involving NO. Reduction of NO to [sup.1]N[O.sup.-] is highly unfavorable, with a predicted reduction potential of about -1.7 ([+ or -] 0.2) V at 1 M versus normal hydrogen electrode. These results represent a substantial revision of the derived and widely cited values of +0.39 V and -0.35 V for the NO/[sup.3]N[O.sup.-] and NO/[sup.1]N[O.sup.-] couples, respectively, and provide support for previous measurements obtained by electrochemical and photoelectrochemical means. With such highly negative reduction potentials, NO is inert to reduction compared with physiological events that reduce molecular oxygen to superoxide. From these reduction potentials, the pKa of [sup.3]N[O.sup.-] has been reevaluated as 11.6 ([+ or -] 3.4). Thus, nitroxyl exists almost exclusively in its protonated form, HNO, under physiological conditions. The singlet state of nitroxyl anion, [sup.1]N[O.sup.-], is physiologically inaccessible. The significance of these potentials to physiological and pathophysiological processes involving NO and [O.sub.2] under reductive conditions is discussed.

The impact of nitric oxide (NO) synthesis on different biological cascades can rapidly change dependent on the rate of NO formation and composition of the surrounding milieu. With this perspective, we used diaminonaphthalene (DAN) and diaminofluorescein (DAF) to examine the nitrosative chemistry derived from NO and superoxide ([O.sup.-.sub.2]) simultaneously generated at nanomolar to low micromolar per minute rates by spermine/NO decomposition and xanthine oxidasecatalyzed oxidation of hypoxanthine, respectively. Fluorescent triazole product formation from DAN and DAF increased as the ratio of [O.sup.-.sub.2] to NO approached equimolar, then decreased precipitously as [O.sup.-.sub.2] exceeded NO. This pattern was also evident in DAF-loaded MCF-7 carcinoma cells and when stimulated macrophages were used as the NO source. Cyclic voltammetry analysis and inhibition studies by using the [N.sub.2][O.sub.3] scavenger azide indicated that DAN- and DAF-triazole could be derived from both oxidative nitrosylation (e.g., DAF radical + NO) and nitrosation (N[O.sup.+] addition). The latter mechanism predominated with higher rates of NO formation relative to [O.sup.-.sub.2]. The effects of oxymyoglobin, superoxide dismutase, and carbon dioxide were examined as potential modulators of reactant availability for the [O.sup.-.sub.2] + NO pathway in vivo. The findings suggest that the outcome of NO biosynthesis in a scavenger milieu can be focused by [O.sup.-.sub.2] toward formation of NO adducts on nucleophilic residues (e.g., amines, thiols, hydroxyl) through convergent mechanisms involving the intermediacy of nitrogen dioxide. These modifications may be favored in microenvironments where the rate of [O.sup.-.sub.2] production is temporally and spatially contemporaneous with nitric oxide synthase activity, but not in excess of NO generation.

3-Nitrotyrosyl adducts in proteins have been detected in a wide range of diseases. The mechanisms by which reactive nitrogen oxide species may impede protein function through nitration were examined by using a unique model system, which exploits a critical tyrosyl residue in the fluorophoric pocket of recombinant green fluorescent protein (GFP). Exposure of purified GFP suspended in phosphate buffer to synthetic peroxynitrite in either 0.5 or 5 [micro]M steps resulted in progressively increased 3-nitrotyrosyl immunoreactivity concomitant with disappearance of intrinsic fluorescence (I[C.sub.50] [approximately equal to] 20 [micro]M). Fluorescence from an equivalent amount of GFP expressed within intact MCF-7 tumor cells was largely resistant to this bolus treatment (I[C.sub.50] > 250 [micro]M). The more physiologically relevant conditions of either peroxynitrite infusion (1 [micro]M/min) or de novo formation by simultaneous, equimolar generation of nitric oxide (NO) and superoxide (e.g., 3-morpholinosydnonimine; NONOates plus xanthine oxidase/hypoxanthine, menadione, or mitomycin C) were examined. Despite robust oxidation of dihydrorhodamine under each of these conditions, fluorescence decrease of both purified and intracellular GFP was not evident regardless of carbon dioxide presence, suggesting that oxidation and nitration are not necessarily coupled. Alternatively, both extra- and intracellular GFP fluorescence was exquisitely sensitive to nitration produced by heme-peroxidase/hydrogen peroxide-catalyzed oxidation of nitrite. Formation of nitrogen dioxide (N[O.sub.2]) during the reaction between NO and the nitroxide 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide indicated that N[O.sub.2] can enter cells and alter peptide function through tyrosyl nitration. Taken together, these findings exemplified that heme-peroxidase-catalyzed formation of N[O.sub.2] may play a pivotal role in inflammatory and chronic disease settings while calling into question the significance of nitration by peroxynitrite. 3-nitrotyrosine | myeloperoxidase | nitric oxide | xanthine oxidase | superoxide

Nitroxyl anion ([NO.sup.-]) is the one-electron reduction product of nitric oxide (NO) and is enzymatically generated by NO synthase in vitro. The physiologic activity and mechanism of action of [NO.sup.-] in vivo remains unknown. The [NO.sup.-] generator Angeli's salt (AS, [Na.sub.2][N.sub.2][O.sub.3]) was administered to conscious chronically instrumented dogs, and pressure-dimension analysis was used to discriminate contractile from peripheral vascular responses. AS rapidly enhanced left ventricular contractility and concomitantly lowered cardiac pre-load volume and diastolic pressure (venodilation) without a change in arterial resistance. There were no associated changes in arterial or venous plasma cGMP. The inotropic response was similar despite reflex blockade with hexamethonium or volume reexpansion, indicating its independence from baroreflex stimulation. However, reflex activation did play a major role in the selective venodilation observed under basal conditions. These data contrasted with the pure NO donor diethylamine/NO, which induced a negligible inotropic response and a more balanced veno/arterial dilation. AS-induced positive inotropy, but not systemic vasodilatation, was highly redox-sensitive, being virtually inhibited by coinfusion of N-acetyl-L-cysteine. Cardiac inotropic signaling by [NO.sup.-] was mediated by calcitonin gene-related peptide (CGRP), as treatment with the selective CGRP-receptor antagonist CGRP-(8-37) prevented this effect but not systemic vasodilation. Thus, [NO.sup.-] is a redox-sensitive positive inotrope with selective venodilator action, whose cardiac effects are mediated by CGRP-receptor stimulation. This fact is evidence linking [NO.sup.-] to redox-sensitive cardiac contractile modulation by nonadrenergic/noncholinergic peptide signaling. Given its cardiac and vascular properties, [NO.sup.-] may prove useful for the treatment of cardiovascular diseases characterized by cardiac depression and elevated venous filling pressures.

Nitroxyl anion (HNO/NO[sup -]), the one-electron reduced form of nitric oxide (NO), induces positive cardiac inotropy and selective venodilation in the normal in vivo circulation. Here we tested whether HNO/NO[sup -] augments systolic and diastolic function of failing hearts, and whether contrary to NO/nitrates such modulation enhances rather than blunts β-adrenergic stimulation and is accompanied by increased plasma calcitonin gene-related peptide (CGRP). HNO/NO[sup -] generated by Angelis' salt (AS) was infused (10 µg/kg per min, i.v.) to conscious dogs with cardiac failure induced by chronic tachycardia pacing. AS nearly doubled contractility, enhanced relaxation, and lowered cardiac preload and afterload (all P < 0.001) without altering plasma cGMP. This contrasted to modest systolic depression induced by an NO donor diethylamine(DEA)/NO or nitroglycerin (NTG). Cardiotropic changes from AS were similar in failing hearts as in controls despite depressed β-adrenergic and calcium signaling in the former. Inotropic effects of AS were additive to dobutamine, whereas DEA/NO blunted β-stimulation and NTG was neutral. Administration of propranolol to nonfailing hearts fully blocked isoproterenol stimulation but had minimal effect on AS inotropy and enhanced lusitropy. Arterial plasma CGRP rose 3-fold with AS but was unaltered by DEA/NO or NTG, supporting a proposed role of this peptide to HNO/NO[sup -] cardiotropic action. Thus, HNO/NO[sup -] has positive inotropic and lusitropic action, which unlike NO/nitrates is independent and additive to β-adrenergic stimulation and stimulates CGRP release. This suggests potential of HNO/NO[sup -] donors for the treatment of heart failure. [ABSTRACT FROM AUTHOR]

Examines the mediation of protein nitration by heme and free metals through Fenton-type chemistry. Details of the chemical origins of nitrated tyrosine residues formed in protein; Cogeneration of nitric oxide; Use of Western blot analysis.

Examines the generation of nitroxyl anion by nitric oxide (NO) synthase in vivo. Administration of NO generator Angeli's salt (AS) to chronologically instrumented dogs; Effects of AS to the contractility of the left ventricle; Absence of associated changes in arterial or venous plasma cGMP.

The authors discuss aspects of the study, entitled "Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling," conducted by T. Ida and colleagues. They say that the researchers describe a new mass spectrometric approach to measure low molecular-weight persulfide (RSSH) and polysulfide content in cells and a proteomic/Tag-Switch assay. Topics discussed include sulfur metabolism, cysteine, and glutathione disulfide (GSSG).