Sonia Donzelli

The chemical biology of nitric oxide: implications in cellular signaling.

Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the past few years, the importance of steady-state NO concentrations has emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose five basic distinct concentration levels of NO activity: cGMP-mediated processes ([NO]<1-30 nM), Akt phosphorylation ([NO] = 30-100 nM), stabilization of HIF-1alpha ([NO] = 100-300 nM), phosphorylation of p53 ([NO]>400 nM), and nitrosative stress (1 microM). In general, lower NO concentrations promote cell survival and proliferation, whereas higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell-cycle arrest profile to a cell survival profile. The resulting reactive nitrogen species that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability, which are referred to as kinetic determinants for molecular target interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.

Superoxide Fluxes Limit Nitric Oxide-induced Signaling

Independently, superoxide (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document}) and nitric oxide (NO) are biologically important signaling molecules. When co-generated, these radicals react rapidly to form powerful oxidizing and nitrating intermediates. Although this reaction was once thought to be solely cytotoxic, herein we demonstrate using MCF7, macrophage, and endothelial cells that when nanomolar levels of NO and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} were produced concomitantly, the effective NO concentration was established by the relative fluxes of these two radicals. Differential regulation of sGC, pERK, HIF-1α, and p53 were used as biological dosimeters for NO concentration. Introduction of intracellular- or extracellular-generated \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} during NO generation resulted in a concomitant increase in oxidative intermediates with a decrease in steady-state NO concentrations and a proportional reduction in the levels of sGC, ERK, HIF-1α, and p53 regulation. NO responses were restored by addition of SOD. The intermediates formed from the reactions of NO with \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} were non-toxic, did not form 3-nitrotyrosine, nor did they elicit any signal transduction responses. H2O2 in bolus or generated from the dismutation of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} by SOD, was cytotoxic at high concentrations and activated p53 independent of NO. This effect was completely inhibited by catalase, suppressed by NO, and exacerbated by intracellular catalase inhibition. We conclude that the reaction of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} with NO is an important regulatory mechanism, which modulates signaling pathways by limiting steady-state levels of NO and preventing H2O2 formation from \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document}.

The Emergence of Nitroxyl (HNO) as a Pharmacological Agent

Once a virtually unknown nitrogen oxide, nitroxyl (HNO) has emerged as a potential pharmacological agent. Recent advances in the understanding of the chemistry of HNO has led to the an understanding of HNO biochemistry which is vastly different from the known chemistry and biochemistry of nitric oxide (NO), the one-electron oxidation product of HNO. The cardiovascular roles of NO have been extensively studied, as NO is a key modulator of vascular tone and is involved in a number of vascular related pathologies. HNO displays unique cardiovascular properties and has been shown to have positive lusitropic and ionotropic effects in failing hearts without a chronotropic effect. Additionally, HNO causes a release of CGRP and modulates calcium channels such as ryanodine receptors. HNO has shown beneficial effects in ischemia reperfusion injury, as HNO treatment before ischemia-reperfusion reduces infarct size. In addition to the cardiovascular effects observed, HNO has shown initial promise in the realm of cancer therapy. HNO has been demonstrated to inhibit GAPDH, a key glycolytic enzyme. Due to the Warburg effect, inhibiting glycolysis is an attractive target for inhibiting tumor proliferation. Indeed, HNO has recently been shown to inhibit tumor proliferation in mouse xenografts. Additionally, HNO inhibits tumor angiogenesis and induces cancer cell apoptosis. The effects seen with HNO donors are quite different from NO donors and in some cases are opposite. The chemical nature of HNO explains how HNO and NO, although closely chemically related, act so differently in biochemical systems. This also gives insight into the potential molecular motifs that may be reactive towards HNO and opens up a novel field of pharmacological development.

The specificity of nitroxyl chemistry is unique among nitrogen oxides in biological systems

The importance of nitric oxide in mammalian physiology has been known for nearly 30 years. Similar attention for other nitrogen oxides such as nitroxyl (HNO) has been more recent. While there has been speculation as to the biosynthesis of HNO, its pharmacological benefits have been demonstrated in several pathophysiological settings such as cardiovascular disorders, cancer, and alcoholism. The chemical biology of HNO has been identified as related to, but unique from, that of its redox congener nitric oxide. A summary of these findings as well as a discussion of possible endogenous sources of HNO is presented in this review.

Generation of nitroxyl by heme protein-mediated peroxidation of hydroxylamine but not N-hydroxy-L-arginine.

The chemical reactivity, toxicology, and pharmacological responses to nitroxyl (HNO) are often distinctly different from those of nitric oxide (NO). The discovery that HNO donors may have pharmacological utility for treatment of cardiovascular disorders such as heart failure and ischemia reperfusion has led to increased speculation of potential endogenous pathways for HNO biosynthesis. Here, the ability of heme proteins to utilize H2O2 to oxidize hydroxylamine (NH2OH) or N-hydroxy-L-arginine (NOHA) to HNO was examined. Formation of HNO was evaluated with a recently developed selective assay in which the reaction products in the presence of reduced glutathione (GSH) were quantified by HPLC. Release of HNO from the heme pocket was indicated by formation of sulfinamide (GS(O)NH2), while the yields of nitrite and nitrate signified the degree of intramolecular recombination of HNO with the heme. Formation of GS(O)NH2 was observed upon oxidation of NH2OH, whereas NOHA, the primary intermediate in oxidation of L-arginine by NO synthase, was apparently resistant to oxidation by the heme proteins utilized. In the presence of NH2OH, the highest yields of GS(O)NH2 were observed with proteins in which the heme was coordinated to a histidine (horseradish peroxidase, lactoperoxidase, myeloperoxidase, myoglobin, and hemoglobin) in contrast to a tyrosine (catalase) or cysteine (cytochrome P450). That peroxidation of NH2OH by horseradish peroxidase produced free HNO, which was able to affect intracellular targets, was verified by conversion of 4,5-diaminofluorescein to the corresponding fluorophore within intact cells.

Hypothesis of the postconcussive vulnerable brain: Experimental evidence of its metabolic occurrence

OBJECTIVE:We evaluated the effects of two consecutive concussive injuries on brain energy metabolism and N-acetylaspartate (NAA) to investigate how the temporal interval between traumatic events influences overall injury severity. METHODS:Rats were injured to induce diffuse traumatic brain injury (TBI) (mild, 450 g/1 m; severe, 450 g/2 m). In two groups, two mild TBIs were delivered in 3- or 5-day intervals. Three additional animal groups were used: single mild TBI, single severe TBI, and sham. All animals were killed 48 hours postinjury. Adenosine 5′-triphosphate (ATP), adenosine diphosphate, and NAA concentrations were analyzed with high-performance liquid chromatography on deproteinized whole brain extracts. RESULTS:In control animals, the NAA concentration was 9.17 ± 0.38 &mgr;mol/g wet weight, the ATP concentration was 2.25 ± 0.21 &mgr;mol/g wet weight, and the ATP-to-adenosine diphosphate ratio was 9.38 ± 1.23. These concentrations decreased to 6.68 ± 1.12 &mgr;mol/g wet weight, 1.68 ± 0.24 &mgr;mol/g wet weight, and 6.10 ± 1.21 &mgr;mol/g wet weight, respectively, in rats that received two mild TBIs at a 5-day interval (P < 0.01; not different from results in rats with single mild TBI). When a second TBI was delivered after 3 days, the NAA concentration was 3.86 ± 0.53 &mgr;mol/g wet weight, the ATP concentration was 1.11 ± 0.18 &mgr;mol/g wet weight, and the ATP-to-adenosine diphosphate ratio was 2.64 ± 0.43 (P < 0.001 versus both controls and 3-day interval; not different from rats receiving a single severe TBI). CONCLUSION:The biochemical modification severity in double TBI is dependent on the interval between traumatic events, which demonstrates the metabolic state of the vulnerable brain after mild TBI. These data support the hypothesis of the application of proton magnetic resonance spectroscopy to measure NAA as a possible tool to monitor the full recovery of brain metabolic functions in the clinical setting, particularly in sports medicine.

Dual Mechanisms of HNO Generation by a Nitroxyl Prodrug of the Diazeniumdiolate (NONOate) Class

Here we describe a novel caged form of the highly reactive bioeffector molecule, nitroxyl (HNO). Reacting the labile nitric oxide (NO)- and HNO-generating salt of structure iPrHN−N(O)=NO−Na+ (1, IPA/NO) with BrCH2OAc produced a stable derivative of structure iPrHN-N(O)=NO−CH2OAc (2, AcOM-IPA/NO), which hydrolyzed an order of magnitude more slowly than 1 at pH 7.4 and 37 °C. Hydrolysis of 2 to generate HNO proceeded by at least two mechanisms. In the presence of esterase, straightforward dissociation to acetate, formaldehyde, and 1 was the dominant path. In the absence of enzyme, free 1 was not observed as an intermediate and the ratio of NO to HNO among the products approached zero. To account for this surprising result, we propose a mechanism in which base-induced removal of the N−H proton of 2 leads to acetyl group migration from oxygen to the neighboring nitrogen, followed by cleavage of the resulting rearrangement product to isopropanediazoate ion and the known HNO precursor, CH3−C(O)−NO. The trappable yield of HNO from 2 was significantly enhanced over 1 at physiological pH, in part because the slower rate of hydrolysis for 2 generated a correspondingly lower steady-state concentration of HNO, thus, minimizing self-consumption and enhancing trapping by biological targets such as metmyoglobin and glutathione. Consistent with the chemical trapping efficiency data, micromolar concentrations of prodrug 2 displayed significantly more potent sarcomere shortening effects relative to 1 on ventricular myocytes isolated from wild-type mouse hearts, suggesting that 2 may be a promising lead compound for the development of heart failure therapies.

Comparing the chemical biology of NO and HNO.

For the past couple of decades nitric oxide (NO) and nitroxyl (HNO) have been extensively studied due to the important role they play in many physiological and/or pharmacological processes. Many researchers have reported important signaling pathways as well as mechanisms of action of these species, showing direct and indirect effects depending on the environment. Both NO and HNO can react with, among others, metals, proteins, thiols and heme proteins via unique and distinct chemistry leading to improvement of some clinical conditions. Understanding the basic chemistry of NO and HNO and distinguishing their mechanisms of action as well as methods of detection are crucial for understanding the current and potential clinical applications. In this review, we summarize some of the most important findings regarding NO and HNO chemistry, revealing some of the possible mechanisms of their beneficial actions.

Nitroxyl exacerbates ischemic cerebral injury and oxidative neurotoxicity

Nitroxyl (HNO) donor compounds function as potent vasorelaxants, improve myocardial contractility and reduce ischemia‐reperfusion injury in the cardiovascular system. With respect to the nervous system, HNO donors have been shown to attenuate NMDA receptor activity and neuronal injury, suggesting that its production may be protective against cerebral ischemic damage. Hence, we studied the effect of the classical HNO‐donor, Angeli’s salt (AS), on a cerebral ischemia/reperfusion injury in a mouse model of experimental stroke and on related in vitro paradigms of neurotoxicity. I.p. injection of AS (40 μmol/kg) in mice prior to middle cerebral artery occlusion exacerbated cortical infarct size and worsened the persistent neurological deficit. AS not only decreased systolic blood pressure, but also induced systemic oxidative stress in vivo indicated by increased isoprostane levels in urine and serum. In vitro, neuronal damage induced by oxygen‐glucose‐deprivation of mature neuronal cultures was exacerbated by AS, although there was no direct effect on glutamate excitotoxicity. Finally, AS exacerbated oxidative glutamate toxicity – that is, cell death propagated via oxidative stress in immature neurons devoid of ionotropic glutamate receptors. Taken together, our data indicate that HNO might worsen cerebral ischemia‐reperfusion injury by increasing oxidative stress and decreasing brain perfusion at concentrations shown to be cardioprotective in vivo.

Nitroxyl in the central nervous system.

Nitroxyl (HNO) is the one-electron-reduced and protonated congener of nitric oxide (NO). Compared to NO, it is far more reactive with thiol groups either in proteins or in small antioxidant molecules either converting those into sulfinamides or inducing disulfide bond formation. HNO might mediate cytoprotective changes of protein function through thiol modifications. However, HNO is a strong oxidant that in vitro reacts with glutathione to form glutathione disulfide and glutathione sulfinamide. The resulting oxidative stress might aggravate tissue damage in inflammatory diseases. In this review, we will summarize the current knowledge of how exogenous HNO affects the central nervous system, especially nerve cells and glia in health and disease. Unlike most other organs, the brain is separated from the circulation by the blood-brain barrier, which limits access of many pharmacological compounds. Given that, we will review what is known about the ability of currently used HNO donors to cross the blood-brain barrier. Moreover, considering that the physiology and composition of the brain has unique properties, for example, expression of brain-specific enzymes like neuronal NO synthase, its high iron content, and increased energy metabolism, we will discuss possible sources of endogenous HNO in the brain.