Dario A. Vitturi

A mitochondria-targeted S-nitrosothiol modulates respiration, nitrosates thiols, and protects against ischemia-reperfusion injury

Nitric oxide (NO•) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO• and S-nitrosated thiol proteins. MitoSNO1-induced NO• production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO• generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO• donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.

SNO-hemoglobin is not essential for red blood cell-dependent hypoxic vasodilation

The coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this oxygen-sensing–NO bioactivity linkage postulates an essential role for the conserved Cys93 residue of the hemoglobin β-chain (βCys93) and, specifically, for S-nitrosation of βCys93 to form S-nitrosohemoglobin (SNO-Hb). The SNO-Hb hypothesis, which conceptually links hemoglobin and NO biology, has been debated intensely in recent years. This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe new mouse models that exclusively express either human wild-type hemoglobin or human hemoglobin in which the βCys93 residue is replaced with alanine to assess the role of SNO-Hb in red blood cell–mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red blood cell S-nitrosothiol abundance but did shift S-nitrosothiol distribution to lower molecular weight species, consistent with the loss of SNO-Hb. Loss of βCys93 resulted in no deficits in systemic or pulmonary hemodynamics under basal conditions and, notably, did not affect isolated red blood cell–dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO bioactivity in vivo.

Conjugated Linoleic Acid is a Preferential Substrate for Fatty Acid Nitration

Background: Nitroalkene fatty acids are electrophilic cell metabolites that mediate anti-inflammatory signaling actions. Results: Conjugated linoleic acid is the preferential unsaturated fatty acid substrate for nitration reactions during oxidative inflammatory conditions and digestion. Conclusion: Nitro-fatty acid formation in vivo occurs during metabolic and inflammatory reactions and modulates cell signaling. Significance: Nitro-conjugated linoleic acid transduces signaling actions of nitric oxide, nitrite, and conjugated linoleic acid. The oxidation and nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that can modulate protein function via post-translational protein modifications. The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined. Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 105 greater extent of nitration products as compared with bis-allylic linoleic acid. Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans and are increased in tissues undergoing episodes of ischemia reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO2-CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic products that can modulate adaptive cell signaling mechanisms.

Olives and Olive Oil Are Sources of Electrophilic Fatty Acid Nitroalkenes

Extra virgin olive oil (EVOO) and olives, key sources of unsaturated fatty acids in the Mediterranean diet, provide health benefits to humans. Nitric oxide (•NO) and nitrite (NO2 −)-dependent reactions of unsaturated fatty acids yield electrophilic nitroalkene derivatives (NO2-FA) that manifest salutary pleiotropic cell signaling responses in mammals. Herein, the endogenous presence of NO2-FA in both EVOO and fresh olives was demonstrated by mass spectrometry. The electrophilic nature of these species was affirmed by the detection of significant levels of protein cysteine adducts of nitro-oleic acid (NO2-OA-cysteine) in fresh olives, especially in the peel. Further nitration of EVOO by NO2 − under acidic gastric digestive conditions revealed that human consumption of olive lipids will produce additional nitro-conjugated linoleic acid (NO2-cLA) and nitro-oleic acid (NO2-OA). The presence of free and protein-adducted NO2-FA in both mammalian and plant lipids further affirm a role for these species as signaling mediators. Since NO2-FA instigate adaptive anti-inflammatory gene expression and metabolic responses, these redox-derived metabolites may contribute to the cardiovascular benefits associated with the Mediterranean diet.

Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine

The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO2-octadeca-9,11-dienoic acid and 12-NO2-octadeca-9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 ± 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl β-oxidation metabolites. High resolution mass determinations and coelution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with mercury chloride. This reaction increases the level of free nitroalkene fraction >10-fold and displays a KD of 7.5 × 10−6 M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive noninvasive index of metabolic and inflammatory status.

Acyloxy Nitroso Compounds as Nitroxyl (HNO) Donors: Kinetics, Reactions with Thiols and Vasodilation Properties

Acyloxy nitroso compounds hydrolyze to nitroxyl (HNO), a nitrogen monoxide with distinct chemistry and biology. Ultraviolet-visible spectroscopy and mass spectrometry show hydrolysis rate depends on pH and ester group structure with the observed rate being trifluoroacetate (3) > acetate (1) > pivalate (2). Under all conditions, 3 rapidly hydrolyzes to HNO. A combination of spectroscopic, kinetic, and product studies show that addition of thiols increases the decomposition rate of 1 and 2, leading to hydrolysis and HNO. Under conditions that favor thiolates, the thiolate directly reacts with the nitroso group, yielding oximes without HNO formation. Biologically, 3 behaves like Angelis salt, demonstrating thiol-sensitive nitric oxide-mediated soluble guanylate cyclase-dependent vasorelaxation, suggesting HNO-mediated vasorelaxation. The slow HNO-donor 1 demonstrates weak thiol-insensitive vasorelaxation, indicating HNO release kinetics determine HNO bioavailability and activity. These results show that acyloxy nitroso compounds represent new HNO donors capable of vasorelaxation depending on HNO release kinetics.

Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation.

Allosteric regulation of nitrite reduction by deoxyhemoglobin has been proposed to mediate nitric oxide (NO) formation during hypoxia. Nitrite is predominantly an anion at physiological pH, raising questions about the mechanism by which it enters the red blood cell (RBC) and whether this is regulated and coupled to deoxyhemoglobin-mediated reduction. We tested the hypothesis that nitrite transport by RBCs is regulated by fractional saturation. Using human RBCs, nitrite consumption was faster at lower fractional saturations, consistent with faster reactions with deoxyheme. A membrane-based regulation was suggested by slower nitrite consumption with intact versus lysed RBCs. Interestingly, upon nitrite addition, intracellular nitrite concentrations attained a steady state that, despite increased rates of consumption, did not change with decreasing oxygen tensions, suggesting a deoxygenation-sensitive step that either increases nitrite import or decreases the rate of nitrite export. A role for anion exchanger (AE)-1 in the control of nitrite export was suggested by increased intracellular nitrite concentrations in RBCs treated with DIDS. Moreover, deoxygenation decreased steady-state levels of intracellular nitrite in AE-1-inhibited RBCs. Based on these data, we propose a model in which deoxyhemoglobin binding to AE-1 inhibits nitrite export under low oxygen tensions allowing for the coupling between deoxygenation and nitrite reduction to NO along the arterial-to-venous gradient.

Current perspectives and challenges in understanding the role of nitrite as an integral player in nitric oxide biology and therapy

Beyond an inert oxidation product of nitric oxide (NO) metabolism, current thinking posits a key role for nitrite as a mediator of NO signaling, especially during hypoxia. This concept has been discussed in the context of nitrite serving a role as an endogenous modulator of NO homeostasis, but also from a novel clinical perspective whereby nitrite therapy may replenish NO signaling and prevent ischemic tissue injury. Indeed, the relatively rapid translation of studies delineating mechanisms of action to ongoing and planned clinical trials has been critical in fuelling interest in nitrite biology, and several excellent reviews have been written on this topic. In this article we limit our discussions to current concepts and what we feel are questions that remain unanswered within the paradigm of nitrite being a mediator of NO biology.

Nitrite and nitrate-dependent generation of anti-inflammatory fatty acid nitroalkenes.

A gap in our understanding of the beneficial systemic responses to dietary constituents nitrate (NO3(-)), nitrite (NO2(-)) and conjugated linoleic acid (cLA) is the identification of the downstream metabolites that mediate their actions. To examine these reactions in a clinical context, investigational drug preparations of (15)N-labeled NO3(-) and NO2(-) were orally administered to healthy humans with and without cLA. Mass spectrometry analysis of plasma and urine indicated that the nitrating species nitrogen dioxide was formed and reacted with the olefinic carbons of unsaturated fatty acids to yield the electrophilic fatty acid, nitro-cLA (NO2-cLA). These species mediate the post-translational modification (PTM) of proteins via reversible Michael addition with nucleophilic amino acids. The PTM of critical target proteins by electrophilic lipids has been described as a sensing mechanism that regulates adaptive cellular responses, but little is known about the endogenous generation of fatty acid nitroalkenes and their metabolites. We report that healthy humans consuming (15)N-labeled NO3(-) or NO2(-), with and without cLA supplementation, produce (15)NO2-cLA and corresponding metabolites that are detected in plasma and urine. These data support that the dietary constituents NO3(-), NO2(-) and cLA promote the further generation of secondary electrophilic lipid products that are absorbed into the circulation at concentrations sufficient to exert systemic effects before being catabolized or excreted.

Modulation of Nitro-fatty Acid Signaling PROSTAGLANDIN REDUCTASE-1 IS A NITROALKENE REDUCTASE

Background: Nitroalkenes are electrophilic anti-inflammatory mediators that signal via Michael addition and are metabolized in vivo. Results: Prostaglandin reductase-1 is identified as a nitroalkene reductase. Conclusion: Prostaglandin reductase-1 reduces fatty acid nitroalkenes to nitroalkanes, inactivating electrophilic reactivity. Significance: A mammalian enzyme is identified that metabolizes fatty acid nitroalkenes in vivo to silence their signaling reactions. Inflammation, characterized by the activation of both resident and infiltrated immune cells, is accompanied by increased production of oxidizing and nitrating species. Nitrogen dioxide, the proximal nitrating species formed under these conditions, reacts with unsaturated fatty acids to yield nitroalkene derivatives. These electrophilic products modulate protein function via post-translational modification of susceptible nucleophilic amino acids. Nitroalkenes react with Keap1 to instigate Nrf2 signaling, activate heat shock response gene expression, and inhibit NF-κB-mediated signaling, inducing net anti-inflammatory and tissue-protective metabolic responses. We report the purification and characterization of a NADPH-dependent liver enzyme that reduces the nitroalkene moiety of nitro-oleic acid, yielding the inactive product nitro-stearic acid. Prostaglandin reductase-1 (PtGR-1) was identified as a nitroalkene reductase by protein purification and proteomic studies. Kinetic measurements, inhibition studies, immunological and molecular biology approaches as well as clinical analyses confirmed this identification. Overexpression of PtGR-1 in HEK293T cells promoted nitroalkene metabolism to inactive nitroalkanes, an effect that abrogated the Nrf2-dependent induction of heme oxygenase-1 expression by nitro-oleic acid. These results situate PtGR-1 as a critical modulator of both the steady state levels and signaling activities of fatty acid nitroalkenes in vivo.