Hemin and iron increase synthesis and trigger export of xanthine oxidoreductase from hepatocytes to the circulation.

We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the circulation was identified as a seminal step in affording protection. However, the cellular signaling and export mechanisms underpinning this process were not identified. Here, we present novel data showing hepatocytes upregulate XOR expression/protein abundance and actively release it to the extracellular compartment following exposure to hemopexin-bound hemin, hemin or free iron. For example, murine (AML-12 cells) hepatocytes treated with hemin (10 µM) exported XOR to the medium in the absence of cell death or loss of membrane integrity (2.0 ± 1.0 vs 16 ± 9 µU/mL p < 0.0001). The path of exocytosis was found to be noncanonical as pretreatment of the hepatocytes with Vaculin-1, a lysosomal trafficking inhibitor, and not Brefeldin A inhibited XOR release and promoted intracellular XOR accumulation (84 ± 17 vs 24 ± 8 hemin vs 5 ± 3 control µU/mg). Interestingly, free iron (Fe2+ and Fe3+) induced similar upregulation and release of XOR compared to hemin. Conversely, concomitant treatment with hemin and the classic transition metal chelator DTPA (20 µM) or uric acid completely blocked XOR release (p < 0.01). Our previously published time course showed XOR release from hepatocytes likely required transcriptional upregulation. As such, we determined that both Sp1 and NF-kB were acutely activated by hemin treatment (∼2-fold > controls for both, p < 0.05) and that silencing either or TLR4 with siRNA prevented hemin-induced XOR upregulation (p < 0.01). Finally, to confirm direct action of these transcription factors on the Xdh gene, chromatin immunoprecipitation was performed indicating that hemin significantly enriched (∼5-fold) both Sp1 and NF-kB near the transcription start site. In summary, our study identified a previously unknown pathway by which XOR is upregulated via SP1/NF-kB and subsequently exported to the extracellular environment. This is, to our knowledge, the very first study to demonstrate mechanistically that XOR can be specifically targeted for export as the seminal step in a compensatory response to heme/Fe overload.

Release of hepatic xanthine oxidase (XO) to the circulation is protective in intravascular hemolytic crisis.

Xanthine oxidase (XO) catalyzes the catabolism of hypoxanthine to xanthine and xanthine to uric acid, generating oxidants as a byproduct. Importantly, XO activity is elevated in numerous hemolytic conditions including sickle cell disease (SCD); however, the role of XO in this context has not been elucidated. Whereas long-standing dogma suggests elevated levels of XO in the vascular compartment contribute to vascular pathology via increased oxidant production, herein, we demonstrate, for the first time, that XO has an unexpected protective role during hemolysis. Using an established hemolysis model, we found that intravascular hemin challenge (40 µmol/kg) resulted in a significant increase in hemolysis and an immense (20-fold) elevation in plasma XO activity in Townes sickle cell phenotype (SS) sickle mice compared to controls. Repeating the hemin challenge model in hepatocyte-specific XO knockout mice transplanted with SS bone marrow confirmed the liver as the source of enhanced circulating XO as these mice demonstrated 100% lethality compared to 40% survival in controls. In addition, studies in murine hepatocytes (AML12) revealed hemin mediates upregulation and release of XO to the medium in a toll like receptor 4 (TLR4)-dependent manner. Furthermore, we demonstrate that XO degrades oxyhemoglobin and releases free hemin and iron in a hydrogen peroxide-dependent manner. Additional biochemical studies revealed purified XO binds free hemin to diminish the potential for deleterious hemin-related redox reactions as well as prevents platelet aggregation. In the aggregate, data herein reveals that intravascular hemin challenge induces XO release by hepatocytes through hemin-TLR4 signaling, resulting in an immense elevation of circulating XO. This increased XO activity in the vascular compartment mediates protection from intravascular hemin crisis by binding and potentially degrading hemin at the apical surface of the endothelium where XO is known to be bound and sequestered by endothelial glycosaminoglycans (GAGs).

Immunomodulatory actions of a kynurenine-derived endogenous electrophile.

The up-regulation of kynurenine metabolism induces immunomodulatory responses via incompletely understood mechanisms. We report that increases in cellular and systemic kynurenine levels yield the electrophilic derivative kynurenine-carboxyketoalkene (Kyn-CKA), as evidenced by the accumulation of thiol conjugates and saturated metabolites. Kyn-CKA induces NFE2 like bZIP transcription factor 2- and aryl hydrocarbon receptor-regulated genes and inhibits nuclear factor κB- and NLR family pyrin domain containing 3-dependent proinflammatory signaling. Sickle cell disease (SCD) is a hereditary hemolytic condition characterized by basal inflammation and recurrent vaso-occlusive crises. Both transgenic SCD mice and patients with SCD exhibit increased kynurenine and Kyn-CKA metabolite levels. Plasma hemin and kynurenine concentrations are positively correlated, indicating that Kyn-CKA synthesis in SCD is up-regulated during pathogenic vascular stress. Administration of Kyn-CKA abrogated pulmonary microvasculature occlusion in SCD mice, an important factor in lung injury development. These findings demonstrate that the up-regulation of kynurenine synthesis and its metabolism to Kyn-CKA is an adaptive response that attenuates inflammation and protects tissues.

Endogenous generation of nitro-fatty acid hybrids having dual nitrate ester (RONO2) and nitroalkene (RNO2) substituents.

Organic nitrate esters, long-recognized therapies for cardiovascular disorders, have not been detected biologically. We characterize in rat stomach unsaturated fatty acid nitration reactions that proceed by generation of nitro-nitrate intermediates (NO2-ONO2-FA) via oxygen and nitrite dependent reactions. NO2-ONO2-lipids represent ∼70% of all nitrated lipids in the stomach and they decay in vitro at neutral or basic pH by the loss of the nitrate ester group (-ONO2) from the carbon backbone upon deprotonation of the α-carbon (pKa ∼7), yielding nitrate, nitrite, nitrosative species, and an electrophilic fatty acid nitroalkene product (NO2-FA). Of note, NO2-FA are anti-inflammatory and tissue-protective signaling mediators, which are undergoing Phase II trials for the treatment of kidney and pulmonary diseases. The decay of NO2-ONO2-FA occurs during intestinal transit and absorption, leading to the formation of NO2-FA that were subsequently detected in circulating plasma triglycerides. These observations provide new insight into unsaturated fatty acid nitration mechanisms, identify nitro-nitrate ester-containing lipids as intermediates in the formation of both secondary nitrogen oxides and electrophilic fatty acid nitroalkenes, and expand the scope of endogenous products stemming from metabolic reactions of nitrogen oxides.

Xanthine Oxidase Drives Hemolysis and Vascular Malfunction in Sickle Cell Disease.

OBJECTIVE: Chronic hemolysis is a hallmark of sickle cell disease (SCD) and a driver of vasculopathy; however, the mechanisms contributing to hemolysis remain incompletely understood. Although XO (xanthine oxidase) activity has been shown to be elevated in SCD, its role remains unknown. XO binds endothelium and generates oxidants as a byproduct of hypoxanthine and xanthine catabolism. We hypothesized that XO inhibition decreases oxidant production leading to less hemolysis. Approach and Results: Wild-type mice were bone marrow transplanted with control (AA) or sickle (SS) Townes bone marrow. After 12 weeks, mice were treated with 10 mg/kg per day of febuxostat (Uloric), Food and Drug Administration-approved XO inhibitor, for 10 weeks. Hematologic analysis demonstrated increased hematocrit, cellular hemoglobin, and red blood cells, with no change in reticulocyte percentage. Significant decreases in cell-free hemoglobin and increases in haptoglobin suggest XO inhibition decreased hemolysis. Myographic studies demonstrated improved pulmonary vascular dilation and blunted constriction, indicating improved pulmonary vasoreactivity, whereas pulmonary pressure and cardiac function were unaffected. The role of hepatic XO in SCD was evaluated by bone marrow transplanting hepatocyte-specific XO knockout mice with SS Townes bone marrow. However, hepatocyte-specific XO knockout, which results in >50% diminution in circulating XO, did not affect hemolysis levels or vascular function, suggesting hepatocyte-derived elevation of circulating XO is not the driver of hemolysis in SCD. CONCLUSIONS: Ten weeks of febuxostat treatment significantly decreased hemolysis and improved pulmonary vasoreactivity in a mouse model of SCD. Although hepatic XO accounts for >50% of circulating XO, it is not the source of XO driving hemolysis in SCD.

Nitro-fatty acids as activators of hSIRT6 deacetylase activity.

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 µm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 µm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.

Nitrite elicits divergent NO-dependent signaling that associates with outcome in out of hospital cardiac arrest.

Brain and heart injury cause most out-of-hospital cardiac arrest deaths but limited pharmacotherapy exists to protect these tissues. Nitrite is a nitric oxide precursor that is protective in pre-clinical models of ischemic injury and safe in Phase I testing. Protection may occur by cGMP generation via the sGC pathway or through S-nitrosothiol and nitrated conjugated linoleic acid (NO2-CLA) formation. We hypothesized that nitrite provided during CPR signals through multiple pathways and that activation of signals is associated with OHCA outcome. To this end, we performed a secondary analysis of a phase 1 study of intravenous nitrite administration during resuscitation in adult out-of-hospital cardiac arrest. Associations between whole blood nitrite and derived plasma signals (cGMP and NO2-CLA) with patient characteristics and outcomes were defined using Chi-square or t-tests and multiple logistic regression. Whole blood nitrite levels correlated inversely with plasma NO2-CLA (p = 0.039) but not with cGMP. Patients with shockable rhythms had higher cGMP (p = 0.027), NO2-CLA (p < 0.0001) and trended towards lower nitrite (p = 0.077). Importantly, plasma cGMP and NO2-CLA levels were higher in survivors (p = 0.033 and 0.019) and in those with good neurological outcome (p = 0.046 and 0.021). Nitrite was lower in patients with good neurologic outcome (p = 0.029). cGMP (OR 4.02; 95% CI 1.04-15.54; p = 0.044) and NO2-CLA (OR 3.74; 95% CI 1.11-12.65; p = 0.034) were associated with survival. Nitrite (OR 0.20; 95% CI 0.05-0.08; p = 0.026) and NO2-CLA (OR 3.96; 95% CI 1.01-15.60; p = 0.049) were associated with favorable neurologic outcome. In summary, nitrite administration was associated with increased plasma cGMP and NO2-CLA formation in selected OHCA patients. Furthermore, patients with the highest levels of cGMP and NO2-CLA were more likely to survive and experience better neurological outcomes.

Hemopexin deficiency promotes acute kidney injury in sickle cell disease.

Acute kidney injury (AKI) is a major clinical concern in sickle cell disease (SCD). Clinical evidence suggests that red cell alarmins may cause AKI in SCD, however, the sterile inflammatory process involved has hitherto not been defined. We discovered that hemopexin deficiency in SCD is associated with a compensatory increase in α-1-microglobulin (A1M), resulting in an up to 10-fold higher A1M-to-hemopexin ratio in SCD compared with healthy controls. The A1M-to-hemopexin ratio is associated with markers of hemolysis and AKI in both humans and mice with SCD. Studies in mice showed that excess heme is directed to the kidneys in SCD in a process involving A1M causing AKI, whereas excess heme in controls is transported to the liver as expected. Using genetic and bone marrow chimeric tools, we confirmed that hemopexin deficiency promotes AKI in sickle mice under hemolytic stress. However, AKI was blocked when hemopexin deficiency in sickle mice was corrected with infusions of purified hemopexin prior to the induction of hemolytic stress. This study identifies acquired hemopexin deficiency as a risk factor of AKI in SCD and hemopexin replacement as a potential therapy.

Nrf2 activation protects against lithium-induced nephrogenic diabetes insipidus.

Lithium (Li) is the mainstay pharmacotherapeutic mood stabilizer in bipolar disorder. Its efficacious use is complicated by acute and chronic renal side effects, including nephrogenic diabetes insipidus (NDI) and progression to chronic kidney disease (CKD). The nuclear factor erythroid-derived 2-related factor 2 (Nrf2) pathway senses and coordinates cellular responses to oxidative and electrophilic stress. Here, we identify that graded genetic activation of Nrf2 protects against Li-induced NDI (Li-NDI) and volume wasting via an aquaporin 2-independent mechanism. Renal Nrf2 activity is differentially expressed on functional segments of the nephron, and its activation along the distal tubule and collecting duct directly modulates ion transporter expression, mimicking paradoxical effects of diuretics in mitigating Li-NDI. In addition, Nrf2 reduces cyclooxygenase expression and vasoactive prostaglandin biosynthesis. Pharmacologic activation of Nrf2 confers protective effects, confirming this pathway as a potentially novel druggable target for the prevention of acute and chronic renal sequelae of Li therapy.

Smooth muscle cytochrome b5 reductase 3 deficiency accelerates pulmonary hypertension development in sickle cell mice.

Pulmonary and systemic vasculopathies are significant risk factors for early morbidity and death in patients with sickle cell disease (SCD). An underlying mechanism of SCD vasculopathy is vascular smooth muscle (VSM) nitric oxide (NO) resistance, which is mediated by NO scavenging reactions with plasma hemoglobin (Hb) and reactive oxygen species that can oxidize soluble guanylyl cyclase (sGC), the NO receptor. Prior studies show that cytochrome b5 reductase 3 (CYB5R3), known as methemoglobin reductase in erythrocytes, functions in VSM as an sGC heme iron reductase critical for reducing and sensitizing sGC to NO and generating cyclic guanosine monophosphate for vasodilation. Therefore, we hypothesized that VSM CYB5R3 deficiency accelerates development of pulmonary hypertension (PH) in SCD. Bone marrow transplant was used to create SCD chimeric mice with background smooth muscle cell (SMC)-specific tamoxifen-inducible Cyb5r3 knockout (SMC R3 KO) and wild-type (WT) control. Three weeks after completing tamoxifen treatment, we observed 60% knockdown of pulmonary arterial SMC CYB5R3, 5 to 6 mm Hg elevated right-ventricular (RV) maximum systolic pressure (RVmaxSP) and biventricular hypertrophy in SS chimeras with SMC R3 KO (SS/R3KD) relative to WT (SS/R3WT). RV contractility, heart rate, hematological parameters, and cell-free Hb were similar between groups. When identically generated SS/R3 chimeras were studied 12 weeks after completing tamoxifen treatment, RVmaxSP in SS/R3KD had not increased further, but RV hypertrophy relative to SS/R3WT persisted. These are the first studies to establish involvement of SMC CYB5R3 in SCD-associated development of PH, which can exist in mice by 5 weeks of SMC CYB5R3 protein deficiency.

Hemoglobin alters vitamin carrier uptake and vitamin D metabolism in proximal tubule cells: implications for sickle cell disease.

Kidney disease, including proximal tubule (PT) dysfunction, and vitamin D deficiency are among the most prevalent complications in sickle cell disease (SCD) patients. Although these two comorbidities have never been linked in SCD, the PT is the primary site for activation of vitamin D. Precursor 25-hydroxyvitamin D [25(OH)D] bound to vitamin D-binding protein (DBP) is taken up by PT cells via megalin/cubilin receptors, hydroxylated to the active 1,25-dihydroxyvitamin D [1,25(OH)2D] form, and released into the bloodstream. We tested the hypothesis that cell-free hemoglobin (Hb) filtered into the PT lumen impairs vitamin D uptake and metabolism. Hb at concentrations expected to be chronically present in the ultrafiltrate of SCD patients competed directly with DBP for apical uptake by PT cells. By contrast, uptake of retinol binding protein was impaired only at considerably higher Hb concentrations. Prolonged exposure to Hb led to increased oxidative stress in PT cells and to a selective increase in mRNA levels of the CYP27B1 hydroxylase, although protein levels were unchanged. Hb exposure also impaired vitamin D metabolism in PT cells, resulting in reduced ratio of 1,25(OH)2D:25(OH)D. Moreover, plasma levels of 1,25(OH)2D were reduced in a mouse model of SCD. Together, our data suggest that Hb released by chronic hemolysis has multiple effects on PT function that contribute to vitamin D deficiency in SCD patients.

Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis.

Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO2-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effects of NO2-OA on intracellular GSH homeostasis were evaluated. In addition to upregulation of GSH biosynthesis, we observed that NO2-OA increased intracellular GSSG in an oxidative stress-independent manner. NO2-OA directly inhibited GR in vitro by covalent modification of the catalytic Cys61, with kon of (3.45 ± 0.04) × 103 M-1 s-1, koff of (4.4 ± 0.4) × 10-4 s-1, and Keq of (1.3 ± 0.1) × 10-7 M. Akin to NO2-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im), bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG.

Electrophilic fatty acid nitroalkenes are systemically transported and distributed upon esterification to complex lipids.

Electrophilic nitro-fatty acids [NO2-FAs (fatty acid nitroalkenes)] showed beneficial signaling actions in preclinical studies and safety in phase 1 clinical trials. A detailed description of the pharmacokinetics (PK) of NO2-FAs is complicated by the capability of electrophilic fatty acids to alkylate thiols reversibly and become esterified in various complex lipids, and the instability of the nitroalkene moiety during enzymatic and base hydrolysis. Herein, we report the mechanism and kinetics of absorption, metabolism, and distribution of the endogenously detectable and prototypical NO2-FA, 10-nitro-oleic acid (10-NO2-OA), in dogs after oral administration. Supported by HPLC-high-resolution-MS/MS analysis of synthetic and plasma-derived 10-NO2-OA-containing triacylglycerides (TAGs), we show that a key mechanism of NO2-FA distribution is an initial esterification into complex lipids. Quantitative analysis of plasma free and esterified lipid fractions confirmed time-dependent preferential incorporation of 10-NO2-OA into TAGs when compared with its principal metabolite, 10-nitro-stearic acid. Finally, new isomers of 10-NO2-OA were identified in vivo, and their electrophilic reactivity and metabolism characterized. Overall, we reveal that NO2-FAs display unique PK, with the principal mechanism of tissue distribution involving complex lipid esterification, which serves to shield the electrophilic character of this mediator from plasma and hepatic inactivation and thus permits efficient distribution to target organs.

Nitro-fatty acids: New drug candidates for chronic inflammatory and fibrotic diseases.

Nitrated oleic acid (NO2-OA) was first identified in 2003, and after the characterization of its formation and thiol reactivity, it was used as a prototypical molecule to investigate the physiological actions of endogenous nitrated fatty acids (NO2-FA). Based on in vitro observations showing significant activation of cytoprotective and anti-inflammatory signaling responses by NO2-FA, experiments were designed to determine their pharmacological potential. Supported by strong intellectual protection and favorable pharmacokinetic and pharmacodynamic data, 10-NO2-OA (CXA-10) underwent pharmaceutical development as a drug to treat fibrotic and inflammatory diseases. NO2-FA are at the intersection of three unconventional drug candidate classes that include 1) fatty acids, 2) metabolic intermediates and 3) electrophilic molecules. These three groups use different scaffolds for drug development, are characterized by broad activities and are individually gaining traction as alternatives to mono-target drug therapies. In particular, NO2-FA share key characteristics with currently approved pharmacological agents regarding reactivity, distribution, and mechanism of action. This review first presents the characteristics, liabilities, and opportunities that these different drug candidate classes display, and then discusses these issues in the context of current progress in the preclinical and clinical development of NO2-FA as drugs. Lessons learned from the novel approaches presented herein were considered early on during development to structurally define and improve NO2-FA and their disease targets.

In situ generation, metabolism and immunomodulatory signaling actions of nitro-conjugated linoleic acid in a murine model of inflammation.

Conjugated linoleic acid (CLA) is a prime substrate for intra-gastric nitration giving rise to the formation of nitro-conjugated linoleic acid (NO2-CLA). Herein, NO2-CLA generation is demonstrated within the context of acute inflammatory responses both in vitro and in vivo. Macrophage activation resulted in dose- and time-dependent CLA nitration and also in the production of secondary electrophilic and non-electrophilic derivatives. Both exogenous NO2-CLA as well as that generated in situ, attenuated NF-κB-dependent gene expression, decreased pro-inflammatory cytokine production and up-regulated Nrf2-regulated proteins. Importantly, both CLA nitration and the corresponding downstream anti-inflammatory actions of NO2-CLA were recapitulated in a mouse peritonitis model where NO2-CLA administration decreased pro-inflammatory cytokines and inhibited leukocyte recruitment. Taken together, our results demonstrate that the formation of NO2-CLA has the potential to function as an adaptive response capable of not only modulating inflammation amplitude but also protecting neighboring tissues via the expression of Nrf2-dependent genes.

Evaluation of 10-Nitro Oleic Acid Bio-Elimination in Rats and Humans.

Nitrated fatty acids are endogenously present in human and animal tissues, as well as in plant-derived oils. In particular, 10-nitro oleic acid (10-NO2-OA) potently induces Nrf2-dependent antioxidant gene expression and inhibits TLR4/NF-κB signaling, thus promoting an overall cyto-protective and anti-inflammatory response. 10-NO2-OA has been extensively tested in animal models and is currently undergoing clinical evaluation in humans. Bio-elimination pathways for 10-NO2-OA were evaluated in rats (30 mg/kg·day) and in humans (0.34 mg/kg) using samples obtained from a double-blind, dose-rising clinical trial. Quantitative radiochromatographic/MS analysis indicated that the renal and fecal pathways are the main routes for 10-NO2-OA excretion in rats, and allowed the identification of 4-nitro-octanedioic acid (NO2-8:0-diCOOH) as the most abundant metabolite in rat urine. In addition, high resolution LC-MS/MS analysis revealed the presence of a novel series of urinary metabolites including ω-carboxylation and ß-oxidation products, as well as N-acetylcysteine, taurine and sulfo-conjugates in both rats and humans. Overall, the findings reported herein not only provide valuable tools for the experimental evaluation of 10-NO2-OA levels in vivo, but importantly they also set the basis for monitoring its metabolism during potential clinical interventions in humans.

The Chemical Basis of Thiol Addition to Nitro-conjugated Linoleic Acid, a Protective Cell-signaling Lipid.

Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and ß-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the ß- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to ß-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions.

Novel Roles for Peroxynitrite in Angiotensin II and CaMKII Signaling.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) oxidation controls excitability and viability. While hydrogen peroxide (H2O2) affects Ca(2+)-activated CaMKII in vitro, Angiotensin II (Ang II)-induced CaMKIIδ signaling in cardiomyocytes is Ca(2+) independent and requires NADPH oxidase-derived superoxide, but not its dismutation product H2O2. To better define the biological regulation of CaMKII activation and signaling by Ang II, we evaluated the potential for peroxynitrite (ONOO(-)) to mediate CaMKII activation and downstream Kv4.3 channel mRNA destabilization by Ang II. In vitro experiments show that ONOO(-) oxidizes and modestly activates pure CaMKII in the absence of Ca(2+)/CaM. Remarkably, this apokinase stimulation persists after mutating known oxidation targets (M281, M282, C290), suggesting a novel mechanism for increasing baseline Ca(2+)-independent CaMKII activity. The role of ONOO(-) in cardiac and neuronal responses to Ang II was then tested by scavenging ONOO(-) and preventing its formation by inhibiting nitric oxide synthase. Both treatments blocked Ang II effects on Kv4.3, tyrosine nitration and CaMKIIδ oxidation and activation. Together, these data show that ONOO(-) participates in Ang II-CaMKII signaling. The requirement for ONOO(-) in transducing Ang II signaling identifies ONOO(-), which has been viewed as a reactive damaging byproduct of superoxide and nitric oxide, as a mediator of GPCR-CaMKII signaling.

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.

Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride.

The current perspective holds that the generation of secondary signaling mediators from nitrite (NO2(-)) requires acidification to nitrous acid (HNO2) or metal catalysis. Herein, the use of stable isotope-labeled NO2(-) and LC-MS/MS analysis of products reveals that NO2(-) also participates in fatty acid nitration and thiol S-nitrosation at neutral pH. These reactions occur in the absence of metal centers and are stimulated by autoxidation of nitric oxide ((•)NO) via the formation of symmetrical dinitrogen trioxide (nitrous anhydride, symN2O3). Although theoretical models have predicted physiological symN2O3 formation, its generation is now demonstrated in aqueous reaction systems, cell models and in vivo, with the concerted reactions of (•)NO and NO2(-) shown to be critical for symN2O3 formation. These results reveal new mechanisms underlying the NO2(-) propagation of (•)NO signaling and the regulation of both biomolecule function and signaling network activity via NO2(-)-dependent nitrosation and nitration reactions.