Peroxynitrite supports a metabolic reprogramming in merlin-deficient Schwann cells and promotes cell survival.

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder characterized by the development of bilateral vestibular schwannomas. The NF2 gene encodes the tumor suppressor merlin, and loss of merlin activity promotes tumorigenesis and causes NF2. Cellular redox signaling has been implicated in different stages of tumor development. Among reactive nitrogen species, peroxynitrite is the most powerful oxidant produced by cells. We recently showed that peroxynitrite-mediated tyrosine nitration down-regulates mitochondrial metabolism in tumor cells. However, whether peroxynitrite supports a metabolic shift that could be exploited for therapeutic development is unknown. Here, we show that vestibular schwannomas from NF2 patients and human, merlin-deficient (MD) Schwann cells have high levels of endogenous tyrosine nitration, indicating production of peroxynitrite. Furthermore, scavenging or inhibiting peroxynitrite formation significantly and selectively decreased survival of human and mouse MD-Schwann cells. Using multiple complementary methods, we also found that merlin deficiency leads to a reprogramming of energy metabolism characterized by a peroxynitrite-dependent decrease of oxidative phosphorylation and increased glycolysis and glutaminolysis. In MD-Schwann cells, scavenging of peroxynitrite increased mitochondrial oxygen consumption and membrane potential, mediated by the up-regulation of the levels and activity of mitochondrial complex IV. This increase in mitochondrial activity correlated with a decrease in the glycolytic rate and glutamine dependence. This is the first demonstration of a peroxynitrite-dependent reprogramming of energy metabolism in tumor cells. Oxidized proteins constitute a novel target for therapeutic development not only for the treatment of NF2 schwannomas but also other tumors in which peroxynitrite plays a regulatory role.

Peroxynitrite-Mediated SIRT (Sirtuin)-1 Inactivation Contributes to Nicotine-Induced Arterial Stiffness in Mice.

Objective- Inhibition of SIRT (sirtuin)-1, a nicotinamide adenine dinucleotide-dependent protein deacetylase, is linked to cigarette smoking-induced arterial stiffness, but the underlying mechanisms remain largely unknown. The aim of the present study was to determine the effects and mechanisms of nicotine, a major component of cigarette smoke, on SIRT1 activity and arterial stiffness. Approach and Results- Arterial stiffness, peroxynitrite (ONOO-) formation, SIRT1 expression and activity were monitored in mouse aortas of 8-week-old C57BL/6 mice (wild-type) or Sirt1-overexpressing ( Sirt1 Super) mice with or without nicotine for 4 weeks. In aortas of wild-type mice, nicotine reduced SIRT1 protein and activity by ≈50% without affecting its mRNA levels. In those from Sirt1 Super mice, nicotine also markedly reduced SIRT1 protein and activity to the levels that were comparable to those in wild-type mice. Nicotine infusion significantly induced collagen I, fibronectin, and arterial stiffness in wild-type but not Sirt1 Super mice. Nicotine increased the levels of iNOS (inducible nitric oxide synthase) and the co-staining of SIRT1 and 3-nitrotyrosine, a footprint of ONOO- in aortas. Tempol, which ablated ONOO- by scavenging superoxide anion, reduced the effects of nicotine on SIRT1 and collagen. Mutation of zinc-binding cysteine 395 or 398 in SIRT1 into serine (C395S) or (C398S) abolished SIRT1 activity. Furthermore, ONOO- dose-dependently inhibited the enzyme and increased zinc release in recombinant SIRT1. Finally, we found SIRT1 inactivation by ONOO- activated the YAP (Yes-associated protein) resulting in abnormal ECM (extracellular matrix) remodeling. Conclusions- Nicotine induces ONOO-, which selectively inhibits SIRT1 resulting in a YAP-mediated ECM remodeling. Visual Overview- An online visual overview is available for this article.

The effect of nitric oxide on mitochondrial respiration.

This article reviews the interactions between nitric oxide (NO) and mitochondrial respiration. Mitochondrial ATP synthesis is responsible for virtually all energy production in mammals, and every other process in living organisms ultimately depends on that energy production. Furthermore, both necrosis and apoptosis, that summarize the main forms of cell death, are intimately linked to mitochondrial integrity. Endogenous and exogenous •NO inhibits mitochondrial respiration by different well-studied mechanisms and several nitrogen derivatives. Instantaneously, low concentrations of •NO, specifically and reversibly inhibit cytochrome c oxidase in competition with oxygen, in several tissues and cells in culture. Higher concentrations of •NO and its derivatives (peroxynitrite, nitrogen dioxide or nitrosothiols) can cause irreversible inhibition of the respiratory chain, uncoupling, permeability transition, and/or cell death. Peroxynitrite can cause opening of the permeability transition pore and opening of this pore causes loss of cytochrome c, which in turn might contribute to peroxynitrite-induced inhibition of respiration. Therefore, the inhibition of cytochrome c oxidase by •NO may be involved in the physiological and/or pathological regulation of respiration rate, and its affinity for oxygen, which depend on reactive nitrogen species formation, pH, proton motriz force and oxygen supply to tissues.

Oxygen radicals, nitric oxide, and peroxynitrite: Redox pathways in molecular medicine.

Oxygen-derived free radicals and related oxidants are ubiquitous and short-lived intermediates formed in aerobic organisms throughout life. These reactive species participate in redox reactions leading to oxidative modifications in biomolecules, among which proteins and lipids are preferential targets. Despite a broad array of enzymatic and nonenzymatic antioxidant systems in mammalian cells and microbes, excess oxidant formation causes accumulation of new products that may compromise cell function and structure leading to cell degeneration and death. Oxidative events are associated with pathological conditions and the process of normal aging. Notably, physiological levels of oxidants also modulate cellular functions via homeostatic redox-sensitive cell signaling cascades. On the other hand, nitric oxide (•NO), a free radical and weak oxidant, represents a master physiological regulator via reversible interactions with heme proteins. The bioavailability and actions of •NO are modulated by its fast reaction with superoxide radical ([Formula: see text]), which yields an unusual and reactive peroxide, peroxynitrite, representing the merging of the oxygen radicals and •NO pathways. In this Inaugural Article, I summarize early and remarkable developments in free radical biochemistry and the later evolution of the field toward molecular medicine; this transition includes our contributions disclosing the relationship of •NO with redox intermediates and metabolism. The biochemical characterization, identification, and quantitation of peroxynitrite and its role in disease processes have concentrated much of our attention. Being a mediator of protein oxidation and nitration, lipid peroxidation, mitochondrial dysfunction, and cell death, peroxynitrite represents both a pathophysiologically relevant endogenous cytotoxin and a cytotoxic effector against invading pathogens.

Role of Peroxynitrite-Induced Activation of Poly(ADP-Ribose) Polymerase (PARP) in Circulatory Shock and Related Pathological Conditions.

Peroxynitrite is a powerful oxidant, formed from the reaction of nitric oxide and superoxide. It is known to interact and modify different biological molecules such as DNA, lipids and proteins leading to alterations in their structure and functions. These events elicit various cellular responses, including cell signaling, causing oxidative damage and committing cells to apoptosis or necrosis. This review discusses nitrosative stress-induced modification in the DNA molecule that results in the formation of 8-nitroguanine and 8-oxoguanine, and its role in disease conditions. Different approaches of cell death, such as necrosis and apoptosis, are modulated by cellular high-energy species, such as ATP and NAD+. High concentrations of peroxynitrite are known to cause necrosis, whereas low concentrations lead to apoptosis. Any damage to DNA activates cellular DNA repair machinery, like poly(ADP-ribose) polymerase (PARP). PARP-1, an isoform of PARP, is a DNA nick-sensing enzyme that becomes activated upon sensing DNA breakage and triggers the cleavage of NAD+ into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins. Peroxynitrite-induced hyperactivation of PARP causes depletion of NAD+ and ATP culminating cell dysfunction, necrosis or apoptosis. This mechanistic pathway is implicated in the pathogenesis of a variety of diseases, including circulatory shock (which is characterized by cellular hypoxia triggered by systemic altered perfusion and tissue oxygen utilization leading end-organ dysfunction), sepsis and inflammation, injuries of the lung and the intestine. The cytotoxic effects of peroxynitrite centering on the participation of PARP-1 and ADP-ribose in previously stated diseases have also been discussed in this review.

Peroxynitrite, a potent macrophage-derived oxidizing cytotoxin to combat invading pathogens.

Macrophages are among the first cellular actors facing the invasion of microorganisms. These cells are able to internalize pathogens and destroy them by means of toxic mediators, many of which are produced enzymatically and have strong oxidizing capacity. Indeed, macrophages count on the NADPH oxidase complex activity, which is triggered during pathogen invasion and leads to the production of superoxide radical inside the phagosome. At the same time, the induction of nitric oxide synthase results in the production of nitric oxide in the cytosol which is able to readily diffuse to the phagocytic vacuole. Superoxide radical and nitric oxide react at diffusion controlled rates with each other inside the phagosome to yield peroxynitrite, a powerful oxidant capable to kill micro-organisms. Peroxynitrite toxicity resides on oxidations and nitrations of biomolecules in the target cell. The central role of peroxynitrite as a key effector molecule in the control of infections has been proven in a wide number of models. However, some microorganisms and virulent strains adapt to survive inside the potentially hostile oxidizing microenvironment of the phagosome by either impeding peroxynitrite formation or rapidly detoxifying it once formed. In this context, the outcome of the infection process is a result of the interplay between the macrophage-derived oxidizing cytotoxins such as peroxynitrite and the antioxidant defense machinery of the invading pathogens.

Bioenergetic deficits in peripheral nerve sensory axons during chemotherapy-induced neuropathic pain resulting from peroxynitrite-mediated post-translational nitration of mitochondrial superoxide dismutase.

Many of the widely used anticancer drugs induce dose-limiting peripheral neuropathies that undermine their therapeutic efficacy. Animal models of chemotherapy-induced painful peripheral neuropathy (CIPN) evoked by a variety of drug classes, including taxanes, vinca alkaloids, platinum-complexes, and proteasome-inhibitors, suggest that the common underlying mechanism in the development of these neuropathies is mitotoxicity in primary nerve sensory axons (PNSAs) arising from reduced mitochondrial bioenergetics [eg adenosine triphosphate (ATP) production deficits due to compromised respiratory complex I and II activity]. The causative mechanisms of this mitotoxicity remain poorly defined. However, peroxynitrite, an important pro-nociceptive agent, has been linked to mitotoxicity in several disease states and may also drive the mitotoxicity associated with CIPN. Our findings reveal that the development of mechano-hypersensitivity induced by paclitaxel, oxaliplatin, and bortezomib was prevented by administration of the peroxynitrite decomposition catalyst Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) without interfering with their anti-tumor effects. Peak CIPN was associated with the nitration and inactivation of superoxide dismutase in the mitochondria, but not in the cytosol, as well as a significant decrease in ATP production within the PNSAs; all of these events were attenuated by MnTE-2-PyP(5+). Our results provide continued support for the role of mitotoxicity in the development of CIPN across chemotherapeutic drug classes, and identify peroxynitrite as a key mediator in these processes, thereby providing the rationale towards development of "peroxynitrite-targeted" therapeutics for CIPN.

Role of peroxynitrite in the cardiovascular dysfunction of septic shock.

The intense systemic inflammatory response characterizing septic shock is associated with an increased generation of free radicals by multiple cell types in cardiovascular and non cardiovascular tissues. The oxygen-centered radical superoxide anion (O2 .-) rapidly reacts with the nitrogen-centered radical nitric oxide (NO.) to form the potent oxidant species peroxynitrite. Peroxynitrite oxidizes multiple targets molecules, either directly or via the secondary generation of highly reactive radicals, resulting in significant alterations in lipids, proteins and nucleic acids, with significant cytotoxic consequences. The formation of peroxynitrite is a key pathophysiological mechanism contributing to the cardiovascular collapse of septic shock, promoting vascular contractile failure, endothelial and myocardial dysfunction, and is also implicated in the occurrence of multiple organ dysfunction in this setting. The recent development of various porphyrin-based pharmacological compounds accelerating the degradation of peroxynitrite has allowed to specifically address these pathophysiological roles of peroxynitrite in experimental septic shock. Such agents, including 5,10,15,20-tetrakis(4- sulfonatophenyl)porphyrinato iron III chloride (FeTTPs), manganese tetrakis(4-N-methylpyridyl)porphyrin (MnTMPyP), Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin) (FP-15) and WW-85, have been shown to improve the cardiovascular and multiple organ failure in small and large animal models of septic shock. Therefore, these findings support the development of peroxynitrite decomposition catalysts as potentially useful novel therapeutic agents to restore cardiovascular function in sepsis.

Biomarkers.

Epidemiologic, clinical, and basic research has identified several antecedent conditions that predispose individuals to heart failure and its predecessor, asymptomatic left ventricular remodeling and dysfunction (stage B heart failure). Many biochemical markers have been described that characterize the remodeling process and the development of cardiac dysfunction. Although natriuretic peptides and cardiac troponin are currently used in the context of diagnosis, risk stratification, and management of stage C and D heart failure, many other biomarkers provide insights into the underlying pathophysiology of left ventricular dysfunction, suggesting new directions for fundamental research or the development of new therapies.

Acetaminophen overdose-induced liver injury in mice is mediated by peroxynitrite independently of the cyclophilin D-regulated permeability transition.

UNLABELLED: Acetaminophen (APAP) is safe at therapeutic dosage but can cause severe hepatotoxicity if used at overdose. The mechanisms of injury are not yet fully understood, but previous reports had suggested that the mitochondrial permeability transition (mPT) may be involved in triggering hepatocellular necrosis. We aimed at inhibiting mitochondrial cyclophilin D (CypD), a key regulator of the mPT, as a potential therapeutic target in APAP hepatotoxicity. Wildtype mice treated with a high dose of APAP (600 mg/kg, intraperitoneal) developed typical centrilobular necrosis, which could not, however, be prevented by cotreatment with the selective CypD inhibitor, Debio 025 (alisporivir, DEB025, a nonimmunosuppressive cyclosporin A analog). Similarly, genetic ablation of mitochondrial CypD in Ppif-null mice did not afford protection from APAP hepatotoxicity. To determine whether APAP-induced peroxynitrite stress might directly activate mitochondrial permeabilization, independently of the CypD-regulated mPT, we coadministered the peroxynitrite decomposition catalyst Fe-TMPyP (10 mg/kg, intraperitoneal, 90 minutes prior to APAP) to CypD-deficient mice. Liver injury was greatly attenuated by Fe-TMPyP pretreatment, and mitochondrial 3-nitrotyrosine adduct levels (peroxynitrite marker) were decreased. Acetaminophen treatment increased both the cytosolic and mitochondria-associated P-JNK levels, but the c-jun-N-terminal kinase (JNK) signaling inhibitor SP600125 was hepatoprotective in wildtype mice only, indicating that the JNK pathway may not be critically involved in the absence of CypD. CONCLUSION: These data support the concept that an overdose of APAP results in liver injury that is refractory to pharmacological inhibition or genetic depletion of CypD and that peroxynitrite-mediated cell injury predominates in the absence of CypD.

Hippocampal neuronal nitric oxide synthase mediates the stress-related depressive behaviors of glucocorticoids by downregulating glucocorticoid receptor.

The molecular mechanisms underlying the behavioral effects of glucocorticoids are poorly understood. We report here that hippocampal neuronal nitric oxide synthase (nNOS) is a crucial mediator. Chronic mild stress and glucocorticoids exposures caused hippocampal nNOS overexpression via activating mineralocorticoid receptor. In turn, hippocampal nNOS-derived nitric oxide (NO) significantly downregulated local glucocorticoid receptor expression through both soluble guanylate cyclase (sGC)/cGMP and peroxynitrite (ONOO(-))/extracellular signal-regulated kinase signal pathways, and therefore elevated hypothalamic corticotrophin-releasing factor, a peptide that governs the hypothalamic-pituitary-adrenal axis. More importantly, nNOS deletion or intrahippocampal nNOS inhibition and NO-cGMP signaling blockade (using NO scavenger or sGC inhibitor) prevented the corticosterone-induced behavioral modifications, suggesting that hippocampal nNOS is necessary for the role of glucocorticoids in mediating depressive behaviors. In addition, directly delivering ONOO(-) donor into hippocampus caused depressive-like behaviors. Our findings reveal a role of hippocampal nNOS in regulating the behavioral effects of glucocorticoids.

Understanding the fate of peroxynitrite in plant cells--from physiology to pathophysiology.

Peroxynitrite (ONOO(-)) is a potent oxidant and nitrating species, generated by the reaction of nitric oxide and superoxide in one of the most rapid reactions known in biology. It is widely accepted that an enhanced ONOO(-) formation contributes to oxidative and nitrosative stress in various biological systems. However, an increasing number of studies have reported that ONOO(-) cannot only be considered as a mediator of cellular dysfunction, but also behaves as a potent modulator of the redox regulation in various cell signal transduction pathways. Although the formation of ONOO(-) has been demonstrated in vivo in plant cells, the relevance of this molecule during plant physiological responses is still far from being clarified. Admittedly, the detection of protein tyrosine nitration phenomena provides some justification to the speculations that ONOO() is generated during various plant stress responses associated with pathophysiological mechanisms. On the other hand, it was found that ONOO(-) itself is not as toxic for plant cells as it is for animal ones. Based on the concepts of the role played by ONOO(-) in biological systems, this review is focused mainly on the search for potential functions of ONOO(-) in plants. Moreover, it is also an attempt to stimulate a discussion on the significance of protein nitration as a paradigm in signal modulation, since the newest reports identified proteins associated with signal transduction cascades within the plant nitroproteome.

Peroxynitrite-driven mechanisms in diabetes and insulin resistance - the latest advances.

Since its discovery, peroxynitrite has been known as a potent oxidant in biological systems, and a rapidly growing body of literature has characterized its biochemistry and role in the pathophysiology of various conditions. Either directly or by inducing free radical pathways, peroxynitrite damages vital biomolecules such as DNA, proteins including enzymes with important functions, and lipids. It also initiates diverse reactions leading eventually to disrupted cell signaling, cell death, and apoptosis. The potential role and contribution of this deleterious species has been the subject of investigation in several important diseases, including but not limited to, cancer, neurodegeneration, stroke, inflammatory conditions, cardiovascular problems, and diabetes mellitus. Diabetes, obesity, insulin resistance, and diabetes-related complications represent a major health problem at epidemic levels. Therefore, tremendous efforts have been put into investigation of the molecular basics of peroxynitrite-related mechanisms in diabetes. Studies constantly seek new therapeutical approaches in order to eliminate or decrease the level of peroxynitrite, or to interfere with its downstream mechanisms. This review is intended to emphasize the latest findings about peroxynitrite and diabetes, and, in addition, to discuss recent and novel advances that are likely to contribute to a better understanding of peroxynitrite-mediated damage in this disease.

[Pathophysiological mechanisms and treatment of septic shock-induced vasoplegia]. / Physiopathologie et traitement de la vasoplégie au cours du sepsis.

The hemodynamics of septic shock is characterized by a primary reduction of vascular tone, which defines vasoplegia. Septic vasoplegia is due to reduced endogenous production of vasopressin, as well as to the overproduction of vasodilating molecules (nitric oxide, prostacyclin, peroxynitrite and kynurenine) and the opening of ATP-sensitive potassium channels. Treatment is supportive and includes primarily alpha-adrenergic catecholamines. Vasopressin may also be useful, although its place is still controversial. Further agents can improve the vascular responsiveness to catecholamines, most notably low doses hydrocortisone, and, to a lesser extent, activated protein C. Further, innovative therapies, based on recent understanding of pathophysiological mechanisms, might become useful agents to treat septic vasoplegia in the future.

Searching for new strategies for the pharmacological treatment of chronic pain.

Chronic pain is poorly managed and represents a huge unmet medical need that affects humans globally. The traditional multifaceted drug regimens for controlling chronic pain are marginally effective, produce highly variable results and display for the most parts unacceptable side effects. Research efforts over the last decade have identified peroxynitrite (PN) as a critical signaling molecule in the development of pain. Targeting PN is a novel evidence-based approach to develop novel therapeutics for managing chronic pain.

Arachidonic acid inhibits neuronal nitric oxide synthase elicited by proinflammatory stimuli and promotes astrocyte survival with both exogenous and endogenous peroxynitrite via different mechanisms.

Cytosolic phospholipase A(2)-inhibited astrocytes respond to the cocktail lipopolysaccharide/interferon-gamma with an immediate formation of peroxynitrite (ONOO(-)) and a delayed lethal response. Low concentrations of arachidonic acid (ARA; i.e., <0.1 microM) cause tyrosine kinase-dependent inhibition of neuronal nitric oxide synthase (nNOS) activity, thereby suppressing formation of ONOO(-) and the ensuing lethal response. ARA promoted its effects only when given to the cultures just prior to, or in parallel with, the proinflammatory mixture. High concentrations of ARA, i.e., >3 microM, promoted cytoprotection when applied to the cultures up to 50 min after the formation of endogenous ONOO(-) had been completed or up to 30 min after addition of exogenous ONOO(-). The mechanism(s) involved in these responses was, however, independent of tyrosine kinase activation and was in fact mediated by ARA metabolites of the lipoxygenase pathway. These results are consistent with a scenario in which astrocytes respond to low or high amounts of ARA with the triggering of different pathways involved in the inflammatory response. Early nNOS inhibition mediated by very low levels of ARA is indeed critical for nuclear factor-kappaB activation, which is otherwise effectively inhibited by constitutive nitric oxide, and for preventing early formation of ONOO(-). Greater ARA concentrations promote survival in astrocytes committed to death by ONOO(-), a species extensively released under inflammatory conditions, via a mechanism dependent on lipoxygenase metabolism and inhibition of downstream events leading to cell demise.

Pathophysiological roles of peroxynitrite in circulatory shock.

Peroxynitrite is a reactive oxidant produced from nitric oxide and superoxide, which reacts with proteins, lipids, and DNA, and promotes cytotoxic and proinflammatory responses. Here, we overview the role of peroxynitrite in various forms of circulatory shock. Immunohistochemical and biochemical evidences demonstrate the production of peroxynitrite in various experimental models of endotoxic and hemorrhagic shock both in rodents and in large animals. In addition, biological markers of peroxynitrite have been identified in human tissues after circulatory shock. Peroxynitrite can initiate toxic oxidative reactions in vitro and in vivo. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na+/K+ ATPase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of peroxynitrite. In addition, peroxynitrite is a potent trigger of DNA strand breakage, with subsequent activation of the nuclear enzyme poly(ADP-ribose) polymerase, which promotes cellular energetic collapse and cellular necrosis. Additional actions of peroxynitrite that contribute to the pathogenesis of shock include inactivation of catecholamines and catecholamine receptors (leading to vascular failure) and endothelial and epithelial injury (leading to endothelial and epithelial hyperpermeability and barrier dysfunction), as well as myocyte injury (contributing to loss of cardiac contractile function). Neutralization of peroxynitrite with potent peroxynitrite decomposition catalysts provides cytoprotective and beneficial effects in rodent and large-animal models of circulatory shock.

Peritonitis-induced peroxynitrite and lung damage depends on c-Jun NH2-terminal kinase signaling of hematopoietic cells.

OBJECTIVES: Abdominal sepsis is a common, life-threatening condition in critically ill patients, and pseudomonas peritonitis remains a serious clinical complication of peritoneal dialysis. This study was performed to determine whether peritonitis induces lung damage through the c-Jun NH2-terminal kinase. DESIGN: : Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Peritonitis models in the mice. INTERVENTIONS: Wild-type, c-Jun NH2-terminal kinase1, and c-Jun NH2-terminal kinase1 mice were subjected to peritonitis. A c-Jun NH2-terminal kinase inhibitor, SP600125 or leflunomide, was administered to mice immediately after peritonitis. MEASUREMENTS AND MAIN RESULTS: The changes of plasma dihydrorhodamine 123 oxidation level, the myeloperoxidase activity, and extravasations of Evans blue dye of lung in wild-type mice with or without c-Jun NH2-terminal kinase inhibitor; c-Jun NH2-terminal kinase1 mice and c-Jun NH2-terminal kinase1 mice; and chimeric mice (wild-type --> wild-type, c-Jun NH2-terminal kinase1 --> wild-type) with Pseudomonas aeruginosa-induced peritonitis were determined to evaluate the role of c-Jun NH2-terminal kinase signaling of the hematopoietic cells in peritonitis-induced lung damage. Our results showed that peritonitis induced dihydrorhodamine 123 oxidation, myeloperoxidase activity, activator protein-1 (AP-1) DNA binding activity, phosphorylated-c-Jun NH2-terminal kinase and inducible nitric oxide synthase expression, and Evans blue dye extravasations in lungs, and administration of specific c-Jun NH2-terminal kinase inhibitor decreased the peritonitis-induced dihydrorhodamine 123 oxidation and lung damage. Also, both c-Jun NH2-terminal kinase1 and c-Jun NH2-terminal kinase1 mice showed a decreased dihydrorhodamine 123 oxidation and lung damage after peritonitis. Finally, dihydrorhodamine 123 oxidation, reactive oxygen species, inducible nitric oxide synthase expression, and lung damage were decreased in c-Jun NH2-terminal kinase1 --> wild-type but not in wild-type --> c-Jun NH2-terminal kinase1 chimeric mice. CONCLUSIONS: Collectively, our data suggest that peritonitis-induced inducible nitric oxide synthase expression, peroxynitrite production, and lung damage depend on the c-Jun NH2-terminal kinase signaling of the hematopoietic cells.

S-nitrosylation of caspase-3 is the mechanism by which adhesion fibroblasts manifest lower apoptosis.

We have previously found that adhesion fibroblasts exhibit lower apoptosis and higher protein nitration as compared with normal peritoneal fibroblasts. In this study, we sought to determine whether the decreased apoptosis observed in adhesion fibroblasts is caused by lower caspase-3 activity due to an increase in caspase-3 S-nitrosylation. For this study, we have utilized primary cultures of fibroblasts obtained from normal peritoneum and adhesion tissues of the same patient(s). Cells were treated with increasing concentrations of peroxynitrite and cell lysates were immunoprecipitated with anti-caspase-3 polyclonal antibody. The biotinylated proteins were detected using a nitrosylation detection kit. Caspase-3 activity and apoptosis were measured by colorimetric and TUNEL assays, respectively. Our results showed that caspase-3 S-nitrosylation is significantly higher in adhesion fibroblasts as compared with normal peritoneal fibroblasts. This increase in S-nitrosylation resulted in a 30% decrease in caspase-3 activity in adhesion fibroblasts. Peroxynitrite treatment resulted in a dose response increase in caspase-3 S-nitrosylation, leading to a decrease in caspase-3 activity and apoptosis in normal peritoneal fibroblasts. We conclude that S-nitrosylation of caspase-3 is the reason for its decreased activity and subsequent decrease in apoptosis of adhesion fibroblasts. The mechanism by which caspase-3 S-nitrosylation occurs is not fully understood. However, the role of hypoxia in the formation of peroxynitrite via superoxide production may suggest a possible mechanism.

Role of peroxynitrite in the redox regulation of cell signal transduction pathways.

Peroxynitrite is a potent oxidant and nitrating species formed from the reaction between the free radicals nitric oxide and superoxide. An excessive formation of peroxynitrite represents an important mechanism contributing to cell death and dysfunction in multiple cardiovascular pathologies, such as myocardial infarction, heart failure and atherosclerosis. Whereas initial works focused on direct oxidative biomolecular damage as the main route of peroxynitrite toxicity, more recent evidence, mainly obtained in vitro, indicates that peroxynitrite also behaves as a potent modulator of various cell signal transduction pathways. Due to its ability to nitrate tyrosine residues, peroxynitrite affects cellular processes dependent on tyrosine phosphorylation. Peroxynitrite also exerts complex effects on the activity of various kinases and phosphatases, resulting in the up- or downregulation of signalling cascades, in a concentration- and cell-dependent manner. Such roles of peroxynitrite in the redox regulation of key signalling pathways for cardiovascular homeostasis, including protein kinase B and C, the MAP kinases, Nuclear Factor Kappa B, as well as signalling dependent on insulin and the sympatho-adrenergic system are presented in detail in this review.