Protein tyrosine phosphorylation and protein tyrosine nitration in redox signaling

Reversible phosphorylation of protein tyrosine residues by polypeptide growth factor-receptor protein tyrosine kinases is implicated in the control of fundamental cellular processes including the cell cycle, cell adhesion, and cell survival, as well as cell proliferation and differentiation. During the last decade, it has become apparent that receptor protein tyrosine kinases and the signaling pathways they activate belong to a large signaling network. Such a network can be regulated by various extracellular cues, which include cell adhesion, agonists of G protein-coupled receptors, and oxidants. It is well documented that signaling initiated by receptor protein tyrosine kinases is directly dependent on the intracellular production of oxidants, including reactive oxygen and nitrogen species. Accumulated evidence indicates that the intracellular redox environment plays a major role in the mechanisms underlying the actions of growth factors. Oxidation of cysteine thiols and nitration of tyrosine residues on signaling proteins are described as posttranslational modifications that regulate, positively or negatively, protein tyrosine phosphorylation (PTP). Early observations described the inhibition of PTP activities by oxidants, resulting in increased levels of proteins phosphorylated on tyrosine. Therefore, a redox circuitry involving the increasing production of intracellular oxidants associated with growth-factor stimulation/cell adhesion, oxidative reversible inhibition of protein tyrosine phosphatases, and the activation of protein tyrosine kinases can be delineated.     

Superoxide generation from nitric oxide synthases

Besides nitric oxide (NO), NO synthases (NOS) also produce superoxide ((*)O(2)()), a primary reactive oxygen species involved in both cell injury and signaling. Neuronal NOS was first found to produce (*)O(2)(-) in vitro. Subsequent studies revealed (*)O(2)(-) generation as a common property of all NOS isoforms. Although NOS was originally shown to produce (*)O(2)(-) under defined conditions such as substrate or cofactor depletion, recent enzymatic studies found that the reduction of oxygen to (*)O(2)(-) is an obligatory step in NO synthesis. Tetrahydrobiopterin appears to play a key role in preventing (*)O(2)(-) release from the NOS oxygenase domain. On the other hand, the NOS reductase domain is also capable of producing significant amounts of (*)O(2)(-). Increasing evidence demonstrates that (*)O(2)(-) generation is involved in both physiological and pathological actions of NOS.     

Role of nitric oxide synthases in elastase-induced emphysema

Nitric oxide (NO) in combination with superoxide produces peroxynitrites and induces protein nitration, which participates in a number of chronic degenerative diseases. NO is produced at high levels in the human emphysematous lung, but its role in this disease is unknown. The aim of this study was to determine whether the NO synthases contribute to the development of elastase-induced emphysema in mice. nNOS, iNOS, and eNOS were quantified and immunolocalized in the lung after a tracheal instillation of elastase in mice. To determine whether eNOS or iNOS had a role in the development of emphysema, mice bearing a germline deletion of the eNOS and iNOS genes and mice treated with a pharmacological iNOS inhibitor were exposed to elastase. Protein nitration was determined by immunofluorescence, protein oxidation was determined by ELISA. Inflammation and MMP activity were quantified by cell counts, RT-PCR and zymography in bronchoalveolar lavage fluid. Cell proliferation was determined by Ki67 immunostaining. Emphysema was quantified morphometrically. iNOS and eNOS were diffusely upregulated in the lung of elastase-treated mice and a 12-fold increase in the number of 3-nitrotyrosine-expressing cells was observed. Over 80% of these cells were alveolar type 2 cells. In elastase-instilled mice, iNOS inactivation reduced protein nitration and increased protein oxidation but had no effect on inflammation, MMP activity, cell proliferation or the subsequent development of emphysema. eNOS inactivation had no effect. In conclusion, in the elastase-injured lung, iNOS mediates protein nitration in alveolar type 2 cells and alleviates oxidative injury. Neither eNOS nor iNOS are required for the development of elastase-induced emphysema.     

Expression of nitric oxide synthases in primary ciliary dyskinesia

Nitric oxide is believed to play a central role in nonspecific defense of upper airways. Patients with primary ciliary dyskinesia have very low concentration of nasal nitric oxide, which may contribute to the chronic upper airway diseases encountered by these patients. The mechanisms underlying this drop of nasal nitric oxide in primary ciliary dyskinesia are still unknown. The goal of the present work was to study nitric oxide synthases expression in upper airway tissues from patients with primary ciliary dyskinesia. For this purpose, 5 patients with primary ciliary dyskinesia and 10 nonallergic age-matched patients without primary ciliary dyskinesia undergoing nasal polypectomy were included. Nasal nitric oxide concentration was measured before polypectomy, and nitric oxide synthase expression and function were studied in nasal polyps. The nasal nitric oxide in patients with primary ciliary dyskinesia was lower than that in patients without primary ciliary dyskinesia (13 [9-16] ppb versus 210 [167-254] ppb, P < .0001). Nitric oxide synthase 2 immunostaining was prominent at the apical part of the ciliated epithelial cells and was similar in both groups. Nitric oxide synthase 3 staining was restricted to endothelial cells in both groups. In addition, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was superimposable to nitric oxide synthases 2 and 3 immunostaining, suggesting a preserved NADPH-activity of nitric oxide synthase. We therefore conclude that the drop in nasal nitric oxide in patients with primary ciliary dyskinesia is not secondary to the loss of nitric oxide synthase expression.     

Mifepristone-induced nitric oxide release and expression of nitric oxide synthases in the human cervix during early pregnancy

BACKGROUND: Nitric oxide (NO) is a factor in cervical ripening, perhaps under the control of progesterone. We studied the effects of the antiprogesterone mifepristone on the release of NO and on the expression of inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) in the uterine cervix of women in early pregnancy. METHODS: Thirteen women were treated with oral mifepristone (200 mg), and 15 women were studied as controls. Cervical fluid samples were collected before treatment then hourly up to 3 h, and the samples were assayed for the concentration of nitric oxide metabolites (NOx). In addition, cervical biopsy samples from six women treated with mifepristone and from six controls were assessed for iNOS and eNOS by immunohistochemistry and Western blotting. RESULTS: In 1-3 h, mifepristone induced 7.4- to 17.2-fold elevations in cervical fluid NOx concentrations; no change was seen in the controls. The expression of both iNOS and eNOS was detected in the cervical cells. The expression of cervical iNOS was strong in five of the six women treated with mifepristone but was not strong in any of the six control women. CONCLUSION: This is the first study to show that mifepristone stimulates the release of NO and the expression of iNOS in cervical cells of women in early pregnancy. This may be one mechanism by which mifepristone initiates cervical ripening.     

Expression of nitric oxide synthases in leukocytes in nasal polyps

BackgroundNitric oxide (NO) has various roles in airway physiology and pathophysiology. Monitoring exhaled NO levels is increasingly common to measure airways inflammation and inhaled NO studied for its therapeutic value in premature infants and adult respiratory distress syndrome. NO is produced by 3 isoforms of NO synthase (NOS1, 2, 3), and each can play distinct and perhaps overlapping roles in the airways. However, the distribution, regulation, and functions of NOS in various cells in the upper airways, particularly in leukocytes, are incompletely understood.ObjectiveTo characterize the expression of NOS isoforms in leukocytes in normal middle turbinate tissues (MT) and in inflammatory nasal tissue (nasal polyps, NP).MethodsNormal MT tissue was collected from surgical specimens that were to be discarded. The NP samples were from surgical tissue archives of 15 patients with chronic rhinosinusitis. Isoforms of NOS in cells were identified by double immunostaining using NOS isoform-specific and leukocyte-specific (mast cell, eosinophil, macrophage, neutrophil, or T cell) antibodies.ResultsThe proportion of total cells below the epithelium that were positive for each isoform of NOS was higher in NP than in MT. Each isoform of NOS was found in all leukocyte populations studied, and there were significant differences in the percentage of leukocytes expressing NOS isoforms between MT and NP.ConclusionAll isoforms of NOS are expressed in leukocytes in MT and NP, and their expression varies among leukocyte types. Our data provide a basis to investigate the regulation, cell distribution, and distinct functions of NOS isoforms in normal and inflamed nasal tissues.     

Effects of nitric oxide synthases in chronic allergic airway inflammation and remodeling

The precise role of each nitric oxide (NO) synthase (NOS) isoform in the pathobiology of asthma is not well established. Our objective was to investigate the contribution of constitutive NO synthase (cNOS) and inducible NOS (iNOS) isoforms to lung mechanics and inflammatory and remodeling responses in an experimental model of chronic allergic pulmonary inflammation. Guinea pigs were submitted to seven ovalbumin exposures with increasing doses (1 approximately 5 mg/ml) for 4 wk. The animals received either chronic L-NAME (N-nitro-L-arginine methyl ester, in drinking water) or 1,400 W (iNOS-specific inhibitor, intraperitoneal) treatments. At 72 h after the seventh inhalation of ovalbumin solution, animals were anesthetized, mechanically ventilated, exhaled NO was collected, and lung mechanical responses were evaluated before and after antigen challenge. Both L-NAME and 1,400 W treatments increased baseline resistance and decreased elastance of the respiratory system in nonsensitized animals. After challenge, L-NAME increased resistance of the respiratory system and collagen deposition on airways, and decreased peribronchial edema and mononuclear cell recruitment. Administration of 1,400 W reduced resistance of the respiratory system response, eosinophilic and mononuclear cell recruitment, and collagen and elastic fibers content in airways. L-NAME treatment reduced both iNOS- and neuronal NOS-positive eosinophils, and 1,400 W diminished only the number of eosinophils expressing iNOS. In this experimental model, inhibition of NOS-derived NO by L-NAME treatment amplifies bronchoconstriction and increases collagen deposition. However, blockage of only iNOS attenuates bronchoconstriction and inflammatory and remodeling processes.     

Augmentation of hypoxia-induced nitric oxide generation in the rat carotid body adapted to chronic hypoxia: an involvement of constitutive and inducible nitric oxide synthases

Acute hypoxia increases the endogenous release of nitric oxide (NO) in rat carotid body and the expression of nitric oxide synthases is modulated by chronic hypoxia. The aim of the study was to examine hypoxia-induced NO generation in rat carotid body adapted to chronic hypoxia with inspired oxygen at 10% for 4 weeks. The concentration of NO was measured electrochemically with a Pt/Nafion/Pd-IrOx/POAP modified electrode inserted into the isolated carotid body superfused with bicarbonate-buffer saline at 35 degrees C. Acute hypoxia increased the concentration of NO by 471.3+/-71.4 nM in the carotid body of chronically hypoxic (CH) rats. The amount of NO release induced by hypoxia was significantly augmented when compared with that of the normoxic control (87.6+/-15.9 nM). The hypoxia-induced NO generation was markedly attenuated by pretreatment with L- NG-nitroarginine methylester (L-NAME; 500 microM), a non-selective nitric oxide synthase (NOS) inhibitor and also by removal of extracellular calcium with the calcium chelator EGTA (5 mM). Additionally, NO generation during hypoxia was reduced by 30% in the CH carotid body treated with S-methylisothiourea (SMT; 50 microM), a specific blocker of inducible NOS (iNOS). Immunohistochemical study revealed that positive iNOS protein immunoreactivity was detected in clusters of glomus cells in the carotid bodies of CH rats, but not in the normoxic group. Thus, chronic hypoxia enhances hypoxia-induced NO generation mediated by calcium-dependent NOSs and iNOS in the carotid body. Extracellular recording of sinus nerve activity of CH carotid bodies showed that L-NAME treatment enhanced the afferent discharge in response to hypoxia, confirming that the generation of NO suppresses the activities of carotid chemoreceptors. Taken together, our results suggest that hypoxia-induced NO production increases in the rat carotid body adapted to chronic hypoxia and that constitutive and inducible NOSs are involved in the NO generation. The enhancement of NO generation may play a physiological role in blunting the hypoxic chemosensitivity during chronic hypoxia.     

Type 2 nitric oxide synthase and protein nitration in chronic lung infection

Inflammation in the lung can lead to increased expression of inducible nitric oxide synthase (iNOS) and enhanced NO production. It has been postulated that the resultant highly reactive NO metabolites may have an important role in host defence, although they might also contribute to tissue damage. However, in a number of inflammatory lung diseases, including bronchiectasis, iNOS expression is increased but no elevation of airway NO can be detected. A potential explanation for this finding is that NO is rapidly scavenged by reaction with superoxide radicals, forming peroxynitrite, which is preferentially metabolized via nitration and nitrosation reactions. To test this hypothesis, anaesthetized, specific pathogen-free rats were inoculated with Pseudomonas aeruginosa incorporated into agar beads (chronically infected group) or sterile agar beads (control group). Ten to 15 days later, the lungs were isolated and fixed. Pseudomonas organisms were isolated from the lungs of the chronically infected group. These lungs showed extensive inflammatory cell infiltration and tissue damage, which were not observed in control lungs. Expression of iNOS was increased in the chronically infected group when compared with the control group. However, the mean number of cells staining for nitrotyrosine in the chronically infected group was not significantly different from that in the controls, nor was there an excess of nitrotyrosine, nitrate, nitrite or nitrosothiol concentrations in the infected lungs. Thus, no evidence was found of increased NO metabolites in chronically infected lungs, including products of the peroxynitrite pathway. These findings suggest that chronic infection does not cause increased iNOS activity in the lung, despite increased expression of iNOS.     

Abnormal pressure-natriuresis in hypertension: role of nitric oxide

The kidneys have a critical role in long-term control of arterial pressure by regulating extracellular fluid and plasma volume. According to the renal body fluid feedback mechanism for long-term control, persistent hypertension can only occur as a result of a reduction in renal sodium excretory function or a hypertensive shift in the pressure-natriuresis relationship. Although an abnormal relationship between renal perfusion pressure and renal sodium excretion has been identified in every type of hypertension where it has been sought, factors responsible for this effect are still unclear. Nitric oxide (NO) is produced within the kidney and plays an important role in the control of many intrarenal processes which regulate the renal response to changes in perfusion pressure and thus, help determine plasma volume and blood pressure. Numerous studies have shown that long-term inhibition of NO synthesis results in a chronic rightward shift and marked attenuation in renal pressure-natriuresis. Recent studies have shown that certain animal models of genetic hypertension and forms of human hypertension areas are associated with a decrease in NO synthesis. Reductions in NO synthesis reduces renal sodium excretory function not only through direct actions on the renal vasculature, but through modulation of other vasoconstrictor processes and through direct and indirect alterations in tubular sodium transport. The causes and consequences of the dysregulation of NO in hypertension and other renal disease processes remain an important area of investigation.     

Defects in activation of nitric oxide synthases occur during delayed angiogenesis in aging

Angiogenesis, the formation of new vessels from pre-existing vasculature, is impaired in aging. This is due, in part, to a lack of regulatory molecules such as nitric oxide (NO). We wished to test the hypothesis that there are deficits in the pathways that mediate NO production during angiogenesis (as defined by fibrovascular invasion into a polyvinyl alcohol (PVA) sponge implant), in aged mice in comparison to young mice. Sponges were implanted subcutaneously in young (6-8 months old, n=11) and aged (23-25 months old, n=13) mice and sampled at 14 and 19 days. Sections from the implants were stained with antibodies against vascular endothelial growth factor receptor 2 (VEGFR-2), Akt, phosphorylated Akt (p-Akt), endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), inducible NOS (iNOS), and 3-nitrotyrosine (3-NT, a marker for nitrosylated proteins). Expression of VEGFR-2 was similar in the sponges of young and aged mice. Moreover, there were no significant differences in levels of Akt or its phosphorylated form in sponges from young and aged mice at 14 and 19 d. In marked contrast, levels of eNOS, p-eNOS and iNOS were significantly decreased in sponges from aged mice relative to young mice (p<0.02 for eNOS, p-eNOS and <0.01 for iNOS between young and aged mice). Concomitantly, there was diminished expression of 3-NT in the sponges from aged mice (p<0.05). Our data indicate that defects in the activation of nitric oxide synthases result in decreased NO production in aged tissues relative to young tissues. We propose that the subsequent lack of NO contributes to impaired angiogenesis in aging.     

GnRH agonist-suppressed expression of nitric oxide synthases and generation of peroxynitrite in adenomyosis

Because overproduction of nitric oxide (NO) and peroxynitrite is known to cause tissue injury, the expression of NO synthases (NOS) and generation of peroxynitrite were investigated in adenomyosis. Immunoreactivities to endothelial and inducible NOS demonstrated phase-dependent changes in normal endometrium, and in eutopic endometrium of adenomyosis. However, NOS were expressed throughout the menstrual cycle in ectopic endometrium from the majority of patients with adenomyosis. Nitrotyrosine, a footprint of peroxynitrite, was detected concomitantly with NOS protein. This suggested that high doses of NO and superoxide are produced in the ectopic endometrium, presumably by stimulation with bioactive molecules such as cytokines and growth factors. The expression of NOS and generation of peroxynitrite were markedly reduced by administration of gonadotrophin-releasing hormone agonists (GnRHa). The suppression of serum concentrations of nitrite/nitrate, stable metabolites of NO, by long-term administration of GnRHa was also demonstrated. The suppression of synthesis of NO and/or peroxynitrite may be part of both the therapeutic and adverse effects of GnRHa therapy.     

Constitutive and inducible nitric oxide synthases after dynorphin-induced spinal cord injury

It has recently been demonstrated that selective inhibition of both neuronal constitutive and inducible nitric oxide synthases (ncNOS and iNOS) is neuroprotective in a model of dynorphin (Dyn) A(1-17)-induced spinal cord injury. In the present study, various methods including the conversion of 3H-L-arginine to 3H-citrulline, immunohistochemistry and in situ hybridization are employed to determine the temporal profiles of the enzymatic activities, immunoreactivities, and mRNA expression for both ncNOS and iNOS after intrathecal injection of a neurotoxic dose (20 nmol) of Dyn A(1-17). The expression of ncNOS immunoreactivity and mRNA increased as early as 30 min after injection and persisted for 1-4 h. At 24-48 h, the number of ncNOS positive cells remained elevated while most neurons died. The cNOS enzymatic activity in the ventral spinal cord also significantly increased at 30 min 48 h, but no significant changes in the dorsal spinal cord were observed. However, iNOS mRNA expression increased later at 2 h, iNOS immunoreactivity and enzymatic activity increased later at 4 h and persisted for 24-48 h after injection of 20 nmol Dyn A(1-17). These results indicate that both ncNOS and iNOS are associated with Dyn-induced spinal cord injury, with ncNOS predominantly involved at an early stage and iNOS at a later stage.     

Endothelial expression of nitric oxide synthases and nitrotyrosine in systemic sclerosis skin.

Although a multisystem disease, systemic sclerosis (SSc) most commonly affects the skin. The skin lesion is characterized by progressive changes, chief amongst which are vascular abnormalities, including endothelial cell (EC) injury and death, and dermal fibrosis. The pathogenesis of the vascular changes, and their relationship to dermal fibrosis, is poorly understood. The purpose of this study was to examine the potential role of nitric oxide (NO)-related free radical production, as part of an assessment of mechanisms leading to endothelial damage. Histologically graded skin biopsies from 33 patients with SSc (ten grade 0, ten grade 1, eight grade 2, and five grade 3) and eight healthy controls were reacted with antibodies against constitutive (eNOS) and inducible (iNOS) forms of nitric oxide synthase and nitrotyrosine. The degree of staining was assessed using a semi-quantitative system and a staining score was developed for the ECs of different vessel types in different areas of dermis at all grades. In biopsies from patients with SSc, superficial microvessel ECs showed a peak of eNOS expression in grade 1 skin which fell as the grade increased. By contrast, iNOS staining increased with the grade of skin lesion, a pattern paralleled by endothelial nitrotyrosine expression. From these findings, it is concluded that a metabolic switch occurs in dermal ECs from endothelial to cytokine inducible forms of NOS during the progression of the skin lesion of SSc. iNOS is a potent inducer of NO production which, in turn, can mediate NO free radical production. At a time of development of the SSc skin lesion when previous studies report evidence of EC damage, the cells express immunodetectable nitrotyrosine, a marker of NO-mediated free radical injury. The data suggest a role for iNOS-induced NO production in EC damage in SSc.     

Nonspecific defence mechanism: the role of nitric oxide.

There is a marked contrast between the extraordinary complexity and specificity of the adaptive immune response and the limited number of effector mechanisms that it can direct. Recently, a great deal of interest has focused on the possible role of nitric oxide (NO) in one of these mechanisms. Here F.Y. Liew and Frank Cox examine the evidence supporting a role for NO in parasitic disease and suggest possible mechanism of NO-mediated parasite damage.     

Nitric oxide in liver fibrosis: The role of inducible nitric oxide synthase

The inducible form of nitric oxide synthase (iNOS) is expressed in hepatic cells in pathological conditions. Its induction is involved in the development of liver fibrosis, and thus iNOS could be a therapeutic target for liver fibrosis. This review summarizes the role of iNOS in liver fibrosis, focusing on 1) iNOS biology, 2) iNOS-expressing liver cells, 3) iNOS-related therapeutic strategies, and 4) future directions.     

Sepsis: emerging role of nitric oxide and selectins

Sepsis--a state of systemic bacterial infection--often leads to multiorgan failure and is associated with high mortality despite the recent advances achieved in intensive care treatment. Many of the ill effects of sepsis are attributed to an abnormally enhanced host inflammatory response that leads to neutrophil recruitment and activation involving selectins, a class of adhesion molecules, in the initial stages. Nitric oxide and its various isoforms have also been implicated in various vascular alterations and directly participate in the cellular toxicity in sepsis. This review briefly describes the role of selectins and nitric oxide in experimental and clinical sepsis as well as the therapeutic outcomes of blocking therapies.