Systemic Biochemical Effects of Inhaled NO in Rats: Increased Expressions of NOS III, Nitrotyrosine-, and Phosphotyrosine-Immunoreactive Proteins in Liver and Kidney Tissues

Inhaled nitric oxide (iNO) has been shown to reduce pulmonary hypertension associated with several disease states. The effects of iNO are thought to be restricted to the pulmonary vasculature because of its rapid inactivation by hemoglobin. Recent data have suggested, however, that iNO can form nitrosothiols, which can be carried throughout the circulation, thus increasing the half life and bioactivity on NO. Other studies have shown that iNO can affect intestinal ischemia and renal hemodynamics. In this study, rats were exposed to 49 +/- 4 ppm or 107 +/- 13 ppm NO for 4 h and the lung, spleen, liver, and kidney tissues were removed and measured for NOS II and NOS III protein, nitrotyrosine (NT), and phosphotyrosine (PT) immunoreactivity. Following 107 ppm iNO, increases in NOS III protein expression, NT, and PT were observed in the liver and kidney, but not in the lung or spleen. No such increases were noted after the lower dose of iNO. These results paralleled those shown for isobutyl nitrite that we reported earlier and indicated that iNO can cause changes in protein chemistry in organs and tissues beyond the lungs. Since iNO produced little systemic hemodynamic effects, it is unlikely that the observed biochemical alterations were derived secondarily from physiological changes.     

Profound biopterin oxidation and protein tyrosine nitration in tissues of ApoE-null mice on an atherogenic diet: contribution of inducible nitric oxide synthase

Diminished nitric oxide (NO) bioactivity and enhanced peroxynitrite formation have been implicated as major contributors to atherosclerotic vascular dysfunctions. Hallmark reactions of peroxynitrite include the accumulation of 3-nitrotyrosine (3-NT) in proteins and oxidation of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH(4)). The present study sought to 1) quantify the extent to which 3-NT accumulates and BH(4) becomes oxidized in organs of apolipoprotein E-deficient (ApoE(-/-)) atherosclerotic mice and 2) determine the specific contribution of inducible NOS (iNOS) to these processes. Whereas protein 3-NT and oxidized BH(4) were undetected or near the detection limit in heart, lung, and kidney of 3-wk-old ApoE(-/-) mice or ApoE(-/-) mice fed a regular chow diet for 24 wk, robust accumulation was evident after 24 wk on a Western (atherogenic) diet. Since 3-NT accumulation was diminished 3- to 20-fold in heart, lung, and liver in ApoE(-/-) mice missing iNOS, iNOS-derived species are involved in this reaction. In contrast, iNOS-derived species did not contribute to elevated protein 3-NT formation in kidney or brain. iNOS deletion also afforded marked protection against BH(4) oxidation in heart, lung, and kidney of atherogenic ApoE(-/-) mice but not in brain or liver. These findings demonstrate that iNOS-derived species are increased during atherogenesis in ApoE(-/-) mice and that these species differentially contribute to protein 3-NT accumulation and BH(4) oxidation in a tissue-selective manner. Since BH(4) oxidation can switch the predominant NOS product from NO to superoxide, we predict that progressive NOS uncoupling is likely to drive atherogenic vascular dysfunctions.     

Irradiation of the rabbit cornea with UVB rays stimulates the expression of nitric oxide synthases-generated nitric oxide and the formation of cytotoxic nitrogen-related oxidants

Until now, the role of nitric oxide (NO) in cornea irradiated with UVB rays remains unknown. Therefore, we investigated nitric oxide synthase isomers (NOS), enzymes that generate NO, nitrotyrosine (NT), a cytotoxic byproduct of NO, and malondialdehyde (MDA), a byproduct of lipid peroxidation, in rabbit corneas repeatedly irradiated with UVB rays (312 nm, 1x daily for 6 days, the dose per day 1.01 J/cm2) using immunohistochemical methods. The biochemical measurement of nitrite and nitrate has been used for the indirect investigation of NO concentration in the aqueous humor. Results show that in contrast to normal corneas, where of the NOS isomers only endothelial nitric oxide synthase (NOS3) was expressed in a significant amount (in the epithelium and endothelium), in irradiated corneas all NOS isomers (also brain nitric oxide synthase, NOS1, and inducible nitric oxide synthase, NOS2) as well as an indirect measure of ONOO-formation and MDA were gradually expressed, first in the epithelium, the endothelium and the keratocytes beneath the epithelium and finally in the cells of all corneal layers and the inflammatory cells that invaded the corneal stroma. This was accompanied by an elevated concentration of NO in the aqueous humor. In conclusion, repeated irradiation with UVB rays evoked the stimulation of NO production, peroxynitrite formation (demonstrated by NT residues) and lipid peroxidation (evaluated by MDA staining).     

Palmitate-induced NO production has a dual action to reduce cell death through NO and accentuate cell death through peroxynitrite formation

The objective of this study was to determine the role of palmitate-induced stimulation of nitric oxide synthase (NOS) on palmitate-induced cell death, specifically distinguishing the effects of the subtype NOS2 from NOS3, defining the effect of NO on mitochondria death pathways, and determining whether palmitate induces peroxynitrite formation which may impact cardiomyocyte cell survival. Cardiomyocytes from embryonic chick hearts were treated with palmitate 300-500 microM. Cell death was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The ability of palmitate to induce NO production and its consequences were tested by using the NOS inhibitor 7-nitroindazole (7-N) and the peroxynitrite scavenger (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) chloride) (FeTPPS). The effect of palmitate on the mitochondria was assessed by Western blotting for cytochrome c release into the cytosol, and assessment of mitochondrial transmembrane potential (DeltaPsi(m)) by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide staining and immunocytochemistry. The NOS inhibitor 7-N, which is selective for NOS2 and not for NOS3, significantly (p<0.05) increased palmitate-induced cell death. In contrast, 7-N did not alter cell death produced by the combination of potassium cyanide and deoxyglucose, which, respectively, inhibit glycolysis and oxidative phosphorylation. The mitochondrial actions of palmitate, specifically palmitate-induced translocation of mitochondrial cytochrome c to cytosol and loss of mitochondrial transmembrane potential, were not altered by pretreatment with 7-N. FeTPPS, which isomerizes peroxynitrite to nitrate and thereby reduces the toxic effects of peroxynitrite, produced a significant reduction in palmitate-induced cell death. In summary, these data suggest that palmitate stimulates NO production, which has a dual action to protect against cell death or to induce cell death. Palmitate-induced cell death is mediated, in part, through NO generation, which leads to peroxynitrite formation. The protective effect of NO is operative through stimulation of NOS2 but not NOS3. The actions of NO on palmitate-induced cell death are independent of mitochondrial cell death pathways.     

Protein tyrosine nitration in cytokine-activated murine macrophages. Involvement of a peroxidase/nitrite pathway rather than peroxynitrite.

Peroxynitrite, formed in a rapid reaction of nitric oxide (NO) and superoxide anion radical (O(2)), is thought to mediate protein tyrosine nitration in various inflammatory and infectious diseases. However, a recent in vitro study indicated that peroxynitrite exhibits poor nitrating efficiency at biologically relevant steady-state concentrations (Pfeiffer, S., Schmidt, K., and Mayer, B. (2000) J. Biol. Chem. 275, 6346-6352). To investigate the molecular mechanism of protein tyrosine nitration in intact cells, murine RAW 264.7 macrophages were activated with immunological stimuli, causing inducible NO synthase expression (interferon-gamma in combination with either lipopolysaccharide or zymosan A), followed by the determination of protein-bound 3-nitrotyrosine levels and release of potential triggers of nitration (NO, O(2)*, H(2)O(2), peroxynitrite, and nitrite). Levels of 3-nitrotyrosine started to increase at 16-18 h and exhibited a maximum at 20-24 h post-stimulation. Formation of O(2) was maximal at 1-5 h and decreased to base line 5 h after stimulation. Release of NO peaked at approximately 6 and approximately 9 h after stimulation with interferon-gamma/lipopolysaccharide and interferon-gamma/zymosan A, respectively, followed by a rapid decline to base line within the next 4 h. NO formation resulted in accumulation of nitrite, which leveled off at about 50 microm 15 h post-stimulation. Significant release of peroxynitrite was detectable only upon treatment of cytokine-activated cells with phorbol 12-myristate-13-acetate, which led to a 2.2-fold increase in dihydrorhodamine oxidation without significantly increasing the levels of 3-nitrotyrosine. Tyrosine nitration was inhibited by azide and catalase and mimicked by incubation of unstimulated cells with nitrite. Together with the striking discrepancy in the time course of NO/O(2) release versus 3-nitrotyrosine formation, these results suggest that protein tyrosine nitration in activated macrophages is caused by a nitrite-dependent peroxidase reaction rather than peroxynitrite.     

Exercise causes a tissue-specific change of NO production in the kidney and lung.

Nitric oxide (NO) is produced in the vascular endothelium and is a potent vasodilator substance that participates in the regulation of local vascular tone. Exercise causes peculiar changes in systemic and regional blood flow, i.e., an increase of systemic blood flow and a redistribution of local tissue blood flow, by which the blood flow is greatly increased in the working muscles, whereas it is decreased in some organs such as the kidney and intestine. Thus we hypothesized that exercise causes a tissue-specific change of NO production in some internal organs. We studied whether exercise affects expression of NO synthase (NOS) mRNA and protein, NOS activity, and tissue level of nitrite/nitrate (stable end products of NO) in the kidneys (in which blood flow during exercise is decreased) and lungs (in which blood flow during exercise is increased with the increase of cardiac output) of rat. Rats ran on a treadmill for 45 min at a speed of 25 m/min. Immediately after this exercise, kidneys and lungs were quickly removed. Control rats remained at rest during this 45-min period. Expression of endothelial NOS (eNOS) mRNA in the kidneys was markedly lower in exercise rats than in control rats, whereas that in the lungs was significantly higher in exercise rats than in control rats. Western blot analysis confirmed down- and upregulation of eNOS protein in the kidney and lung, respectively, after exercise. On the other hand, neither expression of neuronal NOS (nNOS) mRNA and nNOS protein nor inducible NOS (iNOS) mRNA and iNOS protein in the kidneys and lungs differed between exercise and control rats. NOS activity in the kidney was significantly lower in exercise rats than in control rats, whereas that in the lung was significantly higher in exercise rats than in control rats. On the other hand, the iNOS activity in the kidneys and lungs did not differ between exercise rats and control rats. Tissue nitrite/nitrate level in the kidneys was markedly lower in exercise rats, whereas that in the lungs was significantly higher in exercise rats. The present results show that production of NO is markedly and tissue-specifically changed in the kidney and lung by exercise.     

Heme oxygenase-1 induction improves ischemic renal failure: role of nitric oxide and peroxynitrite

The present study evaluated the effects of heme oxygenase-1 (HO-1) induction on the changes in renal outer medullary nitric oxide (NO) and peroxynitrite levels during 45-min renal ischemia and 30-min reperfusion in anesthetized rats. Glomerular filtration rate (GFR), outer medullary blood flow (OMBF), HO and nitric oxide synthase (NOS) isoform expression, and renal low-molecular-weight thiols (-SH) were also determined. During ischemia significant increases in NO levels and peroxynitrite signal were observed (from 832.1 +/- 129.3 to 2,928.6 +/- 502.0 nM and from 3.8 +/- 0.7 to 9.0 +/- 1.6 nA before and during ischemia, respectively) that dropped to preischemic levels during reperfusion. OMBF and -SH significantly decreased after 30 min of reperfusion. Twenty-four hours later, an acute renal failure was observed (GFR 923.0 +/- 66.0 and 253.6 +/- 55.3 microl.min(-1).g kidney wt(-1) in sham-operated and ischemic kidneys, respectively; P < 0.05). The induction of HO-1 (CoCl(2) 60 mg/kg sc, 24 h before ischemia) decreased basal NO concentration (99.7 +/- 41.0 nM), although endothelial and neuronal NOS expression were slightly increased. CoCl(2) administration also blunted the ischemic increase in NO and peroxynitrite (maximum values of 1,315.6 +/- 445.6 nM and 6.3 +/- 0.5 nA, respectively; P < 0.05), preserving postischemic OMBF and GFR (686.4 +/- 45.2 microl.min(-1).g kidney wt(-1)). These beneficial effects of CoCl(2) on ischemic acute renal failure seem to be due to HO-1 induction, because they were abolished by stannous mesoporphyrin, a HO inhibitor. In conclusion, HO-1 induction has a protective effect on ischemic renal failure that seems to be partially mediated by decreasing the excessive production of NO with the subsequent reduction in peroxynitrite formation observed during ischemia.     

Inhaled nitric oxide induced NOS inhibition and rebound pulmonary hypertension: a role for superoxide and peroxynitrite in the intact lamb

Previous in vivo studies indicate that inhaled nitric oxide (NO) decreases nitric oxide synthase (NOS) activity and that this decrease is associated with significant increases in pulmonary vascular resistance (PVR) upon the acute withdrawal of inhaled NO (rebound pulmonary hypertension). In vitro studies suggest that superoxide and peroxynitrite production during inhaled NO therapy may mediate these effects, but in vivo data are lacking. The objective of this study was to determine the role of superoxide in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy in vivo. In control lambs, 24 h of inhaled NO (40 ppm) decreased NOS activity by 40% (P<0.05) and increased endothelin-1 levels by 64% (P<0.05). Withdrawal of NO resulted in an acute increase in PVR (60.7%, P<0.05). Associated with these changes, superoxide and peroxynitrite levels increased more than twofold (P<0.05) following 24 h of inhaled NO therapy. However, in lambs treated with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) during inhaled NO therapy, there was no change in NOS activity, no increase in superoxide or peroxynitrite levels, and no increase in PVR upon the withdrawal of inhaled NO. In addition, endothelial NOS nitration was 18-fold higher (P<0.05) in control lambs than in PEG-SOD-treated lambs following 24 h of inhaled NO. These data suggest that superoxide and peroxynitrite participate in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy. Reactive oxygen species scavenging may be a useful therapeutic strategy to ameliorate alterations in endogenous NO signaling during inhaled NO therapy.     

Nitrite accumulation and nitric oxide emission in relation to cellular signaling in nitrite reductase antisense tobacco

An antisense nitrite reductase (NiR, EC 1.7.7.1) tobacco ( Nicotiana tabacum L.) transformant (clone 271) was used to gain insight into a possible correlation between nitrate reductase (NR, EC 1.6.6.1)-dependent nitrite accumulation and nitric oxide (NO(.)) production, and to assess the regulation of signal transduction in response to stress conditions. Nitrite concentrations of clone 271 leaves were 10-fold, and NO(.) emission rates were 100-fold higher than in wild type leaves. Increased protein tyrosine nitration in clone 271 suggests that high NO(.) production resulted in increased peroxynitrite (ONOO(-)) formation. Tyrosine nitration was also observed in vitro by adding peroxynitrite to leaf extracts. As in mammalian cells, NO(.) and derivatives also increased synthesis of proteins like 14-3-3 and cyclophilins, which are both involved in regulation of activity and stability of enzymes.     

Endotoxaemia in rats: role of leukocyte sequestration in rapid pulmonary nitric oxide synthase-2 expression.

Nitric oxide (NO), depending on the amount, time and source of generation may exert both, protective and deleterious actions during endotoxic acute lung injury (ALI). Evaluation of the expression and localization of NOS isoforms in the lung of lipopolysaccharide (LPS)-treated rats may contribute to understanding the role of NO in pathogenesis of ALI. Tissue samples (lung, heart, liver, kidney and spleen) as well as peripheral blood polymorphonuclear cells (PMNs) were collected from control male Wistar rats and LPS - treated animals, 15, 30, 60, 120 and 180 min after LPS injection (2 mg kg(-1) min(-1) for 10 minutes, i.v.). Levels of NOS-2 and NOS-3 mRNA and protein in tissues and PMNs were estimated by RT-PCR, Northern blotting and Western blotting. Additionally, myeloperoxidase (MPO) activity in tissue samples was assayed. NOS-3 mRNA as well as protein were detected in lungs of control animals; pulmonary NOS-3 expression was not influenced by LPS. The induction of NOS-2 mRNA in rat lungs and in PMNs isolated from peripheral blood was observed 15 minutes after LPS challenge. In contrast, increase of NOS-2 mRNA in the heart, kidneys, liver and spleen was observed 2-3 hours after LPS injection. In all tissues rise in NOS-2 mRNA was followed after 1-2 hours by increase of NOS-2 protein. Importantly, progressive leukocyte sequestration in the lung parenchyma that started as early as 15 min after LPS injection was revealed only in the lungs; in other organs no significant changes in MPO activity were detected up to 180 min after LPS injection. In conclusion, infusion of LPS caused much more rapid expression of NOS-2 in lungs as compared to the heart, kidneys, liver and spleen. Early induction of NOS-2 may depend on the LPS-stimulated rapid neutrophil sequestration within lung vasculature and fast induction of NOS-2 in sequestrated neutrophils.     

Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.     

The overexpression of copper-zinc superoxide dismutase protects NOS III from nitric oxide-mediated inhibition

Previously, we have demonstrated that increased superoxide generation plays a role in the nitric oxide (NO)-mediated inhibition of endothelial NO synthase (NOS III) in endothelial cells (ECs). In this study we demonstrate that the source of the superoxide is likely due to both NADPH oxidase and NOS III itself. Further, this increase appears to be linked to the activation of PKC, as PMA could mimic the increase and PKC inhibition ameliorate the increase. To further investigate this phenomenon we determined the effect of overexpression of copper-zinc superoxide dismutase (CuZn-SOD) and Manganese-SOD (Mn-SOD) on the inhibitory effects of NO. Using adenoviral infection we demonstrated that SOD activity was increased and superoxide levels decreased, in both CuZn-SOD and Mn-SOD overexpressing cells compared to cells infected with an adenovirus expressing bacterial beta-galactosidase protein. However, only the CuZn-SOD overexpression reduced the NO-mediated inhibition of NOS III. In addition, the level of NO-induced peroxynitrite generation and nitrated NOS III protein were reduced only in the CuZn-SOD overexpressing cells. In conclusion, our results indicate that superoxide and peroxynitrite are involved in the inhibition of NOS III by NO, and that the scavenging of superoxide may be necessary to prevent NOS III inhibition during treatments that involve inhaled NO or NO donors.     

Nitric Oxide Production and Perivascular Tyrosine Nitration Following Focal Ischemia in Neonatal Rat

Abstract: Oxygen free radicals and nitric oxide (NO^•) have been proposed to be involved in acute CNS injury produced by cerebral ischemia; however, controversy remains regarding how they cause injury. Because superoxide generation is triggered during reperfusion, the cytotoxic oxidant peroxynitrite could be formed, but it is not known if this occurs. Dot blot and immunohistochemistry studies were performed on the magnitude and time course of tyrosine nitration and inducible NO synthase (NOS2) in the postischemic rat pup brain. Neonatal ischemia was induced by permanent left middle cerebral artery occlusion in association with 1-h occlusion of the left common carotid artery in 7-day-old Wistar pups. Nitrotyrosine (NT) immunoreactivity was evident in the blood vessels close to the cortical infarct at 48–72 h of recovery, and T lymphocytes were involved with this production. NOS2 immunoreactivity was seen in neutrophils in the same vessels and in the parenchyma at 72 h of recirculation. Whereas NT staining decreased with time, NOS2-positive neutrophils could be still detected in arachnoid vessels at 14 days of recirculation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events that lead to brain oxidative stress in neonatal ischemia. Moreover, NO-related species may serve as a signaling function instead of directly mediating toxicity.     

Oxidative stress and inhibition of oxidative phosphorylation induced by peroxynitrite and nitrite in rat brain subcellular fractions

Nitrite and nitrate, two endogenous oxides of nitrogen, are toxic in vivo. Furthermore, the reaction of superoxide (produced by all aerobic cells) with nitric oxide (NO) generates peroxynitrite, a potent oxidizing agent, that can cause biological oxidative stress. Using subcellular fractions from rat brain hemispheres we studied oxidative stress induced by these nitrogen compounds with special emphasis on nitrite. The consumption of Vitamin C (ascorbate) and Vitamin E (alpha tocopherol), two of the important nutritional antioxidants, was followed in synaptosomes (nerve-ending particles) and mitochondria along with changes in parameters of mitochondrial oxidative phosphorylation. Nitrite, but not nitrate, oxidized ascorbate without oxidizing alpha tocopherol in both synaptosomes and mitochondria whereas peroxynitrite oxidized both ascorbate and alpha tocopherol. Functionally, both nitrite and peroxynitrite inhibited mitochondrial oxidative phosphorylation. Nitrite was less potent than peroxynitrite when the effects of equal concentrations of the two were compared. However, since nitrite is much more stable than peroxynitrite the impact of nitrite as an oxidant in vivo could be as much or even more significant than peroxynitrite. Nitrate would not have similar action unless it is reduced to nitrite. It is possible that nitrite may impair oxidative phosphorylation through modulating levels of nitric oxide, changing the activity of heme proteins or a mild uncoupling of mitochondria.     

Nitroxyl oxidizes NADPH in a superoxide dismutase inhibitable manner

Nitric oxide synthases (NOS) convert L-arginine and N(omega)-hydroxy-L-arginine to nitric oxide (*NO) and/or nitroxyl (NO(-)) in a NADPH-dependent fashion. Subsequently, *NO/superoxide (O(2-)-derived peroxynitrite (ONOO(-)) consumes one additional mol NADPH. The related stoichiometry of NO(-) and NADPH is unclear. We here describe that NO(-) also oxidizes NADPH in a concentration-dependent manner. In the presence of superoxide dismutase (SOD), which also converts NO(-) to *NO, nitrite accumulation was almost doubled and no oxidation of NADPH was observed. Nitrate yield from NO(-) was low, arguing against intermediate ONOO(-) formation. Thus, biologically formed NO(-) may function as an effective pro-oxidant unless scavenged by SOD and affect the apparent NADPH stoichiometry of the NOS reaction.     

Spatial mapping of pulmonary and vascular nitrotyrosine reveals the pivotal role of myeloperoxidase as a catalyst for tyrosine nitration in inflammatory diseases

Nitrotyrosine (NO(2)Tyr) formation is a hallmark of acute and chronic inflammation and has been detected in a wide variety of human pathologies. However, the mechanisms responsible for this posttranslational protein modification remain elusive. While NO(2)Tyr has been considered a marker of peroxynitrite (ONOO(-)) formation previously, there is growing evidence that heme-protein peroxidase activity, in particular neutrophil-derived myeloperoxidase (MPO), significantly contributes to NO(2)Tyr formation in vivo via the oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (.NO(2)). Coronary arteries from a patient with coronary artery disease, liver and lung tissues from a sickle cell disease patient, and an open lung biopsy from a lung transplant patient undergoing rejection were analyzed immunohistochemically to map relative tissue distributions of MPO and NO(2)Tyr. MPO immunodistribution was concentrated along the subendothelium in coronary tissue and hepatic veins as well as in the alveolar epithelial compartment of lung tissue from patients with sickle cell disease or acute rejection. MPO immunoreactivity strongly colocalized with NO(2)Tyr formation, which was similarly distributed in the subendothelial and epithelial regions of these tissues. The extracellular matrix protein fibronectin (FN), previously identified as a primary site of MPO association in vascular inflammatory reactions, proved to be a major target protein for tyrosine nitration, with a strong colocalization of MPO, NO(2)Tyr, and tissue FN occurring. Finally, lung tissue from MPO(-/-) mice, having tissue inflammatory responses stimulated by intraperitoneal zymosan administration, revealed less subendothelial NO(2)Tyr immunoreactivity than tissue from wild-type mice, confirming the significant role that MPO plays in catalyzing tissue nitration reactions. These observations reveal that (i) sequestration of neutrophil-derived MPO in vascular endothelial and alveolar epithelial compartments is an important aspect of MPO distribution and action in vivo, (ii) MPO-catalyzed NO(2)Tyr formation occurs in diverse vascular and pulmonary inflammatory pathologies, and (iii) extracellular matrix FN is an important target of tyrosine nitration in these inflammatory processes.     

Sodium orthovanadate induced tyrosine phosphorylation of platelet nitric oxide synthase negatively regulates enzyme activity

The post-translational regulation of platelet nitric oxide synthase (NOS) activity is poorly understood. In the present study we examined how tyrosine phosphorylation of NOS, induced by the tyrosine phosphatase inhibitor sodium orthovanadate (VO4), influenced enzyme activity. Platelet NOS was basally tyrosine phosphorylated, but incubation with VO4 (100-1000 microM) led to a concentration-dependent increase in tyrosine phosphorylation of the enzyme with maximal effects observed at 500 microM. Importantly, we observed no change in serine(1179) or threonine(497) phosphorylation. The increased tyrosine phosphorylation was associated with reduced NOS activity and NO bioavailability, as evidenced by measurement of [(3)H]-L-citrulline and cGMP respectively. The signalling events underlying the effects of VO4 were studied using specific inhibitors to kinases that are known to influence NOS activity. Preincubation of platelets with the Src kinase inhibitor PP2 (20 microM) blocked VO4-induced tyrosine phosphorylation of NOS and abolished the effects of VO4 on cGMP formation. The PKC inhibitor Ro-31-8220 (10 microM) had no effect on VO4-induced tyrosine phosphorylation, but did have a modest but significant effect on cGMP formation. In contrast, the PI-3-kinase inhibitor wortmannin (100 nM) had no effect on either tyrosine phosphorylation or cGMP formation. Our data indicate that tyrosine phosphorylation may act to repress NOS activity. Furthermore, VO4 induces a Src-dependent, and to a lesser degree PKC-dependent, inhibition of platelet NOS.     

Evidence of oxidative stress in human corneal diseases.

This study localized malondialdehyde (MDA, a toxic byproduct of lipid peroxidation), nitrotyrosine [NT, a cytotoxic byproduct of nitric oxide (NO)], and nitric oxide synthase isomers (NOS) in normal and diseased human corneas. Normal corneas (n=11) and those with clinical and histopathological diagnoses of keratoconus (n=26), bullous keratopathy (n=17), and Fuchs' endothelial dystrophy (n=12) were examined with antibodies specific for MDA, NT, eNOS (constitutive NOS), and iNOS (inducible NOS). Normal corneas showed little or no staining for MDA, NT, or iNOS, whereas eNOS was detected in the epithelium and endothelium. MDA was present in all disease groups, with each group displaying a distinct pattern of staining. NT was detected in all keratoconus and approximately one half of Fuchs' dystrophy corneas. iNOS and eNOS were evident in all the diseased corneas. Keratoconus corneas showed evidence of oxidative damage from cytotoxic byproducts generated by lipid peroxidation and the NO pathway. Bullous keratopathy corneas displayed byproducts of lipid peroxidation but not peroxynitrite (MDA but not NT). Conversely, Fuchs' dystrophy corneas displayed byproducts of peroxynitrite with little lipid peroxidation (NT > MDA). These data suggest that oxidative damage occurs within each group of diseased corneas. However, each disease exhibits a distinctive profile, with only keratoconus showing prominent staining for both nitrotyrosine and MDA. These results suggest that keratoconus corneas do not process reactive oxygen species in a normal manner, which may play a major role in the pathogenesis of this disease.     

Regulation of nitric oxide production in limb and ventilatory muscles during chronic exercise training

In this study, we evaluated the differential influence of chronic treadmill training (30 m/min, 15% incline, 1 h/day, 5 days/wk) on nitric oxide (NO) production and NO synthase (NOS) isoform expression as well as 3-nitrotyrosine formation (footprint of peroxynitrite) both in limb (gastrocnemius) and ventilatory (diaphragm) muscles. A group of exercise-trained rats and a control group (no training) were examined after a 4-wk experimental period. Exercise training elicited an approximate fourfold rise in gastrocnemius NOS activity and augmented protein expression of the endothelial (eNOS) and neuronal (nNOS) isoforms of NOS to approximately 480% and 240%, respectively. Qualitatively similar but quantitatively smaller elevations in NOS activity and eNOS and nNOS expression were observed in the diaphragm. No detectable inducible NOS (iNOS) protein expression was found in any of the muscle samples. Training increased the intensity of 3-nitrotyrosine only in the gastrocnemius muscle. We conclude that whole body exercise training enhances both limb and ventilatory muscle NO production and that constitutive and not iNOS isoforms are responsible for increased protein tyrosine nitration in trained limb muscles.