The fine tuning of metabolism, autophagy and differentiation during in vitro myogenesis.

Although the mechanisms controlling skeletal muscle homeostasis have been identified, there is a lack of knowledge of the integrated dynamic processes occurring during myogenesis and their regulation. Here, metabolism, autophagy and differentiation were concomitantly analyzed in mouse muscle satellite cell (MSC)-derived myoblasts and their cross-talk addressed by drug and genetic manipulation. We show that increased mitochondrial biogenesis and activation of mammalian target of rapamycin complex 1 inactivation-independent basal autophagy characterize the conversion of myoblasts into myotubes. Notably, inhibition of autophagic flux halts cell fusion in the latest stages of differentiation and, conversely, when the fusion step of myocytes is impaired the biogenesis of autophagosomes is also impaired. By using myoblasts derived from p53 null mice, we show that in the absence of p53 glycolysis prevails and mitochondrial biogenesis is strongly impaired. P53 null myoblasts show defective terminal differentiation and attenuated basal autophagy when switched into differentiating culture conditions. In conclusion, we demonstrate that basal autophagy contributes to a correct execution of myogenesis and that physiological p53 activity is required for muscle homeostasis by regulating metabolism and by affecting autophagy and differentiation.

A novel approach to study oxidative stress in thyroid diseases: a preliminary study.

BACKGROUND AND OBJECTIVES: Recently, several Authors have emphasized the relationship between oxidative stress and thyroid tumors. Several methods have been proposed in the literature for the measurement of oxidative stress in human tissues, although the high reactivity and short half life of reactive oxygen and nitrogen species make difficult their direct determination. Here we propose a novel approach for the determination of oxidative stress in human tissues, taking into account the relationship between free radicals and thyroid diseases. MATERIALS AND METHODS: Our goal in this preliminary study, was to demonstrate the opportunity to use the coupling of the EPR-spin trapping technique with the hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine, to detect oxidative stress in the human blood of patients with thyroid disease. RESULTS: Our preliminary findings confirm that this is a sensible, precise and valid method to study the oxidative stress and encourage us to continue the project. CONCLUSIONS: Our next goal will be to enroll patients affected by different thyroid diseases and to study the effect of some antioxidants in the management of the disease. This will allow to better understand the pathological path that binds the formation of reactive oxidizing species to the thyroid cancer and eventually to take into account the antioxidant therapy, as a possible additional "therapeutic weapon".

Increased frequency of immunoglobulin (Ig)A-secreting cells following Toll-like receptor (TLR)-9 engagement in patients with Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis affecting mainly infants and children. Human B cells express Toll-like receptor (TLR)-9, whose natural ligands are unmethylated cytosine-guanine dinucleotide (CpG) motifs characteristic of bacterial DNA. The aim of this study was to clarify the pathogenesis of KD analysing the activation status of peripheral blood mononuclear cells (PBMC), focusing on B lymphocyte activation and functions. Ten patients and 10 age-matched healthy donors were recruited from the Bambino Gesù Hospital of Rome, Italy and enrolled into this study. We determined phenotype profile and immunoglobulin (Ig) production of PBMC from KD patients and age-matched controls. We found that the frequency of CD19(+) B lymphocytes and CD19(+) /CD86(+) activated B lymphocytes from KD patients during the acute phase before therapy was increased significantly. Moreover, B lymphocytes of acute-phase KD patients were more prone to CpG oligodeoxynucleotide (ODN) activation compared with the age-matched controls, as assessed by a significant increase of the number of IgA-secreting cells (SC). In the same patients we found a marked increase of IgM, IgG, interleukin (IL)-6 and tumour necrosis factor (TNF)-α production compared with the control group. In addition, in two convalescent KD patients, conventional treatment with intravenous immunoglobulin (IVIG) restored the normal frequency of CD19(+) B cells, the number of IgA-, IgM- and IgG-SC and the production of IL-6 and TNF-α. Our findings indicate that the percentages of peripheral B lymphocytes of acute-phase KD patients are increased and are prone to bacterial activation in terms of increased numbers of IgA-SC and increased production of IL-6 and TNF-α inflammatory cytokines. Thus, our data support the hypothesis of an infectious triggering in KD.

Formation of an adduct by clenbuterol, a beta-adrenoceptor agonist drug, and serum albumin in human saliva at the acidic pH of the stomach: evidence for an aryl radical-based process.

Clenbuterol (CLB) is an antiasthmatic drug used also illegally as a lean muscle mass enhancer in both humans and animals. CLB and amine-related drugs in general are nitrosatable, thus raising concerns regarding possible genotoxic/carcinogenic activity. Oral administration of CLB raises the issue of its possible transformation by salivary nitrite at the acidic pH of gastric juice. In acidic human saliva CLB was rapidly transformed to the CLB arenediazonium ion. This suggests a reaction of CLB with salivary nitrite, as confirmed in aerobic HNO(2) solution by a drastic decrease in nitric oxide, nitrite, and nitrate. In human saliva, both glutathione and ascorbic acid were able to inhibit CLB arenediazonium formation and to react with preformed CLB arenediazonium. The effect of ascorbic acid is particularly pertinent because this vitamin is actively concentrated within the gastric juice. EPR spin trapping experiments showed that preformed CLB arenediazonium ion was reduced to the aryl radical by ascorbic acid, glutathione, and serum albumin, the major protein of saliva. As demonstrated by anti-CLB antibodies and MS, the CLB-albumin interaction leads to the formation of a covalent drug-protein adduct, with a preference for Tyr-rich regions. This study highlights the possible hazards associated with the use/abuse of this drug.

Peroxynitrite-dependent modifications of tyrosine residues in hemoglobin. Formation of tyrosyl radical(s) and 3-nitrotyrosine.

Although peroxynitrite is believed to be one of the most efficient tyrosine-nitrating species of biological relevance so far identified, its nitration efficiency is nevertheless limited. In fact, the nitrating species formed through peroxynitrite decay are caged radicals ((*)OH/(*)NO(2) or, in the presence of carbon dioxide, CO(3)(*-)/(*)NO(2)) and the fraction that escapes from the solvent cage does not exceed 30-35%. One exception may be represented by metal-containing compounds that can enhance the formation of nitrotyrosine through a bimolecular reaction with peroxynitrite. Moreover, if the metal is also regenerated in the reaction, the compound is considered a nitration catalysts and the yield of tyrosine nitration enhanced several fold. Examples of peroxynitrite-dependent nitration catalysts are the Mn-superoxide dismutase, some cytochromes and several metalloporphyrins. On the contrary, it has been claimed that some hemoproteins are scavengers of peroxynitrite and play a role in limiting its biodamaging and bioregulatory activity. In this review, we discuss the case of hemoglobin, which is probably the major target of peroxynitrite in blood. This protein has been reported to protect intracellular and extracellular targets from peroxynitrite-mediated tyrosine nitration. This property is shared with myoglobin and cytochrome c. The possible mechanisms conferring to these proteins a peroxynitrite scavenging role are discussed.

Mechanism of peroxynitrite interaction with ferric hemoglobin and identification of nitrated tyrosine residues. CO(2) inhibits heme-catalyzed scavenging and isomerization.

Hemoproteins are one of the major targets of peroxynitrite in vivo. It has been proposed that the bimolecular heme/peroxynitrite interaction results in both peroxynitrite inactivation (scavenging) and catalysis of tyrosine nitration. In this study, we used spectroscopic techniques to analyze the reaction of peroxynitrite with human methemoglobin (metHb). Although conventional differential spectroscopy did not reveal heme changes, our results suggest that, in the absence of bicarbonate, the heme in metHb reacts bimolecularly with peroxynitrite but is quickly back-reduced by the reaction products. This hypothesis is based on two indirect observations. First, metHb prevents the peroxynitrite-mediated nitration of a target dipeptide, Ala-Tyr, and second, it promotes the isomerization of peroxynitrite to nitrate. Both the scavenging and the isomerization activities of metHb were heme-dependent and inhibited by CO(2). Ferrous cytochrome c was an efficient scavenger of peroxynitrite, but in the ferric form did not show either scavenging or isomerization activities. We found no evidence of an increase in Ala-Tyr nitration with these hemoproteins. Peroxynitrite-treated metHb induced the formation of a long-lived radical assigned to tyrosine by spin-trapping studies. This radical, however, did not allow us to predict an interaction of peroxynitrite with heme. Hb was nitrated by peroxynitrite/CO(2) mainly in tyrosines beta 130, alpha 42, and alpha 140 and, to a lesser extent, alpha 24. The nitration of alpha chain tyrosines more exposed to the solvent (alpha 140 and alpha 24) was higher in CO-Hb and metHb, while nitration of alpha 42, the tyrosine nearest to the heme, was higher in oxyHb. We deduce that the heme/peroxynitrite interaction, which is inhibited in CO-Hb and metHb, affects alpha tyrosine nitration in two opposite ways, i.e., by protecting exposed residues and by promoting nitration of the residue nearest to the heme. Conversely, nitration of beta Tyr 130 was comparable in oxyHb, metHb, and CO-Hb, suggesting a mechanism involving only nitrating species formed during peroxynitrite decay.

Scavenging of peroxynitrite by oxyhemoglobin and identification of modified globin residues.

Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO(2) or hemoproteins, and these reactions affect its fate and toxicity. CO(2) promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO(2) on oxyhemoglobin were determined by mass spectrometry, and we found that alphaTyr42, betaTyr130, and, to a lesser extent, alphaTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with (*)NO(2). Dityrosine (alpha24-alpha42) and disulfides (beta93-beta93 and alpha104-alpha104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species (*)NO(2) mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala-Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala-Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala-Tyr nitration was reduced by 65%. This "sink" function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.

Peroxynitrite induces long-lived tyrosyl radical(s) in oxyhemoglobin of red blood cells through a reaction involving CO2 and a ferryl species.

Peroxynitrite-mediated oxidative chemistry is currently the subject of intense investigation owing to the toxic side effects associated with nitric oxide overproduction. Using direct electron spin resonance spectroscopy (ESR) at 37 degrees C, we observed that in human erythrocytes peroxynitrite induced a long-lived singlet signal at g = 2.004 arising from hemoglobin. This signal was detectable in oxygenated red blood cells and in purified oxyhemoglobin but significantly decreased after deoxygenation. The formation of the g = 2.004 radical required the presence of CO2 and pH values higher than the pKa of peroxynitrous acid (pKa = 6.8), indicating the involvement of a secondary oxidant formed in the interaction of ONOO- with CO2. The g = 2.004 radical yield leveled off at a 1:1 ratio between peroxynitrite and oxyhemoglobin, while CO-hemoglobin formed less radical and methemoglobin did not form the radical at all. These results suggest that the actual oxidant is or is derived from the ONOOCO2- adduct interacting with oxygenated FeII-heme. Spin trapping with 2-methyl-2-nitrosopropane (MNP) of the g = 2.004 radical and subsequent proteolytic digestion of the MNP/hemoglobin adduct revealed the trapping of a tyrosyl-centered radical(s). A similar long-lived unresolved g = 2.004 singlet signal is a common feature of methemoglobin/H2O2 and metmyoglobin/H2O2 systems. We show by spin trapping that these g = 2.004 signals generated by H2O2 also indicated trapping of radicals centered on tyrosine residues. Analysis of visible spectra of hemoglobin treated with peroxynitrite revealed that, in the presence of CO2, oxyhemoglobin was oxidized to a ferryl species, which rapidly decayed to lower iron oxidation states. The g = 2.004 radical may be an intermediate formed during ferrylhemoglobin decay. Our results describe a new pathway of peroxynitrite-dependent hemoglobin oxidation of dominating importance in CO2-containing biological systems and identify the g = 2.004 radical(s) formed in the process as tyrosyl radical(s).

Bilirubin is an effective antioxidant of peroxynitrite-mediated protein oxidation in human blood plasma.

Bilirubin is a bile pigment that may have an important role as an antioxidant. Its antioxidant potential is attributed mainly to the scavenging of peroxyl radicals. We investigated the reaction of bilirubin with peroxynitrite in phosphate buffer and in blood plasma. In phosphate buffer bilirubin was rapidly oxidized by micromolar concentrations of peroxynitrite, and its oxidation yield was higher at alkaline pH with an apparent pKa = 6.9. In contrast, the major oxidation product of bilirubin in plasma was biliverdin, and the pH profile of its oxidation yield showed a slightly increased oxidation at acidic pH without a clear inflection point. The addition of NaHCO3 to bilirubin decreased the peroxynitrite-dependent oxidation, suggesting that the reactive intermediates formed in the reaction between CO2 and peroxynitrite are less efficient oxidants of bilirubin. The antioxidant role of bilirubin was investigated in some peroxynitrite-mediated plasma protein modifications that are enhanced by CO2 (tryptophan oxidation and protein tyrosine nitration) or slightly decreased by CO2 (protein carbonyl groups). Bilirubin in the micromolar concentration range afforded a significant protection against all these oxidative modifications and, notably, protected plasma proteins even when the pigment was added 5 s after peroxynitrite (i.e., when peroxynitrite is completely decomposed). The loss of tryptophan fluorescence triggered by peroxynitrite was a relatively slow process fulfilled only after a few minutes. After this time, bilirubin was unable to reduce the tryptophan loss, and it was unable to reduce previously formed nitrated albumin or previously formed carbonyls. We deduce that bilirubin in plasma cannot react to a significant extent with peroxynitrite, and we suggest that bilirubin, through a hydrogen donation mechanism, participates as a scavenger of secondary oxidants formed in the oxidative process.

Ascorbic acid in human seminal plasma is protected from iron-mediated oxidation, but is potentially exposed to copper-induced damage.

The aim of this study was to assess the interaction of endogenous ascorbate with iron and copper ions in aerobic seminal plasma. The rate of ascorbate consumption was measured by high-performance liquid chromatography and by the concentration of its primary oxidation product, ascorbyl radical (Asc.-) detected by electron spin resonance spectroscopy. The modification in the levels of Asc.- was used to investigate non-invasively and in real time whether metal ions, either present in this fluid or exogenously added, were catalytically active. The Asc.- was detected in seminal plasma as well as in whole semen of all subjects and was unaffected by superoxide dismutase, catalase or metal chelators. These findings and the rapid decrease of Asc.- under nitrogen suggest that Asc.- is probably a result of non-metal-catalysed air auto-oxidation, a reaction generating low levels of reactive oxygen species. Loading of seminal plasma with either Fe2+ or Fe3+ up to a concentration of 50 microM did not increase, or increased only slightly, the rate of ascorbate oxidation. Taking into consideration the concentrations of iron-binding proteins in this fluid, these results suggest that seminal plasma possesses a 'physiological ligand(s)' able to maintain iron ions in a catalytically inactive form. Our results indicate that citrate, which is present in seminal plasma at very high concentrations (10-25 mM), is responsible for the inhibition of iron-dependent catalysis. On the contrary, the loss of ascorbate and the levels of Asc.- were significantly increased by the addition of physiologically relevant concentrations (1 microM) of copper ions (Cu2+ but especially Cu+). We suggest that seminal plasma is potentially exposed to copper-mediated oxidation, a finding that could be of importance in situations of increased copper-loading such as in some pathological conditions or in smoking subjects.

Direct ESR detection or peroxynitrite-induced tyrosine-centred protein radicals in human blood plasma.

Peroxynitrite, the reaction product of O2.- and .NO, is a toxic compound involved in several oxidative processes that modify proteins. The mechanisms of these oxidative reactions are not completely understood. In this study, using direct ESR at 37 degrees C, we observed that peroxynitrite induced in human blood plasma a long-lived singlet signal at g = 2.004 arising from proteins. This signal was not due to a specific plasma protein, because several purified proteins were able to form a peroxynitrite-induced g = 2.004 signal, but serum albumin and IgG showed the most intense signals. Hydroxyurea, a tyrosyl radical scavenger, strongly inhibited the signal, and horseradish peroxidase/H2O2, a radical-generating system known to induce tyrosyl radicals, induced a similar signal. Furthermore peptides containing a Tyr in the central portion of the molecule were able to form a stable peroxynitrite-dependent g = 2.004 signal, whereas peptides in which Tyr was substituted with Gly, Trp or Phe and peptides with Tyr at the N-terminus or near the C-terminus did not form radicals that were stable at 37 degrees C. We suggest that Tyr residues are at least the major radical sources of the peroxynitrite-dependent g = 2.004 signal at 37 degrees C in plasma or in isolated proteins. Although significantly enhanced by CO2/bicarbonate, the signal was detectable in whole plasma at relatively high peroxynitrite concentrations (>2 mM) but, after removal of ascorbate or urate or in dialysed plasma, it was detectable at lower concentrations (100-1000 microM). Our results suggest that the major role of ascorbate and urate is to reduce or 'repair' the radical(s) centred on Tyr residues and not to scavenge peroxynitrite (or nitrosoperoxycarbonate, the oxidant formed in CO2-containing fluids). This mechanism of inhibition by plasma antioxidants may be a means of preserving the physiological functions of peroxynitrite.

One-electron oxidation pathway of peroxynitrite decomposition in human blood plasma: evidence for the formation of protein tryptophan-centred radicals.

Exposure of human blood plasma to peroxynitrite in the presence of 3,5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) resulted in the trapping of a strongly immobilized nitroxide radical adduct. The adduct was due to protein-centred radicals derived not only from serum albumin but also from other major plasma proteins (fibrinogen, IgG, alpha1-antitrypsin and transferrin). Urate significantly protected plasma from the peroxynitrite-induced DBNBS-plasma protein adduct, whereas ascorbate and glutathione were protective at concentrations exceeding those usually found in plasma. Alkylation of plasma -SH groups did not affect the intensity of DBNBS-plasma protein adduct, whereas bicarbonate increased its formation, thus showing a pro-oxidant effect. The DBNBS-plasma protein adduct provided little structural information, but subsequent non-specific-protease treatment resulted in the detection of an isotropic three-line spectrum, indicating the trapping of radicals centred on a tertiary carbon. The nitrogen hyperfine coupling constant of this adduct and its superhyperfine structure were similar to those of DBNBS-tryptophan peptides with the alpha-amino group of tryptophan linked in the amide bond, consistent with a radical adduct formed at C-3 of the indole ring of tryptophan-containing peptides. DBNBS was unable to trap radicals derived from peroxynitrite-treated tyrosine or tyrosine-containing peptides. Methionine treated with peroxynitrite resulted in the trapping of at least two DBNBS-methionine adducts with hyperfine structures different from that of protease-treated DBNBS-plasma proteins. These results demonstrate that peroxynitrite induced in blood plasma the formation of protein radicals centred on tryptophan residues and underline the relevance of the one-electron oxidation pathway of peroxynitrite decomposition in biological fluids.

Role of ascorbate and protein thiols in the release of nitric oxide from S-nitroso-albumin and S-nitroso-glutathione in human plasma.

In this work we investigated the stability in aerobic plasma of two naturally occurring S-nitrosothiols, the S-nitroso adduct of serum albumin (S-NO-albumin) and the S-nitroso adduct of glutathione (S-NO-glutathione). In contrast to their behavior in physiological buffers, in which they are stable, in plasma these S-nitrosothiols showed a slow but continuous release of .NO. In the presence of red blood cells, the .NO was quantitatively oxidized to NO3- with stoichiometric formation of methemoglobin. In the absence of red blood cells, the principal oxidation product was NO2- with small amounts of NO3- (about 1/5 of the amount of NO2-). The release of .NO was also proven by spin trapping experiments with 2-(4-Carboxyphenyl)4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide which, when added to plasma in the presence of S-NO-glutathione, was transformed into 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl. Both dialysable and nondialysable compounds are involved in the release of .NO from S-nitrosothiols. Ascorbate and the thiol group of serum albumin are the plasma components mainly involved in the release of .NO, while endogenous L-cysteine and glutathione play a minor role due to their relative low concentrations. However, in contrast to the thiol-dependent release that is known to induce the formation of disulfides, the ascorbate-dependent release of .NO from S-NO-glutathione resulted in the formation of free sulfhydryls. Our results suggest that in plasma the .NO release from S-NO-albumin and S-NO-glutathione may be regulated by heterolytic NO+ transfer and reductive activation to .NO, rather than by homolytic decomposition of labile S-nitrosothiols.

Nitric oxide-dependent NAD linkage to glyceraldehyde-3-phosphate dehydrogenase: possible involvement of a cysteine thiyl radical intermediate.

Previous studies have demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes NAD(H) linkage to an active site thiol when it comes into contact with .NO-related oxidants. We found that a free-radical generator 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH), which does not release either .NO or .NO-related species, was indeed able to induce the NAD(H) linkage to GAPDH. We performed spin-trapping studies with purified apo-GAPDH to identify a putative thiol intermediate produced by AAPH as well as by .NO-related oxidants. As .NO sources we used .NO gas and two .NO-donors, S-nitroso-N-acetyl-D,L-penicillamine and 3-morpholinosydno-nimine hydrochloride (SIN-1). Because SIN-1 produces .NO and a superoxide radical simultaneously, we also tested the effects of peroxynitrite. All the .NO-related oxidants were able to induce the linkage of NAD(H) to GAPDH and the formation of a protein free-radical identified as a thiyl radical (inhibited by N-ethylmaleimide). .NO gas and the .NO-donors required molecular oxygen to induce the formation of the GAPDH thiyl radical, suggesting the possible involvement of higher nitrogen oxides. Thiyl radical formation was decreased by the reconstitution of GAPDH with NAD+. Apo-GAPDH was a strong scavenger of AAPH radicals, but its scavenging ability was decreased when its cysteine residues were alkylated or when it was reconstituted with NAD+. In addition, after treatment with AAPH, a thiyl radical of GAPDH was trapped at high enzyme concentrations. We suggest that the NAD(H) linkage to GAPDH is mediated by a thiyl radical intermediate not specific to .NO or .NO-related oxidants. The cysteine residue located at the active site of GAPDH (Cys-149) is oxidized by free radicals to a thiyl radical, which reacts with the neighbouring coenzyme to form Cys-NAD(H) linkages. Studies with the NAD+ molecule radio-labelled in the nicotinamide or adenine portion revealed that both portions of the NAD+ molecule are linked to GAPDH.

Morphine affects cytostatic activity of macrophages by the modulation of nitric oxide release.

The serum levels of morphine and its glucuronide metabolites were quantitated in C57BL/6 mice at various intervals following subcutaneous administration of morphine. Since one of the major mechanisms of killing by macrophages is the production of nitric oxide, pharmacokinetics data were correlated with cytostatic activity and the release of NO2- (stable end product of NO metabolism). Morphine and its 3-glucuronide metabolite appear in serum of treated mice, reaching a peak of concentration at 20 min. However, morphine 3'-glucuronide levels were much higher than those of the drug itself, even when the morphine concentration levelled off. Both cytostasis and NO2- production of L1210-activated macrophages were significantly enhanced by opioid treatment immediately after drug injection (peaking after 40 min). In contrast, morphine induced a strong inhibition of both cytostasis and NO2- production 24 h after treatment. The modulation of both cytostasis and NO2- production induced by morphine was completely antagonized by pretreatment of mice with the opioid antagonist naltrexone. The involvement of an inducible isoform of NO synthase was suggested by the inhibitory effects of dexamethasone on NO2- production. These data indicate that in vivo administration of morphine can induce a modulation of the NO biosynthesis of peritoneal macrophages.

Role of thiols in the targeting of S-nitroso thiols to red blood cells.

We compared the nitric oxide (.NO)-releasing characteristics of two NO donors, the S-nitroso adduct of bovine serum albumin (BSANO) and the S-nitroso adduct of L-glutathione (GSNO). In oxygenated phosphate buffer (pH 7.4) and in hemoglobin solution, both NO donors released .NO only in the presence of a low molecular weight thiol (the most active was L-cysteine). The requirement of thiol to release .NO strongly suggests that a transnitrosation reaction occurs between the S-nitroso adduct of the NO donor and the sulfhydryl group of the NO acceptor. The reaction produced a labile S-nitroso-L-cysteine intermediate that released .NO. As shown by spin-trapping experiments, the transnitrosation reaction involved the formation of .NO (trapped by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) and .S radicals (trapped by 5,5'-dimethyl-1-pyrroline N-oxide) of both the NO donors and the NO acceptor (L-cysteine). The reaction leading to .S radical formation was distinct from the transnitrosation reaction, since it was oxygen-dependent. We suggest that .S radicals are formed from oxidizing species produced after a reaction between .NO and molecular oxygen (.NO2 is a likely candidate). As for pure .NO gas, the major oxidation product of NO donors, in phosphate buffer (pH 7.4), was NO2-, with no formation of NO3-. In the presence of oxyhemoglobin, both NO donors produced only NO3-. BSANO and GSNO showed distinct patterns of .NO release both in phosphate buffer and in the presence of hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Antioxidant potential of anaerobic human plasma: role of serum albumin and thiols as scavengers of carbon radicals.

Extracellular fluids contain low-molecular-weight antioxidants that are actively involved in the defense against reactive oxygen species. The antioxidant activity of these compounds is largely due to their ability to trap oxygen radicals. Less known is the ability of extracellular antioxidants to scavenge carbon-centered free radicals (C-radicals). These radicals can be involved in the damage under hypoxic/anoxic conditions as well as in ischemia/reperfusion injury. We studied the reactivity of some plasma antioxidants toward a water-soluble C-radical generated by the azocompound 2,2'-azobis(2-amidinopropane) hydrochloride (AAP) under anaerobic conditions. The AAP C-radical in plasma was trapped by the spin trap 3,5-dibromo-4-nitrosobenzene-sulfonic acid (DBNBS) and produced a DBNBS radical. The scavenging properties of urate, cysteine, glutathione, natural amino acids, and serum albumin were assessed by the inhibition of the intensity of DBNBS radical. The antioxidant activity of ascorbate and that of vitamin E was measured directly by the formation of their free radicals. Urate, vitamin E and non-SH amino acids were ineffective and ascorbate was a poor scavenger of AAP C-radical. At variance, cysteine and glutathione (0.1-1.0 mM) were effective scavengers of AAP C-radicals and, importantly, protected plasma ascorbate from oxidation under both aerobic or anaerobic conditions. Our data show that ascorbate in aerobic plasma can reduce vitamin E radical and the oxidized ascorbate may be recycled by a thiol antioxidant cycle. Low-molecular-weight antioxidants accounted only partially for plasma scavenging activity of C-radicals. Plasma strongly reduced the intensity of DBNBS radical and, after dialysis, its activity was reduced by approximately 10%. Serum albumin showed an antioxidant activity comparable to dialyzed plasma. Also the cysteine residue of serum albumin was an efficient scavenger of C-radicals as shown by approximately 20% decrease in the protein scavenging activity after thiol alkylation. These results suggest that elevation in the concentration of total reduced thiols in plasma may improve its antioxidant activity under hypoxic/anoxic conditions. This may be particularly useful since other important antioxidant mechanisms such as urate, ascorbate, and vitamin E appear to be inefficient.

gp120 HIV envelope glycoprotein increases the production of nitric oxide in human monocyte-derived macrophages.

The effect of recombinant gp120 HIV envelope glycoprotein on the generation of free radicals by monocyte-derived macrophages (MDM) was measured by EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). After 1 day in culture, MDM produced a spin trap adduct of DMPO with hyperfine splitting constants superimposable on those of DMPO-OH. The addition of gp120 to MDM increased the production of DMPO-OH and after 1 h, the amount of DMPO-OH produced by 40 micrograms/ml gp120 was about 300% that of untreated MDM. The use of selective inhibitors suggested the participation of the nitric oxide/L-arginine oxidative pathway, but did not provide evidence for trapping of hydroxyl radical or other oxygen free radicals. The specificity of gp120 was proven by two different anti-gp120 antibodies that either inhibited (polyclonal) or increased (monoclonal) the production of free radicals. Dexamethasone inhibited the effect of gp120, suggesting the possible involvement of an inducible nitric oxide (NO) synthase. Moreover, treatment of MDM with gp120 for 15 h increased in a dose-dependent manner the production of NO2-, a stable end product of NO. Soluble CD4 did not modify the intensity of the DMPO-OH adduct, whereas yeast mannan and Ca(2+)-chelators abolished the increase in the DMPO-OH signal induced by gp120. These data suggest the possible involvement of mannose-specific endocytotic lectin of MDM. The reaction of DMPO with sodium nitroprusside, an organic nitrate that releases NO, also produced DMPO-OH. Our findings indicate that gp120 increases free radical production from MDM as detected by spin-trapping methods, and that the spin trap adduct results from a reaction involving NO or closely related oxidized derivatives.