Altered free radical metabolism in acute mountain sickness: implications for dynamic cerebral autoregulation and blood-brain barrier function.

We tested the hypothesis that dynamic cerebral autoregulation (CA) and blood-brain barrier (BBB) function would be compromised in acute mountain sickness (AMS) subsequent to a hypoxia-mediated alteration in systemic free radical metabolism. Eighteen male lowlanders were examined in normoxia (21% O(2)) and following 6 h passive exposure to hypoxia (12% O(2)). Blood flow velocity in the middle cerebral artery (MCAv) and mean arterial blood pressure (MAP) were measured for determination of CA following calculation of transfer function analysis and rate of regulation (RoR). Nine subjects developed clinical AMS (AMS+) and were more hypoxaemic relative to subjects without AMS (AMS-). A more marked increase in the venous concentration of the ascorbate radical (A(*-)), lipid hydroperoxides (LOOH) and increased susceptibility of low-density lipoprotein (LDL) to oxidation was observed during hypoxia in AMS+ (P < 0.05 versus AMS-). Despite a general decline in total nitric oxide (NO) in hypoxia (P < 0.05 versus normoxia), the normoxic baseline plasma and red blood cell (RBC) NO metabolite pool was lower in AMS+ with normalization observed during hypoxia (P < 0.05 versus AMS-). CA was selectively impaired in AMS+ as indicated both by an increase in the low-frequency (0.07-0.20 Hz) transfer function gain and decrease in RoR (P < 0.05 versus AMS-). However, there was no evidence for cerebral hyper-perfusion, BBB disruption or neuronal-parenchymal damage as indicated by a lack of change in MCAv, S100beta and neuron-specific enolase. In conclusion, these findings suggest that AMS is associated with altered redox homeostasis and disordered CA independent of barrier disruption.

Reaction of peroxynitrite with Mn-superoxide dismutase. Role of the metal center in decomposition kinetics and nitration.

Manganese superoxide dismutase (Mn-SOD), a critical mitochondrial antioxidant enzyme, becomes inactivated and nitrated in vitro and potentially in vivo by peroxynitrite. Since peroxynitrite readily reacts with transition metal centers, we assessed the role of the manganese ion in the reaction between peroxynitrite and Mn-SOD. Peroxynitrite reacts with human recombinant and Escherichia coli Mn-SOD with a second order rate constant of 1.0 +/- 0.2 x 10(5) and 1.4 +/- 0.2 x 10(5) m(-)1 s(-)1 at pH 7.47 and 37 degrees C, respectively. The E. coli apoenzyme, obtained by removing the manganese ion from the active site, presents a rate constant <10(4) m(-)1 s(-)1 for the reaction with peroxynitrite, whereas that of the manganese-reconstituted apoenzyme (apo/Mn) was comparable to that of the holoenzyme. Peroxynitrite-dependent nitration of 4-hydroxyphenylacetic acid was increased 21% by Mn-SOD. The apo/Mn also promoted nitration, but the apo and the zinc-substituted apoenzyme (apo/Zn) enzymes did not. The extent of tyrosine nitration in the enzyme was also affected by the presence and nature (i.e. manganese or zinc) of the metal center in the active site. For comparative purposes, we also studied the reaction of peroxynitrite with low molecular weight complexes of manganese and zinc with tetrakis-(4-benzoic acid) porphyrin (tbap). Mn(tbap) reacts with peroxynitrite with a rate constant of 6.8 +/- 0.1 x 10(4) m(-)1 s(-)1 and maximally increases nitration yields by 350%. Zn(tbap), on the other hand, affords protection against nitration. Our results indicate that the manganese ion in Mn-SOD plays an important role in the decomposition kinetics of peroxynitrite and in peroxynitrite-dependent nitration of self and remote tyrosine residues.

Analysis of superoxide dismutase activity.

Measuring the activity of superoxide dismutases (SODs), the enzymes responsible for maintaining the steady state level of hydrogen peroxide, is challenging because the substrate is unstable at physiological pH and it reacts with itself. Fortunately the rate of reaction with dismutase is far greater than the rate of self reaction. As described in this unit, this activity can be measured indirectly based on competition between SOD and an indicator molecule that reacts avidly with superoxide to produce a measurable change in absorption, thus it is possible to measure total SOD activity or that of CuZn-SOD and MnSOD. The activity can also be measured by an activity stain applied to thin-film agarose or native polyacrylamide gels.

The evolution of free radicals and oxidative stress.

The superoxide free radical has come to occupy an amazingly central role in a wide variety of diseases. Our metabolic focus on aerobic energy metabolism in all cell types, coupled with some chemical peculiarities of the oxygen molecule itself, contribute to the phenomenon. Superoxide is not, as we once thought, just a toxic but unavoidable byproduct of oxygen metabolism. Rather it appears to be a carefully regulated metabolite capable of signaling and communicating important information to the cell's genetic machinery. Redox regulation of gene expression by superoxide and other related oxidants and antioxidants is beginning to unfold as a vital mechanism in health and disease.

The human immunodeficiency virus-1 Tat protein increases cell proliferation, alters sensitivity to zinc chelator-induced apoptosis, and changes Sp1 DNA binding in HeLa cells.

The HIV-1 transcriptional regulatory protein Tat is a pleiotropic factor that represses expression of the human Mn-superoxide dismutase. Tat increases oxidative stress, as shown by decreased glutathione and NADPH levels. These redox changes enhance proliferation and apoptosis and alter the activity of zinc thiolate-containing proteins such as Sp1. Cells stably producing the Tat protein have an increased proliferation rate, which can be inhibited by pretreatment with the antioxidant mercaptopropionylglycine. Conversely, cells exposed to low concentrations of the oxidant paraquat are stimulated to divide. Intermediate and higher paraquat levels result in increased apoptosis or necrosis, respectively, suggesting that the physiological end point depends on the dose of oxidant used. Furthermore, treatment with the zinc chelator (N,N,N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) sensitizes HeLa-tat cells to apoptosis. In these cells, binding of the zinc-containing factor Sp1 to its DNA sequence is higher than in parental cells. Normal DNA binding is partially restored by pretreatment with a compound that mimics superoxide dismutase activity. Interestingly, Sp1-DNA interactions decrease more rapidly in the HeLa-tat cells after TPEN treatment. HeLa cell extracts incubated in the presence of purified Tat protein have increased Sp1 binding, consistent with the results observed in Tat-transfected cells. These results suggest that the Tat protein, via direct or indirect mechanisms, increases proliferation, sensitizes cells to apoptosis, and changes the conformation of Sp1, affecting its ability to bind to its cognate DNA sequence and to retain its zinc.

Transgenic mice with increased copper/zinc-superoxide dismutase activity are resistant to hepatic leukostasis and capillary no-reflow after gut ischemia/reperfusion.

The objectives of this study were to (1) determine whether transgenic (Tg) mice overexpressing copper/zinc-superoxide dismutase (CuZn-SOD) are protected from the deleterious effects of gut ischemia/reperfusion (I/R) and (2) compare the effectiveness of Tg SOD overexpression in attenuating I/R injury to intravascularly administered CuZn-SOD or manganese (Mn)-SOD. The accumulation of fluorescently labeled leukocytes and number of nonperfused sinusoids were monitored by intravital microscopy in livers of wild-type mice (C57BL/6), CuZn-SOD Tg mice, and wild-type mice receiving either CuZn-SOD or Mn-SOD. All parameters were measured for 1 hour after release of the occluded (for 15 minutes) superior mesenteric artery. Gut I/R in wild-type mice led to an increased number of stationary leukocytes, while reducing the number of perfused sinusoids (capillary no-reflow). All of these responses were significantly blunted in CuZn-SOD Tg mice, with a corresponding attenuation of liver enzyme release into plasma. Exogenously administered SOD had little or no effect on gut I/R-induced leukostasis or capillary no-reflow in the liver. These observations suggest a role for superoxide in gut I/R-induced leukostasis and hypoxic stress in the liver. Furthermore, the findings suggest that cellular localization of SOD activity is an important determinant of the protective actions of this enzyme in experimental models of I/R injury.

Iron, free radicals, and oxidative injury.

Iron metabolism and superoxide metabolism are clearly interactive, especially under pathological conditions. Each can exacerbate the toxicity of the other. Iron overload may amplify the damaging effects of superoxide overproduction in a very broad spectrum of inflammatory or ischemia-related conditions. Furthermore, chronic oxidative stress may modulate iron uptake and storage, leading to a self-sustained and ever-increasing spiral of cytotoxic and mutagenic events.

Antiinflammatory activity of tissue plasminogen activator in the carrageenan rat footpad model.

Exogenous plasminogen activators (PAs), such as streptokinase (SK) and tissue plasminogen activator (tPA), have been shown to significantly improve the mortality of patients with acute myocardial infarction. However, reperfusion of the myocardium is associated with neutrophil activation and infiltration into the infarct region. Plasminogen activators influence neutrophil function in vitro, but no data exists regarding the effect of exogenous PAs on inflammation in vivo. Therefore, we evaluated the effect of PAs on inflammation using the carrageenan-induced rat footpad inflammation model. The magnitude of carrageenan-induced inflammation was determined by water-displacement and neutrophil infiltration, following administration of either tPA or SK to Sprague-Dawley rats. tPA (12 mg/kg) inhibited carrageenan-induced inflammation (p < .01). In contrast, administration of SK (40,000 U/kg) enhanced inflammation. These results suggest that exogenous PAs influence the inflammatory process but specific PAs differ in their actions. Ultimately, these differences may influence the efficacy of these agents in the management of acute myocardial infarction and lead to further evaluation of tPA in other inflammatory diseases such as acute respiratory distress syndrome (ARDS) and rheumatoid arthritis (RA), in which neutrophil-mediated injury is likely.

Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase.

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to lambda phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.

A novel Escherichia coli vector for oxygen-inducible high level expression of foreign genes.

By utilizing the oxygen-sensitive Escherichia coli Mn-superoxide dismutase (Mn-SOD) promoter, we have developed a vector system that expresses high levels of cloned foreign genes. The promoter for the bacterial Mn-SOD, as well as both 5'-untranslated and transcriptional termination sequences were ligated to a synthetic linker containing two restriction enzyme cloning sites. The vector also contained the gene for beta-lactamase, which confers ampicillin resistance to the host bacterium and provides a selectable marker. After screening and selection, high level of expression was achieved by exposure to the superoxide-generating agent paraquat (methyl viologen) as the inducer. To test the vector, both native and mutated human Mn-SOD cDNAs were cloned and expressed, respectively. To determine the optimal concentration of inducer necessary for maximal expression, recombinant bacteria were exposed to increasing concentrations of paraquat and subsequently assayed for superoxide dismutase (SOD) activity. The highest expression was induced by 20 microM paraquat, and approached 50% of total soluble protein.

Effects of positive iron status at a cellular level.

The human body is not equipped to eliminate excess iron. As a result, cells continuously store excess absorbed iron in a complex with the protein ferritin. This poses no great threat under normal conditions. However, under certain disease conditions, oxidative processes cause iron to be mobilized and released as Fe(II). This iron is capable of initiating lipid peroxidation, which can lead to loss of membrane structure and function.

Mitochondrial respiration scavenges extramitochondrial superoxide anion via a nonenzymatic mechanism.

We determined that mitochondrial respiration reduced cytosolic oxidant stress in vivo and scavenged extramitochondrial superoxide anion (O2-.) in vitro. First, Saccharomyces cerevisiae deficient in both the cytosolic antioxidant cupro-zinc superoxide dismutase (Cu,Zn-SOD) and electron transport (Rho0 state) grew poorly (P < 0.05) in 21% O2 compared with parent yeast and yeast deficient only in electron transport or Cu,Zn-SOD, whereas anaerobic growth was the same (P > 0.05) in all yeast. Second, isolated yeast and mammalian mitochondria scavenged extramitochondrial O2-. generated by xanthine/xanthine oxidase. Yeast mitochondria scavenged 42% more (P < 0.05) extramitochondrial O2-. during pyruvate/malate-induced respiration than in the resting state. Addition of either antimycin (respiratory chain inhibitor) or FCCP (respiratory chain uncoupler) prevented increased O2-. scavenging. Mitochondria isolated from yeast deficient in the mitochondrial manganous superoxide dismutase (Mn-SOD) increased (P < 0.05) O2-. scavenging 56% during respiration. This apparent SOD activity, expressed in units of SOD activity per milligram of mitochondrial protein, was the same (9 +/- 0.6 vs. 10 +/- 1.0; P = 0.43) as the O2-. scavenging of mitochondria with Mn-SOD, suggesting that respiration-dependent mitochondrial O2-. scavenging was nonenzymatic. Finally, isolated rat liver and lung mitochondria also increased (P < 0.05) O2-. scavenging during respiration. We speculate that respiring mitochondria, via the protonmotive pump, present a polarized, proton-rich surface that enhances nonenzymatic dismutation of extramitochondrial O2-. and that this is a previously unrecognized function of mitochondrial respiration with potential physiological ramifications.

Superoxide radical: controversies, contradictions, and paradoxes.

The study of free radical biology has engendered a great deal of controversy and apparently conflicting observations, particularly with regard to the use of the antioxidant enzyme superoxide dismutase as a protective or therapeutic agent. Slowly, the reasons behind the confusion are beginning to emerge. The superoxide radical, O2.-, has a number of paradoxical physiological and pathophysiological roles. Several examples of the radical's schizophrenic behavior include its roles in bactericidal action versus inflammation, as a modulator of cell division versus malignant transformation and apoptosis, and as both an initiator and a terminator of lipid peroxidation.

Sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite.

Disruption of the zinc-thiolate center at the active site of yeast alcohol dehydrogenase results in inactivation and zinc release. Measurements of activity, zinc release, and thiol/thiolate oxidation were used to assess the effects of biologically relevant oxidants on alcohol dehydrogenase. Alcohol dehydrogenase was inactivated by 1 mM hydrogen peroxide at a rate of 1.3 M-1 s-1. Peroxynitrite, the near diffusion-limited reaction product of nitric oxide and superoxide, inactivated alcohol dehydrogenase with an IC50 = 0.95 microM when catalytic concentrations of alcohol dehydrogenase subunit (0.074 microM) were present. Slow, continuous production of peroxynitrite from decomposition of SIN-1 inactivated alcohol dehydrogenase as effectively as bolus addition. The rate constants for reaction of peroxynitrite with alcohol dehydrogenase at 23 degrees C as determined by two different competition assays were 2.6 x 10(5) M-1 s-1 and 5.2 x 10(5) M-1 s-1. The reaction with alcohol dehydrogenase represents one of the fastest reactions yet determined for peroxynitrite. Hypochlorite inactivated alcohol dehydrogenase at a rate of 4 x 10(3) M-1 s-1. The rate constant for inactivation by taurine choramine, the reaction product of taurine and hypochlorite, was only slightly slower at 2.7 x 10(3) M-1 s-1. Zinc release and thiol/thiolate oxidation were correlated with inactivation by either peroxynitrite or hypochlorite. At the concentrations of peroxynitrite or hypochlorite producing total inactivation, 0.85 zinc atom was released per subunit and 3 thiol/thiolates per subunit were oxidized.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukemia inhibitory factor and tumor necrosis factor induce manganese superoxide dismutase and protect rabbit hearts from reperfusion injury.

Leukemia inhibitory factor (LIF) and tumor necrosis factor (TNF) have been shown to protect animals from radiation, hyperoxia, and endotoxic shock. TNF is also known to induce the expression of manganese superoxide dismutase (MnSOD) in vitro and in vivo. We therefore examined the effects of these cytokines on reperfusion injury in the isolated rabbit heart model. Rabbits were injected intravenously with 10 micrograms of either human TNF-alpha or lymphotoxin (TNF-beta), or murine TNF-alpha or murine LIF dissolved in saline. Control animals were injected with an equal volume of saline. After 24 h, hearts were isolated and perfused. Following an equilibration period, the hearts were subjected to 1 h ischemia and 1 h of reperfusion. All treated groups showed significant increases in percent recovery of developed tension (% preischemic) when compared to saline-treated control hearts. In addition there were significant decreases in lactate dehydrogenase release (LDH), accumulation of thiobarbituric acid reactive substances (TBARS), and accumulation of carbonyl proteins. These results correlate with increases in myocardial MnSOD activity. Thus, the protection from myocardial reperfusion injury seen in the pretreated group may be due to a mechanism that involves the induction of MnSOD.

A site-directed mutant of Cu,Zn-superoxide dismutase modeled after native extracellular superoxide dismutase.

The well-studied cytosolic Cu,Zn-superoxide dismutase (SOD) protects against reperfusion injury, although its short (6 min) plasma half-life and negative charge create undesirable pharmacokinetics. We have designed, cloned, and expressed a genetic variant of SOD with altered pharmacological properties. A fusion gene consisting of the entire coding region of human SOD followed by a positively charged carboxy-terminal (C-terminal) "tail" of eight glycine and six arginine residues was constructed. The tail was modeled after the extracellular SOD (EC-SOD) C-terminal 26-amino acid basic peptide. This EC-SOD tail binds to heparin-like proteoglycans on cell surfaces and contributes to the enzyme's very long (30 h) plasma clearance time. After expression in Escherichia coli, the mutant enzyme was purified and characterized. No differences in specific activity or UV absorption spectrum between the mutant and the native enzyme were found. The thermal stability of the fusion protein was greater than that of native SOD. Although native SOD has no affinity for heparin, the modified enzyme bound to a heparin-agarose column. A "designer" SOD able to bind to cell surfaces may aid in the prevention of superoxide-mediated endothelial damage.

Neutrophil-generated free radicals: possible mechanisms of injury in adult respiratory distress syndrome.

The acute lung injury resulting from adult respiratory distress syndrome (ARDS) is thought to be largely mediated by activated neutrophils. Because activated neutrophils produce the superoxide radical, which is both bacterial and cytotoxic to host cells, this oxygen-derived free radical is likely responsible for at least part of the neutrophil-mediated lung injury. In a rat model of ARDS resulting from intratracheal instillation of interleukin-1, recombinant human manganous superoxide dismutase significantly decreased lung leak. One detrimental action of proteases released by adherent neutrophils may be the degradation of extracellular superoxide dismutase (ECSOD), which normally binds to the heparan sulfate on the surface the endothelium. We found that rabbit ECSOD incubated with either trypsin or activated neutrophils loses affinity for heparin. Furthermore, soluble ECSOD is elevated in the serum of patients with ARDS, consistent with this hypothesis.

The toxicity of high-dose superoxide dismutase suggests that superoxide can both initiate and terminate lipid peroxidation in the reperfused heart.

Recently, we described an anomalous bell-shaped dose-response curve for the protection of the reoxygenated isolated myocardium by superoxide dismutase (SOD). SOD is dramatically protective up to a point (5 micrograms/ml in the perfusate) beyond which it loses its ability to protect and, at very high doses (50 micrograms/ml), exacerbates the injury. We proposed that O2-. may serve as both initiator and terminator of lipid peroxidation, such that over scavenging the radical may increase net lipid peroxidation via increased chain length. We examined the ability of U74389F, a lipid peroxidation inhibitor, to ameliorate the toxicity of high-dose SOD in the isolated perfused rabbit heart preparation. The results show a significant improvement in the percent recovery of developed tension of hearts treated with U74389F and overdosed with MnSOD, as well as a decrease in thiobarbituric acid reactive substances.