Depolymerisation products of hyaluronic acid after exposure to oxygen-derived free radicals.

Preparative chromatographic fractions of human umbilical cord hyaluronic acid (HA) of a molecular weight of 10(6) were subjected to graded oxygen-derived free radical (oxy radical) fluxes produced by: (a) the autoxidation of ferrous ions; (b) the action of xanthine oxidase (XO) on hypoxanthine (HX); and (c) by peripheral blood polymorphonuclear leucocytes that had been stimulated by phorbol myristate acetate (PMA). Analysis by gel chromatography of the products obtained with each of the oxy radical generating systems showed polydispersity in size. The smallest molecules detected had a molecular weight of 10(4). This limiting size was not reduced further by exposure to a second oxy radical flux. The relative proportions of large, medium, and small degradation products were established for various levels of oxy radical flux. Consistently a relatively rapid transition from large to small material was seen on Sepharose 2B chromatography, suggesting an ordered element to the breakdown process. Although the decrease in molecular weight after oxy radical exposure was confirmed by analytical ultracentrifugation, this procedure showed that those samples of lowest viscosity did not have the lowest sedimentation values, possibly reflecting oxy radical-induced repolymerisation. If the size and possibly the conformational characteristics of HA are altered, oxy radical exposure might be expected to alter its biological properties.     

Reactive oxygen intermediates reduce inactivation of serotonin in isolated, perfused rat lungs

Reactive oxygen intermediates such as free radicals have been proposed to mediate lung injury. The present work examined whether or not enzymatically generated oxygen metabolites altered serotonin clearance. Isolated, plasma-perfused rat lungs were exposed to xanthine oxidase (XO) and hypoxanthine (HX). Pulmonary arterial pressure (Ppa) and lung weight were recorded. Fulminant edema was defined as a spontaneous weight increase exceeding 500 mg. Inactivation of serotonin was determined by superfusion bioassay. XO and HX reduced serotonin inactivation from 74 +/- 3% (mean +/- SEM) to 62 +/- 2%. This reduction was inhibited by the scavenger enzymes superoxide dismutase (SOD) and catalase and by allopurinol, an inhibitor of XO. Hydrostatic edema and perfusion per se did not decrease the pulmonary clearance of serotonin. XO and HX did not significantly alter Ppa. Fulminant edema developed in four of six lungs after exposure to XO and HX compared with none in the other groups. It was concluded that reactive oxygen intermediates inhibited serotonin inactivation in isolated rat lungs.     

Influence of hydroxyl radical scavengers on platelet function

The influence of four hydroxil radical (OH.) scavengers on platelet function was investigated. OH. scavengers inhibited ADP, collagen, arachidonic acid, PAF-induced platelet aggregation, and platelet cyclooxygenase pathway activation, which was studied by evaluating platelet malondialdehyde and serum thromboxane A2 formation. The latter was not affected by superoxide dismutase, catalase, or metal ion chelants such as desferioxamine or DETAPAC. The detection of deoxyribose degradation by stimulated platelets suggested that platelets produce OH.. This study shows that activated platelets produce free radicals and that antioxidant agents such as OH. scavengers inhibit platelet function.     

Hydroxyl radical mediates the endothelium-dependent relaxation produced by bradykinin in mouse cerebral arterioles

Bradykinin relaxes arterioles on the brain's surface. This response is endothelium-dependent. The data presented here confirm the hypothesis that hydroxyl free radical mediates this response and may be the endothelium-dependent relaxing factor for bradykinin in this microvascular bed. The response to a locally applied bolus of bradykinin (80 micrograms/ml) was monitored by intravital TV microscopy. The response was significantly inhibited or totally blocked by the presence of superoxide dismutase 60 U/ml, catalase 46 U/ml, or deferoxamine 0.1 or 0.2 mM. The superoxide dismutase scavenges superoxide radical, which is known to enter the subarachnoid space as a consequence of cyclooxygenase activation. Cyclooxygenase is activated by bradykinin. The superoxide can form H2O2, scavenged by catalase, and the two together generate hydroxyl. The formation of hydroxyl radical is catalyzed by iron. Deferoxamine 0.1 mM scavenges the iron, blocking the generation of hydroxyl. Deferoxamine 0.2 mM also directly scavenges the hydroxyl. None of the pharmacologic probes had an effect on arteriolar diameter when locally applied without bradykinin. Since the dilation produced by bradykinin was inhibited or totally blocked by probes that prevented hydroxyl formation or directly scavenged hydroxyl radical, that radical is either an essential mediator of the arteriolar relaxation, or is the endothelium-dependent relaxing factor for bradykinin in pial arterioles.     

Hydrogen peroxide and hydroxyl radical mediation of activated leukocyte depression of cardiac sarcoplasmic reticulum. Participation of the cyclooxygenase pathway

Human peripheral blood leukocytes, activated by phorbol myristate acetate, disrupt canine sarcoplasmic reticulum calcium transport, in vitro, by an oxygen-derived free radical mechanism. Activated leukocytes significantly depress Ca++ uptake activity and Ca++ -stimulated, Mg++ -dependent ATPase activity. The depression is completely inhibited by sodium-azide (0.1 mM) or the combination of superoxide dismutase (10 micrograms/ml) and catalase (10 micrograms/ml). Exogenous hydrogen peroxide (0.441-4.41 mM) uncoupled Ca++ uptake activity from ATP hydrolysis, and this effect was inhibited by catalase. Mannitol alone did not inhibit the effects of activated leukocytes, but superoxide plus mannitol (20-100 mM) resulted in normal ATPase activity, while Ca++ uptake remained depressed. In the presence of indomethacin and ibuprofen, activated leukocytes depressed Ca++ uptake and had no effect on ATPase activity. 2-Amino-methyl-4-t-butyl-6-iodophenol (MK-447) further depressed Ca++ uptake and partially inhibited the effect on ATPase activity. Indomethacin plus catalase completely inhibited the effects of activated leukocytes on cardiac sarcoplasmic reticulum. We conclude, first, that activated leukocytes depress canine cardiac sarcoplasmic reticulum Ca++ transport by an oxygen-free radical mechanism with the generation of hydrogen peroxide and hydroxyl radical. In addition to the classical membrane NADPH oxidase system, significant oxygen radical generation can occur through the cyclooxygenase pathway of arachidonic acid metabolism, and seems to be responsible for the generation of the hydroxyl radical.     

Generation of Reactive Oxygen Species and Reduction of Ferric Chelates by Microsomes in the Presence of a Reconstituted System Containing Ethanol, NAD+ and Alcohol Dehydrogenase

Many of the toxic metabolic actions of ethanol on the liver have been ascribed to the enhanced cellular production of NADH, which arises as a consequence of the oxidation of ethanol by alcohol dehydrogenase (ADH). Experiments were conducted to evaluate whether NADH generated from a reconstituted system containing ethanol plus NAD⁺ plus ADH could interact with ferric chelates to promote microsomal lipid peroxidation and generation of a hydroxyl radical (OH)-like species. In the presence of the reconstituted system and iron, microsomes produced ? OH as assessed by the oxidation of ? OH scavenging agents. This oxidation was inhibited by catalase and competitive ? OH scavengers but not by superoxide dismutase. The ADH-dependent microsomal production of ? OH was effectively catalyzed by ferric-EDTA and -diethylenetriamine pentaacetic acid (-DTPA), but not by ferric-ATP or -citrate. However, all these ferric chelates were reduced by the microsomes in the presence of the reconstituted system. Hydrogen peroxide (H₂O₂) was produced in the presence of ADH and appeared to be a limiting factor for the production of ? OH. The reconstituted system also catalyzed microsomal lipid peroxidation, and the pattern of effectiveness of ferric chelates was opposite that of catalysis of ? OH production. There was little effect by catalase, superoxide dismutase or dimethyl sulfoxide (DMSO) on the ADH-dependent microsomal lipid peroxidation. The reconstituted system was characterized with respect to dependence on NAD⁺ and ADH; ethanol could be replaced by other alcohols, which are substrates for ADH. Pyrazole, a potent inhibitor of ADH, blocked the ability of the reconstituted system to interact with iron and microsomes to produce reactive oxygen species. The overall pattern of response of the ADH-dependent reactions with respect to rates of reactions, catalytic effectiveness of ferric chelates and sensitivity to radical scavengers is similar to that found with a NADPH-generating system. In view of the effectiveness of NADH derived from the oxidation of ethanol by ADH in reducing ferric chelates and promoting microsomal generation of reactive species, increased availability of cellular NADH as a consequence of ethanol oxidation could contribute to the development of oxidative stress and play a role in the toxic actions of ethanol to the liver.     

Damage of cultured chondrocytes by hydrogen peroxide derived from polymorphonuclear leukocytes: a possible mechanism of cartilage degradation

Summary To study the mechanisms of chondrocyte damage, chondrocyte cytotoxicity as shown by chromium-51 release induced by polymorphonuclear leukocytes (PMNLs) was examined. PMNLs significantly enhanced chondrocyte cytotoxicity in the presence of phorbol dibutyrate. This chondrocyte damage was abolished by the addition of catalase, whereas superoxide dismutase and scavengers of hydroxyl radicals and protease inhibitors failed to reverse it. When cartilage matrix components such as hyaluronic acid and various proteoglycans were added to the PMNL-chondrocyte cultures, these components failed to affect the chronium-51 release. These results suggest that the increase in chondrocyte cytotoxicity is due to hydrogen peroxide generated by the PMNLs, and that cartilage matrix components do not prevent it. Hydrogen peroxide from PMNLs may therefore play an important role in cartilage degradation through direct damage of chondrocytes during inflammatory process.     

Hypoxanthine-xanthine oxidase down-regulates GLUT1 transcription via SIRT1 resulting in decreased glucose uptake in human placenta

Appropriate foetal growth and development is dependent on adequate placental glucose uptake. Oxidative stress regulates glucose uptake in various tissues. The effect of oxidative stress on placental glucose transport is not known. Thus, the aim of this study was to determine the effect of oxidative stress on glucose uptake and glucose transporters (GLUTs) in human placenta. Human placenta was incubated in the absence or presence of 0.5?mM hypoxanthine+15?mU/ml xanthine oxidase (HX/XO) for 24?h. Gene and protein expressions of the GLUTs were analysed by quantitative RT-PCR and western blotting respectively. Glucose uptake was measured using radiolabelled ((14)C) glucose. HX/XO significantly decreased GLUT1 gene and protein expression and resultant glucose uptake. There was no effect of the antioxidants N-acetylcysteine, catalase and superoxide dismutase or the NF-κB inhibitor BAY 11-0782 on HX/XO-induced decrease in glucose uptake. However, HX/XO treatment significantly decreased both gene and protein expression of SIRT1. In the presence of the SIRT1 activator resveratrol, the decrease in GLUT1 expression and glucose uptake mediated by HX/XO was abolished. Collectively, the data presented here demonstrate that oxidative stress reduces placental glucose uptake and GLUT1 expression by a SIRT1-dependent mechanism.     

Effect of endothelial injury on the responses of isolated guinea pig pulmonary venules to reduced oxygen tension

The influence of the endothelium on pulmonary venular responses to reduced oxygen tension has not been defined. To examine this question, endothelial injury was induced in small guinea pig pulmonary artery and venule segments (effective lumen radius, 174 +/- 5 and 122 +/- 2 microns, respectively) by perfusion with either a mixture of hypoxanthine (5 mM) and xanthine oxidase (0.05 U/ml) (HX/XO) or collagenase (2 mg/ml). HX/XO significantly (p less than 0.05) reduced the relaxation of precontracted pulmonary arteries by acetylcholine (ACH), bradykinin (BK), and A-23187, and the relaxations were restored by including superoxide dismutase (40 micrograms/ml) in the HX/XO solution. However, neither HX/XO nor collagenase affected vasodilation induced by ACH, BK, and A-23187 in precontracted pulmonary venules. In contrast, HX/XO significantly (p less than 0.05) augmented the sustained contraction of pulmonary venules to hypoxia (HX/XO, 3.2 +/- 1.0 mg/mm; control, 1.0 +/- 0.5 mg/mm) and anoxia (HX/XO, 35.1 +/- 6.6 mg/mm; control, 20.3 +/- 4.0 mg/mm). Collagenase also significantly (p less than 0.05) enhanced the anoxic contractions (collagenase, 36.0 +/- 3.7 mg/mm; control, 20.9 +/- 6.8 mg/mm). Superoxide dismutase (40 micrograms/ml) and catalase (323 micrograms/ml) abolished HX-XO-induced augmentation of the hypoxic and anoxic contractions of pulmonary venules. Collagenase removed 54 +/- 8% of the venular endothelium (control, 5 +/- 1%), whereas HX/XO-exposed endothelial cells contained numerous craters. Neither gossypol (5 microM) nor methylene blue (10 microM) affected pulmonary venular contractions to reduced PO2. Endothelial damage augments the PO2-dependent contractions of the pulmonary venule, and this augmentation does not appear to be due to decreased release of endothelium-derived relaxing factor.     

Role of hydrogen peroxide and hydroxyl radical formation in the killing of Ehrlich tumor cells by anticancer quinones.

The cytotoxicity of the clinically important antineoplastic quinones doxorubicin, mitomycin C, and diaziridinylbenzoquinone for the Ehrlich ascites carcinoma was significantly reduced or abolished by the antioxidant enzymes catalase and superoxide dismutase, the hydroxyl radical scavengers dimethyl sulfoxide, diethylurea, and thiourea, and the iron chelators deferoxamine, 2,2-bipyridine, and diethylenetriaminepentaacetic acid. However, tumor cell killing by 5-iminodaunorubicin, a doxorubicin analog with a modified quinone function that prohibits oxidation-reduction cycling, was not ameliorated by any of the free radical scavengers tested. Furthermore, treatment of intact tumor cells with doxorubicin, mitomycin C, and diaziridinylbenzoquinone but not 5-iminodaunorubicin generated the hydroxyl radical, or a related chemical oxidant, in vitro in a process that required hydrogen peroxide, iron, and intact tumor cells. These results suggest that drug-induced hydrogen peroxide and hydroxyl radical production may play a role in the antineoplastic action of redox active anticancer quinones.     

Neutrophil-derived oxidants mediate formyl-methionyl-leucyl-phenylalanine-induced increases in mucosal permeability in rats

The effects of several free radical scavengers and antioxidant enzymes on neutrophil-mediated changes in mucosal permeability (measured using blood-to-lumen clearance of 51Cr-labeled ethylene-diaminetetraactate) were assessed using ileal loops perfused with N-formyl-methionyl-leucyl-phenylalanine (FMLP). Neither superoxide dismutase nor catalase reduced the FMLP-induced increase in mucosal permeability. However, manganese-loaded desferrioxamine (a superoxide dismutase mimetic), PZ51 (a glutathione peroxidase analogue), desferrioxamine (an iron chelator), or dimethylsulfoxide (a hydroxyl radical scavenger) significantly attenuated FMLP-induced mucosal damage. The results of our experiments indicate that neutrophilic oxidants are responsible for a major portion of the mucosal permeability changes induced by FMLP.     

Effects of reactive oxygen species on intracellular calcium in bovine tracheal epithelium: modulation by nitric oxide

We studied the effects of reactive oxygen species (ROS) on intracellular Ca2+ concentration ([Ca2+]i) and their possible modulation by nitric oxide (NO) in fura-2-loaded cultured bovine tracheal epithelium. Hypoxanthine (HX) and xanthine oxidase (XO), which generate superoxide anion (O2-) and hydrogen peroxide (H2O2), dose dependently increased [Ca2+]i. The increase in [Ca2+]i was reduced in the presence of superoxide dismutase (SOD, 200 U/mL) and catalase (200 U/mL) by 29% and 43%, respectively. The iron chelator o-phenanthroline and the hydroxyl radical (.OH) scavenger dimethylthiourea (DMTU) more potently inhibited the response of [Ca2+]i. H2O2-derived .OH generated by the Fenton reaction caused a marked [Ca2+]i elevation, but exogenous H2O2 did not. Sodium nitroprusside (100 microM), an NO donor, potentiated HX-XO-induced [Ca2+]i rise by 50%, an effect that was abolished in the presence of SOD or DMTU. These results suggest that .OH formed by interaction of O2- and H2O2 in the presence of iron may play a major role in the HX-XO-induced disruption of airway epithelial Ca2+ homeostasis, and that NO potentiates ROS-induced [Ca2+]i response, presumably by reacting with O2- and producing .OH.     

Oxidative mechanisms of monocyte-mediated cytotoxicity.

Human monocytes stimulated with phorbol myristate acetate were able to rapidly destroy autologous erythrocyte targets. Monocyte-mediated cytotoxicity was related to phorbol myristate acetate concentration and monocyte number. Purified preparations of lymphocytes were incapable of mediating erythrocyte lysis in this system. The ability of phorbol myristate acetate-stimulated monocytes to lyse erythrocyte targets was markedly impaired by catalase or superoxide dismutase but not by heat-inactivated enzymes or albumin. Despite a simultaneous requirement for superoxide anion and hydrogen peroxide in the cytotoxic event, a variety of hydroxyl radical and singlet oxygen scavengers did not effect cytolysis. However, tryptophan significantly inhibited cytotoxicity. The myeloperoxidase inhibitor cyanide enhanced erythrocyte destruction, whereas azide reduced it modestly. The inability of cyanide to reduce cytotoxicity coupled with the protective effect of superoxide dismutase suggests that cytotoxicity is independent of the classic myeloperoxidase system. We conclude that monocytes, stimulated with phorbol myristate acetate, generate superoxide anion and hydrogen peroxide, which together play an integral role in this cytotoxic mechanism.     

Superoxide mediates the toxicity of paraquat for Chinese hamster ovary cells.

The roles of superoxide and H2O2 in the cytotoxicity of paraquat were assessed in Chinese hamster ovary cells. Neither catalase nor superoxide dismutase inhibited the loss of ability to form colonies when added to the medium. When introduced into the cells, superoxide dismutase but not catalase inhibited the toxicity of paraquat. That superoxide dismutase acted by its known catalytic action is shown by the loss of inhibition when the enzyme was inactivated by H2O2 before being introduced into the cells. The lack of inhibition by catalase, by dimethyl sulfoxide, and by desferoxamine suggests that the toxicity is not mediated by a reaction between H2O2 and superoxide to engender the hydroxyl radical. Exposure of Chinese hamster ovary cells to paraquat may be a suitable means to determine the effects of superoxide anion in cultured cells and the ways in which cells can resist this toxic action.     

Role of oxygen radicals in cardiac injury due to reoxygenation

The ability of oxygen derived free radicals to induce irreversible cellular injuries during reoxygenation was studied on isolated potassium-arrested heart preparation. Enzymatic scavengers of hydrogen peroxide (H2O2) and superoxide anion (O-2), catalase and superoxide dismutase, were not effective in reversing the cardiac alterations induced by hypoxia. Cellular injuries induced by reoxygenation, 'Oxygen paradox', were partially prevented by scavengers of H2O2 (glutathione reduced form, catalase) and O-2 (superoxide dismutase). The 'oxygen paradox' was associated with a release of malonaldehyde. The inhibition of lipid peroxidation by alpha-tocopherol prevented the toxic effect of molecular oxygen on hypoxic hearts. The specific quenchers of singlet oxygen (histidine) and hydroxyl radical (mannitol) reduced the peroxidation of unsaturated lipids and the intensity of the 'oxygen paradox' phenomenon. The results indicate that in cardiac muscle (i) oxygen derived free radicals are important byproducts of abnormal oxidative metabolism present during the post hypoxic period; (ii) the 'oxygen paradox' phenomenon is related to the formation of lipid hydroperoxides leading to the cellular membrane disruption and to the irreversible alteration of cardiac integrity.     

Effect of hydrogen peroxide administration on life span, superoxide dismutase, catalase, and glutathione in the adult housefly, Musca domestica

The general objective of this study was to further elucidate the relationship between oxidative stress and the aging process. H2O2 is known to be a progenator of reactive oxygen species, such as hydroxyl free radical, by various mechanisms involving, among others, a superoxide anion radical-driven Fenton cycle, or splitting of the 0-0 bond by hemoproteins. Effects of H2O2 administration on life span, activities of superoxide dismutase, catalase, concentrations of endogenous H2O2, and glutathione in the housefly are described. Adult male flies were given various concentrations of H2O2, ranging from 0 to 100 mM H2O2, in their drinking water. Life span was shortened by H2O2 intake except in 10 mM H2O2 administrated flies, which exhibited the longest life span. Flies administered 10 mM H2O2 also contained the highest concentration of reduced glutathione (GSH). Superoxide dismutase and catalase activities were not affected by H2O2 intake. Compensatory elevation in GSH may be responsible for the increase in life span observed in 10 mM H2O2 administered flies.     

Acceleration of Hypertensive Cerebral Injury by the Inhibition of Xanthine-Xanthine Oxidase System in Stroke-Prone Spontaneously Hypertensive Rats

It is well-known that, in ischemic cerebral injury, a free radical and its byproducts are generated by xanthine-xanthine oxidase system and eliminated by scavengers such as superoxide dismutase (SOD), catalase, uric acid and ascorbic acid. To investigate the possible involvement of the xanthine-xanthine oxidase system in hypertensive cerebral injury, we examined chronological changes in uric acid level in the cerebral cortex and the effects of the inhibition of xanthine oxidase or catalase using stroke-prone spontaneously hypertensive rats (SHRSP).     

MnTMPyP, a metalloporphyrin-based superoxide dismutase/catalase mimetic, protects INS-1 cells and human pancreatic islets from an in vitro oxidative challenge

AIMS: Pancreatic islets can be lost early following allotransplantation from oxidative stress. Antioxidant enzyme overexpression could confer a beneficial effect on islets exposed to reactive oxygen species (ROS) and nitrogen species. Here, we tested the effect of MnTMPyP, a superoxide dismutase/catalase mimetic. METHODS: INS-1 insulin-secreting cells or human islets were cultured with MnTMPyP and exposed to a superoxide donor (the hypoxanthine/xanthine oxidase (HX/XO) system), a nitric oxide donor [3-morpholinosydnonimine (SIN-1)] or menadione. Viability of INS-1 cells was assessed by WST-1 colorimetric assay and FACS analysis (Live/Dead test). ROS production was determined using fluorescent probes. Islet viability was estimated by WST-1 assay and endocrine function by static incubation. RESULTS: Following MnTMPyP treatment, ROS production in INS-1 cells was reduced by 4- to 20-fold upon HX/XO challenge and up to 2-fold upon SIN-1 stress. This phenomenon correlated with higher viability measured by WST-1 or Live/Dead test. MnTMPyP preserved islet viability upon exposure to SIN-1 or menadione but not upon an HX/XO challenge. Similarly, decrease in insulin secretion tended to be less pronounced in MnTMPyP-treated islets than in control islet when exposed to SIN-1, but no changes were noticed during an HX/XO stress. CONCLUSIONS: MnTMPyP was able to improve the viability of INS-1 cells and human islets exposed to oxidative challenges in vitro. Protection of INS-1 cells could be as high as 90%. This agent is therefore potentially attractive in situations involving the overproduction of ROS, such as islet transplantation.     

Polymorphonuclear leucocytes as potential source of free radicals in the ischaemic-reperfused myocardium

The feasibility of polymorphonuclear leucocytes as a potential source of free radicals during reperfusion of ischaemic myocardium was evaluated. Isolated rat heart was perfused in the presence of f-Met-Leu-Phe-activated and normal polymorphonuclear leucocytes for 30 min. To judge the degree of cellular injury which might result from activated polymorphonuclear leucocytes during perfusion, isolated hearts were also perfused with superoxide anions, hydroxyl radicals, and hypochlorous acid-generating systems in the absence or presence of their corresponding scavengers, superoxide dismutase plus catalase, dimethylthiourea, and allopurinol, respectively. Activated polymorphonuclear leucocytes stimulated the release of lactate dehydrogenase, a biological marker of cellular injury, and malondialdehyde, a presumptive marker for lipid peroxidation; increased tissue injury, as evidenced by morphologic examinations using light and electron microscopy; decreased dry/wet ratios of heart, signifying oedema formation; and reduced myocardial adenosine triphosphate and creatine phosphate content as well as coronary flow, indicating decreased myocardial performance. These biological, physiological, and morphologic parameters were reversed significantly, but not completely, by treating the heart with scavengers, superoxide dismutase plus catalase or allopurinol, but were reversed completely by simultaneous treatment with superoxide dismutase, catalase, and allopurinol. Comparable results were obtained when the hearts were treated with each of these free radical-generating systems and their corresponding scavengers. Generation of free radicals was confirmed either by cytochrome c reduction or by examining the chemiluminescence response using a luminometer. These results indicate that activated polymorphonuclear leucocytes can cause myocardial cellular injury equivalent to the damage caused by free radicals and oxidants which are present in an ischaemic-reperfused heart, suggesting that polymorphonuclear leucocytes may be a potential source of free radicals in the reperfused heart.     

Effect of reactive oxygen metabolites on endothelial permeability: role of nitric oxide and iron.

OBJECTIVE: We evaluated the effects of the xanthine oxidase (XO)-derived reactive oxygen metabolites on the permeability of bovine pulmonary artery-endothelial monolayers and examined how iron and nitric oxide (NO) participate in these changes in permeability. METHODS: Permeability was measured using a cell-column chromatographic method in which monolayers were exposed to combinations of agents. RESULTS: Exposure of monolayers to a superoxide/peroxide generator, xanthine (X, 0.1 mM)/XO (25 mU/mL), increased solute permeability after 10 minutes, but the same dose of either X or XO alone did not. Exposure of monolayers to peroxide (0.1 mM) also increased permeability, but only after 70 minutes. This X/XO permeability was attenuated by either catalase, superoxide dismutase, methionine (1 mM), an oxy-radical scavenger, or desferrioxamine (0.1 mM), an iron chelator. Spermine NONOate (SNO), an NO donor, attenuated X/XO permeability at 0.1 mM, but this protection was not significant at 0.01 or 1 mM. Spermine NONOate (0.1 mM) did not alter the permeability produced by 0.1 mM peroxide. L-N5-(1-iminoethyl)-ornithine (10 microM), an NO synthase inhibitor, completely blocked peroxide-, and partially attenuated X/XO-mediated permeability. However, 3-morphosynodiomine (SIN-1, 1 mM) plus catalase (1,000 U/mL), a peroxynitrite generator, did not alter permeability. CONCLUSIONS: Xanthine/Xanthine Oxidase permeability involves peroxide, superoxide, oxy-radicals, and iron. Endogenous NO may regulate peroxide-, but not superoxide-mediated permeability. The protective effects of exogenous NO on the X/XO permeability may represent interactions between superoxide, peroxide, and cell surface-bound iron.