Role of xanthine oxidase in passive Heymann nephritis in rats. (1/1226)

Passive Heymann nephritis (PHN) in rats is a model of human membranous nephropathy characterized by formation of subepithelial immune deposits in the glomerular capillary wall and complement activation. Oxygen radicals have been implicated in the subsequent glomerular damage which leads to proteinuria. This study examines the involvement of xanthine oxidase in this process. Xanthine oxidase activity was increased nearly twofold in glomeruli isolated 1 and 12 d after induction of PHN, and this was associated with increased glomerular superoxide anion generation. Analysis of glomerular samples by Northern and Western blotting revealed no quantitative changes in xanthine oxidoreductase expression in PHN, suggesting conversion of xanthine dehydrogenase to the oxidase form as the cause of increased activity. Treatment of rats with tungsten, an inhibitor of xanthine oxidase, before induction of PHN resulted in a marked decrease in glomerular xanthine oxidase activity and superoxide anion generation, and decreased proteinuria by 80% (day 12: 423+/-245 mg/d in PHN versus 78+/-53 mg/d in tungsten-treated PHN animals, P < 0.01). These findings point to a pivotal role of xanthine oxidase in the pathophysiology of PHN and could be of importance in the therapy of human membranous nephropathy.  (+info)

Upregulation of superoxide dismutase and nitric oxide synthase mediates the apoptosis-suppressive effects of shear stress on endothelial cells. (2/1226)

Physiological levels of laminar shear stress completely abrogate apoptosis of human endothelial cells in response to a variety of stimuli and might therefore importantly contribute to endothelial integrity. We show here that the apoptosis-suppressive effects of shear stress are mediated by upregulation of Cu/Zn SOD and NO synthase. Shear stress-mediated inhibition of endothelial cell apoptosis in response to exogenous oxygen radicals, oxidized LDL, and tumor necrosis factor-alpha was associated with complete inhibition of caspase-3-like activity, the central effector arm executing the apoptotic cell death program in endothelial cells. Shear stress-dependent upregulation of Cu/Zn SOD and NO synthase blocks activation of the caspase cascade in response to apoptosis-inducing stimuli. These findings establish the upregulation of Cu/Zn SOD and NO synthase by shear stress as a central protective cellular mechanism to preserve the integrity of the endothelium after proapoptotic stimulation.  (+info)

Inhibition of xanthine oxidase and xanthine dehydrogenase by nitric oxide. Nitric oxide converts reduced xanthine-oxidizing enzymes into the desulfo-type inactive form. (3/1226)

Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were inactivated by incubation with nitric oxide under anaerobic conditions in the presence of xanthine or allopurinol. The inactivation was not pronounced in the absence of an electron donor, indicating that only the reduced enzyme form was inactivated by nitric oxide. The second-order rate constant of the reaction between reduced XO and nitric oxide was determined to be 14.8 +/- 1.4 M-1 s-1 at 25 degrees C. The inactivated enzymes lacked xanthine-dichlorophenolindophenol activity, and the oxypurinol-bound form of XO was partly protected from the inactivation. The absorption spectrum of the inactivated enzyme was not markedly different from that of the normal enzyme. The flavin and iron-sulfur centers of inactivated XO were reduced by dithionite and reoxidized readily with oxygen, and inactivated XDH retained electron transfer activities from NADH to electron acceptors, consistent with the conclusion that the flavin and iron-sulfur centers of the inactivated enzyme both remained intact. Inactivated XO reduced with 6-methylpurine showed no "very rapid" spectra, indicating that the molybdopterin moiety was damaged. Furthermore, inactivated XO reduced by dithionite showed the same slow Mo(V) spectrum as that derived from the desulfo-type enzyme. On the other hand, inactivated XO reduced by dithionite exhibited the same signals for iron-sulfur centers as the normal enzyme. Inactivated XO recovered its activity in the presence of a sulfide-generating system. It is concluded that nitric oxide reacts with an essential sulfur of the reduced molybdenum center of XO and XDH to produce desulfo-type inactive enzymes.  (+info)

Pteridines as inhibitors of xanthine oxidase: structural requirements. (4/1226)

Different pteridine derivatives were investigated for their inhibitory action on xanthine oxidase. From 27 investigated compounds, 13 showed concentration-dependent inhibition of the enzyme. Concentrations necessary for 50% inhibition ranged from <0.1 up to >100 microM. Different types of inhibition were found concerning xanthine and pterin as substrates: competitive, noncompetitive and mixed type. Out of 18 aromatic compounds tested, 12 were inhibitors. Only one out of nine reduced derivatives served as inhibitor. A simple regression model was used to specify the structural requirements for a pteridine to be an inhibitor. The most characteristic features of an inhibitor are aromaticity and no substitution at position 7 of the pteridine ring.  (+info)

Reactive oxygen metabolites increase mitochondrial calcium in endothelial cells: implication of the Ca2+/Na+ exchanger. (5/1226)

In endothelial cells, a bolus of hydrogen peroxide (H2O2) or oxygen metabolites generated by hypoxanthine-xanthine oxidase (HX-XO) increased the mitochondrial calcium concentration [Ca2+]m. Both agents caused a biphasic increase in [Ca2+]m which was preceded by a rise in cytosolic free calcium concentration [Ca2+]c (18 and 6 seconds for H2O2 and HX-XO, respectively). The peak and plateau elevations of [Ca2+] were consistently higher in the mitochondrial matrix than in the cytosol. In Ca2+-free/EGTA medium, the plateau phase of elevated [Ca2+] evoked by H2O2 due to capacitative Ca2+ influx was abolished in the cytosol, but was maintained in the mitochondria. In contrast to H2O2 and HX-XO, ATP which binds the P2Y purinoceptors induced an increase in [Ca2+]m that was similar to that of [Ca2+]c. When cells were first stimulated with inositol 1,4, 5-trisphosphate-generating agonists or the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA), subsequent addition of H2O2 did not affect [Ca2+]c, but still caused an elevation of [Ca2+]m. Moreover, the specific inhibitor of the mitochondrial Ca2+/Na+ exchanger, 7-chloro-3,5-dihydro-5-phenyl-1H-4.1-benzothiazepine-2-on (CGP37157), did not potentiate the effects of H2O2 and HX-XO on [Ca2+]m, while causing a marked increase in the peak [Ca2+]m and a significant attenuation of the rate of [Ca2+]m efflux upon addition of histamine or CPA. In permeabilized cells, H2O2 mimicked the effects of CGP37157 causing an increase in the basal level of matrix free Ca2+ and decreased efflux. Dissipation of the electrochemical proton gradient by carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), and blocade of the Ca2+ uptake by ruthenium red prevented [Ca2+]m increases evoked by H2O2. These results demonstrate that the H2O2-induced elevation in [Ca2+]m results from a transfer of Ca2+ secondary to increased [Ca2+]c, and an inhibition of the Ca2+/Na+ electroneutral exchanger of the mitochondria.  (+info)

The superoxide dismutase activity of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774. (6/1226)

Desulfoferrodoxin (Dfx), a small iron protein containing two mononuclear iron centres (designated centre I and II), was shown to complement superoxide dismutase (SOD) deficient mutants of Escherichia coli [Pianzzola, M.J., Soubes M. & Touati, D. (1996) J. Bacteriol. 178, 6736-6742]. Furthermore, neelaredoxin, a protein from Desulfovibrio gigas containing an iron site similar to centre II of Dfx, was recently shown to have a significant SOD activity [Silva, G., Oliveira, S., Gomes, C.M., Pacheco, I., Liu, M.Y., Xavier, A.V., Teixeira, M., Le Gall, J. & Rodrigues-Pousada, C. (1999) Eur. J. Biochem. 259, 235-243]. Thus, the SOD activity of Dfx isolated from the sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 was studied. The protein exhibits a SOD activity of 70 U x mg-1, which increases approximately 2.5-fold upon incubation with cyanide. Cyanide binds specifically to Dfx centre II, yielding a low-spin iron species with g-values at 2.27 (g perpendicular) and 1.96 (g parallel). Upon reaction of fully oxidized Dfx with the superoxide generating system xanthine/xanthine oxidase, Dfx centres I and II become partially reduced, suggesting that Dfx operates by a redox cycling mechanism, similar to those proposed for other SODs. Evidence for another SOD in D. desulfuricans is also presented - this enzyme is inhibited by cyanide, and N-terminal sequence data strongly indicates that it is an analogue to Cu,Zn-SODs isolated from other sources. This is the first indication that a Cu-containing protein may be present in a sulphate-reducing bacterium.  (+info)

Tissue distribution and characteristics of xanthine oxidase and allopurinol oxidizing enzyme. (7/1226)

Tissue distribution and levels of allopurinol oxidizing enzyme and xanthine oxidase with hypoxanthine as a substrate were compared with supernatant fractions from various tissues of mice and from liver of mice, rats, guinea pigs and rabbits. The allopurinol oxidizing enzyme activities in liver were quite different among the species and the sex difference of the enzyme activity only in mouse liver. In mice, the highest activity of allopurinol oxidizing enzyme was found in the liver with a trace value in lung, but the enzyme activity was not detected in brain, small intestine and kidney, while the highest activity of xanthine oxidase was detected in small intestine, lung, liver and kidney in that sequence. The allopurinol oxidizing enzyme activity in mouse liver supernatant fraction did not change after storage at -20 degrees C or dialysis against 0.1 M Tris-HCl containing 1.15% KCl, but the activity markedly decreased after dialysis against 0.1 M Tris-HCl. On the contrary, the xanthine oxidase was activated 2 to 3 times the usual activity after storage at -20 degrees C or dialysis of the enzyme preparation. These results indicated that allopurinol was hydroxylated to oxipurinol mainly by the enzyme which is not identical to xanthine oxidase in vivo. A possible role of aldehyde oxidase involved in the allopurinol oxidation in liver supernatant fraction was dicussed.  (+info)

Cytosolic Ca2+ movements of endothelial cells exposed to reactive oxygen intermediates: role of hydroxyl radical-mediated redox alteration of cell-membrane Ca2+ channels. (8/1226)

1. The mode of action of reactive oxygen intermediates in cysosolic Ca2+ movements of cultured porcine aortic endothelial cells exposed to xanthine/xanthine oxidase (X/XO) was investigated. 2. Cytosolic Ca2+ movements provoked by X/XO consisted of an initial Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores and a sustained Ca2+ influx through cell-membrane Ca2+ channels. The Ca2+ movements from both sources were inhibited by catalase, cell-membrane permeable iron chelators (o-phenanthroline and deferoxamine), a *OH scavenger (5,5-dimethyl-1-pyrroline-N-oxide), or an anion channel blocker (disodium 4, 4'-diisothiocyano-2, 2'-stilbenedisulphonic acid), suggesting that *O2- influx through anion channels was responsible for the Ca2+ movements, in which *OH generation catalyzed by intracellular transition metals (i.e., Haber-Weiss cycle) was involved. 3. After an initial Ca2+ elevation provoked by X/XO, cytosolic Ca2+ concentration decreased to a level higher than basal levels. Removal of X/XO slightly enhanced the Ca2+ decrease. Extracellular addition of sulphydryl (SH)-reducing agents, dithiothreitol or glutathione, after the removal of X/XO accelerated the decrement. A Ca2+ channel blocker, Ni2+, abolished the sustained increase in Ca2+, suggesting that Ca2+ influx through cell-membrane Ca2+ channels was extracellularly regulated by the redox state of SH-groups. 4. The X/XO-provoked change in cellular respiration was inhibited by Ni2+ or dithiothreitol as well as inhibitors of Haber-Weiss cycle, suggesting that Ca2+ influx was responsible for *OH-mediated cytotoxicity. We concluded that intracellular *OH generation was involved in the Ca2+ movements in endothelial cells exposed to X/XO. Cytosolic Ca2+ elevation was partly responsible for the oxidants-mediated cytotoxicity.  (+info)