The use of acetylated ferricytochrome C for the detection of superoxide radicals produced in biological membranes

Azzi, Angelo; Montecucco, Cesare; Richter, Christoph

doi:10.1016/s0006-291x(75)80188-4

Cited by 332 publications

(127 citation statements)

References 15 publications

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“…We therefore examined NADPH oxidase activity in the renal cortex material derived from animals studied above, by using an acetylated cytochrome c reduction assay. 33 NADPH-dependent, superoxide-producing activity in the membrane fraction was defined as a difference between the reduction rate of acetylated cytochrome c without and with superoxide dismutase (SOD). High salt loading increased NADPH-dependent su- Figure 1.…”

Section: Resultsmentioning

confidence: 99%

l -Arginine Reverses p47phox and gp91phox Expression Induced by High Salt in Dahl Rats

et al. 2003

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Abstract-Derangements in the production and degradation of reactive oxygen species (ROS) as well as nitric oxide (NO) have been implicated in cardiovascular diseases. We explored how supplementation with L-arginine, an NO synthase substrate, restores such derangements of ROS/NO systems in Dahl salt-sensitive, hypertensive (DS) rats. We detected an increase of NADPH oxidase activity, a key enzyme that produces superoxide, in the membrane fraction of the renal cortex derived from DS rats loaded with high salt for 4 weeks; high salt loading also remarkably increased urinary H 2 O 2 , 8-isoprostane, and thromboxane B 2 excretion and decreased plasma NO end products. These changes from high salt loading were counteracted by oral L-arginine supplementation. We further examined expression patterns of NADPH oxidase subunits in renal cortex derived from these animals. High salt loading increased gp91phox and p47phox but not p22phox or Rac1 or mRNA abundance, which were counteracted with L-arginine supplementation. Western blot analyses after subcellular fractionation revealed that L-arginine supplementation distinctly decreases membrane localization of p47phox protein, as it decreases total expression of Rac1 protein in DS rats with high salt loading. These results disclose that high salt loading causes a deficiency in available L-arginine amounts for NO synthases and induces NADPH oxidase activation in the renal cortex of DS rats, which L-arginine supplementation markedly restores. Since superoxide rapidly eliminates NO, which inhibits sodium reabsorption in the cortical collecting duct, superoxide production caused by upregulated NADPH oxidase activity in the renal cortex of high salt-loaded DS rats may accelerate sodium reabsorption and hypertension. Key Words: rats, Dahl Ⅲ arginine Ⅲ hypertension, sodium-dependent Ⅲ nitric oxide Ⅲ cardiovascular diseases R ecent studies have revealed significant associations between the pathogenesis of numerous cardiovascular diseases, including hypertensive states, and the increase in reactive oxygen species (ROS) and/or the decrease in nitric oxide (NO). [1][2][3] Complete understanding of such derangements in ROS/NO systems has been prevented by the fact that mammalian organs are provided with multiple layers of complicated enzymatic as well as nonenzymatic machineries that deal with ROS/NO. 4,5 Among such machineries, NADPH oxidase, which was originally known to produce a large amount of superoxide anion to kill bacteria in neutrophils, 6 has attracted much attention as a potentially important player in a wide array of cardiovascular disorders. Although NADPH oxidase in the nonphagocytic cells has been less well characterized, physiologically relevant low generation of ROS and neutrophillike expression of NADPH oxidase subunits is present in vascular tissues, 7,8 which is activated by pulsatile stretch, 9 platelet-derived growth factor, 10 and angiotensin II. 1,[11][12][13][14][15] These studies, when taken together, suggest that NADPH oxidase may exert broader actions in cardi...

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Section: Resultsmentioning

confidence: 99%

l -Arginine Reverses p47phox and gp91phox Expression Induced by High Salt in Dahl Rats

et al. 2003

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“…Superoxide production was followed spectrophotometrically with partially acetylated cytochrome c (Azzi et al, 1975) and calculated from cytochrome c absorbance at 550-540 nm using extinction coefficient E 550-540 mM ϭ 19.2 cm Ϫ1 . The mitochondrial NADPH reduction state was measured fluorimetrically using an excitation wavelength of 346 nm and an emission wavelength of 460 nm.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial α-Ketoglutarate Dehydrogenase Complex Generates Reactive Oxygen Species

Starkov¹,

Fiskum²,

Chinopoulos³

et al. 2004

J. Neurosci.

638

472

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Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H 2 O 2 production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone, ␣-ketoglutarate supported the highest rate of H 2 O 2 production. In the absence of ADP or in the presence of rotenone, H 2 O 2 production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of ␣-ketoglutarate. Isolated mitochondrial ␣-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H 2 O 2 . NAD ϩ inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H 2 O 2 production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H 2 O 2 than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.

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“…The presence of superoxide radicals in the cell incubation medium was detected and then quanti fied by a second derivative spectrophotometric method based on a modification of the procedure described previously by Ghersi-Egea et al (1991), using acetyl-cytochrome c. Acetyl-cytochrome c was synthesized from acetic anhydride in half saturated sodium acetate, followed by four dialysis steps according to Azzi et al (1975). The concen tration of the acetyl-cytochrome c obtained was cal culated from its reduction spectrum, using an ex tinction coefficient of 19,000 M-1…”

Section: Superoxide Radical Measurementmentioning

confidence: 99%

A Simple Method for Evaluation of Superoxide Radical Production in Neural Cells under Various Culture Conditions: Application to Hypoxia

Daval

Ghersi‐Egea

Oillet³

et al. 1995

J Cereb Blood Flow Metab

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Summary: To evaluate the potential deleterious influence of oxygen-derived free radicals following hypoxia in a model of primary culture of neurons obtained from the fetal rat brain, superoxide radicals were measured as a function of time in the extracellular medium. Neuronal cells were grown for 8 days in the presence or absence of serum, then incubated in a buffered Krebs-Ringer solu tion containing 60 j..l. M acetyl-cytochrome c. The rate of superoxide radical formation was quantified spectropho tometrically by measuring the specific reduction of acetyl-cytochrome c. Under normoxic conditions (95% air-5% CO2), basal production of superoxide that in creased with time was recorded. It was significantly more

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The use of acetylated ferricytochrome C for the detection of superoxide radicals produced in biological membranes

Cited by 332 publications

References 15 publications

l -Arginine Reverses p47phox and gp91phox Expression Induced by High Salt in Dahl Rats

l -Arginine Reverses p47phox and gp91phox Expression Induced by High Salt in Dahl Rats

Mitochondrial α-Ketoglutarate Dehydrogenase Complex Generates Reactive Oxygen Species

A Simple Method for Evaluation of Superoxide Radical Production in Neural Cells under Various Culture Conditions: Application to Hypoxia

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