Calcein-AM is a detector of intracellular oxidative activity

Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent, cell permeant compound, which is converted by intracellular esterases into calcein, an anionic fluorescent form. It is used in microscopy and fluorometry and provides both morphological and functional information of viable cells. In this study we have tested the response of calcein-AM to oxidation. In cell-free fluorometric assays, H₂O₂ and xanthine–xanthine oxidase induced a dose-dependent emission of the AM form but had no effects on calcein. Fluorometric and confocal microscopy tests on human fibroblasts confirmed that the cell permeant AM form is the actual sensor since its removal from culture medium, and its consequent back-diffusion, made the system insensitive to oxidative stimuli. In time-lapse confocal microscopy, calcein-AM detected changes in the intracellular redox state following direct oxidation (H₂O₂, xanthine–xanthine oxidase) and phorbol ester treatment. Comparative tests showed that calcein-AM sensitivity to oxidation is about one order of magnitude higher than other fluorescein derivatives. The absence of leakage, due to the presence of the probe in the extracellular compartment, and its low toxicity allow to perform experiments for prolonged times following the response to the same or different stimuli repeatedly applied. We propose calcein-AM as a sensitive tool for intracellular ROS generation in living cells with useful applications for real-time imaging in confocal microscopy.     

Fluorescence imaging of changes in intracellular chloride in living brain slices.

In brain slice preparations, chloride movements across the cell membrane of living cells are measured traditionally with 36Cl- tracer methods, Cl--selective microelectrodes, or whole-cell recording using patch clamp analysis. We have developed an alternative, noninvasive technique that uses the fluorescent Cl- ion indicator, 6-methoxy-N-ethylquinolinium iodide (MEQ), to study changes in intracellular Cl- by epifluorescence or UV laser scanning confocal microscopy. In brain slices taken from rodents younger than 22 days of age, excellent cellular loading is achieved with the membrane-permeable form of the dye, dihydro-MEQ. Subsequent intracellular oxidation of dihydro-MEQ to the Cl--sensitive MEQ traps the polar form of the dye inside the neurons. Because MEQ is a single-excitation and single-emission dye, changes in intracellular Cl- concentrations can be calibrated from the Stern-Volmer relationship, determined in separate experiments. Using MEQ as the fluorescent indicator for Cl-, Cl- flux through the gamma-aminobutyric acid (GABA)-gated Cl- channel (GABAA receptor) can be studied by dynamic video imaging and either nonconfocal (epifluorescence) or confocal microscopy in the acute brain slice preparation. Increases in intracellular Cl- quench MEQ fluorescence, thereby reflecting GABAA receptor activation. GABAA receptor functional activity can be measured in discrete cells located in neuroanatomically defined populations within areas such as the neocortex and hippocampus. Changes in intracellular Cl- can also be studied under various conditions such as oxygen/glucose deprivation ("in vitro ischemia") and excitotoxicity. In such cases, changes in cell volume may also occur due to the dependence of cell volume regulation on Na+, K+, and Cl- flux. Because changes in cell volume can affect optical fluorescence measurements, we assess cell volume changes in the brain slice using the fluorescent indicator calcein-AM. Determination of changes in MEQ fluorescence versus calcein fluorescence allows one to distinguish between an increase in intracellular Cl- and an increase in cell volume.     

Pretreatment with oxygen species increases the resistance of mammalian cells to hydrogen peroxide and gamma-rays

Pretreatment of Chinese hamster ovary (CHO) or H4 (rat hepatoma) cells with low non-toxic doses of H2O2 or xanthine-xanthine oxidase renders the cells more resistant to the toxic effect of H2O2 and gamma-rays. This increased resistance is observed both in exponentially growing and in plateau-phase cells. Cells pretreated with xanthine-xanthine oxidase are less mutated than control cultures when challenged with ionizing radiation. The number of DNA single-strand breaks (measured by nucleoid sedimentation) induced by a high dose of gamma-rays or H2O2 is lower in cells pretreated with xanthine-xanthine oxidase compared to control cultures. However, the pretreatment does not modify the rate of DNA single-strand breaks rejoining in cells challenged with H2O2 or gamma-rays. The catalase activity is not modified in pretreated cells, but the superoxide dismutase activity is increased about 2-fold.     

Calmodulin participation in oxygen radical-induced cardiac sarcoplasmic reticulum calcium uptake reduction

The effect of scavengers of oxygen radicals on canine cardiac sarcoplasmic reticulum (SR) Ca2+ uptake velocity was investigated at pH 6.4, the intracellular pH of the ischemic myocardium. With the generation of oxygen radicals from a xanthine-xanthine oxidase reaction, there was a significant depression of SR Ca2+ uptake velocity. Xanthine alone or xanthine plus denatured xanthine oxidase had no effect on this system. Superoxide dismutase (SOD), a scavenger of .O2-, or denatured SOD had no effect on the depression of Ca2+ uptake velocity induced by the xanthine-xanthine oxidase reaction. However, catalase, which can impair hydroxyl radical (.OH) formation by destroying the precursor H2O2, significantly inhibited the effect of the xanthine-xanthine oxidase reaction. This effect of catalase was enhanced by SOD, but not by denatured SOD. Dimethyl sulfoxide (Me2SO), a known .OH scavenger, completely inhibited the effect of the xanthine-xanthine oxidase reaction. The observed effect of oxygen radicals and radical scavengers was not seen in the calmodulin-depleted SR vesicles. Addition of exogenous calmodulin, however, reproduced the effect of oxygen radicals and the scavengers. The effect of oxygen radicals was enhanced by the calmodulin antagonists (compounds 48/80 and W-7) at concentrations which showed no effect alone on Ca2+ uptake velocity. Taken together, these findings strongly suggest that .OH, but not .O2-, is involved in a mechanism that may cause SR dysfunction, and that the effect of oxygen radicals is calmodulin dependent.     

The effect of oxidant stress on diamide-treated human granulocytes

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xanthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.     

Interaction of electron leak and proton leak in respiratory chain of mitochondria——Proton leak induced by superoxide from an electron leak pathway of univalent reduction of oxygen

By incubating the isolated rat myocardial mitochondria with xanthine-xanthine oxidase, anexogenous superoxide (O2) generating system, and by ischemia-reperfusion procedure of isolated rat heart as an endogenous O2 generating system, it was found that both sources of O2 showed the same injurious effects on mitochondrial function resulting in (i) increasing proton leak rate, lowering proton pumping activity and Ht/2e ratio of respiratory chain, and (ii) decreasing transmembrane potential of energized mitochondria] inner membrane by succinate oxidation. The injurious effects of O2 on these mitochondrial bioenergitical parameters mentioned above exhibited a dosage- or reaction time-dependent mode. (X has no effects on the electron transfer activity and transmembrane potential of nonenergized mitochondria. Being a superoxide scavenger, 3, 4-dihydroxylphenyl lactate showed obvious protection effects against damage of both exogenous superoxide sources from xanthine-xanthine oxidase system and endogenous Or sou     

Autocrine/paracrine pattern of superoxide production through NAD(P)H oxidase in coronary arterial myocytes

The present study tested the hypothesis that membrane-bound NAD(P)H oxidase in coronary arterial myocytes (CAMs) is capable of producing superoxide (O(2)(*-)) toward extracellular space to exert an autocrine- or paracrine-like action in these cells. Using a high-speed wavelength-switching fluorescent microscopic imaging technique, we simultaneously monitored the binding of dihydroethidium-oxidizing product to exogenous salmon testes DNA trapped outside CAMs and to nuclear DNA as indicators of extra- and intracellular O(2)(*-) production. It was found that a muscarinic agonist oxotremorine (OXO; 80 microM) increased O(2)(*-) levels more rapidly outside than inside CAMs. In the presence of superoxide dismutase (500 U/ml) plus catalase (400 U/ml) and NAD(P)H oxidase inhibitor diphenylene iodonium (50 microM) or apocynin (100 microM), these increases in extra- and intracellular O(2)(*-) levels were substantially abolished or attenuated. The O(2)(*-) increase outside CAMs was also confirmed by detecting oxidation of nitro blue tetrazolium and confocal microscopic localization of Matrigel-trapped OxyBURST H(2)HFF Green BSA staining around these cells. By electron spin resonance spectrometry, the extracellular accumulation of O(2)(*-) was demonstrated as a superoxide dismutase-sensitive component outside CAMs. Furthermore, RNA interference of NAD(P)H oxidase subunits Nox1 or p47 markedly blocked OXO-induced increases in both extra- and intracellular O(2)(*-) levels, whereas small inhibitory RNA of Nox4 only attenuated intracellular O(2)(*-) accumulation. These results suggest that Nox1 represents a major NAD(P)H oxidase isoform responsible for extracellular O(2)(*-) production. This rapid extracellular production of O(2)(*-) seems to be unique to OXO-induced M(1)-receptor activation, since ANG II-induced intra- and extracellular O(2)(*-) increases in parallel. It is concluded that the outward production of O(2)(*-) via NAD(P)H oxidase in CAMs may represent an important producing pattern for its autocrine or paracrine actions.     

The effect of oxidant stress on diamide-treated human granulocytes.

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xamthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.     

A laser scanning confocal microscopy method. Simultaneous detection of intracellular Ca2+ and apoptosis using Fluo-3 and Hoechst 33342

OBJECTIVE: To develop a simple and direct method to simultaneously determine apoptotic cells from a treated population of cells and detect the changes of intracellular Ca2+ in these apoptotic cells, in particular single ones, by confocal microscopy. STUDY DESIGN: MGC-803 cells treated with As2O3 were used as the double-staining cell model with Hoechst 33342 as a DNA probe and Fluo-3AM as a Ca2+ indicator. MGC-803 cell apoptosis induced by As2O3 was first demonstrated by DNA ladder in gel electrophoresis. Based on the difference in DNA stainability with Hoechst 33342 and corresponding fluorescence intensity between live and apoptotic cells, apoptotic cells and the changes in intracellular Ca2+ were detected at the same time by confocal microscopy. No necrotic cells in the group treated with As2O3 were found by the trypan blue exclusion test. RESULTS: The results from confocal microscope detection showed that intact and apoptotic cells were successfully recognized and the changes of intracellular Ca2+ in apoptotic and intact cells were simultaneously detected in the same sample. CONCLUSION: We provided a useful method to exactly detect changes in intracellular Ca2+ in apoptotic cells, especially in single ones, by confocal microscopy and to exclude the artifact effect of necrotic and intact cells.     

Localization of Hydrogen Peroxide Production in Pisum sativum L. Using Epi-Polarization Microscopy to Follow Cerium Perhydroxide Deposition

Cerium is becoming an increasingly popular reagent for histochemical localization of oxidases and phosphatases because it combines directly with reaction products to form fine precipitates of electron-dense materials that can be easily detected using transmission electron microscopy or laser confocal scanning microscopy. We used epi-polarization microscopy to detect cerium perhydroxide deposits formed when H2O2 was produced by diamine oxidase in pea (Pisum sativum L.) epicotyls exposed to exogenous putrescine. Diamine oxidase activity was abundant in cortical cell walls but showed little, if any, association with vascular tissues. Maps of cerium deposition generated using scanning electron microscopy/x-ray microanalysis verified these observations. This study demonstrates the use of epi-polarization microscopy to follow cerium deposition, and the ready accessibility of this microscopy technique should facilitate more widespread use of cerium for plant histochemistry and cytochemistry.     

Nuclear localization of human spermine oxidase isoforms - possible implications in drug response and disease etiology

The recent discovery of the direct oxidation of spermine via spermine oxidase (SMO) as a mechanism through which specific antitumor polyamine analogues exert their cytotoxic effects has fueled interest in the study of the polyamine catabolic pathway. A major byproduct of spermine oxidation is H2O2, a source of toxic reactive oxygen species. Recent targeted small interfering RNA studies have confirmed that SMO-produced reactive oxygen species are directly responsible for oxidative stress capable of inducing apoptosis and potentially mutagenic DNA damage. In the present study, we describe a second catalytically active splice variant protein of the human spermine oxidase gene, designated SMO5, which exhibits substrate specificities and affinities comparable to those of the originally identified human spermine oxidase-1, SMO/PAOh1, and, as such, is an additional source of H2O2. Importantly, overexpression of either of these SMO isoforms in NCI-H157 human non-small cell lung carcinoma cells resulted in significant localization of SMO protein in the nucleus, as determined by confocal microscopy. Furthermore, cell lines overexpressing either SMO/PAOh1 or SMO5 demonstrated increased spermine oxidation in the nucleus, with accompanying alterations in individual nuclear polyamine concentrations. This increased oxidation of spermine in the nucleus therefore increases the production of highly reactive H2O2 in close proximity to DNA, as well as decreases nuclear spermine levels, thus altering the protective roles of spermine in free radical scavenging and DNA shielding, and resulting in an overall increased potential for oxidative DNA damage in these cells. The results of these studies therefore have considerable significance both with respect to targeting polyamine oxidation as an antineoplastic strategy, and in regard to the potential role of spermine oxidase in inflammation-induced carcinogenesis.     

Oxidation by superoxide of tocopherols dispersed in aqueous media with deoxycholate

alpha-Tocopherol dispersed in aqueous media with deoxycholate was found to be oxidized, at a physiological pH, by a xanthine-xanthine oxidase system. This reaction was completely inhibited by the addition of superoxide dismutase, whereas catalase and mannitol (scavenger of hydroxyl radical) did not affect the reaction. This finding indicates that the oxidation of alpha-tocopherol is caused by O2. The reaction product formed was identified as 8 alpha-hydroxy-alpha-tocopherone by thin-layer chromatography and ultraviolet spectroscopy. The product was found to change spontaneously to alpha-tocopherol quinone. beta-, gamma-, and delta-tocopherol dispersed with deoxycholate also reacted with O2. The reaction of tocopherols dispersed in the micellar form may be considered as a model of in vivo reaction of tocopherols, since tocopherols are present in tissues largely in the membranes, where O2 is known to be generated.     

Cytochrome P-450 dependent ethanol oxidation. Kinetic isotope effects and absence of stereoselectivity

Deuterium isotope effects [D(V/K)] and stereoselectivity of ethanol oxidation in cytochrome P-450 containing systems and in the xanthine-xanthine oxidase system were compared with those of yeast alcohol dehydrogenase. The isotope effects were determined by using both a noncompetitive method, including incubation of unlabeled or [1,1-2H2]ethanol at various concentrations, and a competitive method, where 1:1 mixtures of [1-13C]- and [2H6]ethanol or [2,2,2-2H3]- and [1,1-2H2]ethanol were incubated and the acetaldehyde formed was analyzed by gas chromatography/mass spectrometry. The D(V/K) isotope effects of the cytochrome P-450 dependent ethanol oxidation were about 4 with liver microsomes from imidazole-, phenobarbital- or acetone-treated rabbits or with microsomes from acetone- or ethanol-treated rats. Similar isotope effects were reached with reconstituted membranes containing the rabbit ethanol-inducible cytochrome P-450 (LMeb), whereas control rat microsomes and membranes containing rabbit phenobarbital-inducible P-450 LM2 oxidized the alcohol with D(V/K) of about 2.8 and 1.8, respectively. Addition of FeIIIEDTA either to microsomes from phenobarbital-treated rabbits or to membranes containing P-450 LMeb significantly lowered the isotope effect, which approached that of the xanthine-xanthine oxidase system (1.4), whereas desferrioxamine had no significant effect. Incubations of all cytochrome P-450 containing systems or the xanthine-xanthine oxidase systems with (1R)- and (1S)-[1-2H]ethanol, revealed, taking the isotope effects into account, that 44-66% of the ethanol oxidized had lost the 1-pro-R hydrogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyridine nucleotide-dependent generation of hydrogen peroxide by a particulate fraction from human neutrophils

The catalytic oxidation of [14C]-formate to 14CO2 was adapted to measure H2O2 formation in cellfree system. Standard curves employing glucose-glucose oxidase and xanthine-xanthine oxidase demonstrated linearity between 14CO2 evolution and enzyme concentration. A particulate fraction from human neutrophils was capable of oxidizing [14C]-formate; this reaction was dependent upon the presence of catalase, reduced pyridine nucleotide, and cellular material. Reaction increased with time of incubation and protein concentration, although not in a strictly linear fashion. The pH optimum was approximately 5.5 NADPH was a significantly better substrate than NADH, although both were capable of generating H2O2. The particulate fraction derived from phagocytizing cells was more active than a corresponding fraction from resting cells with either substrate. H2O2 production was abnormal in particulate fractions derived from 2 patients with chronic granulomatous disease. H2O2 production was markedly inhibited by superoxide dismutase or cytochrome c (scavengers of superoxide anion) but not by scavengers of singlet oxygen or hydroxyl radical. Reaction was greatly stimulated by the addition of manganous ion. These results are consistent with the hypothesis that the respiratory burst in human neutrophils is initiated by an oxidase that can utilize either NADPH or NADH but exhibits a marked preference for the former. Further, the inhibitor studies strongly support a mechanism involving an initial enzymatic reaction followed by a self-sustaining free radical reaction involving superoxide anion.     

Role of the Phagocyte in Host-Parasite Interactions XI. Relationship Between Stimulated Oxidative Metabolism and Hydrogen Peroxide Formation, and Intracellular Killing

The increased respiratory and hexose monophosphate activities noted in phagocytizing cells results in the formation of hydrogen peroxide. This is brought about by the oxidation of reduced nicotinamide adenine dinucleotide phosphate by its oxidase. Evidence is presented which indicates that this H(2)O(2) is involved in the intracellular killing of bacteria. When molecular oxygen was excluded from phagocytizing leukocytes by anaerobiosis, thus inhibiting H(2)O(2) formation, reduced intracellular killing was observed. In some cases the impairment of leukocytic bactericidal activity by anaerobiosis could be partially reversed by the addition of H(2)O(2). Exogenous catalase also could reduce intracellular killing. In addition, when leukocytic isolates were dialyzed so as to reduce endogenous H(2)O(2), the bactericidal activity of the leukocytes was significantly decreased under both aerobic and anaerobic conditions. These results occurred with both guinea pig and human leukocytes and with several test microorganisms.     

Phagosome-lysosome fusion is a calcium-independent event in macrophages

Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms. Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+]i). Indeed, increases in [Ca2+]i are essential for phagosome- granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes. Since several intracellular pathogens survive in macrophage phagosomes that do not fuse with lysosomes, we examined the regulation of phagosome-lysosome fusion in macrophages. Macrophages (M phi) were treated with 12.5 microM bis-(2-amino-S-methylphenoxy) ethane- N,N,N'',N'',-tetraacetic acid tetraacetoxymethyl ester (MAPT/AM), a cell- permeant calcium chelator which reduced resting cytoplasmic [Ca2+]; from 80 nM to < or = 20 nM and completely blocked increases in [Ca2+]i in response to multiple stimuli, even in the presence of extracellular calcium. Subsequently, M phi phagocytosed serum-opsonized zymosan, staphylococci, or Mycobacterium bovis. Microbes were enumerated by 4'',6- diamidino-2-phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LAMP-1) as a membrane marker and rhodamine dextran as a content marker for lysosomes. Confirmation of phagosome-lysosome fusion by electron microscopy validated the fluorescence microscopy findings. We found that phagosome-lysosome fusion in M phi occurs noramlly at very low [Ca2+]i (< or = 20 nM). Kinetic analysis showed that in M phi none of the steps leading from particle binding to eventual phagosome-lysosome fusion are regulated by [Ca2+]i in a rate- limiting way. Furthermore, confocal microscopy revealed no difference in the intensity of LAMP-1 immunofluorescence in phagolysosome membranes in calcium-buffered vs. control macrophages. We conclude that neither membrane recognition nor fusion events in the phagosomal pathway in macrophages are dependent on or regulated by calcium.     

Cytokinin interaction with free radical metabolism and senescence: Effects on endogenous lipoxygenase and purine oxidation

A typical system comprising xanthine-xanthine oxidase, which produces superoxide free radicals, significantly increased endogenous levels of the senescence-associated lipoxygenase enzyme while cytokinin reversed this effect. It is suggested that in its interaction with free radicals cytokinin may have a dual effect: a) it may inhibit purine oxidation by the formation of a 2,8 dihydroxy purine which lowers the substrate affinity of xanthine oxidase; b) it may act as a direct free radical scavenger by virtue of H abstraction from the α-carbon atom in the amine bond.     

H_2O_2介导H_2S诱导的拟南芥气孔关闭

以拟南芥（Arabidopsis thaliana）为材料,研究了过氧化氢（H2O2）在硫化氢（H2S）调控气孔运动信号转导中的作用。结果表明,光下H2S的供体硫氢化钠（NaHS）能够诱导拟南芥气孔关闭;且能够显著提高叶片和保卫细胞胞质H2O2含量;H2O2的清除剂AsA和H2O2合成酶的抑制剂可不同程度地抑制NaHS诱导的拟南芥气孔关闭及叶片和保卫细胞胞质H2O2水平的升高;NaHS对AtrbohD、AtrbohF、Atpao2和Atpao4突变体气孔关闭、叶片和保卫细胞胞质H2O2水平升高的诱导作用要明显的小于野生型,但对AtPAO2和AtPAO4过表达株系叶片和保卫细胞H2O2水平的升高较野生型显著。据此推测,来源于NADPH氧化酶、细胞壁过氧化物酶和多胺氧化酶途径的H2O2参与H2S诱导的拟南芥气孔关闭。     

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-alpha.

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O2- and H2O2, in endothelial cells. Then we examined the influence of tumor necrosis factor-alpha and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O2- and H2O2. We determined a basal intracellular H2O2 concentration of 32.8 +/- 10.7 pM in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 +/- 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 +/- 3.79 mU/mg protein. Only 0.48 +/- 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H2O2 release was 75 +/- 12 pmol.min-1.mg cell protein-1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 +/- 0.75 microU/mg with 47.1 +/- 11.7% in the oxidase form. Intracellular purine levels of 1.19 +/- 1.04 nmol hypoxanthine/mg protein, 0.13 +/- 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 microgram/ml lipopolysaccharide (LPS) for 1-12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H2O2 in the direct endothelial toxicity of TNF and LPS.     

The microbial metabolism of C1 compounds. The stoicheiometry of respiration-driven proton translocation in Pseudomonas AM1 and in a mutant lacking cytochrome c

This paper clarifies the role of cytochrome c in Pseudomonas AM1 by measuring the stoicheiometry of proton translocation driven by respiration of endogenous or added substrates in wild-type bacteria and in a mutant lacking cytochrome c (mutant PCT76). The maximum -->H(+)/O ratio (protons translocated out of the bacteria per atom of oxygen consumed during respiration) was about 4 and, except when respiration was markedly affected, this ratio was similar in mutant and wild-type bacteria. The -->H(+)/O ratios were unaltered when the usual oxidase (cytochrome a(3)) was inhibited by 300mum-KCN and respiration involved the single cytochrome b functioning as an alternative oxidase. Ratios measured in cells respiring endogenous substrate and in cells loaded with malate or 3-hydroxybutyrate suggest that there are two proton-translocating segments operating during the oxidation of NADH. By contrast, during oxidation of formaldehyde or methylamine only one pair of protons is translocated. Proton translocation could not be measured with methanol as substrate, because its oxidation was inhibited (90-95%) by 5mm-KSCN. It is tentatively proposed that the electron-transport chain for NADH oxidation in Pseudomonas AM1 is arranged such that the NADH-ubiquinone oxidoreductase forms one proton-translocating segment and the second segment consists of ubiquinone and cytochromes b and a/a(3). The cytochrome c appears to be essential only for respiration and proton translocation from methanol (and possibly from methylamine); there is no conclusive evidence that cytochrome c ever mediates between cytochromes b and a/a(3) in Pseudomonas AM1.