Biomedical subjects

H Sies

Publications and source records attributed to H Sies.

At least 271 records · Page 15Linked to original sources

PubMed1988

Hydroperoxide-induced chemiluminescence in rabbit lungs: role of arachidonic acid enzymes.

Low-level chemiluminescence (C) is thought to be an index of oxidant stress. We measured the relationship between low-level C, pulmonary arterial pressure, and perfusate concentration of thromboxane B2 (TxB2) in isolated perfused rabbit lungs during challenge with tert-butyl hydroperoxide (t-bu-OOH). We also measured glutathione release as another index of oxidant stress. We found that C was correlated with each variable, suggesting that oxidant stress measured by C and by glutathione release stimulated TxB2 production and pulmonary vasoconstriction. We also investigated the contribution of active O2 metabolites produced by prostaglandin (PG) peroxidase to oxidant stress by studying the effects of t-bu-OOH before and after the use of cyclooxygenase and lipoxygenase inhibitors. We found that C was augmented after inhibition, perhaps due to metabolism of t-bu-OOH by peroxidases of both arachidonic acid (AA) metabolic pathways in the absence of their normal substrates. We studied phenylbutazone, thought to inhibit peroxidases, and AA. C during t-bu-OOH administration was not augmented after phenylbutazone and was markedly inhibited after AA administration perhaps because AA competes with t-bu-OOH. To further study the role of peroxidases we pretreated the lungs with the antioxidant dithiothreitol, which inhibits peroxidases involved in both the cyclooxygenase and lipoxygenase pathways. Dithiothreitol nearly abolished C produced by t-bu-OOH and also prevented the increased light caused by eicosatetrynoic acid. We directly tested the hypothesis that C occurred as a result of the interaction of t-bu-OOH and the cyclooxygenase and lipoxygenase enzymes; we measured C when t-bu-OOH was added to purified PGH2 synthase or soybean lipoxygenase. The combination of t-bu-OOH with PGH2 synthase or lipoxygenase led to C that was inhibited by dithiothreitol and by the antioxidant phenol. These results suggest that enzymes involved in AA metabolism can interact with t-bu-OOH and that the action of these enzymes on t-bu-OOH leads to C. The results may mean that lipid peroxides can indirectly contribute to tissue oxidant stress due to production of active O2 metabolites as by-products of their metabolism by AA peroxidases.

Animals↗

PubMed1988

Energy dependence of enzyme release from hypoxic isolated perfused rat heart tissue.

There is a sudden release of intracellular constituents upon reoxygenation of isolated perfused hypoxic heart tissue (O2 paradox) or on perfusion with calcium-free medium after a period of hypoxia. Rat hearts were perfused by the method of Langendorff (Pfluegers Arch. 61: 291-332, 1895) with Krebs-Henseleit medium containing 10 mM glucose. Hearts were equilibrated for 30 min, followed by 90 min of hypoxia or 60 min of hypoxia and 30 min of reoxygenation. The massive enzyme release observed upon reoxygenation after 60 min of hypoxia was prevented by infusing 0.5 or 5 mM cyanide 5 min before reoxygenation. Lactate dehydrogenase (LDH) release commenced immediately upon withdrawal of cyanide. Hearts perfused with calcium-free medium throughout hypoxia did not release increased amounts of LDH at reoxygenation. Perfusing heart tissue with medium containing 0 or 25 microM calcium, but not 0.25 or 2.5 mM, after 50 min of hypoxia initiated a release of cardiac LDH, which was not further enhanced by reoxygenation. Enzyme release was significantly inhibited when the calcium-free perfusion medium included 10 mM 2-deoxyglucose (replacing glucose), 0.5 mM dinitrophenol, or 2.5 mM cyanide. Histologically, hearts perfused with calcium-free medium after 50 min of hypoxia showed areas of severe necrosis and contracture without any evidence of the contraction bands that were seen in hearts reoxygenated in the presence of calcium. Cardiac ATP and creatine phosphate (PCr) levels were significantly decreased after 50-60 min of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1987

Cholestasis and changes in the microcirculation of perfused rat liver caused by the calcium ionophore A23187 and type I antiarrhythmic drugs.

The calcium-ionophore A23187 causes a reversible increase of the hydrostatic pressure in the portal vein of perfused rat liver. Concomitantly, hepatic functions like the production of bile and the transhepatic movement of the bile acid taurocholate are diminished, mainly due to decreased uptake. These phenomena are partly explained by changes in the microcirculation of the liver, visualized by Trypan blue staining. Both the increase in portal pressure and the major part of the decrease of biliary excretion of taurocholate and bile flow are prevented by the addition of the vasodilator papaverine. The type I antiarrhythmic drugs quinidine and N-propylajmaline bitartrate (NPA), at high concentrations, also induce a rise in portal pressure and act as a cholestatic agent. The rise in portal pressure caused by NPA requires the presence of extracellular calcium and is counteracted by papaverine. In contrast to A23187, the cholestasis caused by NPA is not influenced by papaverine. While NPA decreases the hepatic uptake and biliary excretion of taurocholate, papavarine is able to restore only the uptake and not the excretion. The concentration of taurocholate in the bile is not significantly changed by NPA.

Animals↗

PubMed1987

pH control of hepatic glutamine degradation. Role of transport.

Glutamine uptake is decreased in isolated perfused rat liver when the extracellular pH is lowered. This is also observed in the presence of ammonia concentrations nearly 20-fold above that required for half-maximal stimulation of glutaminase, indicating that the effect is not explained by a submaximal ammonium activation of the enzyme. In livers perfused with a physiological glutamine concentration (0.6 mM), the tissue glutamine but not glutamate content is strongly dependent on the extracellular pH and increases from 2.9 mumol/g to 4.7 mumol/g liver when the extracellular pH is increased from 7.3 to 7.5. Subfractionation of the livers revealed that the mitochondrial glutamine concentration increases from about 15 mM to 50 mM, when the extracellular pH is raised from 7.3 to 7.7, whereas the cytosolic glutamine concentration increases only slightly. Simultaneously the cytosolic and mitochondrial pH values are largely unaffected, being 7.25 and 7.7 respectively. Thus, the pH gradient between mitochondria and cytosol remains unchanged when the extracellular pH varies. Amiloride (2 mM) inhibits glutamine uptake by the liver and abolishes the extra/intracellular pH gradient. With amiloride present, tissue glutamine levels are no longer dependent on extracellular pH and are only about 2 mumol/g liver. It is concluded that pH control of glutaminase flux is also mediated by variations of the mitochondrial glutamine concentration pointing to a regulatory role of the glutamine carrier in the mitochondrial membrane for hepatic glutamine breakdown.

Animals↗

PubMed1987

Generation of photoemissive species by mitomycin C redox cycling in rat liver microsomes.

Generation of reactive oxygen species during redox cycling is thought to be involved in the chemotherapeutic action of quinone anticancer drugs. A clinically used agent which contains a quinone moiety is mitomycin C (Mit C). With isolated rat liver microsomes we detected photoemissive species during Mit C-induced redox cycling. After addition of reduced glutathione (GSH) a large increase in Mit C-induced chemiluminescence was observed. The increase of photoemission in deuterium oxide as well as greater than 90% of intensity at wavelengths greater than 610 nm suggest that singlet oxygen is a photoemissive species generated by this system. Glutathione disulfide (GSSG) accumulates during the reaction. We propose that superoxide anion radicals formed during redox cycling of Mit C react with GSH. Generation of glutathionyl radicals followed by oxygen addition then leads to the formation of photoemissive species and GSSG.

Animals↗

PubMed1987

Inhibition of taurocholate efflux from rat hepatic canalicular membrane vesicles by glutathione disulfide.

In right-side out rat hepatic canalicular membrane vesicles glutathione disulfide (GSSG) inhibited the efflux of taurocholate approx. 70% in the presence or approx. 55% in the absence of a valinomycin-mediated K+ diffusion potential; maximal inhibition occurred at 5 mM GSSG. The inhibition by GSSG was abolished by dithioerythritol. Neither dithioerythritol alone nor GSH inhibited taurocholate efflux. S-(2,4-Dinitrophenyl)glutathione and N-ethylmaleimide showed intermediate inhibitory effects.

Animals↗

PubMed1987

Perivascular nerve stimulation and phenylephrine responses in rat liver. Metabolic effects, Ca2+ and K+ fluxes.

Electrical stimulation of perivascular nerves (20 Hz/2 ms/20 V) in perfused rat liver led to a transient increase of 14CO2 production from [1-14C]glutamate, glutathione and thiol efflux, an increase in the lactate/pyruvate and the 3-hydroxybutyrate/acetoacetate ratio, glucose release and of portal pressure. These metabolic effects were accompanied by a Ca2+ release from the liver within the initial 2 min, being followed by Ca2+ reuptake, which lasted about 3 min. The initial Ca2+ release was 67 nmol/g liver and was smaller than that observed after phenylephrine (5 microM) addition (156 nmol/g liver). Hepatic Ca2+ release following nerve stimulation or phenylephrine was not significantly affected when the hemodynamic changes were largely prevented by sodium nitroprusside (10 microM). Although the amounts of Ca2+ released were different, the glycogenolytic responses, but not the other metabolic effects, were quantitatively similar with nerve stimulation and phenylephrine. Within the first 3 min of nerve stimulation there was a K+ uptake by the liver being followed by a K+ release over the next 5-6 min and a subsequent slow K+ uptake phase. These changes resembled those observed with phenylephrine. Phentolamine, an alpha-adrenergic antagonist, abolished the Ca2+ and K+ movements following nerve stimulation as well as glucose release and the hemodynamic changes. During continuous infusion of phenylephrine, nerve stimulation led still to an increase of portal pressure; however, the effects of nerve stimulation on Ca2+ and K+ fluxes and glucose release were largely suppressed. It is concluded that the metabolic effects of electrical nerve stimulation are mediated by a redistribution of cellular Ca2+ following alpha-receptor activation. Nerve stimulation involves Ca2+ and K+ fluxes across the plasma membrane. The metabolic effects are qualitatively similar to those induced by phenylephrine. The quantitative difference between nerve stimulation and phenylephrine is explained by a differential subacinar response, with fewer cells being reached by nerve stimulation than cells containing alpha-receptors. The hemodynamic changes of nerve stimulation point to the existence of sphincters near the inflow of the sinusoidal bed.

Animals↗

PubMed1987

Direct protective effect of NAD(P)H:quinone reductase against menadione-induced chemiluminescence of postmitochondrial fractions of mouse liver.

In the presence of NADPH and oxygen, menadione (2-methyl-1,4-naphthoquinone) elicits low level red chemiluminescence from rodent liver preparations. This chemiluminescence is believed to arise from the formation of active oxygen species that are generated when the quinone undergoes oxidative cycling. The obligatory two-electron reduction of quinones to hydroquinones catalyzed by NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) has been implicated in the suppression of this photoemission by competing with oxidative cycling (Wefers, H., Komai, T., Talalay, P., and Sies, H. (1984) FEBS Lett. 169, 63-66 and references therein). Thus, in previous studies, we showed that treatment of mice with BHA (2(3)-tert-butyl-4-hydroxyanisole), which elevates cytosolic quinone reductase activity about 10-fold, reduced menadione-dependent chemiluminescence of hepatic post-mitochondrial supernatant fractions, whereas inhibition of quinone reductase by dicoumarol greatly intensified light emission. We demonstrate here that addition of pure quinone reductase to this preparation suppresses menadione-dependent chemiluminescence, and that the protective effect of 2(3)-tert-butyl-4-hydroxyanisole treatment can be accounted for completely by the induction of this specific enzyme. These results provide conclusive evidence that in this system the protective action of anticarcinogenic antioxidants is entirely attributable to the elevation of the level of an electrophile-processing enzyme.

Animals↗

PubMed1987

Sustained oscillations in extracellular calcium concentrations upon hormonal stimulation of perfused rat liver.

Sustained oscillations in extracellular free Ca2+ were shown to occur on addition of vasopressin, phenylephrine or angiotensin II in isolated rat liver perfused with low (10 microM)-Ca2+ medium. The amplitude and frequency of oscillation depend on hormone concentration. In contrast, Ca2+ releases on addition of ATP, t-butyl hydroperoxide or arachidonate do not exhibit oscillatory behaviour. The vasopressin-induced oscillations were suppressed by glucagon and dibutyryl 3',5'-cyclic AMP, but not by dibutyryl 3',5'-cyclic GMP. These observations in the extracellular space complement observations by Woods, Cuthbertson & Cobbold [(1986) Nature (London) 319, 600-602] on oscillations in intracellular free Ca2+ in single liver cells.

Adenosine Triphosphate↗

PubMed1987

Loss of transforming activity of plasmid DNA (pBR322) in E. coli caused by singlet molecular oxygen.

Plasmid DNA pBR322 in aqueous solution was exposed to singlet molecular oxygen (1O2) generated by microwave discharge. DNA damage was detected as loss of transforming activity of pBR322 in E. coli (CMK) dependent on the time of exposure. DNA damage was effectively decreased by singlet-oxygen quenchers such as sodium azide and methionine. Replacement of water in the incubation buffer by D2O led to an increase in DNA damage. 9,10-Bis(2-ethylene)anthracene disulfate was used as a chemical trap for 1O2 quantitation by HPLC analysis of the endoperoxide formed.

DNA Damage↗

PubMed1987

Intact organ spectrophotometry and single-photon counting.

In toxicology, it is of interest not only to assess enzyme levels and capacities for potential fluxes, but it is also useful to develop methods for determining actual concentrations and fluxes in the intact cell and organ. To this end, several noninvasive techniques have been developed over the years. Our interest has been largely in photometric techniques. Transmission spectrophotometry through solid organs permits monitoring of the cytochromes of the mitochondrial respiratory chain and cytochrome P-450 as well as other pigments of biological interest. Furthermore, the steady state level of catalase Compound I in liver provides information on rates of H2O2 production. These are in the nM to microM concentration range. More recently, the monitoring of photoemission from intact organs has been useful in toxicological problems. The major photoemissive species, singlet molecular oxygen and excited carbonyls, can now be monitored with good signal/noise ratio. Redox cycling of quinones and the generation of photoemissive species were studied in menadione metabolism. Inhibition of phase II led to a significant increase in the steady state level of singlet oxygen, as did the inhibition of two-electron reduction by using the inhibitor dicoumarol for DT diaphorase. Conversely, the induction of DT diaphorase by pretreatment with BHA protected by decreasing the level of reactive oxygen species.

Radiation↗

PubMed1987

Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation.

An oxystat system was employed in conjunction with a single-photon counting apparatus for simultaneous monitoring of oxygen uptake, oxidative decomposition of membrane lipids, and occurrence of electronically excited species during microsomal lipid peroxidation. During NADPH/ADP-iron-promoted lipid peroxidation at a steady state oxygen partial pressure (pO2) of 30 mm Hg, complex time relationships among oxygen uptake, malondialdehyde (MDA) formation, and low-level chemiluminescence were observed. While the first two parameters occurred nearly simultaneously, low-level chemiluminescence occurred with a significant delay. A decrease of the steady state pO2 to 3 mm Hg led to significant increases of the lag phases of all three parameters and a further enhancement of the time displacement of low-level chemiluminescence in relation to oxygen uptake and MDA formation. At a pO2 of 0.5 mm Hg, the lowest pO2 maintained during this study, no low-level chemiluminescence was observed while oxygen uptake and MDA formation were still detected. In contrast, during NADPH/CCl4-promoted lipid peroxidation at a pO2 of 0.5 mm Hg a sudden drastic rise of low-level chemiluminescence accompanying oxygen uptake and MDA formation was observed. At pO2 between 0.5 and 3 mm Hg all three parameters occurred nearly concomitantly during the entire incubation. At pO2 levels above 3 mm Hg all three parameters showed principally the same behavior. However, the respective maxima of low-level chemiluminescence were reached with some delay. The present observations support the assumption that the decomposition of membrane lipid peroxyl radicals to MDA and the formation of electronically excited species proceed via different pathways. The time displacement between oxygen uptake and MDA formation, on the one hand, and low-level chemiluminescence, on the other hand, depends on the type of initiating radical system and on the steady state pO2 level. It is suggested that the differences are due to distinct subsets (chemical or spatial) of secondary peroxyl radicals in the membrane.

Adenosine Diphosphate↗

PubMed1987

Hormones, glutathione status and protein S-thiolation.

The formation of mixed disulfides between proteins and glutathione has been discussed as a potentially interesting metabolic signal. The S-thiolation of proteins with glutathione has been observed in several systems in vitro. We have correlated the increase in glutathione disulfide (GSSG) with the amount of protein mixed disulfides. The methodological aspects are briefly presented; normal values for protSSG are about 20-30 nmol per g wet weight of liver. Several processes have been related to changes in the thiol redox state. The stimulation of flux through the pentose phosphate pathway during the metabolism of t-butyl hydroperoxide is presented, and the increase in cellular activity of glucose-6-phosphate dehydrogenase is correlated with the increase in the level of protSSG. Hormonal stimulation of GSH efflux from the liver by vasopressin or by alpha-adrenergic agonists such as phenylephrine or epinephrine is presented and discussed in relation to physiological states of peripheral (non hepatic) GSH utilization. Preliminary work relates the release of GSH to the perturbations in thiol redox state in inflammation and in exercise.

Animals↗

PubMed1987

Generation of excited species catalyzed by horseradish peroxidase or hemin in the presence of reduced glutathione and H2O2.

Horseradish peroxidase (HRP) (EC 1.11.1.7) catalyzes the oxidation of reduced glutathione. This reaction is accompanied by light emission, which is attributed to the generation of singlet oxygen. The chemiluminescence is directly related to thiyl radical formation, as deduced from the correlation between the time course of HRP-compound II formation and light emission in the presence of different amounts of H2O2. Superoxide dismutase has an inhibitory effect on the chemiluminescence without affecting the HRP-compound II formation. This indicates the direct involvement of superoxide radicals in the production of photoemissive species. Replacement of HRP by hemin is also accompanied by chemiluminescence.

Glutathione↗

PubMed1987

Intracellular and intercellular liver heterogeneity: an overview.

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Animals↗

PubMed1987

Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium.

Salmonella typhimurium strains containing deletions of oxyR, a positive regulator of defenses against oxidative stress, show 10- to 55-fold higher frequencies of spontaneous mutagenesis compared to otherwise isogenic oxyR+ control strains. The high spontaneous-mutation frequency in oxyR deletion strains is decreased by a factor of 3 when the strains are grown anaerobically. oxyR deletion strains show an increase in small deletion mutations and at least three of the six possible base-substitution mutations (T.A to A.T, C.G to T.A, and C.G to A.T). However, the largest increase in mutation frequency is observed for T.A to A.T transversions (40- to 146-fold), the base-substitution mutation most frequently caused by chemical oxidants. The introduction into oxyR deletion strains of multicopy plasmids carrying the oxyR-regulated genes for catalase (katG) or alkyl hydroperoxide reductase (ahp) results in overexpression of the respective enzyme activities and decreases the number of spontaneous mutants to wild-type levels. The introduction into oxyR deletions of a plasmid carrying the gene for superoxide dismutase (sodA) decreases the mutation frequency by a factor of 5 in some strain backgrounds. Strains that contain a dominant oxyR mutation and overexpress proteins regulated by oxyR show lower spontaneous-mutation frequencies by a factor of 2. These results indicate that oxyR and oxyR-regulated genes play a significant role in defense against spontaneous oxidative DNA damage. The role of oxidative damage to DNA in "spontaneous" mutagenesis is discussed.

Anaerobiosis↗

PubMed1987

Antioxidant activity in cells and organs.

Antioxidant activity in cells occurs at several levels. The present work focuses on some aspects of enzymatic antioxidant defense. In particular, the so-called ancillary enzymes and transport systems are presented. Recent aspects on the control of the levels of antioxidant enzymes include the oxyR regulon in microorganisms as well as the response to DNA hypomethylation in livers of mice. Ebselen is a novel selenoorganic compound with antioxidant activity capable of conferring protection against oxidative challenge. Its activity to catalyse the GSH-peroxidase reaction is thought to mediate the effect in intact cells. The nonenzymatic antioxidant defense (e.g., vitamins E and C and other natural or chemically synthesized compounds) form an important complementary part of cellular antioxidant defense.

Animals↗

PubMed1987

Xanthine oxidase is not responsible for reoxygenation injury in isolated-perfused rat heart.

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger et al., Gastroenterology, 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60 min of hypoxia and 30 minutes of reoxygenation. The infusion of 250 microM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60 min of hypoxia. Protection from 30 min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30 mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100 microM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allopurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.

Allopurinol↗