Biomedical subjects

H Sies

Publications and source records attributed to H Sies.

At least 307 records · Page 17Linked to original sources

PubMed1985

Excited species generation in horseradish peroxidase-mediated oxidation of glutathione.

Photoemissive excited species are produced by the horseradish peroxidase (HRP)-catalyzed oxidation of reduced glutathione (GSH), without exogenously added hydroperoxide under aerobic conditions. The emitted low-level chemiluminescence consisted of two phases. Light emission occurred at wavelengths beyond 610 nm (greater than or equal to 90% intensity), indicative of singlet oxygen 1O2. Deuterium oxide enhanced photoemission 4.4-fold. Ascorbate inhibited chemiluminescence completely. In the absence of GSH or when GSH was replaced by the disulfide, no red chemiluminescence was observed. The glutathionyl radical GS. is most likely to be involved in both phases of light emission. Further, the superoxide radical plays a role, as substantiated by the inhibitory effect of superoxide dismutase. Both phases of photoemission were abolished by glutathione peroxidase; thus hydroperoxides are regarded as essential intermediates for the formation of excited species. Catalase abolished phase I and did not affect phase II. In contrast, glutathione S-transferase 1-2 (showing peroxidase activity towards organic hydroperoxides but not towards H2O2) inhibited phase II, whereas phase I was still present. Glutathione sulfonate and the disulfide GSSG were detected as oxidation products from GSH under conditions where phase II chemiluminescence was observed. HRP Compound III accumulated during the reaction. It is concluded that phase I is dependent on exogenously added or endogenously generated H2O2, whereas phase II does not require H2O2 but an organic peroxy species. A mechanism based on chain reactions involving oxygen addition to the thiyl radical is proposed. Sulfenyl peroxy species are suggested as transient intermediates in reactions finally leading to the generation of excited states such as singlet molecular oxygen.

Free Radicals↗

PubMed1985

Functional hepatocyte heterogeneity in ammonia metabolism. The intercellular glutamine cycle.

In the rat liver acinus area synthesis and glutaminase activity are predominantly localized in the periportal area, whereas glutamine synthetase activity and the transport system for glutamate in the plasma membrane are perivenous. Detoxification of ammonium ions at low concentrations occurs exclusively by glutamine synthesis, but not by urea formation. Therefore, the two pathways of ammonia detoxification in the liver acinus represent the sequence of a low-affinity, but high-capacity system (urea synthesis) and a high-affinity system (glutamine synthesis). In agreement with this finding, obtained in experiments with the metabolically- and structurally-intact perfused rat liver, is also an almost complete inhibition of perivenous glutamine synthesis without impairment of periportal urea synthesis. This was shown after induction of a perivenous liver cell necrosis following CCl4 pretreatment with the consequence of a diminished hepatic ammonia extraction. Periportal glutaminase and perivenous glutamine synthetase are simultaneously active, resulting in an intercellular (as opposed to intracellular) glutamine cycle, being under the control of hormones, pH and portal ammonia and glutamine concentrations. The intercellular glutamine cycle provides an effective means for almost complete conversion of portal ammonium ions to urea by additional substrate supply of periportal urea synthesis, by periportal glutamine degradation and by the perivenous re-synthesis of glutamine from ammonia which escaped urea synthesis. Because urea synthesis, in contrast to glutamine synthesis, is a major pathway for the removal of bicarbonate, the switching of ammonia detoxification from urea synthesis to glutamine synthesis in acidosis or vice versa in alkalosis points to an important role of the liver in maintaining pH homeostasis. The acid-base-induced changes of the route of hepatic ammonia detoxification and therefore bicarbonate removal are performed by the regulatory properties of the enzymes of the intercellular glutamine cycle.

Acid-Base Equilibrium↗

PubMed1984

Generation of low-level chemiluminescence during the metabolism of 1-naphthol by rat liver microsomes.

The metabolism of 1-naphthol in rat liver microsomal fractions supplemented with NADPH is accompanied by low-level chemiluminescence which reflects the formation of molecular excited states. Photoemission consists of two phases which both are dependent on microsomal protein and 1-naphthol concentration. The involvement of cytochrome P-450 in the microsomal metabolism of 1-naphthol was indicated by an inhibition of chemiluminescence by aminopyrine or metyrapone. Oxygen is required for light emission. Whereas phase I is hardly influenced by superoxide dismutase, phase II is suppressed. Chemiluminescence was not associated with malondialdehyde accumulation, in contrast to NADPH-dependent lipid peroxidation in microsomal fractions in the absence of 1-naphthol. Phase I of chemiluminescence appears to directly reflect cytochrome P-450-dependent hydroxylation, and phase II is attributed to redox cycling of products arising from these reactions, e.g. the 1,4- and/or 1,2-naphthoquinones as oxidation products of the corresponding dihydroxynaphthalenes.

Aminopyrine↗

PubMed1984

Evaluation of alpha-tocopherol antioxidant activity in microsomal lipid peroxidation as detected by low-level chemiluminescence.

The significance of microsomal vitamin E in protecting against the free-radical process of lipid peroxidation was evaluated with the low-level-chemiluminescence technique in microsomal fractions from vitamin E-deficient and control rats. The induction period that normally precedes the ascorbate/ADP/Fe3+-induced lipid peroxidation was taken as reflecting the microsomal vitamin E content and was found to be 5-6-fold decreased in microsomal fractions from vitamin E-deficient rats. Supplementation of microsomal fractions from vitamin E-deficient rats with exogenous vitamin E partially restores the induction period observed in that from control rats. The decrease in chemiluminescence intensity and the increase in the induction period both correlate linearly with the amount of vitamin E added. However, the efficiency of exogenous vitamin E is about 50-fold lower than that exerted by the naturally occurring vitamin E in microsomal membranes. These observations are discussed in terms of the process of re-incorporation of vitamin E into membranes, the experimental model for lipid peroxidation selected, and the method to evaluate lipid peroxidation, namely low-level chemiluminescence.

Animals↗

PubMed1984

A novel biologically active seleno-organic compound--I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen).

a synthetic seleno-organic compound, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (PZ 51), exhibits GSH peroxidase-like activity in vitro, in contrast to its sulfur analog, PZ 25. In addition, PZ 51 behaves as an antioxidant shown by a temporary protection of rat liver microsomes against ascorbate/ADP-Fe-induced lipid peroxidation, an effect also elicited by PZ 25 but to a smaller extent. This protection against lipid peroxidation is independent of GSH and of P-450 monooxygenase activity.

Animals↗

PubMed1984

Subcellular glutathione contents in isolated hepatocytes treated with L-buthionine sulfoximine.

The glutathione contents of the mitochondrial and cytosolic fractions and extracellular space of isolated hepatocytes decrease when glutathione synthesis is inhibited with L-buthionine sulfoximine. Mitochondrial glutathione is depleted to 50% of its initial value whereas the cytosolic pool is completely emptied after 2 h incubation in the presence of inhibitor. The mitochondrial glutathione content was only fully depleted when L-buthionine sulfoximine was added together with phorone (2,6-dimethyl-2,5-heptadiene-4-one), a substrate of the glutathione S-transferases (E.C. 2.5.1.18).

Animals↗

PubMed1984

Effect of phenylephrine on glutamate and glutamine metabolism in isolated perfused rat liver.

Addition of phenylephrine to isolated perfused rat liver is followed by an increased 14CO2 production from [1-14C]glutamate, [1-14C]glutamine, [U-14C]proline and [3-14C]pyruvate, but by a decreased 14CO2 production from [1-14C]pyruvate. Simultaneously, there is a considerable decrease in tissue content of 2-oxoglutarate, glutamate and citrate. Stimulation of 14CO2 production from [1-14C]glutamate is also observed in the presence of amino-oxyacetate, suggesting a stimulation of glutamate dehydrogenase and 2-oxoglutarate dehydrogenase fluxes by phenylephrine. Inhibition of pyruvate dehydrogenase flux by phenylephrine is due to an increased 2-oxoglutarate dehydroxygenase flux. Phenylephrine stimulates glutaminase flux and inhibits glutamine synthetase flux to a similar extent, resulting in an increased hepatic glutamine uptake. Whereas the effects of NH4+ ions and phenylephrine on glutaminase flux were additive, activation of glutaminase by glucagon was considerably diminished in the presence of phenylephrine. The reported effects are largely overcome by prazosin, indicating the involvement of alpha-adrenergic receptors in the action of phenylephrine. It is concluded that stimulation of gluconeogenesis from various amino acids by phenylephrine is due to an increased flux through glutamate dehydrogenase and the citric acid cycle.

Animals↗

PubMed1984

Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.

Acyl Coenzyme A↗

PubMed1984

Biliary transport of glutathione disulfide studied with isolated rat-liver canalicular-membrane vesicles.

Canalicular plasma membrane vesicles isolated from rat liver (right side out) were used to study glutathione disulfide (GSSG) transport. GSSG is transported into an osmotically sensitive compartment. Extrapolation to an external osmolarity of infinity indicates 35% binding after 10 min of incubation, e.g. as mixed disulfides between glutathione and SH-groups in membrane proteins. Glutathione disulfide uptake occurred with equal rates in the presence of KCl or NaCl. After equilibration, no significant GSSG concentration gradient across the canalicular membrane was found. The temperature dependence of uptake is consistent with a carrier-mediated transport for GSSG (Q10 X 1.6) and seems to exclude simple diffusion. Concentration dependence of GSSG uptake shows Michaelis-Menten kinetics at concentrations up to 1 mM; a Km of 0.4 mM and a V of 1.1 nmol X min-1 X mg protein-1 is calculated. At higher concentrations a linear dependence is observed without saturation kinetics. It is concluded that transport of glutathione disulfide is mediated by a carrier present in the canalicular membrane of the hepatocyte.

Animals↗

PubMed1984

Relation between glutathione redox changes and biliary excretion of taurocholate in perfused rat liver.

The influence of the intracellular glutathione status on bile acid excretion was studied in the perfused rat liver. Perturbation of the thiol redox state by short term additions of diamide (100 microM) or hydrogen peroxide (250 microM) or t-butyl hydroperoxide (250 microM) led to a reversible inhibition of biliary taurocholate release without affecting hepatic uptake; inhibition amounted to 45% for diamide and 90% for the hydroperoxides. Concomitantly, the bile acid accumulated intracellularly. Bile flow increased from 1.3 to 2.0 microliters X min-1 X g liver-1 upon infusion of taurocholate (10 microM); the latter value was suppressed to 1.2 microliters X min-1 X g liver-1 by the addition of t-butyl hydroperoxide (250 microM). Similarly, the hepatic disposition of another bile constituent, bilirubin, was suppressed by 70% upon addition of hydrogen peroxide. While the addition of hydrogen peroxide inhibited also the endogenous release of bile acids almost completely, endogenous bile flow was much less affected, decreasing from 1.3 to 1.0 microliters X min-1 X g liver-1. Measurement of [14C]erythritol clearance showed bile/perfusate ratios of about unity both in the absence and presence of hydrogen peroxide, suggesting canalicular origin of the bile under both conditions. In livers from Se-deficient rats low in Se-GSH peroxidase (less than 5% of controls), hydrogen peroxide inhibited taurocholate transport substantially less, providing evidence for the involvement of glutathione in mediating the inhibition observed in normal livers. The percentage inhibition of taurocholate release and intracellular glutathione disulfide (GSSG) content were closely correlated. The addition of t-butyl hydroperoxide caused a several-fold increase of biliary GSSG release, whereas biliary GSH release was even decreased. The results establish a role of glutathione in canalicular taurocholate disposition.

Animals↗

PubMed1984

Biliary transport of glutathione S-conjugate by rat liver canalicular membrane vesicles.

Transport of S-dinitrophenyl glutathione, a model compound of glutathione S-conjugates, was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited time-dependent uptake of [2-3H]glycine-glutathione conjugate into an osmotically sensitive intravesicular space. Inactivation of vesicle-associated gamma-glutamyltransferase by affinity labeling with L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid had no effect on the initial rate of transport. Chemical analysis revealed that the intact glutathione conjugate accounted for most vesicle-associated radioactivity, reflecting the low transferase activity in the liver and membrane vesicles. The initial rate of transport followed saturation kinetics with respect to conjugate concentrations; an apparent Km of 1.0 mM and Vmax of 1.7 nmol/mg of protein X 20 s were calculated. These results indicate that transport of the glutathione S-conjugate across the canalicular membranes is a carrier-mediated process. Sodium chloride in the transport medium could be replaced by KCl, LiCl, or choline chloride without any changes in transport activity. The rate of conjugate transport was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). The rate of conjugate uptake was enhanced by replacing KCl in the transport medium with K gluconate, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. These data indicate that conjugate transport is electrogenic and involves the transfer of negative charge. Transport of S-dinitrophenyl glutathione was inhibited by S-benzyl glutathione, oxidized glutathione, or reduced glutathione. This transport system in canalicular membranes may function in biliary secretion of glutathione S-conjugates of xenobiotics whose synthesis in hepatocytes requires glutathione S-transferases.

Animals↗

PubMed1984

The isozyme pattern of glutathione S-transferases in rat heart.

The isozyme pattern of glutathione S-transferases of rat heart differs markedly from that of liver. Heart lacks the majority of basic isozymes recognized in rat liver, and isoelectric focusing of glutathione S-transferases obtained from affinity chromatography on S-hexylglutathione-linked Sepharose 6B revealed that 3 major acidic isozymes with pI values of 6.8, 6.3 and 4.9 are present in heart, at least two of which were not found in liver. In addition, multiple bands were found in the pI range 5.0-5.5 for glutathione S-transferase activity and protein staining.

Animals↗

PubMed1984

Mitochondrial and cytosolic glutathione after depletion by phorone in isolated hepatocytes.

The glutathione content of cytosol and mitochondria of isolated hepatocytes was depleted by addition of a low concentration of phorone (0.5 mM) by 75% and 40% respectively. Different rates of replenishment indicate metabolic separation of cytosolic and mitochondrial glutathione pools. The release from hepatocytes occurred at a rate of about 8 nmol/g wet weight/min, both in controls and after phorone depletion.

Animals↗

PubMed1984

Protection against reactive oxygen species by NAD(P)H: quinone reductase induced by the dietary antioxidant butylated hydroxyanisole (BHA). Decreased hepatic low-level chemiluminescence during quinone redox cycling.

Menadione elicits low-level chemiluminescence (lambda greater than 620 nm) associated with redox cycling of the quinone in mouse hepatic postmitochondrial fractions. This photoemission is suppressed when the animals are fed a diet containing the anticarcinogenic antioxidant, 2[3]-(tert-butyl)-4-hydroxyanisole (BHA), which leads to a 13-fold increase in NAD(P)H: quinone reductase (EC 1.6.99.2). Inhibition of the enzyme by dicoumarol completely abolishes the protective effect of BHA treatment and leads to higher chemiluminescence, reaching similar photoemission for BHA-treated and control animals. These findings indicate that the two-electron reduction promoted by quinone reductase prevents redox cycling and that BHA protects against reactive oxygen species by elevating the activity of this enzyme.

Animals↗

PubMed1984

Cardiac transport of glutathione disulfide and S-conjugate. Studies with isolated perfused rat heart during hydroperoxide metabolism.

The relationship between the intracellular GSSG level and the rate of GSSG release was studied in the isolated perfused heart. GSSG and GSH were released from the heart into the effluent perfusion fluid at rates of 110 and 370 pmol X min-1 X g of heart-1, respectively, much lower than for similar conditions in liver. Perfusion with t-butyl hydroperoxide led to an increase in GSSG release associated with an elevated level of intracellular GSSG. Saturation kinetics was found for the rate of GSSG release with respect to the intracellular GSSG level with an apparent Km value of 30 nmol X g of heart-1 and a maximal rate of 7.5 nmol X min-1 X g of heart-1. The maximal rate of GSSG release was temperature-sensitive with a temperature coefficient of 1.8. When 1-chloro-2,4-dinitrobenzene was infused during perfusion with t-butyl hydroperoxide, mutual competition was found in the release of GSSG and the glutathione-S-conjugate, S-(2,4-dinitrophenyl)-glutathione. The maximal rate for the glutathione-S-conjugate transport was estimated to be 40 nmol X min-1 X g of heart-1. These results are consistent with the existence of a transport system for GSSG in rat heart, its capacity being substantially lower than that for the S-conjugate in heart and that for GSSG in liver. Thus, the sensitivity of heart to oxidative stress may be explained, in part, by a low capacity of GSSG export.

Animals↗

PubMed1984

Compartmentation of high-energy phosphates in resting and working rat skeletal muscle.

The subcellular distribution of high-energy phosphates in various types of skeletal muscle of the rat was analysed by subfractionation of tissues in non-aqueous solvents. Different glycolytic and oxidative capacities were calculated from the ratio of phosphoglycerate kinase and citrate synthase activities, ranging from 25 in m. soleus to 130 in m. tensor fasciae latae. In the resting state, the subcellular contents of ATP, creatine phosphate and creatine were similar in m. soleus, m. vastus intermedius, m. gastrocnemius and m. tensor fasciae latae but, significantly, a higher extramitochondrial ADP-content was found in m. soleus. A similar observation was made in isometrically and isotonically working m. gastrocnemius. The extramitochondrial, bound ADP accounted fully for actin-binding sites in resting fast-twitch muscles, but an excess of bound ADP was found in m. soleus and working m. gastrocnemius. The amount of non-actin-bound ADP reached maximal values of approx. 1.2 nmol/mg total protein. It could not be enhanced further by prolonged isotonic stimulation or by increased isometric force development. It is suggested that non-actin-bound ADP is accounted for by actomyosin-ADP complexes generated during the contraction cycle. Binding of extramitochondrial ADP to actomyosin complexes in working muscles thus acts as a buffer for cytosolic ADP in addition to the creatine system, maintaining a high cytosolic phosphorylation potential also at increasing rates of ATP hydrolysis during muscle contraction.

Adenosine Diphosphate↗

PubMed1984

Hepatic uptake of cadmium and its biliary release as affected by dithioerythritol and glutathione.

Net cadmium uptake in the isolated perfused rat liver was half-maximal at 5 microM, and the maximal rate of uptake was 22 nmoles/min per gram liver wet weight. Uptake was augmented when a permeable thiol, dithioerythritol, was infused, whereas it was restricted when glutathione as a nonpermeable thiol or also when bovine serum albumin were infused. The ratio of extra cadmium taken up versus dithioerythritol added was 1:2. Uptake of cadmium was insensitive to anoxia or to the infusion of cyanide. Biliary cadmium release in the perfused liver was not augmented by dithioerythritol but was rather suppressed, whereas bile flow or the release of added 3H-taurocholate were unaffected.

Animals↗

PubMed1984

Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies.

S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.

Animals↗