Cyclic AMP-dependent control of the rat hepatic glutathione disulfide-sulfhydryl ratio.

The disulfide-sulfhydryl ratio of rat hepatic tissue has been found to vary diurnally lowest in the early morning and highest in the early evening (Isaacs, J. (1976) Fed. Proc. 35, 1472, and Isaacs, J. and Binkley, F. (1977) Biochim. Biophys. Acta 497, 192-204). Intraperitoneal injections of dibutyryl cyclic AMP induces an increase in hepatic glutathione protein mixed disulfides (GSSProt) combined with a corresponding decrease in reduced glutathione (GSH) and protein sulfhydryl (ProtSH). Also, dibutyryl cyclic AMP caused hepatic catalase activity to decrease and to increase hepatic production of peroxide molecules. A decrease in catalase activity directs more of the increased peroxide into the glutathione peroxidase pathway. This leads to increased amounts of oxidized glutathione (GSSG) which ultimately results in increased levels of GSSProt. Therefore cyclic AMP may mediate its effect on the disulfide-sulfhydryl ratio via control over catalase and peroxide generation. Support for this idea is provided by the close temporal correlation between the diurnal variations in cyclic AMP, hepatic catalase, peroxide generation and GSSProt-GSH levels.     

Glutathione dependent control of protein disulfide-sulfhydryl content by subcellular fractions of hepatic tissue

The disulfide-sulfhydryl (SS/SH) ratios of subcellular fractions of rat hepatic tissue were found to vary diurnally with the ratio lowest in the early morning and highest in the early evening. These changes were found in the nuclear, microsomal and cytosol fractions. The primary reaction is the reversible formation of mixed disulfides of glutathione with proteins. This formation is controlled by the activity of thiol transferase and the level of oxidized glutathione (GSSG) as substrate. Several enzymes including mitochondrial and microsomal oxidases, glutathione reductase and peroxidase and glucose-6-phosphate dehydrogenase were found to control the levels of GSSG. An NADPH-dependent microsomal oxidase system, inhibited by GSSG, was found to produce activated oxygen which served as substrate for flutathione peroxidase. Evidence is presented for the concept that the formation of mixed disulfides of proteins with glutathione is a mechanism for maintenance of a disulfide-sulfhydryl ratio such that the integrity of particulate membranes is maintaine during oxidative and reductive stresses on the hepatic cells.     

Fluidizing effect of endogenous ubiquinone in bovine heart mitochondrial membranes

Exposure of isolated rat liver cells to glucagon or dibutyryl cyclic AMP leads to a prompt decrease in the rate of cellular peroxide generation as evidenced by (i) a reduced rate of [14C]formate oxidation and (ii) a lowered steady-state concentration of catalase Compound I.     

Effect of in vitro cultivation and Mycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures.

Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains of Mycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels.     

Effects of new antioxidant compounds PNU-104067F and PNU-74389G on antioxidant defense in normal and diabetic rats

Diabetes mellitus and its complications are associated with elevated oxidative stress, leading to much interest in antioxidant compounds as possible therapeutic agents. Two new classes of antioxidant compounds, the pyrrolopyrimidines and the 21-aminosteroids, are known to inhibit lipid peroxidation and other biomolecular oxidation. We hypothesized that in the presence of excess oxidants or the impaired antioxidant defense seen in diabetes mellitus, administration of antioxidants such as these may reverse the effects of diabetes on antioxidant parameters. This study measured the effects of subchronic (14 day) treatment with a pyrrolopyrimidine (PNU-104067F) or a 21-aminosteroid (PNU-74389G) in normal and diabetic Sprague-Dawley rats. Activity levels of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, concentrations of oxidized and reduced glutathione, and lipid peroxidation were used as measures of antioxidant defense in liver, kidney, heart, and brain tissue. In normal rats, the only effect was a 43% increase in cardiac lipid peroxidation after treatment with PNU-104067F. In diabetic rats, the only reversals of the effects of diabetes were a 30% decrease in hepatic glutathione peroxidase activity after PNU-74389G treatment and a 33% increase in cardiac glutathione disulfide concentration after PNU-104067F treatment. In contrast to these effects, increased cardiac glutathione peroxidase and catalase activities, increased brain glutathione peroxidase activity, increased hepatic lipid peroxidation, decreased hepatic glutathione content, and decreased hepatic catalase activity were seen in diabetic rats, reflecting an exacerbation of the effects of diabetes.     

The correlation of cyclic AMP and protein kinase activity in adipocytes with lipolysis stimulated by ACTH: the effect of adenosine deaminase and actinomycin D.

ACTH at levels as low as 0.05 mU/ml stimulated lipolysis, protein kinase and cyclic AMP accumulation in isolated fat cells from fed and fasted rats. Changes in cyclic AMP levels and in the protein kinase activity ratio were well correlated temporally. The protein kinase activity ratio was potentiated by adenosine deaminase. A sudden increase or decrease in either ACTH or dibutyryl cyclic AMP concentration was associated with a rapid and corresponding change in the rate of glycerol production. With ACTH, the changes in glycerol production were accompanied by appropriate changes in cyclic AMP levels. Actinomycin-D (10 UM) did not affect lipolysis or cyclic AMP accumulation activated by ACTH in fat cells.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Is cyclic AMP the regulator of hepatic ornithine decarboxylase activity?

The role of cyclic AMP in the regulation of hepatic ornithine decarboxylase (ODC) activity in the rat was studied in the whole animal and in the perfused organ. Dibutyryl cyclic AMP or butyrate given to intact rats increased ODC activity; this increase was abolished by hypophysectomy 1 h prior to administering ether compound. Administration of 1 mg 1-methyl-3-isobutylxanthine (MIX) to intact rats increased ODC activity within 4 hours whereas hypophysectomy 1 h before treatment prevented this increase. No change in hepatic cyclic AMP content was seen in either intact or hypophysectomized rats following MIX. Perfusion with 0.5 mM dibutyryl cyclic AMP decreased ODC activity in isolated livers whereas perfusion with 0.5 mM 8-bromocyclic GMP produced a small increase in ODC activity. These data suggest that the effect of dibutyryl cyclic AMP in intact animals may be a property of the butyrate and that this action as well as the action of MIX may be mediated through the permissive effect of pituitary and/or adrenal hormones. The normal hepatocyte does not increase its ornithine decarboxylase activity after direct exposure to dibutyryl cyclic AMP.     

Plasma membrane vesicles from isolated hepatocytes retain the increase of amino acid transport induced by dibutyryl cyclic AMP in intact cells

We have investigated the effect of cyclic AMP on hepatic amino acid transport by measuring the uptake of L-alanine in plasma membrane vesicles purified from hepatocytes incubated without or with dibutyryl cyclic AMP. The application of an Na+ gradient to vesicles from hepatocytes exposed to dibutyryl cyclic AMP, compared to control, resulted in a greater transient accumulation of L-alanine, whereas in the presence of a K+ gradient a similar slow equilibration of L-alanine was observed. Kinetic analysis of L-alanine influx revealed that the increased uptake resulted from an increased capacity (Vmax) with no change in the affinity (Km) of the carriers for L-alanine. These results strongly suggest that dibutyryl cyclic AMP induces stable changes at the membrane level probably by increasing the number of amino acid carrier molecules.     

Reactive oxygen species and induction of lignin peroxidase in Phanerochaete chrysosporium

We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O(2)) of Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free exchange of atmospheric air [control cultures]). A significant increase in the intracellular peroxide concentration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed when the fungus was exposed to pure O(2) gas. The specific activities of manganese superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher in cultures exposed to pure O(2) (oxygenated cultures) than in cultures grown with atmospheric air. Significantly higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures. A hydroxyl radical scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression at the mRNA and protein levels. This effect was confirmed by in situ generation of hydroxyl radicals via the Fenton reaction, which significantly enhanced LIP expression. The level of intracellular cyclic AMP (cAMP) was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP levels were obtained at high O(2) concentrations and in cultures grown with atmospheric air. These results suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis. Thus, the induction of LIP expression by O(2) is at least partially mediated by the intracellular ROS.     

Hormonal regulation of four urea cycle enzymes in postnatal rat liver in organ culture

The administration of hydrocortisone to 3- to 15-day-old rats increased the levels of hepatic argininosuccinate synthetase (ASS) and arginase. In 13-day-old rat liver explants maintained in organ culture, ornithine carbamoyltransferase (OTC), carbamoylphosphate synthetase (CPS) and arginase were stimulated by betamethasone. Actinomycin D prevented the responses of the latter two enzymes. Dibutyryl cyclic AMP raised OTC, CPS, ASS and arginase in vitro. The responses of the latter three enzymes were blocked by cycloheximide and puromycin and partially inhibited by actinomycin D. The simultaneous presence of betamethasone and dibutyryl cyclic AMP in the culture medium raised CPS and OTC in an additive manner. The sequential treatment of the cultures with betamethasone followed by dibutyryl cyclic AMP increased CPS and arginase synergistically and amplified the response of ASS to dibutyryl cyclic AMP.     

Expression of catalase and glutathione peroxidase in renal insufficiency

Chronic renal failure (CRF) is associated with oxidative stress, the precise mechanism of which is yet to be elucidated. The present study was undertaken to investigate in renal insufficiency the expression of catalase and glutathione peroxidase, which play a critical role in antioxidant defense system by catalyzing detoxification of hydrogen peroxide (H2O2) and organic hydroperoxides. Rats were randomly assigned to the CRF (5/6 nephrectomized) and sham-operated control groups and observed for 6 weeks. Renal and thoracic aortic catalase and glutathione peroxidase protein abundance was measured by Western blotting. The enzyme activities in the renal and aortic extracts, hepatic glutathione levels, blood pressure and urinary nitric oxide metabolites (NO(x)) excretion were also measured. Blood pressure and urinary nitric oxide metabolite (NO(x)) excretion were also measured. The CRF group showed a significant down-regulation of both immunodetectable catalase and glutathione peroxidase proteins in the remnant kidney. Catalase activity was also significantly decreased in the remnant kidney whereas glutathione peroxidase activity was not significantly affected. Furthermore, the protein abundance of catalase was unchanged whereas the enzyme activity was significantly decreased in the thoracic aorta of CRF animals compared to the sham-operated controls. By contrast, both the protein abundance and the enzyme activity of glutathione peroxidase were not significantly affected in the aorta of CRF animals compared to the sham-operated controls. This was coupled with marked arterial hypertension, significant reduction of hepatic glutathione levels and urinary NO(x) excretion pointing to increased inactivation and sequestration of NO by superoxide. These events point to the role of impaired antioxidant defense system in the pathogenesis of oxidative stress in CRF.     

Cyclic AMP inhibition of phosphoinositide turnover in human neutrophils

The effect of increased intracellular levels of cyclic AMP on phosphoinositide metabolism was studied in human neutrophils stimulated with fMet-Leu-Phe. Intracellular cyclic AMP was raised by preincubation either with dibutyryl cyclic AMP and theophylline or with prostaglandin E1. Concentrations of dibutyryl cyclic AMP and theophylline fully inhibitory for the metabolic responses inhibited phosphoinositide breakdown and phosphatidic acid formation to a large extent. The accumulation of the water-soluble inositol phosphates was also measured. In agreement with the data obtained on the phospholipids, inositol phosphate generation was found to be severely, though not completely, reduced. Treatment with dibutyryl cyclic AMP and theophylline also inhibited resynthesis of membrane inositol lipids. Treatment with prostaglandin E1 had a similar, though less, marked effect on inositol lipid turnover, which was parallel with a smaller inhibition of metabolic responses. We therefore suggest that the elevation of intracellular cyclic AMP mainly affects neutrophil responses by inhibiting the phosphoinositide cycle.     

Dehydroepiandrosterone improves hepatic antioxidant reserve and stimulates Akt signaling in young and old rats

This study examined, in the liver of young and old (3- and 24-month-old, respectively) healthy Wistar rats, the in vivo effect of dehydroepiandrosterone (DHEA) (10mg/kg body weight) administered subcutaneously for 5 weeks. Reduced (GSH) and oxidized (GSSG) glutathione levels, glucose-6-phosphate dehydrogenase (G6PDH), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) activities, hydrogen peroxide concentration, GST and p-Akt/Akt immunocontent ratio were assessed in hepatic tissue. DHEA treatment significantly increased total glutathione content (17%) and GSH (22%) in 3- and 24-month-old treated groups when compared to control groups. The aging factor increased G6PDH (51%) and GPx (22%) activities as well as the hydrogen peroxide concentration (33%), independently of treatment. DHEA treatment increased p-Akt (54%) and p-Akt/Akt ratio (36%) immunocontents in both treated groups. Increased serum levels of alanine aminotransferase (ALT) in aged rats were reduced by DHEA treatment (34%).     

Effect of flooding on the activities of some enzymes of activated oxygen metabolism,the levels of antioxidants,and lipid peroxidation in senescing tobacco leaves

Effects of flooding on the activities of some enzymes of activated oxygen metabolism, the levels of antioxidants, and lipid peroxidation in senescing leaves of tobacco were investigated. As judged by the decrease in chlorophyll and protein levels, flooding accelerated the senescence of tobacco leaves. Total peroxide and the lipid peroxidation product, malondialdehyde, increased in both control and flooding-treated leaves with increasing duration of the experiment. Throughout the duration of the experiment, flooded leaves had higher levels of total peroxide and malondialdehyde than did control leaves. Flooding resulted in an increase in peroxidase and ascorbate peroxidase activities and a reduction of superoxide dismutase activity in the senescing leaves. Glycolate oxidase, catalase, and glutathione reductase activities were not affected by flooding. Flooding increased the levels of total ascorbate and dehydroascorbate. Total glutathione, reduced form glutathione, or oxidized glutathione levels in flooded leaves were lower than in control leaves during the first two days of the experiment, but were higher than in control leaves at the later stage of the experiment. Our work suggests that senescence of tobacco induced by flooding may be a consequence of lipid peroxidation possibly controlled by superoxide dismutase activity. Our results also suggest that increased rates of hydrogen peroxide in leaves of flooded plants could lead to increased capacities of the scavenging system of hydrogen peroxide.Abbreviations GSH reduced form glutathione - GSSG oxidized form glutathione - GSSG reductase glutathione reductase - MDA malondialdehyde - SOD superoxide dismutase     

Effect of in vitro cultivation andMycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures

Summary Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains ofMycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels. This study was supported in part by Grant AI 12559 from the National Institutes of Health. The supply of cholera toxin from Dr. R. A. Finkelstein is most appreciated as are the assistance and advice of J. A. Engelhardt and Y. D. B. Stahl.     

Decrease of hepatic catalase level by treatment with diallyl sulfide and garlic homogenates in rats and mice

Diallyl sulfide (DAS) is a flavor compound derived from garlic and is active in the inhibition of chemically induced cytotoxicity and carcinogenicity in animal models. This study was conducted to examine the effects of the treatment of DAS and garlic homogenates on the activities of catalase, glutathione peroxidase, and superoxide dismutase. Male Sprague-Dawley rats were treated with DAS i.g. at daily doses of 50 or 200 mg/kg for 8 days, causing the hepatic catalase activity to decrease by 55 and 95%, respectively. Such a decrease in hepatic catalase activity was also observed when the DAS treatment was extended to 29 days. Western blot analysis showed that the DAS treatments resulted in corresponding decreases in the liver catalase protein level. No significant change in the catalase activity in the kidney, lung, and brain was observed with the treatments, but a slight decrease in heart catalase activity was observed. These treatments did not cause significant changes in superoxide dismutase and glutathione peroxidase activities in these tissues. Treatment with DAS at a daily dose of 200 mg/kg for 1-7 days resulted in a gradual decrease in the liver catalase activity to 5% of the control level, but it did not decrease the erythrocyte catalase activity. Treatment of rats with fresh garlic homogenates (2 or 4 g/kg, i.g., daily for 7 days) caused a 35% decrease in liver catalase activity. A/J mice treated with DAS and garlic homogenates also showed a decrease in the liver catalase activity. Diallyl sulfone (DASO2), a DAS metabolite, however, did not effectively decrease catalase activity in mice. The catalase activity was not inhibited by either DAS or DASO2 in vitro. The present results demonstrate that treatment with DAS and garlic homogenates decrease the hepatic catalase level in rats and mice.     

Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes

Glutamine synthetase specific activity increases greater than 100-fold during the insulin-mediated differentiation of confluent 3T3-L1 cells into adipocytes. Incubation of the adipocytes for 22 h with 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline, 0.2 mM 8-bromo-cyclic AMP, 10 micro M epinephrine, or 1 microgram of alpha 1-24 adrenocorticotropic hormone/ml decreased glutamine synthetase by greater than 60%. During the same incubation period, there was no effect of these compounds on protein or on the specific activities of glucose-6-P dehydrogenase or hexokinase. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase activity was half-maximal at 50 micro M dibutyryl cyclic AMP. Furthermore, between 10 micro M and 5 mM dibutyryl cyclic AMP, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase was similar in the absence or presence of 1 microgram of insulin/ml. Immunotitration of glutamine synthetase activity from 3T3 adipocytes indicates that the dibutyryl cyclic AMP-mediated decrease in the activity is due to a decrease in the cellular content of glutamine synthetase molecules. We studied the effects of dibutyryl cyclic AMP on the synthesis and degradation of glutamine synthetase. Synthesis rate was estimated from the incorporation of L-[35S]methionine into glutamine synthetase during a 60-min incubation period. Degradation rate was estimated from the first order disappearance of radioactivity from glutamine synthetase in 3T3 adipocytes previously incubated with L-[35S]methionine. Glutamine synthetase was isolated by immunoprecipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Incubation of 3T3 adipocytes with dibutyrl cyclic AMP resulted in a rapid decline in the apparent synthesis rate of glutamine synthetase. In addition, dibutyryl cyclic AMP treatment increased the initial rate of glutamine synthetase degradation. The half-life of glutamine synthetase was 24.5 h in control cultures and 16 h in dibutyryl cyclic AMP-treated cultures. In contrast, dibutyryl cyclic AMP had little effect on the synthesis or degradation of soluble protein. Our data indicate that the dibutyryl cyclic AMP-mediated decrease in 3T3 adipocyte glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the initial degradation rate of the enzyme.     

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10–100 μM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 μM. In contrast, concentrations of dibutyryl cyclic AMP between 200 μM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 μM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 μM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent either treatment alone. It is conclude that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.