Effects of glucagon on the redox states of cytochromes in mitochondria in situ in perfused rat liver

The effects of glucagon on the respiratory function of mitochondria in situ were investigated in isolated perfused rat liver. Glucagon at the concentrations higher than 20 pM and cyclic AMP (75 microM) stimulated hepatic respiration, and shifted the redox state of pyridine nucleotide (NADH/NAD) in mitochondria in situ to a more reduced state as judged by organ fluorometry and beta-hydroxybutyrate/acetoacetate ratio. The organ spectrophotometric study revealed that glucagon and cyclic AMP induced the reduction of redox states of cytochromes a(a3), b and c+c1. Atractyloside (4 micrograms/ml) abolished the effects of glucagon on these parameters and gluconeogenesis from lactate. These observations suggest that glucagon increases the availability of substrates for mitochondrial respiration, and this alteration in mitochondrial function is crucial in enhancing gluconeogenesis.     

Effects of ethanol-derived acetaldehyde on the phosphorylation potential and on the intramitochondrial redox state in intact rat liver

The role of acetaldehyde (AcH) in the ethanol-induced shift toward reduction of the cytosolic and mitochondrial free NAD⁺/free NADH ratios and its effect on the phosphorylation potential was investigated in livers of fed, intact rats given ethanol (1 g/kg ip). Calcium cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase, was administered to block predominantly intramitochondrial NADH production from AcH oxidation. Compared with ethanol alone, cyanamide almost totally reversed the elevation of the β-OH-butyrate/acetoacetate ratio but only slightly reduced the lactate/ pyruvate ratio, which was calculated to be in near equilibrium with the hepatic ethanol/ AcH ratio after cyanamide. Ethanol or cyanamide alone had no effect on ATP, ADP, or P_i, but together they significantly decreased the $\text{ATP}\text{ADP}\text{· P}{}_{\mathrm{<mtext>i</mtext>}}$ ratio by increasing both ADP and P_i levels. No association between changes in the phosphorylation potential and the redox states was, however, observed. An ethanol-induced increase in AMP was abolished by cyanamide. The results demonstrate that the effect of ethanol on the mitochondrial redox state requires active AcH oxidation and suggest that moderate AcH accumulation likely to occur during alcohol-aversive drug treatment significantly lowers the cellular phosphorylation potential.     

Effects of binge ethanol on lipid homeostasis and oxidative stress in a rat model of nonalcoholic fatty liver disease

Excess fat accumulation renders the liver more vulnerable to ethanol, but it is still unclear how alcohol enhances lipid dysmetabolism and oxidative stress in a pre-existing steatosis condition. The effects produced by binge ethanol consumption in the liver of male Wistar rats fed a standard (Ctrl) or a high-fat diet HFD were compared. The liver status was checked through tissue histology and standard serum parameters. Alteration of hepatic lipid homeostasis and consequent oxidative unbalance were assessed by quantifying the mRNA expression of the lipid-regulated peroxisome proliferator-activated receptors (PPARs), of the cytochromes CYP2E1 and CYP4A1, and of some antioxidant molecules such as the metallothionein isoforms MT1 and MT2 and the enzymes catalase and superoxide dismutase. The number of adipose differentiation-related protein (ADRP)-positive lipid droplets (LDs) was evaluated by immunohistochemical staining. As a response to the double insult of diet and ethanol the rat liver showed: (1) a larger increase in fat accumulation within ADRP-positive LDs; (2) stimulation of lipid oxidation in the attempt to limit excess fat accumulation; (3) induction of antioxidant proteins (MT2, in particular) to protect the liver from the ethanol-induced overproduction of oxygen radicals. The data indicate an increased susceptibility of fatty liver to ethanol and suggest that the synergistic effect of diet and ethanol on lipid dysmetabolism might be mediated, at least in part, by PPARs and cytochromes CYP4A1 and CYP2E1.     

The para isomer of dinitrobenzene disrupts redox homeostasis in liver and kidney of male wistar rats

BackgroundPara-Dinitrobenzene (p-DNB) is one of the isomers of dinitrobenzene which have been detected as environmental toxicants. Skin irritation and organ toxicities are likely for industrial workers exposed to p-DNB. This study evaluated the effect of sub-chronic exposure of rats to p-DNB on cellular redox balance, hepatic and renal integrity.MethodsForty eight male Wistar rats weighing 160–180 g were administered 50, 75, 1000 and 2000 mg/kg b.wt (body weight) of p-DNB or an equivalent volume of vehicle (control) orally and topically for 14 days. After the period of treatment, the activities of kidney and liver catalase (CAT), alkaline phosphatase (ALP) and superoxide dismutase (SOD) as well as extent of renal and hepatic lipid peroxidation (LPO) were determined. Serum ALP activity and plasma urea concentration were also evaluated.ResultsCompared with control animals, p-DNB -administered rats showed decrease in the body and relative kidney and liver weights as well as increased renal and hepatic hydrogen peroxide and lipid peroxidation levels accompanied by decreased superoxide dismutase and catalase activities. However, p-DNB caused a significant increase in plasma urea concentration and serum, liver and kidney ALP activities relative to control. In addition, p-DNB caused periportal infiltration, severe macro vesicular steatosis and hepatic necrosis in the liver.ConclusionsOur findings show that sub-chronic oral and sub-dermal administration of p-DNB may produce hepato-nephrotoxicity through oxidative stress.     

Effects of heme oxygenase-1 expression on sterol homeostasis in rat astroglia

Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol metabolism are characteristic of Alzheimer-diseased (AD) neural tissues. Central oxidation of cholesterol to oxysterols has been implicated in neuroembryogenesis, synaptic plasticity, and membrane repair. In the current study, we demonstrated that transient transfection of rat astroglia with human (h)ho-1 cDNA for 3 days significantly decreased intracellular cholesterol concentrations and increased levels of four oxysterol species (measured by GC/MS) compared to untreated control cultures and HO-1-transfected cells exposed to the HO inhibitor, tin mesoporphyrin (SnMP). Relative to control preparations, oxidative stress was augmented in mitochondria (isolated by subcellular fractionation) and culture media derived from HO-1-transfected astrocytes, as evidenced by enhanced oxidation of the synthetic reporter molecules, linoleoyl tyrosine (LT), linoleoyl tyrosine cholesterol ester (LTC), or linoleoyl tyrosine deoxyguanosyl ester (LTG; measured by GC/MS and LC/MS/MS). We also observed enhanced oxidation of exogenous LTC in human neuroblastoma (M17) cells exposed for 18 h to conditioned media collected from HO-1-transfected astrocytes relative to control media. In AD and other pathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g., beta-amyloid) into altered patterns of glial sterol metabolism which, in turn, may affect neuronal membrane turnover, survival, and adaptability.     

Coumarin impairs redox homeostasis in wheat aleurone layers

Many plant families produce coumarin (COU) and its derivatives as secondary metabolites via the phenylpropanoid biosynthetic pathway. This ubiquitous group of phytochemicals was shown to have diverse physiological effects on cellular, tissue, and organ levels. So far, research dealing with the hormonal like behavior of COU and its interaction with the activity and/or transport of phytohormones is very limited. In the current study, the impact of COU on redox homeostasis in aleurone layers of wheat grains was investigated. Aleurone layers were incubated in either 1000 μM COU or 5 μM gibberellic acid (GA₃) alone or in combination with 5 μM abscisic acid (ABA). Results revealed that both COU and GA₃ treatments induced the production of α-amylase but inhibited the activities of superoxide dismutase, catalase and ascorbate peroxidase. The downregulation of antioxidant enzymes that is provoked by COU and GA₃ was accompanied by significant accumulation of both H₂O₂ and malondialdehyde. In contrast with the effect of ABA, both COU and GA₃ treatments resulted in a significant reduction in cell viability as revealed by trypan blue staining. These results suggest that COU could disrupt the redox balance in aleurone layers through downregulation of the enzymatic antioxidant system. Therefore, the current study provides evidence for the gibberellin like activity of COU.     

Cadmium affects the redox state of rat liver glucocorticoid receptor

It has previously been documented that cadmium displays high affinity for protein thiol groups and induces an impairment of glucocorticoid receptor (GR) cellular functions. The present study examined the possibility that cadmium exerts these effects on GR activity by disturbing the receptor's redox equillibrium. To that end, the influence of cadmium on the rat liver GR potential to form intramolecular and intermolecular disulfide bonds under nonreducing conditions and under oxidizing conditions produced by the addition of hydrogen peroxide (H₂O₂) to the cytosol was examined by nonreducing SDS-PAGE and immunoblotting. The results show that cadmium inhibits formation of disulfide bonds within the GR both in the absence and in the presence of H₂O₂. The creation of intermolecular disulfide linkages between the apo-GR and associated heat shock proteins Hsp90 and Hsp70, which was evident in the presence of H₂O₂, was also significantly impaired after cadmium administration. These observations are consistent with the assumption that cadmium affects the redox state of the receptor, possibly by binding to its sulfhydryl groups. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of rat liver cyclic AMP-dependent protein kinase by flavonoids.

Rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK), assayed using the synthetic peptide substrate, LRRASLG, is inhibited by a range of plant-derived flavonoids. In general, maximal inhibitory effectiveness (IC50 values 1 to 2 microM) requires 2,3-unsaturation and polyhydroxylation involving at least two of the three flavonoid rings. 3-Hydroxyflavone (IC50 value 4 microM), 3,5,7,2',4'-pentahydroxyflavone (IC50 = 10 microM) and 5,7,4'-trihydroxyflavone (IC50 = 7 microM) represent somewhat less active variations from this pattern. Flavonoid O-methylation or O-glycosylation greatly decreases inhibitory effectiveness, as does 2,3-saturation. Various flavonoid-related compounds, notably gossypol (IC50 = 10 microM), also inhibit cAK. Flavonoids and related compounds are in general much better inhibitors of cAK than of avian Ca(2+)-calmodulin-dependent myosin light chain kinase or of plant Ca(2+)-dependent protein kinase. Tricetin (IC50 = 1 microM) inhibits cAK in a fashion that is non-competitive with respect to both peptide substrate and ATP (Ki value 0.7 microM). When histone III-S is used as a substrate, inhibition of cAK requires much higher flavonoid concentrations.     

Properties and regulation of a trans-plasma membrane redox system in rat liver

1. Regulation of the reduction of ferricyanide by the isolated perfused rat liver was studied. 2. The rate of reduction was dependent on the rate of supply of ferricyanide and independent of perfusate oxygen concentration. 3. The effect of pH was also examined; the rate of reduction was optimal at pH 7.4 and was inhibited to a greater extent by pH values below 7.4 than those above 7.4. 4. The effects of substrates on the rate of ferricyanide reduction was assessed. Reductants of the cytosolic and mitochondrial NADH/NAD⁺ couple were tested. 2-Hydroxybutyrate (10mm), lactate (10mm), glycerol (10mm) and ethanol (10mm) each had no effect. Dihydroxyacetone (10mm) stimulated the rate. 5. Dehydroascorbate (1mm), stimulated the rate of ferricyanide reduction; the stimulation did not appear to be attributable to the production of reduced substances that were excreted to reduce extracellular ferricyanide. 6. The effects of glucagon and cyclic AMP on the rate of ferricyanide reduction were examined. Glucagon inhibited the rate by approx. 30% and half-maximal inhibition occurred at 0.1 nm, corresponding to the concentration at which half-maximal stimulation of glucose release occurred. Cyclic AMP stimulated glucose release but had no significant effect on the rate of ferricyanide reduction. It is concluded that the trans-plasma membrane redox system of liver that reduces extracellular ferricyanide is regulated by glucagon. The rate is also altered by the substrate dihydroxyacetone. The effect of glucagon may be direct as it cannot be mimicked by cyclic AMP and it occurs directly following exposure to the hormone.     

The integration of glutathione homeostasis and redox signaling

Formation of reactive oxygen species (ROS) is a common feature of abiotic and biotic stress reactions. ROS need to be detoxified to avoid deleterious reactions, but at the same time, the increased formation of ROS can also be exploited for redox signaling. Glutathione, as the most abundant low-molecular weight thiol in the cellular redox system, is used for both detoxification of ROS and transmission of redox signals. Detoxification of H(2)O(2) through the glutathione-ascorbate cycle leads to a transient change in the degree of oxidation of the cellular glutathione pool, and thus a change in the glutathione redox potential. The shift in the glutathione redox potential can be sensed by glutaredoxins (GRXs), small ubiquitous oxidoreductases, which reversibly transfer electrons between the glutathione redox buffer and thiol groups of target proteins. While very little is known about native GRX target proteins and their behavior in vivo, it is shown here that reduction-oxidation-sensitive GFP (roGFP), when expressed in plants, is an artificial target protein of GRXs. The specific interaction of roGFP with GRX results in continuous formation and release of the roGFP disulfide bridge depending on the actual redox potential of the cellular glutathione buffer. Ratiometric analysis of redox-dependent fluorescence allows dynamic imaging of the glutathione redox potential. It was hypothesized that a similar equilibration occurs between the glutathione buffer and native target proteins of GRXs. As a consequence, even minor deviations in the glutathione redox potential due to either depletion of reduced glutathione (GSH) or increasing oxidation can be exploited for fine tuning the activity of target proteins. The integration of the glutathione buffer with redox-active target proteins is a local reaction in specific subcellular compartments. This observation emphasizes the importance of subcellular compartmentalization in understanding the biology of the cellular redox system in plants.     

Iron homeostasis and methionine-centred redox cycle in nasal polyposis

Abstract

Nasal polyposis is a multifactorial disease with a strong inflammatory component. Its pathogenesis is often associated with ROS production catalysed by redox-active iron. This study aimed to characterize the roles of iron homeostasis and redox status in the pathogenesis of polyposis. Nasal polyps (NP) from asthmatics and non-asthmatics and turbinates from controls and NP-patients were analysed for ferritin, ferritin-bound iron (FBI) and levels of methionine-centred redox cycle proteins. The ferritin content in both NPs was significantly higher than in adjacent turbinates. No differences in FBI were observed between both NP groups and both turbinates groups, while in NPs it was significantly higher. In NP-turbinates the highest levels of redox proteins were observed. In conclusion, re-distribution of iron occurs upon the development of NP. While FBI is elevated in NPs, the adjacent turbinate remain iron-poor and low-inflammatory, suggesting the formation of virtual boundary between these tissues.     

Protein disulfide isomerase in redox cell signaling and homeostasis

Thiol proteins may potentially act as redox signaling adaptor proteins, adjusting reactive oxygen species intermediates to specific signals and redox signals to cell homeostasis. In this review, we discuss redox effects of protein disulfide isomerase (PDI), a thioredoxin superfamily oxidoreductase from the endoplasmic reticulum (ER). Abundantly expressed PDI displays ubiquity, interactions with redox and nonredox proteins, versatile effects, and several posttranslational modifications. The PDI family contains >20 members with at least some apparent complementary actions. PDI has oxidoreductase, isomerase, and chaperone effects, the last not directly dependent on its thiols. PDI is a converging hub for pathways of disulfide bond introduction into ER-processed proteins, via hydrogen peroxide-generating mechanisms involving the oxidase Ero1α, as well as hydrogen peroxide-consuming reactions involving peroxiredoxin IV and the novel peroxidases Gpx7/8. PDI is a candidate pathway for coupling ER stress to oxidant generation. Emerging information suggests a convergence between PDI and Nox family NADPH oxidases. PDI silencing prevents Nox responses to angiotensin II and inhibits Akt phosphorylation in vascular cells and parasite phagocytosis in macrophages. PDI overexpression spontaneously enhances Nox activation and expression. In neutrophils, PDI redox-dependently associates with p47phox and supports the respiratory burst. At the cell surface, PDI exerts transnitrosation, thiol reductase, and apparent isomerase activities toward targets including adhesion and matrix proteins and proteases. Such effects mediate redox-dependent adhesion, coagulation/thrombosis, immune functions, and virus internalization. The route of PDI externalization remains elusive. Such multiple redox effects of PDI may contribute to its conspicuous expression and functional role in disease, rendering PDI family members putative redox cell signaling adaptors.     

Effects of flavonoids on vascular smooth muscle of the isolated rat thoracic aorta

The potency, structure-activity relationship, and mechanism of vasorelaxation of a series of flavonoids, representing different subclasses (flavonols: fisetin, rutin, quercetin; flavones: chrysin, flavone, baicalein; flavanones: naringenin, naringin; isoflavones: diadzein and flavanes: epigallo catechin gallate), were examined in the isolated rat aorta. Most of the flavonoids tested showed concentration dependent relaxant effects against K+ (80 mM) and phenylephrine (PE, 0.1 microM)-induced contractions with a greater inhibition of the responses to the alpha1-adrenoceptor agonist. The relaxant effects of most of the flavonoids involve in part the release of nitric oxide and prostaglandins from the endothelium as pretreatment with L-NAME and indomethacin attenuated the responses. In addition, the relaxant action of the flavonoids includes inhibition of Ca+2 influx and release of Ca+2 from intracellular stores. A structure-activity relationship amongst the flavonoids was suggested.     

Iron homeostasis and methionine-centred redox cycle in nasal polyposis

Nasal polyposis is a multifactorial disease with a strong inflammatory component. Its pathogenesis is often associated with ROS production catalysed by redox-active iron. This study aimed to characterize the roles of iron homeostasis and redox status in the pathogenesis of polyposis. Nasal polyps (NP) from asthmatics and non-asthmatics and turbinates from controls and NP-patients were analysed for ferritin, ferritin-bound iron (FBI) and levels of methionine-centred redox cycle proteins. The ferritin content in both NPs was significantly higher than in adjacent turbinates. No differences in FBI were observed between both NP groups and both turbinates groups, while in NPs it was significantly higher. In NP-turbinates the highest levels of redox proteins were observed. In conclusion, re-distribution of iron occurs upon the development of NP. While FBI is elevated in NPs, the adjacent turbinate remain iron-poor and low-inflammatory, suggesting the formation of virtual boundary between these tissues.     

Glucagon-stimulated calcium efflux in the isolated perfused rat liver is dependent on cellular redox potential

In the absence of any exogenous substrates, glucagon (1 X 10(-9) M) stimulated 45Ca2+ efflux from perfused livers derived from fed rats but not in livers of 24-h-fasted animals. In livers of 24-h-fasted animals perfused under conditions which would decrease cellular NAD(P)H/NAD(P)+ ratio (pyruvate (2.0 mM) or acetoacetate (10.0 mM], glucagon (1 X 10(-9) M) did not stimulate 45Ca2+ efflux. Similarly, in livers of 24-h-fasted animals perfused with substrates which increase cellular NAD(P)H content (lactate (2.0 mM) or beta-hydroxybutyrate (10.0 mM], glucagon (1 X 10(-9) M) did not increase 45Ca2+ efflux. Glucagon (1 X 10(-9) M) elicited an increase in 45Ca2+ efflux from livers of 24-h-fasted animals, only when the livers were perfused with [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate] ratios similar to those reported for livers of fed rats. Stimulation of 45Ca2+ efflux elicited by either 8-CPT-cAMP, a cAMP analog, or high glucagon concentrations (1 X 10(-8) M) was not affected whether livers were perfused with pyruvate (2.0 mM) or lactate (2.0 mM). Administration of isobutylmethylxanthine (50 microM) alone, or glucagon (1 X 10(-9) M) in the presence of isobutylmethylxanthine (50 microM) stimulated 45Ca2+ efflux from livers of 24-h-fasted animals perfused with pyruvate (2.0 mM) but not from livers perfused with lactate (2.0 mM). The ability of glucagon (1 X 10(-9) M) to elevate tissue cAMP levels was also regulated by the oxidation-reduction state of the livers. The data indicate that glucagon-stimulated 45Ca2+ efflux from perfused livers is mediated via cAMP and is dependent on the oxidation-reduction state of the livers.