Pyruvate restores contractile function and antioxidant defenses of hydrogen peroxide-challenged myocardium

PURPOSE: Pyruvate, a natural energy-yielding fuel in myocardium, neutralizes peroxides by a direct decarboxylation reaction, and indirectly augments the glutathione (GSH) antioxidant system by generating NADPH reducing power via citrate formation. The possibility that pyruvate's antioxidant actions could mediate its enhancement of contractile performance in prooxidant-challenged myocardium was investigated in isolated working guinea-pig hearts reversibly injured by hydrogen peroxide. METHODS: Hearts were challenged by 10 min perfusion with 100 microM H(2)O(2), followed by 90 min H(2)O(2)-free perfusion. Metabolic and antioxidant treatments (each 5m M) were administered at 30-90 min post-H(2)O(2). Phosphocreatine phosphorylation state, GSH/glutathione disulfide redox potential (GSH/GSSG) and key enzyme activities were measured in snap-frozen myocardium. RESULTS: H(2)O(2) exposure depleted myocardial energy and antioxidant reserves and produced marked contractile impairment that persisted throughout the H(2)O(2) washout period. Relative to untreated post-H(2)O(2) myocardium, pyruvate restored contractile performance, increased GSH/GSSG 52% and maintained phosphocreatine phosphorylation state; in contrast, lactate lowered cardiac performance and phosphorylation state. Neither the pharmacological antioxidant N -acetylcysteine (NAC) nor the pyruvate analog alpha-ketobutyrate increased cardiac function; both treatments increased GSH/GSSG but lowered phosphocreatine potential. H(2)O(2) partially inactivated aconitase, creatine kinase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), but all three enzymes spontaneously recovered during H(2)O(2) washout. Pyruvate did not further activate these enzymes and unexpectedly inhibited GAPDH by 60-70%. CONCLUSION: Pyruvate promoted robust contractile recovery of H(2)O(2)-challenged myocardium by the combination of citrate-mediated antioxidant mechanisms and maintenance of myocardial energy reserves.     

Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes

Formation of mixed disulfides between glutathione and the cysteines of some proteins (glutathionylation) has been suggested as a mechanism through which protein functions can be regulated by the redox status. The aim of this study was to identify the proteins of T cell blasts that undergo glutathionylation under oxidative stress. To this purpose, we radiolabeled cellular glutathione with (35)S, exposed T cells to oxidants (diamide or hydrogen peroxide), and performed nonreducing, two-dimensional electrophoresis followed by detection of labeled proteins by phosphorimaging and their identification by mass spectrometry techniques. We detected several proteins previously not recognized to be glutathionylated, including cytoskeletal proteins (vimentin, myosin, tropomyosin, cofilin, profilin, and the already known actin), enzymes (enolase, aldolase, 6-phosphogluconolactonase, adenylate kinase, ubiquitin-conjugating enzyme, phosphoglycerate kinase, triosephosphate isomerase, and pyrophosphatase), redox enzymes (peroxiredoxin 1, protein disulfide isomerase, and cytochrome c oxidase), cyclophilin, stress proteins (HSP70 and HSP60), nucleophosmin, transgelin, galectin, and fatty acid binding protein. Based on the presence of several protein isoforms in control cells, we suggest that enolase and cyclophilin are heavily glutathionylated under basal conditions. We studied the effect of glutathionylation on some of the enzymes identified in the present study and found that some of them (enolase and 6-phosphogluconolactonase) are inhibited by glutathionylation, whereas the enzymatic activity of cyclophilin (peptidylprolyl isomerase) is not. These findings suggest that protein glutathionylation might be a common mechanism for the global regulation of protein functions.     

Reactive oxygen species play an essential role in IGF-I signaling and IGF-I-induced myocyte hypertrophy in C2C12 myocytes

IGF-I induces skeletal muscle hypertrophy by stimulating protein synthesis and suppressing the protein degradation pathway; the downstream signaling pathways Akt-mammalian target of rapamycin (mTOR)-p70-kDA-S6-kinase (p70S6K), and Forkhead box O1 (FoxO1) play essential roles in this regulation. Reactive oxygen species (ROS) modulate the signaling of various growth factors via redox regulation. However, the role of ROS in IGF-I signaling is not fully understood. In this study, we investigated whether ROS regulate the signaling and biological action of IGF-I in C2C12 myocytes. We found that IGF-I induces ROS in C2C12 myocytes. While treatment with H(2)O(2) significantly enhanced IGF-I-induced phosphorylation of the IGF-I receptor (IGF-IR), IGF-IR phosphorylation was markedly attenuated when cells were treated with antioxidants. The downstream signaling pathway, Akt-mTOR-p70S6K was subsequently down-regulated. Furthermore, the phosphorylation of FoxO1 by IGF-I decreased concomitantly with the restoration of the expression of its target genes, Atrogin-1 and muscle RING finger 1, which are related to muscle atrophy. Nox4 knockdown, which is reportedly to produce ROS in insulin signaling, attenuated IGF-I-induced IGF-IR phosphorylation, indicating that Nox4 is involved in the regulation of IGF-I signaling. Importantly, antioxidant treatments inhibited IGF-I-induced myocyte hypertrophy, demonstrating that ROS are necessary for IGF-I-induced myocyte hypertrophy in vitro. These results indicate that ROS play an essential role in the signaling and biological action of IGF-I in C2C12 myocytes.     

The role of redox signaling in cardiac hypertrophy induced by experimental hyperthyroidism

This study was conducted to test whether oxidative stress activates the intracellular protein kinase B (AKT1) signaling pathway, which culminates with cardiac hypertrophy in experimental hyperthyroidism. Male Wistar rats were divided into four groups: control, vitamin E, thyroxine (T(4)), and T(4)+vitamin E. Hyperthyroidism was induced by T(4) administration (12 mg/l in drinking water for 28 days). Vitamin E treatment was given during the same period via s.c. injections (20 mg/kg per day). Morphometric and hemodynamic parameters were evaluated at the end of the 4-week treatment period. Protein oxidation, redox state (reduced glutathione, GSH/glutathione dissulfide, GSSG), vitamin C, total radical-trapping antioxidant potential (TRAP), hydrogen peroxide (H2O2), and nitric oxide metabolites (NO(X)) were measured in heart homogenates. The p-AKT1/AKT1 ratio, p-glycogen-synthase kinase (GSK)3B/GSK3B ratio, FOS, and JUN myocardial protein expression were also quantified by western blot after 4 weeks. Increases in biochemical parameters, such as protein oxidation (41%), H2O2 (62%), and NO(X) (218%), and increase in the left ventricular end-diastolic pressure were observed in the T(4) group. T(4) treatment also caused a decrease in GSH/GSSG ratio (83%), vitamin C (34%), and TRAP (55%). These alterations were attenuated by vitamin E administration to the hyperthyroid rats. Expression of p-AKT1/AKT1, p-GSK3B/GSK3B, FOS, and JUN were elevated in the T(4) group (by 69, 37, 130, and 33% respectively), whereas vitamin E administration promoted a significant reduction in their expression. These results indicate that oxidative stress plays an important role in cardiac hypertrophy, and suggest redox activation of AKT1 and JUN/FOS signaling pathways with H2O2 acting as a possible intracellular mediator in this adaptive response to experimental hyperthyroidism.     

Polaprezinc protects human colon cells from oxidative injury induced by hydrogen peroxide: relevant to cytoprotective heat shock proteins

INTRODUCTION Polaprezinc [N-(3-aminopropionyl)-L-histidinato zinc], an antiulcer drug, is a chelate compound consisting of zinc ion, L-carnosine, dipeptide of β-alanine, and L-histidine and has an antioxidant effect and anti-H pylori activity[1-4]. It ha…     

Green fluorescent protein selectively induces HSP70-mediated up-regulation of COX-2 expression in endothelial cells

Reporter genes, including green fluorescent protein (GFP), have been used to monitor the expression of transgenes introduced into vascular cells by gene transfer vectors. Here, we demonstrate that GFP by itself can selectively induce expression of certain genes in endothelial cells. Elevation of the cytoplasmic concentration of GFP in endothelial cells, specifically, resulted in a robust upregulation of heat shock protein 70 (HSP70). GFP induced both mRNA and protein expression of HSP70 in a dose-dependent manner. GFP-mediated up-regulation of HSP70 resulted in induction of cyclooxygenase-2 (COX-2) followed by prostaglandin E2 (PGE2) production. GFP-mediated up-regulation of HSP70 is independent of mitogen-activated protein kinase and phosphatidylinositol-3-kinase signaling cascades because inhibition of these pathways had no effect on HSP70 increases. Adenoviral delivery of GFP into murine vasculature significantly enhanced blood flow, suggesting that sufficient PGE2 is produced to induce vasodilation. Identification of the molecular partners that interact with GFP will increase our understanding of the vascular-specific factors that regulate stress angiogenesis and hemostasis.     

Effect of melatonin on temporal changes of reactive oxygen species and glutathione after MPP(+) treatment in human astrocytoma U373MG cells

1-Methyl-4-phenylpyridinium (MPP(+)) ion, a toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, is produced by monoamine oxidase B in astrocytes. MPP(+) causes a selective dopaminergic neurodegeneration, the pathophysiologic hallmark of Parkinson disease. However, the toxic effect of MPP(+) on astrocytes remains unclear. Here, we examined the effect of MPP(+) on human astrocytoma U373MG cells, with particular attention to the temporal interaction of glutathione (GSH) and reactive oxygen species (ROS) (H2O2 and O). MPP(+) induced astrocyte apoptosis in a dose-dependent manner 48 hr after treatment. Distinctive early (<6 hr) and late (24-48 hr) responses were observed. ROS production and the oxidized GSH (GSSG)/GSH ratio, indicators of oxidative stress, rose dramatically after 24 hr of MPP(+) exposure, whereas the H2O2 level transiently decreased at 6 hr. ROS overproduction and GSH dysfunction were concomitantly associated with caspase-3 activation and finally led to cell apoptosis. Moreover, GSH depletion by diethyl maleate, but not buthionine sulfoximine, caused cells to die quickly and potentiated the cytotoxicity of MPP(+). Co-treatment with melatonin, a known antioxidant secreted by the pineal gland, significantly prevented cell apoptosis by inhibiting oxidative stress and caspase-3 activation, but it did not affect that the early changes due to MPP(+) treatment. Our results demonstrate that in astrocytes, GSH is involved in the early decrease and late increase in ROS levels induced by MPP(+) treatment. Melatonin remedies the dysfunction of GSH system to block caspase-3 activation and cell apoptosis induced by oxidative stress during the long-term exposure of MPP(+).     

Protein glutathionylation increases in the liver of patients with non-alcoholic fatty liver disease

Background and Aim:  Oxidative stress is an important pathophysiological mechanism in non-alcoholic steatohepatitis, where hepatocyte apoptosis is significantly increased correlating with disease severity. Protein glutathionylation occurs as a response to oxidative stress, where an increased concentration of oxidized glutathione modifies post-translational proteins by thiol disulfide exchange. In this study, we analyzed the protein glutathionylation in non-alcoholic fatty liver disease (NAFLD) and evaluated a potential association between glutathionylation, fibrosis, and vitamin E treatment.
Methods:  Protein glutathionylation was studied in the livers of 36 children (mean age 12.5 years, range 4–16 years) subdivided into three groups according to their NAFLD activity score (NAS) by Western blot analysis and immunohistochemistry, using a specific monoclonal antibody. In addition, we identified the hepatocyte ultrastructures involved in glutathionylation by immunogold electron microscopy.
Results:  Our findings showed that protein glutathionylation increases in the livers of patients with NAFLD and it is correlated with steatohepatitis and liver fibrosis. Its increase appears mainly in nuclei and cytosol of hepatocytes, and it is reversed by antioxidant therapy with reduced fibrosis.
Conclusion:  Protein glutathionylation significantly increases in livers with NAFLD, strongly suggesting that oxidative injury plays a crucial role in this disease. Furthermore, the marked increase of protein glutathionylation, in correlation with collagen VI immunoreactivity, suggests a link between the redox status of hepatic protein thiols and fibrosis.     

Glutathione redox cycle is an important defense system of endothelial cells against chronic hyperoxia

Exposure of cultured pulmonary artery endothelial cells to 95% O2 resulted in the following sequence of events: decrease in [3H]thymidine incorporation after 24 h; increase of intracellular glutathione (GSH) and loss of cellular protein after 48 h; increase of spontaneous and decrease of provoked prostacyclin formation as well as increased release of cellular LDH after 72 h. This oxygen toxicity model was used to study the following 2 questions. (1) What is the relative importance of the GSH redox cycle compared to catalase as antioxidative defense against hyperoxia? Endothelial cells were grown in selenium-depleted medium to inhibit glutathione peroxidase activity. Endothelial GSH biosynthesis was inhibited by buthionine sulfoximine. Catalase activity was reduced by aminotriazole. Endothelial cells with an impaired GSH redox cycle were easily killed by hyperoxia within 24 h, while inhibition of catalase did not enhance the susceptibility of endothelial cells to hyperoxia. (2) Can endothelial GSH content be increased by exogenous sulfhydryl reagents and does this result in an increase of endothelial cells' resistance to hyperoxia? Exogenous GSH, N-acetylcysteine, cysteine, and L-2-oxothiazolidine-4-carboxylate (L-2-oxo) increased intracellular GSH. All sulfhydryl reagents (with the exception of L-2-oxo) protected endothelial cells from hyperoxia. Concentrations of exogenous GSH and N-acetylcysteine that did not increase intracellular GSH reduced hyperoxia-induced endothelial cell injury. Thus the capacity of the GSH redox cycle rather than intracellular GSH levels or catalase determines endothelial cells' resistance to hyperoxia.     

Extracellular redox state: refining the definition of oxidative stress in aging

Oxidative stress in aging can result from an imbalance of prooxidants and antioxidants with excessive, destructive free radical chemistry. Thiol systems are important in the control of these processes, both by protecting against damage and serving in redox signaling mechanisms to sense danger and repair the damage. Studies by a number of research groups in collaboration with the Emory Clinical Biomarkers Laboratory show that the redox state of the central tissue antioxidant, glutathione (GSH), can be measured in human plasma and provides a quantitative systemic indicator of oxidative stress. Plasma GSH/GSSG redox in humans becomes oxidized with age, in response to chemotherapy, as a consequence of cigarette smoking, and in association with common age-related diseases (e.g., type 2 diabetes, cardiovascular disease). However, the GSH/GSSG redox is not equilibrated with the larger plasma cysteine/cystine (Cys/CySS) pool, and the Cys/CySS redox varies with age in a pattern that is distinct from that of GSH/GSSG redox. Furthermore, in vitro studies show that variation in Cys/CySS redox over the range found in vivo affects signaling pathways, which control cell proliferation and oxidant-induced apoptosis. The results point to the conclusion that free radical scavenging antioxidants are of increased importance when thiol/disulfide redox states are oxidized. Because thiol/disulfide redox states, per se, function in redox signaling and control as well as antioxidant protection, GSH/GSSG and Cys/CySS redox states may provide central parameters to link environmental influences and progression of changes associated with aging.     

Mechanisms of pyruvate inhibition of oxidant-induced apoptosis in human endothelial cells

We have recently demonstrated that the redox reactant pyruvate prevents hydrogen peroxide (H2O2)-induced endothelial apoptosis and that its anti-apoptotic feature is mediated partially through the mitochondrial compartment. However, little is known about molecular signal pathways that mediate the anti-apoptotic feature of pyruvate. A biochemical approach to elucidate such signal pathways was attempted in human umbilical vein endothelial cells (HUVECs). Effects of antioxidant pyruvate were compared with those of cytosolic reductant L-lactate, redox-neutral acetate, and malate-aspartate shuttle blocker aminooxyacetate. Various indices of endothelial apoptosis were correlated with cell viability. Submillimolar H2O2 caused >50% cell killing, as manifested by its oxidant insult. The massive cell death induced by H2O2 was inhibited by pyruvate but not by L-lactate or aminooxyacetate, suggesting a role of cytosolic NADH reducing equivalents, possibly via stimulated oxidant generation. The induction and nuclear translocation of p53 by H2O2 was blocked by pyruvate and appeared to be somewhat enhanced by L-lactate or aminooxyacetate in association with oxidant generation. Nuclear translocation of p53 accompanied the transactivation of bax and downregulation of bcl-2. The pyruvate-related redox manipulation inhibited the H2O2-induced p53 activation, restored the downregulated bcl-2 and the upregulated bax, and hence enhanced the bcl-2/bax expression ratio. In contrast, L-lactate, acetate, or aminooxyacetate had no such effect. These results indicate that pyruvate could modulate key regulatory signal pathways in cytosol and mitochondrial matrix, thereby inactivating endothelial death pathways. Furthermore, it is suggested that stabilizing the expression of bcl-2 and bax genes by metabolic antioxidants may be an effective strategy for endothelial protection against oxidative stress.