Detection of Ras GTPase protein radicals through immuno-spin trapping

Over the past decade immuno-spin trapping (IST) has been used to detect and identify protein radical sites in numerous heme and metalloproteins. To date, however, the technique has had little application toward nonmetalloproteins. In this study, we demonstrate the successful application of IST in a system free of transition metals and present the first conclusive evidence of (?)NO-mediated protein radical formation in the HRas GTPase. HRas is a nonmetalloprotein that plays a critical role in regulating cell-growth control. Protein radical formation in Ras GTPases has long been suspected of initiating premature release of bound guanine nucleotide. This action results in altered Ras activity both in vitro and in vivo. As described herein, successful application of IST may provide a means for detecting and identifying radical-mediated Ras activation in many different cancers and disease states in which Ras GTPases play an important role.     

Obesity-induced tissue free radical generation: an in vivo immuno-spin trapping study

Assessment of tissue free radical production is routinely accomplished by measuring secondary by-products of redox reactions and/or diminution of key antioxidants such as reduced thiols. However, immuno-spin trapping, a newly developed immunohistochemical technique for detection of free radical formation, is garnering considerable interest as it allows for the visualization of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-adducted molecules. Yet, to date, immuno-spin trapping reports have utilized in vivo models in which successful detection of free radical adducts required exposure to lethal levels of oxidative stress not reflective of chronic inflammatory disease. To study the extents and anatomic locations of more clinically relevant levels of radical formation, we examined tissues from high-fat (HF) diet-fed mice, a model of low-grade chronic inflammation known to demonstrate enhanced rates of reactive species production. Mice subjected to 20 weeks of HF diet displayed increased free radical formation (anti-DMPO mean fluorescence staining) in skeletal muscle (0.863±0.06 units vs 0.512±0.07 units), kidney (0.076±0.0036 vs 0.043±0.0025), and liver (0.275±0.012 vs 0.135±0.014) compared to control mice fed normal laboratory chow (NC). Western blot analysis of tissue homogenates confirmed these results showing enhanced DMPO immunoreactivity in HF mice compared to NC samples. The obesity-related results were confirmed in a rat model of pulmonary hypertension and right heart failure in which intense immunodetectable radical formation was observed in the lung and right ventricle of monocrotaline-treated rats compared to saline-treated controls. Combined, these data affirm the utility of immuno-spin trapping as a tool for in vivo assessment of altered extents of macromolecule oxidation to radical intermediates under chronic inflammatory conditions.     

Identification of the myoglobin tyrosyl radical by immuno-spin trapping and its dimerization

5,5-Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO.     

Effect of low flow ischemia-reperfusion injury on liver function

The release of liver enzymes is typically used to assess tissue damage following ischemia-reperfusion. The present study was designed to determine the impact of ischemia-reperfusion on liver function and compare these findings with enzyme release. Isolated, perfused rat livers were subjected to low flow ischemia followed by reperfusion. Alterations in liver function were determined by comparing rates of oxygen consumption, gluconeogenesis, ureagenesis, and ketogenesis before and after ischemia. Lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) activities in effluent perfusate were used as markers of parenchymal and endothelial cell injury, respectively. Trypan blue staining was used to localize necrosis. Total glutathione (GSH + GSSG) and oxidized glutathione (GSSG) were measured in the perfusate as indicators of intracellular oxidative stress. LDH activity was increased 2-fold during reperfusion compared to livers kept normoxic for the same time period whereas PNP activity was elevated 5-fold under comparable conditions. Rates of oxygen consumption, gluconeogenesis, and ureagenesis were unchanged after ischemia, but ketogenesis was decreased 40% following 90 min ischemia. During reperfusion, the efflux rates of total glutathione and GSSG were unchanged from pre-ischemic values. Significant midzonal staining of hepatocyte nuclei was observed following ischemia-reperfusion, whereas normoxic livers had only scattered staining of individual cells. Reperfusion of ischemic liver caused release of hepatic enzymes and midzonal cell death, however, several major liver functions were unaffected under these experimental conditions. These data indicate that there were negligible changes in liver function in this model of ischemia and reperfusion despite substantial enzyme release from the liver and midzonal cell death.     

In vivo detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin trapping

Free radicals are known to play a major role in sepsis. Combined immuno-spin trapping and molecular magnetic resonance imaging (MRI) was used to detect in vivo and in situ levels of free radicals in murine septic encephalopathy after cecal ligation and puncture (CLP). DMPO (5,5-dimethyl pyrroline N-oxide) was injected over 6 h after CLP, before administration of an anti-DMPO probe (anti-DMPO antibody bound to albumin–gadolinium–diethylene triamine pentaacetic acid–biotin MRI targeting contrast agent). In vitro assessment of the anti-DMPO probe in oxidatively stressed mouse astrocytes significantly decreased T₁ relaxation (p < 0.0001) compared to controls. MRI detected the presence of anti-DMPO adducts via a substantial decrease in %T₁ change within the hippocampus, striatum, occipital, and medial cortex brain regions (p < 0.01 for all) in septic animals compared to shams, which was sustained for over 60 min (p < 0.05 for all). Fluorescently labeled streptavidin was used to target the anti-DMPO probe biotin, which was elevated in septic brain, liver, and lungs compared to sham. Ex vivo DMPO adducts (qualitative) and oxidative products, including 4-hydroxynonenal and 3-nitrotyrosine (quantitative, p < 0.05 for both), were elevated in septic brains compared to shams. This is the first study that has reported on the detection of in vivo and in situ levels of free radicals in murine septic encephalopathy.     

Theaflavin attenuates ischemia-reperfusion injury in a mouse fatty liver model

The incidence of non-alcoholic fatty liver disease (NAFLD) has been increasing, and there is a shortage of liver donors, which has led to the acceptance of steatotic livers for transplantation. However, steatotic livers are known to experience more severe acute ischemia-reperfusion (I/R) injury than normal livers upon transplantation. In the present study, we investigated the role of theaflavin, a polyphenol substance extracted from black tea, in attenuating acute I/R injury in a fatty liver model. We induced I/R in normal and steatotic livers treated with or without theaflavin. We also separated primary hepatocytes from the normal and steatotic livers, and applied RAW264.7 cells, a mouse macrophage cell line, that was pretreated with theaflavin. We observed that liver steatosis, oxidative stress, inflammation and hepatocyte apoptosis were increased in the steatotic liver compared to the normal liver, however, these changes were significantly decreased by theaflavin treatment. In addition, theaflavin significantly diminished the ROS production of steatotic hepatocytes and TNF-α production by LPS-stimulated RAW264.7 cells. We concluded that theaflavin has protective effects against I/R injury in fatty livers by anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.     

Glutathione and ischemia-reperfusion injury in the perfused rat liver.

Using the isolated perfused rat liver, we investigated the relationship of glutathione (GSH) with reactive oxygen species (ROS) generation and liver cell damage during ischemia/reperfusion in normal and GSH-depleted conditions. Lucigenin-enhanced chemiluminescence was used as a sensitive index of tissue ROS generation. After 30 minutes of equilibration, livers were subjected to global ischemia for various times (60 or 90 minutes) and then reperfused for another 120 minutes. Intracellular ROS levels increased sharply at the onset of reperfusion and then declined slowly. After 30 to 60 minutes of reperfusion, ROS levels started to increase progressively in a linear fashion. However, sinusoidal glutathione disulfide release did not increase during reperfusion in the same livers, suggesting that intracellular ROS generation is too low to cause a significant increase in GSH oxidation. Pretreatment with phorone (300 mg/kg intrapentoneally [ip]), which reduced hepatic GSH by 90%, did not cause any difference in intracellular ROS generation compared with the control livers. There were also no significant differences in lactate dehydrogenase and thiobarbituric acid reactive substances (TBARS) release between the control and phorone-treated livers during reperfusion after various times of ischemia. These data indicate that ROS generation in the normal isolated perfused liver during ischemia/reperfusion is extremely low and intracellular GSH does not serve as a major intracellular defense system against such a low oxidative stress.     

Mechanisms of interleukin-6 protection against ischemia-reperfusion injury in rat liver

Numerous animal studies simulating liver injury have demonstrated that interleukin-6 (IL-6) exerts a protective effect. This study was designed to further analyze the molecular mechanisms underlying the protective role of IL-6 in a rat model of liver ischemia/reperfusion injury. We show that IL-6: (i) at high doses reduces cell damage which occurs in ischemic-reperfused liver, while at low doses displays only a limited protective capacity, (ii) anticipates and enhances hepatocyte compensatory proliferation seen in ischemic-reperfused liver also at a low, more pharmacologically acceptable dose, (iii) sustains the acute phase response which is dampened in ischemic-reperfused liver, (iv) strengthens the heat shock-stress response shown by ischemic-reperfused liver and (v) overcomes the dysfunctions of the unfolding protein response found in ischemic-reperfused liver. We also show that IL-6-enhanced STAT3 activation probably plays a crucial role in the potentiation of the different protective pathways activated in ischemic-reperfused liver. Our data confirm that IL-6 is a potential therapeutic in liver injury of different etiologies and reveal novel mechanisms by which IL-6 sustains liver function after ischemia/reperfusion injury.     

Carbon monoxide protects against ischemia-reperfusion injury in vitro via antioxidant properties

Carbon monoxide (CO) is believed to mediate many of the cytoprotective effects attributed to the activation of heme oxygenase (HO-1), the enzyme responsible for CO production. Recently, the study of CO-releasing molecules (CO-RMs) has provided a new approach for the delivery of CO. In the present study, we examined whether the cardioprotective properties of CO-RM2 in isolated rat hearts subjected to an ischemia-reperfusion (I/R) sequence were associated with the presence of CO. In addition, the antioxidant properties of CO-RM2 were evaluated. In hearts pretreated with CO-RM2, the improvement in contractile function at the end of the reperfusion period after 20 min of global total ischemia was significantly greater than in controls. These beneficial effects were accompanied by a reduction in 1) LDH activity release 2) infarct size 3) ventricular superoxide production. The improvement in myocardial function and the reduction in oxidative stress were not observed when hearts were pretreated with inactivated CO-RM2 (iCO-RM2). Additionally, CO-RM2, but not iCO-RM2, was found to exert antioxidant properties. These results suggest that the production of CO is a necessary factor in the cardioprotective and antioxidant actions of CO-releasing compound. These results may open up new ground for a novel class of cardioprotective compounds.     

Alpha-lipoic acid preconditioning reduces ischemia-reperfusion injury of the rat liver via the PI3-kinase/Akt pathway

In liver resection and transplantation ischemia-reperfusion injury (IRI) is one of the main causes of organ dys- or nonfunction. The aim of the present study was to determine whether alpha-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA (+/-wortmannin), followed by ischemia (1 h, 37 degrees C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-kappaB and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 micromol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-kappaB and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway.     

内质网应激预处理条件下大鼠肝脏缺血再灌注损伤模型的建立

目的:通过建立了内质网应激预处理条件下的大鼠肝脏缺血再灌注损伤模型,探讨内质网应激预处理在体内的应用.方法:以衣霉素为内质网应激诱导剂,采用大鼠肝脏70%缺血再灌注损伤模型.按照不同给药剂量分为50μ g/kg组、100μ g,kg组、200μ g/kg组、对照组,观察不同给药剂量条件下,血清转氨酶水平的变化.结果:给药100μ g/kg体重、诱导5天条件下大鼠术后转氨酶水平显著低于对照组.其它组与对照组无统计学差异.肝组织病理切片证实该预处理条件对肝脏缺血再灌注损伤有显著保护作用.结论:在特定的给药剂量条件下,衣霉素诱导的内质网应激预处理对大鼠肝脏缺血再灌注损伤有显著的保护作用.     

Measurement of intracellular chloride activity in mouse liver slices with microelectrodes

Steady-state membrane potential (V_m) and intracellular Cl⁻ activity (a_Clⁱ) were measured with double-barreled Cl⁻-selective microelectrodes in mouse liver slices. In bathing solutions (33.8° C) containing pyruvate, glutamate, fumarate, and glucose, V_m and a_Clⁱ were −27.6 ± 1.0 mV and 32.6 ± 1.5 mM, respectively. This apparent value of a_Clⁱ exceeded the level required for passive distribution of this ion (a_Cl^eq = 26.4 ± 1.3 mM) by 6.2 ± 1.0 mM. This difference was essentially unchanged in experiments where (i) Na⁺ was replaced by choline, (ii) HCO₃⁻ was removed, and (iii) Cl⁻ was replaced by gluconate. These data argue against the presence of Na⁺- or HCO₃⁻-coupled Cl⁻ transport mechanisms in the plasma membrane of mouse liver cells. This implies that a_Clⁱ is in fact at equilibrium and interference with the response of Cl⁻-selective microelectrodes by intracellular anions is responsible for the apparent difference between a_Clⁱ and a_Cl^eq. We found that Cl⁻-selective microelectrodes containing Corning 477315 ligand are sensitive to taurocholate, a representative bile salt. Their selectivity to taurocholate is about 60-times their selectivity towards Cl⁻. This suggests that interference of bile acids at concentrations normally present in hepatocytes with determinations of a_Clⁱ can account for the apparent difference a_Clⁱ − a_Cl^eq.     

Ischemic postconditioning attenuates liver warm ischemia-reperfusion injury through Akt-eNOS-NO-HIF pathway

Background  

Ischemic postconditioning (IPO) has been demonstrated to attenuate ischemia/reperfusion (I/R) injury in the heart and brain, its roles to liver remain to be defined. The study was undertaken to determine if IPO would attenuate liver warm I/R injury and its protective mechanism.     

The effects of resveratrol administration on lipid oxidation in experimental renal ischemia-reperfusion injury in rats

ABSTRACT

We investigated how resveratrol affects lipid oxidation during experimental renal ischemia-reperfusion injury in rats. We used 48 adult male rats assigned to five groups: group 1, control; group 2, renal ischemia; group 3, renal ischemia + reperfusion; group 4, resveratrol + renal ischemia; group 5, resveratrol + renal ischemia + reperfusion. Plasma and renal tissue malondialdehyde (MDA), and erythrocyte and renal tissue glutathione (GSH) levels were measured and histologic changes in the renal tissue were examined. Ischemia-reperfusion affected the MDA-GSH balance adversely and caused histopathological changes in the renal tissue of the ischemia and ischemia + reperfusion groups. Resveratrol treatment normalized MDA and GSH levels as well as the histopathology that occurred in the renal tissue of the ischemia and ischemia + reperfusion groups.     

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein-associated O-GlcNAc

Increased levels of protein O-linked N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to determine whether in isolated neonatal rat ventricular myocytes (NRVMs) an increase in protein O-GlcNAcylation resulted in improved survival and viability following ischemia-reperfusion (I/R). NRVMs were exposed to 4 h of ischemia and 16 h of reperfusion, and cell viability, necrosis, apoptosis, and O-GlcNAc levels were assessed. Treatment of cells with glucosamine, hyperglycemia, or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate(PUGNAc), an inhibitor of O-GlcNAcase, significantly increased O-GlcNAc levels and improved cell viability, as well as reducing both necrosis and apoptosis compared with untreated cells following I/R. Alloxan, an inhibitor of O-GlcNAc transferase, markedly reduced O-GlcNAc levels and exacerbated I/R injury. The improved survival with hyperglycemia was attenuated by azaserine, which inhibits glucose metabolism via the hexosamine biosynthesis pathway. Reperfusion in the absence of glucose reduced O-GlcNAc levels on reperfusion compared with normal glucose conditions and decreased cell viability. O-GlcNAc levels significantly correlated with cell viability during reperfusion. The effects of glucosamine and PUGNAc on cellular viability were associated with reduced calcineurin activation as measured by translocation of nuclear factor of activated T cells, suggesting that increased O-GlcNAc levels may attenuate I/R induced increase in cytosolic Ca²⁺. These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection. hexosamine biosynthesis; calcium; protein O-glycosylation     

Identification of protein radicals formed in the human neuroglobin-H2O2 reaction using immuno-spin trapping and mass spectrometry

Neuroglobin (Ngb) is a recently discovered protein that shows only minor sequence similarity with myoglobin and hemoglobin but conforms to the typical 3-over-3 alpha-helical fold characteristic of vertebrate globins. An intriguing feature of Ngb is its heme hexacoordination in the absence of external ligands, observed both in the ferrous and in the ferric (met) forms. In Ngb, the imidazole of a histidine residue (His-64) in the distal position, above the heme plane, provides the sixth coordination bond. In this work, a valine residue was introduced at position 64 (H64V variant) to clarify the possible role(s) of the distal residue in protecting the heme iron of Ngb from attack by strong oxidants. SDS-PAGE analyses revealed that the oxidation of the H64V variant of metNgb by H 2O 2 resulted in the formation of dimeric and trimeric products in contrast to the native protein. Dityrosine cross-links were shown by their fluorescence to be present in the oligomeric products. When the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was included in the reaction mixture, nitrone adducts were detected by immuno-spin trapping. The specific location of the DMPO adducts on the H64V variant protein was determined by a mass spectrometry method that combines off-line immuno-spin trapping and chromatographic procedures. This method revealed Tyr-88 to be the site of modification by DMPO. The presence of His-64 in the wild-type protein results in the nearly complete loss of detectable radical adducts. Together, the data support the argument that wild-type Ngb is protected from attack by H 2O 2 by the coordinated distal His.     

Rb1 postconditioning attenuates liver warm ischemia-reperfusion injury through ROS-NO-HIF pathway

AimsGinsenoside Rb1 could prevent ischemic neuronal death and focal cerebral ischemia, but its roles to liver warm I/R injury remain to be defined. We determined if Rb1 would attenuate warm I/R injury in mice.Main methodsMice were divided into sham, I/R, Rb1 + I/R (Rb1 postconditioning, 20 mg/kg, i.p. after ischemia), sham + L-NAME, I/R + L-NAME, and Rb1 + I/R + L-NAME groups using 60 min of the liver median and left lateral lobes ischemia. Serum levels of alanine aminotransferase (ALT) were measured and morphology changes of livers were evaluated. Contents of nitric oxide (NO) and nitric oxide synthase (NOS), malondialdehye (MDA) and activity of superoxide dismutase (SOD) were measured. Expressions of Akt, p-Akt, iNOS, HIF-1alpha, tumor necrosis factor-a (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were also determined by western blot or immunohistochemistry.Key findingsRb1 postconditioning attenuated the dramatically functional and morphological injuries. The levels of ALT were significantly reduced in Rb1 group (p < 0.05). Rb1 upregulated the concentrations of NO, iNOS in serum, iNOS, and activity of SOD in hepatic tissues (p < 0.05), while it dramatically reduced the concentration of MDA (p < 0.05). Protein expressions of p-Akt, iNOS and HIF-1alpha were markedly enhanced in Rb1 group. Protein and mRNA expressions of TNF-α and ICAM-1 were markedly suppressed by Rb1 (p < 0.05).SignificanceWe found that Rb1 postconditioning could protect liver from I/R injury by upregulating the content of NO and NOS, and also HIF-1alpha protein expression. These protective effects could be abolished by L-NAME. These findings suggested Rb1 may have the therapeutic potential through ROS-NO-HIF pathway for management of liver warm I/R injury.