ABSTRACT
We tested the impact of A1 adenosine receptor (AR) deletion on injury and oxidant damage in mouse hearts subjected to 25-min ischemia/45-min reperfusion (I/R). Wild-type hearts recovered approximately 50% of contractile function and released 8.2 +/- 0.7 IU/g of lactate dehydrogenase (LDH). A1AR deletion worsened dysfunction and LDH efflux (15.2 +/- 2.6 IU/g). Tissue cholesterol and native cholesteryl esters were unchanged, whereas cholesteryl ester-derived lipid hydroperoxides and hydroxides (CE-O(O)H; a marker of lipid oxidation) increased threefold, and alpha-tocopherylquinone [alpha-TQ; oxidation product of alpha-tocopherol (alpha-TOH)] increased sixfold. Elevations in alpha-TQ were augmented by two- to threefold by A1AR deletion, whereas CE-O(O)H was unaltered. A(1)AR deletion also decreased glutathione redox status ([GSH]/[GSSG + GSH]) and enhanced expression of the antioxidant response element heme oxygenase-1 (HO-1) during I/R: fourfold elevations in HO-1 mRNA and activity were doubled by A1AR deletion. Broad-spectrum AR agonism (10 microM 2-chloroadenosine; 2-CAD) countered effects of A1AR deletion on oxidant damage, HO-1, and tissue injury, indicating that additional ARs (A(2A), A(2B), and/or A3) can mediate similar actions. These data reveal that local adenosine engages A1ARs during I/R to limit oxidant damage and enhance outcome selectively. Control of alpha-TOH/alpha-TQ levels may contribute to A1AR-dependent cardioprotection.
Subject(s)
Adenosine/metabolism , Myocardial Reperfusion Injury/metabolism , Oxidative Stress/physiology , Receptor, Adenosine A1/metabolism , 2-Chloroadenosine/pharmacology , Adenosine A1 Receptor Agonists , Animals , Cholesterol/metabolism , Cholesterol Esters/metabolism , Chromatography, High Pressure Liquid , Glutathione/metabolism , Heme Oxygenase-1/metabolism , Hydroxides/metabolism , L-Lactate Dehydrogenase/metabolism , Lipid Peroxides/metabolism , Male , Mice , Myocardial Reperfusion Injury/genetics , Oxidation-Reduction , Oxidative Stress/drug effects , Oxidative Stress/genetics , Polymerase Chain Reaction , Receptor, Adenosine A1/genetics , Vitamin E/analogs & derivatives , Vitamin E/metabolismABSTRACT
After acute myocardial infarction (AMI), infiltrating proinflammatory cells generate two-electron oxidants such as hypochlorous acid (HOCl). Myoglobin (Mb) is present at approximately 0.3 mM in cardiomyocytes and, therefore, represents a significant target for oxidation. Exposure of horse Mb (50 microM) to reagent HOCl (0-500 microM) or activated human neutrophils (4-40x10(6) cells/ml) yielded oxidized Mb (Mb(ox)) as judged by amino acid analysis and peptide mass mapping. HOCl/Mb ratios of 1-5 mol/mol gave Mb(ox) with up to four additional oxygen atoms. Hydrolysis of Mb(ox) followed by amino acid analysis indicated that methionine (Met) and tryptophan (Trp) residues were modified by HOCl. Peptide mass mapping revealed that Met55 was oxidized at a lower HOCl/Mb ratio than Met131 and this preceded Trp7/14 modification (susceptibility Met55>Met131>Trp7>Trp14). Incubation of Mb with activated neutrophils and physiological chloride anion yielded Mb(ox) with a composition similar to that determined with HOCl/Mb ratios <2 mol/mol, with oxidation of Met, but not Trp, detected. These data indicate that Mb undergoes site-specific oxidation depending on the HOCl/protein ratio. As Mb is released from necrotic cardiomyocytes into the vasculature after AMI, HOCl-modified Mb may be a useful surrogate marker to gauge the extent of myocardial inflammation.
Subject(s)
Hypochlorous Acid/pharmacology , Methionine/metabolism , Myoglobin/pharmacology , Tryptophan/metabolism , Amino Acid Sequence , Chloramines/metabolism , Chromatography, High Pressure Liquid , Humans , In Vitro Techniques , Molecular Sequence Data , Myoglobin/chemistry , Myoglobin/metabolism , Neutrophil Activation , Neutrophils/metabolism , Spectrometry, Mass, Electrospray IonizationABSTRACT
Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. Cultured human neuronal cells exposed to experimental hypoxia-re-oxygenation (H/R) injury responded with an increased production of reactive oxygen species (ROS) and a significant decrease in intracellular ATP. Expression of genes encoding for hypoxia-inducible factor 1-alpha (HIF1-alpha), inducible haemoxygenase-1 (HO-1), glucose transporter-1 (Glut-1), the oxygen-sensor neuroglobin (Nb) and Cu,Zn-superoxide dismutase (SOD1), catalase (CAT) and glutathione peroxidase-1 (Gpx-1) increased significantly in response to the insult. Enhanced expression of HO-1, SOD1 and CAT correlated with an increase in the corresponding protein activity. Despite the cellular response to bolster antioxidant capacity, apoptosis and necrosis increased following H/R injury. In contrast, ROS accumulation, the endogenous gene response and cell death was limited in neuronal cells pre-incubated with 50 or 100, but not 10 microM of the phenolic antioxidant 3,3',5,5'-tetra-t-butyl-biphenyl-4,4'-diol (BP) prior to H/R injury. These data indicate that the early endogenous gene response to H/R injury is unable to inhibit neuronal dysfunction and that increasing cellular antioxidant capacity with a synthetic polyphenol (>10 microM) is potentially neuro-protective.
Subject(s)
Flavonoids/administration & dosage , Hyperbaric Oxygenation , Neurons/drug effects , Oxidative Stress/drug effects , Phenols/administration & dosage , Adenosine Triphosphate/metabolism , Analysis of Variance , Annexins/metabolism , Caspases/metabolism , Catalase/metabolism , Cell Differentiation , Cell Hypoxia/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Flavonoids/chemistry , GAP-43 Protein/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Heme Oxygenase-1/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neuroblastoma/pathology , Neurons/metabolism , Phenols/chemistry , Polyphenols , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Superoxides/metabolism , Time FactorsABSTRACT
Hydrogen peroxide (H(2)O(2)) is an oxidant implicated in cell signalling and various pathologies, yet relatively little is known about its impact on endothelial cell function. Herein we studied the functional and biochemical changes in aortic vessels and cultured porcine aortic endothelial cells (PAEC) exposed to H(2)O(2). Exposure of aortic rings to 25 or 50 microM, but not 10 microM, H(2)O(2) for 60 min prior to constriction significantly decreased subsequent relaxation in response to acetylcholine (ACh), but not the nitric oxide ((.)NO) donor sodium nitroprusside. Treatment of PAEC with 50 microM H(2)O(2) significantly decreased ACh-induced accumulation of (.)NO, as measured with a (.)NO-selective electrode, yet such treatment increased nitric oxide synthase activity approximately 3-fold, as assessed by conversion of L-arginine to L-citrulline. Decreased (.)NO bioavailability was reflected in decreased cellular cGMP content, associated with increased superoxide anion radical (O(2)(-.)), and overcome by addition of polyethylene glycol superoxide dismutase. Increased cellular O(2)(-.) production was inhibited by allopurinol, diphenyliodonium and rotenone in an additive manner. The results show that exposure of endothelial cells to H(2)O(2) decreases the bioavailability of agonist-induced (.)NO as a result of increased production of O(2)(-.) likely derived from xanthine oxidase, NADPH-oxidase and mitochondria. These processes could contribute to H(2)O(2)-induced vascular dysfunction that may be relevant under conditions of oxidative stress such as inflammation.
