Endothelial dysfunction in a rat model of endotoxic shock. Importance of the activation of poly (ADP-ribose) synthetase by peroxynitrite (original) (raw)
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Cardiovascular toxicology, 2016
Peroxynitrite is a powerful oxidant, formed from the reaction of nitric oxide and superoxide. It is known to interact and modify different biological molecules such as DNA, lipids and proteins leading to alterations in their structure and functions. These events elicit various cellular responses, including cell signaling, causing oxidative damage and committing cells to apoptosis or necrosis. This review discusses nitrosative stress-induced modification in the DNA molecule that results in the formation of 8-nitroguanine and 8-oxoguanine, and its role in disease conditions. Different approaches of cell death, such as necrosis and apoptosis, are modulated by cellular high-energy species, such as ATP and NAD(+). High concentrations of peroxynitrite are known to cause necrosis, whereas low concentrations lead to apoptosis. Any damage to DNA activates cellular DNA repair machinery, like poly(ADP-ribose) polymerase (PARP). PARP-1, an isoform of PARP, is a DNA nick-sensing enzyme that beco...
British Journal of Pharmacology, 1997
1. Peroxynitrite is a toxic oxidant species produced from nitric oxide (NO) and superoxide. We have recently observed that the cell-permeable superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) inhibits the suppression of mitochondrial respiration elicited by authentic peroxynitrite in vitro. Here we have investigated the relative potency of MnTBAP and a range of related compounds in terms of inhibition of peroxynitrite-induced oxidation and cytotoxicity. In addition, we tested the effects of MnTBAP on the vascular and the cellular energetic failure in rodent models of endotoxic shock. 2. We observed a dose-related inhibition of the peroxynitrite-induced oxidation of dihydrorhodamine 123 to rhodamine by MnTBAP, ZnTBAP and FeTBAP, but not by MnTMPyP [(5,10,15,20-tetrakis(N-methyl-4'-pirydyl)porphinato)-mangan ese (III)]. In addition, MnTBAP, ZnTBAP and FeTBAP, but not MnTMPyP prevented the suppression of mitochondrial respiration by authentic peroxynitrite in cultured J774 macrophages. 3. In rat cultured aortic smooth muscle cells, MnTBAP protected against the suppression of mitochondrial respiration in response to authentic peroxynitrite, immunostimulation and nitric oxide (NO) donor compounds. MnTBAP slightly reduced the amount of nitrite/nitrate produced in response to immunostimulation in these cells. 4. Administration of MnTBAP, 15 mg kg-1 i.v., before the administration of endotoxin (15 mg kg-1, i.v.) to rats ameliorated the development of vascular hyporeactivity and the development of endothelial dysfunction in the thoracic aorta ex vivo. 5. MnTBAP also prevented the endotoxin-induced decrease in mitochondrial respiration, the development of DNA single strand breaks, and the depletion of intracellular NAD+ in peritoneal macrophages ex vivo. 6. MnTBAP did not inhibit the expression by endotoxin of the inducible NO synthase in lung samples. 7. MnTBAP did not alter survival rate in mice challenged with high dose endotoxin. 8. Our findings, taken together with previous data demonstrating protective effects of NO synthase inhibitors against the endotoxin-induced contractile and energetic failure in the models of shock used in the current study, and with the known ability of peroxynitrite to cause cellular energy depletion, suggest a role for peroxynitrite in the pathogenesis of cellular energetic failure and contractile dysfunction in endotoxin shock.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2005
Objective-Peroxynitrite, a potent oxidant generated by the reaction of NO with superoxide, has been implicated in the promotion of atherosclerosis. We designed this study to determine whether peroxynitrite induces its proatherogenic effects through induction of endoplasmic reticulum (ER) stress. Methods and Results-Human vascular endothelial cells treated with Sin-1, a peroxynitrite generator, induced the expression of the ER chaperones GRP78 and GRP94 and increased eIF2␣ phosphorylation. These effects were inhibited by the peroxynitrite scavenger uric acid. Sin-1 caused the depletion of ER-Ca 2ϩ , an effect known to induce ER stress, resulting in the elevation of cytosolic Ca 2ϩ and programmed cell death (PCD). Sin-1 treatment was also found, via 3-nitrotyrosine and GRP78 colocalization, to act directly on the ER. Adenoviral-mediated overexpression of GRP78 in endothelial cells prevented Sin-1-induced PCD. Consistent with these in vitro findings, 3-nitrotyrosine was observed and colocalized with GRP78 in endothelial cells of early atherosclerotic lesions from apolipoprotein E-deficient mice. Conclusions-Peroxynitrite is an ER stress-inducing agent. Its effects include the depletion of ER-Ca 2ϩ , a known mechanism of ER stress induction. The observation that 3-nitrotyrosine-containing proteins colocalize with markers of ER stress within early atherosclerotic lesions suggests that peroxynitrite contributes to atherogenesis through a mechanism involving ER stress. (Arterioscler Thromb Vasc Biol. 2005;25:2623-2629.)
British Journal of Pharmacology, 1997
Zymosan is a wall component of the yeast Saccharomyces Cerevisiae. Injection of zymosan into experimental animals is known to produce an intense in¯ammatory response. Recent studies demonstrated that the zymosan-induced in¯ammatory response in vivo can be ameliorated by inhibitors of nitric oxide (NO) biosynthesis. The cytotoxic eects of NO are, in part, mediated by the oxidant preoxynitrite and subsequent activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS). In the present in vitro study, we have investigated the cellular mechanisms of vascular failure elicited by zymosan-activated plasma and the contribution of peroxynitrite production and activation of PARS to the changes. 2 Incubation of rat aortic smooth muscle cells with zymosan-activated plasma (ZAP) induced the production of nitrite, the breakdown product of NO, due to the expression of the inducible isoform of NO synthase (iNOS) over 6 ± 24 h. In addition, ZAP triggered the production of peroxynitrite in these cells, as measured by the oxidation of the¯uorescent dye dihydrorhodamine 123 and by nitrotyrosine Western blotting. 3 Incubation of the smooth muscle cells with ZAP induced DNA single strand breakage and PARS activation. These eects were reduced by inhibition of NOS with N G-methyl-L-arginine (L-NMA, 3 mM), and by glutathione (3 mM), a scavenger of peroxynitrite. The PARS inhibitor 3-aminobenzamide (1 mM) inhibited the ZAP-induced activation of PARS. 4 Incubation of thoracic aortae with ZAP in vitro caused a reduction of the contractions of the blood vessels to noradrenaline (vascular hyporeactivity) and elicited a reduced responsiveness to the endothelium-dependent vasodilator acetylcholine (endothelial dysfunction). 5 Preincubation of the thoracic aortae with L-NMA (1 mM), glutathione (3 mM) or by the PARS inhibitor 3-aminobenzamide (1 mM) prevented the development of vascular hyporeactivity in response to ZAP. Moreover, glutathione and 3-aminobenzamide treatment protected against the ZAP-induced development of endothelial dysfunction. The PARS-related loss of the vascular contractility was evident at 30 min after incubation in endothelium-intact, but not in endothelium-denuded vessels and also manifested at 6 h after incubation with ZAP in endothelium-denuded rings. The acute response is probably related, therefore, to peroxynitrite formation (involving the endothelial NO synthase), whereas the delayed response may be related to the expression of iNOS in the smooth muscle. 6 The data obtained suggest that zymosan-activated plasma causes vascular dysfunction by inducing the simultaneous formation of superoxide and NO. These radicals combine to form peroxynitrite, which, in turn causes DNA injury and PARS activation. The protective eect of 3-aminobenzamide demonstrates that PARS activation contributes both to the development of vascular hyporeactivity and endothelial dysfunction during the vascular failure induced by ZAP.
Peroxynitrite Induces Haem Oxygenase-1 In Vascular Endothelial Cells: a Link to Apoptosis.
Roberta FORESTI, Padmini SARATHCHANDRA, James E. CLARK, Colin J. GREEN and Roberto MOTTERLINI, 1999
Peroxynitrite (ONOO−) is a potent oxidizing agent generated by the interaction of nitric oxide (NO) and the superoxide anion. In physiological solution, ONOO− rapidly decomposes to a hydroxyl radical, one of the most reactive free radicals, and nitrogen dioxide, another species able to cause oxidative damage. In the present study we investigated the effect of ONOO− on the expression of haem oxygenase-1 (HO-1), an inducible protein that is highly up-regulated by oxidative stress. Exposure of bovine aortic endothelial cells to ONOO− (250±1000 µM) produced a concentration-dependent increase in haem oxygenase activity and HO-1 protein expression. This effect was completely abolished by the ONOO− scavengers uric acid and N-acetylcysteine, and partly attenuated by 1,3-dimethyl-2-thiourea, a scavenger of hydroxyl radicals. ONOO− also produced a concentration-dependent increase in apoptosis and cytotoxicity, which were considerably decreased by uric acid and N-acetylcysteine. A 70%decrease in apoptosis was observed when cells were exposed to ONOO− in the presence of 10 µM tin protoporphyrin IX (SnPPIX), an inhibitor of haem oxygenase activity. When SnPPIX was added 5 min after ONOO−, apoptosis decreased by only 40%, which suggests that an interaction between ONOO− and the protoporphyrin occurs in our system. Increased haem oxygenase activity by pretreatment of cells with haemin resulted in elevated bilirubin production and was associated with a substantial decrease (35%) in ONOO−-mediated apoptosis. These results indicate the ability of ONOO− to modulate the expression of the stress protein HO-1 and suggest that the haem oxygenase pathway contributes to protection against the cytotoxic action of ONOO−.
Indian Journal of Clinical Biochemistry, 2015
Peroxynitrite is formed in biological systems when nitric oxide and superoxide rapidly interact at near equimolar ratio. Peroxynitrite, though not a free radical by chemical nature, is a powerful oxidant which reacts with proteins, DNA and lipids. These reactions trigger a wide array of cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. The present review outlines the various peroxynitrite-induced DNA modifications with special mention to the formation of 8-nitroguanine and 8-oxoguanine as well as the induction of DNA single strand breakage. Low concentrations of peroxynitrite cause apoptotic death, whereas higher concentrations cause necrosis with cellular energetics (ATP and NAD ?) serving as control between the two modes of cell death. DNA damage induced by peroxynitrite triggers the activation of DNA repair systems. A DNA nick sensing enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) becomes activated upon detecting DNA breakage and it cleaves NAD ? into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins. Over-activation of PARP induced by peroxynitrite consumes NAD ? and consequently ATP decreases, culminating in cell dysfunction, apoptosis or necrosis. This mechanism has been implicated in the pathogenesis of various diseases like diabetes, cardiovascular diseases and neurodegenerative diseases. In this review, we have discussed the cytotoxic effects (apoptosis and necrosis) of peroxynitrite in the etiology of the mentioned diseases, focusing on the role of PARP in DNA repair in presence of peroxynitrite.
British Journal of Pharmacology, 1998
Peroxynitrite, a cytotoxic oxidant formed from the reaction of nitric oxide (NO) and superoxide is a mediator of cellular injury in ischaemia/reperfusion injury, shock and in¯ammation. Here we investigated whether L-buthionine-(S,R)-sulphoximine (BSO), an inhibitor of g-glutamylcysteine synthetase, alters endothelial and vascular smooth muscle injury in response to peroxynitrite in vitro and during endotoxic shock in vivo. 2 In human umbilical vein endothelial cells and in rat aortic smooth muscle cells, BSO (1 mM, for 24 h) enhanced, whereas glutathione (3 mM) or glutathione ethyl ester (3 mM) attenuated the peroxynitrite (100 ± 1000 mM)-induced suppression of mitochondrial respiration (measured by the conversion of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan), formation of nitrotyrosine (detected by Western blotting), protein oxidation (measured by detection of 2,4 dinitrophenylhydrazinereactive carbonyls), and DNA single strand breakage and activation of the nuclear enzyme poly (ADPribose) synthetase (PARS) (measured by the incorporation of radiolabelled NAD + into nuclear proteins and by the alkaline unwinding assay, respectively). Glutathione ethyl ester treatment reduced the BSOinduced enhancement of peroxynitrite-induced cytotoxicity. 3 In rat isolated thoracic aortic rings, BSO treatment (in vivo, at 1 g kg 71 intraperitoneally (i.p.) for 24 h) enhanced, whereas pretreatment with glutathione (in vitro, 3 mM) attenuated the peroxynitriteinduced reduction of the contractions to noradrenaline, and the peroxynitrite-induced impairment of the endothelium-dependent relaxations to acetylcholine. 4 In BSO-pretreated rats, treatment with bacterial lipopolysaccharide (LPS, 15 mg kg 71 , i.p., for 6 h) caused a more pronounced vascular hyporeactivity and endothelial dysfunction ex vivo. BSO pretreatment also increased the degree of nitrotyrosine staining (detected by imunohistochemistry) in the aorta after LPS treatment. 5 In conclusion, our results demonstrate that L-buthionine-(S,R)-sulphoximine, an inhibitor of gglutamylcysteine synthetase enhances peroxynitrite-and endotoxic shock-induced vascular failure. Based on these ®ndings, we suggest that endogenous glutathione plays an important protective role against peroxynitrite-and LPS-induced vascular injury.
Role of the Peroxynitrite - Poly (ADP-Ribose) Polymerase Pathway in the Pathogenesis of Liver Injury
Current Pharmaceutical Design, 2006
Throughout the last 2 decades, experimental evidence from in vitro studies and preclinical models of disease has demonstrated that reactive oxygen and nitrogen species, including the reactive oxidant peroxynitrite, are generated in parenchymal, endothelial, and infiltrating inflammatory cells during stroke, myocardial and other forms of reperfusion injury, myocardial hypertrophy and heart failure, cardiomyopathies, circulatory shock, cardiovascular aging, atherosclerosis and vascular remodeling after injury, diabetic complications, and neurodegenerative disorders. Peroxynitrite and other reactive species induce oxidative DNA damage and consequent activation of the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1), the most abundant isoform of the PARP enzyme family. PARP overactivation depletes its substrate NAD ؉ , slowing the rate of glycolysis, electron transport, and ATP formation, eventually leading to functional impairment or death of cells, as well as up-regulation of various proinflammatory pathways. In related animal models of disease, peroxynitrite neutralization or pharmacological inhibition of PARP provides significant therapeutic benefits. Therefore, novel antioxidants and PARP inhibitors have entered clinical development for the experimental therapy of various cardiovascular and other diseases. This review focuses on the human data available on the pathophysiological relevance of the peroxynitrite-PARP pathway in a wide range of disparate diseases, ranging from myocardial ischemia/reperfusion injury, myocarditis, heart failure, circulatory shock, and diabetic complications to atherosclerosis, arthritis, colitis, and neurodegenerative disorders.
CARDIOVASCULAR EFFECTS OF PEROXYNITRITE
Clinical and Experimental Pharmacology and Physiology, 2007
both exist as free radicals. By itself, PN is not a free radical, but it can generate nitrogen dioxide (·NO 2 ) and carbonate radical ( ·) upon reaction with CO 2 . 2. The reaction of CO 2 constitutes a major pathway for the disposition of PN produced in vivo and this is based on the rapid reaction of PN anion with CO 2 and the availability of CO 2 in both intra-and extracellular fluids. The free radicals ·NO 2 and ·, in combination with ·NO, generated from nitric oxide synthase, can bring about oxidation of critical biological targets resulting in tissue injury. However, the reactions of ·NO 2 , · and ·NO with carbohydrates, protein and non-protein thiols, phenols, indoles and uric acid could result in the formation of a number of nitration and nitrosation products in the vasculature. These products serve as long-acting ·NO donors and, therefore, contribute to vasorelaxant properties, protective effects on the heart, inhibition of leucocyte-endothelial cell interactions and reduction of reperfusion injury.