title: Melatonin ameliorates ischemic-like injury-evoked nitrosative stress: Involvement of HtrA2/PED pathways in endothelial cells.
abstract: Peroxynitrite contributes to diverse cellular stresses in the pathogenesis of ischemic complications. Here, we investigate the downstream effector signaling elements of nitrosative stress which regulate ischemia-like cell death in endothelial cells and protective effect of melatonin. When the mitochondrial membrane potential (ΔΨm) of oxygen-glucose deprivation (OGD)-treated cells was assessed using the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol -carbocyanine iodide, we observed spontaneous changes in peroxynitrite formation. Concomitantly, western blot and confocal microscopy analyses indicated that prolonged OGD exposure initiates the release of mitochondrial HtrA2 and dramatically decreases phosphoprotein enriched in astrocytes (PED or PEA-15) protein levels. Consistently, cultured endothelial cells treated with peroxynitrite (1-50 μm) exhibited a concentration-dependent release of mitochondrial HtrA2 and concomitant PED degradation in vitro. Notably, HtrA2 activation coincided with increased nitrotyrosine immunoreactivity in microvessels of rats following microsphere embolism. Additionally, the protective effect of PED overexpression in OGD-induced apoptosis was abolished by transfection with the PED(S104A/S116A) mutant. Furthermore, the effect of melatonin, an potential antioxidant, on endothelial apoptotic cascade was examined in OGD-evoked nitrosative stress. Our data showed that the application of melatonin provided significant protection against OGD-induced peroxynitrite formation and mitochondrial HtrA2 release, accompanied with a decrease in degradation PED and x-linked inhibitor of apoptosis protein, which is associated with activation of the caspase cascade. Taken together, the protective effect of melatonin is likely mediated, in part, by inhibition of peroxynitrate-mediated nitrosative stress, which in turn relieves imbalance of mitochondrial HtrA2-PED signaling and endothelial cell death.
authors.affiliation: Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, Zhejiang University, Hangzhou, China. changhuahan@zju.edu.cn
authors.forename: Feng
authors.initials: F
authors.lastname: Han
authors.forename: Rong-rong
authors.initials: RR
authors.lastname: Tao
authors.forename: Gen-sheng
authors.initials: GS
authors.lastname: Zhang
authors.forename: Ying-mei
authors.initials: YM
authors.lastname: Lu
authors.forename: Lu-lu
authors.initials: LL
authors.lastname: Liu
authors.forename: Ying-xian
authors.initials: YX
authors.lastname: Chen
authors.forename: Yi-jia
authors.initials: YJ
authors.lastname: Lou
authors.forename: Kohji
authors.initials: K
authors.lastname: Fukunaga
authors.forename: Ze-Hui
authors.initials: ZH
authors.lastname: Hong
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins; D002545:Brain Ischemia / Q000188:drug therapy* / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008550:Melatonin; D053078:Membrane Potential, Mitochondrial; D018613:Microscopy, Confocal; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D009419:Nerve Tissue Proteins; C092039:PEA15 protein, human; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; C490770:Tra2b protein, rat; D030421:Peroxynitrous Acid; D000068103:Serine-Arginine Splicing Factors; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2; D008550:Melatonin