scholarly journals 5-Hydroxytryptamine1A receptor/Giβγ stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of Src in Chinese hamster ovary fibroblasts

2000 ◽  
Vol 347 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Yurii V. MUKHIN ◽  
Maria N. GARNOVSKAYA ◽  
Georgiann COLLINSWORTH ◽  
Jasjit S. GREWAL ◽  
DeKisha PENDERGRASS ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Reactive Oxygen ◽  
Mitogen Activated Protein Kinase ◽  
Oxygen Species ◽  
Erk Activation ◽  
Activation Pathway

The hypothesis of this work is that the ‘serotonin’ or 5-hydroxytryptamine (5-HT)1A receptor, which activates the extracellular signal-regulated kinase (ERK) through a Giβγ-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT1A receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT1A receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT1A receptor in Chinese hamster ovary (CHO) cells downstream of Giβγ subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT1A receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide.

Enhanced survival effect of pyruvate correlates MAPK and NF-κB activation in hydrogen peroxide-treated human endothelial cells

2004 ◽  
Vol 96 (2) ◽  
pp. 793-801 ◽  
Author(s):  
Yong-Jin Lee ◽  
Il-Jun Kang ◽  
Rolf Bünger ◽  
Young-Hee Kang
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Therapeutic Potential ◽  
Cell Model ◽  
Mapk Pathway ◽  
Umbilical Vein ◽  
Reactive Oxygen ◽  
Antioxidant Potential ◽  
Mitogen Activated Protein Kinase ◽  
Oxygen Species

We recently reported that pyruvate inhibited translocation and activation of p53 caused by DNA damage due to oxidant injury (Lee YJ, Kang IJ, Bünger R, and Kang YH. Microvasc Res 66: 91-101, 2003); this was associated with increased expression of apoptosis-related bcl-2 and decreased expression of bax gene. This study attempted to delineate possible regulatory sites and mechanisms of antiapoptotic pyruvate, focusing on reactive oxygen species-mediated signaling in a human umbilical vein endothelial cell model. We compared the effects of the cytosolic reductant l-lactate and malate-aspartate shuttle blocker aminooxyacetate, both of which increase cytosolic NADH, on the downstream signaling pathway. Hydrogen peroxide (0.5 mM H2O2) depleted intracellular total glutathione that was prevented by pyruvate but not by l-lactate or aminooxyacetate. Activation of caspase-3 and the cleavage of procaspase-6 and procaspase-7 were strongly inhibited by pyruvate but markedly enhanced by l-lactate and aminooxyacetate, implicating redox-related antiapoptotic mechanisms of pyruvate. Western blot analysis and immunochemical data revealed that H2O2-induced transactivation of nuclear factor-κB (NF-κB) was also inhibited by pyruvate but not by l-lactate or aminooxyacetate. In addition, H2O2 downregulated extracellular signal-regulated kinase (ERK1/2) and phosphorylated p38 mitogen-activated protein kinase (MAPK), effects that were fully reversed by pyruvate within 2 h. Collectively, these findings indicate that pyruvate can protect cellular glutathione, thus enhancing cellular antioxidant potential, and that enhanced antioxidant potential can desensitize NF-κB transactivation due to reactive oxygen species, suggesting possible metabolic redox relations to NF-κB. Furthermore, pyruvate blocked the p38 MAPK pathway and activated the ERK pathway in an apparently redox-sensitive manner, which may regulate expression of genes believed to prevent apoptosis and promote cell survival. Thus pyruvate may have therapeutic potential for reducing endothelial dysfunction and improving survival during oxidative stress.

scholarly journals A Novel Fluorescent Sensor Protein for Visualization of Redox States in the Cytoplasm and in Peroxisomes

2010 ◽  
Vol 30 (15) ◽  
pp. 3758-3766 ◽  
Author(s):  
Taisuke Yano ◽  
Masahide Oku ◽  
Natsuko Akeyama ◽  
Akinori Itoyama ◽  
Hiroya Yurimoto ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cho Cells ◽  
Redox State ◽  
Fluorescent Sensor ◽  
Chinese Hamster ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Wild Type ◽  
Efficient System ◽  
Redox States

ABSTRACT Reactive oxygen species are generated within peroxisomes during peroxisomal metabolism. However, due to technological difficulties, the intraperoxisomal redox state remain elusive, and the effect of peroxisome deficiency on the intracellular redox state is controversial. A newly developed, genetically encoded fluorescence resonance energy transfer (FRET) probe, Redoxfluor, senses the physiological redox state via its internal disulfide bonds, resulting in a change in the conformation of the protein leading to a FRET response. We made use of Redoxfluor to measure the redox states at the subcellular level in yeast and Chinese hamster ovary (CHO) cells. In wild-type peroxisomes harboring an intact fatty acid β-oxidation system, the redox state within the peroxisomes was more reductive than that in the cytosol, despite the fact that reactive oxygen species were generated within the peroxisomes. Interestingly, we observed that the redox state of the cytosol of cell mutants for peroxisome assembly, regarded as models for a neurological metabolic disorder, was more reductive than that of the wild-type cells in yeast and CHO cells. Furthermore, Redoxfluor was utilized to develop an efficient system for the screening of drugs that moderate the abnormal cytosolic redox state in the mutant CHO cell lines for peroxisome assembly without affecting the redox state of normal cells.

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