共查询到20条相似文献,搜索用时 15 毫秒
1.
Gelain DP De Souza LF Ribeiro GR Zim M Jardim FR Moreira JC Bernard EA 《Free radical research》2004,38(1):37-47
Extracellular purines are involved in the regulation of a wide range of physiological processes, including cytoprotection, ischemic preconditioning, and cell death. These actions are usually mediated via triggering of membrane purinergic receptors, which may activate antioxidant enzymes, conferring cytoprotection. Recently, it was demonstrated that the oxidative stress induced by cisplatin up-regulated A1 receptor expression in rat testes, suggesting an involvement of purinergic signaling in the response of testicular cells to oxidant injury. In this article, we report the effect of hydrogen peroxide on purinergic agonist release by cultured Sertoli cells. Extracellular inosine levels are strongly increased in the presence of H2O2, suggesting an involvement of this nucleoside on Sertoli cells response to oxidant treatment. Inosine was observed to decrease H2O2-induced lipoperoxidaton and cellular injury, and it also preserved cellular ATP content during H2O2 exposure. These effects were abolished in the presence of nucleoside uptake inhibitors, indicating that nucleoside internalisation is essential for its action in preventing cell damage. 相似文献
2.
Extracellular ATP and adenosine modulation of MAPKs is well described in different cells types, but few studies have addressed the effects of extracellular inosine on these kinases. Previous results showed that hydrogen peroxide and TNF-alpha increase extracellular inosine concentration in cultured Sertoli cells and this nucleoside protects Sertoli cells against hydrogen peroxide induced damage and participates in TNF-alpha induced nitric oxide production. In view of the fact that MAPKs are key mediators of the cellular response to a large variety of stimuli, we investigated the effect of extracellular inosine on the phosphorylation of ERK 1/2 and p38 MAPKs in cultured Sertoli cells. The involvement of this nucleoside in the activation of ERK 1/2 by TNF-alpha was also investigated. Inosine and the selective A1 adenosine receptor agonist R-PIA increases the phosphorylation of ERK 1/2 and p38, and this was blocked by the selective A1 adenosine receptors antagonists, CPT and DPCPX. These antagonists also inhibited TNF-alpha increase in the phosphorylation of ERK 1/2. TNF-alpha also rapidly augmented extracellular inosine concentration in cultured Sertoli cells. These results show that extracellular inosine modulates ERK 1/2 and p38 in cultured Sertoli cells, possible trough A1 adenosine receptor activation. This nucleoside also participates in TNF-alpha modulation of ERK 1/2. 相似文献
3.
Bhavya B. Chandrika Cheng Yang Yang Ou Xiaoke Feng Djamali Muhoza Alexandrea F. Holmes Sue Theus Sarika Deshmukh Randy S. Haun Gur P. Kaushal 《PloS one》2015,10(10)
We examined whether endoplasmic reticulum (ER) stress-induced autophagy provides cytoprotection from renal tubular epithelial cell injury due to oxidants and chemical hypoxia in vitro, as well as from ischemia-reperfusion (IR) injury in vivo. We demonstrate that the ER stress inducer tunicamycin triggers an unfolded protein response, upregulates ER chaperone Grp78, and activates the autophagy pathway in renal tubular epithelial cells in culture. Inhibition of ER stress-induced autophagy accelerated caspase–3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy. Compared to wild-type cells, autophagy-deficient MEFs subjected to ER stress had enhanced caspase–3 activation and cell death, a finding that further supports the cytoprotective role of ER stress-induced autophagy. Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H2O2 and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A. In contrast, inhibition of ER stress-induced autophagy or autophagy-deficient cells markedly enhanced cell death in response to oxidant injury and chemical hypoxia. In mouse kidney, similarly to renal epithelial cells in culture, tunicamycin triggered ER stress, markedly upregulated Grp78, and activated autophagy without impairing the autophagic flux. In addition, ER stress-induced autophagy markedly ameliorated renal IR injury as evident from significant improvement in renal function and histology. Inhibition of autophagy by chloroquine markedly increased renal IR injury. These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo. 相似文献
4.
This study was undertaken to investigate the inducibility of glutathione (GSH), glutathione reductase (GR) and glutathione peroxidase (GPx) by 3H-1,2-dithiole-3-thione (D3T) in beta-cells, and the resultant cytoprotection against oxidant injury. Incubation of the insulin-secreting RINm5F cells with D3T led to significant induction of GSH, GR and GPx. D3T-mediated induction of GSH was abolished by buthionine sulfoximine (BSO), suggesting a critical involvement of γ-glutamylcysteine ligase (γGCL). Consistently, incubation of RINm5F cells with D3T resulted in increased expression of γGCL protein and mRNA. Pretreatment of RINm5F cells with D3T provided remarkable protection against oxidant-elicited cytotoxicity. On the other hand, depletion of cellular GSH by BSO sensitized RINm5F cells to oxidant injury. Furthermore, cotreatment of RINm5F cells with BSO to reverse D3T-mediated GSH induction abolished the cytoprotective effects of D3T on oxidant injury. Taken together, this study demonstrates that upregulation of glutathione system by D3T is effective for protecting against oxidative beta-cell injury. 相似文献
5.
Diego Varela Antonello Penna Felipe Simon Ana Luisa Eguiguren El��as Leiva-Salcedo Oscar Cerda Francisco Sala Andr��s Stutzin 《The Journal of biological chemistry》2010,285(10):7566-7574
Volume-sensitive outwardly rectifying (VSOR) Cl− channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H2O2 plays an essential role in the activation of these channels and that H2O2 per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H2O2-induced and hypotonicity-mediated VSOR Cl− activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H2O2-induced and hypotonicity-mediated VSOR Cl− current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl− current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 μm H2O2 VSOR Cl− current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 μm H2O2, exogenous addition of ATP in the presence of extracellular Ca2+ resulted in a decrease in the half-time for VSOR Cl− current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl− current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl− current onset in a extracellular Ca2+-dependent manner. 相似文献
6.
Gonzalez A Santofimia-Castaño P Rivera-Barreno R Salido GM 《Journal of physiology and biochemistry》2012,68(2):181-191
This work was designed in order to gain an insight on the mechanisms by which antioxidants prevent pancreatic disorders. We
have examined the properties of cinnamtannin B-1, which belongs to the class of polyphenols, against the effect of hydrogen
peroxide (H2O2) in mouse pancreatic acinar cells. We have studied Ca2+ mobilization, oxidative state, amylase secretion, and cell viability of cells treated with cinnamtannin B-1 in the presence
of various concentrations of H2O2. We found that H2O2 (0.1–100 μM) increased CM-H2DCFDA-derived fluorescence, reflecting an increase in oxidation. Cinnamtannin B-1 (10 μM) reduced H2O2-induced oxidation of CM-H2DCFDA. CCK-8 induced oxidation of CM-H2DCFDA in a similar way to low micromolar concentrations of H2O2, and cinnamtannin B-1 reduced the oxidant effect of CCK-8. In addition, H2O2 induced a slow and progressive increase in intracellular free Ca2+ concentration ([Ca2+]c). Cinnamtannin B-1 reduced the effect of H2O2 on [Ca2+]c, but only at the lower concentrations of the oxidant. H2O2 inhibited amylase secretion in response to cholecystokinin, and cinnamtannin B-1 reduced the inhibitory action of H2O2 on enzyme secretion. Finally, H2O2 reduced cell viability, and the antioxidant protected acinar cells against H2O2. In conclusion, the beneficial effects of cinnamtannin B-1 appear to be mediated by reducing the intracellular Ca2+ overload and intracellular accumulation of digestive enzymes evoked by ROS, which is a common pathological precursor that
mediates pancreatitis. Our results support the beneficial effect of natural antioxidants in the therapy against oxidative
stress-derived deleterious effects on cellular physiology. 相似文献
7.
This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+]o) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas. (Mol Cell Biochem 269: 165–173, 2005) 相似文献
8.
SummaryReactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion (O2?), and hydroxyl radical (OH?) have been implicated in mediating various pathological events such as cancer, atherosclerosis, diabetes, ischemia, inflammatory diseases, and the aging process. The glutathione (GSH) redox cycle and antioxidant enzymes—superoxide dismutase (SOD) and catalase (CAT)—play an important role in scavenging ROS and preventing cell injury. Pycnogenol has been shown to protect endothelial cells against oxidant-induced injury. The present study determined the effects of pycnogenol on cellular metabolism of H2O2 and O2? and on glutathione-dependent and -independent antioxidant enzymes in bovine pulmonary artery endothelial cells (PAEC). Confluent monolayers of PAEC were incubated with pycnogenol, and oxidative stress was triggered by hypoxanthine and xanthine oxidase or H2O2. Pycnogenol caused a concentration-dependent enhancement of H2O2 and O2? clearance. It increased the intracellular GSH content and the activities of GSH peroxidase and GSH disulfide reductase. It also increased the activities of SOD and CAT. The results suggest that pycnogenol promotes a protective antioxidant state by upregulating important enzymatic and nonenzymatic oxidant scavenging systems. 相似文献
9.
S. R. Mallery C. L. Ng-Bautista L. E. Lantry G. M. Ness A. K. Hegtvedt A. Lazo R. T. Bailer B. L. Hout R. E. Stephens G. P. Brierley 《Journal of cellular biochemistry》1994,56(4):568-581
Features of AIDS-related Kaposi's sarcoma (AIDS-KS), such as the multifocal presentation at mucosal and epidermal sites subjected to trauma, suggest that AIDS-KS is initially a reactive hyperplasia that subsequently progresses to a neoplasia. It is recognized that there is an association between sustained states and the subsequent development of neoplasia (e.g., ulcerative colitis/colonic adenocarcinoma). Furthermore, patients who develop AIDS-KS experience both a constant immune stimulation due to sustained high levelsof virus-induced cytokines and, because of a sparing effect on their phagoctic cells, retention of the phagocytic inflammatory response. A component of phygocytic activation is the initiation of the oxidative brust, resulting in the generation of reactive oxygen species (ROS), which can be mutagenic to host cells if released beyond the phagolysosome and not inactivated. Our results demonstrate that cultured AIDS-KS cells possess drcreased cytoprotective capabilities. Relativeto either dermal fibroblasts, or human microvascular endothelial cells (HMECs), AIDS-KS cells contained significantly lower levels of glutathione, a tripeptide integral in both cytoprotection and maintenance of cellular thiol status. While HMECs increased catalase activity during culture in the cytokine-rich KS milieu (control medium supplement with conditioned medium from MOT, an TLV II-infected cell line), AIDS-KS cells demonstrated reduced catalase function under these conditions. Furthermore, HMEC cultures showed in inherent biochemical responsiveness, by increasing catalase activity following exposure to exaogenous H2(O2). In contrast, the catalase activity of AIDS-KS cells decreased following (H2O2) challenge. Our results show that an inherent deficiency in cellular cytoprotection is present in AIDS-KS cells and suggest that oxidant stress may function in the development and progression AIDS-KS. 相似文献
10.
The effect of hydrogen peroxide (H2O2) on cytosolic free calcium concentration ([Ca2+]c) as well as its effect on glutamate secretion in rat hippocampal astrocytes have been the aim of the present research. Our results show that 100 μM H2O2 induces an increase in [Ca2+]c, that remains at an elevated level while the oxidant is present in the perfusion medium, due to its release from intracellular stores as it was observed in the absence of extracellular Ca2+, followed by a significant increase in glutamate secretion. Ca2+-mobilization in response to the oxidant could only be reduced by thapsigargin plus FCCP, indicating that the Ca2+-mobilizable pool by H2O2 includes both endoplasmic reticulum and mitochondria. We conclude that ROS in hippocampal astrocytes might contribute to an elevation of resting [Ca2+]c which, in turn, could lead to a maintained secretion of the excitatory neurotransmitter glutamate, which has been considered a situation potentially leading to neurotoxicity in the hippocampus. 相似文献
11.
Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are constantly generated as by-products of normal metabolic cellular pathways and can be overproduced in response to stress. In this study, we investigated ROS production and localization of H2O2 after salt (200 mM KCl) and osmotic (iso-osmotic sorbitol concentration) stress in the unicellular green alga Micrasterias. By means of the dye H2DCFDA and confocal laser scanning microscopy, most ROS production could be detected in KCl-treated cells when compared to sorbitol-exposed cells and controls. For ultrastructural detection of H2O2, CeCl3, which reacts with H2O2 and produces cerium perhydroxide deposits, has been used. Cerium was identified by transmission electron microscopy (TEM)-coupled electron energy loss spectroscopy (EELS) in organelles of KCl- and sorbitol-treated cells and in controls. Statistical measurements of the presence of the cerium M4,5 edge were performed in mitochondria, chloroplasts, cell walls, and cytoplasmic sites of five individual cells after each treatment. The most pronounced increase in H2O2 production was found in chloroplasts of KCl- and sorbitol-treated cells. This shows that the chloroplast reveals the strongest response in H2O2 production after stress induction in Micrasterias. Significant elevation of H2O2 production also occurred in mitochondria and cytoplasm, whereas H2O2 levels remained unchanged or even slightly decreased in cell walls of treated cells. Additionally, TEM micrographs and EELS analyses provided indirect evidence for an increased H2O2 production at the plasma membrane of KCl-treated cells, indicating an involvement of the plasma membrane NADPH oxidase in H2O2 generation. 相似文献
12.
Jian Sun Xuan Zhang Shurong Deng Chunlan Zhang Meijuan Wang Mingquan Ding Rui Zhao Xin Shen Xiaoyang Zhou Cunfu Lu Shaoliang Chen 《PloS one》2012,7(12)
Extracellular ATP (eATP) has been implicated in mediating plant growth and antioxidant defense; however, it is largely unknown whether eATP might mediate salinity tolerance. We used confocal microscopy, a non-invasive vibrating ion-selective microelectrode, and quantitative real time PCR analysis to evaluate the physiological significance of eATP in the salt resistance of cell cultures derived from a salt-tolerant woody species, Populus euphratica. Application of NaCl (200 mM) shock induced a transient elevation in [eATP]. We investigated the effects of eATP by blocking P2 receptors with suramin and PPADS and applying an ATP trap system of hexokinase-glucose. We found that eATP regulated a wide range of cellular processes required for salt adaptation, including vacuolar Na+ compartmentation, Na+/H+ exchange across the plasma membrane (PM), K+ homeostasis, reactive oxygen species regulation, and salt-responsive expression of genes related to K+/Na+ homeostasis and PM repair. Furthermore, we found that the eATP signaling was mediated by H2O2 and cytosolic Ca2+ released in response to high salt in P. euphratica cells. We concluded that salt-induced eATP was sensed by purinoceptors in the PM, and this led to the induction of downstream signals, like H2O2 and cytosolic Ca2+, which are required for the up-regulation of genes linked to K+/Na+ homeostasis and PM repair. Consequently, the viability of P. euphratica cells was maintained during a prolonged period of salt stress. 相似文献
13.
Abstract: The mechanism for hydrogen peroxide (H2O2)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled PC12 cells. In the presence of butanol, H2O2 caused a great accumulation of [3H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H2O2 of cell lysates exerted no effect on PLD activity. Treatment with H2O2 had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H2O2-induced PLD activity. Thus, H2O2-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H2O2-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H2O2 treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca2+ abolished H2O2-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca2+ potentiated H2O2-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca2+-dependent protein tyrosine kinase(s) somehow participates in H2O2-induced PLD activation in PC12 cells. 相似文献
14.
Maggie K. Diamond-Stanic Elizabeth M. Marchionne Mary K. Teachey David E. Durazo John S. Kim Erik J. Henriksen 《Biochemical and biophysical research communications》2011,(3):2645
Increased cellular exposure to oxidants may contribute to the development of insulin resistance and type 2 diabetes. Skeletal muscle is the primary site of insulin-dependent glucose disposal in the body; however, the effects of oxidative stress on insulin signaling and glucose transport activity in mammalian skeletal muscle are not well understood. We therefore studied the effects of a low-level in vitro oxidant stress (30–40 μM H2O2) on basal and insulin-stimulated (5 mU/ml) glucose transport activity and insulin signaling at 2, 4, and 6 h in isolated rat soleus muscle. H2O2 increased basal glucose transport activity at 2 and 4 h, but not at 6 h. This low-level oxidant stress significantly impaired insulin-stimulated glucose transport activity at all time points, and was associated with inhibition of insulin-stimulated phosphorylation of Akt Ser473 and GSK-3β Ser9. In the presence of insulin, H2O2 decreased total protein expression of IRS-1 at 6 h and IRS-2 at 4 and 6 h. Phosphorylation of p38 MAPK Thr180/Tyr182 was transiently increased by H2O2 in the presence and absence of insulin at 2 and 4 h, but not at 6 h. Selective inhibition of p38 MAPK with A304000 partially rescued the H2O2-induced reduction in insulin-stimulated glucose transport activity. These results indicate that direct in vitro exposure of isolated mammalian skeletal muscle to a low-level oxidant stress impairs distal insulin signaling and insulin-stimulated glucose transport activity, at least in part, due to a p38 MAPK-dependent mechanism. 相似文献
15.
Toshinori Suzuki Asuka Nakamura Michiyo Inukai 《Bioorganic & medicinal chemistry》2013,21(13):3674-3679
Hypobromous acid (HOBr) is formed by eosinophil peroxidase and myeloperoxidase in the presence of H2O2, Cl?, and Br? in the host defense system of humans, protecting against invading bacteria. However, the formed HOBr may cause damage to DNA and its components in the host. When a guanine nucleoside (3′,5′-di-O-acetyl-2′-deoxyguansoine) was treated with HOBr at pH 7.4, spiroiminodihydantoin, guanidinohydantoin/iminoallantoin, dehydro-iminoallantoin, diimino-imidazole, amino-imidazolone, and diamino-oxazolone nucleosides were generated in addition to an 8-bromoguanine nucleoside. The major products were spiroiminodihydantoin under neutral conditions and guanidinohydantoin/iminoallantoin under mildly acidic conditions. All the products were formed in the reaction with HOCl in the presence of Br?. These products were also produced by eosinophil peroxidase or myeloperoxidase in the presence of H2O2, Cl?, and Br?. The results suggest that the products other than 8-bromoguanine may also have importance for mutagenesis by the reaction of HOBr with guanine residues in nucleotides and DNA. 相似文献
16.
Autophagy, a self-eating process, is responsible for degradation of long-lived proteins and damaged cellular proteins/organelles.
Double-membrane autophagosomes, formed during the process, engulf proteins/organelles and fuse with lysosomes to degrade the
contents. It is important to maintain cell homeostasis and many physiological processes including cellular responses to oxidative
stress. Oxidative stress induced by myocardial infarction is a major factor of heart failures. In this study, we examined
how propofol modulates hydrogen peroxide (H2O2)-induced autophagic cell death in H9c2 cardiomyocytes. H2O2 dramatically induced cell death, which was similarly reduced in the presence of either propofol or autophagy inhibitors (e.g.,
wortmannin), suggesting that propofol has a protective effect in H2O2-induced autophagic cell death. Acidic autophagic vacuoles were elevated in H2O2-treated H9c2 cells, but they were largely decreased in the presence of propofol. Furthermore, many autophagy-related proteins
such as LC3-II, ATG proteins, p62, AMPK, and JNK were activated in H2O2-treated H9c2 cells and were significantly deactivated in the presence of propofol. These results show that propofol regulates
oxidative stress-induced autophagic cell death in cardiomyocytes. We further suggest that propofol can act as a cardioprotectant
in heart diseases. 相似文献
17.
Agnes W. Boots Milena Hristova David I. Kasahara Guido R. M. M. Haenen Aalt Bast Albert van der Vliet 《The Journal of biological chemistry》2009,284(26):17858-17867
Activation of the NADPH oxidase homolog dual oxidase 1 (DUOX1) within the airway epithelium represents a key mechanism of innate airway host defense, through enhanced production of H2O2, which mediates cellular signaling pathways that regulate the production of various inflammatory mediators. Production of the CXC chemokine interleukin (IL)-8/CXCL8 forms a common epithelial response to many diverse stimuli, including bacterial and viral triggers, environmental oxidants, and other biological mediators, suggesting the potential involvement of a common signaling pathway that may involve DUOX1-dependent H2O2 production. Following previous reports showing that DUOX1 is activated by extracellular ATP and purinergic receptor stimulation, this study demonstrates that airway epithelial IL-8 production in response to several bacterial stimuli involves ATP release and DUOX1 activation. ATP-mediated DUOX1 activation resulted in the activation of ERK1/2 and NF-κB pathways, which was associated with epidermal growth factor receptor (EGFR) ligand shedding by ADAM17 (a disintegrin and metalloproteinase-17). Although ATP-mediated ADAM17 activation and IL-8 release were not prevented by extracellular H2O2 scavenging by catalase, these responses were attenuated by intracellular scavengers of H2O2 or related oxidants, suggesting an intracellular redox signaling mechanism. Both ADAM17 activation and IL-8 release were suppressed by inhibitors of EGFR/ERK1/2 signaling, which can regulate ADAM17 activity by serine/threonine phosphorylation. Collectively, our results indicate that ATP-mediated DUOX1 activation represents a common response mechanism to several environmental stimuli, involving H2O2-dependent EGFR/ERK activation, ADAM17 activation, and EGFR ligand shedding, leading to amplified epithelial EGFR activation and IL-8 production. 相似文献
18.
Summary Oxidant injury of the vascular endothelium is considered an early event in the pathogenesis of atherosclerosis. The model
of oxidant injury is crucial to the investigation of antioxidants. In the present study, a convenient in vitro model of oxidant
injury induced by hydrogen peroxide (H2O2) was developed using bovine pulmonary artery endothelial cells (PAEC). Viability of PAEC grown in 96-well culture plates
was determined with methylthiazol tetrazolium (MTT) colorimetric assay. Cell membrane integrity was measured by lactate dehydrogenase
(LDH) release from PAEC grown in 24-well plates. Malondialdehyde (MDA, a product of lipid peroxidation) in PAEC grown in 6-well
plates was detected by a thiobarbituric acid fluorometric assay. Incubation of H2O2 with PAEC caused a dose-dependent decrease of cell viability, an increase of LDH release, and an elevation of MDA production.
MTT assay was convenient, quantitative, non-radioactive, and suitable for testing a large number of samples. The fluorometric
assay for measuring MDA production in endothelial cells used 6-well plates instead of 80-cm2 flasks employed by previous investigators. The use of multiwell culture plates in these assays made it possible for more
samples to be tested in any single experiment. The three assays are reproducible with low intraplate and interplate coefficients
of variation. This in vitro model is suitable for screening antioxidants and for studying pharmacodynamics at the cellular
level. 相似文献
19.
Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca2+-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30 °C to 37 °C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca2+ ([Ca2+]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe2+-accumulation following pretreatment with FeSO4. Thus intracellular Fe2+-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe2+-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe2+-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37 °C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe2+-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe2+-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2 channels by Fe2+ may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe2+ is released by heme degradation under intracerebral hemorrhage. 相似文献
20.
Hitoshi Tanigawa Futoshi Toyoda Kosuke Kumagai Noriaki Okumura Tsutomu Maeda Hiroshi Matsuura Shinji Imai 《Purinergic signalling》2018,14(3):245-258
Extracellular ATP regulates various cellular functions by engaging multiple subtypes of P2 purinergic receptors. In many cell types, the ionotropic P2X7 receptor mediates pathological events such as inflammation and cell death. However, the importance of this receptor in chondrocytes remains largely unexplored. Here, we report the functional identification of P2X7 receptor in articular chondrocytes and investigate the involvement of P2X7 receptors in ATP-induced cytotoxicity. Chondrocytes were isolated from rabbit articular cartilage, and P2X7 receptor currents were examined using the whole-cell patch-clamp technique. ATP-induced cytotoxicity was evaluated by measuring caspase-3/7 activity, lactate dehydrogenase (LDH) leakage, and prostagrandin E2 (PGE2) release using microscopic and fluorimetric/colorimetric evaluation. Extracellular ATP readily evoked a cationic current without obvious desensitization. This ATP-activated current was dose related, but required millimolar concentrations. A more potent P2X7 receptor agonist, BzATP, also activated this current but at 100-fold lower concentrations. ATP-induced currents were largely abolished by selective P2X7 antagonists, suggesting a predominant role for the P2X7 receptor. RT-PCR confirmed the presence of P2X7 in chondrocytes. Heterologous expression of a rabbit P2X7 clone successfully reproduced the ATP-induced current. Exposure of chondrocytes to ATP increased caspase-3/7 activities, an effect that was totally abrogated by P2X7 receptor antagonists. Extracellular ATP also enhanced LDH release, which was partially attenuated by the P2X7 inhibitor. The P2X7 receptor-mediated elevation in apoptotic caspase signaling was accompanied by increased PGE2 release and was attenuated by inhibition of either phospholipase A2 or cyclooxygenase-2. This study provides direct evidence for the presence of functional P2X7 receptors in articular chondrocytes. Our results suggest that the P2X7 receptor is a potential therapeutic target in chondrocyte death associated with cartilage injury and disorders including osteoarthritis. 相似文献