Nitrite‐mediated protection against hypochlorous acid–induced chondrocyte toxicity: A novel cytoprotective role of nitric oxide in the inflamed joint?

Whiteman, Matthew; Rose, Peter; Siau, Jia Ling; Halliwell, Barry

doi:10.1002/art.11284

Cited by 38 publications

(32 citation statements)

References 91 publications

(166 reference statements)

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“…Chondrocytes were treated with authentic ONOO -(50 µM; A), SIN-1 (1 mM; B), or staurosporine (0.5 µM; C) cell lysates collected at the times stated, and caspase activity was measured as described in Materials and Methods by using fluorescent substrates (53,54). Chondrocytes were treated with various caspase inhibitors (55) prior to ONOO -/SIN-1 addition, and cell death was analyzed by MTT reduction and LDH leakage (D) and the formation of subG1 cell populations (E) measured after 18 h. The effects of ONOO -(50 µM) and SIN-1 (1 mM) on caspase activity in staurosporine (0.5 µM)-treated cells (F). Experiments were conducted as described in Materials and Methods, and data are expressed as mean ± standard deviation of six or more separate experiments.…”

Section: Discussionmentioning

confidence: 99%

Peroxynitrite mediates calcium‐dependent mitochondrial dysfunction and cell death via activation of calpains

et al. 2004

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Chondrocyte cell death is a hallmark of inflammatory and degenerative joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), but the molecular and cellular mechanisms involved have yet to be elucidated. Because 3-nitrotyrosine, a marker for reactive nitrogen species such as peroxynitrite, has been observed in OA and RA cartilage and has been associated with chondrocyte cell death, we investigated the mechanisms by which peroxynitrite induces cell death in human articular chondrocytes. The earliest biochemical event observed, subsequent to treatment with either peroxynitrite or the peroxynitrite generator SIN-1, was a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death. Although, chondrocyte death exhibited several classical hallmarks of apoptosis, including annexin V labeling, increased fraction of cells with subG1 DNA content and DNA condensation, we did not find evidence for caspase involvement either by Western blotting, fluorimetric assays, or caspase inhibition. Additionally, peroxynitrite did not inhibit cellular caspase activity. Furthermore, using other established assays of cell viability, including the MTT assay and release of lactate dehydrogenase, we found that the predominant mode of cell death involved calcium-dependent cysteine proteases, otherwise known as calpains. Our data show, for the first time, that peroxynitrite induces mitochondrial dysfunction in cells via a calcium-dependent process that leads to caspase-independent apoptosis mediated by calpains.

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Section: Discussionmentioning

confidence: 99%

Peroxynitrite mediates calcium‐dependent mitochondrial dysfunction and cell death via activation of calpains

et al. 2004

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“…All samples were assayed in duplicate and results show insulin concentration in mg/ml. Cellular ATP, glutathione (GSH) and cell viability assay Intracellular ATP and GSH were assayed as described previously (Whiteman et al 2003). Briefly, cells were seeded in 24-well plates at a density of 0 .…”

Section: Insulin Assaymentioning

confidence: 99%

Hydrogen sulphide reduces insulin secretion from HIT-T15 cells by a KATP channel-dependent pathway

Ali¹,

Whiteman²,

Low³

et al. 2007

Journal of Endocrinology

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Hydrogen sulphide (H 2 S), a naturally occurring gas exerts physiological effects by opening K ATP channels. Anti-diabetic drugs (e.g. glibenclamide) block K ATP channels and abrogate H 2 S-mediated physiological responses which suggest that H 2 S may also regulate insulin secretion by pancreatic b-cells. To investigate this hypothesis, insulin-secreting (HIT-T15) cells were exposed to NaHS (100 mM) and the K ATP channeldriven pathway of insulin secretion was tracked with various fluorescent probes. The concentration of insulin released from HIT-T15 cells decreased significantly after NaHS exposure and this effect was reversed by the addition of glibenclamide (10 mM). Cell viability and intracellular ATP and glutathione (GSH) levels remained unchanged, suggesting that changes in insulin secretion were not ATP linked or redox dependent. Through fluorescence imaging studies, it was found that K C efflux occurs in cells exposed to NaHS. The hyperpolarised cell membrane, a result of K C leaving the cell, prevents the opening of voltage-gated Ca 2C channels. This subsequently prevents Ca 2C influx and the release of insulin from HIT-T15 cells. This data suggest that H 2 S reduces insulin secretion by a K ATP channel-dependent pathway in HIT-T15 cells. This study reports the molecular mechanism by which H 2 S reduces insulin secretion and provides further insight into a recent observation of increased pancreatic H 2 S production in streptozotocin-diabetic rats.

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“…Cells were seeded overnight at a density of 4.0 · 10 4 cells/well in 96-well microplates (Rose et al 2003;Whiteman et al 2003b). ONOO --mediated intracellular protein nitration (Cambridge Biosciences) and protein oxidation (Zenith Technology) were performed using commercial ELISAs as described (Buss et al 1997;Whiteman et al 2003b). …”

Section: Measurement Of Onoo --Induced Cytotoxicitymentioning

confidence: 99%

The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’?

Whiteman¹,

Armstrong²,

Chu³

et al. 2004

Journal of Neurochemistry

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387

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Hydrogen sulfide (H 2 S) is a well-known cytotoxic gas. Recently it has been shown to stimulate N-methyl-D-aspartate (NMDA) receptors to enhance long-term potentiation suggesting a novel neuromodulatory role in vivo. Endogenous levels of H 2 S in the brain are reported to range between 10 and 160 lM. Considerably lower H 2 S levels are reported in the brains of Alzheimer's disease (AD) patients, where levels of brain protein nitration (probably mediated by peroxynitrite) are markedly increased. Activation of NMDA receptors leads to intracellular tyrosine nitration by peroxynitrite. Because H 2 S and peroxynitrite are important mediators in brain function and disease, we investigated the effects of the H 2 S 'donor', sodium hydrogen sulfide (NaSH) on peroxynitritemediated damage to biomolecules and to cultured human SH-SY5Y cells. H 2 S significantly inhibited peroxynitrite-mediated tyrosine nitration and inactivation of a 1 -antiproteinase to a similar extent to reduced glutathione at each concentration tested (30-250 lM). H 2 S also inhibited peroxynitrite-induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH-SY5Y cells. These data suggest that H 2 S has the potential to act as an inhibitor of peroxynitrite-mediated processes in vivo and that the potential antioxidant action of H 2 S deserves further study, given that extracellular GSH levels in the brain are very low.

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Nitrite‐mediated protection against hypochlorous acid–induced chondrocyte toxicity: A novel cytoprotective role of nitric oxide in the inflamed joint?

Cited by 38 publications

References 91 publications

Peroxynitrite mediates calcium‐dependent mitochondrial dysfunction and cell death via activation of calpains

Peroxynitrite mediates calcium‐dependent mitochondrial dysfunction and cell death via activation of calpains

Hydrogen sulphide reduces insulin secretion from HIT-T15 cells by a KATP channel-dependent pathway

The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’?

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