169,775 research outputs found
Peroxynitrite mobilizes calcium ions from ryanodine-sensitive stores, a process associated with the mitochondrial accumulation of the cation and the enforced formation of species mediating cleavage of genomic DNA.
No full textPeroxynitrite does not directly cause strand scission of genomic DNA. Rather, as we previously reported, the DNA cleavage is largely mediated by H2O2 resulting from the dismutation of superoxide generated in the mitochondria upon peroxynitrite-dependent inhibition of complex III. The present study demonstrates that this process is strictly controlled by the availability of Ca2+ in the mitochondrial compartment. Experiments using intact as well as permeabilized U937 cells showed that the DNA-damaging response evoked by peroxynitrite is enhanced by treatments causing an increase in mitochondrial Ca2+ uptake and remarkably reduced under conditions leading to inhibition of mitochondrial Ca2+ accumulation. An additional, important observation was that the source of the Ca2+ mobilized by peroxynitrite is the ryanodine receptor; preventing the mobilization of Ca2+ with ryanodine suppressed the mitochondrial formation of reactive oxygen species and the ensuing DNA strand scission. Identical results were obtained using PC12, C6, and THP-1 cells. These results, along with our previous findings indicating that the DNA damage induced by peroxynitrite is also suppressed by inhibition of the electron flow through complex I, e.g., by rotenone, or by the respiration-deficient phenotype, demonstrate that the mitochondrial formation of DNA-damaging species is critically regulated by the inhibition of complex III and by the availability of Ca2+
Different signalling pathways mediate the opposite effects of endogenous versus exogenous nitric oxide on hydroperoxide toxicity in CHP100 neuroblastoma cells
No full textThe results presented in this study indicate that the toxic response brought about by increasing concentrations of tert-butylhydroperoxide in CHP100 cells was mitigated significantly by exogenously added nitric oxide donors via a cyclic GMP-independent mechanism, In contrast with these results, endogenous nitric oxide generated by the Ca2+-mobilizing agent caffeine was found to increase hydroperoxide toxicity. Under these conditions, nitric oxide was not directly toxic to the cells, Rather, nitric oxide was found to promote the caffeine-mediated release of Ca2+ from ryanodine-sensitive Ca2+ stores via a cyclic GMP-independent mechanism. Release of the cation from ryanodine-sensitive Ca2+ stores was causally linked with the caffeine/nitric oxide-mediated enhancement of tert-butylhydroperoxide toxicity, It is concluded that endogenous and exogenous nitric oxide activate diverging signalling pathways independent of cyclic GMP formation and causing opposite effects on the toxic response evoked by tert-butylhydroperoxide in CHP100 cells
Nitric oxide controls fat deposition in dystrophic skeletal muscle by regulating fibro-adipogenic precursor differentiation
No full textDuchenne muscular dystrophy (DMD) is an hereditary disease characterized by loss of muscle fibers and their progressive substitution by fat and fibrous tissue. Mesenchymal fibro-adipogenic progenitors (FAPs) expressing the platelet-derived growth factor receptor alpha (PDGFRα) are an important source of fibrosis and adipogenesis in dystrophic skeletal muscle. Among the therapies suggested for dystrophy are those based on nitric oxide (NO) donating drugs, the administration of which slows disease progression. NO has been shown to act by enhancing the regenerative potential of the diseased muscle. Whether it acts also by inhibiting fibrosis and adipogenesis was not known. Here, we show in vitro that NO regulates FAP fate through inhibition of their differentiation into adipocytes. In mdx mice, an animal model of DMD, treatment with the NO donating drug molsidomine reduced the number of PDGFRα(+) cells as well as the deposition of both skeletal muscle fat and connective tissues. Inhibition of adipogenesis was due to NO-induced increased expression of miR-27b leading to downregulation of peroxisome proliferator-activated receptors gamma (Pparγ1) expression in a pathway independent of cGMP generation. These findings reveal an additional effect of NO in dystrophic muscle that conceivably synergizes with its known effects on regeneration improvement and explain why NO-based therapies appear effective in the treatment of muscular dystrophy
Fat deposition and accumulation in the damaged and inflamed skeletal muscle : cellular and molecular players
No full textThe skeletal muscle has the capacity to repair damage by the activation and differentiation of fiber sub-laminar satellite cells. Regeneration impairment due to reduced satellite cells number and/or functional capacity leads to fiber substitution with ectopic tissues including fat and fibrous tissue and to the loss of muscle functions. Muscle mesenchymal cells that in physiological conditions sustain or directly contribute to regeneration differentiate in adipocytes in patients with persistent damage and inflammation of the skeletal muscle. These cells comprise the fibro-adipogenic precursors, the PW1-expressing cells and some interstitial cells associated with vessels (pericytes, mesoangioblasts and myoendothelial cells). Resident fibroblasts that are responsible for collagen deposition and extracellular matrix remodeling during regeneration yield fibrotic tissue and can differentiate into adipose cells. Some authors have also proposed that satellite cells themselves could transdifferentiate into adipocytes, although recent results by lineage tracing techniques seem to put this theory to discussion. This review summarizes findings about muscle resident mesenchymal cell differentiation in adipocytes and recapitulates the molecular mediators involved in intramuscular adipose tissue deposition
Endogenous and exogenous nitric oxide enhance the DNA strand scission induced by tert-butylhydroperoxide in PC12 cells via peroxynitrite-dependent and independent mechanisms, respectively.
No full textA short-term exposure to tert-butylhydroperoxide (tB-OOH) promoted a concentration-dependent formation of DNA single-strand breaks in PC12 cells. These events were paralleled by an increase in the cytosolic concentration of Ca2+ that was in part cleared by the mitochondria. Unlike the extent of Ca2+ mobilization and/or mitochondrial Ca2+ clearance, the DNA strand scission evoked by the hydroperoxide was markedly reduced by the nitric oxide (NO) scavenger 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (PTIO) or by the NO synthase inhibitor N-nitro-L-arginine methylester (L-NAME). Inhibitors of electron transport (rotenone and myxothiazol), ruthenium red (RR, a polycation which inhibits the calcium uniporter of mitochondria), or peroxynitrite scavengers (Trolox and L-methionine) were as effective as PTIO or L-NAME in inhibiting the DNA-damaging response mediated by tB-OOH. Rotenone, RR or peroxynitrite scavengers did not further reduce the residual DNA cleavage observed following treatment with tB-OOH in L-NAME-supplemented cells. Exogenous NO also increased the DNA damage caused by tB-OOH in L-NAME-supplemented cells and this response was blunted by RR or by inhibitors of electron transport but was insensitive to peroxynitrite scavengers. We conclude that both endogenous and exogenous NO enhance the DNA cleavage generated by tB-OOH in PC12 cells. However, only endogenous NO set the bases for an involvement of peroxynitrite in this DNA-damaging response
Calcium-dependent mitochondrial formation of species mediating DNA single strand breakage in U937 cells exposed to sublethal concentrations of tert-butylhydroperoxide.
No full textTreatment of U937 cells with a sublethal albeit DNA-damaging concentration of tert-butylhydroperoxide (tB-OOH) enhanced mitochondrial Ca++ uptake and ruthenium red (RR), a polycation that inhibits the calcium uniporter of mitochondria, significantly reduced the extent of DNA cleavage generated by the hydroperoxide. Release of Ca++ From the ryanodine(Ry)/caffeine(Cf)-sensitive stores further increased mitochondrial Ca++ uptake and elicited a parallel enhancement in DNA strand scission induced by tB-OOH that was prevented by both Ry and RR. DNA damage caused by tB-OOH alone or associated with either Cf or RR was prevented by iron chelators, insensitive to antioxidants and repaired with kinetics superimposable with those observed after treatment with H2O2. Cf enhanced the DNA-damaging effects of tB-OOH in permeabilized cells as well, and similar effects were observed upon addition of CaCl2. Cf did not further increase the formation of DNA lesions elicited by tB-OOH in the presence of CaCl2. The enhancing effects of Cf were prevented by RR and ryanodine, whereas those mediated by exogenous calcium were prevented only by RR. DNA strand scission caused by tB-OOH alone or associated with Cf in the permeabilized cell system was severely inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid. The mechanism(s) whereby Ca++ promotes the mitochondrial formation of species that will ultimately result in the formation of DNA lesions was subsequently analyzed using intact as well as permeabilized cells. Hydrogen peroxide was identified to be one of these species
Peroxynitrite - An ugly biofactor ?
No full textCellular damage occurring under oxidative conditions has been ascribed mainly to the formation of peroxynitrite (ONOOH/ONOO-) that originates from the reaction of NO• with O2•-. The detrimental effects of peroxynitrite are exacerbated by the reaction with CO2 that leads to ONOOC(O)O-, which further decays to the strong oxidant radicals NO2• and CO3•-. The reaction with CO2, however, may redirect peroxynitrite specificity. An excessive formation of peroxynitrite represents an important mechanism contributing to the DNA damage, the inactivation of metabolic enzymes, ionic pumps, and structural proteins, and the disruption of cell membranes. Because of its ability to oxidize biomolecules, peroxynitrite is implicated in an increasing list of diseases, including neurodegenerative and cardiovascular disorders, inflammation, pain, autoimmunity, cancer, and aging. However, peroxynitrite displays also protective activities: (i) at high concentrations, it shows anti-viral, anti-microbial, and anti-parasitic actions; and (ii) at low concentrations, it stimulates protective mechanisms in the cardiovascular, nervous, and respiratory systems. The detrimental effects of peroxynitrite and related reactive species are impaired by (pseudo-) enzymatic systems, mainly represented by heme-proteins (e.g., hemoglobin and myoglobin). Here, we report biochemical aspects of peroxynitrite actions being at the root of its biomedical effects. Copyrigh
The mechanism of the nitric oxide-mediated enhancement of tert-butylhydroperoxide-induced DNA single strand breakage.
No full text1 Caffeine (Cf) enhances the DNA cleavage induced by tert-butylhydroperoxide (tB-OOH) in U937 cells via a mechanism involving Ca2+-dependent mitochondrial formation of DNA-damaging species (Guidarelli el al., 1997b). Nitric oxide (NO) is not involved in this process since U937 cells do not express the constitutive nitric oxide synthase (cNOS).
2 Treatment with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP, 10 mu M), or S-nitrosoglutathiane (GSNO, 300 mu M), however, potentiated the DNA strand scission induced by 200 mu M tB-OOH. The DNA lesions generated by tB-OOH alone, or combined with SNAP, were repaired with superimposable kinetics and were insensitive to anti-oxidants and peroxynitrite scavengers but suppressed by iron chelators.
3 SNAP or GSNO did not cause mitochondrial Ca2+ accumulation but their enhancing effects on the tB-OOH-induced DNA strand scission were prevented by ruthenium red, an inhibitor of the calcium uniporter of mitochondria. Furthermore, the enhancing effects of both SNAP and GSNO were identical to and not additive with those promoted by the Ca2+-mobilizing agents Cf or ATP.
4 The SNAP- or GSNO-mediated enhancement of the tB-OOH-induced DNA cleavage was abolished by the respiratory chain inhibitors rotenone and myxothiazol and was not apparent in respiration-deficient cells.
5 It is concluded that, in cells which do not express the enzyme cNOS, exogenous NO enhances the accumulation of DNA single strand breaks induced by tB-OOH via a mechanism involving inhibition of complex III
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