Abstract
The purpose of this study was to determine the reactive species that is responsible for cytokine-mediated β-cell death. Inhibitors of inducible nitric oxide synthase prevent this death, and addition of exogenous nitric oxide using donors induces β-cell death. The reaction of nitric oxide with superoxide results in the generation of peroxynitrite, and this powerful oxidant has been suggested to be the mediator of β-cell death in response to cytokine treatment. Recently, coumarin-7-boronate has been developed as a probe for the selective detection of peroxynitrite. Using this reagent, we show that addition of the NADPH oxidase activator phorbol 12-myristate 13-acetate to nitric oxide-producing macrophages results in peroxynitrite generation. Using a similar approach, we demonstrate that cytokines fail to stimulate peroxynitrite generation by rat islets and insulinoma cells, either with or without phorbol 12-myristate 13-acetate treatment. When forced to produce superoxide using redox cyclers, this generation is associated with protection from nitric oxide toxicity. These findings indicate that: (i) nitric oxide is the likely mediator of the toxic effects of cytokines, (ii) β-cells do not produce peroxynitrite in response to cytokines, and (iii) when forced to produce superoxide, the scavenging of nitric oxide by superoxide is associated with protection of β-cells from nitric oxide-mediated toxicity.
Highlights
The species that mediates cytokine-induced -cell death, be it nitric oxide or peroxynitrite, is unknown
The damaging actions of cytokines on INS832/13 cells are not modified in the presence of the peroxynitrite scavenger phenylalanine boronate [40]; inhibition of inducible nitric oxide synthase (iNOS) activity with L-NG-monomethyl arginine (L-NMMA) completely prevents the loss of INS832/13 cell viability in response to IL-1 ϩ IFN␥ (Fig. 1, A and B)
These findings indicate that a product of iNOS mediates the loss of -cell viability in response to cytokine treatment and that a scavenger of peroxynitrite does not prevent this damage
Summary
The species that mediates cytokine-induced -cell death, be it nitric oxide or peroxynitrite, is unknown. Results: Cytokines stimulate the formation of nitric oxide but fail to stimulate peroxynitrite production by -cells. The reaction of nitric oxide with superoxide results in the generation of peroxynitrite, and this powerful oxidant has been suggested to be the mediator of -cell death in response to cytokine treatment. Coumarin-7-boronate has been developed as a probe for the selective detection of peroxynitrite. Using this reagent, we show that addition of the NADPH oxidase activator phorbol 12-myristate 13-acetate to nitric oxide-producing macrophages results in peroxynitrite generation. We demonstrate that cytokines fail to stimulate peroxynitrite generation by rat islets and insulinoma cells, either with or without phorbol 12-myristate 13-acetate treatment. These findings indicate that: (i) nitric oxide is the likely mediator of the toxic effects of cytokines, (ii) -cells do not produce peroxynitrite in response to cytokines, and (iii) when forced to produce superoxide, the scavenging of nitric oxide by superoxide is associated with protection of -cells from nitric oxide-mediated toxicity
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