Inhibition of the cytokine-mediated inducible nitric oxide synthase expression in rat insulinoma cells by phenyl N-tert-butylnitrone

Cytokines and nitric oxide (NO) have been implicated in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). We have shown that the spin-trapping agent phenyl N-tert-butylnitrone (PBN) protects against streptozotocin (STZ)-induced IDDM in mice. In order to gain more insights into the mechanism(s) of the protective action of PBN against IDDM, we have investigated the effect of this compound on the cytokine-induced NO generation (measured as nitrite) in rat insulinoma RIN-SF cells. Our results demonstrate that PEN cotreatment prevents the generation of nitrite by RIN-5F cells induced by treatment with tumor necrosis factor-alpha, interleukin 1 beta, and interferon-gamma in a dose-dependent fashion. The generation of NO as a result of cytokine treatment and the inhibitory effect of PEN were further confirmed by electron paramagnetic resonance spectroscopy. Aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), abolished the cytokine-induced nitrite generation whereas N-nitro-L-arginine, an inhibitor more selective for other NOS isoforms, was significantly less effective. Western and Northern analyses demonstrated that PEN inhibits the cytokine-mediated expression of iNOS at the transcriptional level. Cytokine-induced nitrite formation was also inhibited by the two antioxidant agents alpha-lipoic acid and N-acetylcysteine. These results indicate that PBN protects against IDDM at least in part by prevention of cytokine-induced NO generation by pancreatic beta-cells. (C) 2000 Academic Press.