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-tr apping agent phenyl N-tert-butylnitrone (PBN) protects against streptozotoc in (STZ)-induced IDDM in mice. In order to gain more insights into the mech anism(s) of the protective action of PBN against IDDM, we have investigated the effect of this compound on the cytokine-induced NO generation (measure d 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 inte rferon-gamma in a dose-dependent fashion. The generation of NO as a result of cytokine treatment and the inhibitory effect of PEN were further confirm ed by electron paramagnetic resonance spectroscopy. Aminoguanidine, a selec tive inhibitor of inducible nitric oxide synthase (iNOS), abolished the cyt okine-induced nitrite generation whereas N-nitro-L-arginine, an inhibitor m ore selective for other NOS isoforms, was significantly less effective. Wes tern and Northern analyses demonstrated that PEN inhibits the cytokine-medi ated expression of iNOS at the transcriptional level. Cytokine-induced nitr ite formation was also inhibited by the two antioxidant agents alpha-lipoic acid and N-acetylcysteine. These results indicate that PBN protects agains t IDDM at least in part by prevention of cytokine-induced NO generation by pancreatic beta-cells. (C) 2000 Academic Press.