Positive modulation by Ras of interleukin-1 beta-mediated nitric oxide generation in insulin-secreting clonal beta (HIT-T15) cells

In the present study, we have shown that exposure of insulin-secreting clon al beta (HIT-T15) cells to interleukin-lo (IL-1 beta) results in a time- an d concentration-dependent increase in nitric oxide (NO) release. These effe cts by IL-1 beta on NO release were mediated by induction of inducible nitr ic oxide synthase (iNOS) from the cells. Preincubation of HIT cells with Cl ostridium sordellii lethal toxin-82, which irreversibly glucosylates and in activates small G-proteins, such as Ras, Rap, Ral, and Rac, but not Cdc42, completely abolished IL-1 beta -induced NO release. Pre-exposure of HIT cel ls to C. sordellii lethal toxin-9048, which monoglucosylates and inhibits R as, Cdc42, Rac, and Rap, but not Ral, also attenuated IL-1 beta -mediated N O release. These data indicate that activation of Ras and/or Rac may be nec essary for IL-1 beta -mediated NO release. Preincubation of HIT cells with C. difficile toxin-B, which monoglucosylates Rac, Cdc42, and Rho, had no de monstrable effects on IL-mediated NO release, ruling out the possibility th at Rac may be involved in this signaling step. Further, two structurally di ssimilar inhibitors of Ras function, namely manumycin A and damnacanthal, i nhibited, in a concentration-dependent manner, the IL-1 beta -mediated NO r elease from these cells. Together, our data provide evidence, for the first time, that Ras activation is an obligatory step in IL-1 beta -mediated NO release and, presumably, the subsequent dysfunction of the pancreatic beta cell. Our data also provide a basis for future investigations to understand the mechanism of cytokine-induced beta cell death leading to the onset of insulin-dependent diabetes mellitus. (C) 2001 Elsevier Science Inc. All rig hts reserved.