Positive modulation by Ras of interleukin-1beta-mediated nitric oxide generation in insulin-secreting clonal beta (HIT-T15) cells.
In the present study, we have shown that exposure of insulin-secreting clonal beta (HIT-T15) cells to interleukin-1beta (IL-1beta) results in a time- and concentration-dependent increase in nitric oxide (NO) release. These effects by IL-1beta on NO release were mediated by induction of inducible nitric oxide synthase (iNOS) from the cells. Preincubation of HIT cells with Clostridium sordellii lethal toxin-82, which irreversibly glucosylates and inactivates small G-proteins, such as Ras, Rap, Ral, and Rac, but not Cdc42, completely abolished IL-1beta-induced NO release. Pre-exposure of HIT cells to C. sordellii lethal toxin-9048, which monoglucosylates and inhibits Ras, Cdc42, Rac, and Rap, but not Ral, also attenuated IL-1beta-mediated NO release. These data indicate that activation of Ras and/or Rac may be necessary for IL-1beta-mediated NO release. Preincubation of HIT cells with C. difficile toxin-B, which monoglucosylates Rac, Cdc42, and Rho, had no demonstrable effects on IL-mediated NO release, ruling out the possibility that Rac may be involved in this signaling step. Further, two structurally dissimilar inhibitors of Ras function, namely manumycin A and damnacanthal, inhibited, in a concentration-dependent manner, the IL-1beta-mediated NO release from these cells. Together, our data provide evidence, for the first time, that Ras activation is an obligatory step in IL-1beta-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.
Tannous
M
M
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, 619 Shapero Hall, Wayne State University, Detroit, MI 48202, USA.
Amin
R
R
Popoff
M R
MR
Fiorentini
C
C
Kowluru
A
A
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007375:Interleukin-1 / Q000494:pharmacology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D013997:Time Factors; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D007328:Insulin; D007375:Interleukin-1; D009569:Nitric Oxide; D020744:rac GTP-Binding Proteins; D018631:ras Proteins; D020741:rho GTP-Binding Proteins