Cytokine- or chemically derived nitric oxide alters the expression of proteins detected by two-dimensional gel electrophoresis in neonatal rat isletsof Langerhans

Interleukin-1 beta (IL-1 beta) treatment of neonatal rat islets for 24 h in duces changes in the expression of 105 of 2,200 proteins, as determined pre viously by two-dimensional (2D) gel electrophoresis, Nitric oxide (NO) has been implicated as one of the mediators of IL-1 beta effects in insulin-con taining cell lines and rat islets, The aims of this study were 1) to determ ine the involvement of NO in IL-1 beta -induced alterations in protein expr ession and 2) to investigate the effects of chemically generated NO on prot ein expression by 2D gel electrophoresis of neonatal rat islet samples. IL- 1 beta -induced NO production was prevented by incubation of islets in argi nine-free medium supplemented with the arginine analog N-G-nitro-L-arginine . [S-35]methionine-labeled islet proteins were separated using 2D gel elect rophoresis and analyzed using the BioImage computer program. Analysis revea led that the expression Levels of 23 protein spots of the 105 protein spots , altered by prior treatment with IL-1 beta (60 U/ml) alone, were significa ntly affected (P < 0.01 [n = 4] and P < 0.05 [n = 19]) when NO production w as prevented. The effects of chemically generated NO were investigated by e xposing islets to the NO donor GSNO (100 mu mol/l) for 24 h before labeling with [S-35]methionine and 2D gel electrophoresis, Computer-based analysis identified alterations in the expression of 19 of a total of 1,600 detectab le proteins in GSNO-treated islets (P < 0.01). We conclude 1) that the expr ession of up to 42 proteins is altered by cytokine-induced or chemically ge nerated NO in the precise experimental conditions chosen and 2) that the ma jority of proteins altered by prior treatment with IL-1<beta> may be the re sult of NO-independent IL-1 beta -mediated regulation of gene expression, T his study demonstrates that the combination of 2D gel electrophoresis and m ass spectrometry is a powerful tool in the identification of beta -cell pro teins involved in the response to toxic mediators.