Exogenous nitric oxide (NO) generation or IL-1 beta-induced intracellular NO production stimulates inhibitory auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase in RINm5F cells.
Nitric oxide (NO) stimulates the auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which results in the inhibition of enzyme activity. In the present work we show that addition of exogenous NO or IL-1 beta-induced intracellular NO generation cause GAPDH ADP-ribosylation and inhibition of enzyme activity. Incubation of RINm5F cells with sodium nitroprusside (SNP) for 18 h caused a time- and dose-dependent inhibition of GAPDH activity. Half-maximal inhibition of GAPDH activity was observed with 80 microM of the NO donor, with maximal inhibition after roughly 6 h of incubation. In parallel, SNP induced endogenous ADP-ribosylation of GAPDH measured by a decreased incorporation of [32P]ADP-ribose from [32P]NAD+ in the cytosol of the SNP-treated cells. Stimulation of endogenous NO production by inducing the NO synthase by exposure to the cytokine IL-1 beta results in decreased GAPDH activity. IL-1 beta (10(-9) M) inhibited GAPDH activity about 55%, compared with control values. Production of nitrite and inhibition of GAPDH was reversed by the NO synthase inhibitor NG-monomethyl-L-arginine, indicating that endogenous generated NO was the effective molecule. Again, GAPDH inhibition was associated with NO-stimulated endogenous ADP-ribosylation of the enzyme. Western blot analysis of GAPDH excluded degradation of GAPDH by NO. NO-stimulated auto-ADP-ribosylation resulted in inhibition of the glycolytic enzyme GAPDH and may be relevant as a cytotoxic effect of NO. In concert with its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain NO may mediate autocytotoxic effect in beta-cells.
Dimmeler
S
S
Universität Konstanz, Fakultät für Biologie, Germany.
Ankarcrona
M
M
Nicotera
P
P
Brüne
B
B
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D007340:Insulinoma / Q000201:enzymology* / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology*; D051381:Rats; D014407:Tumor Cells, Cultured
D007375:Interleukin-1; D000246:Adenosine Diphosphate Ribose; D009569:Nitric Oxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases