Inhibition of glucocorticoid receptor binding by nitric oxide

Septic shock is a dangerous condition with high mortality rates. In sepsis, the inducible form of nitric oxide (NO) synthase is induced, releasing hig h amounts of NO. Glucocorticoids have potent anti-inflammatory properties a nd are very effective in inhibiting the induction of this enzyme if adminis tered before the shock onset. It is known that glucocorticoid receptor (GR) has critical cysteine residues for steroid binding in its hormone-binding and DNA-binding domains. It has also been reported that NO reacts with -SH groups, forming S-nitrosothiols. Therefore, we examined the potential effec t of NO on the ligand-binding ability of GR. NO donors (S-nitroso-acetyl-DL -penicillamine, S-nitroso-DL-penicillamine, or S-nitroso-glutathione) decre ased, in a time- and dose-dependent manner, the binding of [H-3]triamcinolo ne to immunoprecipitated GR from mouse L929 fibroblasts. The nonnitrosylate d parent molecules, N-acetyl-DL-penicillamine, and reduced gluthatione were without effect. Scatchard plots revealed that the number of ligand binding sites and K-d were reduced (50%) by NO donors. Western blot analysis ruled out the possibility that dissociation of GR/heat shock protein 90 heteroco mplex or decrease in GR protein would account for the inhibitory effect of NO. Decreased ligand binding to GR was found when NO donors were incubated with intact fibroblasts. Incubation with NO donors also decreased the stero id-induced reduction in [3H]uridine incorporation into RNA. All of these NO effects were inhibited by the thiol-protecting agent dithiothreitol. There fore, S-nitrosylation of critical -SH groups in GR by NO with consequent de creases in binding and affinity may be the mechanisms which explain the fai lure of glucocorticoids to exert their antiinflammatory effects in septic s hock.