Induction of glutathione peroxidase in response to inactivation by nitric oxide

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatme nt of lipopolysaccharide (LPS, 1 mug/ml) and tumor necrosis factor-alpha (T NF-alpha, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inh ibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and a rginase. iNOS expression was detected by RT-PCR in the treated cells. GPX w as inactivated irreversibly when the cells had been homogenized before expo sure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNT-ce exerted a transient effect on the GPX activity . The treatment with SNAP (200 muM) or sodium nitroprusside (200 muM) enhan ced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4, 5,5,-tetramethyhmidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-alpha, and aminoguanidine blocked the effec t. In cells metabolically labeled with Se-75, LPS + TNF-alpha accelerated t he incorporation of radioactivity into GPX molecule by 2.1-fold. These resu lts suggest that inactivation of GPX by NO triggers a signal for inducing G PX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.