Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

I We have examined whether modulation of the polyamine biosynthetic pathway , through inhibition by alpha-difluoromethylornithine (DFMO) of the rate li miting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J77 4 macrophages. 2 DFMO potentiated LPS-stimulated nitrite production in both a concentratio n- and time-dependent manner, increasing nitrite levels by 48+/-5% at 10 mM . This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production. 3 Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10 mM) to potentiate LPS-indu ced NO synthesis. 4 Metabolism of L-[H-3]arginine to L-[H-3]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by eit her DFMO (1-10 mM) or by putrescine (0.001-1 mM), spermidine (0.001-1 mM) o r spermine (0.001-1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70+/-0.07%) by L-NMMA (100 mu M). 5 Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (similar to 2 fold) iNOS protein expression. 6 These results show that DFMO potentiates LPS-induced nitrite production i n the murine macrophage cell line J774. Since the only known mechanism of a ction of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we con clude that expression of iNOS can be critically regulated by endogenous pol yamines.