EFFECT OF ANTIFUNGAL IMIDAZOLES ON MESSENGER-RNA LEVELS AND ENZYME-ACTIVITY OF INDUCIBLE NITRIC-OXIDE SYNTHASE

1 Experiments were performed to examine the effects of anti-fungal imidazole compounds (clotrimazole, econazole and miconazole) on the induction of nitric oxide (NO) synthase and subsequent production of NO in the cultured murine monocyte/macrophage cell line J774 using a specific cDNA probe for inducible NO synthase mRNA and by monitoring nitrite production. 2 Stimulation of J774 cells with lipopolysaccharide (LPS, 10 mug ml-1) resulted in the induction of NO synthase activity as determined by nitrite accumulation in the culture medium (48 +/- 3 nmol per 10(6) cells over 24 h). Production of nitrite was inhibited by co-incubation of cells with LPS (10 mug ml-1) and either dexamethasone (10 muM) or N(G)-monomethyl-L-arginine (L-NMMA; 0.1 mM), however, only L-NMMA was an effective inhibitor of nitrite production when added after induction of NO synthase had occurred. 3 Co-incubation of J774 cells with LPS (10 mug ml-1) and either clotrimazole, econazole or miconazole (1-10 muM) resulted in a concentration-dependent inhibition of nitrite production over the subsequent 24 h without any evidence for a cytotoxic effect. However, addition of these imidazoles after induction of NO synthase did not inhibit nitrite production. 4 Messenger RNA for inducible NO synthase was not detected in unstimulated J774 cells. Treatment with LPS (10 mug ml-1) for 4 h resulted in significant expression of mRNA for inducible NO synthase which was not altered in the presence of econazole (10 muM) but was reduced significantly by dexamethasone (10 muM). 5 These results demonstrate that anti-fungal imidazoles inhibit the production of nitric oxide by cultured J774 cells by a mechanism which appears to differ from that of dexamethasone and substrate-type inhibitors of NO synthase. Furthermore, the presence of mRNA for NO synthase does not indicate the presence of functionally active NO synthase.