Effect of anti-fungal imidazoles on mRNA 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 micrograms 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 micrograms ml-1) and either dexamethasone (10 microM) or NG-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 micrograms ml-1) and either clotrimazole, econazole or miconazole (1-10 microM) 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 micrograms 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 microM) but was reduced significantly by dexamethasone (10 microM). 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.
Bogle
R G
RG
Department of Pharmacology and Clinical Pharmacology, St. George's Hospital Medical School, London.
Whitley
G S
GS
Soo
S C
SC
Johnstone
A P
AP
Vallance
P
P
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D003022:Clotrimazole / Q000494:pharmacology*; D004464:Econazole / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008825:Miconazole / Q000494:pharmacology*; D019001:Nitric Oxide Synthase; D009573:Nitrites / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*
D009573:Nitrites; D012333:RNA, Messenger; D004464:Econazole; D008825:Miconazole; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D003022:Clotrimazole