TETRACYCLINE INHIBITS THE NITRIC-OXIDE SYNTHASE ACTIVITY-INDUCED BY ENDOTOXIN IN CULTURED MURINE MACROPHAGES

Here we investigate the effects of tetracycline base and of a semi-syn thetic tetracycline derivative, doxycycline, on the induction of induc ible nitric oxide synthase and, hence, on the production of nitric oxi de (NO) by lipopolysaccharide in J774 macrophage cultured in vitro. Th e treatment of J774 line with tetracycline base (6.25-250 mu M) or dox ycycline (5-50 mu M) dose-dependently decreased the lipopolysaccharide -stimulated (1 mu g/ml) inducible NO synthase activity and, consequent ly, nitrite formation. For instance, the inhibition was 70% for tetrac ycline base at 250 mu M and 68% for doxycycline at 50 mu M. The inhibi tory effect of tetracyclines was due neither to a reduction in the via bility of the cells, studied as colorimetric -[4,5-dimethylthiazol-2yl ]-2,5-diphenyltetrazolium bromide (MTT) reduction assay, nor to an ind iscriminate inhibition of total protein synthesis, but to a specific d ecrease in inducible NO synthase protein content in the cells, as atte sted by the significant reduction of the expression of inducible NO sy nthase, assayed by sodium-dodecyl sulphate-polyacrylamide gel electrop horesis (SDS-PAGE) and Western blot. However, no effect of tetracyclin es on inducible NO synthase mRNA accumulation could be demonstrated in lipopolysaccharide-stimulated macrophage line, suggesting that the in hibitory effect of tetracyclines on NO synthesis involves post-transcr iptional events. The reduction in lipopolysaccharide-stimulated nitrit e accumulation produced by tetracyclines was significantly less when t hey were applied 6 h after lipopolysaccharide and absent 12 h after li popolysaccharide, indicating that tetracyclines modify an early event in inducible NO synthase activation operating after mRNA transcription . The findings presented in this study indicate that the modulation of NO synthesis is another possible pathway by which tetracyclines may f unction as anti-inflammatory compounds. (C) 1998 Elsevier Science B.V.