INDUCTION OF NITRIC-OXIDE SYNTHASE IN HUMAN VASCULAR SMOOTH-MUSCLE - INTERACTIONS BETWEEN PROINFLAMMATORY CYTOKINES

Objective: We have attempted to demonstrate the induction of inducible nitric oxide synthase in human vascular tissue and define the capacit y of different cytokines to induce this enzyme. Methods: Segments of h uman arteries were stimulated with lipopolysaccharide (10 mu g/ml), in terleukin-1 beta (5 U/ml), tumor necrosis factor-alpha (10 U/ml), and interferon-gamma (200 U/ml). Cytokines were either used alone or in ce rtain combinations, as well as in the presence of L-N-G-monomethyl-arg inine (100 mu mol/l) or cycloheximide (1 mu mol/l). Induction was asse ssed by measurement of mRNA expression, immunocytochemical localisatio n of the expressed protein, nitric oxide synthase activity and levels of nitrite, a product of nitric oxide formation. Results: PCR analysis showed the presence of mRNA for iNOS in stimulated samples which coul d be inhibited by cycloheximide. There was positive staining with an a ntibody against human iNOS in the media of stimulated vessel segments. Stimulated segments were also shown to contain Ca2+-independent nitri c oxide synthase activity. The cytokines and Lipopolysaccharide togeth er gave a significant rise in levels of nitrite in the medium after 36 and 48 h, which was inhibited by L-N-G-monomethyl-arginine and cycloh eximide. Only interferon-gamma incubated alone was capable of increasi ng nitrite levels. This effect was enhanced by co-incubation with eith er interleukin-1 beta, tumor necrosis factor-alpha or lipopolysacchari de. Conclusion: We have shown that increased production of nitrite by human vascular tissue in response to cytokines is associated with indu ction of iNOS as shown at the molecular and protein levels, and furthe r supported by the presence of increased Ca2+-independent nitric oxide synthase activity following cytokine stimulation. (C) 1998 Elsevier S cience B.V. All rights reserved.