CAMP inhibits inducible nitric oxide synthase expression and NF-KB-Bindingactivity in cultured rat hepatocytes

Background. The inducible nitric oxide synthase, (iNOS) is strongly express ed following inflammatory stimuli. Adenosine 3 ' ,5 ' -cyclic monophosphate (cAMP) increases iNOS expression and activity in a number of cell types bu t decreases cytokine-stimulated iNOS expression in hepatocytes. The mechani sms for this effect are unknown. Methods. Rat hepatocytes were stimulated with cytokines to induce iNOS and cultured with cAMP agonists dibutyryl-cAMP (dbcAMP), 8-bromo-cAMP, and fors kolin (FSK). Nitric oxide synthesis was assessed by supernatant nitrite lev els and iNOS expression was measured by Northern and Western blot analyses. Nuclear factor kappaB binding was assessed by electromobility shift assay. Results. Cyclic AMP dose dependently decreased NO synthesis in response to a combination of proinflammatory cytokines or interleukin-1 beta (IL-1 beta ) alone. The adenylate cyclase inhibitor SQ 22,536 increased cytokine- or I L-1 beta -stimulated NO synthesis. dbcAMP decreased iNOS mRNA expression an d iNOS protein expression. Both dbcAMP and glucagon decreased iNOS promoter activity in rat hepatocytes transfected with the murine iNOS promoter and decreased DNA binding of the transcription factor NF-kappaB. Conclusion. These data suggest that cAMP is important in hepatocyte iNOS ex pression and agents that alter cAMP levels may profoundly alter the respons e of hepatocytes to inflammatory stimuli through effects on the iNOS promot er region and NF-kappaB (C) 2001 Academic Press.