Abstract
To investigate the role of the cytochrome P-450 system in NO synthesis, cytochrome P-450IIIA, IIE and IA activities were specifically inhibited by cimetidine (IIIA), clotrimazole (IIIA), benzoflavone (IA) and disulfiram (IIE) in a model of cultured rat hepatocytes. Cytokine-induced NO synthesis was significantly decreased in the presence of cimetidine and clotrimazole. Kinetic analysis revealed a non-competitive mode of inhibition ( K i = 21 mM, cimetidine; K i = 13 μ M, clotrimazole). Reverse transcriptase-PCR and immunoblot analysis revealed no significant change in steady state levels of iNOS mRNA and protein expression with P-450IIIA inhibition. Purified iNOS enzyme activity was not altered. These data suggest that cytokinemediated hepatocyte synthesis of NO is dependent upon P-450IIIA activity, which functions in a post-translational capacity.