Nitric oxide is a recently discovered biomolecule with a broad range of actions. The present study investigated the regulation of nitric oxide synthase activity by dexamethasone and the cofactor tetrahydrobiopterin in murine macrophages. The influence of the tetrahydrobiopterin biosynthesis inhibitors 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, and phenprocoumon, an inhibitor of sepiapterin reductase, on the synthesis of nitric oxide was investigated. Dexamethasone decreased the nitric oxide production due to direct inhibition of the induction of nitric oxide synthase and of GTP cyclohydrolase. Substitution of tetrahydrobiopterin via sepiapterin could not overcome the dexamethasone-mediated inhibition. 2,4-Diamino-6-hydroxypyrimidine abolished nitric oxide synthesis and synergized with dexamethasone, completely eliminating nitric oxide production. Phenprocoumon inhibited~ production of nitric oxide via interference with later steps of tetrahydrobiopterin biosynthesis. An exogenous supply of tetrahydrobiopterin through sepiapterin led to a further increase of nitric oxide production, even in fully activated macrophages. The 'amount of nitric oxide produced by murine macrophages is therefore limited by the amount of tetrahydrobiopterin present in the cells. Inhibitors of tetrahydrobiopterin biosynthesis could provide a novel approach for therapy of pathological conditions mediated by nitric oxide, such as septic shock.Nitric oxide has recently been brought into focus as an effector molecule involved in non-oxidative killing of bacteria, protozoa, fungi and tumour cells by rodent macrophages [l, 21. Furthermore, NO is suggested to account for the action of endothelium-derived relaxing factor, a product of vascular endothelial cells [3], and as a signaling agent in neurons [4]. NO is synthesized by NO synthase through oxidation of either one of the two equivalent terminal guanidin0 nitrogen atoms from L-arginine, yielding L-citrulline and the active radical. The NO synthases characterized so far exist in two forms, (a) as a constitutive form, which is Ca2+/ calmodulin dependent, expressed in brain [5] and endothelial cells [6]. (b) The NO synthase expressed in activated macrophages [7] is inducible by cytokines and bacterial endotoxin. This form was previously thought to be Ca2+ independent, but was recently shown to contain calmodulin as a tightly . It is endogenously synthesized by the pathway shown in Fig. 1. The complex reaction mechanism of NO synthases, addressing the BH, requirement, has recently been partially elucidated Glucocorticoids have a substantial effect on the natural resistence against a broad range of bacteria and fungi [14]. Recently, it has been reported that dexamethasone reduces the activity of the inducible NO synthase in the murine macrophage-like tumor cell line, J774 [15].The present study investigated the influence of dexamethasone on L-arginine metabolism and biosynthesis of BH, in respect to NO production in murine peritoneal macrophages. The regulatory role o...