Regulation of the L‐arginine‐dependent and tetrahydrobiopterin‐dependent biosynthesis of nitric oxide in murine macrophages

Abstract

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...

2,4‐diamino‐6‐hydroxypyrimidine, an inhibitor of tetrahydrobiopterin synthesis, downregulates the expression of iNOS protein and mRNA in primary murine macrophages

“…In accordance with previous studies [8,11,14], DAHP at the concentrations used did not decrease the viability of the macrophages as indicated by three different sets of experimental evidence. First, the total yield of protein from DAHP-treated Duplicate cultures were processed for the determination of iNOS activity or intra-and extracellular biopterin levels.…”

“…The NO production of RAW 264.7 cells stimulated with IFN-y plus LPS was not suppressed by coincubation with DAHP, and even after pretreatment of the cells with DAHP the inhibition was weak [13]. Finally, DAHP down-regulated the release of NO by IFN-y/ LPS-activated murine peritoneal exudate macrophages, but data which would allow to ascribe the effect to inhibition of BH, synthesis were not presented [14].…”

“…Inducible nitric oxide synthase (INOS), the enzyme responsible for high outputproduction of NO from t_-arginine and molecular oxygen in cytokine-activated cells (reviewed in [14]), requires tetrahydrobiopterin (BH,) as a cofactor for activity [5,6]. In mouse fibroblasts, murine brain endothelial cells, rat vascular smooth muscle cells and the mouse macrophage cell line RAW264.7, the induction of NO release by interferon-y (IFN-y), tumor necrosis factor-a and/or lipopolysaccharide (LPS) is paralleled by an enhanced formation of BH, and an up-regulation of GTP-*Corresponding author.…”

Endothelial Dysfunction of Coronary Resistance Arteries Is Improved by Tetrahydrobiopterin in Atherosclerosis

“…An increase of both endogenous tetrahydrobiopterin production and NO synthesis through application of sepiapterin has previously been demonstrated by other authors. 12,13,28 Our choice of agonists for the investigation of endothelial function in human vessels was based on pilot experiments in which we determined the NO dependence of the different agonists by pretreatment with N G -nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis. L-NAME nearly completely inhibited the dilatory response of human arterioles to histamine, induced constriction to serotonin, and significantly increased vasoconstriction to acetylcholine.…”

Characterization and ontogenesis of nitric oxide synthase activity in pig enterocytes

“…In macrophages, the amount of NO produced is limited by the amount of tetrahydrobiopterin present in the cells [24]. Furthermore, in cerebellum, it has been shown that tetrahydrobiopterin does not function as a reactant in NO synthase catalysis but in that case, the role of the cofactor may be allosteric […”