Pharmacological characterization of guanidinoethyldisulphide (GED), a novel inhibitor of nitric oxide synthase with selectivity towards the inducible isoform

1 . Guanidines, amidines, S‐alkylisothioureas, and recently, mercaptoalkylguanidines have been described as inhibitors of the generation of nitric oxide (NO) from L‐arginine by NO synthases (NOS). We have recently demonstrated that guanidinoethyldisulphide (GED), formed from the dimerisation of mercaptoethylguanidine (MEG), is a novel inhibitor of nitric oxide synthases. Here we describe the pharmacological properties of GED on purified NOS isoforms, various cultured cell types, vascular ring preparations, and in endotoxin shock. 2 . GED potently inhibited NOS activity of purified inducible NOS (iNOS), endothelial NOS (ecNOS), and brain NOS (bNOS) enzymes with Ki values of 4.3, 18 and 25 μm, respectively. Thus, GED has a 4 fold selectivity for iNOS over ecNOS at the enzyme level. The inhibitory effect of GED on ecNOS and iNOS was competitive vs. L‐arginine and non‐competitive vs. tetrahydrobiopterin. 3 . Murine J774 macrophages, rat aortic smooth muscle cells, murine lung epithelial cells, and human intestinal DLD‐1 cells were stimulated with appropriate mixtures of pro‐inflammatory cytokines or bacterial lipopolysaccharide to express iNOS. In these cells, GED potently inhibited nitrite formation (EC50 values: 11,9, 1 and 30 μm, respectively). This suggests that uptake of GED may be cell type‐ and species‐ dependent. The inhibitory effect of GED on nitrite production was independent of whether GED was given together with immunostimulation or 6 h afterwards, indicating that GED does not interfere with the process of iNOS induction. 4 . GED caused relaxations in the precontracted vascular ring preparations (EC50: 20 μm). Part of this relaxation was endothelium‐dependent, but was not blocked by methylene blue (100 μm), an inhibitor of soluble guanylyl cyclase. In precontracted rings, GED enhanced the acetylcholine‐induced, endothelium‐dependent relaxations at 10 μm and caused a slight inhibition of the relaxations at 100 μm. The vascular studies demonstrate that the inhibitory potency of GED on ecNOS in the ring preparations is considerably lower than its potency against iNOS in the cultured cells. These data suggest that the selectivity of GED towards iNOS may lie, in part, at the enzyme level, as well as differential uptake by cells expressing the various isoforms of NOS. 5 . In a rat model of endotoxin shock in vivo, administration of GED, at 3 mg kg−1 bolus followed by 10 mg kg−1 h−1 infusion, starting at 90 min after bacterial lipopolysaccharide (LPS, 15