PHARMACOLOGICAL CHARACTERIZATION OF GUANIDINOETHYLDISULPHIDE (GED), ANOVEL INHIBITOR OF NITRIC-OXIDE SYNTHASE WITH SELECTIVITY TOWARDS THEINDUCIBLE ISOFORM

1 Guanidines, amidines, S-alkylisothioureas, and recently, mercaptoalk ylguanidines have been described as inhibitors of the generation of ni tric oxide (NO) from L-arginine by NO synthases (NOS). We have recentl y demonstrated that guanidinoethyldisulphide (GED), formed from the di merisation of mercaptoethylguanidine (MEG), is a novel inhibitor of ni tric 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 N OS activity of purified inducible NOS (iNOS), endothelial NOS (ecNOS), and brain NOS (bNOS) enzymes with K-i values of 4.3, 18 and 25 mu M, respectively. Thus, GED has a 4 fold selectivity for iNOS over ecNOS a t the enzyme level. The inhibitory effect of GED on ecNOS and iNOS was competitive vs. L-arginine and non-competitive vs. tetrahydrobiopteri n. 3 Murine J774 macrophages, rat aortic smooth muscle cells, murine l ung 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 inhib ited nitrite formation (EC(50) values: 11,9, 1 and 30 mu M, respective ly). This suggests that uptake of GED may be cell type- and species-de pendent. The inhibitory effect of GED on nitrite production was indepe ndent 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 vascula r ring preparations (EC(50): 20 mu M). Part of this relaxation was end othelium-dependent, but was not blocked by methylene blue (100 mu M), an inhibitor of soluble guanylyl cyclase. In precontracted rings, GED enhanced the acetylcholine-induced, endothelium-dependent relaxations at 10 mu M and caused a slight inhibition of the relaxations at 100 mu M. The vascular studies demonstrate that the inhibitory potency of GE D on ecNOS in the ring preparations is considerably lower than its pot ency against iNOS in the cultured cells. These data suggest that the s electivity of GED towards iNOS may lie, in part, at the enzyme level, as well as differential uptake by cells expressing the various isoform s 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 mg kg(- 1), i.v.), prevented the delayed fall in mean arterial blood pressure, prevented the development of the vascular hyporeactivity to noradrena line of the thoracic aorta ex vivo and protected against the impairmen t of the endothelium-dependent relaxations associated with this model of endotoxaemia; The same bolus-and infusion of the inhibitor did not alter blood pressure or ex vivo vascular reactivity in normal animals over 90 min. 6 Administration of GED (10 mg kg(-1), i.p.) given at 2 h after LPS (120 mg kg(-1), i.p.) and every 6 h thereafter caused a sig nificant improvement in, the survival rate in a lethal model of endoto xin shock in mice between 12 and 42 h. 7 In conclusion, we found that GED is a competitive inhibitor of iNOS activity. Its selectivity towar ds iNOS may lie both at the enzyme level and at the level of cell upta ke. GED has beneficial effects in models of endotoxin shock that are d riven by iNOS. GED or its derivatives may be useful tools in the exper imental therapy of inflammatory conditions associated with NO overprod uction duel to iNOS expression.