Effects of a novel guanylyl cyclase inhibitor on the actions of nitric oxide and peroxynttrite in immunostimulated smooth muscle cells and in endotoxic shock

Introduction: Nitric oxide (NO), produced by the inducible isoform of NO syntfuue (iNOS) in circulatory shock exerts cytotoxk and vasodilator effects. Pan of these effects are mediated by formation of peroxynitrite. a toxic oxidant produced by the rapid reaction of NO and Superoxide. Other part of the vascular actions of NO in shock are thought to be mediated by the action of NO on the soluble guanylyl cyclase (GQ in the smooth muscle, and subsequent decrease in the intracellular calcium levels. Using lH-(1.2,4] oxadiazolo{4,3-alquinoxalin- 1-one (ODQ). a potent inhibitor of GC, we studied the rote of GC activation in the NO- and peroxynitrite-related vascular alterations. Methods aad results: In vitro, stimulation of rat aortic smooth muscle cells with bacterial upoporysaccharide (LPS) and pmma-inlerferon (IFN) cauMd the expression of iNOS, production of nitrite and nitrate (breakdown products of NO) aad suppression of mitochondria! respiration over 24-48h. The amounts of NO produced, and the suppression of mitochondria! respiration were not affected by pretreatmeat with ODQ (1100 uM). ODQ did not affect the degree of suppression of mitochondria! respiration in response to NO donor agents or to peroxynitrite. Exposure to LPS (10 |lg/ml) for 6h caused a time-dependent relaxation of ncnpinephrine-precontncied eodotheüum-denuded thoracic aortic rings. This response is due to the expression of iNOS, and can be blocked by pharmacological inhibitors of NOS such as NG-methyl-L-argmiiie. ODQ (1 uM) caused a significant protection against the LPS-induced relaxations in this experimental system. Similar to the in vitro responses, there was a significant suppression of the norepinephrine-induced contractions in ex vivo experiments, where rings were taken from animals treated in vivo with LPS (10 mg/kg for 3h). ODQ treatment in vitro (1 uM) caused a significant restoration of the contractile responses. In mice ehalkBgnd with 60 mg/kg LPS (i.p.). ODQ (20 mg/kg) improved survival at 24 and 48h. Conclusions: These studies indicate that GC activation does not play a role in the expression of iNOS, does not contribute to NO- or peroxynitrite cytotoxkity, bat does contribute to die vascular hyporeactivity associated with endotoxk shock. GC inhibition alone is sufficient to influence survival in this model of sepsis. Nevertheless, it is expected that GC inhibition can only interfere with part of the NO-reJated cytotoxk and cardiovascular derangements. Therefore, appropriate inhibitors of NO biosynthesis may be superior to GC inhibitors in endotoxic shock.