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

Objective: Nitric oxide (NO), produced by the inducible isoform of NO synth ase (NOS) in circulatory shock exerts cytotoxic and vasodilator effects. Pa rt of these effects are mediated by formation of peroxynitrite, a toxic oxi dant produced by the rapid reaction of NO and superoxide. Other parts of th e vascular actions of NO in shock are thought to be mediated by the action of NO on the soluble guanylyl cyclase (GC) in the smooth muscle and subsequ ent decrease in the intracellular calcium levels. Using 1H-(1,2,4)oxadiazol o(4,3-alpha)quinoxalin-1-one (ODQ), a potent inhibitor of GC, we studied th e role of GC activation in the NO- and peroxynitrite-related vascular alter ations. Design: In vitro: Controlled experiment using cultured rat aortic smooth mu scle cells. In vivo: Prospective, randomized, controlled animal study. Setting: Experimental laboratory. Subjects: Male Wistar rats and male Swiss mice. Interventions: In vitro: a) Stimulation of rat aortic smooth muscle cells w ith bacterial lipopolysaccharide (LPS) and gamma-interferon, measurement of the production of nitrite and nitrate (breakdown products of NO), and supp ression of mitochondrial respiration for 24 to 48 hrs, in the presence or a bsence of ODQ; and b) in norepinephrine-precontracted endothelium-denuded t horacic aortic rings, exposure to LPS (10 ng/mL) in the presence or absence of ODQ. In vivo: Rats treated in vivo with LPS (10 mg/kg iv for 3 hrs) and mice challenged with 60 mg/kg LPS ip, in the presence or absence of ODQ. Measurements and Main Results: Stimulation of rat aortic smooth muscle cell s with bacterial LPS and gamma-interferon induced the production of nitrite and nitrate (breakdown products of NO) and suppression of mitochondrial re spiration for 24 to 48 hrs. The amount of NO produced was slightly enhanced with ODQ (10-100 mu M), whereas the suppression of mitochondrial respirati on was not affected by ODQ (1-100 mu M). ODQ did not affect the degree of s uppression of mitochondrial respiration in response to NO donor agents or t o peroxynitrite. Exposure to LPS (10 ng/mL) for 6 hrs caused a time-depende nt relaxation of norepinephrine-precontracted endothelium-denuded thoracic aortic rings. This response was caused by the expression of inducible NOS a nd could be blocked by pharmacologic inhibitors of NOS such as N-G-methyl-L -arginine, ODQ (1 mu M) prevented the LPS-induced loss of vascular tone in this experimental system. Similar to the in vitro responses, there was a si gnificant suppression of the norepinephrine-induced contractions in ex vivo experiments, in which rings were taken from animals treated in vivo with L PS (10 mg/kg for 3 hrs). ODQ treatment in vitro (1 mu M) caused a complete restoration of the contractile responses. In mice challenged with 60 mg/kg LPS ip, ODQ (20 mg/kg), given either as a pretreatment or as a 4-hr posttre atment, improved survival at 24-144 hrs. Conclusion: These studies indicate that GC activation does not contribute t o NO- or peroxynitrite-induced cytotoxicity but does contribute to the vasc ular hyporeactivity induced by endotoxin in vitro and in vibro. GC inhibiti on alone is sufficient to influence survival in a murine model of severe se psis.