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 synthase (NOS) in circulatory shock exerts cytotoxic and vasodilator effects. Part of these effects are mediated by formation of peroxynitrite, a toxic oxidant produced by the rapid reaction of NO and superoxide. Other parts of the 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 subsequent decrease in the intracellular calcium levels. Using 1H-(1,2,4)oxadiazolo(4,3-alpha)quinoxalin-1 -one (ODQ), a potent inhibitor of GC, we studied the role of GC activation in the NO- and peroxynitrite-related vascular alterations.


METHODS
In vitro: Controlled experiment using cultured rat aortic smooth muscle cells. In vivo: Prospective, randomized, controlled animal study.


METHODS
Experimental laboratory.


METHODS
Male Wistar rats and male Swiss mice.


METHODS
In vitro: a) Stimulation of rat aortic smooth muscle cells with bacterial lipopolysaccharide (LPS) and gamma-interferon, measurement of the production of nitrite and nitrate (breakdown products of NO), and suppression of mitochondrial respiration for 24 to 48 hrs, in the presence or absence of ODQ; and b) in norepinephrine-precontracted endothelium-denuded thoracic 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.


RESULTS
Stimulation of rat aortic smooth muscle cells with bacterial LPS and gamma-interferon induced the production of nitrite and nitrate (breakdown products of NO) and suppression of mitochondrial respiration for 24 to 48 hrs. The amount of NO produced was slightly enhanced with ODQ (10-100 EM), whereas the suppression of mitochondrial respiration was not affected by ODQ (1-100 microM). ODQ did not affect the degree of suppression of mitochondrial respiration in response to NO donor agents or to peroxynitrite. Exposure to LPS (10 ng/mL) for 6 hrs caused a time-dependent relaxation of norepinephrine-precontracted endothelium-denuded thoracic aortic rings. This response was caused by the expression of inducible NOS and could be blocked by pharmacologic inhibitors of NOS such as N(G)-methylL-arginine. ODQ (1 microM) prevented the LPS-induced loss of vascular tone in this experimental system. Similar to the in vitro responses, there was a significant suppression of the norepinephrine-induced contractions in ex vivo experiments, in which rings were taken from animals treated in vivo with LPS (10 mg/kg for 3 hrs). ODQ treatment in vitro (1 microM) 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 posttreatment, improved survival at 24-144 hrs.


CONCLUSIONS
These studies indicate that GC activation does not contribute to NO- or peroxynitrite-induced cytotoxicity but does contribute to the vascular hyporeactivity induced by endotoxin in vitro and in vivo. GC inhibition alone is sufficient to influence survival in a murine model of severe sepsis.
Zingarelli
B
B
Division of Critical Care, Children's Hospital Medical Center, Cincinnati, OH, USA.
Haskó
G
G
Salzman
A L
AL
Szabó
C
C
D000818:Animals; D001011:Aorta / Q000201:enzymology; D019069:Cell Respiration / Q000187:drug effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors*; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010069:Oxadiazoles / Q000494:pharmacology*; D016877:Oxidants / Q000378:metabolism*; D011810:Quinoxalines / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012772:Shock, Septic / Q000201:enzymology*; D016019:Survival Analysis; D014664:Vasodilation / Q000187:drug effects
C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D009566:Nitrates; D009573:Nitrites; D010069:Oxadiazoles; D016877:Oxidants; D011810:Quinoxalines; C039642:peroxynitric acid; D009569:Nitric Oxide; D007371:Interferon-gamma; D006162:Guanylate Cyclase