The selective guanylate cyclase inhibitor ODQ reduces multiple organ injury in rodent models of Gram-positive and Gram-negative shock.
OBJECTIVE
An enhanced formation of endogenous nitric oxide contributes to the circulatory failure caused by endotoxin (lipopolysaccharide). Many of the biological actions of nitric oxide are mediated by the guanylate cyclase/cyclic guanosine 3prime;,5'-monophosphate system. We recently discovered that two cell wall components, namely lipoteichoic acid and peptidoglycan of the Gram-positive bacterium Staphylococcus aureus, synergize to cause shock and multiple organ dysfunction syndrome in the rat. Here we investigate the effects of a selective guanylate cyclase inhibitor, 1H-(1,2,4)oxadiazole(4,3-alpha)quinoxaline-1-one (ODQ), on the circulatory failure and multiple organ dysfunction syndrome (kidney, liver, lung) caused by a) coadministration of lipoteichoic acid and peptidoglycan (Gram-positive shock) or b) lipopolysaccharide (Gram-negative shock) in the anesthetized rat. Furthermore, we investigated whether ODQ scavenges superoxide anions and/or hydroxyl radicals.


METHODS
The in vivo portion of the study was a prospective, randomized, controlled animal study. The in vitro portion included a) cultured ventricular myoblasts of the rat, H9c2(2-1) cells, and b) a cell free superoxide anion assay system.


METHODS
University-based research laboratory.


METHODS
Seventy-five anesthetized, male Wistar rats were used for the in vivo study.


METHODS
For the in vivo portion of the study, after surgical preparation, anesthetized rats were observed for 6 hrs. All rats were pretreated and received an intravenous infusion of saline (1.5 mL.kg-1.hr-1), which was maintained throughout the experiment. The rats were assigned to nine groups. Group 1 contained control rats (sham) subjected to 2 mL/kg saline intraperitoneally, 2 hrs before the experiment (n = 7). Group 2 contained control rats (sham) that received 2 mg/kg ODQ intraperitoneally, 2 hrs before the experiment (n = 9). Group 3 contained control rats (sham) that received 2 mL/kg dimethyl sulfoxide, 30% v/v in saline intraperitoneally, as a vehicle for ODQ, 2 hrs before the experiment (n = 6). In group 4 rats, Gram-positive shock was induced by coadministration of lipoteichoic acid (3 mg/kg intravenously) and peptidoglycan (10 mg/kg intravenously) (n = 10). In group 5, rats were pretreated with ODQ (as described previously) before lipoteichoic acid/peptidoglycan (n = 9). In group 6, rats were pretreated with dimethyl sulfoxide (as described previously) before lipoteichoic acid/peptidoglycan (n = 7). In group 7, Gram-negative shock was induced by lipopolysaccharide (6 mg/kg intravenously) (n = 11). In group 8, rats were pretreated with ODQ (as described previously) before lipopolysaccharide (n = 8). In group 9, rats were pretreated with dimethyl sulfoxide (as described previously) before lipopolysaccharide (n = 8). For the in vitro portion of the study, rat cells were preincubated with vehicle (saline and/or dimethyl sulfoxide) and ODQ (0.1 microM to 1 mM) for 2 hrs. The cells then were exposed to H2O2 (1 mM) for 4 hrs at 37 degrees C, after which time cell viability was determined by measuring the mitochondrial-dependent reduction of 3-(4,5-di-methyliazol-2-yl)-2,5-diphenyltetrazolium bromide to blue formazan. Next, an aqueous solution was incubated with ODQ (as described previously), and superoxide anions were produced by using a hypoxanthine/xanthine-oxidase assay. The chemiluminescence assay was used to evaluate any potential antioxidative effects of ODQ.


RESULTS
In vivo, administration of lipoteichoic acid/peptidoglycan or lipopolysaccharide resulted within 6 hrs in hypotension, acute renal dysfunction, hepatocellular injury, and lung injury. Pretreatment of rats with ODQ attenuated the renal dysfunction, lung injury, and hepatocellular injury caused by lipoteichoic acid/peptidoglycan or lipopolysaccharide. In vitro, administration of H2O2 (for 4 hrs) to rat cardiomyoblasts decreased mitochondrial respiration attributable to generation of hydroxyl radicals. Pretreatment of cells with ODQ did not abolish this cell injury. In addition, ODQ did not scavenge superoxide anions.


CONCLUSIONS
These results imply that ODQ, an inhibitor of guanylate cyclase, reduces the multiple organ injury and dysfunction caused by wall fragments of Gram-positive or Gram-negative bacteria in the anesthetized rat. The observed protective effects of ODQ are not attributable to the ability of ODQ to reduce the formation or the effects of superoxide anions or hydroxyl radicals.
Zacharowski
K
K
William Harvey Research Institute, St. Bartholomew's and The Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, United Kingdom. k.zacharowski@mds.qmw.ac.uk
Berkels
R
R
Olbrich
A
A
Chatterjee
P K
PK
Cuzzocrea
S
S
Foster
S J
SJ
Thiemermann
C
C
D000818:Animals; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000627:therapeutic use*; D004927:Escherichia coli Infections / Q000150:complications / Q000188:drug therapy*; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors*; D006439:Hemodynamics / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors / Q000633:toxicity; D007668:Kidney / Q000187:drug effects / Q000473:pathology; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008168:Lung / Q000187:drug effects / Q000473:pathology; D008297:Male; D009102:Multiple Organ Failure / Q000209:etiology / Q000378:metabolism / Q000517:prevention & control*; D010069:Oxadiazoles / Q000627:therapeutic use*; D011810:Quinoxalines / Q000627:therapeutic use*; D051381:Rats; D017208:Rats, Wistar; D012772:Shock, Septic / Q000150:complications / Q000188:drug therapy*; D013203:Staphylococcal Infections / Q000150:complications / Q000188:drug therapy*
C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D004791:Enzyme Inhibitors; D010069:Oxadiazoles; D011810:Quinoxalines; D006861:Hydrogen Peroxide; D006162:Guanylate Cyclase