Electron paramagnetic resonance investigations of nitrosyl complex formation during endotoxin tolerance

The prior administration of low dose endotoxin induces a state of hyporesponsiveness or tolerance to the lethal effects of endotoxin. It is generally accepted that macrophages are main cellular components in the development of tolerance, hence, nitric oxide (.NO) as one of the macrophage mediators may play a role in host defense mechanisms during tolerance. In this study, we utilized EPR spectroscopy to directly detect nitrosyl complexes as products of .NO in whole blood, livers and intestines of lipopolysaccharide (LPS)-tolerant rats. Male Sprague-Dawley rats were injected with a "low dose" LPS (0.5 mg/kg) 12-168 h prior to a "high dose" LPS (3 mg/kg), then sacrificed 6 h later. EPR signals of nitrosyl hemoprotein complexes were detected in specimens after high dose LPS. The post-LPS EPR signals of nitrosyl complexes from all samples were attenuated by a prior injection of low dose LPS. The signals of dinitrosyl-iron-dithiolate became apparent in samples from tolerant rats as signals of nitrosyl hemoprotein decreased. The maximal tolerance in terms of diminished .NO production was observed when low dose LPS was given 48-96 h prior to high dose LPS. Hemoglobin concentrations in the intestine used as biomarkers of hemorrhagic damage, were concomitantly attenuated in the jejunum of tolerant rats. These results together with our previous studies indicate that suppression of .NO production may contribute to the amelioration of hepatic and intestinal injury during endotoxin tolerance.