Oxidation, tyrosine nitration and cytostasis induction in the absence of inducible nitric oxide synthase

In the present study, we evaluated the impact of the lack of the gene for inducible nitric oxide synthase (iNOS) on oxidation, tyrosine nitration and cytotoxicity reactions triggered by immunostimulation. In mice injected with E. coli endotoxin (bacterial lipopolysaccharide, LPS, 50 mg/kg i.p.), there was a significant increase in the degree of oxidation of dihydrorhodamine 123 to rhodamine 123. This response was attenuated by inhibition of NO biosynthesis with NG-methyl-L-arginine (L-NMA, 30 mg/kg i.p.). In mice lacking functional iNOS gene (iNOS knock-out mice), the degree of the LPS-induced, L-NMA inhibitable increase in dihydrorhodamine oxidation was decreased, but not completely abolished. LPS stimulation induced a marked increase in the immunoreactivity for nitrotyrosine (an indicator of peroxynitrite formation), as measured in the aorta and lung. An L-NMA inhibitable increase in nitrotyrosine staining induced by LPS was also observed in the tissues of the iNOS knockout animals. LPS treatment induced the appearance of DNA single strand breakage and a suppression of mitochondrial respiration in peritoneal macrophages ex vivo. A significant degree of LPS-induced DNA single strand breakage and suppression of mitochondrial respiration was still observed in the peritoneal macrophages obtained from the iNOS knockout animals. Macrophages from wild-type mice stimulated with LPS and interferon-gamma suppressed the proliferation of various target cells (P815 mastocytoma, L929 fibrosarcoma and embryonic lung fibroblast cell line): this effect was abolished by in vitro treatment with L-NMA (1 mM). Macrophages from the iNOS knockout animals exhibited a reduced degree of target cell cytostatic activity. The remainder of the cytostasis in iNOS knockout macrophages was abolished by preventing cell contact and neutralizing tumor necrosis factor á. The present results demonstrate that the lack of iNOS gene does not fully abolish oxidation, tyrosine nitration and cytostatic activity in response to immunostimulation. The current findings may have implications for the development of NO-based approaches for the experimental therapy of inflammation.