OXIDATION, TYROSINE NITRATION AND CYTOSTASIS INDUCTION IN THE ABSENCEOF 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 nitr ation and cytotoxicity reactions triggered by immunostimulation. In mi ce injected with E. coil endotoxin (bacterial lipopolysaccharide, LPS, 50 mg/kg i.p.), there was a significant increase in the degree of oxi dation of dihydrorhodamine 123 to rhodamine 123. This response was att enuated by inhibition of NO biosynthesis with N-G-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 abolis hed. LPS stimulation induced a marked increase in the immunoreactivity for nitrotyrosine (an indicator of peroxynitrite formation), as measu red in the aorta and lung. An L-NMA inhibitable increase in nitrotyros ine staining induced by LPS was also observed in the tissues of the iN OS knockout animals. LPS treatment induced the appearance of DNA singl e strand breakage and a suppression of mitochondrial respiration in pe ritoneal macrophages ex vivo. A significant degree of LPS-induced DNA single strand breakage and suppression of mitochondrial respiration wa s 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 ce lls (P815 mastocytoma, L929 fibrosarcoma and embryonic lung fibroblast cell line): this effect was abolished by in vitro treatment with L-NM A (1 mM). Macrophages from the iNOS knockout animals exhibited a reduc ed degree of target cell cytostatic activity. The remainder of the cyt ostasis in iNOS knockout macrophages was abolished by preventing cell contact and neutralizing tumor necrosis factor ct. The present results demonstrate that the lack of iNOS gene does not fully abolish oxidati on, tyrosine nitration and cytostatic activity in response to immunost imulation. The current findings may have implications for the developm ent of NO-based approaches for the experimental therapy of inflammatio n.