Abstract-Superoxide anions (O 2 Ϫ) are supposedly involved in the pathogenesis of endothelial dysfunction. We investigated whether the enhanced formation of O 2 Ϫ is involved in the attenuation of endothelium-dependent relaxation induced by lipopolysaccharide (LPS). Rats were injected with LPS (10 mg/kg IP), the aorta was removed after 12 or 30 hours, and generation of O 2 Ϫ , H 2 O 2 , and ONOO Ϫ was measured using chemiluminescence assays. Protein tyrosine nitration and expression of xanthine oxidase (XO), NAD(P)H oxidase, and manganese superoxide dismutase were determined by Western or Northern blotting, and endothelium-dependent relaxation in aortic rings was studied. LPS treatment increased vascular O 2 Ϫ (from 35Ϯ2 cpm/ring at baseline to 166Ϯ21 cpm/ring at 12 hours and 225Ϯ16 cpm/ring at 30 hours) and H 2 O 2 formation, which was partially sensitive to the NAD(P)H oxidase inhibitor diphenylene iodonium at both time points studied and to the XO inhibitor oxypurinol only 30 hours after LPS treatment. Expression of XO and NAD(P)H oxidase (p22phox, p67phox, and gp91phox) were increased by LPS in a time-dependent manner, as were protein tyrosine nitration and ONOO Ϫ formation. LPS also induced expression of the oxidative stress-sensitive protein manganese superoxide dismutase. Endotheliumdependent relaxation was impaired after LPS treatment and could not be restored by inhibition of inducible NO synthase. Inhibition of O 2Ϫ with superoxide dismutase, oxypurinol, tiron, or the superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride did not restore but further deteriorated the relaxation of LPS-treated rings. In summary, treatment of rats with LPS enhances vascular expression of XO and NAD(P)H oxidase and increases formation of O 2 Ϫ and ONOO Ϫ is at least partially due to activation of vascular NAD(P)H oxidase and xanthine oxidase (XO). 1 Severe hypotension and hyporesponsiveness to vasoconstrictors and endothelium-dependent dilator agents are hallmarks of sepsis, eg, after exposure to bacterial lipopolysaccharide (LPS). 2 Although LPS-induced vascular hyporesponsiveness to constrictor agents may be partially due to excessive generation of NO by inducible NO synthase 2 (iNOS), the mechanism underlying LPS-induced endothelial dysfunction is not clear. 3 We investigated whether enhanced formation of O 2 Ϫ could be involved in the development of endothelial dysfunction after exposure to LPS. Methods MaterialsDiphenylene iodonium (DPI), U44069, and aminohydromethylthiazine (AMT) were obtained from Alexis. Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) was obtained from Calbiochem. Recombinant human superoxide dismutase (rhSOD) was a gift from Grünenthal Inc. Rat vascular smooth muscle cell p22phox cDNA was a kind gift from K.K. Griendling, Emory University. XO cDNA was provided by M. Saksela, University of Helsinki. The mouse

Abstract-Endogenously produced nitric oxide (NO) modulates nitrovasodilator-induced relaxation. We investigated the underlying mechanism in wild-type (WT) mice and endothelial NO synthase knockout (eNOS Ϫ/Ϫ ) mice to determine whether a chronic lack of endothelial NO alters the soluble guanylyl cyclase (sGC) pathway. In aortic segments from eNOS Ϫ/Ϫ mice, the vasodilator sensitivity to sodium nitroprusside (SNP) was significantly greater than that in WT mice. There was no difference in sensitivity to the G-kinase I activator 8-para-chlorophenylthio-cGMP or to cromakalim. N -Nitro-L-arginine had no effect on the SNP-induced relaxation in eNOS Ϫ/Ϫ but increased the sensitivity in WT mice so it was no longer different than that of eNOS Ϫ/Ϫ . Basal cGMP levels in aortic rings were significantly lower in eNOS Ϫ/Ϫ mice than in WT mice. SNP (300 nmol/L) induced a significantly greater cGMP accumulation in eNOSmice than in WT mice. The maximal SNP-induced (10 mol/L) increase in cGMP was similar in both strains. SNP-stimulated sGC activity was significantly greater in eNOS Ϫ/Ϫ mice than in WT mice. Incubation of aortic segments from WT mice with N -nitro-L-arginine increased sGC activity, an effect prevented by coincubation with SNP (10 mol/L). The aortic expressions of the sGC ␣1 and ␤1 subunits in WT and eNOS Ϫ/Ϫ mice were identical as determined with Western blot analysis. These data suggest that chronic exposure to endothelium-derived NO, as well as acute exposure to nitrovasodilator-derived NO, desensitizes sGC to activation by NO but does not alter sGC expression. Both the acute cessation of endothelial NO formation in WT mice and the chronic deficiency of NO in eNOS Ϫ/Ϫ mice restore the NO sensitivity of sGC and enhance vascular smooth muscle relaxation in response to nitrovasodilator agents. Key Words: nitric oxide Ⅲ mice Ⅲ genes Ⅲ vasodilator agents V ascular relaxation responses elicited by nitrovasodilator agents and by endothelium-derived nitric oxide (NO) are mainly mediated via the activation of soluble guanylyl cyclase (sGC) and a subsequent increase in intracellular cGMP levels. 1 Interactions between endogenous and exogenous NO have been reported to modulate vasodilatory responsiveness. 2-5 Different mechanisms may underlie this phenomenon, such as the downregulation of sGC, sGC desensitization, or inhibition of the cGMP-dependent signal transduction cascade. In the present study, we investigated the mechanism by which endothelium-derived NO affects the nitrovasodilator-induced relaxation in wild-type (WT) mice and in endothelial NO synthase knockout mice (eNOS Ϫ/Ϫ ). Methods Animals and Tissue PreparationsWT c57 black b6 mice were purchased from Charles River, and eNOS Ϫ/Ϫ mice and age-matched control animals were obtained from the Department of Physiology at Heinrich Heine Universität Düssel-dorf. 6 Mice were housed under conditions that conformed with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication No. 85-23).Mice were killed by cervical dislocation. T...