ABSTRACT
Superoxide anions (O2-) are supposedly involved in the pathogenesis of endothelial dysfunction. We investigated whether the enhanced formation of O2- 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 O2-, H2O2, and ONOO- 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 O2- (from 35+/-2 cpm/ring at baseline to 166+/-21 cpm/ring at 12 hours and 225+/-16 cpm/ring at 30 hours) and H2O2 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- formation. LPS also induced expression of the oxidative stress-sensitive protein manganese superoxide dismutase. Endothelium-dependent relaxation was impaired after LPS treatment and could not be restored by inhibition of inducible NO synthase. Inhibition of O2- 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 O2- and ONOO-. Because removal of O2- compromised rather than restored endothelium-dependent relaxation, a direct role of O2- in the induction of endothelial dysfunction is unlikely. Other mechanisms, such as prolonged protein tyrosine nitration by peroxynitrite (which is formed from NO and O2-) or downregulation of the NO effector pathway, are more likely to be involved.
Subject(s)
Endothelium, Vascular/physiopathology , Endotoxemia/physiopathology , Escherichia coli Infections/physiopathology , NADPH Oxidases/physiology , Superoxides/metabolism , Xanthine Oxidase/physiology , Analysis of Variance , Animals , Aorta/metabolism , Aorta/physiology , Blotting, Northern , Down-Regulation , Enzyme Inhibitors/pharmacology , Escherichia coli , Immunoblotting , In Vitro Techniques , Lipopolysaccharides/administration & dosage , Luminescent Measurements , Muscle Relaxation , Muscle, Smooth, Vascular/physiology , NADPH Oxidases/antagonists & inhibitors , Nitric Oxide/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Onium Compounds/pharmacology , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Superoxide Dismutase/physiologyABSTRACT
The hypothesis was tested whether postinfarction hypertrophy/congestive heart failure in rats is associated with endothelial dysfunction and increased vascular generation of reduced oxygen species. Myocardial infarction was induced in Sprague-Dawley rats by ligation of the left coronary artery. After 16 weeks, endothelium-dependent (with acetylcholine) and -independent (with sodium nitro-prusside) relaxations were studied in isolated aortic rings, and isolated rings from the femoral and mesenteric arteries. The generation of superoxide, hydrogenperoxide, and peroxynitrite was measured in arteries using lucigenin- and luminol-enhanced chemiluminescence techniques. Systolic blood pressure decreased over the 16 week study period as compared to shamoperated control rats; organ weights (lungs, right and left ventricles) significantly increased in coronary artery ligated rats indicating development of congestive heart failure. Surprisingly, concentration response curves with acetylcholine and sodium nitroprusside were almost identical in myocardial infarction rats as compared to control animals, irrespective of which type of vessel was studied (aorta, femoral or mesenteric arteries). In addition, no differences in the production of reduced radical species were found in aortic tissue from heart failure rats as compared to control rats.
