Abstract-Angiotensin II infusion causes endothelial dysfunction by increasing NAD(P)H oxidase-mediated vascular superoxide production. However, it remains to be elucidated how in vivo angiotensin II treatment may alter the expression of the gp91 phox isoforms and the endothelial nitric oxide synthase (NOS III) and subsequent signaling events and whether, in addition to the NAD(P)H oxidase, NOS III contributes to vascular superoxide formation. We therefore studied the influence of in vivo angiotensin II treatment (7 days) in rats on endothelial function and on the expression of the NAD(P)H oxidase subunits p22 phox , nox1, nox4, and gp91 phox and NOS III. Further analysis included the expression of NO-downstream targets, the soluble guanylyl cyclase (sGC), the cGMP-dependent protein kinase I (cGK-I), and the expression and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) at Ser239 (P-VASP). Angiotensin II caused endothelial dysfunction and increased vascular superoxide. Likewise, we found an increase in vascular protein kinase C (PKC) activity, in the expression of nox1 (6-to 7-fold), gp91 phox (3-fold), p22 phox (3-fold), NOS III mRNA, and protein. NOS-inhibition with N G -nitro-L-arginine decreased superoxide in vessels from angiotensin II-treated animals, compatible with NOS-uncoupling. Vascular NO assessed with electron paramagnetic resonance was markedly reduced. Likewise, a decrease in sGC-expression and P-VASP levels was found. In vivo PKC-inhibition with chelerythrine reduced angiotensin II-induced superoxide production and markedly inhibited upregulation of NAD(P)H oxidase subunits. We therefore conclude that angiotensin II-induced increases in the activity and the expression of NAD(P)H oxidase are at least in part PKC-dependent. NADPH oxidase-induced superoxide production may trigger NOS III uncoupling, leading to impaired NO/cGMP signaling and to endothelial dysfunction in this animal model. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2002;90:e58-e65.) Key Words: angiotensin II Ⅲ nitric oxide synthase uncoupling Ⅲ nox expression Ⅲ cGMP-dependent protein kinase Ⅲ vasodilator-stimulated phosphoprotein

1 The aim of this work was to investigate the mechanism of vasorelaxation induced by red wine polyphenolic compounds (RWPC) and two de®ned polyphenols contained in wine, leucocyanidol and catechin. The role of the endothelium, especially endothelium-derived nitric oxide (NO), was also investigated. 2 Relaxation produced by polyphenols was studied in rat aortic rings with and without functional endothelium, pre-contracted to the same extent with noradrenaline (0.3 and 0.1 mM, respectively). RWPC and leucocyanidol, but not catechin, produced complete relaxation of vessels with and without endothelium. However, 1000 fold higher concentrations were needed to relax endothelium-denuded rings compared to those with functional endothelium. 3 High concentrations of catechin (in the range of 10 71 g l 71 ) only produced partial relaxation (maximum 30%) and had the same potency in rings with and without endothelium. 4 The NO synthase inhibitor, N o -nitro-L-arginine-methyl-ester (L-NAME, 300 mM) completely abolished the endothelium-dependent but not the endothelium-independent relaxations produced by all of the polyphenolic compounds. 5 In contrast to superoxide dismutase (SOD, 100 u ml 71 ), neither RWPC nor leucocyanidol a ected the concentration-response curve for the NO donor, SIN-1 (3-morpholino-sydnonimine) which also produces superoxide anion (O 2 7 ). 6 In aortic rings with endothelium, RWPC (10 72 g l 71 ) produced a 7 fold increase in the basal production of guanosine 3' : 5'-cyclic monophosphate (cyclic GMP) which was prevented by L-NAME (300 mM). 7 Electron paramagnetic resonance (e.p.r.) spectroscopy studies with Fe 2+ -diethyldithiocarbamate as an NO spin trap demonstrated that RWPC and leucocyanidol increased NO levels in rat thoracic aorta about 2 fold. This NO production was entirely dependent on the presence of the endothelium and was abolished by L-NAME (300 mM). 8 These results show that RWPC and leucocyanidol, but not the structurally closely related polyphenol catechin, induced endothelium-dependent relaxation in the rat aorta. They indicate that this e ect results from enhanced synthesis of NO rather than enhanced biological activity of NO or protection against breakdown by O 2 7 . It is concluded that some polyphenols, with speci®c structure, contained in wine possess potent endothelium-dependent vasorelaxing activity.

Abstract-During the past decade, it has become apparent that reactive oxygen species play a critical role in the genesis of many vascular diseases. The superoxide anion is among the most important of these, not only because of its rapid reaction with NO but also because it serves as a progenitor for many other reactive oxygen species. Although there are many approaches to detecting and quantifying superoxide in chemical systems, its detection in intact tissues is more difficult. The validity of the most popular and frequently used assay for this purpose, lucigenin-enhanced chemiluminescence, has been recently questioned. It has been suggested that lucigenin itself, especially at high concentrations (Ͼ50 mol/L), may act as a source for superoxide via redox cycling. Lower lucigenin concentrations (5 mol/L) do not participate in redox cycling to an important extent in intact tissues and, therefore, provide an accurate assessment of the rate of superoxide production in such samples. Other useful assays for superoxide include those using the fluorescent dye dihydroethidine, 2-methyl-6-phenyl-3,7-dihydroimidazo(1,2-␣)pyrazin-3-one (CLA), and 2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl) 8-benzylimidazo[1,2-␣]pyrazin-3-one (coelenterazine). The chemiluminescent compound 5-amino-2,3-dihydroxy-1,4-phthalayineidone (luminol) may also be used to detect various reactive oxygen species and may be made specific for various oxidants, such as hydrogen peroxide, superoxide, and peroxynitrite, by altering the experimental conditions. Although each of these methods may be associated with potential artifacts, the use of Ն2 different techniques that yield similar results provides a reliable approach for the study of reactive oxygen species in intact vascular tissues.

Mitochondrial aldehyde dehydrogenase (ALDH-2) was recently identified to be essential for the bioactivation of glyceryl trinitrate (GTN). Here we assessed whether other organic nitrates are bioactivated by a similar mechanism. The ALDH-2 inhibitor benomyl reduced the vasodilator potency, but not the efficacy, of GTN, pentaerythritol tetranitrate (PETN), and pentaerythritol trinitrate in phenylephrine-constricted rat aorta, whereas vasodilator responses to isosorbide dinitrate, isosorbide-5-mononitrate, pentaerythritol dinitrate, pentaerythritol mononitrate, and the endothelium-dependent vasodilator acetylcholine were not affected. Likewise, benomyl decreased GTN-and PETN-elicited phosphorylation of the cGMP-activated protein kinase substrate vasodilator-stimulated phosphoprotein (VASP) but not that elicited by other nitrates. The vasodilator potency of organic nitrates correlated with their potency to inhibit ALDH-2 dehydrogenase activity in mitochondria from rat heart and increase mitochondrial superoxide formation, as detected by chemiluminescence. In contrast, mitochondrial ALDH-2 esterase activity was not affected by PETN and its metabolites, whereas it was inhibited by benomyl, GTN applied in vitro and in vivo, and some sulfhydryl oxidants. The bioactivation-related metabolism of GTN to glyceryl-1,2-dinitrate by isolated RAW macrophages was reduced by the ALDH-2 inhibitors benomyl and daidzin, as well as by GTN at concentrations Ͼ1 M. We conclude that mitochondrial ALDH-2, specifically its esterase activity, is required for the bioactivation of the organic nitrates with high vasodilator potency, such as GTN and PETN, but not for the less potent nitrates. It is interesting that ALDH-2 esterase activity was inhibited by GTN only, not by the other nitrates tested. This difference might explain why GTN elicits mitochondrial superoxide formation and nitrate tolerance with the highest potency.Organic nitrates such as nitroglycerin (glyceryl trinitrate; GTN) are widely used in the therapy of cardiovascular diseases such as stable and unstable angina (Abrams, 1995).The anti-ischemic effects of organic nitrates are largely caused by venous and coronary artery dilation as well as the improvement of collateral blood flow, which all decrease myocardial oxygen consumption. Their use, however, is limited because of the rapid development of tolerance and crosstolerance characterized by decreased sensitivity of the vasculature to the organic nitrates and to endothelium-dependent vasodilators, respectively (Mangione and Glasser,

Abstract-Nebivolol is a ␤ 1 -receptor antagonist with vasodilator and antioxidant properties. Because the vascular NADPH oxidase is an important superoxide source, we studied the effect of nebivolol on endothelial function and NADPH oxidase activity and expression in the well-characterized model of angiotensin II-induced hypertension. Angiotensin II infusion (1 mg/kg per day for 7 days) caused endothelial dysfunction in male Wistar rats and increased vascular superoxide as detected by lucigenin-derived chemiluminescence, as well as dihydroethidine staining. Vascular NADPH oxidase activity, as well as expression at the mRNA and protein level, were markedly upregulated, as well as NOS III uncoupled, as evidenced by NO synthase III inhibitor experiments and dihydroethidine staining and by markedly decreased hemoglobin-NO concentrations. Treatment with the ␤-receptor blocker nebivolol but not metoprolol (10 mg/kg per day for each drug) normalized endothelial function, reduced superoxide formation, increased NO bioavailability, and inhibited upregulation of the activity and expression of the vascular NADPH oxidase, as well as membrane association of NADPH oxidase subunits (Rac1 and p67 phox ). In addition, NOS III uncoupling was prevented. In vitro treatment with nebivolol but not atenolol or metoprolol induced a dissociation of p67 phox and Rac1, as well as an inhibition of NADPH oxidase activity assessed in heart membranes from angiotensin II-infused animals, as well as in homogenates of Nox1 and cytosolic subunit-transfected and phorbol ester-stimulated HEK293 cells. These findings indicate that nebivolol interferes with the assembly of NADPH oxidase. Thus, inhibitory effects of this ␤-blocker on vascular NADPH oxidase may explain, at least in part, its beneficial effect on endothelial function in angiotensin II-induced hypertension. (Hypertension. 2006;48:677-684.) Key Words: angiotensin II Ⅲ nitric oxide synthase Ⅲ endothelium Ⅲ oxidative stress Ⅲ vasodilation B oth arterial hypertension and coronary artery disease are associated with an activation of the circulating and local renin-angiotensin system and increased oxidative stress within the vascular wall. 1,2 Angiotensin II (Ang II) treatment has been shown to cause endothelial dysfunction, which is at least in part mediated by increased vascular superoxide levels. 3,4 Superoxide sources involved may include the NADPH oxidases 3,5 and an uncoupled endothelial NO synthase (NOS III). 4,6,7 Increased vascular superoxide production and endothelial dysfunction are also accompanied by increased NOS III expression but decreased vascular NO production, downregulation of the target enzyme soluble guanylyl cyclase, and by a decrease in the activity of the cGMP-dependent kinase (cGK-I). 4,8 Interestingly, these phenomena seemed to be linked, at least in part, to an activation of protein kinase C (PKC), because the inhibition of increased vascular PKC activity in vitro and in vivo partially inhibited superoxide production and improved NO/soluble guanylyl cyclase/cGK-I ...

No abstract

Abstract-Nitroglycerin (glyceryl trinitrate, GTN) relaxes blood vessels primarily via activation of the soluble guanylyl cyclase (sGC)/cGMP/cGMP-dependent protein kinase (cGK-I) pathway. Although the precise mechanism of sGC activation by GTN in the vascular wall is unknown, the mediatory role of nitric oxide (NO) has been postulated. We tested the GTN/NO hypothesis in different types of isolated rat and rabbit blood vessels using two novel approaches:(1) EPR spin trapping using colloid Fe(DETC) 2 and (2) analysis of cGK-I-dependent phosphorylation of the vasodilator-stimulated phosphoprotein at Ser239 (P-VASP). For comparison, another organic nitrate, isosorbide dinitrate (ISDN), and endothelium-dependent vasodilator, calcium ionophore A23187, were tested. We found a marked discrepancy between GTN's strong vasoactivity (vasodilation and augmentation of P-VASP) and its poor NO donor properties. In aortas precontracted with phenylephrine, GTN, ISDN, and A23187 induced nearly full relaxations (Ͼ80%) and doubling of vascular P-VASP content at concentrations of 100 nmol/L, 100 mol/L, and 1 mol/L, respectively. GTN applied in vasorelaxant concentrations (10 to 1000 nmol/L) did not significantly increase the basal vascular NO production, in contrast to ISDN and A23187. The absence of GTN-derived NO was confirmed in rabbit vena cava and renal artery. A significant increase in vascular NO formation was observed only at suprapharmacological GTN concentrations (Ͼ10 mol/L). The concentration dependency of NO formation from GTN was comparable to that of ISDN, although the latter exhibits 100-folds lower vasorelaxant potency. We conclude that GTN activates the sGC/cGMP/cGK-I pathway and induces vasorelaxation without intermediacy of the free radical NO. 2 It is believed that the beneficial therapeutic effect of GTN is due to selective vasodilation of coronary arteries and venous capacitance vessels with minimal effect on arteriolar tone. 3 Several evidences indicate that the principal mechanism of GTN-induced smooth muscle relaxation is the activation of the intracellular enzyme, soluble guanylyl cyclase (sGC), and subsequent elevation of the cyclic guanosine-3Ј,-5Ј-monophosphate (cGMP) levels 4 -12 (see reviews 13,14 ). Among other effects, the elevated cGMP level leads to activation of cGMP-dependent protein kinase (cGK-I), which in turn mediates vasorelaxation via phosphorylation of several proteins regulating intracellular Ca 2ϩ mobilization. 15 However, the precise mechanism by which GTN activates vascular sGC is still controversially discussed. 16 -19 According to the hypothesis presently favored, GTN undergoes intracellular bioconversion into the putative metabolite nitric oxide (NO), which is known as a direct activator of sGC and as a smooth muscle relaxant. 5,8,20 -23 After the discovery of endothelium-derived NO, 24 -26 the idea of NO being the active principle of GTN has been widely accepted by the scientific community, and it has been often speculated that GTN can replace the compromised endothelial NO produ...

Objectives-Nebivolol, in contrast to other selective ␤ 1 -adrenergic receptor antagonists like atenolol, improves endothelial function in patients with oxidative stress within vascular tissue. With the present studies we sought to determine whether ␤ receptor blockade with nebivolol may improve endothelial function in hyperlipidemia and whether this is attributable to reductions in vascular oxidative stress. Methods and Results-Watanabe heritable hyperlipidemic rabbits (WHHL) were treated with nebivolol (10 mg/kg per day for 8 weeks). New Zealand white rabbits (NZWR) served as controls. Nebivolol improved endothelial function, reduced vascular superoxide and vascular macrophage infiltration, and prevented NO synthase uncoupling in WHHL. Nebivolol treatment did not modify the expression of sGC or cGK-I but improved cGK-I activity (assessed by the phosphorylation state of the VAsodilator Stimulated Phosphoprotein at serine 239 , P-VASP). NAD(P)H oxidase activity in whole blood and isolated neutrophils was dose-dependently inhibited by nebivolol, whereas atenolol, metoprolol, and carvedilol were markedly less effective. Key Words: nebivolol Ⅲ NO synthase Ⅲ superoxide Ⅲ neutrophils Ⅲ NADPH oxidase ␤ -Adrenergic receptor blockers have become a mainstay in the management of unstable and stable angina and acute myocardial infarction as well as in the treatment of patients with hypertension and chronic congestive heart failure. Third-generation ␤ receptor blockers comprise substances that block selectively the ␤ 1 -receptor and that also have vasodilator properties attributable to simultaneous ␣-receptor blocking effects. Interestingly, the vasodilatory properties of the third-generation ␤-blocker nebivolol revealed to be mediated by the release of the endotheliumderived relaxing factor NO. Nebivolol-induced vasodilation was almost completely blocked by the inhibitors of the NO synthase L-NMMA. 1 In vitro studies revealed that this phenomenon is at least in part attributable to stimulation of ␤ 2 receptors on endothelial cells by nebivolol metabolites, leading to an increase in endothelial [Ca 2ϩ ] levels and subsequently to NO synthase (NOS) III activation. 2 Chronic treatment with nebivolol has also been shown to improve endothelial function in patients with essential hypertension. 3 Interestingly, endothelial function in the setting of hypertension has recently been shown to be improved by the antioxidant vitamin C, suggesting that this phenomenon may be partly attributable to increased production of superoxide. 4 The beneficial effects on endothelial function in hypertensive patients may therefore indicate that nebivolol treatment inhibits vascular superoxide production. Indeed, recent studies revealed that antihypertensive doses of nebivolol decreased oxidative stress in healthy volunteers, reflected by a decrease in the formation of the isoprostane 8-iso-PGF 2␣ . 5 Indirect evidence for antioxidative properties of nebivolol was recently provided by in vitro experiments. Incubation of cultured endothelial as well...