Central role of mitochondrial aldehyde dehydrogenase and reactive oxygen species in nitroglycerin tolerance and cross-tolerance.

Recent studies suggest that mitochondrial aldehyde dehydrogenase (ALDH-2) plays a central role in the process of nitroglycerin (glyceryl trinitrate, GTN) biotransformation in vivo and that its inhibition accounts for mechanism-based tolerance in vitro. The extent to which ALDH-2 contributes to GTN tolerance (impaired relaxation to GTN) and cross-tolerance (impaired endothelium-dependent relaxation) in vivo remain to be elucidated. Rats were treated for three days with GTN. Infusions were accompanied by decreases in vascular ALDH-2 activity, GTN biotransformation, and cGMP-dependent kinase (cGK-I) activity. Further, whereas in control vessels, multiple inhibitors and substrates of ALDH-2 reduced both GTN-stimulation of cGKI and GTN-induced vasodilation, these agents had little effect on tolerant vessels. A state of functional tolerance (in the GTN/cGMP pathway) was recapitulated in cultured endothelial cells by knocking down mitochondrial DNA (rho(0) cells). In addition, GTN increased the production of reactive oxygen species (ROS) by mitochondria, and these increases were associated with impaired relaxation to acetylcholine. Finally, antioxidants/reductants decreased mitochondrial ROS production and restored ALDH-2 activity. These observations suggest that nitrate tolerance is mediated, at least in significant part, by inhibition of vascular ALDH-2 and that mitochondrial ROS contribute to this inhibition. Thus, GTN tolerance may be viewed as a metabolic syndrome characterized by mitochondrial dysfunction.

Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling.

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.

Mechanisms of increased vascular superoxide production in an experimental model of idiopathic dilated cardiomyopathy.

OBJECTIVE: In the present study, we sought to identify mechanisms underlying increased oxidative stress in vascular tissue in an experimental animal model of chronic congestive heart failure (CHF). METHODS AND RESULTS: Superoxide and nitric oxide (NO) was measured in vessels from cardiomyopathic hamsters (CHF hamsters) and golden Syrian hamsters. We also determined expression of endothelial nitric oxide synthase (NOSIII), the soluble guanylyl cyclase, the cGMP-dependent kinase, and the NADPH oxidase. To analyze the contribution of the renin-angiotensin system to oxidative stress, CHF hamsters were treated with the angiotensin-converting enzyme inhibitor captopril for 200 days (120 mg . kg(-1) . d(-1)). CHF led to increased superoxide production by NOSIII and the NADPH oxidase. Decreased NO production in CHF was associated with a decrease in the expression of NOSIII and an inhibition of NO downstream signaling in the aorta. NOSIII expression was increased within the left ventricle. Captopril treatment normalized NOSIII expression in vessels and the myocardium, reduced superoxide levels, and prevented NOSIII uncoupling. Accordingly, endothelial function, NO production, and downstream signaling were improved in CHF vessels. CONCLUSIONS: Oxidative stress in CHF is mediated by NADPH oxidase and an uncoupled NOSIII secondary to an activation of the renin-angiotensin system leading to impaired NO downstream signaling.

Role for peroxynitrite in the inhibition of prostacyclin synthase in nitrate tolerance.

OBJECTIVES: We tested whether in vivo nitroglycerin (NTG) treatment causes tyrosine nitration of prostacyclin synthase (PGI(2)-S), one of the nitration targets of peroxynitrite, and whether this may contribute to nitrate tolerance. BACKGROUND: Long-term NTG therapy causes tolerance secondary to increased vasoconstrictor sensitivity and increased vascular formation of reactive oxygen species. Because NTG releases nitric oxide (NO), NTG-induced stimulation of superoxide production should increase vascular nitrotyrosine levels, compatible with increased formation of peroxynitrite, the reaction product from NO and superoxide. METHODS: New Zealand White rabbits and Wistar rats were treated with NTG (0.4 mg/h for 3 days). Tolerance was assessed with isometric tension studies. Vascular peroxynitrite levels were quantified with luminol-derived chemiluminescence (LDCL) and peroxynitrite scavengers, such as uric acid and ebselen. As a surrogate parameter for the assessment of the activity of cyclic guanosine monophosphate-dependent kinase-I (cGK-I; the final signaling pathway for NO), the phosphorylation of the vasodilator-stimulated phosphoprotein (P-VASP) at serine 239 was analyzed. RESULTS: Nitroglycerin treatment increased LDCL, and the inhibitory effect of uric acid and ebselen on LDCL was augmented in tolerant rings. Immunoprecipitation of 3-nitrotyrosine-containing proteins and immunohistochemistry analysis identified PGI(2)-S as a tyrosine-nitrated protein. Accordingly, conversion of ((14)C)-PGH(2) into 6-keto-PGF(1 alpha) (=PGI(2)-S activity) was strongly inhibited. In vitro incubation of tolerant rings with ebselen and uric acid markedly increased the depressed P-VASP levels and improved NTG sensitivity of the tolerant vasculature. CONCLUSIONS: Nitroglycerin-induced vascular peroxynitrite formation inhibits the activity of PGI(2)-S as well as NO, cGMP, and cGK-I signaling, which may contribute to vascular dysfunction in the setting of tolerance.

Nebivolol prevents vascular NOS III uncoupling in experimental hyperlipidemia and inhibits NADPH oxidase activity in inflammatory cells.

OBJECTIVE: Nebivolol, in contrast to other selective beta1-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 beta 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 serine239, 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. CONCLUSIONS: Nebivolol therapy effectively prevents NO synthase III uncoupling and prevents activation of the neutrophil NAD(P)H oxidase and infiltration of inflammatory cells. These novel antioxidative stress actions of this compound may explain partly the beneficial effects on endothelial function in patients with enhanced vascular oxidative stress.

Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012.

In the present study we sought to determine the ability of the chemiluminescence dye 8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012) to detect superoxide in different biological systems. In human whole blood or isolated leukocytes, the sensitivity of the luminol analogue L-012 to detect superoxide was higher as compared with luminol, lucigenin, coelenterazine, and the fluorescence dye dihydroethidine. In isolated leukocytes as well as aortic rings from control (New Zealand White) and hyperlipidemic (Watanabe heritable hyperlipidemic) rabbits, L-012-enhanced chemiluminescence was successful in detecting differences in superoxide formation under basal conditions and on stimulation with the direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu). The effects of PDBu were abrogated by gliotoxin and inhibitors of protein kinase C such as chelerythrine, identifying NAD(P)H oxidase as the significant superoxide source. Experiments using electron paramagnetic resonance and the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide revealed that in contrast to lucigenin, L-012 is not subject to redox cycling. These findings indicate that L-012-enhanced chemiluminescence represents a sensitive and reliable probe to detect superoxide in whole blood, inflammatory cells, and vascular tissue.

Clinical aspects of reactive oxygen and nitrogen species.

Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolaemia, hypertension, diabetes mellitus and chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species in endothelial and/or smooth muscle cells and the adventitia, and the subsequent decrease in vascular bioavailability of NO. Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include NAD(P)H-oxidase, xanthine oxidase and endothelial nitric oxide synthase in an uncoupled state. Recent studies indicate that endothelial dysfunction of peripheral and coronary resistance and conductance vessels represents a strong and independent risk factor for future cardiovascular events. Ways to reduce endothelial dysfunction include risk-factor modification and treatment with substances that have been shown to reduce oxidative stress and, simultaneously, to stimulate endothelial NO production, such as inhibitors of angiotensin-converting enzyme or the statins. In contrast, in conditions where increased production of reactive oxygen species, such as superoxide, in vascular tissue is established, treatment with NO, e.g. via administration of nitroglycerin, results in a rapid development of endothelial dysfunction, which may worsen the prognosis in patients with established coronary artery disease.

Detection of superoxide and peroxynitrite in model systems and mitochondria by the luminol analogue L-012.

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10-100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.

Mechanisms underlying dysfunction of carotid arteries in genetically hyperlipidemic rabbits.

In the present study we compared the vascular reactivity and integrity of the nitric oxide (NO)-cyclic 3',5'-guanosine monophopsphate (cGMP) pathway in carotid arteries of hyper- and normolipidemic rabbits. Vasodilation to acetylcholine, nitroglycerin, and sodium nitroprusside was desensitized in hyperlipidemia, but the nitroprusside-induced relaxation was normalized by an NO synthase inhibitor in endothelium-intact and -denuded vessels. Hyperlipidemic carotid arteries exhibited increased basal NO (detected by EPR spin-trapping) and reactive oxygen species formation (detected by chemiluminescence), whereas acetylcholine-induced NO formation was nearly abolished. Hyperlipidemia increased NADPH-dependent superoxide formation in carotid membranes, and carotid cryosections stained with the fluorescent dye dihydroethidium revealed increased endothelial and medial reactive oxygen species formation. Hyperlipidemia elicited macrophage invasion into the carotid wall, as detected by a dot-immunoblot. The basal activity of cGMP-dependent proteinkinase, the nitroprusside-stimulated activity of soluble guanylyl cyclase, and its protein expression were decreased by hyperlipidemia. The cGMP phosphodiesterase activity was marginally increased by hyperlipidemia, such that the ratio of cGMP-forming vs. -degrading capacity was decreased by 2-fold. Hyperlipidemia triggers infiltration of macrophages into the carotid wall and endothelial as well as smooth muscle superoxide formation. Consequently, relaxation of the carotid arteries are impaired due to smooth muscle and endothelial dysfunction.

Reversal of endothelial nitric oxide synthase uncoupling and up-regulation of endothelial nitric oxide synthase expression lowers blood pressure in hypertensive rats.

OBJECTIVES: We sought to examine the hypothesis that a pharmacologic up-regulation of endothelial nitric oxide synthase (eNOS) combined with a reversal of eNOS uncoupling provides a protective effect against cardiovascular disease. BACKGROUND: Many cardiovascular diseases are associated with oxidant stress involving protein kinase C (PKC) and uncoupling of eNOS. METHODS: Messenger ribonucleic acid (mRNA) expression was analyzed with RNase protection assay or quantitative real-time polymerase chain reaction, vascular nitric oxide (NO) with spin trapping, and reactive oxygen species (ROS) with dihydroethidium fluorescence. RESULTS: Aortas of spontaneously hypertensive rats (SHR) showed an elevated production of ROS when compared with aortas of Wistar-Kyoto rats (WKY). The aortic expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits (Nox1, Nox2, Nox4, and p22phox) was higher in SHR compared with WKY. In SHR, aortic production of ROS was reduced by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), indicating eNOS "uncoupling" in hypertension. Oral treatment with the PKC inhibitor midostaurin reduced aortic Nox1 expression, diminished ROS production, and reversed eNOS uncoupling in SHR. Aortic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) were significantly reduced in SHR compared with WKY. Midostaurin normalized BH4 levels in SHR. In both WKY and SHR, midostaurin increased aortic expression of eNOS mRNA and protein, stimulated bioactive NO production, and enhanced relaxation of the aorta to acetylcholine. Midostaurin lowered blood pressure in SHR and, to a lesser extent, in WKY; the compound did not change blood pressure in WKY made hypertensive with L-NAME. CONCLUSIONS: Pharmacologic interventions that combine eNOS up-regulation and reversal of eNOS uncoupling can markedly increase bioactive NO in the vasculature and produce beneficial hemodynamic effects such as a reduction of blood pressure.