cyclic-gmp and acetovanillone

A reduction of the nitric oxide (NO) action in vascular smooth muscle cells (VSMC) could play a role in the vascular damage induced by the glycaemic excursions occurring in diabetic patients; in this study, we aimed to clarify whether a short-term incubation of cultured VSMC with high glucose reduces the NO ability to increase cGMP and the cGMP ability to phosphorylate VASP at Ser-239. We observed that a 180 min incubation of rat VSMC with 25 mmol/L glucose does not impair the NO-induced cGMP increase but reduces VASP phosphorylation in response to both NO and cGMP with a mechanism blunted by antioxidants. We further demonstrated that high glucose increases radical oxygen species (ROS) production and that this phenomenon is prevented by the PKC inhibitor chelerythrine and the NADPH oxidase inhibitor apocynin. The following sequence of events is supported by these results: (i) in VSMC high glucose activates PKC; (ii) PKC activates NADPH oxidase; (iii) NADPH oxidase induces oxidative stress; (iv) ROS impair the signalling of cGMP, which is involved in the antiatherogenic actions of NO. Thus, high glucose, via oxidative stress, can reduce the cardiovascular protection conferred by the NO/cGMP pathway via phosphorylation of the cytoskeleton protein VASP in VSMC.

Topics: Acetophenones; Animals; Antioxidants; Benzophenanthridines; Cell Adhesion Molecules; Cells, Cultured; Cyclic GMP; Glucose; Male; Microfilament Proteins; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidases; Nitric Oxide; Oxidative Stress; Phosphoproteins; Phosphorylation; Protein Kinase C; Rats; Rats, Zucker; Reactive Oxygen Species; Serine

INTRODUCTION.: Prevalence of erectile dysfunction (ED) increases progressively with aging, but the ED pathophysiology at its early stages is still poorly investigated. AIM.: This study aimed to evaluate the functional and molecular alterations of erectile function at middle age, focusing on the contribution of oxidative stress in erectile tissue for the ED. METHODS.: Young (3.5-month) and middle-aged (10-month) male Wistar rats were used. Rat corpus cavernosum (RCC) was dissected free and mounted in 10-mL organ baths containing Krebs solution. Intracavernosal pressure (ICP) in anesthetized rats was evaluated. MAIN OUTCOME MEASURES.: Concentration-response curves to endothelium-dependent and endothelium-independent agents, as well as to electrical field stimulation (EFS), were obtained in RCC strips. Measurement of cyclic guanosine monophosphate (cGMP) and expressions of neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), gp91(phox) and superoxide dismutase-1 (SOD-1) expressions in RCC were evaluated. RESULTS.: ICP was significantly reduced in middle-aged compared with young rats. RCC relaxations to acetylcholine (10(-8) to 10(-2)  M), sodium nitroprusside (10(-8) to 10(-2)  M), sildenafil (10(-9) to 10(-5)  M), BAY 41-2272 (10(-9) to 10(-5)  M), and EFS (4-32 Hz) were decreased in middle-aged group, which were nearly normalized by apocynin (NADPH oxidase inhibitor; 10(-4)  M) or SOD (75 U/mL). Prolonged treatment with apocynin (85 mg/rat/day, 4 weeks) also restored the impaired relaxations in middle-aged rats. Relaxations to 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (8-Br-cGMP; 10(-8) to 3 × 10(-4)  M) remained unchanged between groups. Basal and stimulated cGMP production were lower in middle-aged group, an effect fully restored by apocynin and SOD. Protein expression of nNOS and phosphorylated eNOS (p-eNOS) (Ser-1177) reduced, whereas gp(91phox) mRNA expression increased in RCC from middle-aged rats. CONCLUSIONS.: ED in middle-aged rats is associated with decreased NO bioavailability in erectile tissue due to upregulation of NADPH oxidase subunit gp91(phox) and downregulation of nNOS/p-eNOS. Antioxidant therapies may be a good pharmacological approach to prevent ED at its early stages.

Topics: Acetophenones; Acetylcholine; Aging; Animals; Blood Pressure; Cyclic GMP; Down-Regulation; Electric Stimulation; Endothelium, Vascular; Enzyme Inhibitors; Erectile Dysfunction; Free Radical Scavengers; Male; Membrane Glycoproteins; Muscle Relaxation; NADPH Oxidase 2; NADPH Oxidases; Nitric Oxide Synthase; Nitroprusside; Penile Erection; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Pyrazoles; Pyridines; Rats; RNA, Messenger; Sildenafil Citrate; Sulfones; Superoxide Dismutase; Superoxide Dismutase-1; Up-Regulation; Vasodilator Agents

NADPH oxidase is a major source of superoxide anions in the pulmonary arteries (PA). We previously reported that intratracheal SOD improves oxygenation and restores endothelial nitric oxide (NO) synthase (eNOS) function in lambs with persistent pulmonary hypertension of the newborn (PPHN). In this study, we determined the effects of the NADPH oxidase inhibitor apocynin on oxygenation, reactive oxygen species (ROS) levels, and NO signaling in PPHN lambs. PPHN was induced in lambs by antenatal ligation of the ductus arteriosus 9 days prior to delivery. Lambs were treated with vehicle or apocynin (3 mg/kg intratracheally) at birth and then ventilated with 100% O(2) for 24 h. A significant improvement in oxygenation was observed in apocynin-treated lambs after 24 h of ventilation. Contractility of isolated fifth-generation PA to norepinephrine was attenuated in apocynin-treated lambs. PA constrictions to NO synthase (NOS) inhibition with N-nitro-l-arginine were blunted in PPHN lambs; apocynin restored contractility to N-nitro-l-arginine, suggesting increased NOS activity. Intratracheal apocynin also enhanced PA relaxations to the eNOS activator A-23187 and to the NO donor S-nitrosyl-N-acetyl-penicillamine. Apocynin decreased the interaction between NADPH oxidase subunits p22(phox) and p47(phox) and decreased the expression of Nox2 and p22(phox) in ventilated PPHN lungs. These findings were associated with decreased superoxide and 3-nitrotyrosine levels in the PA of apocynin-treated PPHN lambs. eNOS protein expression, endothelial NO levels, and tetrahydrobiopterin-to-dihydrobiopterin ratios were significantly increased in PA from apocynin-treated lambs, although cGMP levels did not significantly increase and phosphodiesterase-5 activity did not significantly decrease. NADPH oxidase inhibition with apocynin may improve oxygenation, in part, by attenuating ROS-mediated vasoconstriction and by increasing NOS activity.

Topics: Acetophenones; Animals; Animals, Newborn; Biopterins; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Endothelium, Vascular; Hypertension, Pulmonary; Lung; NADPH Oxidases; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Norepinephrine; Pulmonary Artery; Reactive Oxygen Species; Sheep; Superoxides; Tyrosine; Vasoconstriction; Vasodilation

The vasodegenerative phase of diabetic retinopathy is likely caused by endothelial dysfunction and reduced endothelial repair. Migration of endothelial progenitor cells (EPCs) into areas of vascular injury is critical to vascular repair. This key function, often defective in diabetes, is largely mediated by nitric oxide (NO), which is known to be inactivated by superoxide produced by NADPH oxidase. The authors tested the hypothesis that either increasing eNOS expression or inhibiting NADPH oxidase would restore the reparative function in diabetic EPCs.. Peripheral blood was obtained from healthy (n = 27) and diabetic (n = 31) persons, and CD34(+) cells were isolated. Expression and activation of eNOS and NADPH oxidase and intracellular levels of NO, superoxide, and peroxynitrite were evaluated. cGMP production and migration to SDF-1α were also determined. Reparative function was evaluated in a mouse model of retinal ischemia-reperfusion injury.. Diabetic EPCs demonstrate reduced eNOS expression and decreased NO bioavailability and migration in response to SDF-1α. Increasing eNOS expression in diabetic cells by AVE3085 resulted in increased peroxynitrite levels and, therefore, did not enhance NO-mediated functions in vitro and in vivo. Expression of Nox2, NADPH oxidase activity, and superoxide levels were higher in diabetic than in nondiabetic EPCs. Pretreatment with apocynin or gp91ds-tat increased NO bioavailability without increasing eNOS activity in response to SDF-1α. Ex vivo NADPH oxidase inhibition in diabetic cells restored migratory function in vitro and enhanced their homing to ischemic retinal vasculature in vivo.. The NADPH oxidase system is a promising target for correcting vasoreparative dysfunction in diabetic EPCs.

Topics: Acetophenones; Adult; Animals; Antigens, CD34; Benzodioxoles; Chemokine CXCL12; Cyclic GMP; Diabetic Retinopathy; Disease Models, Animal; Endothelium, Vascular; Enzyme Inhibitors; Female; Glycoproteins; Humans; Indans; Male; Mice; Middle Aged; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type III; Peroxynitrous Acid; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Superoxides; Young Adult

Oxidative stress is well known to lead to vascular dysfunction. Thioredoxin reductase (TrxR) catalyzes the reduction of oxidized thioredoxin. Reduced thioredoxin plays a role in cellular antioxidative defense and in decreasing S-nitrosylation. It is not known whether TrxR affects vascular reactivity. We hypothesized that TrxR inhibition decreases vascular relaxation via increased oxidative stress and S-nitrosylation. Aortic rings from C57BL/6 mice were treated with the TrxR inhibitor, 1-chloro-2,4-dinitrobenzene (DNCB), or auranofin for 30 minutes. Vascular relaxation to acetylcholine was measured in the rings contracted with phenylephrine. DNCB and auranofin reduced relaxation compared with vehicle (vehicle Emax = 71 ± 3%, DNCB Emax = 53 ± 3%; P < 0.05). The antioxidants, apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor), and tempol (superoxide dismutase mimetic) normalized impaired relaxation by DNCB in aorta (DNCB Emax = 53 ± 3%, DNCB + tempol Emax = 66 ± 3%; P < 0.05). In addition, DNCB reduced sodium nitroprusside-induced relaxation. DNCB increased soluble guanylyl cyclase (sGC) S-nitrosylation and decreased sGC activity. These data suggest that TrxR regulates vascular relaxation via antioxidant defense and sGC S-nitrosylation. TrxR may be an enzyme to approach for treatment of vascular dysfunction and arterial hypertension.

Topics: Acetophenones; Acetylcholine; Animals; Antioxidants; Aorta, Thoracic; Auranofin; Cyclic GMP; Cyclic N-Oxides; Dinitrochlorobenzene; Enzyme Inhibitors; Guanylate Cyclase; Hydrogen Peroxide; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Nitroprusside; Oxidative Stress; Pyrazoles; Pyridines; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; S-Nitrosothiols; Soluble Guanylyl Cyclase; Spin Labels; Thioredoxin-Disulfide Reductase; Vasodilation; Vasodilator Agents

A positive association between renin-angiotensin system, especially AT1 receptor, and oxidative stress in the pathogenesis of hypertension and cardiovascular/renal diseases has been suggested. However, the role of oxidative stress, especially superoxide radicals in renal sodium handling in response to AT1 and AT2 receptors, is not known. Therefore, the present study was designed to investigate the role of NAD(P)H oxidase (NOX), a major superoxide radical producing enzyme, in AT1 and AT2 receptor function on natriuresis/diuresis in Sprague-Dawley rats. The rats under anesthesia were intravenously infused with NOX inhibitor apocynin (3.5 μg·kg(-1)·min(-1)), the AT1 receptor antagonist candesartan (100 μg/kg; bolus), and the AT2 receptor agonist CGP-42112A (1 μg·kg(-1)·min(-1)) alone and in combinations. Candesartan alone significantly increased urinary flow (UF; μl/30 min) by 53 and urinary Na excretion (U(Na)V; μmol/min) by 0.4 over basal. Preinfusion of apocynin had no effect on the net increase in UF or U(Na)V in response to candesartan. On the other hand, apocynin preinfusion caused profound increases in CGP-42112A-induced UF by 72, U(Na)V by 1.14, and fractional excretion of Na by 7.8. Apocynin and CGP-42112A alone did not cause significant increase in UF or U(Na)V over the basal. CGP-42112A infusion in the presence of apocynin increased urinary nitrite/nitrates and cGMP over basal. The infusion of candesartan, apocynin, and CGP-42112A alone or in combinations had no effect on the blood pressure or the glomerular filtration rate, suggesting tubular effects on natriuresis/diuresis. The data suggest that NOX may have an antagonistic role in AT2 receptor-mediated natriuresis/diuresis possibly via neutralizing nitric oxide and thereby influence fluid-Na homeostasis.

Topics: Acetophenones; Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Biphenyl Compounds; Blood Pressure; Cyclic GMP; Glomerular Filtration Rate; Homeostasis; Male; NADPH Oxidases; Natriuresis; Nitric Oxide; Oligopeptides; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Sodium; Tetrazoles

BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine) relaxes mesenteric arteries (MA) in a synergistic fashion with nitric oxide (NO). We hypothesized that the relaxation to BAY 41-2272 is decreased in spontaneously hypertensive rats (SHR) because of the reduced NO bioavailability in this strain and that relaxation would be improved by inhibiting the oxidative stress. We aimed to evaluate the influence of oxidative stress in BAY 41-2272-induced vasorelaxation in isolated MA from SHR.. MA function was evaluated by concentration-response curves to BAY 41-2272. We measured protein expression of endothelial NO synthase (eNOS), soluble guanylyl cyclase (sGC) and human-antigen R (HuR) (sGC mRNA-stabilizing protein), sGC activity and plasma levels of superoxide dismutase (SOD), and total antioxidant status (TAS).. Cyclic guanosine monophosphate (cGMP)-dependent and -independent relaxation induced by BAY 41-2272 (0.0001-1 micromol/l) was impaired in SHR compared with Wistar-Kyoto (WKY). We observed reduced expression of eNOS, sGC and HuR, and decreased sGC activity in SHR. Plasma levels of SOD and TAS were also diminished in SHR. Incubation with SOD or indomethacin increased relaxation to BAY 41-2272 in SHR. Furthermore, acetylcholine (ACh)-induced relaxation was increased in the presence of BAY 41-2272 or SOD, apocynin, or indomethacin.. Augmented oxidative stress in SHR impaired cGMP-dependent and -independent relaxation induced by BAY 41-2272, by decreasing NO bioavailability and sGC expression and by increasing contractile activity. Inhibiton of oxidative stress improved the relaxation of BAY 41-2272 in SHR. BAY 41-2272 might be an alternative therapeutic tool for hypertension if administrated with antioxidant compounds.

Topics: Acetophenones; Acetylcholine; Animals; Antioxidants; Bridged Bicyclo Compounds, Heterocyclic; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Fatty Acids, Unsaturated; Guanylate Cyclase; Hydrazines; Nitric Oxide Synthase Type III; Nitroprusside; Oxidative Stress; Pyrazoles; Pyridines; Rats; Rats, Inbred SHR; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Superoxide Dismutase; Vasodilation; Vasodilator Agents

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is a protective principle in the vasculature. Many cardiovascular diseases are associated with reduced NO bioactivity and eNOS uncoupling due to oxidative stress. Compounds that reverse eNOS uncoupling and increase eNOS expression are of therapeutic interest. Zizyphi Spinosi semen (ZSS) is one of the most widely used traditional Chinese herbs with protective effects on the cardiovascular system. In human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells, an extract of ZSS increased eNOS promoter activity, eNOS mRNA and protein expression, and NO production in a concentration- and time-dependent manner. Major ZSS constituents include saponins, such as jujuboside A and B, and pentacyclic triterpenes, such as betulin and betulinic acid. Jujuboside A, jujuboside B, or betulin had no significant effect on eNOS expression, whereas betulinic acid increased eNOS mRNA and protein expression in HUVEC and EA.hy 926 cells. Interestingly, betulinic acid also attenuated the expression of NADPH oxidase subunits Nox4 and p22phox, thereby reducing oxidative stress and improving eNOS function. Consequently, betulinic acid-treated endothelial cells showed an increased production of bioactive NO (as indicated by a higher efficacy in stimulating cGMP generation in RFL-6 reporter cells). Thus, betulinic acid possesses combined properties of eNOS up-regulation and NADPH oxidase down-regulation. Compounds such as betulinic acid may have a therapeutic potential in cardiovascular disease.

Topics: Acetophenones; Betulinic Acid; Blotting, Western; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Endothelial Cells; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; NADPH Oxidase 4; NADPH Oxidases; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Pentacyclic Triterpenes; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Saponins; Spermine; Triterpenes; Ziziphus

Dietary (-)-epicatechin is known to improve bioactivity of (*)NO in arterial endothelium of humans, but the mode of action is unclear. We used the fluorophore 4,5-diaminofluorescein diacetate to visualize the (*)NO level in living human umbilical vein endothelial cells (HUVEC). Untreated cells showed only a weak signal, whereas pretreatment with (-)-epicatechin (10 microM) or apocynin (100 microM) elevated the (*)NO level. The effects were more pronounced when the cells were treated with angiotensin II with or without preloading of the cells with (*)NO via PAPA-NONOate. While (-)-epicatechin scavenged O2(*-), its O-methylated metabolites prevented O2(*-) generation through inhibition of endothelial NADPH oxidase activity, even more strongly than apocynin. From the effect of 3,5-dinitrocatechol, an inhibitor of catechol-O-methyltransferase (COMT), on HUVEC it is concluded that (-)-epicatechin serves as 'prodrug' for conversion to apocynin-like NADPH oxidase inhibitors. These data indicate an (*)NO-preserving effect of (-)-epicatechin via suppression of O2(*-)-mediated loss of (*)NO.

Topics: Acetophenones; Angiotensin II; Catechin; Cells, Cultured; Cyclic GMP; Endothelial Cells; Enzyme Inhibitors; Humans; Methylation; Molecular Structure; NADPH Oxidases; Nitric Oxide; Signal Transduction; Umbilical Cord

Although hyperhomocysteinaemia is a risk factor for cardiovascular disease, the mechanisms underlying this association have not been elucidated. It has been demonstrated, however, that copper augments the inhibitory effect of homocysteine on nitric oxide (NO)-mediated relaxation of the rat aorta through increased superoxide formation, which reacts with NO thereby reducing the bioavailability of NO. Since it follows that the administration of a copper chelator may blunt the pathogenic impact of hyperhomocysteinaemia, in vivo, the effect of penicillamine administration on NO-dependent relaxation and superoxide formation in the aortae of hyperhomocysteinaemic rabbits was studied. New Zealand White rabbits were fed a methionine-rich (20 g/kg chow) diet for 1 month+/-penicillamine administered orally (10 mg/kg/day) and aortic relaxation elicited with acetylcholine and superoxide measured. The role of NADPH oxidase was also studied using a range of inhibitors and western analysis of gp47(phox) (a catalytic subunit of NADPH oxidase). The methionine-rich diet markedly increased plasma total homocysteine levels. In hyperhomocysteinaemic rabbits there was a marked reduction of acetylcholine-stimulated relaxation and an increase in superoxide formation that were both inhibited with superoxide dismutase and apocynin, an NADPH oxidase inhibitor. Gp47(phox) expression was also increased in aortae from methionine fed rabbits. Penicillamine administration significantly reduced plasma total copper in methionine-fed rabbits compared to controls. Impaired acetylcholine-stimulated relaxation, increased superoxide formation and increased gp47(phox) expression in aortae from methionine-fed rabbits was reversed by penicillamine administration. These data indicate that hyperhomocysteinaemia augments the formation of arterial superoxide through an increase in NADPH oxidase expression/activity which in turn reduces NO bioavailability. Since these effects were reversed by penicillamine, these data consolidate the hypothesis that copper plays a role in mediating homocysteine-induced vasculopathy.

Topics: Acetophenones; Acetylcholine; Administration, Oral; Animals; Aorta; Blotting, Western; Cyclic GMP; Dietary Supplements; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Homocysteine; Hyperhomocysteinemia; In Vitro Techniques; Methionine; NADPH Oxidases; Nitric Oxide Donors; Nitroprusside; Onium Compounds; Penicillamine; Rabbits; Superoxides; Time Factors; Vasodilation; Vasodilator Agents

The cardiovascular hormone atrial natriuretic peptide (ANP) exerts anti-inflammatory effects on tumor necrosis factor-alpha-activated endothelial cells by inducing mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). The underlying mechanisms are as yet unknown. We aimed to elucidate the signaling pathways leading to an induction of MKP-1 by ANP in primary human endothelial cells. By using antioxidants, generation of reactive oxygen species (ROS) was shown to be crucially involved in MKP-1 upregulation. ANP was found to increase ROS formation in cultured cells as well as in the endothelium of intact rat lung vessels. We applied NAD(P)H oxidase (Nox) inhibitors (apocynin and gp91ds-tat) and revealed this enzyme complex to be crucial for superoxide generation and MKP-1 expression. Moreover, by performing Nox2/4 antisense experiments, we identified Nox2 as the critically involved Nox homologue. Pull-down assays and confocal microscopy showed that ANP activates the small Rho-GTPase Rac1. Transfection of a dominant-negative (RacN17) and constitutively active Rac1 mutant (RacV12) indicated that ANP-induced superoxide generation and MKP-1 expression are mediated via Rac1 activation. ANP-evoked production of superoxide was found to activate c-Jun N-terminal kinase (JNK). Using specific inhibitors, we linked ANP-induced JNK activation to MKP-1 expression and excluded an involvement of protein kinase C, extracellular signal-regulated kinase, and p38 MAPK. MKP-1 induction was shown to depend on activation of the transcription factor activator protein-1 (AP-1) by using electrophoretic mobility shift assay and AP-1 decoys. In summary, our work provides insights into the mechanisms by which ANP induces MKP-1 and shows that ANP is a novel endogenous activator of endothelial Rac1 and Nox/Nox2.

Topics: Acetophenones; Animals; Atrial Natriuretic Factor; Capillaries; Cell Cycle Proteins; Cells, Cultured; Cyclic GMP; Cycloheximide; DNA, Antisense; Dual Specificity Phosphatase 1; Endothelial Cells; Endothelium, Vascular; Enzyme Induction; Glycoproteins; Guanylate Cyclase; Humans; Immediate-Early Proteins; JNK Mitogen-Activated Protein Kinases; Lung; MAP Kinase Kinase 4; MAP Kinase Signaling System; Membrane Glycoproteins; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; NADPH Oxidase 1; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidase 5; NADPH Oxidases; Oligonucleotides, Antisense; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Atrial Natriuretic Factor; Recombinant Fusion Proteins; RNA, Messenger; Transcription Factor AP-1; Transfection; Umbilical Veins

We have recently shown that superoxide and hydrogen peroxide are putative inducers of angiogenesis in vivo, possibly through up regulation of inducible nitric oxide synthase (NOS) and increased production of endogenous nitric oxide (NO). The aim of the present work was to elucidate the implication of reactive oxygen species in endothelial cell functions, using cultures of human umbilical vein endothelial cells (HUVEC). Superoxide dismutase (SOD), tempol (membrane permeable SOD mimetic) and the NADPH oxidase inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride and apocynin, but not allopurinol, inhibited HUVEC proliferation and migration, as well as activity of endothelial NOS (eNOS). Catalase and the intracellular hydrogen peroxide scavenger sodium pyruvate decreased, while hydrogen peroxide increased HUVEC proliferation, migration and activity of eNOS. Dexamethasone induced the proliferation and migration of HUVEC and activated eNOS. Nomega-nitro-L-arginine methyl ester (L-NAME), but not Nomega-nitro-D-arginine methyl ester, decreased endothelial cell functions and reversed the effects of dexamethasone and hydrogen peroxide. N5-(1-iminoethyl)-L-ornithine dihydrochloride, but not the inducible NOS specific inhibitor N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride also decreased endothelial cell functions, similarly to L-NAME. The guanylate cyclase inhibitor 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one inhibited HUVEC proliferation in a concentration-dependent manner and completely reversed hydrogen peroxide-induced proliferation, migration and cGMP accumulation. In conclusion, superoxide and hydrogen peroxide seem to play a significant role in promoting endothelial cell proliferation and migration, possibly through regulation of eNOS activity.

Topics: Acetophenones; Antioxidants; Catalase; Cell Movement; Cell Proliferation; Cells, Cultured; Cyclic GMP; Cyclic N-Oxides; Dexamethasone; Dose-Response Relationship, Drug; Down-Regulation; Endothelial Cells; Enzyme Inhibitors; Humans; Hydrogen Peroxide; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Phosphorylation; Pyruvates; Spin Labels; Stereoisomerism; Sulfones; Superoxide Dismutase

Recent studies suggest that adipose tissue hormone, leptin, is involved in the pathogenesis of arterial hypertension. However, the mechanism of hypertensive effect of leptin is incompletely understood. We investigated whether antioxidant treatment could prevent leptin-induced hypertension. Hyperleptinemia was induced in male Wistar rats by administration of exogenous leptin (0.25 mg/kg twice daily s.c. for 7 days) and separate groups were simultaneously treated with superoxide scavenger, tempol, or NAD(P)H oxidase inhibitor, apocynin (2 mM in the drinking water). After 7 days, systolic blood pressure was 20.6% higher in leptin-treated than in control animals. Both tempol and apocynin prevented leptin-induced increase in blood pressure. Plasma concentration and urinary excretion of 8-isoprostanes increased in leptin-treated rats by 66.9% and 67.7%, respectively. The level of lipid peroxidation products, malonyldialdehyde + 4-hydroxyalkenals (MDA+4-HNE), was 60.3% higher in the renal cortex and 48.1% higher in the renal medulla of leptin-treated animals. Aconitase activity decreased in these regions of the kidney following leptin administration by 44.8% and 45.1%, respectively. Leptin increased nitrotyrosine concentration in plasma and renal tissue. Urinary excretion of nitric oxide metabolites (NO(x)) was 57.4% lower and cyclic GMP excretion was 32.0% lower in leptin-treated than in control group. Leptin decreased absolute and fractional sodium excretion by 44.5% and 44.7%, respectively. Co-treatment with either tempol or apocynin normalized 8-isoprostanes, MDA+4-HNE, aconitase activity, nitrotyrosine, as well as urinary excretion of NO(x), cGMP and sodium in rats receiving leptin. These results indicate that oxidative stress-induced NO deficiency is involved in the pathogenesis of leptin-induced hypertension.

Topics: Acetophenones; Aconitate Hydratase; Aldehydes; Animals; Antioxidants; Blood Pressure; Body Weight; Creatine; Cyclic GMP; Cyclic N-Oxides; Drinking; Eating; Hypertension; Isoprostanes; Kidney; Leptin; Male; Malondialdehyde; Natriuresis; Nitric Oxide; Rats; Rats, Wistar; Reactive Nitrogen Species; Sodium; Spin Labels; Tyrosine

Hypercholesterolaemia promotes erectile dysfunction through increased superoxide formation and negation of nitric oxide (NO) bioactivity in cavernosal tissue. The source of superoxide has not been clearly defined, however. Sildenafil (Viagra), the standard therapy for erectile dysfunction, may also be rendered more effective by the presence of an NO donor. One drug that intrinsically fulfils this criterion is sildenafil nitrate (NCX 911), an NO donating derivative of sildenafil. The objective of this study, therefore, was to determine the source of superoxide and its effect on erectile function in corpus cavernosum from hypercholesterolaemic rabbits and to determine whether NCX 911 confers an improvement over sildenafil citrate in this model. Hypercholesterolaemia elicited an increase in superoxide formation by rabbit cavernosal tissue and a reduction of carbachol-stimulated relaxation both of which were reversed by diphenylene iodonium chloride and apocynin (NADPH oxidase inhibitors). In response to sodium nitroprusside, hypercholesterolaemia also caused an attenuation of cavernosal relaxation which was not reversed with NADPH oxidase inhibitors. Both sildenafil citrate and NCX 911 significantly reversed impaired carbachol-stimulated relaxation and inhibited superoxide formation by cavernosal tissue from hypercholesterolaemic rabbits, NCX 911 being more potent. NCX 911 also augmented cavernosal cGMP levels, an effect blocked by the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo {4,3-a}quinoxalin-1-one (ODQ). These data demonstrate that hypercholesterolaemia promotes erectile dysfunction through an augmentation of superoxide derived from NADPH oxidase in cavernosal tissue. It also indicates that NO donating sildenafil may be therapeutically more beneficial than conventional sildenafil in treating erectile dysfunction with an oxidative stress-related aetiology.

Topics: Acetophenones; Allopurinol; Animals; Carbachol; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypercholesterolemia; In Vitro Techniques; Male; Muscle Relaxation; NADPH Oxidases; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Onium Compounds; Oxadiazoles; Penis; Phosphodiesterase Inhibitors; Piperazines; Purines; Quinoxalines; Rabbits; Rotenone; Sildenafil Citrate; Sulfones; Superoxides; Uncoupling Agents; Xanthine Oxidase