Reactive oxygen species (ROS) from NADPH and xanthine oxidase modulate the cutaneous local heating response in healthy humans

Local cutaneous heating produces vasodilation that is largely nitric oxide (NO) dependent. We showed that angiotensin II (ANG II) attenuates this by an ANG II receptor, type 1 (AT1R)-dependent mechanism that is reversible with the antioxidant ascorbate, indicating oxidative stress. Reactive oxygen species (ROS) produced by ANG II employ NADPH and xanthine oxidase pathways. To determine whether these mechanisms pertain to skin, we measured cutaneous local heating with 10 μM ANG II, using apocynin to inhibit NADPH oxidase and allopurinol to inhibit xanthine oxidase. We also inhibited superoxide with tempol, and H(2)O(2) with ebselen. We heated the skin of the calf in 8 healthy volunteers (24.5-29.9 yr old) to 42°C and measured local blood flow to assess the percentage of maximum cutaneous vascular conductance. We remeasured while perfusing allopurinol, apocynin, ebselen, and tempol through individual microdialysis catheters. This was then repeated with ANG II combined with antioxidant drugs. tempol and apocynin alone had no effect on the heat response. Allopurinol enhanced the entire response (125% of heat alone), while ebselen suppressed the heat plateau (76% of heat alone). ANG II alone caused significant attenuation of the entire heat response (52%). When added to ANG II, Allopurinol partially reversed the ANG II attenuation. Heat with ebselen and ANG II were similar to heat and ANG II; ebselen only partially reversed the ANG II attenuation. Apocynin and tempol each partially reversed the attenuation caused by ANG II. This suggests that ROS, produced by ANG II via NADPH and xanthine oxidase pathways, modulates the response of skin to the application of heat, and thus contributes to the control of local cutaneous blood flow.     

Endostatin uncouples NO and Ca2+ response to bradykinin through enhanced O2*- production in the intact coronary endothelium

The present study tested the hypothesis that endostatin stimulates superoxide (O2*-) production through a ceramide-mediating signaling pathway and thereby results in an uncoupling of bradykinin (BK)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) from nitric oxide (NO) production in coronary endothelial cells. With the use of high-speed, wavelength-switching, fluorescence-imaging techniques, the [Ca2+]i and NO levels were simultaneously monitored in the intact endothelium of freshly isolated bovine coronary arteries. Under control conditions, BK was found to increase NO production and [Ca2+]i in parallel. When the arteries were pretreated with 100 nM human recombinant endostatin for 1 h, this BK-induced NO production was reduced by 89%, whereas [Ca2+]i was unchanged. With the conversion rate of L-[3H]arginine to L-[3H]citrulline measured, endostatin had no effect on endothelial NO synthase (NOS) activity, but it stimulated ceramide by activation of sphingomyelinase (SMase), whereby O2*-. production was enhanced in endothelial cells. O2*-. scavenging by tiron and inhibition of NAD(P)H oxidase by apocynin markedly reversed the effect of endostatin on the NO response to BK. These results indicate that endostatin increases intracellular ceramide levels, which enhances O2*-. production through activation of NAD(P)H oxidase. This ceramide-O2*-. signaling pathway may contribute importantly to endostatin-induced endothelial dysfunction.     

Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine induces vascular endothelial superoxide production: implication of NADPH oxidase

Modified low-density lipoprotein (LDL) induces reactive oxygen species (ROS) production by vascular cells. It is unknown if specific oxidized components in these LDL particles such as oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) can stimulate ROS production. Bovine aortic endothelial cells (BAEC) were incubated with ox-PAPC (50 microg/ml). At 4 h, ox-PAPC significantly enhanced the rate of O2- production. Pretreatment of BAEC in glucose-free Dulbecco's modified Eagle's medium plus 10 mM 2-deoxyglucose (2-DOG), the latter being an antimetabolite that blocks NADPH production by the pentose shunt, significantly reduced the rate of O2- production. The intensity of NAD(P)H autofluorescence decreased by 28 +/- 12% in BAEC incubated with ox-PAPC compared to untreated cells, with a further decrease in the presence of 2-DOG. Ox-PAPC also increased Nox4 mRNA expression by 2.4-fold +/- 0.1 while pretreatment of BAEC with the small interfering RNA (siNox4) attenuated Nox4 RNA expression. Ox-PAPC further reduced the level of glutathione while pretreatment with apocynin (100 microM) restored the GSH level (control = 22.54 +/- 0.23, GSH = 18.06 +/- 0.98, apocynin = 22.55 +/- 0.60, ox-PAPC + apocynin = 21.17 +/- 0.36 nmol/10(6) cells). Treatment with ox-PAPC also increased MMP-2 mRNA expression accompanied by a 1.5-fold increase in MMP-2 activity. Ox-PAPC induced vascular endothelial OO2-(.) production that appears to be mediated largely by NADPH oxidase activity.     

Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries

We tested the hypothesis that ceramide induces endothelial dysfunction in small coronary arteries via NADPH oxidase-mediated superoxide and resulting peroxynitrite formation. With the use of dihydroethidium as a superoxide indicator, C(2)-ceramide was found to increase superoxide production in the endothelial cells of small coronary arteries, which was inhibited by the NADPH oxidase inhibitors N-vanillylnonanamide, apocynin, and diphenylene iodonium. NADPH oxidase expression was confirmed in endothelial cells, as indicated by the immunoblotting of its subunits gp91(phox) and p47(phox). C(2)-ceramide increased NADPH oxidase activity by 52%, which was blocked by NADPH oxidase inhibitors but not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport chain enzymes. By Western blot analysis, ceramide-induced NADPH oxidase activation was found to be associated with the translocation of p47(phox) to the membrane. In isolated and pressurized small coronary arteries, N-vanillylnonanamide, apocynin, or uric acid, a peroxynitrite scavenger, largely restored the inhibitory effects of ceramide on bradykinin- and A-23187-induced vasorelaxation. With the use of nitrotyrosine as a marker, C(2)-ceramide was found to increase peroxynitrite in small coronary arteries, which could be blocked by uric acid. We conclude that NADPH oxidase-mediated superoxide production and subsequent peroxynitrite formation mediate ceramide-induced endothelial dysfunction in small coronary arteries.     

Hydrogen peroxide promotes endothelial dysfunction by stimulating multiple sources of superoxide anion radical production and decreasing nitric oxide bioavailability.

Hydrogen peroxide (H(2)O(2)) is an oxidant implicated in cell signalling and various pathologies, yet relatively little is known about its impact on endothelial cell function. Herein we studied the functional and biochemical changes in aortic vessels and cultured porcine aortic endothelial cells (PAEC) exposed to H(2)O(2). Exposure of aortic rings to 25 or 50 microM, but not 10 microM, H(2)O(2) for 60 min prior to constriction significantly decreased subsequent relaxation in response to acetylcholine (ACh), but not the nitric oxide ((.)NO) donor sodium nitroprusside. Treatment of PAEC with 50 microM H(2)O(2) significantly decreased ACh-induced accumulation of (.)NO, as measured with a (.)NO-selective electrode, yet such treatment increased nitric oxide synthase activity approximately 3-fold, as assessed by conversion of L-arginine to L-citrulline. Decreased (.)NO bioavailability was reflected in decreased cellular cGMP content, associated with increased superoxide anion radical (O(2)(-.)), and overcome by addition of polyethylene glycol superoxide dismutase. Increased cellular O(2)(-.) production was inhibited by allopurinol, diphenyliodonium and rotenone in an additive manner. The results show that exposure of endothelial cells to H(2)O(2) decreases the bioavailability of agonist-induced (.)NO as a result of increased production of O(2)(-.) likely derived from xanthine oxidase, NADPH-oxidase and mitochondria. These processes could contribute to H(2)O(2)-induced vascular dysfunction that may be relevant under conditions of oxidative stress such as inflammation.     

gp91phox-containing NAD(P)H oxidase mediates attenuation of nitric oxide-dependent control of myocardial oxygen consumption by ANG II

We have previously reported that ANG II stimulation increased superoxide anion (O2-) through the activation of NAD(P)H oxidase and inhibited nitric oxide (NO)-dependent control of myocardial oxygen consumption (MVo2) by scavenging NO. Our objective was to investigate the role of NAD(P)H oxidase, especially the gp91phox subunit, in the NO-dependent control of MVo2. MVo2 in mice with defects in the expression of gp91phox [gp91(phox)(-/-)] was measured with a Clark-type oxygen electrode. Baseline MVo2 was not significantly different between wild-type (WT) and gp91(phox)(-/-) mice. Stimulation of NO production by bradykinin (BK) induced significant decreases in MVo2 in WT mice. BK-induced reduction in MVo2 was enhanced in gp91(phox)(-/-) mice. BK-induced reduction in MVo2 in WT mice was attenuated by 10(-8) mol/l ANG II, which was restored by coincubation with Tiron or apocynin. In contrast to WT mice, BK-induced reduction in MVo2 in gp91(phox)(-/-) mice was not altered by ANG II. There was a decrease in lucigenin (5 x 10(-6) mol/l)-detectable O2- in gp91(phox)(-/-) mice compared with WT mice. ANG II resulted in significant increases in O2- production in WT mice, which was inhibited by coincubation with Tiron or apocynin. However, ANG II had no effect on O2- production in gp91(phox)(-/-) mice. Histological examination showed that the development of abscesses and/or the invasion of inflammatory cells occurred in lungs and livers but not in hearts and kidneys from gp91(phox)(-/-) mice. These results indicate that the gp91(phox) subunit of NAD(P)H oxidase mediates O2- production through the activation of NAD(P)H oxidase and attenuation of NO-dependent control of MVo2 by ANG II.     

Perivascular nitric oxide and superoxide in neonatal cerebral hypoxia-ischemia

Decreased cerebral blood flow (CBF) has been observed following the resuscitation from neonatal hypoxic-ischemic injury, but its mechanism is not known. We address the hypothesis that reduced CBF is due to a change in nitric oxide (NO) and superoxide anion O(2)(-) balance secondary to endothelial NO synthase (eNOS) uncoupling with vascular injury. Wistar rats (7 day old) were subjected to cerebral hypoxia-ischemia by unilateral carotid occlusion under isoflurane anesthesia followed by hypoxia with hyperoxic or normoxic resuscitation. Expired CO(2) was determined during the period of hyperoxic or normoxic resuscitation. Laser-Doppler flowmetry was used with isoflurane anesthesia to monitor CBF, and cerebral perivascular NO and O(2)(-) were determined using fluorescent dyes with fluorescence microscopy. The effect of tetrahydrobiopterin supplementation on each of these measurements and the effect of apocynin and N(omega)-nitro-L-arginine methyl ester (L-NAME) administration on NO and O(2)(-) were determined. As a result, CBF in the ischemic cortex declined following the onset of resuscitation with 100% O(2) (hyperoxic resuscitation) but not room air (normoxic resuscitation). Expired CO(2) was decreased at the onset of resuscitation, but recovery was the same in normoxic and hyperoxic resuscitated groups. Perivascular NO-induced fluorescence intensity declined, and O(2)(-)-induced fluorescence increased in the ischemic cortex after hyperoxic resuscitation up to 24 h postischemia. L-NAME treatment reduced O(2)(-) relative to the nonischemic cortex. Apocynin treatment increased NO and reduced O(2)(-) relative to the nonischemic cortex. The administration of tetrahydrobiopterin following the injury increased perivascular NO, reduced perivascular O(2)(-), and increased CBF during hyperoxic resuscitation. These results demonstrate that reduced CBF follows hyperoxic resuscitation but not normoxic resuscitation after neonatal hypoxic-ischemic injury, accompanied by a reduction in perivascular production of NO and an increase in O(2)(-). The finding that tetrahydrobiopterin, apocynin, and L-NAME normalized radical production suggests that the uncoupling of perivascular NOS, probably eNOS, due to acquired relative tetrahydrobiopterin deficiency occurs after neonatal hypoxic-ischemic brain injury. It appears that both NOS uncoupling and the activation of NADPH oxidase participate in the changes of reactive oxygen concentrations seen in cerebral hypoxic-ischemic injury.     

Effect of NADPH oxidase inhibition on cardiopulmonary bypass-induced lung injury

Cardiopulmonary bypass (CPB) causes acute lung injury. Reactive oxygen species (ROS) from NADPH oxidase may contribute to this injury. To determine the role of NADPH oxidase, we pretreated pigs with structurally dissimilar NADPH oxidase inhibitors. Low-dose apocynin (4-hydroxy-3-methoxy-acetophenone; 200 mg/kg, n = 6), high-dose apocynin (400 mg/kg, n = 6), or diphenyleneiodonium (DPI; 8 mg/kg) was compared with diluent (n = 8). An additional group was treated with indomethacin (10 mg/kg, n = 3). CPB was performed for 2 h with deflated lungs, complete pulmonary artery occlusion, and bronchial artery ligation to maximize lung injury. Parameters of pulmonary function were evaluated for 25 min following CPB. Blood chemiluminescence indicated neutrophil ROS production. Electron paramagnetic resonance determined the effect of apocynin and DPI on in vitro pulmonary endothelial ROS production following hypoxia-reoxygenation. Both apocynin and DPI attenuated blood chemiluminescence and post-CPB hypoxemia. At 25 min post-CPB with Fi(O(2)) = 1, arterial Po(2) (Pa(o(2))) averaged 52 +/- 5, 162 +/- 54, 335 +/- 88, and 329 +/- 119 mmHg in control, low-dose apocynin, high-dose apocynin, and DPI-treated groups, respectively (P < 0.01). Indomethacin had no effect. Pa(O(2)) correlated with blood chemiluminescence measured after drug administration before CPB (R = -0.60, P < 0.005). Neither apocynin nor DPI prevented the increased tracheal pressure, plasma cytokine concentrations (tumor necrosis factor-alpha and IL-6), extravascular lung water, and pulmonary vascular protein permeability observed in control pigs. NADPH oxidase inhibition, but not xanthine oxidase inhibition, significantly blocked endothelial ROS generation following hypoxia-reoxygenation (P < 0.05). NADPH oxidase-derived ROS contribute to the severe hypoxemia but not to the increased cytokine generation and pulmonary vascular protein permeability, which occur following CPB.     

(-)-Epicatechin elevates nitric oxide in endothelial cells via inhibition of NADPH oxidase

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.     

Aldosterone stimulates superoxide production in macula densa cells

Two major factors which regulate tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole are release of superoxide (O(2)(-)) and nitric oxide (NO) by macula densa (MD) cells. MD O(2)(-) inactivates NO; however, among the factors that increase MD O(2)(-) release, the role of aldosterone is unclear. We hypothesize that aldosterone activates the mineralocorticoid receptor (MR) on MD cells, resulting in increased O(2)(-) production due to upregulation of cyclooxygenase-1 (COX-2) and NOX-2, and NOX-4, isoforms of NAD(P)H oxidase. Studies were performed on MMDD1 cells, a renal epithelial cell line with properties of MD cells. RT-PCR and Western blotting confirmed the expression of MR. Aldosterone (10(-8) mol/l for 30 min) doubled MMDD1 cell O(2)(-) production, and this was completely blocked by MR inhibition with 10(-5) mol/l eplerenone. RT-PCR, real-time PCR, and Western blotting demonstrated aldosterone-induced increases in COX-2, NOX-2, and NOX-4 expression. Inhibition of COX-2 (NS398), NADPH oxidase (apocynin), or a combination blocked aldosterone-induced O(2)(-) production to the same degree. These data suggest that aldosterone-stimulated MD O(2)(-) production is mediated by COX-2 and NADPH oxidase. Next, COX-2 small-interfering RNA (siRNA) specifically decreased COX-2 mRNA without affecting NOX-2 or NOX-4 mRNAs. In the presence of the COX-2 siRNA, the aldosterone-induced increases in COX-2, NOX-2, and NOX-4 mRNAs and O(2)(-) production were completely blocked, suggesting that COX-2 causes increased expression of NOX-2 and NOX-4. In conclusion 1) MD cells express MR; 2) aldosterone increases O(2)(-) production by activating MR; and 3) aldosterone stimulates COX-2, which further activates NOX-2 and NOX-4 and generates O(2)(-). The resulting balance between O(2)(-) and NO in the MD is important in modulating TGF.     

17beta-Estradiol reverses shear-stress-mediated low density lipoprotein modifications

Within arterial bifurcations or branching points, oscillatory shear stress (OSS) induces oxidative stress mainly via the reduced nicotinamide adenine dinucleodtide phosphate (NADPH) oxidase system. It is unknown whether 17beta-estradiol (E(2)) can regulate OSS-mediated low-density lipoprotein (LDL) modifications. Bovine aortic endothelial cells were pretreated with E(2) at 5 nmol/L, followed by exposure to OSS (0 +/- 3.0 dynes/cm(2) s and 60 cycles/min) in a flow system. E(2) decreased OSS-mediated NADPH oxidase mRNA expression, and E(2)-mediated (.-)NO production was mitigated by the NO synthase inhibitor N(G)-nitro-l-argenine methyl ester. The rates of O(2)(-.) production in response to OSS increased steadily as determined by superoxide-dismutase-inhibited ferricytochrome c reduction; whereas, pretreatment with E(2) decreased OSS-mediated O(2)(-.) production (n = 4, p < 0.05). In the presence of native LDL (50 microg/mL), E(2) also significantly reversed OSS-mediated LDL oxidation as determined by high-performance liquid chromatography. In the presence of O(2)(-.) donor, xanthine oxidase (XO), E(2) further reversed XO-induced LDL lipid peroxidation (n = 3, p < 0.001). Mass spectra acquired in the m/z 400-1800 range, revealed XO-mediated LDL protein nitration involving tyrosine 2535 in the alpha-2 domains, whereas pretreatment with E(2) reversed nitration, as supported by the changes in nitrotyrosine intensities. Thus, E(2) plays an indirect antioxidative role. In addition to upregulation of endothelial (.-)NO synthase and downregulation of Nox4 expression, E(2) influences LDL modifications via lipid peroxidation and protein nitration.     

Is reduction of the sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide a reliable measure of intracellular superoxide production?

The sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide (XTT) is advantageous in that it yields a water-soluble formazan, unlike most other available tetrazoliums. XTT is reducible by superoxide, as are other tetrazoliums, but is not directly reduced by xanthine oxidase plus xanthine or by glucose oxidase plus glucose. This led to the suggestion that XTT reduction might serve as a reliable index of intracellular O(2)(-) production. We now show that soluble extracts of Escherichia coli contain two NADPH:XTT reductases that act aerobically or anaerobically. That being the case, XTT reduction is not a reliable measure of intracellular O(2)(-).     

Apocynin improves oxygenation and increases eNOS in persistent pulmonary hypertension of the newborn

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.     

Endothelium-specific sepiapterin reductase deficiency in DOCA-salt hypertension

The endothelial nitric oxide synthase (eNOS) requires tetrahydrobiopterin (H(4)B) as a cofactor and, in its absence, produces superoxide (O(2)(·-)) rather than nitric oxide (NO(·)), a condition referred to as eNOS uncoupling. DOCA-salt-induced hypertension is associated with H(4)B oxidation and uncoupling of eNOS. The present study investigated whether administration of sepiapterin or H(4)B recouples eNOS in DOCA-salt hypertension. Bioavailable NO(·) detected by electron spin resonance was markedly reduced in aortas of DOCA-salt hypertensive mice. Preincubation with sepiapterin (10 μmol/l for 30 min) failed to improve NO(·) bioavailability in hypertensive aortas while it augmented NO(·) production from control vessels, implicating a hypertension-associated deficiency in sepiapterin reductase (SPR), the rate-limiting enzyme for sepiapterin conversion to H(4)B. Indeed, a decreased SPR expression was observed in aortic endothelial cells, but not in endothelium-denuded aortic remains, implicating an endothelium-specific SPR deficiency. Administration of hypertensive aortas with H(4)B (10 μmol/l, 30 min) partially restored vascular NO(·) production. Combined administration of H(4)B and the NADPH oxidase inhibitor apocynin (100 μmol/l, 30 min) fully restored NO(·) bioavailability while reducing O(2)(·-) production. In angiotensin II-induced hypertension, however, aortic endothelial SPR expression was not affected. In summary, administration of sepiapterin is not effective in recoupling eNOS in DOCA-salt hypertension, due to an endothelium-specific loss in SPR, whereas coadministration of H(4)B and apocynin is highly efficient in recoupling eNOS. This is consistent with our previous observations that in angiotensin II hypertension, endothelial deficiency in dihydrofolate reductase is alternatively responsible for uncoupling of eNOS. Taken together, these data indicate that strategies specifically targeting at different H(4)B metabolic enzymes might be necessary in restoring eNOS function in different types of hypertension.     

A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis

Inorganic nitrite (NO(2)(-)) is emerging as a regulator of physiological functions and tissue responses to ischemia, whereas the more stable nitrate anion (NO(3)(-)) is generally considered to be biologically inert. Bacteria express nitrate reductases that produce nitrite, but mammals lack these specific enzymes. Here we report on nitrate reductase activity in rodent and human tissues that results in formation of nitrite and nitric oxide (NO) and is attenuated by the xanthine oxidoreductase inhibitor allopurinol. Nitrate administration to normoxic rats resulted in elevated levels of circulating nitrite that were again attenuated by allopurinol. Similar effects of nitrate were seen in endothelial NO synthase-deficient and germ-free mice, thereby excluding vascular NO synthase activation and bacteria as the source of nitrite. Nitrate pretreatment attenuated the increase in systemic blood pressure caused by NO synthase inhibition and enhanced blood flow during post-ischemic reperfusion. Our findings suggest a role for mammalian nitrate reduction in regulation of nitrite and NO homeostasis.     

Early determinants of H2O2-induced endothelial dysfunction

Reactive oxygen species (ROS) can stimulate nitric oxide (NO(*)) production from the endothelium by transient activation of endothelial nitric oxide synthase (eNOS). With continued or repeated exposure, NO(*) production is reduced, however. We investigated the early determinants of this decrease in NO(*) production. Following an initial H(2)O(2) exposure, endothelial cells responded by increasing NO(*) production measured electrochemically. NO(*) concentrations peaked by 10 min with a slow reduction over 30 min. The decrease in NO(*) at 30 min was associated with a 2.7-fold increase in O(2)(*-) production (p < 0.05) and a 14-fold reduction of the eNOS cofactor, tetrahydrobiopterin (BH(4), p < 0.05). Used as a probe for endothelial dysfunction, the integrated NO(*) production over 30 min upon repeated H(2)O(2) exposure was attenuated by 2.1-fold (p = 0.03). Endothelial dysfunction could be prevented by BH(4) cofactor supplementation, by scavenging O(2)(*-) or peroxynitrite (ONOO(-)), or by inhibiting the NADPH oxidase. Hydroxyl radical (()OH) scavenging did not have an effect. In summary, early H(2)O(2)-induced endothelial dysfunction was associated with a decreased BH(4) level and increased O(2)(*-) production. Dysfunction required O(2)(*-), ONOO(-), or a functional NADPH oxidase. Repeated activation of the NADPH oxidase by ROS may act as a feed forward system to promote endothelial dysfunction.     

Diabetes induces pulmonary artery endothelial dysfunction by NADPH oxidase induction

Recent data suggest that diabetes is a risk factor for pulmonary hypertension. The aim of the present study was to analyze whether diabetes induces endothelial dysfunction in pulmonary arteries and the mechanisms involved. Male Sprague-Dawley rats were randomly divided into a control (saline) and a diabetic group (70 mg/kg(-1) streptozotocin). After 6 wk, intrapulmonary arteries were mounted for isometric tension recording, and endothelial function was tested by the relaxant response to acetylcholine. Protein expression and localization were measured by Western blot and immunohistochemistry and superoxide production by dihydroethidium staining. Pulmonary arteries from diabetic rats showed impaired relaxant response to acetylcholine and reduced vasoconstrictor response to the nitric oxide (NO) synthase inhibitor L-NAME, whereas the response to nitroprusside and the expression of endothelial NO synthase remained unchanged. Endothelial dysfunction was reversed by addition of superoxide dismutase or the NADPH oxidase inhibitor apocynin. An increase in superoxide production and increased expression of the NADPH oxidase regulatory subunit p47(phox) were also found in pulmonary arteries from diabetic rats. In conclusion, the pulmonary circulation is a target for diabetes-induced endothelial dysfunction via enhanced NADPH oxidase-derived superoxide production.     

High-fat diet-induced reduction in nitric oxide-dependent arteriolar dilation in rats: role of xanthine oxidase-derived superoxide anion

Obesity frequently leads to the development of hypertension. We hypothesized that high-fat diet (HFD)-induced obesity impairs the endothelium-dependent dilation of arterioles. Male Wistar rats were fed with normal (control) or HFD (60% of saturated fat, for 10 wk). In rats with HFD, body weight, mean arterial blood pressure, and serum insulin, cholesterol, and glucose were elevated. In isolated gracilis muscle arterioles (diameter: approximately 160 microm) of HFD, rat dilations to ACh (at 1 microM, maximum: 83 +/- 3%) and histamine (at 10 microM, maximum: 16 +/- 4%) were significantly (P < 0.05) decreased compared with those of control responses (maximum: 90 +/- 2 and 46 +/- 4%, respectively). Dilations to the NO donor sodium nitroprusside were similar in the two groups. Inhibition of NO synthesis by N(omega)-nitro-l-arginine methyl ester reduced ACh- and histamine-induced dilations in control arterioles but had no effect on microvessels of HFD rats. The superoxide dismutase mimetic Tiron or xanthine oxidase inhibitor allopurinol enhanced ACh (maximum: 90 +/- 2 and 93 +/- 2%, respectively)- and histamine (maximum: 30 +/- 7 and 37 +/- 8%, respectively)-induced dilations in HFD arterioles, whereas the NAD(P)H oxidase inhibitor apocynin had no significant effect. Correspondingly, in carotid arteries of HFD rats, an enhanced superoxide production was shown by lucigenin-enhanced chemiluminescence, in association with an increased xanthine oxidase, but not NAD(P)H oxidase activity. In addition, a marked xanthine oxidase immunostaining was detected in the endothelial layer of the gracilis arterioles of HFD, but not in control rats. These findings suggest that, in obese rats, NO mediation of endothelium-dependent dilation of skeletal muscle arterioles is reduced because of an enhanced xanthine oxidase-derived superoxide production. These alterations demonstrate substantial dysregulation of arteriolar tone by the endothelium in HFD-induced obesity, which may contribute to disturbed tissue blood flow and development of increased peripheral resistance.     

NADPH oxidase contributes to renal damage and dysfunction in Dahl salt-sensitive hypertension

The goal of this study was to test the hypothesis that NADPH oxidase contributes importantly to renal cortical oxidative stress and inflammation, as well as renal damage and dysfunction, and increases in arterial pressure. Fifty-four 7- to 8-wk-old Dahl salt-sensitive (S) or R/Rapp strain rats were maintained for 5 wk on a high sodium (8%) or high sodium + apocynin (1.5 mmol/l in drinking water). Arterial and venous catheters were implanted on day 21. By day 35 in the high-Na S rats, mRNA expression of renal cortical gp91phox, p22phox, p47phox, and p67phox NADPH subunits in S rats increased markedly, and treatment of high-Na S rats with the NADPH oxidase inhibitor apocynin resulted in significant decreases in mRNA expression of these NADPH oxidase subunits. At the same time, in apocynin-treated S rats 1) renal cortical GSH/GSSG ratio increased, 2) renal cortical O2(.-) release and NADPH oxidase activity decreased, and 3) renal glomerular and interstitial damage markedly fell. Apocynin also decreased renal cortical monocyte/macrophage infiltration, and apocynin, but not the xanthine oxidase inhibitor allopurinol, attenuated decreases in renal hemodynamics and lowered arterial pressure. These data suggest that NADPH oxidase plays an important role in causing renal cortical oxidative stress and inflammation, which lead to decreases in renal hemodynamics, renal cortical damage, and increases in arterial pressure.