Effect of the NADPH oxidase inhibitor apocynin on ischemia-reperfusion lung injury

Apocynin (4-hydroxy-3-methoxy-acetophenone) inhibits NADPH oxidase in activated polymorphonuclear (PMN) leukocytes, preventing the generation of reactive oxygen species. To determine if apocynin attenuates ischemia-reperfusion lung injury, we examined the effects of apocynin (0.03, 0.3, and 3 mM) in isolated in situ sheep lungs. In diluent-treated lungs, reperfusion with blood (180 min) after 30 min of ischemia (ventilation 28% O(2), 5% CO(2)) caused leukocyte sequestration in the lung and increased vascular permeability [reflection coefficient for albumin (sigma(alb)) 0.47 +/- 0.10, filtration coefficient (K(f)) 0.14 +/- 0.03 g. min(-1). mmHg(-1). 100 g(-1)] compared with nonreperfused lungs (sigma(alb) 0.77 +/- 0. 03, K(f) 0.03 +/- 0.01 g. min(-1). mmHg(-1). 100 g(-1); P < 0.05). Apocynin attenuated the increased protein permeability at 0.3 and 3 mM (sigma(alb) 0.69 +/- 0.05 and 0.91 +/- 0.03, respectively, P < 0. 05); K(f) was decreased by 3 mM apocynin (0.05 +/- 0.01 g. min(-1). mmHg(-1). 100 g(-1), P < 0.05). Diphenyleneiodonium (DPI, 5 microM), a structurally unrelated inhibitor of NADPH oxidase, worsened injury (K(f) 0.32 +/- 0.07 g. min(-1). mmHg(-1). 100 g(-1), P < 0.05). Neither apocynin nor DPI affected leukocyte sequestration. Apocynin and DPI inhibited whole blood chemiluminescence and isolated PMN leukocyte-induced resazurin reduction, confirming NADPH oxidase inhibition. Apocynin inhibited pulmonary artery hypertension and perfusate concentrations of cyclooxygenase metabolites, including thromboxane B(2). The cyclooxygenase inhibitor indomethacin had no effect on the increased vascular permeability, suggesting that cyclooxygenase inhibition was not the explanation for the apocynin results. Apocynin prevented ischemia-reperfusion lung injury, but the mechanism of protection remains unclear.     

Effect of eicosapentaenoic acid and docosahexaenoic acid on oxidative stress and inflammatory markers in treated-hypertensive type 2 diabetic subjects

n-3 fatty acids reduce the risk of cardiovascular disease via a number of possible mechanisms. Despite this, there has been concern that these fatty acids may increase lipid peroxidation. The data in vivo are inconclusive, due in part to limitations in the methodologies. In this regard, the measurement of F2-isoprostanes provides a reliable assessment of in vivo lipid peroxidation and oxidant stress. This study aimed to assess the effects of supplementation with purified eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), the two major n-3 fatty acids, on urinary F2-isoprostanes and markers of inflammation, in type 2 diabetic patients. In a double-blind, placebo controlled trial of parallel design, 59 nonsmoking, treated-hypertensive, type 2 diabetic subjects, were randomized to 4 g daily of purified EPA, DHA, or olive oil for 6 weeks, while maintaining their usual diet. F2-isoprostanes, measured using gas chromatography-mass spectrometry in 24 h urines and C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), were measured before and after intervention. Thirty-nine men and 12 women aged 61.2 +/- 1.2 years, with body mass index (BMI), 29.5 +/- 0.5 kg/m2; 24 h blood pressure, 138/73 mmHg; HbA1c, 7.3 +/- 0.1% and fasting glucose, 7.9 +/- 0.2 mmol/l completed the intervention. Baseline urinary F2-isoprostanes were positively associated with HbA1c (p=.011) and fasting glucose (p=.032). Relative to the olive oil group, postintervention urinary F2-isoprostanes were decreased 19% by EPA (p=.017) and 20% by DHA (p=.014). There were no significant changes in CRP, IL-6, and TNF-alpha following EPA or DHA supplementation. In regression analysis, Delta F2-isoprostanes were positively associated with Delta HbA1c (p=.007) independent of treatment group; and with Delta TNF-alpha (p=.034) independent of age, gender, BMI, and treatment group. There were no associations with Delta CRP or Delta IL-6. This study is the first report demonstrating that either EPA or DHA reduce in vivo oxidant stress without changing markers of inflammation, in treated hypertensive, type 2 diabetic subjects.     

Superoxide mediates acute renal vasoconstriction produced by angiotensin II and catecholamines by a mechanism independent of nitric oxide

NAD(P)H oxidases (NOX) and reactive oxygen species (ROS) are involved in vasoconstriction and vascular remodeling during hypertension produced by chronic angiotensin II (ANG II) infusion. These effects are thought to be mediated largely through superoxide anion (O(2)(-)) scavenging of nitric oxide (NO). Little is known about the role of ROS in acute vasoconstrictor responses to agonists. We investigated renal blood flow (RBF) reactivity to ANG II (4 ng), norepinephrine (NE, 20 ng), and alpha(1)-adrenergic agonist phenylephrine (PE, 200 ng) injected into the renal artery (ira) of anesthetized Sprague-Dawley rats. The NOX inhibitor apocynin (1-4 mg.kg(-1).min(-1) ira, 2 min) or the superoxide dismutase mimetic Tempol (1.5-5 mg.kg(-1).min(-1) ira, 2 min) rapidly increased resting RBF by 8 +/- 1% (P < 0.001) or 3 +/- 1% (P < 0.05), respectively. During NO synthase (NOS) inhibition (N(omega)-nitro-l-arginine methyl ester, 25 mg/kg iv), the vasodilation tended to increase (apocynin 13 +/- 4%, Tempol 10 +/- 1%). During control conditions, both ANG II and NE reduced RBF by 24 +/- 4%. Apocynin dose dependently reduced the constriction by up to 44% (P < 0.05). Similarly, Tempol blocked the acute actions of ANG II and NE by up to 48-49% (P < 0.05). In other animals, apocynin (4 mg.kg(-1).min(-1) ira) attenuated vasoconstriction to ANG II, NE, and PE by 46-62% (P < 0.01). During NOS inhibition, apocynin reduced the reactivity to ANG II and NE by 60-72% (P < 0.01), and Tempol reduced it by 58-66% (P < 0.001). We conclude that NOX-derived ROS substantially contribute to basal RBF as well as to signaling of acute renal vasoconstrictor responses to ANG II, NE, and PE in normal rats. These effects are due to O(2)(-) rather than H(2)O(2), occur rapidly, and are independent of scavenging of NO.     

Apocynin attenuates oxidative stress and hypertension in young spontaneously hypertensive rats independent of ADMA/NO pathway

Both NADPH oxidase-derived reactive oxygen species (ROS) and asymmetric dimethylarginine (ADMA) are increased in hypertension. Apocynin, an NADPH oxidase inhibitor, could inhibit ROS, thus we tested whether apocynin can block NADPH oxidase and prevent increases of ADMA and blood pressure (BP) in spontaneously hypertensive rats (SHRs). SHRs and Wistar Kyoto (WKY) rats, aged 4 weeks, were assigned to four groups: untreated SHRs and WKY rats, SHRs and WKY rats that received 2.5 mM apocynin for 8 weeks. BP was significantly higher in SHRs compared to WKY rats, which was attenuated by apocynin. Apocynin prevented p47phox translocation in SHR kidneys, but not the increase of superoxide and H(2)O(2). Additionally, apocynin did not protect SHRs against increased ADMA. Apocynin blocks NADPH oxidase to attenuate hypertension, but has little effect on the ADMA/nitric oxide (NO) pathway in young SHRs. The reduction of ROS and the preservation of NO simultaneously might be a better approach to restoring ROS-NO balance to prevent hypertension.     

Ozone-induced increase in exhaled 8-isoprostane in healthy subjects is resistant to inhaled budesonide

The aim of this study was to quantify lung oxidant stress after short-term ozone exposure as reflected by 8-isoprostane concentrations in exhaled breath condensate (EBC) and to investigate the effects of inhaled budesonide on this response. 8-Isoprostane is a prostaglandin-F(2 alpha) isomer that is formed in vivo by free radical-catalyzed peroxidation of arachidonic acid. EBC is a noninvasive method to collect airway secretions. We undertook a double-blind, randomized, placebo-controlled, crossover study with inhaled budesonide (800 microg) or placebo twice daily for 2 weeks prior to ozone exposure (400 parts per billion) for 2 h in nine healthy nonsmokers. Exhaled 8-isoprostane was measured by an enzyme immunoassay. 8-Isoprostane was increased 4 h after ozone exposure compared to pre-exposure values in both placebo (36.9 +/- 3.9 pg/ml, mean +/- SEM, vs. 16.9 +/- 0.7 pg/ml; p <.001) and budesonide groups (33.4 +/- 2.6 pg/ml vs. 15.8 +/- 0.3 pg/ml; p <.001). Pretreatment with budesonide did not affect the increases in 8-isoprostane (mean differences 3.4 pg/ml, 95% CI -8.9 to 15.7, p =.54). Short-term ozone exposure causes acute increase in lung oxidative stress as reflected by exhaled 8-isoprostane. This increase is resistant to pretreatment with a high dose of inhaled budesonide.     

Apocynin improves diaphragmatic function after endotoxin administration.

Free radicals are known to play an important role in modulating the development of respiratory muscle dysfunction during sepsis. Moreover, neutrophil numbers increase in the diaphragm after endotoxin administration. Whether or not superoxide derived from infiltrating white blood cells contributes to muscle dysfunction during sepsis is, however, unknown. The purpose of the present study was to examine the effect of apocynin, an inhibitor of the superoxide-generating neutrophil NADPH complex, on endotoxin-induced diaphragmatic dysfunction. We studied groups of rats given saline, endotoxin, apocynin, or both endotoxin and apocynin. Animals were killed 18 h after injection, a portion of the diaphragm was used to assess force generation, and the remaining diaphragm was used for determination of 4-hydroxynonenal (a marker of lipid peroxidation) and nitrotyrosine levels (a marker of free radical-mediated protein modification). We found that endotoxin reduced diaphragm force generation and that apocynin partially prevented this decrease [e.g., force in response to 20 Hz was 23 +/- 1 (SE), 12 +/- 2, 23 +/- 1, and 19 +/- 1 N/cm(2), respectively, for saline, endotoxin, apocynin, and endotoxin/apocynin groups; P < 0.001]. Apocynin also prevented endotoxin-mediated increases in diaphragm 4-hydroxynonenal and nitrotyrosine levels (P < 0.01). These data suggest that neutrophil-derived free radicals contribute to diaphragmatic dysfunction during sepsis.     

The effect of peroxynitrite on the catalytic activity of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is a key enzyme of the *NO/cGMP pathway. Many cardiovascular disorders are associated with reduced *NO-mediated effects, while vascular superoxide (O(2)*(-)) production is increased. Both radicals rapidly react to peroxynitrite. We investigated whether peroxynitrite affects the activity and protein expression of sGC in intact vascular preparations. Catalytic sGC activity and expression of the sGC-beta(1) subunit was measured by conversion of radiolabeled GTP and western blot, respectively, using cytosolic extracts from rat aorta that had been incubated for 4 h with *NO/O(2)*(-) systems (devoid of free *NO) generating either 0.13 microM or 7.5 microM peroxynitrite/min. Incubation of rat aorta with 0.13 microM peroxynitrite/min had no effect. In striking contrast, incubation with 7.5 microM peroxynitrite/min resulted in a shift of the concentration-response curve obtained with a *NO donor (p =.0004) and a reduction of maximal specific activity from 3579 +/- 495 to 2422 +/- 265 pmol cGMP/mg/min (p =.036). The expression of the sGC-beta(1) subunit was unchanged. Exposure of aorta to the O(2)*(-) component had no effect, while exposure to the *NO-component reduced sGC expression to 58.8 +/- 7% (p <.001) and maximal sGC activity from 4041 +/- 992 to 1429 +/- 491 pmol cGMP/mg/min (p =.031). These data suggest that continuous generation of extracellular peroxynitrite might interfere with the *NO/cGMP signaling in vascular cells.     

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.     

Role of oxidative stress in multiparity-induced endothelial dysfunction

Multiparity is associated with increased risk of cardiovascular disease. We tested whether multiparity induces oxidative stress in rat vascular tissue. Coronary arteries and thoracic aorta were isolated from multiparous and age-matched virgin rats. Relaxation to ACh and sodium nitroprusside (SNP) was measured by wire myography. We also tested the effect of the superoxide dismutase mimetic MnTE2PyP (30 microM), the NADPH oxidase inhibitor apocynin (10 microM), and the peroxynitrite scavenger FeTPPs (10 microM) on ACh-mediated relaxation in coronary arteries. Vascular superoxide anion was measured using the luminol derivative L-012 and nitric oxide (NO) generation by the Griess reaction. Multiparity reduced maximal response and sensitivity to ACh in coronary arteries [maximal relaxation (E(max)): multiparous 49+/-3% vs. virgins 95%+/-3%; EC(50): multiparous 135+/-1 nM vs. virgins 60+/-1 nM], and in aortic rings (E(max): multiparous 38+/-3% vs. virgins 79+/-4%; EC(50): multiparous 160+/-2 nM vs. virgins 90+/-3 nM). Coronary arteries from the two groups relaxed similarly to SNP. Superoxide anions formation was significantly higher in both coronary arteries (2.8-fold increase) and aorta (4.1-fold increase) from multiparous rats compared with virgins. In multiparous rats, incubation with MnTE2PyP, apocynin, and FeTPPs improved maximal relaxation to ACh (MnTE2PyP: 74+/-5%; vehicle: 41+/-5%; apocynin: 73+/-3% vs. vehicle: 41+/-3%; FeTPPs: 72+/-3% vs. vehicle: 46+/-3%) and increased sensitivity (EC(50): MnTE2PyP: 61+/-0.5 nM vs. vehicle: 91+/-1 nM; apocynin: 45+/-3 nM vs. vehicle: 91+/-6 nM; FeTPP: 131 +/- 2 nM vs. vehicle: 185+/-1 nM). Multiparity also reduced total nitrate/nitrite levels (multiparous: 2.5+/-2 micromol/mg protein vs. virgins: 7+/-1 micromol/mg protein) and endothelial nitric oxide synthase protein levels (multiparous: 0.53+/-0.1 protein/actin vs. virgins: 1.0+/-0.14 protein/actin). These data suggest that multiparity induces endothelial dysfunction through decreased NO bioavailability and increased reactive oxygen species formation.     

Effect of thromboxane antagonists on ozone-induced airway responses in dogs

Airway hyperresponsiveness after inhaled ozone in dogs may occur as a result of thromboxane release in the airway. In this study, two thromboxane receptor antagonists, L-655,240 and L-670,596, were used in doses that inhibit the response to an inhaled thromboxane mimetic, U-46619, to determine further the role of thromboxane in ozone-induced airway hyperresponsiveness. Dogs were studied on 2 days separated by 1 wk. On each day, the dogs inhaled ozone (3 ppm) for 30 min. On one randomly assigned day, 10 dogs received an infusion of L-655,240 (5 mg.kg-1.h-1) and 5 dogs received an infusion of L-670,596 (1 mg.kg-1.h-1); on the other day dogs received a control infusion. Airway responses to doubling doses of acetylcholine were measured before and after inhalation of ozone and were expressed as the concentration of acetylcholine giving a rise in resistance of 5 cmH2O.l-1.s from baseline (acetylcholine provocation concentration). The development of airway hyperresponsiveness after ozone was not inhibited by the thromboxane antagonists. The mean log difference in the acetylcholine provocative concentration before and after ozone on the L-655,240 treatment day was 0.62 +/- 0.12 (SE) and on the control day was 0.71 +/- 0.12 (P = 0.48); on the L-670,596 treatment day the mean log difference was 0.68 +/- 0.15 (SE) and on the control day it was 0.75 +/- 0.19 (P = 0.45). These results do not support an important role for thromboxane in causing ozone-induced airway hyperresponsiveness.     

Vildagliptin in drug-na?ve patients with type 2 diabetes: a 24-week, double-blind, randomized, placebo-controlled, multiple-dose study.

This 24-week double-blind, randomized, multicenter, placebo-controlled, parallel-group study was performed in 632 drug-na?ve patients with type 2 diabetes to assess efficacy and tolerability of vildagliptin (50 mg qd, 50 mg bid, or 100 mg qd). HbA1c decreased modestly in patients receiving placebo (Delta=-0.3+/-0.1%) and to a significantly greater extent in patients receiving vildagliptin 50 mg qd (Delta=-0.8+/-0 .1%), 50 mg bid (Delta=-0.8+/-0.1%), or 100 mg qd (Delta=-0.9+/-0.1%, p<0.01 for all groups VS. placebo) from an average baseline of 8.4%. In patients diagnosed >or=3 months before enrollment, HbA1c increased with placebo (Delta=+0.2+/-0.2%) and between-treatment differences (vildagliptin-placebo) were -0.8+/-0.2% (p<0.001), -0.7+/-0.2% (p=0.003), and -0.9+/-0.2% (p<0.001) with vildagliptin 50 mg qd, 50 mg bid, and 100 mg qd, respectively. There was no apparent dose-response in the overall population; however, in patients with high baseline HbA1c, there were greater reductions with either 100 mg dose regimen (Delta=-1.3+/-0.2% and -1.4+/-0.2%) compared to 50 mg qd (Delta=-0.8+/-0.1%). Body weight decreased modestly in all groups (by 0.3 to 1.8 kg). The incidence of adverse events was similar across all groups and 相似文献   

The oxidation of apocynin catalyzed by myeloperoxidase: proposal for NADPH oxidase inhibition

Apocynin has been used as an efficient inhibitor of the NADPH oxidase complex and its mechanism of inhibition is linked to prior activation through the action of peroxidases. Here we studied the oxidation of apocynin catalyzed by myeloperoxidase (MPO) and activated neutrophils. We found that apocynin is easily oxidized by MPO/H2O2 or activated neutrophils and has as products dimer and trimer derivatives. Since apocynin impedes the migration of the cytosolic component p47phox to the membrane and this effect could be related to its conjugation with essential thiol groups, we studied the reactivity of apocynin and its MPO-catalyzed oxidation products with glutathione (GSH). We found that apocynin and its oxidation products do not react with GSH. However, this thiol compound was efficiently oxidized by the apocynin radical during the MPO-catalyzed oxidation. We suggest that the reactivity of apocynin radical with thiol compounds could be involved in the inhibitory effect of this methoxy-catechol on NADPH oxidase complex.     

Apocynin inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH oxidase. Since it acts as a potent inhibitor in studies with neutrophils and macrophages, no inhibitory effect can often be found in non-phagocyte cells. In our experiments, apocynin even stimulated reactive oxygen species (ROS) production by vascular fibroblasts. Even when added to macrophages, apocynin initially caused an increase in ROS production. The inhibition of ROS formation followed, suggesting that in the presence of leukocyte myeloperoxidase and hydrogen peroxide, apocynin is converted to another compound. Apocynin pre-activated with H2O2 and horseradish peroxidase (HRP) inhibited ROS production immediately. In non-phagocytes, apocynin stimulated ROS production and no inhibition was observed even after 60 min. Apocynin treated with H2O2 and HRP, however, decreased ROS production in the same manner as in macrophages. The stimulatory effect on ROS production can be abolished by tiron and superoxide dismutase (SOD), suggesting that superoxide was the produced species. The effect of apocynin was inhibited by diphenylene iodinium (DPI), a non-scavenging NADPH oxidase inhibitor. It can be summarized that apocynin stimulates cell superoxide production. In the presence of peroxidase and hydrogen peroxide, however, it is converted into another compound that acts as an inhibitor of superoxide production. It strongly suggests that under conditions in vivo, apocynin can have opposite effects on phagocytes and non-phagocyte cells. It acts as an inhibitor of phagocyte NADPH oxidase but also as a ROS production stimulator in non-phagocyte cells.     

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.     

Effect of indomethacin on allergen-induced asthmatic responses

Previous studies have suggested that inhibition of the cyclooxygenase pathway of arachidonic acid metabolism may suppress the late asthmatic responses to inhaled allergen. Both human and animal studies have suggested that prostanoids may also be involved in increases in airway responsiveness after ozone and allergen. We studied seven atopic subjects, who had a dual asthmatic response to inhaled allergen, during a control period and then after pretreatment with indomethacin (50 mg) or placebo twice daily for 2 days, administered in a randomized, double-blind manner. Indomethacin had no significant effect on the base-line airway responsiveness to histamine (P = 0.22) or the allergen-induced early or late asthmatic response (P = 0.49). However, indomethacin inhibited the increase in airway responsiveness (express as histamine PC20) after allergen inhalation. The log difference in preallergen to postallergen histamine PC20 was 0.49 +/- 0.08 (SE) during the control period, 0.46 +/- 0.08 (SE) after placebo (P = 0.81), and 0.22 +/- 0.10 (SE) after indomethacin (P = 0.02). Although indomethacin is useful for examining the role of cyclooxygenase products in asthmatic responses, it should not be considered in the treatment of asthma. We conclude that cyclooxygenase products are not significant mediators of allergen-induced early or late asthmatic responses but are involved in the pathogenesis of airway hyperresponsiveness after allergen inhalation.     

The effects of interval training on oxygen pulse and performance in supra-threshold runs

The aim of this study was to examine (i) the effects of a severe interval training period on oxygen pulse kinetics (O2-p, the ratio between VO2 and heart rate), and (ii) to study the consequences of these effects on the variation of performance (time to exhaustion) during severe runs. Seven athletes were tested before and after an eight-weeks period of a specific intermittent training at v Delta 50, i.e., the intermediate velocity between the lactate threshold (vLT) and the velocity associated with VO2max (vVO2max ). During the test sessions, athletes performed an incremental test and an all-out test at the pretraining v Delta 50. After the training period they also completed an additional all-out test at the posttraining v Delta 50 (v Delta 50bis). Results showed that after training there was i) an increase in the O2-p maximal value during the incremental test (22.7 +/- 1.5 mlO2.b-1 vs. 20.6 +/- 1.5 mlO2.b-1; p < 0.04), ii) a decrease in the time to reach the O2-p steady state (TRO2-p ) at the same absolute v Delta 50 (33 +/- 7 s vs. 60 +/- 27 s; p < 0.04) and iii) an increase in the O2-p steady state duration (TSSO2-p) at the same absolute v Delta 50 (552 +/- 201 s vs. 407 +/- 106 s; p < 0.04). However, there was no relationship between the improvement of these two O 2 -p kinetics parameters (TRO2-p and TSS O2-p) and those of the performance. This study found that after an individualised interval-training program conducted at the same absolute velocity, the O2-p kinetics reached a steady state quicker and for a longer duration than before training. This is however not related with the improvement of performance.     

Apocynin stimulates osteoblast differentiation and inhibits bone-resorbing mediators in MC3T3-E1 cells

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH-oxidase. In the present study, the effect of apocynin on the function of osteoblastic MC3T3-E1 cells was studied. Apocynin caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and mineralization in the cells (P < 0.05). Antimycin A (AMA), which inhibits complex III of the electron transport system, has been used as a reactive oxygen species (ROS) generator in biological systems. We exposed cultured osteoblastic MC3T3-E1 cells to AMA with or without pretreatment with apocynin. Apocynin significantly (P < 0.05) increased cell survival, calcium deposition, and osteoprotegerin release and decreased the production of ROS and osteoclast differentiation inducing factors such as TNF-α, IL-6, and receptor activator of nuclear factor-kB ligand (RANKL) in the presence of AMA. These results demonstrate that apocynin can protect osteoblasts from mitochondrial dysfunction-induced toxicity and may have positive effects on skeletal structure.     

Inorganic arsenic activates reduced NADPH oxidase in human primary macrophages through a Rho kinase/p38 kinase pathway

Inorganic arsenic is an immunotoxic environmental contaminant to which millions of humans are chronically exposed. We recently demonstrated that human primary macrophages constituted a critical target for arsenic trioxide (As(2)O(3)), an inorganic trivalent form. To specify the effects of arsenic on macrophage phenotype, we investigated in the present study whether As(2)O(3) could regulate the activity of NADPH oxidase, a major superoxide-generating enzymatic system in human phagocytes. Our results show that superoxide levels were significantly increased in a time-dependent manner in blood monocyte-derived macrophages treated with 1 muM As(2)O(3) for 72 h. Concomitantly, As(2)O(3) induced phosphorylation and membrane translocation of the NADPH oxidase subunit p47(phox) and it also increased translocation of Rac1 and p67(phox). Apocynin, a selective inhibitor of NADPH oxidases, prevented both p47(phox) translocation and superoxide production. NADPH oxidase activation was preceded by phosphorylation of p38-kinase in As(2)O(3)-treated macrophages. The p38-kinase inhibitor SB-203580 prevented phosphorylation and translocation of p47(phox) and subsequent superoxide production. Pretreatment of macrophages with the Rho-kinase inhibitor Y-27632 was found to mimic inhibitory effects of SB-203580 and to prevent As(2)O(3)-induced phosphorylation of p38 kinase. Treatment with As(2)O(3) also resulted in an increased secretion of the proinflammatory chemokine CCL18 that was fully inhibited by both apocynin and SB-203580. Taken together, our results demonstrate that As(2)O(3) induced a marked activation of NADPH oxidase in human macrophages, likely through stimulation of a Rho-kinase/p38-kinase pathway, and which may contribute to some of the deleterious effects of inorganic arsenic on macrophage phenotype.     

Bioavailability of apocynin through its conversion to glycoconjugate but not to diapocynin

Apocynin (4-hydroxy-3-methoxyacetophenone) is a major active ingredient from the rhizomes of Picrorhiza kurroa, a botanical plant used as an herbal medicine for treatment of a number of inflammatory diseases. Recently, apocynin is regarded as a specific inhibitor for NADPH oxidase in cell and animal models. In vitro studies indicated conversion of apocynin to diapocynin in the presence of peroxidases, e.g., myloperoxidase, posing the possibility that diapocynin also contributes to the anti-oxidative action of apocynin. The objectives of this study are to examine the bioavailability of apocynin to plasma, liver and brain tissue after intraperitoneal (i.p.) injection, and to examine whether apocynin is converted to diapocynin in vivo. Diapocynin was chemically synthetized and characterized by NMR and IR. Apocynin (5 mg/kg body wt) was injected i.p. to adult male Sprague-Dawley rats and plasma, liver and brain were collected at different times (30 min, 1 and 2 h) after injection. Samples were treated with β-glucuronidase to hydrolyze the glycosyl linkage and analyzed by HPLC/MS. At 30 min and 1 h after injection, approximately 50% of apocynin was converted to its glycosyl derivative and was distributed in plasma, liver and brain. No diapocynin was detected in any samples. These results indicate rapid glycosylation of apocynin and its transport to blood and other organs but no apparent conversion to diapocynin in vivo.     

Selective inhibition by apocynin of the proliferation and adhesion to fibronectin of v-H-ras-transformed 3Y1 cells

We determined the effects of apocynin, a representative inhibitor of NADPH oxidase, on the proliferative and adhesive properties of 3Y1 rat fibroblasts and the 3Y1 v-H-ras-transformed derivative, HR-3Y1-2. Apocynin inhibited the proliferation of HR-3Y1-2 but not 3Y1 cells at 10 μM and 100 μM. Apocynin also decreased the intracellular reactive oxygen species (ROS) level in HR-3Y1-2 but not 3Y1 cells. We also evaluated the effects of apocynin on cell adhesion to fibronectin and found decreased adhesion of HR-3Y1-2 cells to fibronectin-coated plates. Our results indicate that apocynin selectively down-regulated β1-integrin cell surface expression on the HR-3Y1-2 cells. It also inhibited the migration and invasion of these cells. These data suggest that reducing the production of NADPH oxidase-mediated ROS could be an effective means for ameliorating the abnormal growth, adhesion and motility of v-H-ras-transformed cells.