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.     

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.     

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.     

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.     

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.     

Possible usefulness of apocynin, an NADPH oxidase inhibitor, for nitrate tolerance: prevention of NO donor-induced endothelial cell abnormalities

The long-term benefits of nitroglycerin therapy are limited by tolerance development. Understanding the precise nature of mechanisms underlying nitroglycerin-induced endothelial cell dysfunction may provide new strategies to prevent tolerance development. In this line, we tested interventions to prevent endothelial dysfunction in the setting of nitrate tolerance. When bovine aortic endothelial cells (BAECs) were continuously treated with nitric oxide (NO) donors, including nitroglycerin, over 2-3 days, basal production of nitrite and nitrate (NO(x)) was diminished. The diminished basal NO(x) levels were mitigated by intermittent treatment allowing an 8-h daily nitrate-free interval during the 2- to 3-day treatment period. Addition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin restored the basal levels of NO(x) that were decreased by continuous nitroglycerin treatment of BAECs. Apocynin caused significant improvement of increased mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in BAECs given nitroglycerin continuously over the treatment period. Apocynin also reduced endothelial production of reactive oxygen species (ROS) after continuous nitroglycerin treatment. These results showed an essential similarity to the effects of a nitrate-free interval. Application of the NOS inhibitor N(omega)-nitro- l-arginine methyl ester caused a recovery effect on basal NO(x) and eNOS expression but was without effect on ROS levels in continuously NO donor-treated BAECs. In conclusion, the present study characterized abnormal features and functions of endothelial cells following continuous NO donor application. We suggest that inhibition of NADPH oxidase, by preventing NO donor-induced endothelial dysfunction, may represent a potential therapeutic strategy that confers protection from nitrate tolerance development.     

Lymphocyte migration through monolayers of endothelial cell lines involves VCAM-1 signaling via endothelial cell NADPH oxidase

Lymphocytes migrate from the blood across endothelial cells to reach foreign substances sequestered in peripheral lymphoid organs and inflammatory sites. To study intracellular signaling in endothelial cells during lymphocyte migration, we used murine endothelial cell lines that promote lymphocyte migration and constitutively express VCAM-1. The maximum rate of resting splenic lymphocyte migration across monolayers of the endothelial cells occurred at 0-24 h. This migration was inhibited by anti-VCAM-1 or anti-alpha4 integrin, suggesting that VCAM-1 adhesion was required for migration. To determine whether signals within the endothelial cells were required for migration, irreversible inhibitors of signal transduction molecules were used to pretreat the endothelial cell lines. Inhibitors of NADPH oxidase activity (diphenyleneiodonium and apocynin) blocked migration >65% without affecting adhesion. Because NADPH oxidase catalyzes the production of reactive oxygen species (ROS), we examined whether ROS were required for migration. Scavengers of ROS inhibited migration without affecting adhesion. Furthermore, VCAM-1 ligand binding stimulated NADPH oxidase-dependent production of ROS by the endothelial cells lines and primary endothelial cell cultures. Finally, VCAM-1 ligand binding induced an apocynin-inhibitable actin restructuring in the endothelial cell lines at the location of the lymphocyte or anti-VCAM-1-coated bead, suggesting that an NADPH oxidase-dependent endothelial cell shape change was required for lymphocyte migration. In summary, VCAM-1 signaled the activation of endothelial cell NADPH oxidase, which was required for lymphocyte migration. This suggests that endothelial cells are not only a scaffold for lymphocyte adhesion, but play an active role in promoting lymphocyte migration.     

Ischemia-activated microglia induces neuronal injury via activation of gp91phox NADPH oxidase

Although glial cells play a major role in the pathogenesis of many neurological diseases by exacerbating neuronal and non-neuronal cell death, the mechanisms involved are unclear. We examined the effects of microglia-(MCM) or astrocyte-(ACM) conditioned media obtained by chemical ischemia on the neuronal injury in SH-SY5Y cells. Chemical ischemia was induced by the treatment with NaN₃ and 2-deoxy-d-glucose for 2 h. MCM-treated SH-SY5Y cells showed reduced the viability, increased caspase-3 activity, decreased Bcl-2/Bax ratio, and increased cytochrome c release, increased inflammatory cytokines, and increased reactive oxygen species (ROS) generation. MCM also increased gp91phox nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which was inhibited by NADPH oxidase inhibitor, apocynin, and gp91phox siRNA. However, ACM did not show any significant changes. The results suggest that microglia activated by ischemic insult may increase reactive oxygen species generation via activation of gp91phox NADPH oxidase, resulting in neuronal injury.     

Reactive oxygen species are messengers in maintenance of human and guinea pig gallbladder tonic contraction

The tonic contraction of human and guinea pig gallbladder (GB) is dependent on basal levels of PGE(2) and thromboxane A(2) (TxA(2)). The pathway involved in the genesis of these prostaglandins has not been elucidated. We aimed to examine the source of reactive oxygen species (ROS) and whether they contribute to the genesis of GB tonic contraction by generating basal prostaglandin levels. Tonic contraction was studied in human and guinea pig GB muscle strips treated with ROS scavengers (Tiron and catalase), apocynin (an inhibitor of NADPH oxidase), and NOX-1 small interference RNA (siRNA). The subunits of NADPH oxidase and their functional roles were determined with specific antibodies in GB muscle cells. ROS scavengers reduced the GB tonic contraction and H(2)O(2) and PGE(2) levels. Apocynin also inhibited the tonic contraction. Antibodies against subunits of NADPH oxidase present in GB muscle cells lowered H(2)O(2) and PGE(2) levels. NOX-1 siRNA transfection reduced the tonic contraction, NOX-1 expression, and levels of H(2)O(2) and PGE(2). Tiron and apocynin inhibited the expected increase in tension and H(2)O(2) levels induced by stretching of muscle strips. H(2)O(2) increased the levels of PGE(2) and TxA(2) by increasing platelet-activating factor-like lipids that phosphorylate p38 and cPLA(2) sequentially. H(2)O(2) generated by NADPH oxidase participates in a signal transduction pathway that maintains the GB tonic contraction by activating PAF, p38, and cPLA(2) to generate prostaglandins.     

Subchronic apocynin treatment attenuates methamphetamine-induced dopamine release and hyperactivity in rats

Aims

The effects of methamphetamine are linked to stimulation of dopaminergic neurons, which can be accompanied by production of reactive oxygen species (ROS). Apocynin (4-hydroxy-3-methoxy-acetophenone) is a nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (NOX) inhibitor shown to mitigate oxidative stress in a number of models. The present study aimed at testing whether apocynin suppresses the dopamine-releasing and locomotor-activating properties of methamphetamine.

Main methods

(1) Apocynin (0.01–100 μM) was applied to rat striatal slices preloaded with [³H]dopamine and its efficacy to evoke [³H]overflow and to alter methamphetamine (3 μM)-evoked [³H]overflow was measured. (2) Groups of rats received apocynin (15 or 50 mg/kg/day) or vehicle injection for seven consecutive days, and the efficacy and potency of methamphetamine to evoke [³H]overflow were determined. (3) Groups of apocynin-treated rats were administered methamphetamine (0.5 or 1 mg/kg) or saline to determine the effect of apocynin on stimulant-induced hyperactivity.

Key findings

(1) Apocynin applied to striatal slices did not evoke [³H]overflow or alter methamphetamine-evoked [³H]overflow. (2) However, subchronic apocynin treatment significantly and dose-dependently decreased methamphetamine's potency and efficacy to evoke [³H]overflow. (3) Subchronic apocynin treatment also decreased the locomotor activity evoked by methamphetamine.

Significance

Subchronic apocynin treatment diminished methamphetamine induced dopamine-release and its locomotor-activating properties. The pattern of results indicates that apocynin is more effective after repeated, rather than after acute, treatment. The findings also suggest that NOX inhibitors or agents suppressing oxidative stress may constitute a new area for research to understand how methamphetamine produces its deleterious and neurotoxic outcomes in the brain.     

Angiopoietin-1-induced angiogenesis is modulated by endothelial NADPH oxidase

Reactive oxygen species (ROS) play a central role in the pathogenesis of many cardiovascular diseases, such as atherosclerosis and hypertension. Endothelial NADPH oxidase is the major source of intracellular ROS. The present study investigated the role of endothelial NADPH oxidase-derived ROS in angiopoietin-1 (Ang-1)-induced angiogenesis. Exposure of porcine coronary artery endothelial cells (PCAECs) to Ang-1 (250 ng/ml) for periods up to 30 min led to a transient and dose-dependent increase in intracellular ROS. Thirty minutes of pretreatment with the NADPH oxidase inhibitors diphenylene iodinium (DPI, 10 microM) and apocynin (200 microM) suppressed Ang-1-stimulated ROS. Pretreatment with either DPI or apocynin also significantly attenuated Ang-1-induced Akt and p44/42 MAPK phosphorylation. In addition, inhibition of NADPH oxidase significantly suppressed Ang-1-induced endothelial cell migration and sprouting from endothelial spheroids. Using mouse heart microvascular endothelial cells from wild-type (WT) mice and mice deficient in the p47(phox) component of NADPH oxidase (p47(phox-/-)), we found that although Ang-1 stimulated intracellular ROS, Akt and p42/44 MAPK phosphorylation, and cell migration in WT cells, the responses were strikingly suppressed in cells from the p47(phox-/-) mice. Furthermore, exposure of aortic rings from p47(phox-/-) mice to Ang-1 demonstrated fewer vessel sprouts than WT mice. Inhibition of the Tie-2 receptor inhibited Ang-1-induced endothelial migration and vessel sprouting. Together, our data strongly suggest that endothelial NADPH oxidase-derived ROS play a critical role in Ang-1-induced angiogenesis.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which is inhibited by resveratrol

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. Hyperglycemia-induced oxidative stress is implicated in the etiology of diabetic nephropathy. Resveratrol has potent antioxidative and protective effects on diabetic nephropathy. We aimed to examine whether high glucose (HG)-induced NADPH oxidase activation and reactive oxygen species (ROS) production contribute to glomerular mesangial cell proliferation and fibronectin expression and the effect of resveratrol on HG action in mesangial cells. By using rat mesangial cell line and primary mesangial cells, we found that NADPH oxidase inhibitor (apocynin) and ROS inhibitor (N-acetyl cysteine) both inhibited HG-induced mesangial cell proliferation and fibronectin expression. HG-induced elevation of NADPH oxidase activity and production of ROS in mesangial cells was inhibited by apocynin. These results suggest that HG induces mesangial cell proliferation and fibronectin expression through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunits p22(phox) and p47(phox) expression through JNK/NF-κB pathway, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced mesangial cell proliferation and fibronectin expression through inhibiting HG-induced JNK and NF-κB activation, NADPH oxidase activity elevation and ROS production. These results demonstrate that HG enhances mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide novel therapeutic targets for diabetic nephropathy.     

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.     

Endogenous reactive oxygen species are essential for proliferation of neural stem/progenitor cells

It is widely thought that accumulation of reactive oxygen species (ROS) causes injury to cells. In this study, we investigated the effect of endogenous ROS on the proliferation of neural stem/progenitor cells derived from the hippocampus of embryonic mice. The cells were treated with free radical-scavenging agents [3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone) or 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol)], an NADPH oxidase inhibitor (apocynin), catalase, a nitric oxide synthase inhibitor [N^ω-nitro-l-arginine methyl ester hydrochloride (L-NAME)] or a peroxynitrite generator (SIN-1) during the culture period. Edaravone and tempol had the ability to decrease endogenous ROS in the cells exposed for periods from 1 to 24 h, with attenuation of the proliferation activity of the cells during culture. Apocynin and L-NAME were also effective in attenuating cell proliferation but not cellular damage. Conversely, SIN-1 was capable of promoting the proliferation activity. However, catalase had no effect on the proliferation activity of the cells during culture. Furthermore, tempol significantly decreased the level of NFκB p65, phospho-cyclic AMP response element-binding protein, and β-catenin within the nucleus of the cells. These data suggest that endogenous ROS and nitric oxide are essential for the proliferation of embryonic neural stem/progenitor cells.     

Effect of apocynin on ozone-induced airway hyperresponsiveness to methacholine in asthmatics.

Apocynin is an inhibitor of NADPH oxidase present in inflammatory cells such as eosinophils and neutrophils. We investigated the effect of inhaled apocynin on ozone-induced bronchial hyperresponsiveness in vivo. Seven mild atopic asthmatics participated in a placebo-controlled, cross-over study with two exposures to O(3) at 2-week intervals. Apocynin (3 ml of 0.5 mg/ml) was inhaled 2 times before and 6 times after O(3) exposure at hourly intervals. At 36 h before and 16 h after O(3) exposure, methacholine inhalation challenge tests (Mch) were performed, and PC(20) and maximal % fall from baseline (MFEV(1)) were calculated from dose-response curves. O(3)-induced change in PC(20) (Delta PC(20)) after placebo treatment was -1.94 +/- 0.39 DD (mean +/- SEM doubling dose Mch) (p =.001) and apocynin was -0.6 +/- 0.33 DD (p =.17). The difference between apocynin and placebo treatment was 1.3 DD +/- 0.42 (p =.02). O(3)-induced Delta MFEV(1) was 11.9 +/- 1.5% (p =.008) during placebo inhalation and 3.85 +/- 1.8% during apocynin (p =.47). Apocynin reduced the Delta MFEV(1) by 8.05% compared to placebo (p =.025). We conclude that apocynin markedly reduced O(3)-induced hyperreactivity for Mch as well as maximal airway narrowing. The results suggest that apocynin may have a role in preventing ozone-induced exacerbations of asthma.     

NADPH oxidase controls EGF-induced proliferation via the ERK1/2-independent mechanism

Using HyPer, a ratiometric GFP-based biosensor, the dynamics of H2O2 in living cells has been studied. It was found that activation of the receptor of the epidermal growth factor (EGF) in epithelial cells leads to sustained generation of intracellular H2O2, which is blocked by apocynin, an inhibitor of the assembly of plasma membrane NADPH oxidase. Apocynin also blocked HeLa cell proliferation induced by EGF, indicating that NADPH oxidase should be involved in the process. However, apocynin failed to alter the kinetics of the EGF-stimulated ERK1/2 activation. It was concluded that NADPH oxidase and intracellular H2O2 are the important downstream targets of the EGF receptor, which mediate the proliferation response by mechanisms distinct from the activation of the classical ERK1/2 MAP-kinase pathway.