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PubMed2002

Heterologous expression and characterization of mouse spermine oxidase.

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.

Amino Acid Sequence↗
PubMed2003

Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells.

Superoxide production by phagocytes involves activation of a multi-component NADPH oxidase. Recently, several homologues of the catalytic component of the phagocyte oxidase, gp91phox, were identified in various tissues. Here we describe two proteins, p41 and p51, with significant homology to two cytosolic components of the phagocytic oxidase, p47phox and p67phox. Like p47phox, p41 contains an amino-terminal Phox homology domain, two SH3 domains, and a conserved carboxyl-terminal, proline-rich motif. Similarly, p51 is homologous to p67phox, containing four amino-terminal tetratrico-peptide repeats, a conserved "activation domain" motif, a PB1 domain, and a carboxyl-terminal SH3 domain. The highest levels of p41 transcript are detected in the colon and in other gastrointestinal tissues that express Nox1, the predominant gp91phox homologue in these tissues. In contrast, the p51 transcript showed a more widespread expression pattern, suggesting that it may support other tissue-specific oxidases. Mouse colon in situ hybridization detected both transcripts in the epithelial cells of colon crypts. Heterologous co-expression of p41 and p51 significantly enhances the superoxide-generating activity of Nox1-expressing cells; thus, p41 and p51 appear to be novel regulators of Nox1. These proteins also support the activity of gp91phox, albeit at much lower levels than the cytosolic phox counterparts. Our results suggest colon epithelial cells contain a multi-component NAD(P)H oxidase with a molecular architecture similar to the phagocytic oxidase.

Amino Acid Sequence↗
PubMed2003

Human monocytes use Rac1, not Rac2, in the NADPH oxidase complex.

Phagocyte NADPH oxidase is critical for defense against pathogens and contributes to inflammatory tissue injury. One component of the NADPH oxidase complex is the small GTP-binding protein Rac. There are two isoforms of Rac, and Rac2 is the predominant isoform in neutrophils and has been shown to be essential for NADPH oxidase activity. In primary human monocytes we report that in contrast to neutrophils, Rac1 is the predominantly expressed isoform. Upon monocyte activation by a variety of agents, we found that Rac1 dissociates from Rho GDP dissociation inhibitor (RhoGDI) and translocates to the membrane. We also found that Rac1 interacts with two other NADPH oxidase components, p67phox and p47phox, upon monocyte activation. These data indicate that Rac1, and not Rac2, is a component of the activated NADPH oxidase in monocytes. This finding suggests that it may be possible to selectively interfere with either monocyte or neutrophil NADPH oxidase activity, thereby selectively targeting chronic versus acute inflammatory processes.

Blotting, Western↗
PubMed1997

Effect of covalent modification on coproporphyrinogen oxidase from chicken red blood cells.

The biosynthesis of heme is a complex multi-step pathway requiring the efforts of eight enzymes. The initial enzymes in the heme biosynthetic pathway have been well characterized in relation to their mechanisms. Coproporphyrinogen oxidase (Copro'gen oxidase) is one of the last three enzymes in the pathway and is one of the least well understood. Copro'gen oxidase converts coproporphyrinogen III to protoporphyrinogen IX via oxidative decarboxylation of the 3- and 8-propionic side chain moieties. To further our understanding of the recognition and binding of substrate, Copro'gen oxidase was partially purified from chicken red blood cell hemolysates then incubated with covalent modifiers of specific amino acids. Incubation with tetranitromethane, p-hydroxyphenylglyoxal, N-acetylimidazole, or trinitrobenzenesulfonic acid resulted in substantial reduction of Copro'gen oxidase activity implying the presence of critical tyrosine, arginine and lysine residues. We conclude that these amino acids play important roles in the enzymic mechanism (for both binding and catalysis) of Copro'gen oxidase.

Animals↗
PubMed1986

Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue.

The localization of lysyl oxidase was examined in calf and rat aortic connective tissue at the ultrastructural level using polyclonal chicken anti-lysyl oxidase and gold conjugated rabbit anti-chicken immunoglobulin G to identify immunoreactive sites. Electron microscopy of calf aortic specimens revealed discrete gold deposits at the interface between extracellular bundles of amorphous elastin and the microfibrils circumferentially surrounding these bundles. The antibody did not react with microfibrils which were distant from the interface with elastin. There was negligible deposition of gold within the bundles of amorphous elastin and those few deposits seen at these sites appeared to be associated with strands of microfibrils. Lysyl oxidase was similarly localized in newborn rat aorta at the interface between microfibrils and nascent elastin fibers. Gold deposits were not seen in association with extracellular collagen fibers even after collagen-associated proteoglycans had been degraded by chondroitinase ABC. However, the antibody did recognize collagen-bound lysyl oxidase in collagen fibers prepared from purified collagen to which the enzyme had been added in vitro. No reaction product was seen if the anti-lysyl oxidase was preadsorbed with purified lysyl oxidase illustrating the specificity of the antibody probe. The present results are consistent with a model of elastogenesis predicting the radial growth of the elastin fiber by the deposition and crosslinking of tropoelastin units at the fiber-microfibril interface.

Amino Acid Oxidoreductases↗
PubMed2003

Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes.

Cytosolic phospholipase A2 (cPLA2)-generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.

Cell Membrane↗
PubMed1996

Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox.

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox-p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.

Cytosol↗
PubMed1957

The equilibrium between cytochrome oxidase and carbon monoxide.

An evolution argument which attempted to trace the development of hemoglobins from such respiratory pigments as cytochrome oxidase presupposed that the latter possesses, in addition to its high affinity for oxygen, an approximately hyperbolic equilibrium function, and little if any Bohr effect (decline in affinity for oxygen with rise in acidity). Since cytochrome oxidase, unlike hemoglobin, is irreversibly oxidized by oxygen, the present experiments examine its combination with carbon monoxide, with which, like hemoglobin, it yields a true equilibrium. In all known hemoglobins the form of the equilibrium function and the vigor of the Bohr effect are similar with carbon monoxide and with oxygen, so that observations involving the former gas are relevant to the relations of the latter. The equilibrium function of cytochrome oxidase with carbon monoxide-percentage saturation vs. partial pressure of CO-is slightly inflected (in the Hill equation n = 1.26; for a hyperbola, n = 1). No Bohr effect is present in the range of pH 7-8. The pressure of carbon monoxide at which half-saturation occurs (p(50)) is about 0.17 mm. at 10-13 degrees C. The affinity for carbon monoxide is therefore higher than commonly supposed. These properties are consistent with the evolution argument. They are important also for the physiological functioning of cytochrome oxidase, the nearly hyperbolic equilibrium function facilitating a high degree of saturation, and the lack of Bohr effect making this enzyme impervious to hyperacidity. The slight inflection of the equilibrium function shows that the Fe-porphyrin units of cytochrome oxidase interact to a degree, hence that the enzyme must contain more than one such unit per molecule. It is suggested that in cytochrome oxidase two Fe-porphyrin groups may unite with one oxygen in the manner Fe(++)-O(2)-Fe(++); and that the evolution of hemoglobins proceeded over a first stage in which the hemes were separated so that each combines with only one molecule of oxygen, so tending to remain reduced; to a further stage in which the separated hemes interact through the protein to facilitate one another in combining with oxygen.

Carbon Monoxide↗
PubMed2003

Role of NADPH oxidases in the control of vascular gene expression.

All vascular cells, including endothelial cells and smooth muscle cells, express components of the leukocyte NADPH oxidase such as p22phox, p47phox, and Rac. Endothelial cells and fibroblasts also express the leukocyte NADPH oxidase subunit gp91phox/nox2, whereas in smooth muscle cells nox1 and nox4 are found. The different vascular NADPH oxidases represent important sources for the basal as well as the agonist-induced superoxide anion (O(2) .-) generation in the vasculature. In vascular smooth muscle cells, activation of the NADPH oxidases and the subsequent formation of O(2) .- has been demonstrated for various agents including angiotensin II, thrombin, lysophosphatidylcholine, and tumor necrosis factor alpha. By influencing the activity of p38 mitogen-activated protein kinase and AKT, NADPH oxidase-derived O(2) .- increases the expression of several pro-arteriosclerotic genes, such as monocyte chemoattractant protein-1, tissue factor, and vascular endothelial growth factor. Thus, the vascular NADPH oxidases play an important role in mediating the signal transduction cascade of pro-arteriosclerotic stimuli.

Animals↗
PubMedDate not supplied

Single-cell optical imaging of the phagocyte NADPH oxidase.

The phagocyte NADPH oxidase is a key component of the innate immune response against invading microorganisms, because the generation of superoxide (O(2)(-)) inside the phagocytic vacuole by this enzyme is responsible for microbial killing by mechanisms that are directly or indirectly dependent on reactive oxygen species (ROS) formation. Most of what is known about the membrane-embedded and cytosolic NADPH oxidase subunits and their intricate network of interactions on assembly and activation has been derived from biochemical and biophysical studies involving subcellular fractionation or reconstituted cell-free systems. Such investigations can be complemented by single-cell microscopy on phagocytes, which may reveal spatial and/or temporal details about NADPH oxidase assembly that cannot be obtained from fractionated-cell assays. In recent years, we have investigated the NADPH oxidase in neutrophils using two complementary optical imaging techniques: Raman microscopy, a vibrational spectroscopic technique that does not require protein labeling, and live-cell fluorescence microscopy, which sheds light on the dynamics of NADPH oxidase assembly in individual cells. Here, we briefly introduce these techniques, compare their characteristics, and show their potential for studying NADPH oxidase at the single-cell level. New microscopy data are presented to illustrate the versatility of Raman and fluorescence microscopy on intact neutrophils.

Adult↗
PubMedDate not supplied

NADPH oxidases in the kidney.

NADPH oxidases have a distinct cellular localization in the kidney. Reactive oxygen species (ROS) are produced in the kidney by fibroblasts, endothelial cells (EC), vascular smooth muscle cells (VSMC), mesangial cells (MCs), tubular cells, and podocyte cells. All components of the phagocytic NADPH oxidase, as well as the Nox-1 and -4, are expressed in the kidney, with a prominent expression in renal vessels, glomeruli, and podocytes, and cells of the thick ascending limb of the loop of Henle (TAL), macula densa, distal tubules, collecting ducts, and cortical interstitial fibroblasts. NADPH oxidase activity is upregulated by prolonged infusion of angiotensin II (Ang II) or a high salt diet. Since these are major factors underlying the development of hypertension, renal NADPH oxidase may have an important pathophysiological role. Indeed, recent studies with small interference RNAs (siRNAs) targeted to p22( phox ) implicate p22( phox ) in Ang II-induced activation of renal NADPH oxidase and the development of oxidative stress and hypertension, while studies with apocynin implicate activation of p47( phox ) in the development of nephropathy in a rat model of type 1 diabetes mellitus (DM). Experimental studies of the distribution, signaling, and function of NADPH oxidases in the kidney are described.

Animals↗
PubMed2004

Isolation and characterization of the human monoclonal antibodies C10 in single-chain fragment variable (scFv) format to glucose oxidase from Aspergillus niger.

Despite biotechnological and clinical applications very few monoclonal antibodies (MAbs) directed to the enzyme glucose oxidase, have been produced so far because of the heavy side effects of the immunization schedule for conventional MAb preparation. In contrast, the phage display method allows for the selection of monoclonal human antibody fragments against any antigens, including toxic proteins. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human fragments recognizing glucose oxidase, we used the large synthetic ETH-2 library based on the principle of protein design. Phage displaying glucose oxidase reactive scFvs were obtained after three rounds of selection on glucose oxidase-coated immunotubes and subsequent amplification in TG1 E. coli cells. Eventually, one high reactive scFv clone was selected and further examined. The anti-glucose oxidase scFv C10 was found suitable for Western blot; Biacore analysis showed that the binding affinity of the glucose oxidase-reactive scFv is almost equal that of MAbs prepared with conventional hybridoma technology. Finally, the cDNA sequence of this human scFv may be exploited to generate bispecific antibodies to target in the tumor environment-specific toxic enzymatic reaction.

Antibodies, Monoclonal↗
PubMed1984

Effect of molybdenum-deficient and low iron diets on xanthine oxidase activity and iron status in rats.

We tested the hypothesis proposed by Topham, Woodruff and Walker that intestinal xanthine oxidase is important for iron absorption. We made weanling rats xanthine oxidase-deficient and measured their growth and iron status. There were no significant differences between control and xanthine oxidase-depleted rats in growth or iron absorption or a variety of measures of iron metabolism, except that xanthine oxidase-depleted rats accumulated nonheme iron in the liver. Iron deficiency caused a loss in intestinal xanthine oxidase activity, but also caused an increase in hepatic xanthine oxidase activity. This result may be important for understanding changes in purine and protein metabolism during iron deficiency.

Animals↗
PubMed1988

Induction of cytosolic activation factor for NADPH oxidase in differentiated HL-60 leukemia cells.

NADPH oxidase was induced in HL-60 human promyelocytic leukemia cells when these cells were treated with 10(-7) M 1,25-dihydroxyvitamin D3 (VD3) for 4 days. The treated cells were disrupted by sonication, and the postnuclear fraction was separated into 48,000 X g supernatant (cytosol) and precipitate (membrane) fractions. Membrane-bound NADPH oxidase was activated in vitro with SDS and cytosol. However, the cytosol from untreated HL-60 cells could not activate NADPH oxidase. The cytosolic activity was induced 2 days after VD3 treatment and fully expressed on day 4. The activity was heat-sensitive and destroyed by trypsin. The possibility that the cytosolic activation factor is a protein kinase C (PKC) was then tested. Ca2+- and phospholipid-dependent PKC activity was low in the cytosol of untreated HL-60 cells but increased in the cytosols of VD3-treated cells 4 and 11 times, respectively, 2 days and 4 days after treatment. H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], inhibited PKC activity in a dose-dependent manner at 1-100 microM. Cytosolic activity of NADPH oxidase was not inhibited at all at those concentrations. Furthermore, PKC activity was lost when Ca2+ was omitted from the assay mixture, but NADPH oxidase was activated in the presence of EGTA. These results indicated that the cytosolic factor is not a PKC, and that NADPH oxidase in this cell-free system is activated by a mechanism that does not involve PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2001

Ectopic expression of an Arabidopsis calmodulin-like domain protein kinase-enhanced NADPH oxidase activity and oxidative burst in tomato protoplasts.

Among plant defense responses to pathogen attack, the release of active oxygen species (AOS), termed the oxidative burst, may affect the attacking pathogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxidase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsis protein phosphatase 2A abolished this enhancement, whereas Arabidopsis dual-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect tMEK2MUT, a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity when overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2MUT, also enhanced production of AOS in intact protoplasts. Our results show that ectopic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.

Arabidopsis↗
PubMed1997

Xanthine oxidase mediates myocardial injury after hepatoenteric ischemia-reperfusion.

OBJECTIVES: To determine if myocardial injury results from hepatoenteric ischemia-reperfusion. We also proposed to determine if this remote heart injury is mediated by a xanthine oxidase-dependent mechanism. DESIGN: Randomized, controlled animal study. SETTING: University-based animal research facility. SUBJECTS: Thirty-six New Zealand white male rabbits, weighing 1.8 to 3 kg. INTERVENTIONS: Anesthetized rabbits were randomly assigned to one of four groups (n = 9 per group): a) a sham-operated group; b) a sham-operated group pretreated with sodium tungstate (xanthine oxidase inactivator); c) an aorta occlusion group; and d) an aorta occlusion group pretreated with sodium tungstate. Descending thoracic aorta occlusion was maintained for 40 mins with a 4-Fr Fogarty embolectomy catheter, followed by 2 hrs of reperfusion. MEASUREMENTS AND MAIN RESULTS: Myocardial injury, manifested by increased circulating creatine kinase-MB fraction activity, was significantly associated with aortic occlusion and reperfusion (p < .05). Sodium tungstate pretreatment significantly (p < .05) reduced circulating and myocardial xanthine oxidase activity. Xanthine oxidase inactivation by sodium tungstate significantly decreased circulating creatine kinase-MB fraction activity after hepatoenteric ischemia-reperfusion (p < .05). Finally, circulating creatine kinase-MB fraction activity was significantly associated with circulating xanthine oxidase activity (r2 = .85; p < .001). CONCLUSIONS: We conclude that remote myocardial injury is caused by hepatoenteric ischemia-reperfusion. The pathoetiology of this myocardial injury involves a xanthine oxidase-dependent mechanism.

Animals↗
PubMed2002

Increased free radical production in hypertension due to increased expression of the NADPH oxidase subunit p22(phox) in lymphoblast cell lines.

OBJECTIVES: To confirm increased production of reactive oxygen species (ROS) in hypertension, to demonstrate the source of ROS and to analyse NADPH oxidase subcomponent expression in hypertension. DESIGN: A lymphoblast model was used, as this has previously been used in the study of hypertension and of NADPH oxidase. Chemiluminescence (CL) was chosen to assay ROS production, as it is simple and sensitive. METHODS: Lymphocytes from 12 hypertensive patients (HT), and 12 age- and sex-matched normotensive (NT) subjects, were immortalized. Luminol, isoluminol and Cypridina luciferin analogue (CLA) CL were used to assay ROS production. NADPH oxidase subunits were measured by Western blot analysis. RESULTS: Stimulation with 50 micromol/l arachidonic acid (AA) resulted in increased ROS production in HT cell lines with luminol, CLA and isoluminol CL. Stimulation with 500 nmol/l 12-O-tetradecanoylphorbol-13-acetate (TPA) produced a detectable increase in HT ROS production with luminol and with CLA, whereas there was no significant difference with isoluminol. The ROS production was abolished by diphenyleneiodonium chloride (DPI) but not by rotenone, indicating that a non-mitochondrial flavoprotein such as NADPH oxidase is the source of ROS. Analysis of NADPH oxidase subcomponents revealed an increase in p22(phox) in HT subjects. CONCLUSIONS: We have shown there is increased ROS production in lymphoblasts derived from hypertensive subjects, probably originating from NADPH oxidase. As the ROS production persists in transformed cells, this suggests a genetic predisposition to increased ROS production. Increased expression of p22(phox) in HT lymphoblasts may account for some of the increased ROS.

Arachidonic Acid↗
PubMed2004

Role of NAD(P)H oxidase on vascular alterations in angiotensin II-infused mice.

OBJECTIVES: Angiotensin (Ang) II stimulates vascular reactive oxygen species generation via NAD(P)H oxidase activation. We investigated whether vascular NAD(P)H oxidase influences structure and function of resistance arteries from Ang II-infused mice. METHODS: Mice received Ang II alone (400 ng/kg per min, subcutaneously), Ang II + apocynin (NAD(P)H oxidase inhibitor, 2.5 mg/day, in the food), apocynin alone or Ang II + hydralazine (50 mg/kg per day) for 14 days. Systolic blood pressure (SBP) was measured by tail-cuff methodology and function and structure of small mesenteric arteries were studied in pressurized vessels. Vascular collagen type I/III content was evaluated by confocal immunofluorescence microscopy and by immunoblotting. RESULTS: The rise in SBP induced by Ang II (P < 0.001) was prevented by apocynin and hydralazine. Media/lumen ratio increase in Ang II-infused mice (P < 0.01) was prevented by apocynin. Acetylcholine-mediated relaxation, which was impaired in Ang II-infused mice (P < 0.001), was improved by apocynin. Confocal microscopy and immunoblotting demonstrated increased collagen type I/III content in mesenteric arteries from Ang II-infused mice. Apocynin, but not hydralazine, prevented the increase in collagen abundance in Ang II-infused mice. The increase in vascular NAD(P)H oxidase activity by Ang II (P < 0.001) was prevented by apocynin. CONCLUSIONS: The NAD(P)H oxidase inhibitor apocynin reduced blood pressure elevation and prevented structural alterations, endothelial dysfunction, and collagen deposition in the media of small arteries in Ang II-infused mice. Although hydralazine also decreased blood pressure, it had no effects on vascular collagen content. Our findings suggest that NAD(P)H oxidase activity plays an important role in vascular functional and structural changes and in the composition of the vascular wall in Ang II-dependent hypertension.

Acetophenones↗

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