This Article Abstract Full Text (PDF) All Versions of this Article: 97/10/1055    most recent 01.RES.0000189301.10217.87v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miller, A. A. Articles by Sobey, C. G. Search for Related Content PubMed PubMed Citation Articles by Miller, A. A. Articles by Sobey, C. G. Related Collections Cell signalling/signal transduction Brain Circulation and Metabolism Other Vascular biology

(Circulation Research. 2005;97:1055.)
© 2005 American Heart Association, Inc.

Integrative Physiology

NADPH Oxidase Activity and Function Are Profoundly Greater in Cerebral Versus Systemic Arteries

Alyson A. Miller, Grant R. Drummond, Harald H.H.W. Schmidt, Christopher G. Sobey

From the Department of Pharmacology, University of Melbourne, Parkville (A.A.M., C.G.S.), and the Department of Pharmacology, Monash University, Clayton (G.R.D., H.H.H.W.S), Victoria, Australia.

Correspondence to Dr Christopher G. Sobey, Department of Pharmacology, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail cgsobey{at}unimelb.edu.au

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent studies suggest that the superoxide generating enzyme NADPH oxidase may play a functional role in regulating cerebral vascular tone. We tested whether the activity, function, and expression of NADPH oxidase differs between rat cerebral and systemic arteries. Superoxide production by basilar (BA), middle cerebral (MCA), carotid (CA), renal (RA), and mesenteric (MA) arteries and aorta (AO) was measured using lucigenin-enhanced chemiluminescence. Superoxide production from NADPH oxidase was localized and semiquantified using dihydroethidium. Vascular functional responses were assessed in a myograph or organ bath. Vascular Nox4 protein expression was measured using Western blotting. Superoxide production (basal or in response to NADPH or angiotensin II) in the intracranial arteries, BA, and MCA was 10- to 100-fold greater than in AO, CA, RA, or MA. Similar results were found using either intact vessels or arterial homogenates, and were associated with 10-fold greater expression of Nox4 in the BA versus AO, CA, and MA. Superoxide production was attenuated by the NADPH oxidase inhibitors, diphenyleneiodonium, apocynin, and gp91ds-tat. NADPH and H₂O₂ were strong relaxing stimuli in the BA, where the H₂O₂ scavenger catalase, as well as apocynin, attenuated these relaxations and also augmented contractions to angiotensin II. NADPH oxidase activity is markedly higher in intracranial versus systemic arteries, in association with higher Nox4 expression. In cerebral arteries, endogenous H₂O₂ derived from NADPH oxidase activation appears to cause relaxation and is able to offset angiotensin II-induced constriction. These data are consistent with the concept that NADPH oxidase-derived reactive oxygen species modulate cerebral vascular tone under physiological conditions.

Key Words: cerebral • NADPH oxidase • reactive oxygen species • angiotensin II • hydrogen peroxide

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Emerging evidence indicates that reactive oxygen species (ROS) are important molecules in the control of vascular reactivity.¹ Indeed, the superoxide anion (O₂^–) increases vascular tone via the inactivation of endothelium-derived nitric oxide (NO). Recent studies, however, have revealed that the by-product of O₂^– metabolism, hydrogen peroxide (H₂O₂), is a powerful endogenous vasodilator within the cerebral circulation.^2–5 Thus, ROS may represent important signaling molecules for regulating local cerebral blood flow.

Numerous investigators have reported that NADPH oxidases are the primary generators of ROS within the vasculature.^6–9 These enzymes are membrane-associated and generate O₂^– by transferring electrons to molecular oxygen via a flavin-containing "Nox" catalytic subunit.¹⁰ In the systemic circulation, NADPH oxidase has been studied extensively, and it is now believed that excessive O₂^– generation by NADPH oxidase contributes to endothelial dysfunction associated with numerous cardiovascular diseases.¹¹ Although it has been recently established that NADPH oxidase is expressed and active in the cerebral circulation,^12–14 its function in this vascular bed is less clear. Application of the substrates for NADPH oxidase, NADPH or NADH, increases O₂^– production by cerebral arteries.^12–14 Interestingly, activation of NADPH oxidase using NADPH elicits profound cerebral vasodilatation in vitro¹² and in vivo,^13,14 raising the possibility that in contrast with the systemic circulation, NADPH oxidase-derived ROS may normally play a functional role in vasodilator responses in the cerebral circulation. No study, however, has directly compared the activity and function of NADPH oxidase between systemic and cerebral circulations. Therefore, the aim of the present study was to test whether NADPH oxidase activity and function is normally greater in rat cerebral versus systemic arteries.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
All procedures were approved by the institutional animal ethics committee. In total, 145 male, department-bred Sprague-Dawley rats (300±5 g) were studied. Rats were euthanized by inhalation of 80% CO₂/20% O₂.

O₂^– Production by NADPH Oxidase in Intact Cerebral and Systemic Arteries
Basilar, middle cerebral, common carotid, mesenteric (2nd order branch of the superior mesenteric artery), and renal arteries and thoracic aorta were excised and cut into 5 mm-long ring segments. O₂^– production was measured by 5 µmol/L lucigenin-enhanced chemiluminescence in the absence and presence of NADPH (100 µmol/L) as previously described.¹⁵ In some experiments, arteries were treated with the Cu²⁺-chelating agent diethyldithiocarbamate (DETCA; 3 mmol/L) for 30 minutes before start of assay (to inhibit endogenous Cu²⁺/Zn²⁺ superoxide dismutase [SOD] activity), in the presence and absence of angiotensin II (0.1 µmol/L) and/or the NADPH oxidase inhibitors diphenyleneiodonium (DPI; 5 µmol/L), apocynin (300 µmol/L), and gp91ds-tat (RKKRRQRRRCSTRIRRQL-CONH2; 1 µmol/L). In experiments using apocynin and gp91ds-tat, arteries were incubated with drugs for 1 hour before addition of NADPH and DETCA. Additionally, experiments in NADPH-treated basilar arteries were performed in the presence of the NO synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME; 100 µmol/L). Background counts were subtracted and O₂^– production normalized for dry tissue weight.

O₂^– Production by NADPH Oxidase in Cerebral and Systemic Artery Homogenates
Basilar, common carotid, and mesenteric (2nd order branch of the superior mesenteric artery) arteries and thoracic aorta were excised and cut into 5 mm-ring segments. Arteries were homogenized in either 70 µL (basilar and mesenteric) or 90 µL (carotid and aorta) of lysis buffer (composition in mmol/L: sucrose 250, HEPES 10) with protease inhibitors (Roche Complete Mini, #1836153) using 0.2 mL glass homogenizers. O₂^– production was measured by 5 µmol/L lucigenin-enhanced chemiluminescence in the presence of NADPH (100 µmol/L) and DETCA (3 mmol/L). Background counts were subtracted and values were normalized to protein content (as measured using the Bradford protein assay; BioRad).

Localization and Semiquantification of O₂^– by Dihydroethidium
Dihydroethidium (DHE; 2 µmol/L) was used to localize and measure O₂^– production in frozen sections (16-µm) of basilar and common carotid arteries and thoracic aorta as previously described.^15,16 In the presence of O₂^–, DHE is oxidized to ethidium and oxyethidium, which intercalate between DNA strands, producing a nuclear staining pattern. Arterial sections were treated with NADPH (100 µmol/L) for 30 minutes before treatment with DHE. Some sections were treated with polyethylene glycol-SOD (PEG-SOD, 250 U/mL) or native Cu²⁺/Zn²⁺-SOD (250 U/mL) for 30 minutes before incubation with DHE. Fluorescent images were acquired as 8-bit (256 intensity levels) and were analyzed with Image-Pro Plus software (version 5.0.1.11, Media Cypernetics Inc). For each arterial section, pixels in the upper 50% intensity level were counted, background fluorescence was subtracted, and data were presented as total fluorescent units per mm² of cross sectional artery wall area.

Expression of Nox4
Expression of Nox4 in basilar, common carotid, and mesenteric arteries (2nd order branch of the superior mesenteric artery) and thoracic aorta was measured by Western blotting. Anti-Nox4 rabbit polyclonal antibodies were raised against the Nox4 peptide (aa 84 to 101; RGSQKVPSRRTRRLLDKS). We have previously demonstrated that preincubation of Nox4 antibody sera with Nox4 peptide (10 µg/mL) results in undetectable immuoreactive bands, implying specificity of the Nox4 antibody.¹⁷ Arteries were homogenized in ice-cold lysis buffer (composition in mmol/L: sucrose 250; HEPES 10) with protease inhibitors (Roche Complete Mini, #1836153) using 0.2 mL glass homogenizers. Homogenates were cleared by centrifugation (10 000 relative centrifugal force; 5 minutes) and protein concentration was determined using the Bradford protein assay (BioRad). For blotting, gel sample buffer was added to homogenates and boiled for 5 minutes. Equal amounts of protein were loaded onto a 12% polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked in 5% skim milk for 1 hour at room temperature and then incubated overnight (4°C) with the Nox4 primary antibody (1:1000). Membranes were then incubated with a horseradish peroxidase anti-rabbit immunoglobulin for 1 hour at room temperature (1:1000; Chemicon Australia Pty). Equal protein loading was verified by -actin immunostaining (monoclonal mouse -actin antibody). Immunoreactive bands were detected by enhanced chemiluminescence (Progen Biosciences) and quantitated densitometrically with the use of a Kodak Image Station 440CF (Perkin-Elmer Life Sciences).

In Vitro Preparation
Basilar, common, carotid and mesenteric (2nd order branch of the superior mesenteric artery) arteries and thoracic aorta were excised and placed in cold, carbogen-bubbled (95% O₂, 5% CO₂) Krebs-bicarbonate solution (composition, in mmol/L; NaCl 118, KCl 4.5, MgSO₄ 0.45, KH₂PO₄ 1.03, NaHCO₃ 25, glucose 11.1, CaCl₂ 2.5). Arteries were cut into 5-mm rings. Ring segments of basilar, carotid, and mesenteric arteries were then threaded onto 2 wires and mounted in a Mulvany-Halpern myograph (Danish Myo Technology A/S), and resting tension was increased to either 5 mN (basilar and mesenteric) or 15 mN (carotid). Segments of aorta were mounted in organ baths with a resting tension of 0.5 g. All rings were continuously bathed in warm (37°C) carbogen-bubbled Krebs-bicarbonate solution. After an equilibration period of 60 minutes, rings were exposed to a high potassium physiological salt solution (KPSS) containing 122.7 mmol/L KCl (equimolar replacement of NaCl with KCl) to induce vascular contraction defined as 100% of KPSS. In all rings, endothelial integrity was assessed by measuring acetylcholine-induced (ACh, 10 µmol/L) relaxations of serotonin (0.1 to 1 µmol/L; for basilar, aorta, mesenteric) or U46619-induced (0.03 µmol/L; for carotid) tone (50% to 60% of contraction to KPSS).

In Vitro Protocol
Rings were again contracted submaximally (50% to 60% of contraction to KPSS) with either serotonin (basilar, aorta, and mesenteric) or U46619 (carotid). Once contractions were stable, the effects of the following agonists on vascular tone were investigated: NADPH (10 and 100 µmol/L), sodium nitroprusside (SNP; 0.01 to 1 µmol/L), and H₂O₂ (10 to 1000 µmol/L). Responses were examined in the presence and absence of apocynin (300 µmol/L) or the H₂O₂ scavenger catalase (1000 U/mL). In some experiments, arteries were contracted submaximally (20% of contraction to KPSS) with KCl (20 mmol/L). Once contractions were stable, the effect of angiotensin II (100 nmol/L) on vascular tone in the presence and absence of catalase (1000 U/mL) or apocynin (300 µmol/L) was investigated. Arteries were treated with apocynin for 90 minutes before experiments commenced.

Drugs
H₂O₂ was purchased from Merck, DHE from Molecular Probes, angiotensin II from Auspep, apocynin from Fluka, and gp91ds-tat from SynPep Corporation. All other drugs were purchased from Sigma. DPI, DHE, and apocynin were prepared at 10 mmol/L in dimethyl sulfoxide (DMSO) and diluted in Krebs-HEPES solution such that the final concentration of DMSO was <0.05% to 0.001%. All other drugs were dissolved and diluted in either Krebs-HEPES (lucigenin/DHE experiments) or Krebs-bicarbonate (myograph/organ bath experiments) solution.

Data Analysis
All results are expressed as mean±SEM. Statistical comparisons were performed using either 1-way ANOVA with Bonferroni multiple comparisons post hoc-test or using Student paired or unpaired t test, as appropriate. P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
O₂^– Production by NADPH Oxidase in Cerebral and Systemic Arteries
Basal O₂^– production (counts measured in the absence of NADPH) by intact basilar and middle cerebral arteries was higher (up to 120-fold) than levels generated by the aorta and carotid, mesenteric, and renal arteries (P<0.05, Figure 1A). Similarly, in the presence of NADPH, O₂^– production by basilar and middle cerebral arteries was markedly higher (10- to 90-fold) than levels generated by aorta and carotid, mesenteric, and renal arteries (P<0.001, Figure 1B). When arteries were treated with the Cu²⁺/Zn²⁺-SOD inhibitor DETCA (3 mmol/L), O₂^– levels in all vessels were augmented; however, O₂^– levels in basilar and middle cerebral arteries remained profoundly greater than levels in systemic arteries (P<0.01, Figure 1B). In arterial homogenates, NADPH-stimulated O₂^– production (in the presence of DETCA) was also substantially higher in basilar arteries than in the aorta and carotid and mesenteric arteries (P<0.01, Figure 1C). The NADPH oxidase inhibitor DPI virtually abolished NADPH-stimulated O₂^– production in all intact arteries (P<0.01, Figure 1B). Moreover, in basilar arteries, the selective NADPH oxidase inhibitors apocynin and gp91ds-tat inhibited NADPH-stimulated O₂^– production (P<0.05, Figure 1D). The NOS inhibitor L-NAME (100 µmol/L) had no significant effect on NADPH-stimulated O₂^– production by basilar arteries (control, 5.9±0.6; L-NAME 6.6±0.6x10³ counts/mg of dry tissue; n=6, P>0.05, 1-way ANOVA), suggesting that NADPH oxidase (and not NOS) was the primary source of vascular O₂^–. In the presence of NADPH (100 µmol/L) and DETCA (3 mmol/L), angiotensin II (30 minutes, 0.1 µmol/L) increased O₂^– production in all arteries. Angiotensin II-induced increases in O₂^– production were 30- to 120-fold greater in basilar arteries than in similarly-treated aorta and carotid and mesenteric arteries (P<0.01, Figure 2).

View larger version (21K): [in this window] [in a new window]   Figure 1. Vascular O2– production by cerebral and systemic arteries as measured by lucigenin-enhanced chemiluminescence. A, Basal O2– production (in the absence of NADPH) in intact middle cerebral (MCA), basilar (BA), common carotid (CA), mesenteric (MA), and renal arteries (RA) and thoracic aorta (AO). B, The effect of NADPH (100 µmol/L) on O2– production by intact arteries in the absence and presence of the Cu2+/Zn2+-SOD inhibitor, DETCA (3 mmol/L), and the NADPH oxidase inhibitor DPI (5 µmol/L). C, The effect of NADPH (100 µmol/L) on O2– production by arterial homogenates in the presence of DETCA (3 mmol/L). D, The effect of apocynin (300 µmol/L) and gp91ds-tat (1µmol/L) on NADPH-stimulated O2– production (control; in the presence of DETCA) by intact basilar arteries. Values are given as mean±SEM (103 counts per mg of dry tissue; n=5 to 6). *P<0.05 vs O2– production in similarly-treated AO, CA, MA, and RA; P<0.05 vs NADPH/DETCA-treated arteries; # P<0.05 vs control (1-way ANOVA).

View larger version (13K): [in this window] [in a new window]   Figure 2. Angiotensin II (Ang II, 100 nmol/L)-induced increase in O2– production by intact basilar (BA), carotid (CA), and mesenteric (MA) arteries and thoracic aorta (AO). Experiments were performed in the presence of NADPH (100 µmol/L) and the Cu2+/Zn2+-SOD inhibitor DETCA (3 mmol/L). The data represent counts in angiotensin II-treated rings minus counts in vehicle-treated rings from the same animal. Vascular O2– production was assayed using lucigenin-enhanced chemiluminescence. Values are given as mean±SEM (103 counts per mg of dry tissue; n=6). *P<0.05 vs AO, CA, and MA (1-way ANOVA).

Localization of O₂^– Production by NADPH Oxidase
O₂^– production by NADPH oxidase was localized and measured in arterial sections of basilar and carotid arteries and aorta using DHE. In the presence of NADPH, ethidium/oxyethidium fluorescence was observed throughout the vascular wall of all arteries, but was substantially higher in basilar arteries than in carotid arteries or aorta (P<0.001, Figure 3A through 3C). In basilar arteries, fluorescence intensity was uniform throughout all cell types, whereas in carotid arteries and aorta, fluorescence was more intense in endothelial and adventitial cells than in vascular smooth muscle cells (Figure 3A through 3C). Treatment of sections with PEG-SOD inhibited fluorescence in all arteries (P<0.01, Figure 3D through 3F), confirming that O₂^– was the major ROS detected by DHE. By contrast, treatment with native Cu²⁺/Zn²⁺-SOD did not attenuate the signal (data not shown), indicating that the DHE fluorescence reflected intracellular O₂^–.

View larger version (12K): [in this window] [in a new window]   Figure 3. In situ detection of NADPH-stimulated (100 µmol/L) O2– production by ethidium/oxyethidium fluorescence in sections of basilar arteries (A), aorta (B), and carotid (C) arteries. All experiments were performed in the presence of NADPH (100 µmol/L). PEG-SOD inhibited fluorescence in all arteries (D through F). The intensity of red fluorescence represents the amount of O2– generated. e indicates the orientation of the endothelium. Magnification, x400; scale bar, 50 µm. G, Summary of quantitative data for ethidium/oxyethidium fluorescence, presented as fluorescent units per mm2 of artery wall. Values are given as mean±SEM (n=3 to 6). *P<0.05 vs fluorescence intensity in aorta and carotid arteries; P<0.05 vs NADPH-treated arteries.

Nox4 Expression
Expression of Nox4 protein was 10-fold greater in basilar arteries than in the aorta (Figure 4; P<0.05). Expression of Nox4 protein in common carotid and mesenteric arteries was similar to that found in aorta (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 4. Representative Western blot showing expression of the NADPH oxidase catalytic subunit Nox4 in basilar and thoracic aorta (top). Also shown is a summary of densitometric data (bottom). The values are expressed as relative intensity/µg of protein and are given as mean±SEM (n=5). *P<0.05 vs aorta (paired t test).

Effect of NADPH on Vascular Tone
ACh (10 µmol/L) elicited similar relaxation responses in all arteries (basilar, 82±4%; aorta, 82±3%; carotid, 92±2%; mesenteric, 87±3%), indicating a functional endothelium. NADPH (10 to 100 µmol/L) elicited concentration-dependent relaxations of all arteries (Figure 5A). NADPH-induced relaxations of basilar arteries, however, were significantly greater than responses of carotid arteries and aorta (P<0.01, 1-way ANOVA). This enhanced relaxation was selective for NADPH, as relaxations to SNP were similar in all 4 arteries (Figure 5B). Furthermore, apocynin (300 µmol/L) virtually abolished NADPH-induced relaxation in the basilar artery but had no effect on ACh-induced (0.1 µmol/L) relaxation, suggesting that NADPH oxidase activity was responsible for mediating these effects (P<0.05, paired t test; Figure 5C and 5D; data shown only for 100 µmol/L NADPH).

View larger version (29K): [in this window] [in a new window]   Figure 5. Effect of NADPH (A; 10 to 100 µmol/L) and SNP (B; 0.01 to 1 µmol/L) on vascular tone of thoracic aorta (AO) and common carotid (CA), mesenteric (MA), and basilar (BA) arteries. AO, MA, and BA were pre-contracted (&50% of contraction to KPSS) with serotonin and CA with U46619 before addition of NADPH or SNP. Also shown is the effect of apocynin (300 µmol/L) on responses of pre-contracted BA to NADPH (C; 100 µmol/L) and ACh (D; 10 µmol/L). Values are expressed as percent relaxation of serotonin- or U46619-induced tone and are given as mean±SEM (n=5 to 8). *P<0.05 vs relaxations in AO and CA; P<0.0.05 vs relaxations in AO (1-way ANOVA); #P<0.0.05 vs control (paired t test).

Vascular Responsiveness to Endogenous and Exogenous H₂O₂
In basilar, carotid, and mesenteric arteries, relaxations to NADPH (10 to 100 µmol/L) were either abolished or inhibited by catalase (1000 U/mL; P<0.01; Figure 6A; data shown only for 100 µmol/L NADPH). In contrast, NADPH-induced relaxations of the aorta were potentiated by catalase (P<0.01). Interestingly, in the presence of catalase, NADPH elicited small, transient contractions of basilar and carotid arteries (&20% of pre-contracted tone, Figure 6A). Catalase had no effect on relaxations to SNP (1 µmol/L, Figure 6B), however. These experiments suggest that relaxations to NADPH are mediated by endogenous H₂O₂, presumably derived from NADPH oxidase, in all arteries except the aorta.

View larger version (15K): [in this window] [in a new window]   Figure 6. The effect of catalase (1000 U/mL) on relaxations of thoracic aorta (AO) and common carotid (CA), mesenteric (MA), and basilar (BA) arteries to (A) NADPH (100 µmol/L) and (B) SNP (0.1 µmol/L). Values are expressed as percent relaxation of serotonin- or U46619-induced tone and are given as mean± SEM (n=6 to 7). *P<0.05 vs control (1-way ANOVA).

We next compared the sensitivity of cerebral and systemic arteries to exogenous H₂O₂. In all arteries, exogenous H₂O₂ (10 to 1000 µmol/L) induced a biphasic response: A small transient contraction followed by a large, sustained relaxation. There were no differences between arteries with respect to H₂O₂-induced contractions (data not shown); however, relaxations to H₂O₂ (100 to 1000 µmol/L) were greater in basilar and mesenteric arteries when compared with aorta and carotid arteries (P<0.01, Figure 7A). In all arteries, catalase abolished relaxations to H₂O₂ (P<0.01, Figure 7B).

View larger version (18K): [in this window] [in a new window]   Figure 7. A, The effect of H2O2 (10 to 1000 µmol/L) on the tone of thoracic aorta (AO) and common carotid (CA), mesenteric (MA), and basilar (BA) arteries. *P<0.05 vs relaxations in AO and CA; P<0.0.05 vs relaxations in AO (one-way ANOVA).B, The effect of catalase (100 U/mL) on relaxations to H2O2 (100 µmol/L). *P<0.05 vs control. Values are expressed either as percent relaxation of serotonin- or U46619-induced tone and are given as mean±SEM (n=6).

Effect of Angiotensin II on Vascular Tone
Angiotensin II (100 nmol/L) elicited contractions of all arteries (Figure 8A), but those contractions were smaller in the basilar artery than in systemic arteries (P<0.05, 1-way ANOVA). Treatment with catalase resulted in a 3-fold increase in angiotensin II-induced contractions of the basilar artery, but not the systemic arteries (P<0.01, 1-way ANOVA; Figure 8A). Similarly, in the presence of apocynin, there was a 3-fold increase in angiotensin II-induced contractions of the basilar artery (P<0.05, paired t test; Figure 8B).

View larger version (13K): [in this window] [in a new window]   Figure 8. A, The effect of angiotensin II (100 nmol/L) on the vascular tone of thoracic aorta (AO) and common carotid (CA), mesenteric (MA), and basilar (BA) arteries in the absence and presence of catalase (1000 U/mL). *P<0.05 vs contractions to angiotensin II in AO, CA, and MA; P<0.0.05 vs BA control (one-way ANOVA). B, Effect of apocynin (300 µmol/L) on responses of BA to angiotensin II (100 nmol/L). All arteries were pre-contracted (&20% of contraction to KPSS) with KCl before addition of angiotensin II. Values are expressed as a percent of maximal contraction to KPSS and are given as mean±SEM (n=6 to 8). #P<0.05 vs control (paired t test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The findings of this study reveal striking differences between intracranial cerebral arteries and a range of systemic arteries with respect to the activity, function, and expression of the ROS-generating enzyme NADPH oxidase. Specifically, using 2 separate techniques, we found that basal and stimulated O₂^– generation from NADPH oxidase is 1 to 2 orders of magnitude higher in cerebral arteries than in systemic arteries. The higher level of NADPH oxidase-derived O₂^– in the basilar artery was associated with a 10-fold higher expression of the catalytic subunit protein Nox4. Activation of NADPH oxidase in vitro elicited relaxation of all arteries, but this response was greatest in cerebral arteries. Thus, we report for the first time that the activity, expression, and function of NADPH oxidase is profoundly greater in cerebral than systemic arteries. Like NADPH, angiotensin II was found to be a much stronger stimulus for NADPH oxidase activity in the basilar artery, where its contractile efficacy appears to be modulated by endogenous H₂O₂. These findings support the concept that NADPH oxidase-derived ROS may play a physiological role in regulating cerebral blood flow.

NADPH Oxidase Activity and Cerebral Arteries
NADPH oxidase is normally expressed and functional in both the cerebral and systemic vasculature.^{6–8,12–14} When increased to supranormal levels by cardiovascular risk factors, NADPH oxidase activity is thought to be linked to endothelial dysfunction associated with various cardiovascular diseases.¹¹ However, it is not known what role, if any, is played by NADPH oxidase-derived ROS in modulating vascular function in the systemic circulation under normal physiological conditions. In contrast, findings from recent studies have raised the possibility that NADPH oxidase-derived ROS may serve as an important physiological vasodilator mechanism in the cerebral circulation.^13,14 Using the chemiluminescent probe lucigenin, we found that either angiotensin II or the substrate for NADPH oxidase, NADPH, increased O₂^– production in intact cerebral and systemic arteries, and this effect was inhibited by the NADPH oxidase inhibitors DPI, apocynin, and gp91ds-tat. Moreover, inhibition of NOS had no effect on O₂^– production in basilar arteries, providing strong evidence that NADPH oxidase was the primary source of O₂^– detected by this assay. A striking observation in these experiments was that O₂^– production by NADPH oxidase was profoundly greater in intracranial cerebral arteries (ie, basilar and middle cerebral) versus systemic arteries (ie, aorta, carotid, renal, and mesenteric). Furthermore, a considerably higher NADPH oxidase activity was also found in arterial homogenates of cerebral versus non-cerebral vessels, confirming that this phenomenon is not dependent on the presence of whole arterial tissue or even intact cells, and is consistent with a regional difference in enzyme subunit expression levels (see below). Because detected levels in cerebral arteries remained up to 75-fold higher even when Cu²⁺/Zn²⁺-SOD was inhibited by DETCA, it seems likely that this regional difference is, in large part, due to differing levels of NADPH oxidase activity and not simply varying degrees of O₂^– inactivation. In addition to the quantitative chemiluminescence data, we localized and measured O₂^– production in NADPH-treated basilar arteries, aorta, and carotid arteries using the oxidative fluorescent dye dihydroethidium. Ethidium/oxyethidium fluorescence was visible in endothelial, vascular smooth muscle, and adventitial cells of basilar and carotid arteries and the aorta, indicating that NADPH oxidase is expressed and active throughout the vasculature of all these arteries. Although the range and resolution of O₂^– detection may differ between the 2 methodologies, making it difficult to strictly compare quantitative findings from each approach, using both assays we found markedly greater levels of O₂^– production in basilar arteries versus systemic and/or non-intracranial arteries. Thus, together these results provide strong evidence that NADPH oxidase activity is substantially greater in the cerebral circulation versus systemic vasculature under physiological conditions.

Nox4 Expression
We have previously reported that Nox4 mRNA expression is abundant in the rat basilar artery and is augmented during chronic hypertension.¹³ We now report that Nox4 protein expression is markedly higher in the basilar artery than in systemic arteries. Although the associated higher expression of Nox4 protein in cerebral versus systemic arteries seems likely to contribute to the higher relative production of ROS by cerebrovascular NADPH oxidase, we cannot exclude the possibility that other Nox subunits, such as Nox2 and Nox1, might also contribute substantially to this regional difference. Of relevance here is our finding that the inhibitors of the association of cytosolic and membrane-associated NADPH oxidase components, apocynin and gp91ds-tat, both inhibited the NADPH-induced superoxide production by &50% in the basilar artery. Furthermore, a recent study has reported that when expressed in cultured cells, Nox4, unlike Nox2, does not appear to require cytosolic components to function.¹⁸ Thus, it is conceivable that other Nox proteins, including Nox2 and Nox1, may also contribute to the molecular basis of higher NADPH oxidase activity in cerebral arteries.

NADPH Oxidase Function and Cerebral Arteries
We next tested for any regional differences in the regulation of vascular tone associated with these differing levels of NADPH oxidase activity. Recent studies in rats, rabbits, and mice have shown that NADPH causes cerebral vasodilatation in vivo and in vitro.^12–14 We confirmed here that application of NADPH to isolated basilar arteries elicits powerful cerebral vascular relaxation. Because in systemic arteries activation of NADPH oxidase and generation of O₂^– is generally thought to increase vascular tone via the inactivation of endothelium-derived NO,^19,20 it was somewhat surprising to find that NADPH consistently elicited relaxations of all 3 systemic arteries. Most notable, however, was the finding that cerebral (basilar) arteries were more responsive in terms of relaxation to NADPH than systemic arteries. This enhanced vasodilatation was relatively specific for NADPH, as relaxations to the endothelium-dependent vasodilator ACh and the endothelium-independent vasodilator SNP were similar in all artery types. Of importance, apocynin virtually abolished NADPH-induced relaxation of the basilar artery, providing strong evidence that the relaxant effect of NADPH is NADPH oxidase-dependent. Interestingly, apocynin appears to inhibit NADPH-induced cerebral relaxation more effectively than it inhibits NADPH-induced O₂^– production. This may reflect differences in the accessiblity of apocynin to NADPH oxidase in the different experimental procedures, or may perhaps suggest that only apocynin-sensitive Nox homologues contribute to the vasodilatation.

The identity of the NADPH oxidase-derived ROS responsible for mediating cerebral vasodilatation to NADPH is not well defined. We previously reported that NADPH-induced dilatation of rat basilar arteries in vivo were inhibited by catalase and DETCA, suggesting that H₂O₂ (or a downstream ROS) generated from the dismutation of O₂^– by Cu²⁺/Zn²⁺-SOD was responsible for the vasodilatation.¹³ In contrast, a recent study in mice found that O₂^– and not H₂O₂ was partially responsible for cerebral vasodilatation to NADPH.¹⁴ In the present study, we found that catalase abolished NADPH-induced relaxation of cerebral arteries without affecting responses to SNP, indicating that the effect of NADPH was mediated by endogenous H₂O₂ (or a downstream ROS thereof) and that the effects of catalase were independent of contractile agents used in arterial preparations. We confirmed here previous reports that H₂O₂ is a powerful endogenous cerebral vasodilator.^3,4 Thus, cerebral arteries appear to be not only very sensitive to the relaxant effect of H₂O_2, but also likely to generate much higher amounts of H₂O₂ in response to stimuli of NADPH oxidase by virtue of a much higher activity of this enzyme relative to systemic arteries. The greater cerebral vasodilator response to NADPH is therefore likely to be a consequence of both higher ROS generation by NADPH oxidase and greater sensitivity to H₂O₂ of cerebral arteries.

The present findings using extracellular NADPH as an activator of NADPH oxidase do, however, raise an important question: How does extracellular NADPH increase NADPH oxidase activity when the substrate binding domain is believed to be intracellular?²¹ One potential explanation is that application of NADPH to the extracellular milieu results in elevated intracellular levels of the pyridine nucleotide. NADPH is a relatively large, highly charged molecule, however, and is therefore unlikely to cross the plasma membrane by simple diffusion. Hence, to influence the intracellular concentration of NADPH, extracellular NADPH would either have to be actively transported across the plasma membrane or pass through a channel. Alternatively, NADPH could conceivably act as a reducing equivalent for regeneration of intracellular NADPH from NADP⁺ by donating electrons either to a plasma membrane spanning electron transport protein or to a membrane permeable electron acceptor (ie, analogous to the mitochondrial citrate/pyruvate shunt). To date, there is no information on the existence of such mechanisms in vascular cells, or indeed in any other mammalian cell types, nor is there any published evidence on whether extracellular NADPH influences the intracellular concentration of the pyridine nucleotide. Nevertheless, the observations that NADPH-induced ROS production and cerebral vasodilatation in vitro and in vivo can be attenuated by specific NADPH oxidase inhibitors and gene knockdown approaches (present study and others^{9,12–14,22)} suggest that NADPH, either directly or indirectly, triggers NADPH oxidase activation.

Potential Functional Consequences of Higher Cerebral NADPH Oxidase Activity
It is well established that the activity of NADPH oxidase is increased by vasoactive hormones such as angiotensin II.⁸ Indeed, we found here that angiotensin II potentiated vascular O₂^– production, and this effect was markedly greater (100-fold) in basilar arteries compared with aorta. Interestingly, this is analogous to our previous finding in spontaneously hypertensive rats, where O₂^– production from NADPH oxidase was selectively enhanced in basilar arteries.¹³ Thus, vasodilator ROS production stimulated by angiotensin II in cerebral arteries would be expected to offset and hence protect against angiotensin II-induced cerebral vasospasm. Consistent with this hypothesis is our present finding that catalase and apocynin can substantially and selectively augment cerebral vasoconstriction by angiotensin II. Mechanical forces, including cyclic stretch and laminar and oscillatory shear stress, have also been shown to increase NADPH oxidase activity in endothelial cells,^23,24 and we have recently reported that NADPH oxidase-derived ROS partly mediate flow-dependent responses of the basilar artery in vivo.²⁵ Thus, it is possible that NADPH oxidase-derived ROS serve as important secondary messengers and/or paracrine signaling factors in endothelial cells of the cerebral circulation, increasing blood flow in response to hormonal and physical stimuli. It is therefore noteworthy that the present results support the concept that the phenomenon of high NADPH oxidase activity in cerebral arteries is physiologically relevant and is not limited to studies using exogenous NADPH as a stimulus.

In summary, this study has demonstrated that under normal conditions, NADPH oxidase activity is profoundly greater in cerebral versus systemic arteries. Activation of NADPH oxidase in cerebral arteries can cause powerful vasodilatation and offset angiotensin II-induced vasoconstriction via H₂O₂ derived from the dismutation of NADPH oxidase-derived O₂^–. These findings are associated with markedly higher expression of the Nox4 catalytic subunit of NADPH oxidase in cerebral than in systemic arteries. Our findings raise the prospect that NADPH oxidase-derived ROS may play an important role in the control of cerebral vascular tone. If so, caution may need to be taken in the future use of therapies to inhibit the effect of ROS in systemic vascular disease so as not to inadvertently compromise cerebral blood flow.

	Acknowledgments

 
These studies were supported by a Project Grant from the National Health and Medical Research Council of Australia (NHMRC) (ID 350477). C.G.S. is a Senior Research Fellow of the NHMRC. G.R.D. is the recipient of a Monash Fellowship from Monash University.

	Footnotes

 
C.G.S. and G.R.D. are consultants for and have significant ownership interests in Radical Biotechnology Pty Ltd of Australia.

Original received March 10, 2005; resubmission received August 8, 2005; revised resubmission received September 14, 2005; accepted September 28, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Droge W. Free radicals in the physiological control of cell function. Physiol Rev. 2002; 82: 47–95.[Abstract/Free Full Text]

2. Paravicini TM, Drummond GR, Sobey CG. Reactive oxygen species in the cerebral circulation: physiological roles and therapeutic implications for hypertension and stroke. Drugs. 2004; 64: 2143–2157.[Medline] [Order article via Infotrieve]

3. Sobey CG, Heistad DD, Faraci FM. Potassium channels mediate dilatation of cerebral arterioles in response to arachidonate. Am J Physiol. 1998; 275: H1606–H1612.[Medline] [Order article via Infotrieve]

4. Sobey CG, Heistad DD, Faraci FM. Mechanisms of bradykinin-induced cerebral vasodilatation in rats: evidence that reactive oxygen species activate K⁺ channels. Stroke. 1997; 28: 2290–2295.[Abstract/Free Full Text]

5. Kontos HA, Wei EP, Povlishock JT, Christman CW. Oxygen radicals mediate the cerebral arteriolar dilation from arachidonate and bradykinin in cats. Circ Res. 1984; 55: 295–303.[Abstract/Free Full Text]

6. Mohazzab KM, Kaminski PM, Wolin MS. NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol. 1994; 266: H2568–H2572.[Medline] [Order article via Infotrieve]

7. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J Clin Invest. 1996; 97: 1916–1923.[Medline] [Order article via Infotrieve]

8. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.[Abstract/Free Full Text]

9. Ellmark SH, Dusting GJ, Fui MN, Guzzo-Pernell N, Drummond GR. The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle. Cardiovasc Res. 2005; 65: 495–504.[Abstract/Free Full Text]

10. Bengtsson SH, Gulluyan LM, Dusting GJ, Drummond GR. Novel isoforms of NADPH oxidase in vascular physiology and pathophysiology. Clin Exp Pharmacol Physiol. 2003; 30: 849–854.[CrossRef][Medline] [Order article via Infotrieve]

11. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840–844.[Abstract/Free Full Text]

12. Didion SP, Faraci FM. Effects of NADH and NADPH on superoxide levels and cerebral vascular tone. Am J Physiol Heart Circ Physiol. 2002; 282: H688–H695.[Abstract/Free Full Text]

13. Paravicini TM, Chrissobolis S, Drummond GR, Sobey CG. Increased NADPH oxidase activity and Nox4 expression during chronic hypertension is associated with enhanced cerebral vasodilatation to NADPH in vivo. Stroke. 2004; 35: 584–589.[Abstract/Free Full Text]

14. Park L, Anrather J, Zhou P, Frys K, Wang G, Iadecola C. Exogenous NADPH increases cerebral blood flow through NADPH oxidase-dependent and -independent mechanisms. Arterioscler Thromb Vasc Biol. 2004; 24: 1860–1865.[Abstract/Free Full Text]

15. Paravicini TM, Gulluyan LM, Dusting GJ, Drummond GR. Increased NADPH oxidase activity, gp91phox expression, and endothelium-dependent vasorelaxation during neointima formation in rabbits. Circ Res. 2002; 91: 54–61.[Abstract/Free Full Text]

16. Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.[Abstract/Free Full Text]

17. Wingler K, Wunsch S, Kreutz R, Rothermund L, Paul M, Schmidt HH. Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo. Free Radic Biol Med. 2001; 31: 1456–1464.[CrossRef][Medline] [Order article via Infotrieve]

18. Martyn KD, Frederick LM, von Loehneysen K, Dinauer MC, Knaus UG. Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases. Cell Signal. 2006; 18: 69–82.[CrossRef][Medline] [Order article via Infotrieve]

19. Souza HP, Laurindo FR, Ziegelstein RC, Berlowitz CO, Zweier JL. Vascular NAD(P)H oxidase is distinct from the phagocytic enzyme and modulates vascular reactivity control. Am J Physiol Heart Circ Physiol. 2001; 280: H658–H667.[Abstract/Free Full Text]

20. Di Wang H, Hope S, Du Y, Quinn MT, Cayatte A, Pagano PJ, Cohen RA. Paracrine role of adventitial superoxide anion in mediating spontaneous tone of the isolated rat aorta in angiotensin II-induced hypertension. Hypertension. 1999; 33: 1225–1232.[Abstract/Free Full Text]

21. Li JM, Shah AM. Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells. J Biol Chem. 2002; 277: 19952–19960.[Abstract/Free Full Text]

22. Gorlach A, Brandes RP, Nguyen K, Amidi M, Dehghani F, Busse R. A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall. Circ Res. 2000; 87: 26–32.[Abstract/Free Full Text]

23. Howard AB, Alexander RW, Nerem RM, Griendling KK, Taylor WR. Cyclic strain induces an oxidative stress in endothelial cells. Am J Physiol. 1997; 272: C421–C427.[Medline] [Order article via Infotrieve]

24. De Keulenaer GW, Chappell DC, Ishizaka N, Nerem RM, Alexander RW, Griendling KK. Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: role of a superoxide-producing NADH oxidase. Circ Res. 1998; 82: 1094–1101.[Abstract/Free Full Text]

25. Paravicini TM, Miller AA, Drummond GR, Sobey CG Flow-induced cerebral vasodilatation in vivo involves activation of phosphatidylinositol 3-kinase (PI3-K), NADPH oxidase, and nitric oxide synthase. J Cereb Blood Flow Metab. In press.

This article has been cited by other articles:

				C. Vecchione, A. Frati, A. Di Pardo, G. Cifelli, D. Carnevale, M. T. Gentile, R. Carangi, A. Landolfi, P. Carullo, U. Bettarini, et al. Tumor Necrosis Factor-{alpha} Mediates Hemolysis-Induced Vasoconstriction and the Cerebral Vasospasm Evoked by Subarachnoid Hemorrhage Hypertension, July 1, 2009; 54(1): 150 - 156. [Abstract] [Full Text] [PDF]

				T. M. De Silva, B. R.S. Broughton, G. R. Drummond, C. G. Sobey, and A. A. Miller Gender Influences Cerebral Vascular Responses to Angiotensin II Through Nox2-Derived Reactive Oxygen Species Stroke, April 1, 2009; 40(4): 1091 - 1097. [Abstract] [Full Text] [PDF]

				C. R.W. Kuhlmann, L. Librizzi, D. Closhen, T. Pflanzner, V. Lessmann, C. U. Pietrzik, M. de Curtis, and H. J. Luhmann Mechanisms of C-Reactive Protein-Induced Blood-Brain Barrier Disruption * Supplemental Methods Stroke, April 1, 2009; 40(4): 1458 - 1466. [Abstract] [Full Text] [PDF]

				C. Iadecola, L. Park, and C. Capone Threats to the Mind: Aging, Amyloid, and Hypertension Stroke, March 1, 2009; 40(3_suppl_1): S40 - S44. [Abstract] [Full Text] [PDF]

				A. A. Miller, G. R. Drummond, T. M. De Silva, A. E. Mast, H. Hickey, J. P. Williams, B. R. S. Broughton, and C. G. Sobey NADPH oxidase activity is higher in cerebral versus systemic arteries of four animal species: role of Nox2 Am J Physiol Heart Circ Physiol, January 1, 2009; 296(1): H220 - H225. [Abstract] [Full Text] [PDF]

				A. Starr, R. Graepel, J. Keeble, S. Schmidhuber, N. Clark, A. Grant, A. M. Shah, and S. D. Brain A reactive oxygen species-mediated component in neurogenic vasodilatation Cardiovasc Res, April 1, 2008; 78(1): 139 - 147. [Abstract] [Full Text] [PDF]

				H. Girouard, A. Lessard, C. Capone, T. A. Milner, and C. Iadecola The neurovascular dysfunction induced by angiotensin II in the mouse neocortex is sexually dimorphic Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H156 - H163. [Abstract] [Full Text] [PDF]

				M. Mittal, M. Roth, P. Konig, S. Hofmann, E. Dony, P. Goyal, A.-C. Selbitz, R. T. Schermuly, H. A. Ghofrani, G. Kwapiszewska, et al. Hypoxia-Dependent Regulation of Nonphagocytic NADPH Oxidase Subunit NOX4 in the Pulmonary Vasculature Circ. Res., August 3, 2007; 101(3): 258 - 267. [Abstract] [Full Text] [PDF]

				A. A. Miller, G. R. Drummond, A. E. Mast, H. H.H.W. Schmidt, and C. G. Sobey Effect of Gender on NADPH-Oxidase Activity, Expression, and Function in the Cerebral Circulation: Role of Estrogen Stroke, July 1, 2007; 38(7): 2142 - 2149. [Abstract] [Full Text] [PDF]

				K. Bedard and K.-H. Krause The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology Physiol Rev, January 1, 2007; 87(1): 245 - 313. [Abstract] [Full Text] [PDF]

				J. Kitayama, C. Yi, F. M. Faraci, and D. D. Heistad Modulation of Dilator Responses of Cerebral Arterioles by Extracellular Superoxide Dismutase Stroke, November 1, 2006; 37(11): 2802 - 2806. [Abstract] [Full Text] [PDF]

				F. M. Faraci Hydrogen peroxide: watery fuel for change in vascular biology. Arterioscler Thromb Vasc Biol, September 1, 2006; 26(9): 1931 - 1933. [Full Text] [PDF]

				A. N. Lyle and K. K. Griendling Modulation of vascular smooth muscle signaling by reactive oxygen species. Physiology, August 1, 2006; 21: 269 - 280. [Abstract] [Full Text] [PDF]

				C. G. Sobey and A. A. Miller Radicals spark interest in cerebral vasodilator mechanisms. Focus on "TNF-{alpha} dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation" Am J Physiol Cell Physiol, April 1, 2006; 290(4): C950 - C951. [Full Text] [PDF]

				H. Girouard, L. Park, J. Anrather, P. Zhou, and C. Iadecola Angiotensin II Attenuates Endothelium-Dependent Responses in the Cerebral Microcirculation Through Nox-2-Derived Radicals Arterioscler Thromb Vasc Biol, April 1, 2006; 26(4): 826 - 832. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 97/10/1055    most recent 01.RES.0000189301.10217.87v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miller, A. A. Articles by Sobey, C. G. Search for Related Content PubMed PubMed Citation Articles by Miller, A. A. Articles by Sobey, C. G. Related Collections Cell signalling/signal transduction Brain Circulation and Metabolism Other Vascular biology