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Cardiovascular Research 2007 73(1):73-81; doi:10.1016/j.cardiores.2006.10.005
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Copyright © 2006, European Society of Cardiology

Endothelial nitric oxide synthase activation leads to dilatory H2O2 production in mouse cerebral arteries

Annick Drouina, Nathalie Thorin-Trescasesa, Edith Hamelb, John R. Falckc and Eric Thorina,*

aUniversité de Montréal, Department of Surgery and Research Center, Institut de Cardiologie de Montréal, Montréal, Québec, Canada
bInstitut Neurologique de Montréal, McGill University, Montréal, Québec, Canada
cDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, U.S.A.

* Corresponding author. Institut de Cardiologie de Montréal, centre de recherche, 5000, rue Bélanger, Montréal, Québec, Canada, H1T 1C8. Tel.: +1 514 376 3330; fax: +1 514 376 1355. Email address: eric.thorin{at}umontreal.ca

Received 24 June 2006; revised 20 September 2006; accepted 6 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Hydrogen peroxide (H2O2) produced by the vascular endothelium is a signaling molecule regulating vascular tone. We hypothesized that H2O2 derived from eNOS activity could play a physiological role in endothelium-dependent dilation of mouse cerebral arteries.

Methods: Simultaneous endothelium-dependent dilation and fluorescence-associated free radical (DCF-DA) or NO (DAF-2) production were recorded in isolated and pressurized (60 mm Hg) cerebral artery of C57Bl/6 male mice.

Results: Without synergism, N-nitro-L-arginine (L-NNA) or the H2O2 scavengers catalase, PEG-catalase and pyruvate reduced (P<0.05) by 50% the endothelium-dependent dilation induced by acetylcholine (ACh). Simultaneously with the dilation, H2O2 – but not NO – production, sensitive to either L-NNA or catalase, was detected. In cerebral arteries from C57Bl/6·eNOS–/– mice, catalase had no effect on ACh-induced dilation and no H2O2-associated fluorescence was observed. In C57Bl/6 mice, silver diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor, but not the specific NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (PTIO), prevented ACh-induced dilation and H2O2 production suggesting that eNOS-derived superoxide is an intermediate in the production of H2O2. The catalase-sensitive ACh-induced dilation was restored by the eNOS cofactor tetrahydrobiopterin (BH4). This reversal was associated with a NO-associated fluorescence sensitive to PTIO but not to catalase. Soluble guanylate cyclase inhibition with 1H-[1,2,4]-oxadiazole-4,3-aquinoxalin-1-one (ODQ) prevented the dilation induced by ACh and by exogenous H2O2. Lastly, L-NNA, PTIO and ODQ – but not DETC, catalase or pyruvate – increased the pressure-dependent myogenic tone, suggesting that eNOS produces NO at rest, but leads to H2O2 during muscarinic stimulation.

Conclusion: H2O2-dependent dilation in mouse cerebral arteries appears to be a physiological eNOS-derived mechanism.

KEYWORDS Endothelial function; Nitric oxide; Microcirculation; Oxygen radicals

This article is referred to in the Editorial by Yokoyama and Hirata (pages 8–9) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Hydrogen peroxide (H2O2), formed from superoxide (O2–) as a result of the activity of various superoxide dismutases (SOD), is an important regulator of the function of the cerebrovascular wall [1,2]. Although not a free radical, H2O2 is a reactive oxidative species (ROS). It has been proposed to be an endothelium-derived hyperpolarizing factor (EDHF) both in cerebral [1] and peripheral [3–5] arteries. H2O2 may also dilate arteries by alternative unknown means [6–8], by stimulating the production of prostanoids [9] or the activation of soluble guanylate cyclase (sGC) [10].

Under physiological conditions, the origin of O2– that leads to the formation of H2O2 is unsettled. Endothelial NOS (eNOS) however, generates O2– during enzymatic cycling [11,12]. The eNOS-dependent generation of O2– is proposed to be functionally significant only in pathological conditions and related to the limited availability of L-arginine, the substrate of eNOS, as well as of its essential cofactor tetrahydrobiopterin (BH4) [13–15]. This concept has been challenged in a recent review article by Rabelink and Lüscher [16] where they proposed that eNOS-dependent O2– generation leading to the formation of H2O2 may be relevant in physiological conditions for host defense. It is unclear however, if this concept applies to the regulation of vascular tone. Since H2O2 is an important regulator of cerebrovascular tone [17], we have investigated the role of endogenous H2O2 during endothelial stimulation and its dependence on eNOS activation. We hypothesized that eNOS produces physiologically relevant levels of free radicals leading to H2O2-dependent dilation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animals and tissues preparation
Our study has been approved by our institutional ethical committee and conforms to the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Experiments were conducted on cerebral arteries isolated from 3 month-old male mice of C57BL/6 (29±1 g, n=66; Charles River Laboratories, Quebec, Canada) and C57Bl/6·129P2-Nos3tm1Unc/J (eNOS–/–, 24±1 g, n=6; Jackson Laboratory, Maine, U.S.A.) using a method previously described [18]. Mice were anesthetized by CO2 inhalation and the brain was rapidly removed from the cranial cavity and placed in ice-cold physiological salt solution (PSS) of the following composition (mmol/L): NaCl 130, KCl 4.7, CaCl2 1.6, MgSO4 1.17, NaHCO3 14.9, KH2PO4 1.18, EDTA 0.026, glucose 10. In all experiments, the PSS was oxygenated by a gas mixture containing 12% O2, 5% CO2 and the balance of N2 generating a pO2 of 150±10 mm Hg (n=5; measured using the Nova Stat Profile M apparatus). In one series of experiments, the balance of N2 was replaced by O2 to obtain a gas mixture of 95% O2 and 5% CO2 generating a pO2 of 410±11 mm Hg (n=5). Cerebral arteries (anterior, posterior and posterior communicating cerebral arteries) as well as the gracilis arteries were carefully isolated, cannulated at both ends and pressurized at 60 mm Hg (cerebral arteries) or 80 mm Hg (gracilis arteries, Ref. [18]) in no-flow condition (internal diameter of 80–140 µm).

2.2. Reactivity studies
An equilibration period of 40 min was allowed before starting the experiment and the myogenic tone was measured. Similar vessel pre-constrictions (reduction of 50% of the maximal diameter) with phenylephrine (PE; 10 to 30 µmol/L) were obtained before each experiment. A single cumulative concentration–response curve to acetylcholine (ACh; 1 nmol/L to 30 µmol/L), bradykinin (0.1 nmol/L to 0.3 µmol/L) or exogenous hydrogen peroxide (H2O2; 1 nmol/L to 30 µmol/L) was performed on each segment. We used catalase (100 U/mL) or PEG-catalase (50 U/mL), N{omega}-nitro-L-arginine (L-NNA; 10 µmol/L), pyruvate (3 mmol/L), a H2O2 scavenger [19], silver diethyldithiocarbamate (DETC; 1 mmol/L), a superoxide dismutase (SOD) inhibitor, 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (PTIO; 100 µmol/L), a NO scavenger, indomethacin (10 µmol/L), 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE; 1 µmol/L), a 11,12 epoxyeicosatrienoic acid (11,12 EET) receptor antagonist [20], tetrahydrobiopterin (BH4; 10 µmol/L and 1 mmol/L) and 1H-[1,2,4]oxadiazolo-[4,3a]quinoxalin-1-one (ODQ; 10 µmol/L), a soluble guanylate cyclase (sGC) inhibitor. Apamin (1 µmol/L), charybdotoxin (0.1 µmol L) and iberiotoxin (0.1 µmol/L) were used to block small-conductance Ca2+-sensitive K+ channels (SKCa), intermediate-conductance (I) KCa and big-conductance (B) KCa, respectively. All antagonists and inhibitors were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada) except for EEZE that was synthesized by Dr. JR Falck. All drugs were directly added to the bath chamber (extraluminally) and the final concentration of ethanol or DMSO never exceeded 0.01%.

2.3. Fluorescence studies
Pressurized cerebral arteries were incubated in oxygenated PSS (37 °C) containing either 5 µmol/L of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (DCF-DA, a ROS-reacting fluorescent dye; Molecular Probe, OR, U.S.A.; Ref. [6,21]) or 10 µmol/L of 4,5-diaminofluorescein diacetate (DAF-2, a fluorescent dye more selective to NO; Calbiochem, CA, U.S.A.; Ref. [21,22]) 30 min before the beginning of the experiment with or without inhibitors. Vessels were then washed with PSS, pre-constricted with PE and dilated with ACh (1 µmol/L) while recording simultaneously the changes in diameter and in fluorescence intensities of dichlorofluorescein retained intracellularly after cleavage of the acetate moieties. Fluorescence intensities at 492–495 nm (excitation) were measured at 520 nm with an IonOptix Acquire system (IonOptix, MA, U.S.A.). Before each experiment, basal fluorescence intensity was recorded. Results represent differences between stimulated and basal intensity.

2.4. Endothelial cell culture
Basilar artery of 3 month-old C57Bl/6 mice was isolated and endothelial cells (EC) were cultured as previously described [23].

2.5. Determination of eNOS monomers and dimers
After 30 min incubation with or without BH4 (1 mmol), cultured EC were resuspended for 30 min in a lysis buffer and proteins separated in non-reducing gels. In reducing conditions, 5 mmol/L of dithiothreitol was added. Rabbit antibody against eNOS (BD Biosciences, Mississauga, ON, Canada) diluted 1:100 was used.

2.6. Statistics
n refers to the number of animals used in each protocol. Half-maximum effective concentrations (EC50) of ACh and exogenous H2O2 were measured as previously described [18]. The pD2 value is the –log of the EC50. Continuous variables are expressed as means±standard error of the mean (SEM). The maximal diameter (Dmax) was determined by changing the PSS to a Ca2+-free PSS [18]. Myogenic tone is expressed as percentage of the Dmax. Dilations are expressed as percentage of Dmax. ANOVA were performed to compare concentration–response curves. Differences were considered to be statistically significant when the P-value was <0.05 (Scheffe's F test).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Implication of H2O2 and eNOS in cerebral vasodilation
The NOS inhibitor N{omega}-nitro-L-arginine (L-NNA, 10 µmol/L) reduced the endothelium-dependent dilation to ACh of cerebral arteries (Table 1, Fig. 1A). Catalase (100 U/mL) and the cell permeable PEG-catalase (50 U/mL) likewise reduced ACh-induced dilation (Table 1, Fig. 1A and B). Combination of L-NNA and catalase had no additive inhibitory effects on the dilatory response induced by ACh (Fig. 1A). In addition, bradykinin (0.1 nmol/L–1 µmol/L)-induced endothelium-dependent dilations (Emax=41±3%) were also significantly reduced by catalase (Emax=28±4%, n=3, P<0.05).


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  		Table 1 Myogenic tone (MT), efficacy (Emax) and potency (pD2) to acetylcholine of cerebral arteries isolated from C57Bl/6 and eNOS–/– male mice

 

Figure 1
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  		Fig. 1 ACh-induced endothelium-dependent dilations compared to denuded arteries (–endothelium) of C57Bl/6 pressurized cerebral arteries: (A) sensitivity to L-NNA (10 µmol/L) and catalase (100 U/mL) without synergism. (B) NO scavenging with PTIO (100 µmol/L) did not prevent ACh-induced dilation, while scavenging of H2O2 by pyruvate (3 mmol/L) or PEG-catalase (50 U/mL) or SOD inactivation by DETC (1 mmol/L) limited the dilation induced by ACh. *P<0.05 compared to control (n numbers are in Table 1).

 
Addition of pyruvate (3 mmol/L), a H2O2 scavenger, or DETC (1 mmol/L), a SOD inhibitor, reduced ACh-induced maximal dilation (Table 1, Fig. 1B). The NO scavenger PTIO (100 µmol/L) however, had no inhibitory effect on the dilation induced by ACh (Table 1, Fig. 1B).

To confirm these pharmacological data, H2O2 production was assessed in pressurized vessels after incorporation of the fluorescent ROS-reactive dye, DCF-DA [24]. ACh-induced dilation was associated with an increase in fluorescent intensity (Fig. 2A and C): ROS-dependent signals were abolished by L-NNA, catalase, DETC and pyruvate, but not by PTIO (Fig. 2C), demonstrating the specificity of the dye for H2O2 in our experimental conditions.


Figure 2
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  		Fig. 2 Representative changes in fluorescence intensities induced by ACh in pressurized mouse cerebral arteries loaded with (A) DCF-DA (5 µmol/L) and (B) DAF-2 (10 µmol/L). Graphical representations of the increase in fluorescence intensities of DCF-DA (C) and DAF-2 (D) during ACh-induced dilation (n=3 to 5 per group). Cat: catalase; Pyr: pyruvate. *P<0.05 compared to control; {dagger}P<0.05 compared to BH4 or combined with catalase.

 
3.2. ACh-induced dilation of cerebral arteries of eNOS–/– mice and gracilis arteries of C57Bl/6 mice
To confirm that H2O2 originated from eNOS, cerebral arteries were isolated from C57Bl/6·eNOS–/– mice: ACh induced a dilation that was neither affected by L-NNA nor by catalase (Fig. 3A). In addition, ACh-induced dilation did not significantly increase the fluorescence intensity of DCF-DA (+36±10 a.u. in eNOS–/– compared to +189±33 a.u. in cerebral arteries from C57Bl/6 mice; n=4 per group, P<0.05).


Figure 3
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  		Fig. 3 Effect of L-NNA (10 µmol/L) and catalase (100 U/mL) on ACh-induced dilation in (A) cerebral arteries isolated from eNOS–/– mice (n=6) and (B) gracilis arteries isolated from C57Bl/6 mice (n=3). *P<0.05 compared to control.

 
Similar experiments were performed in pressurized mouse gracilis arteries (80–140 µm) from C57Bl/6 mice. ACh-induced dilation was sensitive to L-NNA but insensitive to catalase (Fig. 3B). ACh-induced dilation was not associated with a rise in DCF-associated fluorescence intensity (+44±12 a.u. to ACh, 1 µmol/L) suggesting that H2O2 is not a ubiquitous pathway in the vasculature.

3.3. Effects of BH4 on eNOS activity
BH4 (1 mmol/L) per se did not affect ACh-induced dilation but restored the reduction of ACh-induced dilation by catalase (Table 1, Fig. 4A). Excess of BH4 led to the production of NO, as revealed by the apparition of a strong DAF-2-associated fluorescence (Fig. 2B and D). This signal was insensitive to catalase (Fig. 2D) but was prevented by PTIO (Fig. 2D) alone with no further inhibition of the signal by combination with pyruvate. Providing an excess of BH4 however, also strongly increased the production of H2O2 as revealed by the rise in DCF-associated fluorescence intensity (Fig. 2C): This increase in fluorescence was sensitive to catalase and L-NNA (Fig. 2C). As a functional consequence, ACh-induced dilation in the presence of BH4 was decreased by a combination of PTIO and pyruvate but not by single NO or H2O2 scavenging (Fig. 4C). In pressurized gracilis arteries, BH4 addition had however no effect on H2O2 production (+101±40 a.u. and 95±1 a.u. with and without BH4, respectively, n=4 per group).


Figure 4
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  		Fig. 4 Effects of BH4 alone or in combination with catalase on the endothelium-dependent dilation to ACh (A) (n numbers are in Table 1). In (B), representative Western blot and graphical representation showing the lack of influence of BH4 on the formation of monomers (140 kd) and homodimers (280 kd) of eNOS in reducing (R) and non-reducing (NR) conditions. (C) Effect of catalase, pyruvate and PTIO on ACh-induced dilation in the presence of exogenous addition of BH4 (1 mmol/L). *P<0.05 compared to control (experiments reproduced with 3 primary cultures).

 
In the presence of catalase, the threshold concentration of BH4 that promoted eNOS-dependent NO-mediated dilation was found to be 1 mmol/L. At a concentration of 10 µmol/L of BH4, ACh (1 µmol/L)-induced dilation (13±1%) was similar to that obtained in the presence of catalase alone. At 1 mmol/L, however, the dilation was significantly greater (35±1%) and normalized the dilation to ACh in the presence of catalase (Fig. 4). DAF-2-dependent fluorescence measured in the presence of catalase, increased with the concentration of BH4 (0, 10 µmol/L and 1 mmol/L) by +60±16, +153±14 and +525±62 (arbitrary units), respectively in response to ACh (1 µmol/L).

Cultured endothelial cells isolated from the mouse basilar artery were incubated with or without BH4 (1 mmol/L). Both monomers and homodimers of eNOS proteins were detected by Western blot, and the relative expression of monomers versus dimers was not altered by exposure to BH4 (Fig. 4B).

3.4. Effect of high O2 tension on ACh-induced dilation
In order to determine whether eNOS-dependent H2O2 production was sensitive to pro-oxidative high O2 tension, a series of experiments was performed in PSS aerated with a mixture of 95% O2 and 5% CO2 generating a pO2 of 410±11 mm Hg (n=5) compared to 150±10 mm Hg (n=5) in 12% O2 (without affecting pH). In conditions of high O2 tension, the maximal dilation induced by ACh (1 µmol/L) was (P<0.05) reduced (Fig. 5A). More importantly, eNOS activation led to a greater (P<0.05) DAF signal and a lower (P<0.05) DCF-DA signal (Fig. 5B), suggestive of a greater NO production and a lower H2O2 production, in conditions with high O2 tension. This was confirmed functionally, as catalase no longer reduced (P<0.05) the dilation induced by ACh (Fig. 5A). L-NNA (10 µmol/L) however, completely prevented the dilation induced by ACh in PSS aerated with 95% O2 (data not shown).


Figure 5
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  		Fig. 5 Effects of 95% O2 (pO2=410±11 mm Hg, n=5) compared to the normal conditions in 12% O2 (pO2=150±10 mm Hg, n=5) on (A) the maximal dilation (n=6) and (B) the simultaneous production of H2O2 (DCF-DA, n=3) and NO (DAF, n=3) of isolated mouse cerebral arteries induced by ACh (1 µmol/L). Data are expressed as mean±SEM. *P<0.05 compared to 12% O2; {dagger}P<0.05 compared to control.

 
3.5. Dilatory mechanism of action of H2O2
ODQ (1 µmol/L), an inhibitor of the sGC, reduced the dilatory response triggered by ACh as efficiently as catalase (Table 1, Fig. 6A). Exogenous H2O2 induced an endothelium-independent dilation (Fig. 6B), which was abolished in the presence of ODQ. Likewise, catalase prevented exogenous H2O2-induced dilation.


Figure 6
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  		Fig. 6 Effects of catalase (100 U/mL) and ODQ (10 µmol/L) on the dilation induced (A) by ACh (n=6 per group) and (B) by exogenous H2O2 (with or without endothelium; n=5 per group) in cerebral arteries from C57Bl/6 mice. *P<0.05 compared to control.

 
3.6. Impact of eNOS activity on myogenic tone
L-NNA, PTIO and ODQ strongly increased myogenic tone (Table 1). In contrast, addition of catalase, pyruvate and DETC did not affect myogenic tone (Table 1). BH4 did not reduce the myogenic response.

3.7. Other factors involved in ACh-induced dilation of C57Bl/6 mouse cerebral arteries
In C57Bl/6 mouse cerebral arteries, indomethacin, a cyclooxygenase (COX) inhibitor, reduced the dilatory effect of ACh (Table 1). The involvement of EDHF was first tested in the presence of apamin and charybdotoxin (Table 1). In our experimental conditions, the combination of the two toxins did not significantly limit the dilatory response triggered by ACh (Table 1). In the presence of iberiotoxin however, the dilation induced by ACh was reduced. Activation of BKCa by endothelium-derived arachidonic acid metabolite 11,12 epoxyecosatrienoic acids (11,12 EET) hyperpolarizes smooth muscle [20]. 14,15-epoxyeicosa-5(Z)-enoic acid (EZEE), a 11,12 EET receptor antagonist [18,20] reduced ACh-induced dilation similarly to iberiotoxin. Altogether, co-addition of indomethacin, catalase and EZEE abolished ACh-induced dilation (Table 1) as efficiently as removal of the endothelium (Fig. 1A).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The results of the present study suggest that H2O2 derived from eNOS activity is an EDRF in pressurized cerebral arteries isolated from young and healthy C57Bl/6 mice. ACh-induced dilation was sensitive without synergism, to eNOS inhibition and H2O2 scavengers and associated with H2O2 production, which was prevented by L-NNA, catalase, DETC and pyruvate. This H2O2-dependent response was absent in cerebral arteries from eNOS–/– mice. This supports the concept that in C57Bl/6 mouse cerebral arteries, activation of eNOS-dependent dilation is partly mediated by H2O2 and that this pathway is physiologically relevant. This confirms the seminal report of Rosenblum [25] who demonstrated that in vivo, bradykinin-induced cerebrovascular dilation was sensitive to catalase in mice. On the other hand, our findings are in contrast with the eNOS-dependent generation of O2–, which has only been observed in pathological states [26].

Inhibition of the eNOS limited the dilation as well as H2O2 scavengers, which strongly suggests that H2O2 originates from stimulated eNOS activity. This hypothesis is supported by the absence of H2O2-associated fluorescence in cerebral arteries incubated in the presence of L-NNA as well as in cerebral arteries isolated from eNOS–/– mice. In the present study, eNOS–/– mice imposed to us as the model of choice to validate our hypothesis that eNOS was the source of H2O2 despite the fact that we [27] and others [28] have reported that these knockout mice develop compensatory mechanisms to minimize the effect of eNOS disruption on the regulation of the cerebral blood flow.

The specificity of the two fluorescent dyes used in the study for H2O2 and NO (DCF-DA and DAF, respectively) is illustrated by the data presented in Fig. 2. While DCF signals were abolished by H2O2 scavenging, they were insensitive to PTIO suggesting that in these healthy arteries and in our experimental conditions peroxynitrites are not produced. Peroxynitrites would have been detected by DCF [24] and their production prevented by PTIO (Fig. 2C). This interaction between O2– and NO occurs however, at much higher levels of O2– unlikely to be reached in physiologically healthy vessels [2,13–16]. Likewise, additional sources of O2– have been reported in diseased arteries [1,2], such as NADP(H) oxidase. This pathway however, has never been shown to be of any significance in the endothelium of healthy arteries. Most importantly, NADP(H) oxidase is not sensitive to activation by ACh.

The generation of H2O2 from eNOS-derived O2– most likely requires endothelial superoxide dismutases (SOD) as revealed by the inhibitory effects of DETC. Our demonstration of the critical role of SOD in the regulation of vascular tone confirms previous reports using human coronary and submucosal intestinal arteries [4,8]. In vivo, the dilation to ACh of cerebral arterioles of Mn SOD+/– mice is also impaired [17]. Although in SOD deficient mice, excess free radicals may have damaged the endothelium and perturbed NO availability, our study further supports the concept for a physiological role of H2O2: a significant part of the endothelium-dependent dilation induced by ACh is reduced by H2O2 scavenging.

The production of O2– by eNOS is generally referred to as "eNOS uncoupling" [13,15], which is believed to be associated to the pro-oxidative environment of cardiovascular diseases [2]. Re-coupling of eNOS to produce NO is achieved by exposing the endothelium to BH4 [29], a cofactor of eNOS. It has been shown that the cerebrum of healthy mice contains an average level of BH4 of 90–100 pmol/g of tissue, a value 3 to 4 times lower than other tissues [30]. It is unlikely however, that a limited availability of BH4 is responsible for the eNOS-dependent production of O2– in these healthy vessels. First, eNOS activation in the presence of BH4 induces the production of NO which is also associated with a great production of H2O2. There is a functional consequence: the inhibitory effect of catalase on the dilation, observed in the absence of exogenous BH4, is compensated by NO and conversely, scavenging of only NO by PTIO is insufficient to prevent the dilation induced by ACh due to the simultaneous increased production of H2O2. This is why both scavengers are required to prevent the dilation induced by ACh in the presence of BH4. Second, in the presence of a high O2 tension, ACh-induced dilation became NO-dependent, while the DCF-DA signal was reduced, an effect therefore independent of the concentration of BH4. Mouse blood pO2 is slightly over 140 mm Hg [31] and was closely matched by our normal experimental conditions using 12% O2. Other teams studying small resistance artery physiology use either 10% O2 [32,33] or 21% O2 [34,35]. Hence, our data promote the concept that the production of H2O2 originating from eNOS activation may represent a "physiological eNOS uncoupling" state in mouse cerebral arteries. The "eNOS uncoupling" that leads to O2– production has been associated with a predominance of eNOS proteins in the monomer configuration [36]. In our hands, exposure to BH4 of endothelial cells isolated from mouse basilar arteries did not favor an allosteric remodeling towards the homodimer configuration of eNOS proteins. This supports therefore the concept proposed by Rabelink and Lüscher [16] that eNOS uncoupling may occur physiologically and suggests that in mouse cerebral arteries, eNOS can function in a coupled state, responsible for the resting and constitutive release of NO as evidenced by the NO-dependent regulation of myogenic tone, and in an uncoupled state, during agonist-dependent stimulation. Our results suggest therefore that BH4 directly modifies eNOS-dependent activation rather than its dimerization, as proposed by other studies [37,38].

Providing an excess of BH4 was not indeed associated with a direct activation of eNOS: the myogenic tone was not decreased by the cofactor. On the other hand, L-NNA, PTIO and ODQ, unlike catalase, pyruvate and DETC, strongly increased myogenic tone supporting the current knowledge that in resting conditions, eNOS produces NO that regulates basal vascular tone [1].

The dilatory mechanisms of action of H2O2 were first investigated by challenging the vessels with exogenous H2O2, which induced an endothelium-independent dilation as previously reported in human coronary arteries [4]. In our hands, the dilation was initiated at low sub-nanomolar concentrations, while sub-micromolar concentrations are required in peripheral arteries [3,4]. This further demonstrates the high sensitivity of mouse cerebral arteries to peroxide. This dilation was prevented by ODQ, the soluble guanylate cyclase inhibitor, suggesting that H2O2 shares a similar dilatory pathway with NO [10]. Likewise, ACh-induced dilation was prevented by ODQ.

The dilation induced by ACh was not completely blocked by catalase, suggesting the involvement of other endothelium-derived relaxing factors. Cyclooxygenase inhibition reduced significantly the efficacy of ACh likely by preventing the production of prostacyclin. In addition to H2O2 and prostacyclin, the involvement of a hyperpolarizing factor in cerebral arteries had to be expected [1]. As reported by others in the cerebral circulation [39], we observed that inhibition of 11,12 EET receptors by EZEE [18] significantly impaired the dilation induced by ACh. Arachidonic acid-derived 11,12 EET hyperpolarizes smooth muscle cells by activating iberiotoxin-sensitive BKCa channels as previously shown [20,40]. Our data are therefore in line with the literature.

In conclusion, our results suggest that ACh-dependent eNOS activation leads to a dilation essentially triggered by H2O2 in pressurized cerebral arteries isolated from C57Bl/6 mice. Like NO, H2O2 activates the soluble guanylate cyclase. This "physiological eNOS uncoupling" can be overcome by providing an excess of BH4 although eNOS produces NO in resting conditions. This eNOS-dependent pathway is not universally distributed in the vasculature but may be a key endothelium-dependent pathway of dilation in the brain.


   Acknowledgements
 
This work has been supported in part by the Foundation of the Montreal Heart Institute, the Heart and Stroke Foundation of Quebec and the Canadian Institute for Health Research (MOP14496), the NIH (GM31278, JRF) and the Robert A. Welch Foundation (JRF). E. Thorin is a senior scholar of the Fonds de la Recherche en Santé du Québec.


   Notes
 
Time for primary review 30 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Faraci F.M. Reactive oxygen species: influence on cerebral vascular tone. J Appl Physiol (2006) 100:739–743.[Abstract/Free Full Text]
  2. Cai H. Hydrogen peroxide regulation of endothelial function: origins, mechanisms, and consequences. Cardiovasc Res (2005) 68:26–36.[Abstract/Free Full Text]
  3. Matoba T., Shimokawa H., Nakashima M., Hirakawa Y., Mukai Y., Hirano K., et al. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice. J Clin Invest (2000) 106:1521–1530.[ISI][Medline]
  4. Miura H., Bosnjak J.J., Ning G., Saito T., Miura M., Gutterman D.D. Role for hydrogen peroxide in flow-induced dilation of human coronary arterioles. Circ Res (2003) 92:e31–e40.[Abstract/Free Full Text]
  5. Matoba T., Shimokawa H. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in animals and humans. J Pharmacol Sci (2003) 92:1–6.[CrossRef][ISI][Medline]
  6. Chaytor A.T., Edwards D.H., Bakker L.M., Griffith T.M. Distinct hyperpolarizing and relaxant roles for gap junctions and endothelium-derived H2O2 in NO-independent relaxations of rabbit arteries. Proc Natl Acad Sci U S A (2003) 100:15212–15217.[Abstract/Free Full Text]
  7. Gluais P., Edwards G., Weston A.H., Vanhoutte P.M., Félétou M. Hydrogen peroxide and endothelium-dependent hyperpolarization in the guinea-pig carotid artery. Eur J Pharmacol (2005) 513:219–224.[CrossRef][ISI][Medline]
  8. Hatoum O.A., Binion D.G., Miura H., Telford G., Otterson M.F., Gutterman D.D. Role of hydrogen peroxide in ACh-induced dilation of human submucosal intestinal microvessels. Am J Physiol (2004) 288:H48–H54.[ISI]
  9. Iida Y., Katusic Z.S. Mechanisms of cerebral arterial relaxations to hydrogen peroxide. Stroke (2000) 31:2224–2230.[Abstract/Free Full Text]
  10. Iesaki T., Gupte S.A., Kaminski P.M., Wolin M.S. Inhibition of guanylate cyclase stimulation by NO and bovine arterial relaxation to peroxynitrite and H2O2. Am J Physiol (1999) 46:H978–H985.
  11. Porasuphatana S., Tsai P., Rosen G.M. The generation of free radicals by nitric oxide synthase. Comp Biochem Physiol, Part C Pharmacol Toxicol (2003) 134:281–289.[CrossRef]
  12. Stroes E., Hijmering M., van Zandvoort M., Wever R., Rabelink T.J., Faassen E.E. Origin of superoxide production by endothelial nitric oxide synthase. FEBS Lett (1998) 438:161–164.[CrossRef][ISI][Medline]
  13. Alp N.J., Chanon K.M. Regulation of endothelial nitric oxide synthase by tetrahydrobiopterin in vascular disease. Arterioscler Thromb Vasc Biol (2004) 24:1–9.[Free Full Text]
  14. Kawashima S., Yokoyama M. Dysfunction of endothelial nitric oxide synthase and atherosclerosis. Arterioscler Thromb Vasc Biol (2004) 24:998–1005.[Abstract/Free Full Text]
  15. Katusic Z.S., d'Uscio L.V. Tetrahydrobiopterin: mediator of endothelial protection. Arteroscler Thromb Vasc Biol (2004) 24:397–398.[Free Full Text]
  16. Rabelink T.J., Lüscher T.F. Endothelial nitric oxide synthase. Host defense enzyme of the endothelium? Arterioscler Thromb Vasc Biol (2006) 26:267–271.[Abstract/Free Full Text]
  17. Faraci F.M., Modrick M.L., Lynch C.M., Didion L.A., Fegan P.E., Didion S.P. Selective cerebral vascular dysfunction in Mn-SOD deficient mice. J Appl Physiol (2006) 100(6):2089–2093.[Abstract/Free Full Text]
  18. Krummen S., Falck J.R., Thorin E. Two distinct pathways account for EDHF-dependent dilatation in the gracilis artery of dyslipidaemic hApoB+/+ mice. Br J Pharmacol (2005) 145:264–270.[CrossRef][ISI][Medline]
  19. Mallet R.T., Sun J., Knott E.M., Sharma A.B., Olivencia-Yurvati A.H. Metabolic cardioprotection by pyruvate: recent progress. Exp Biol Med (2005) 230:435–443.[Abstract/Free Full Text]
  20. Gauthier K.M., Deeter C., Krishna U.M., Reddy Y.K., Bondlela M., Falck J.R., et al. 14,15-Epoxyeicosa-5(Z)-enoic acid: a selective epoxyeicosatrienoic acid antagonist that inhibits endothelium-dependent hyperpolarization and relaxation in coronary arteries. Circ Res (2002) 90:1028–1036.[Abstract/Free Full Text]
  21. Hatoum O.A., Otterson M.F., Kopelman D., Miura H., Sukhotnik I., Larsen B.T., et al. Radiation induces endothelial dysfunction in murine intestinal arterioles via enhanced production of reactive oxygen species. Arterioscler Thromb Vasc Biol (2006) 26:287–294.[Abstract/Free Full Text]
  22. Sylvester F.A., Stepp D.W., Frisbee J.C., Lombard J.H. High-salt diet depresses acetylcholine reactivity proximal to NOS activation in cerebral arteries. Am J Physiol (2002) 283:H353–H363.[ISI]
  23. Thorin E., Shatos M.A., Shreeve S.M., Walters C.L., Bevan J.A. Human vascular endothelium heterogeneity. A comparative study of cerebral and peripheral cultured vascular endothelial cells. Stroke (1997) 28:375–381.[Abstract/Free Full Text]
  24. Bevers L.M., Braam B., Post J.A., van Zonneveld A.J., Rabelink T.J., Koomans H.A., et al. Tetrahydrobiopterin, but not L-arginine, decreases NO synthase uncoupling in cells expressing high levels of endothelial NO synthase. Hypertension (2006) 47:87–94.[Abstract/Free Full Text]
  25. Rosenblum W.I. Hydroxyl radical mediates the endothelium-dependent relaxation produced by bradykinin in mouse cerebral arterioles. Circ Res (1987) 61:601–603.[Abstract/Free Full Text]
  26. Forstermann U., Munzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation (2006) 113(13):1708–1714.[Abstract/Free Full Text]
  27. Thorin E., Huang P.L., Fishman M.C., Bevan J.A. Nitric oxide inhibits alpha2-adrenoceptor-mediated endothelium-dependent vasodilation. Circ Res (1998) 82(12):1323–1329.[Abstract/Free Full Text]
  28. Meng W., Ayata C., Waeber C., Huang P.L., Moskowitz M.A. Neuronal NOS-cGMP-dependent ACh-induced relaxation in pial arterioles of endothelial NOS knockout mice. Am J Physiol (1998) 274:H411–H415.[ISI][Medline]
  29. Vasquez-Vivar J., Kalyanaraman B., Martasek P., Hogg N., Masters B.S., Karoui H., et al. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A (1998) 95:9220–9225.[Abstract/Free Full Text]
  30. Yoshida Y.I., Eda S., Masada M. Alteration of tetrahydrobiopterin biosynthesis and pteridine levels in mouse tissues during growth and aging. Brain Develop (2000) 22:S45–S49.[CrossRef][Medline]
  31. Didion S.P., Lynch C.M., Baumbach G.L., Faraci F.M. Impaired endothelium-dependent responses and enhanced influence of Rho-kinase in cerebral arterioles in type II diabetes. Stroke (2005) 36:342–347.[Abstract/Free Full Text]
  32. Bagi Z., Erdei N., Toth A., Li W., Hintze T.H., Koller A., et al. Type 2 diabetic mice have increased arteriolar tone and blood pressure: enhanced release of COX-2-derived constrictor prostaglandins. Arterioscler Thromb Vasc Biol (2005) 25:1610–1616.[Abstract/Free Full Text]
  33. Gokina N.I., Park K.M., McElroy-Yaggy K., Osol G. Effects of Rho kinase inhibition on cerebral artery myogenic tone and reactivity. J Appl Physiol (2005) 98:1940–1948.[Abstract/Free Full Text]
  34. Huang A., Sun D., Kaley G., Koller A. Superoxide released to high intra-arteriolar pressure reduces nitric oxide-mediated shear stress- and agonist-induced dilations. Circ Res (1998) 83:960–965.[Abstract/Free Full Text]
  35. Sato A., Sakuma I., Gutterman D.D. Mechanism of dilation to reactive oxygen species in human coronary arterioles. Am J Physiol (2003) 285:H2345–H2354.[ISI]
  36. Zou M.H., Shi C., Cohen R.A. Oxidation of the zinc–thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest (2002) 109:817–826.[CrossRef][ISI][Medline]
  37. Cai S., Alp N.J., McDonald D., Smith I., Kay J., Canevari L., et al. GTP cyclohydrolase I gene transfer augments intracellular tetrahydrobiopterin in human endothelial cells: effects on nitric oxide synthase activity, protein levels and dimerisation. Cardiovasc Res (2002) 55:838–849.[Abstract/Free Full Text]
  38. Rodriguez-Crespo I., Counts Gerber N., Ortiz de Montellano P.R. Endothelial nitric-oxide synthase. J Biol Chem (1996) 271(19):11462–11467.[Abstract/Free Full Text]
  39. Campbell W.B., Gebremedhin D., Pratt P.F., Harder D.R. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res (1996) 78:415–423.[Abstract/Free Full Text]
  40. Archer S.L., Gragasin F.S., Wu X., Wang S., McMurtry S., Kim D.H., et al. Endothelium-derived hyperpolarizing factor in human internal mammary artery is 11,12-epoxyeicosatrienoic acid and causes relaxation by activating smooth muscle BK(Ca) channels. Circulation (2003) 107:769–776.[Abstract/Free Full Text]

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