Cardiovascular Research

Cardiovascular Research 2003 59(2):488-500; doi:10.1016/S0008-6363(03)00424-3
© 2003 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation

Sibel Ülker, Dorothy McMaster, Pascal P. McKeown and Ulvi Bayraktutan^*

Department of Medicine, Institute of Clinical Science, Block B, Queen's University Belfast, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland, UK

^* Corresponding author. Tel.: +44-2890-263-178; fax: +44-2890-329-899. u.bayraktutan{at}qub.ac.uk

Received 22 October 2002; accepted 28 April 2003

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: Enhanced oxidative stress is involved in mediating the endothelial dysfunction associated with hypertension. The aim of this study was to investigate the relative contributions of pro-oxidant and anti-oxidant enzymes to the pathogenesis of endothelial dysfunction in genetic hypertension. Methods: Dilator responses to endothelium-dependent and endothelium-independent agents such as acetylcholine (ACh) and sodium nitroprusside were measured in the thoracic aortas of 28-week-old spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar Kyoto rats (WKY). The activity and expression (mRNA and protein levels) of endothelial nitric oxide synthase (eNOS), p22-phox, a membrane-bound component of NAD(P)H oxidase, and antioxidant enzymes, namely, superoxide dismutases (CuZn- and Mn-SOD), catalase and glutathione peroxidase (GPx), were also investigated in aortic rings. Results: Relaxant responses to ACh were attenuated in phenylephrine-precontracted SHR aortic rings, despite a 2-fold increase in eNOS expression and activity. Although the activity and/or expression of SODs, NAD(P)H oxidase (p22-phox) and GPx were elevated in SHR aorta, catalase activity and expression remained unchanged compared to WKY. Pretreatment of SHR aortic rings with the inhibitor of xanthine oxidase, allopurinol, and the inhibitor of cyclooxygenase, indomethacin, significantly potentiated ACh-induced relaxation. Pretreatment of SHR rings with catalase and Tiron, a superoxide anion (O₂^–) scavenger, increased the relaxant responses to the levels observed in WKY rings whereas pyrogallol, a O₂^–-generator, abolished relaxant responses to ACh. Conclusion: These data demonstrate that dysregulation of several enzymes, resulting in oxidative stress, contributes to the pathogenesis of endothelial dysfunction in SHR and indicate that the antioxidant enzyme catalase is of particular importance in the reversal of this defect.

KEYWORDS Endothelium; Antioxidants; Nitric oxide synthase; Nitric oxide; Hypertension

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Nitric oxide (NO) is generated within normal endothelium by eNOS in the presence of cofactors tetrahydrobiopterin (H₄B), NADPH and O₂ and plays a pivotal role in the regulation of vascular tone and blood pressure [1].The endothelium is altered in several disease states, including hypertension, leading to a phenomenon termed ‘endothelial dysfunction’, which is characterized by impaired endothelium-derived vasodilatation [2]. So far, several factors, including reduced availability of cofactor H₄B [3], diminished production of NO due to inefficent utilization of substrate L-arginine [4] or impaired expression and activity of eNOS protein [5] have been implicated in the etiology of endothelial dysfunction. Moreover, excessive release of endothelium-constrictor prostanoid (PGH₂) [6] and increased synthesis of vasoconstrictive cyclooxygenase products such as thromboxane A₂ (TXA₂) along with higher expression of endothelin-1 have also been associated with the pathogenesis of endothelial dysfunction in SHR and stroke prone SHR (SHRSP), respectively [7,8]. However, a single unifying mechanism has yet to emerge.

Excessive vascular production or diminished metabolism of reactive oxygen species (ROS) has recently been implicated in the pathogenesis of endothelial dysfunction in diabetes mellitus [9] and in SHRSP [10]. O₂^– is known as the foundation radical, formed by the reduction of O₂ that is converted to other ROS such as H₂O₂ by SODs [11]. H₂O₂ mediates vascular tone by inducing either contraction [12] or relaxation [13], depending on the vascular bed and experimental conditions as well as produces endothelial barrier by either directly altering endothelial function [14] or enhancing the membrane permeability to macromolecules [15]. The principal sources of ROS, at a vascular level, are cyclooxygenases, mitochondrial NAD(P)H oxidases, xanthine oxidases. NAD(P)H oxidase and the uncoupled state of eNOS as a result of its activation at suboptimal concentrations of H₄B, have received much attention in recent years. However, the actual contribution of these enzymes to endothelial dysfunction, that develops prior to or secondary to cardiovascular disease such as hypertension, remains to be determined.

The present study focused on hypertensive endothelial dysfunction and was designed to: (1) investigate the underlying molecular mechanisms of endothelial dysfunction in the thoracic aortic homogenates of SHR compared to normotensive WKY with reference to expression and activity of eNOS, NAD(P)H oxidase and aforementioned antioxidant enzymes; (2) study the putative enzymatic source(s) of excess ROS in hypertensive aorta and their relevance to impaired endothelium-dependent relaxation; and (3) investigate the efficacy of antioxidant agents such as O₂^–-scavengers and enzymes in reversing the endothelial dysfunction observed in SHR aorta.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The investigation conforms with 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).

2.1 Animals and thoracic aorta preparation
The studies were conducted using thoracic aortas obtained from 28-week-old male SHR and their matched normotensive counterparts, WKY. Thoracic aortas of rats were removed under deep pentobarbitone sodium anesthesia (26 mg/kg body weight, i.p.) and carefully cleaned of adhering tissue. The aortas were then either homogenized in a glass to glass homogenizer for total RNA/protein isolation or enzyme activity assays or were cut into four transverse rings (3 mm in length) for vascular reactivity experiments.

2.2 Vascular reactivity studies
Rings were equilibrated for 90 min under 2-g resting tension in organ baths filled with carbonated (95% O₂/5% CO₂) Krebs buffer [in (mM): NaCl 118.3, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.22, CaCl₂ 2.5, NaHCO₃ 25, glucose 11.1, pH 7.4]. Changes in isometric tension were detected by a force transducer and recorded via an 8 channel transducer data acquisition system (LE-TR201 and PowerLab/8S, respectively, ADInstruments). Subsequently, concentration–response curves to phenylephrine (PE, 0.001–30 µM) were obtained. The rings were then serially washed to baseline and equilibrated prior to contraction with submaximal concentration of PE to obtain approximately 80% of maximal contractile response. At the plateau of contraction, relaxant responses to acetylcholine (ACh, 0.001–30 µM) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.001–10 µM) were investigated to evaluate endothelium-dependent and endothelium-independent relaxations, respectively. The relaxant responses to ACh and SNAP were calculated as a percentage of initial preconstriction to PE. This experimental pattern was repeated throughout the whole study and only one vasorelaxant agent was used on each ring.

2.3 RT-PCR
Total RNA was isolated from aortic homogenates using guanidinium thiocyanate–phenol–chloroform extraction method [16]. RT-PCRs were carried out using Superscript one-step RT-PCR kit (Invitrogen) according to the manufacturer's instructions to prepare the probes for Southern and Northern analyses. The gene specific primers used to amplify eNOS, CuZn-SOD, Mn-SOD, catalase, p22-phox, GPx and GAPDH are provided in Table 1.

View this table: [in this window] [in a new window]   Table 1 Primers used in RT-PCR reactions

 
2.4 Preparation of cDNA probes
Gel purified PCR products were ligated into a TA cloning vector (Invitrogen) and transformed into E. coli INVF' competent cells. Plasmids containing inserts were selected via -complementation and automated DNA sequencing was conducted on an ABI Prism 377 DNA sequencer (Perkin-Elmer). Comparisons to template cDNA sequences were made using the Match-Box web server 1.2 database available on the internet [17].

2.5 Northern blotting
First, 20 µg of total RNA were electrophoresed through 1% formaldehyde–agarose gel and transferred onto Hybond-NX nylon membrane (Amersham Pharmacia). The blots were then hybridized with the appropriate fluorescein-labeled cDNA probe. Quantification of autoradiograms was performed by densitometric analysis and values were normalized against GAPDH.

2.6 Southern blotting
The amplified eNOS and Mn-SOD PCR products were size fractionated using agarose gel electrophoresis. Southern blotting and quantification of bands were performed as previously described [18].

2.7 Protein extraction and Western blotting
Methodology for protein extraction and Western blotting was as previously described [19]. The primary antibodies to eNOS, SODs and catalase were obtained from Transduction Laboratories, Chemicon and RDI, respectively. EAR antibody that recognizes p22-phox was a kind gift of Dr Mark Quinn (Bozeman, Texas, USA). Since a species-specific antibody for GPx protein could not be obtained, Western analysis for this protein was not performed.

2.8 Nitrite detection
Aortic homogenates were treated with nitrate dehydrogenase to convert nitrate to nitrite prior to mixing equal volumes of aortic homogenate and Griess reagent (sulfanilamide 1% w/v, naphthylethylenediamine dihydrochloride 0.1% w/v, and orthophosphoric acid 2.5% v/v). The samples were incubated at room temperature for 10 min and the absorbances were determined at 540 nm wavelength and compared to those of known concentrations of sodium nitrite. The amount of nitrite formed was normalized to the protein content of the respective aorta.

2.9 NAD(P)H oxidase assay
Aortic homogenates were prepared on ice in lysis buffer containing 1 mM EGTA, 20 mM monobasic potassium phosphate (pH 7.0), 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 10 µg/ml aprotinin and 0.5 mM phenylmethylsulfonyl fluoride. Oxidase activity was measured and calculated as previously described using 5 µM lucigenin in the reaction [19]. In some experiments, inhibitors of the ROS-generating enzymes i.e. L-NAME (0.1 mM), rotenone (50 µM), allopurinol (100 µM) or indomethacin (50 µM) were added to aortic homogenates for 60 min before determining O₂^– generation. Control rings from the same animals were analyzed simultaneously in parallel experiments.

2.10 SOD assay
Total SOD activity was measured by a reaction dependent upon the inhibition in reduction of cytochrome C by endogenous SOD in aortic homogenates using a Cobas Fara centrifugal analyzer. The O₂^–, required for reduction, was generated by a reaction of xanthine–xanthine oxidase (XO). One unit of XO activity was defined by the amount of homogenate required to inhibit, by 50%, the rate of cytochrome c reduction. This was followed by the measurement of Mn-SOD activity, which is resistant to incubation at room temperature with 3 mM potassium cyanide (BDH Chemicals) for 45 min. CuZn-SOD activity was subsequently calculated by the subtraction of Mn-SOD activity from total SOD activity.

2.11 Catalase assay
The activity of catalase was determined in homogenized aortic supernatants using a method adapted for automation on the Cobas Fara centrifugal analyzer [20]. Briefly, the activity was determined by monitoring the decomposition of H₂O₂ at 240 nm in the presence of methanol which produces formaldehyde which in turn reacts with Purpald (4-amino-3-hydazino-5-mercapto-1,2,4-triazole) and potassium periodate to produce a chromophore. Quantification was performed in comparison to the results that were obtained with catalase and formaldehyde standards.

2.12 Glutathione peroxidase (GPx) assay
The activity of GPx was determined in homogenized aortic supernatants using a method developed by McMaster et al. [21]. Briefly, a fresh solution containing 0.3 U/ml glutathione reductase, 1.25 mM reduced glutathione and 0.19 mM NADPH in 50 mM potassium buffer (pH 7.4) was prepared. Homogenates of 100 µg total protein were added to this solution and incubated for 3 min prior to addition of 12 mM t-butylhydroperoxide to commence the reaction. Absorbances were read at 340 nm for 4 min. Activities were calculated as nmol glutathione/mg protein/min.

2.13 Statistical analysis
Results are presented as mean±S.E.M. Each of the vascular reactivity and molecular biological experiments including enzyme activity assays was performed using a minimum of 10 and five rats, respectively. Comparison analyses were performed by ANOVA or paired and unpaired Student's t-tests where appropriate. The Bonferroni correction for multiple comparisons was used to determine the statistical significance of the P values.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 General data
The heart weight/body weight ratio (mg/g) (3.53±0.17 vs. 3.06±0.15, P<0.05, n = 60) and systolic blood pressure (mmHg) (188±13 vs. 140±11, P<0.05, n = 10) were significantly higher in SHR compared to WKY. However, there was no significant difference in body weight (g) (SHR 395±34, WKY 384±22, P>0.05, n = 60) or basal heart rate (bpm) (SHR 246±17, WKY 234±21, P>0.05, n = 60) between the two groups.

3.2 Relative abundance of mRNA and protein contents of eNOS, p22-phox and antioxidant enzymes
RT-PCR–Southern blot (for eNOS and Mn-SOD) and Northern blot (for CuZn-SOD, catalase, GPx and p22-phox) analyses detected a single transcript for each gene (Fig. 1A). The size of the mRNA transcripts for p22-phox, CuZn-SOD, catalase and GPx were 0.8, 0.7, 2.4 and 1.2 kb, respectively. The densitometric analysis of autoradiograms revealed an approximately 4-fold increase in GPx but no change in catalase mRNA expressions after normalizing to GAPDH mRNA levels in SHR aorta. The mRNA expression of other genes also revealed almost 2-fold increase in SHR aortas compared to those of WKY (Fig. 1B).

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 (A) Representative RT-PCR–Southern blots (eNOS and Mn-SOD) and Northern blots (p22-phox, CuZn-SOD, catalase, GPx and GAPDH) showing the mRNA expressions in the thoracic aortas of 28-week-old WKY and SHR. (B) Summary of the changes in SHR aortic gene expressions relative to those of WKY, after normalization of autoradiograms by the levels of GAPDH reference gene. Data from five separate experiments are expressed as mean±S.E.M.

 
Since the alterations in mRNA levels may not necessarily reflect the changes in corresponding proteins, the levels of protein expression were investigated by Western analyses (Fig. 2A). Analyses of the Western autoradiograms also revealed 2-fold increases in eNOS, p22-phox, CuZn-SOD and Mn-SOD but no difference in catalase protein level in SHR aorta compared to WKY (Fig. 2B).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 (A) Expression of eNOS, p22-phox and antioxidant enzyme proteins in the thoracic aortas of 28-week-old WKY and SHR as determined by Western blot. (B) Summary of the alterations in SHR aortic protein levels compared those of WKY. Data from five separate experiments are expressed as mean±S.E.M.

 
3.3 Enzyme activity levels
In accordance with the observed increases in mRNA and protein levels, the activities of eNOS, NADH oxidase, NADPH oxidase and both isoforms of SOD were found to be significantly higher in SHR aortic homogenates. However, both catalase and GPx activities were similar in SHR and WKY (Table 2).

View this table: [in this window] [in a new window]   Table 2 Enzyme activities in aortic homogenates

 
3.4 Vasodilator responses in thoracic aorta
In preliminary experiments, rings were contracted with a standard dose of KCl (60 mM) in order to provide an internal reference for contractile responsiveness between SHR and WKY aortas. The 2- and 2.5-g resting tensions were determined to be optimal resting tensions (RP_o) for WKY and SHR rings, respectively. However, to standardize the experimental protocol, 2-g resting tension was used for aortas obtained from both strains. Although this slightly decreased the contractile responses of SHR rings to PE, it did not alter overall percent relaxation rate or sensitivity (pD₂) to ACh compared to their counterparts from WKY. pD₂ values for WKY and SHR were 5.93±0.09 vs. 5.97±0.08, respectively.

In PE-precontracted aortic rings, ACh elicited a concentration-dependent relaxation in WKY. In contrast, ACh, at higher concentrations, induced dose-dependent vasoconstriction in SHR aortic rings indicating a defect at endothelial level (Fig. 3A). However, no difference was observed in the magnitude of endothelium-independent relaxant responses to incremental concentrations of SNAP (Fig. 3B). The pharmacological inactivation of endothelium by treatment with a NOS inhibitor, L-NAME (20–100 µM), or its denudation by mechanical force completely inhibited endothelium-derived vascular relaxation in both SHR and WKY (Fig. 3C,D).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A,B) Acetylcholine (ACh)- and S-nitroso-N-acetyl-D,L-penicillamine (SNAP)-induced relaxation of phenylephrine-preconstricted SHR and WKY aortic rings. (C,D) Denudation of endothelium by L-NAME (100 µM) or mechanical force completely abolished relaxant responses to ACh in both strains. Results are expressed as mean±S.E.M. from 10 separate experiments. *P<0.01 SHR vs. WKY, #P<0.01 compared to relaxation in endothelium-denuded rings.

 
3.5 Effect of superoxide anion and its scavengers on vasodilatation
To specifically determine a role for O₂^– in endothelial function, relaxant responses were recorded in PE-precontracted SHR and WKY aortic rings following 20 min incubation with a superoxide generator (pyrogallol, 50–150 µM) or O₂^– scavengers, namely, Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid, 1–10 µM) or MPG (mercaptopropionylglycine, 1–10 µM) (Fig. 4A–C). Pyrogallol almost completely abolished ACh-induced relaxation which was improved by Tiron and MPG in a dose dependent manner in both strains. To determine whether the vasodilatory effects of O₂^– scavengers are mimicked by SOD, the relaxant responses of SHR and WKY rings were determined following treatment with exogenous SOD (150–500 U/ml) for 20 min. SOD did not improve relaxant responses in any of the rings (Fig. 5A). Furthermore, the pretreatment of SHR or WKY rings with an irreversible endogenous SOD inhibitor diethylthiocarbamate (DETCA, 1–10 mM) for 20 min did not lead to further deterioration in endothelial function (Fig. 5B). In contrast, the incubation of rings with catalase (100–250 U/ml) or SOD plus catalase (20 and 100 U/ml, respectively) for 20 min significantly improved endothelial function. However, the relaxant responses obtained in response to combination of SOD plus catalase were not greater than those obtained with catalase alone (Fig. 5C,D). The incubation of rings with another antioxidant enzyme GPx alone (10–50 U/ml) or in combination with SOD (20 U/ml) did not significantly improve endothelial function in SHR or WKY aortic rings.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Acetylcholine-induced relaxation in WKY and SHR thoracic aorta, in the absence and presence of 100 µM pyrogallol (A), 10 µM Tiron (B) and 10 µM MPG (C). Results are expressed as mean±S.E.M. from 10 separate experiments. *P<0.01 SHR vs. WKY, #P<0.01 compared to relaxation in the presence of pyrogallol, Tiron and MPG.

 

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Acetylcholine-induced relaxation in WKY and SHR thoracic aorta, in the absence and presence of 150 U/ml SOD (A), 10 mM DETCA, an inhibitor of SOD (B), 100 U/ml catalase (Cat) (C) and a combination of 20 U/ml SOD plus 100 U/ml catalase (D). Results are expressed as mean±S.E.M. from 10 to 12 separate experiments. *P<0.01 SHR vs. WKY, #P<0.01 compared to relaxation in the presence of SOD, DETCA, catalase and SOD plus catalase.

 
3.6 Enzymatic sources of superoxide in genetic hypertension
To determine the enzymatic source of the O₂^– generated in hypertensive aorta, the rings were incubated for 20 min with the selective inhibitors of potential O₂^–-generating enzymes, namely, rotenone (10–50 µM, mitochondrial NADH dehydrogenase), allopurinol (100–300 µM, xanthine oxidase) or indomethacin (10–50 µM, cyclooxygenase). In separate experiments, the aortas were treated with either a flavoprotein inhibitor diphenyleneiodonium (DPI) (0.3–10 µM) or one of the two inhibitors of NAD(P)H oxidase system, namely, phenylarsine oxide (PAO) (1–10 µM) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) (1–10 µM, NAD(P)H oxidase inhibitors) prior to detection of relaxant responses to ACh. Although rotenone did not alter vascular relaxation of SHR and WKY to ACh, both allopurinol and indomethacin significantly improved these responses in SHR rings (Fig. 6A–C). The relaxant responses to ACh were abolished by DPI in both SHR and WKY rings. Other NAD(P)H oxidase inhibitors i.e. PAO (1–10 µM) and AEBSF (1–10 µM) did not improve relaxant responses to ACh (Fig. 7A–C).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Acetylcholine-induced relaxation in WKY and SHR thoracic aorta, in the absence and presence of 10 µM rotenone (A), 300 µM allopurinol (B), and 10 µM indomethacin (C). Data are expressed as mean±S.E.M. from 10 to 12 separate experiments. *P<0.01 SHR vs. WKY, #P<0.01 compared to relaxation in the presence of rotenone, allopurinol, indomethacin and DPI.

 

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Acetylcholine-induced relaxation in WKY and SHR thoracic aorta, in the absence and presence of 0.3 µM DPI (A), 10 µM AEBSF (B), and 10 µM PAO (C). Data are expressed as mean±S.E.M. from 10 to 12 separate experiments. *P<0.01 SHR vs. WKY, #P<0.01 compared to relaxation in the presence of DPI, AEBSF and PAO.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
NO, generated by eNOS, within vascular endothelium plays pivotal roles in the maintenance of blood pressure and vascular tone [1]. Although eNOS is constitutively expressed, many physiological stimuli like sex hormones may regulate its expression and hence NO generation [22]. Although a relative deficiency in NO has been associated with genetic hypertension, the data as to regulation of eNOS in this model are somewhat conflicting in that both decreased and increased eNOS expression has been reported [23,24]. Although the underlying causes of this discrepancy are unknown, it may possibly be due to use of SHR in different hypertensive stages, i.e. prehypertensive (up to 15 weeks) or hypertensive (15 weeks) for different studies [25]. In this study, 28-week-old male SHR and matched normotensive WKY were used. SNAP, an endothelium-independent relaxant, did not alter the magnitude of vasorelaxation in the thoracic aortas of both strains thereby suggesting the presence of an intact and functional vascular smooth muscle cell (VSMC) layer in both strains. However, the current study does not rule out that there may be functional differences in VSMC between WKY and SHR as has been shown by other investigators [26]. Results of the current study simply and clearly show that there is endothelial dysfunction in SHR.

ACh, an endothelium-dependent relaxant, produced vasorelaxation in WKY aortic rings in a dose-dependent manner. Similarly, ACh induced vasodilatation in SHR aortic rings at lower doses but elicited vasoconstriction at higher doses, therefore, suggesting the presence of a defect in endothelium and hence stimulation of the muscarinic receptors on VSMC [27]. Mechanical or pharmacological denudation of endothelium in SHR and WKY rings, by forceps and L-NAME treatments, respectively, also yielded vasoconstrictive responses to ACh. However, our study reveals that these attenuated relaxations in SHR aortic rings are not due to a decrease in eNOS mRNA/protein expressions or NO generation, as these parameters were found to be elevated at least 2-fold in SHR compared to WKY. It is noteworthy that the measurements of mRNA/protein levels and enzyme activities of eNOS and other enzymes reflect a variety of cells in the aortas. Therefore, the changes observed in these parameters cannot solely be ascribed to the endothelium despite the presence of an intact VSMC in both strains at 28 weeks of age.

Enhanced production of ROS, in particular O₂^–, has recently been implicated in the pathogenesis of endothelial dysfunction in a number of disease states including diabetes mellitus and SHRSP [9,10]. Although O₂^– has been reported to scavenge NO within the vascular wall to reduce its biological half-life, its direct relevance to attenuated endothelium-derived vasodilatation has remained to be determined [28]. In the present study, some rings were therefore pretreated with different concentrations of O₂^–-generator pyrogallol or O₂^–-scavengers namely, Tiron or MPG. The former treatment completely abolished vasodilatation whereas the latter treatments improved endothelial function in both SHR and WKY rings in a dose-dependent manner supporting the inverse correlation between the levels of O₂^– and potency of vasodilatation. Since, several enzymes such as xanthine oxidase, cyclooxygenase, eNOS, NAD(P)H oxidase and mitochondrial dehydrogenase have been associated with vascular O₂^– overproduction, some rings were treated with the specific inhibitors of these enzymes to investigate their relative contribution to O₂^– generation. Rotenone, an inhibitor of mitochondrial NADH dehydrogenase, did not have any effect on vascular relaxation while DPI attenuated relaxant responses to ACh in SHR and WKY rings. Although the underlying mechanism(s) of attenuation of relaxant responses by DPI were not investigated in the present study, previous studies have shown that these effects of DPI may be due to its ability to inhibit NO stimulation through reducing the heme moiety of soluble guanylate cyclase [29]. Although DPI is a broad-spectrum flavoprotein inhibitor that targets several enzymes such as eNOS, xanthine oxidase and cytochrome P450 reductase, it is commonly used to inhibit phagocytic and nonphagocytic NAD(P)H oxidase [19,30]. Hence, to further investigate whether the attenuation of vascular relaxation was due to an inhibitory effect of DPI on NAD(P)H oxidase, different concentrations of two additional NAD(P)H oxidase inhibitors, namely, PAO and AEBSF were used in separate experiments. PAO reversibly and completely inhibits NAD(P)H oxidase in neutrophils while AEBSF, a serine protease inhibitor, inhibits the binding of p47-phox to membrane-bound components, i.e. p22-phox and gp91-phox in an irreversible manner [31,32]. It was shown that PAO and AEBSF did not alter vascular relaxation to ACh. These effects may be explained by the recent findings showing that PAO and AEBSF do not increase NO availability despite decreasing the release of O₂^– [33]. In contrast, apocynin that has similar effects to AEBSF has been shown to increase NO availability in 3–4-month-old SHRSP when used at higher doses, i.e. 3 mM [34]. However, this drug was reported to be less effective at lower doses and in older (9–12 months) SHRSP, an age group that is closer to the animals used in the present study. Besides, apocynin produced similar anti-proliferative and anti-migratory responses to AEBSF in three different human endothelial cells at the doses used in our study [34]. Hence, it is plausible to hypothesize that the attenuated relaxant responses generated with PAO and AEBSF were not due to the choice of the inhibitors but due to limited contribution of NAD(P)H oxidase system to vascular O₂^– production despite a 2-fold increase in the mRNA and protein expression of NAD(P)H membrane-bound component, p22-phox and overall NAD(P)H oxidase activity in SHR aortas.

It is noteworthy in this context that several recent reports have demonstrated that O₂^– can also be generated by eNOS as a consequence of relative deficiency of its cofactor H₄B [3]. Indeed, H₄B has been implicated in the formation and stabilization of eNOS and its absence has been linked to uncoupling of the L-arginine–NO pathway and hence generation of O₂^– instead [35]. However, in our study we revealed a significant elevation in the levels of nitrite, an index of enhanced NO generation, in SHR aortic homogenates which not only suggests increased activity of eNOS but also indicates comparable levels of H₄B in SHR aortas compared to those of matched WKY. In support of these findings, we have recently shown that eNOS but not iNOS enzyme activity was also two-fold higher in SHR aortic homogenates compared those of WKY as measured by the conversion of L-arginine to L-citrulline [36].

On the other hand, the incubation of SHR rings with indomethacin and allopurinol, which inhibit cyclooxygenase and xanthine oxidase enzymes, respectively, improved ACh-induced vascular relaxations to the levels observed in WKY. Although, the current study did not investigate the underlying mechanisms of action for these enzymes in SHR, it is possible that indomethacin might have improved vascular relaxations due to inhibition of synthesis of vasoconstrictive cyclooxygenase products, such as PGH₂ and thromboxane A₂ (TXA₂). Indeed, it has been reported that the inhibition of thromboxane receptor normalizes endothelial dysfunction in adult SHR [29].

Deficiency or inactivation of SODs that dismutate O₂^– to H₂O₂ may also lead to increased levels of O₂^– in intact blood vessels. Although, a reduced SOD activity has previously been reported in SHR [37], our study did not confirm this report and revealed elevated mRNA/protein expressions and activities of CuZn-SOD and Mn-SOD in SHR aortas. Increased expression of SOD protein has also been reported in aortas of rabbits with coarctation-induced hypertension [38]. In support of these findings, it was shown in the present study that pretreatment of SHR and WKY rings with exogenous SOD did not improve relaxant responses to ACh possibly indicating sufficient availability of SOD in the vascular system. However, as the cell-impermeable exogenous SOD can only be effective in scavenging externally generated O₂^–, the validity of this hypothesis was investigated in experiments where an irreversible endogenous CuZn-SOD inhibitor, DETCA was used. Treatment of vascular rings with DETCA did not elicit any further impairment in endothelium-dependent relaxant responses thereby suggesting that the attenuated relaxation to ACh is not a consequence of SOD inhibition.

In contrast to exogenous SOD, pretreatment of vascular rings with catalase, a scavenger of H₂O₂, improved endothelium-dependent relaxation in SHR to the levels obtained in WKY rings. A recent study designed to investigate whether ROS mediate endothelium-dependent contractions to ACh in the aorta of SHR has shown that dismutation of endothelial O₂^– production into hydroxyl radicals and H₂O₂ plays a key role in endothelium-dependent contractile responses [12]. Indeed, a previous study had shown that exogenous H₂O₂ could cause contractions in aortas from SHR and WKY and removal of endothelium augmented contractions considerably in SHR compared to WKY in a COX pathway mediated manner [11]. The increase in H₂O₂ levels may have significant proatherogenic properties. H₂O₂ is known to be more stable than its precursor O₂^– and can readily cross VSMC membrane due to its uncharged nature where it promotes hypertrophy and stimulates several enzyme systems such as matrix metalloproteases [39]. H₂O₂ elicits irreversible endothelial damage, despite initially stimulating NO production. Indeed, homocysteine-induced endothelial cell injury has been associated with H₂O₂ and has been reduced by catalase [40]. Furthermore, SOD and catalase were found to inhibit hypertension-dependent vascular permeability and related cell damage in Ang II-induced hypertensive rats, suggesting the involvement of O₂^– and H₂O₂, respectively [41]. However, catalase produced better vasorelaxation in SHR aorta and rabbit lung compared to SOD. In our study, the effect of physiological concentrations of GPx, another enzyme that degrades H₂O₂ to H₂O, on vascular relaxations has also been investigated and revealed no significant improvement in endothelial function.

In conclusion, the accentuation of vasorelaxant responses to catalase suggests that a significant part of vasoconstriction in thoracic aortas of SHR may be due to concomitant excess generation of H₂O₂. Considering the enhanced expressions and activities of CuZn-SOD and Mn-SOD in SHR aorta and inability of DETCA, a SOD inhibitor, to cause further deterioration of endothelial function, we hypothesize that excess production of O₂^– cannot solely account for the pathogenesis of endothelial dysfunction in SHR. The potentiation of vasorelaxant responses by the selective inhibitors of xanthine oxidase and cyclooxygenase but not NAD(P)H oxidase indicates that increased vascular availability of O₂^– may be due to the involvement of more than one enzymatic system.

Time for primary review 20 days.

	Acknowledgements

 
This work was supported by the Heart Trust Fund (Royal Victoria Hospital) and the Royal Society, UK.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

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