The bimodal regulation of vascular function by superoxide anion: role of endothelium

Reactive oxygen species (ROS) are implicated in vascular ho-meostasis. This study investigated whether O 2. 󰠏 , the foundation molecule of all ROS, regulates vasomotor function. Hence, vascular reactivity was measured using rat thoracic aortas exposed to an O 2. 󰠏 generator (pyrogallol) which dose-dependently regulated both α -adrenergic agonist-mediated contractility to phenylephrine and endothelium-dependent relaxations to acetylcholine. Pyrogallol improved and attenuated responses to acetylcholine at its lower (10 nM - 1 μ M) and higher (10 - 100 μ M) concentrations, respectively while producing the inverse effects with phenylephrine. The endothelial inactivation by L-NAME abolished acetylcholine-induced vasodilatations but increased phenylephrine and KCl-induced vasoconstrictions regardless of the pyrogallol dose used. Relaxant responses to sodium nitroprusside, a nitric oxide donor, were not affected by pyrogallol. Other ROS i.e. peroxynitrite and H 2 O 2 that may be produced during experiments did not alter vascular functions. These findings suggest that the nature of O 2. 󰠏 -evoked vascular function is determined by its local concentration and the presence of a functional endothelium. [BMB reports 2008; 41(3): 223-229]


INTRODUCTION
The endothelium plays a pivotal role in the maintenance of normal vascular tone. However, its properties in pathological conditions change leading to a phenomenon termed "endothelial dysfunction" which is characterized by impaired endothelium-dependent relaxation (1). Endothelial dysfunction has been reported in conduit and resistance arteries of patients with vascular diseases including hypertension and in various animal models of human diseases (2)(3)(4). Despite current data on the etiology of endothelial dysfunction being rather diverse, much of the recent data have implicated oxidative stress in its patho-genesis (5,6). Oxidative stress is associated with enhanced production of reactive oxygen species (ROS) in particular superoxide anion (O2 . ) that is normally converted to hydrogen peroxide (H2O2) by superoxide dismutases (SODs). H2O2 is in turn metabolized to H2O by antioxidant enzymes; catalase and glutathione peroxidase (7). However, in pathological states the balance between the synthesis and metabolism of ROS may be impaired thus leading to generation of oxidative stress. O2 . can elicit direct vasoconstriction and scavenge nitric oxide (NO), the most potent endogenous vasodilator, to produce peroxynitrite (OONO . ) which oxidizes proteins, breaks DNA strands and reduces intracellular antioxidants such as glutathione and cysteine (1,8). Contrary to these findings, other studies have revealed that low levels of O2 . are required for normal vascular cell proliferation and migration and contribute to endothelium-dependent vasodilatation in some vascular beds particularly after dismutation to H2O2 (9,10).
In the light of these contradictory findings, the aim of the current study was to investigate whether local concentrations of O2 . determine the nature of vascular functions using aortic rings from Sprague Dawley (SD) rats. To this end, relaxant responses to endothelium-(in)dependent agents and contractile responses to an α-adrenergic agonist phenylephrine (PE) and receptor-independent constrictor KCl were investigated in vessels exposed to incremental concentrations of an O2 . -generating agent, pyrogallol. The requirement for an intact endothelium and the involvement of other ROS including H2O2 and OONO . in putative functional changes were also examined in the study.

Effects of pyrogallol on levels of O2 . in aortic rings
Pyrogallol produced dose-dependent increases in the levels of aortic O2 . as detected by lucigenin enhanced-chemiluminescence, cytochrome C reduction and DHE oxidation assays without altering the level of LDH release, an indicator of cellular damage (supplemental Table 1).

Effects of O2 . on vasorelaxations
In PE-precontracted aortic rings, both acetylcholine (ACh) and SNP produced concentration-dependent relaxations where enhttp://bmbreports.org  dothelial inactivation by mechanical (by rubbing) or pharmacological (via incubation with 50 μM of L-NAME) means selectively inhibited endothelium-dependent but not endothelium-independent vascular relaxations (Supplemental Fig. 1). Pyrogallol regulated endothelium-dependent relaxant responses in aortic rings in a dose-dependent manner in that it improved and attenuated ACh-mediated relaxations at its lower (10 nM -1 μM) and higher (10 and 100 μM) concentrations, respectively ( Fig. 1A-B). However, pyrogallol did not affect relaxant responses to SNP. It is of note that the inactivation of endothelium selectively abrogated ACh-but not SNP-mediated relaxant responses in the presence of pyrogallol (data not shown).
To investigate whether pyrogallol-induced effects are attributable to its O2 . -releasing function, the aortic rings were pretreated with one of the two structurally different O2 . scavengers, namely Tiron (4, 5-dihydroxy-1, 3-benzenedisulfonic acid, 1 μM) or MPG (mercaptopropionylglycine, 1 μM) for 20 min. Both agents prevented pyrogallol-mediated (10 μM) endothelium-dependent relaxations without affecting the basal relaxations determined in the absence of pyrogallol ( Fig. 2A-B). To support these findings, experiments with exogenous SOD (150 U / ml) or an endogenous inhibitor of SOD, namely dieth-yldithiocarbamate (DETCA, 1 -10 mM) were conducted. Pretreatments with either agent alone for 20 min failed to alter the ACh-mediated relaxant responses while the combination of DETCA and 10 μM pyrogallol worsened these responses and the SOD and pyrogallol combination was found to be ineffective ( Fig. 2C-D). To establish whether the effects of low concentrations of pyrogallol are also mediated by O2 . , similar experiments were conducted in the presence of 10 nM of pyrogallol where Tiron and MPG suppressed low dose pyrogallol-induced increases in ACh-mediated relaxant responses by almost 22% while DETCA and exogenous SOD failed to alter these responses in a significant manner.
Experiments performed under identical conditions with Tiron, MPG, SOD or DETCA revealed that none of these treatments had an impact on endothelium-independent relaxant responses.

Putative involvement of other ROS in O2
.

-mediated vasorelaxations
To examine the relative contributions of hydroxyl radical ( . OH), H2O2 or OONO . that may be generated during incubation with pyrogallol to relaxant changes, the aortic rings were treathttp://bmbreports.org BMB reports OH, H2O2 and OONO . . It was revealed that none of these agents displayed significant effects on vasorelaxations. Fig. 3A-C selectively show the effects of DMSO, catalase and Mn(III)TMPyP on ACh-mediated relaxations in the presence of 10 μM of pyrogallol. To ensure whether pyrogallol treatments led to increases in intracellular levels of OONO . or H2O2, the levels of these ROS were measured in aortic vessels which showed steady increases to incremental concentrations of pyrogallol (supplemental Table 2). Given that OONO . or H2O2 (10 nM -100 μM for each agent) per se produced dose-dependent increases in vascular relaxation in vessels with or without endothelium (Supplemental Fig. 2), it was safe to suggest that the pyrogallol-mediated increases in OONO . or H2O2 did not reach the lower levels that were used in the latter experiments. Similar experiments with the listed inhibitors revealed their inefficacy in altering endothelium-independent relaxant responses to SNP.
The receptor-independent contractile responses of pyrogallol were determined through use of KCl (120 mM) that produced similar contractions in isolated aortic rings with or without endothelium in the absence or presence of pyrogallol ( Fig. 4D-E). However, endothelial inactivation by L-NAME dramatically enhanced KCl-induced contractile responses in the presence of pyrogallol (Fig. 4F).

Putative involvement of other ROS in O2
.

-mediated vascular contractility
Tiron and MPG did not affect basal contractility to PE. However, they diminished pyrogallol-induced (10 μM) contractile responses to PE by almost 50 % (P ＜ 0.05). Inactivation of endothelium with L-NAME (50 μM) did not influence inhibitory effects of these components on pyrogallol-evoked contractions (Supplemental Fig. 3). In contrast to O2 . scavengers, treatments with exogenous SOD, DETCA, catalase, DMSO and Mn(III) TMPyP in the presence of pyrogallol (100 nM or 10 μM) did not significantly affect PE-mediated contractile responses and generated between 3 to 5 % difference in these responses.

DISCUSSION
Several factors including diminished production of NO due to inefficient utilization of substrate L-arginine or impaired activity of endothelial NO synthase have been implicated in the pathogenesis of endothelial dysfunction (11,12). However, oxidative stress characterized by the excessive vascular presence of O2 . , has recently been associated with the pathogenesis of this phenomenon in a variety of diseases with vascular complications such as hypertension and hypercholesterolemia (2, 6). O2 . under pathological conditions may trigger the formation of other ROS such as OONO . , .
Since the majority of the previous studies investigating the effects of ROS on vascular functions have focused mainly on exogenous H2O2 (10,(13)(14)(15)(16), the current study aimed to investigate the direct effects of O2 . , the foundation molecule of all ROS, on vasomotor functions using aortas from SD rats. This study primarily demonstrated that pyrogallol, a potent O2 . -generator, produced significant dose-dependent increases in intracellular levels of O2 . without affecting the tissue viability as determined by the measurements of LDH release. These findings were particularly important to ascertain that the putative differences that might be observed in the study would not be due to cellular damage. It also demonstrated that both ACh and SNP produced dose-dependent vasodilatations in aortic rings and that the inactivation of endothelium by mechanical or pharmacological means selectively abolished relaxations to ACh thereby indicating the existence of a functional vascular smooth muscle layer following endothelial damage by either means. The investigation of the impact of O2 . on vasomotor function revealed that the endothelium-derived vasodilatations to ACh were improved and attenuated by lower (10 nM -1 μM) and higher (10-100 μM) concentrations of pyrogallol, respectively and also that the inactivation of endothelium abrogated solely ACh-mediated responses regardless of the concentration of pyrogallol. Taken together these data implied a prerequisite for endothelial presence in modulation of beneficial activities of pyrogallol. This hypothesis was supported by the findings illustrating that the endothelial inactivation dramatically increased PE-induced contractile responses irrespective of the pyrogallol dose used despite initially recording significant decreases with lower concentrations (10 nM -1μM) of pyrogallol in PE-evoked contractility.
It is noteworthy here that the combination of PE and pyrogallol generated similar effects in endothelium-intact and -denuded rings, findings were in agreement with previous ones suggesting that the smooth muscle cells or α1-mediated responses may be the sites of action of O2 . (17,18). Moreover, the additional contractility studies with KCl (120 mM) alone or with different doses of pyrogallol (10 nM -100 μM) produced similar contractions in vascular rings with or without endothelium. However, pretreatment of aortic rings with L-NAME significantly enhanced KCl-mediated contractile responses to pyrogallol selectively in endothelium-intact segments whereby indicated that the accentuation of pyrogallol-mediated contractility was due to endothelial inactivation but not to L-NAME itself. These data were rather surprising given that KCl induces smooth muscle contractions by depolarizing the cell membrane via a mechanism depending on calcium influx through the voltagedependent channels (19).
It has been well-documented that NO is involved in much of the endothelium-derived vasodilatation and is readily neutralized by O2 . (1,8,20). The direct correlation between O2 . http://bmbreports.org BMB reports and vascular reactivity was further assessed in this study using O2 . scavengers i.e. Tiron and MPG which prevented pyrogallolinduced impairments in ACh-mediated relaxant responses without altering the basal endothelium-dependent relaxations. Nonetheless, while the beneficial effects of O2 . scavengers were not mimicked by exogenous SOD, an irreversible inhibitor of endogenous SOD i.e. DETCA worsened relaxant responses when used in combination with pyrogallol. These findings suggest that an increase in endogenous O2 . stemmed from the inhibition of SOD alone is not sufficient to neutralize intracellular NO to deteriorate vascular relaxations.
It is inevitable that the incubation of aortic rings with pyrogallol would lead to generation of other ROS including and H2O2 on vasculature showed that both agents could produce relaxations in a dose-dependent manner in vessels with or without endothelium thereby indicating direct vasodilatory effects of these compounds on smooth muscle cells. In previous studies, OONO . -mediated relaxations have been assigned to elevation of cGMP levels, interference with Ca 2+ movement and membrane hyperpolarization via K + -channel activation while those of H2O2 have been attributed to the NO synthesis and activation of smooth muscle K + channels (21,22). Although, the elucidation of the molecular mechanisms involved in O2 . -mediated vascular responses was out of the scope of the current study, it is possible that some of these mechanisms may be associated with O2 . -induced relaxant responses. In the current study, the inability of the ROS inhibitors to alter vascular responses may be explained by the existence of a delicate balance between their relaxant and contractile effects e.g. OONO . , in addition to its vasodilator effects, also generates vascular contractions by reducing intracellular antioxidants including glutathione and oxidizing tetrahydrobiopterin, a critical cofactor for NO synthesis (1).
In summary, the major conclusions to be drawn from this study are firstly that the vascular relaxant and contractile functions are regulated by O2 . in a dose-dependent fashion. The similar bimodal regulation of vascular function has previously been reported with H2O2 in endothelium-intact and endothelium-denuded rat aortas (21). Secondly, a functional endothelium is required for the regulation of O2 . -mediated dilator and contractile responses to ACh and PE, respectively. Finally, O2 . may exert a direct effect on smooth muscle cells via stimulation of α1-adrenergic receptors.

MATERIALS AND METHODS
All the experiments described in this article were performed in accordance with the UK "Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986".

Drugs and chemicals
All chemicals, other than Mn(III)TMPyP (IDS Ltd., UK), used in this study were from Sigma Chemical Company (UK).

Animals
The studies were conducted using the thoracic aortas obtained from 12-14 week old male SD rats. The aortas were removed from anticoagulated rats (100 U heparin, IV) under deep pentobarbitone sodium anesthesia (100 mg / kg body, IP) and carefully cleaned of adhering tissue.

Vascular reactivity studies
Thoracic aortas were cut into six 2-3 mm rings and mounted horizontally on stainless hooks in 25 ml 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]. Rings were equilibrated for 90 min under 2 g resting tension and only one vasodilator was used for each ring experiment. Changes in isometric tension were detected and recorded by a force transducer and an 8 channel transducer data acquisition system (LE-TR201 and PowerLab / 8S, ADInstruments), respectively. After initial equilibration period, ordinary Krebs solution was replaced with fresh solution containing 1 μM indomethacin to dismiss the relaxant effects of prostanoids. All experiments were performed in the latter solution.
At the beginning of each experiment dose-response curves to PE (0.003-10 μM) were obtained. Rings were then washed and equilibrated prior to contraction with submaximal concentration of PE before detecting endothelium-dependent or endothelium-independent relaxant responses to acetylcholine (ACh, 0.03-1 μM) and sodium nitroprusside (SNP, 0.01-10 μM), respectively. For contractility studies, the rings were incubated with cumulative concentrations of PE (0.003-10 μM) or a single dose of KCl (120 mM) until the respective contractions reached a plateau. For pyrogallol experiments, rings that were used in the abovementioned experiments were washed and equilibrated prior to 20 min incubation with the required concentration of pyrogallol [C6H6O3; 1,2,3-trihydroxybenzene] and repetition of the same experiments. This experimental pattern allowed each vascular ring to serve as its own control for a given chemical.
At the end of each experiment, the rings were blotted dry, weighed and the lengths were measured to calculate tension as normalized for cross-sectional area as previously reported (23).

Detection of O2
. O2 generation in aortic rings was measured using three differ-ent approaches. Firstly, thoracic aortic rings (~10 mm) were placed in Krebs solution for 30 min at 37 o C prior to transfer into Krebs solution containing 5 μM of lucigenin. The chemiluminescence was recorded every 60 sec for 10 min using a luminometer and the levels of O2 . were calculated by taking into account the differences in the readings before and after addition of the aortic segments to the buffer (24).
Secondly, level of O2 . was measured by SOD-inhibitable cytochrome C reduction assay. Briefly, aortic homogenates were incubated with 50 μM cytochrome C for 60 min at 37 o C in Krebs buffer with / out SOD (125 U / ml). Absorbances were recorded using a GENios plate reader (TECAN) at 550 nm with 60 sec intervals for 8 min. Production of O2 . was calculated as previously described and results were expressed as pmole O2 . per mg tissue (25).
Finally, the level of O2 . was measured using dihydroethidium (DHE) assay. DHE is oxidized by O2 . thereby yielding a fluorescent compound whose emission relates to the amount of O2 . present in the system. This assay was performed in 96well microplates. Each well contained 100 μl of homogenate obtained from aortas (~10 mm) exposed to different concentrations of pyrogallol (10 nM -100 μM) in phosphate buffer (pH 7.8) plus 10 μM of DHE in a volume of 100 μl of phosphate buffer. The reaction was performed at 25 o C and the extent of DHE oxidation was followed by measuring the increase of the fluorescence using a microplate reader for 60 min (excitation = 485 nm and emission = 595 nm). Three independent experiments were performed for each concentration of pyrogallol. The percentage generation of fluorescence with aortic homogenates was calculated using the pyrogallol-untreated aortic homogenates as positive controls.

Detection of H2O2
H2O2 synthesis was measured using 2',7'-dichlorodihydrofluorescein (DCHF) diacetate (DA) as a fluorescent probe for intracellular H2O2 measurement (26). DCHF-DA diffuses readily to the intracellular compartment, where it is desacetylated to the non-membrane-permeable DCHF. Then, during the cellular production of H2O2, DCHF is oxidized and emits a fluorescent signal. To this end, the normal and pyrogallol-incubated aortic segments were homogenized and loaded with 20 μM DCHF-DA for 30 min at 37 o C and fluorescence generation at 488 nm (excitation) and 525 nm (emission) was detected. The fluorescent signal was registered as a function of the time.

Detection of OONO .
Thoracic aortas were incubated with different concentrations of pyrogallol (10 nM -100 μM) in the presence of 20 μM 123dihydrorhodamine (123-DHR) for 2 h at 37 o C in Krebs solution. Following incubation, 123-DHR conversion to 123-rhodamine was measured by fluorometric analysis at excitation / emission wavelengths of 485 and 530 nm, respectively. Fluorescence due to autooxidation of 123-DHR was deducted from the original measurements (27).

LDH measurement
The vascular rings were exposed to different concentrations of pyrogallol in a 3 mL of phenol-free cell culture medium (DMEM) for 3 h at 37 o C in a humidified tissue culture incubator (5 % CO2 / 95 % air) prior to removal of an 100 μl of aliquot. The LDH release was measured spectrophotometrically from the changes in absorbance at 560 nm using 0.25 mM NADH and 0.75 mM pyruvate as substrates following subtraction of the background values obtained from the tubes without rings (28).

Statistical analysis
Data are expressed as mean ± SEM. Each of the vascular reactivity was performed using 8 rats. Dose-response curves were fitted by non-linear regression with simplex algorithm and Emax values, the maximum contractile response of the tissue that indicates the potency, were calculated. Relaxant responses were given as the percentages of PE precontraction. Comparisons of dose-response curves were evaluated by two-way analysis of variance (ANOVA) for repeated measures. Molecular biological experiments were performed using aortic samples from 3 different rats. Statistical significances for LDH release and ROS production were assessed by two-way ANOVA followed by Bonferroni-Dunn's post hoc analysis. P values less than 0.05 were considered to be statistically significant.