Journal of Cardiovascular Pharmacology:
Effect of Nebivolol on Endothelial Nitric Oxide and Peroxynitrite Release in Hypertensive Animals: Role of Antioxidant Activity
Mason, R Preston PhD*‡; Kubant, Ruslan MD, PhD†; Jacob, Robert F PhD‡; Walter, Mary F PhD‡; Boychuk, Bohdan MD, PhD†; Malinski, Tadeusz PhD†
From the *Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; †Department of Biochemistry, Ohio University, Athens, Ohio; and ‡Elucida Research, Beverly, Massachusetts. RPM has served as a consultant to Mylan Pharmaceuticals.
Received for publication February 7, 2006; accepted June 2, 2006.
Reprints: Tadeusz Malinski, PhD, Ohio University, 350 W. State Street, Athens, OH 45701 (e-mail: firstname.lastname@example.org).
We tested the activity of nebivolol, a β1-selective blocker with respect to nitric oxide (NO) and peroxynitrite (ONOO−) generation in the endothelium of normotensive Wistar Kyoto (WKY rats) and spontaneously hypertensive rats (SHR). The endothelial effects of nebivolol and its 2 optical enantiomers were correlated with its antioxidant activity and compared to another β-blocker, atenolol, and 2 agonists of nitric oxide synthase (eNOS), calcium ionophore (CI) and acetylcholine (ACh). The effects of nebivolol on the bioavailability of NO and ONOO−, indicators of endothelial function and dysfunction, respectively, were measured in vitro using nanosensors placed in mesenteric arteries. Compared with WKY rats, treatment of SHR vessels either with ACh (1 μmol/L) or CI (1 μmol/L) showed marked deficiencies (>40%, P < 0.01) in bioavailable NO concomitant with increased ONOO− levels (>50%, P < 0.01). The [NO]/[ONOO−] ratio measured after stimulation with CI was 2.77 ± 0.05 in WKY rats and much lower (1.14 ± 0.11) in SHR indicating significant eNOS uncoupling and endothelial dysfunction in hypertensive animals. Treatment with nebivolol (10 μmol/L) inhibited eNOS uncoupling and reduced endothelial dysfunction in SHR, as evidenced by an increase in the [NO]/[ONOO−] ratio to 3.09 ± 0.04. The basis for nebivolol activity is attributed to its unique membrane interactions as determined by small-angle x-ray diffraction, as well as its antioxidant activity at nanomolar to micromolar levels. The antioxidant effects of nebivolol and its enantiomers were not reproduced by atenolol. These results demonstrate that nebivolol inhibits endothelial dysfunction through a potent antioxidant mechanism attributed to its physicochemical interactions with the membrane, independent of β1-blockade activity.
The contribution of endothelial dysfunction to arteriosclerosis and its clinical manifestations (coronary artery disease, heart failure) have been well characterized.1-3 Key risk factors for arteriosclerosis, including dyslipidemia, smoking, and diabetes, can be specifically linked to abnormalities in nitric oxide (NO)-mediated endothelial vasodilation.1-3 A reduction in NO bioavailability also contributes to elevated vascular resistance and loss of sensitivity to stimulation of vasodilation, hallmark features of hypertension.4-6 Beyond vasodilation, NO has well-characterized vascular benefits, including inhibition of smooth muscle cell proliferation and migration, adhesion of leukocytes to the endothelium, and platelet aggregation.7 In patients at higher risk for cardiovascular disease, such as African Americans, and from clinical consequences, there is evidence for reduced NO-mediated vasodilation associated with increased superoxide generation in endothelial cells.8-10 Thus, agents that directly stimulate NO release may have important therapeutic advantages in the prevention and treatment of cardiovascular disease.
Nebivolol is a new β-blocker with highly selective β1-adrenergic receptor blocking and direct vasodilatory properties.11 The mechanism of vasodilation is attributed to stimulation of endothelial-dependent NO synthase, an effect that can be inhibited with NG-monomethyl-L-arginine.12 Of its 2 enantiomers, d-nebivolol shows β1-selectivity, whereas l-nebivolol lacks β1-adrenergic activity. However, both enantiomers stimulate endothelial-dependent NO release, with evidence for greater activity with l-nebivolol, despite its absence of high β1-selectivity.11 In cellular and animal models, nebivolol has been demonstrated to affect vasodilation through endothelial β2-adrenergic receptor-mediated NO production and/or adenosine triphosphate efflux with consequent stimulation of P2Y-purinoceptor-mediated NO release.13,14 It has also been reported that nebivolol inhibits NO synthase uncoupling and produces systemic antioxidant effects.15,16 However, the molecular mechanism(s) by which nebivolol reduces oxidative stress and inhibits eNOS uncoupling in the cell is not well understood.
In this study, we tested the hypothesis that nebivolol can restore NO bioavailability in a genetic animal model of hypertension and reverse endothelial dysfunction. This hypothesis was tested using isolated mesenteric arteries from stroke-prone SHR characterized by reduced NO bioavailability despite increased eNOS enzymatic activity.17 The basis for this paradox in endothelial function in SHR vessels is attributed to excessive superoxide (O2−) generation, leading to reduced levels of NO and formation of the highly reactive product, peroxynitrite (ONOO−).18 This is consistent with other hypertension studies that have linked increased O2− formation to loss of endothelial function caused by eNOS uncoupling and increased NAD(P)H oxidase activity.19,20 In this article, we report the endothelial-dependent effects of nebivolol, along with its separate enantiomers, in SHR and normotensive Wistar-Kyoto (WKY) rat vessels. The effects of nebivolol were also compared to another β-blocker, atenolol, using nanotechnological approaches. The antioxidant mechanism for nebivolol was evaluated in model lipid vesicles enriched with polyunsaturated fatty acids and correlated with its physicochemical interactions, as directly determined by small-angle x-ray diffraction approaches. These studies provide new insights into mechanisms of hypertension and the role of a lipophilic β1-selective agent with antioxidant actions.
MATERIALS AND METHODS
Dilinoleoylphosphatidylcholine (DLPC) and natural lipids extracted from bovine cardiac tissue (bovine cardiac phosphatidylcholine) were obtained from Avanti Polar Lipids (Alabaster, AL), dissolved in high-performance liquid chromatography-grade chloroform (10.0 mg/mL), and stored at -80°C. The phospholipid fatty acid composition was determined by gas and liquid chromatography. The overall ratio of saturated to unsaturated fatty acids was 0.8:1, with the primary constituents being 18:2 linoleic acid (30%), 16:0 palmitic acid (22%), 18:1 oleic acid (13%), and 20:4 arachidonic acid (11%). Atenolol, nebivolol racemate, l-nebivolol, and d-nebivolol were supplied by Elucida Research (Beverly, MA). Trolox, probucol, calcium ionophore (CI, A23187), and acetylcholine (ACh) were purchased from Sigma (St. Louis, MO). CHOD-iodide color reagent (stock) was prepared according to a procedure modified from El-Saadani et al.21 and consisted of 0.2 mol/L K2HPO4, 0.12 mol/L KI, 0.15 mmol/L NaN3, 10 μmol/L ammonium molybdate, and 0.1 g/L benzalkonium chloride. Before experimental use, the CHOD reagent was activated by adding 24 μmol/L ethylenediaminetetraacetic acid, 20 μmol/L butylated hydroxytoluene, and 0.2% Triton X-100.
WKY rats and stroke-prone SHR of either sex were obtained from inbred colonies (National Institutes of Health, Bethesda, MD) and used at 15 weeks of age.
Preparation of Mesenteric Resistance Arteries
Rats were killed with pentothal (150 mg/kg) injected intraperitoneally, and the mesentery was removed and placed in modified Hanks' balanced salt solution (4°C, pH 7.4) containing: 137 mmol/L NaCl, 10 mmol/L Tris-HCl, 1 mmol/L MgCl2, 5 mmol/L KCl, 0.9 mmol/L CaCl2, 0.8 mmol/L MgSO4, 0.33 mmol/L KH2PO4, and 0.1 mmol/L L-arginine. A small segment of the third-order branch of the mesenteric artery (≈3-mm long and ≈200-μm internal diameter) was isolated and cleaned of adhering tissue under a dissection microscope (M3C, Wild AG, Gais, Switzerland).
NO and ONOO− Nanosensors
Concurrent measurements of NO and ONOO− were performed with 2 electrochemical nanosensors combined into 1 working unit. Their design was based on previously developed and well-characterized chemically modified carbon-fiber technology.22 Each of the nanosensors was made by depositing a sensing material on the tip of a carbon fiber (length 4-5 μm, diameter 100-200 nm). We used a conductive film of polymeric Ni(II) tetrakis (3-methoxy-4-hydroxyphenyl) porphyrin and conductive polymeric film of Mn(III) [2.2] paracyclophanylporphyrin for the NO and ONOO− sensors, respectively.
The amperometric method (with a response time of 0.1 ms) provided a quantitative signal (current) directly proportional to changes in NO or ONOO− concentration from their basal levels. The basal level of NO or ONOO− was measured in separate experiments by differential pulse voltammetry (response time 3 s).23 The experiments were performed with (Gamry III) double-channel potentiostat.
Ex Vivo Measurements of NO and ONOO−
All measurments of NO and ONOO− were performed on the intact endothelial cell. Isolated ring segments of mesenteric arteries from WKY rats and SHR were placed vertically and immobilized on Sylgord film in an organ chamber combining oxygenated fresh Hanks' balanced salt solution, 2 mL, at 37°C. A module of NO/ONOO− nanosensors (total diameter ≈400 nm) was inserted into the lumen of the resistance artery ring and positioned near (5 ± 2 μm) intact endothelial cells with the help of a computer-controlled micromanipulator (x-y-z resolution 0.2 μm) under a microscope with a CD camera. After a background current was established, CI, ACh, nebivolol and its enantiomers, or atenolol were individually injected directly into the organ, with a picoinjector or nanoinjector. A rapid change of current (proportional to generated NO or ONOO− concentration) were observed (300-900 ms) after addition of drugs and were monitored continuously for 60 s.
Preparation of Multilamellar Lipid Vesicles (MLVs) for X-ray Diffraction and Lipid Peroxidation Analyses
Aliquots of lipid (1.0-5.0 mg) were transferred to 13 × 100-mm test tubes and dried down under a steady stream of N2 gas while vortexing vigorously for 3 min. Residual solvent was removed under vacuum in low-light conditions. Lipid samples were then resuspended in diffraction buffer (0.5 mmol/L HEPES, 154.0 mmol/L NaCl, pH 7.3) and vortex mixed at ambient temperature to form MLVs, as described elsewhere.24 For x-ray diffraction analyses, MLVs were prepared from binary mixtures of bovine cardiac phosphatidylcholine and cholesterol; the final phospholipid concentration was 2.5 mg/mL, and the cholesterol-to-phospholipid (C/P) mole ratio was adjusted to 1:5. Drugs were tested in this system at a drug-to-phospholipid mole ratio of 1:30, resulting in a final concentration of <1%, by mass. For peroxidation studies, MLVs were prepared from DLPC (1.0 mg/mL) in the absence or presence of drugs (nebivolol racemate, atenolol) and antioxidants (Trolox, probucol) at various concentrations.
Small-angle X-ray Diffraction Analysis of Drug/Membrane Structure
MLVs were oriented for x-ray diffraction analysis as described previously.25 Briefly, aliquots containing 250 μg of MLVs (based on phospholipid) were transferred to custom-designed Lucite sedimentation cells, each containing an aluminum foil substrate upon which to collect a single membrane pellet. Samples were then loaded into a Sorvall AH-629 swinging bucket ultracentrifuge rotor (DuPont, Wilmington, DE) and centrifuged at 35,000g for 50 min at 5°C. Following orientation, the supernatants were aspirated and the aluminum foil substrates, which supported the membrane pellets, were removed from the sedimentation cells and mounted onto curved glass slides. The samples were then placed in hermetically sealed brass canisters in which temperature and relative humidity were controlled during x-ray diffraction experiments. Potassium tartrate (K2C4H4O6 · ½H2O) was used to establish a relative humidity level of 74% in these experiments. Lipid vesicles were analyzed by transmission electron microscopy and shown to exist as stable lipid bilayers.
The oriented membrane samples were aligned at grazing incidence with respect to a collimated, monochromatic x-ray beam produced by a Rigaku Rotaflex RU-200, high-brilliance microfocus generator (Rigaku-MSC, The Woodlands, TX). Analytical x-rays are generated by electron bombardment of a rotating copper anode and are filtered through a thin nickel foil to provide monochromatic CuKα radiation (Kα1 and Kα2 unresolved; λ = 1.54 Å). Collimation of the x-ray beam was achieved using a single Frank's mirror. Diffraction data were collected on a 1-dimensional, position-sensitive electronic detector (Hecus X-ray Systems, Graz, Austria) using a sample-to-detector distance of 150 mm. In addition to direct calibration, crystalline cholesterol monohydrate was used to verify the calibration of the detector, as previously described.26
This technique allows for precise measurement of the unit cell periodicity, or d-space, of the membrane lipid bilayer, which is the distance from the center of 1 lipid bilayer to the next, including surface hydration. The d-space for any given membrane multibilayer is calculated from Bragg's law, hλ = 2 d sin θ, where h is the diffraction order, λ is the wavelength of the x-ray radiation (1.54 Å), d is the membrane lipid bilayer unit cell periodicity, and θ is the Bragg angle equal to one half the angle between the incident beam and scattered beam. Fourier transformation of the collected x-ray diffraction data provides the time-averaged electron density distribution (distance, Å, vs electrons/Å) associated with the membrane lipid bilayer, as previously described.25 Changes in electron density distribution that occur in the presence of a drug allow for the measurement of its location in the bilayer.
Lipid Peroxidation Analysis
DLPC-enriched MLVs were subjected to time-dependent autoxidation by incubating at 37°C in an uncovered shaking water bath. After a 48-h incubation period to allow for autooxidation, 100 μL aliquots of the samples were removed and combined with 1.0 mL of active CHOD-iodide color reagent. Test samples were immediately covered with foil and incubated at room temperature for >4 h in the absence of light. Sample absorbances were then measured against a CHOD blank at 365 nm using a Beckman DU-640 spectrophotometer.
The CHOD colorimetric assay is based on the oxidation of iodide (I−) by lipid hydroperoxides to form triiodide anion (I3−), which has a molar absorptivity value (ϵ) of 2.46 × 104 mol1 cm−1 at 365 nm. The molar quantity of I3− liberated in this reaction is directly proportional to the amount of lipid hydroperoxides that is formed in the process of lipid peroxidation.21
Data are presented as mean ± SD. The significance of differences between results from independent experimental conditions was tested using the 2-tailed Student t test. A value of P < 0.05 was considered significant.
Release of NO and ONOO− from WKY Rats and SHR Mesenteric Arteries
Endothelial NO and ONOO− were measured simultaneously after stimulation with a receptor-dependent (ACh) and receptor-independent (CI) agonist. The maximum of NO release was observed at 1 μmol/L concentration of CaI or ACh, and therefore, this concentration was used throughout the studies. In the absence of exogenous eNOS agonists a minute basal concentration of NO near the endothelial surface was detected (7 ± 3 nmol/L for WKY rats and 3 ± 2 nmol/L for SHR) by the nanosensors operating in a differential pulse voltammetry mode. After stimulation with CI, NO increased significantly from its basal level. Amperometric measurements showed CI stimulated maximal NO concentration on the level of 520 ± 43 nmol/L and 312 ± 38 nmol/L for WKY rats and SHR, respectively (Fig. 1A). The basal concentration of ONOO− was 4 ± 2 for WKY rats and 10 ± 3 nmol/L for SHR. After stimulation with CI, maximal ONOO− reached 188 ± 19 nmol/L for WKY rats and a much higher level (273 ± 15 nmol/L) for SHR (Fig. 1B). As expected, ACh (receptor-dependent eNOS agonist) produced ≈30% less NO and also ≈35% less of ONOO− as compared with the maximal production of these 2 molecules observed after CI stimulation in both WKY rats and SHR.
Comparative Effects of Nebivolol, Its Enantiomers, and Atenolol on NO Release from WKY Rats and SHR Intact Arteries
We observed that the β1-selective antagonist, nebivolol racemate, along with its separate enantiomers also stimulated NO release in small resistance arteries of both WKY rats and SHR in a concentration-dependent fashion. Figure 2 compares peak NO release levels from arteries isolated from control WKY rats following treatment with nebivolol racemate, l-nebivolol or d-nebivolol. The order of activity was l-nebivolol > nebivolol racemate ≫ d-nebivolol. Maximum NO release by the compounds was achieved at a concentration of 10 μmol/L. At this concentration, the concentration of NO was 340 ± 30 nmol/L, 284 ± 24 nmol/L, and 180 ± 21 nmol/L for l-nebivolol, nebivolol racemate, and d-nebivolol, respectively. In SHR arteries, the dose-response curve for nebivolol and nebivolol enantiomers-stimulated NO was uniformly lower (24% ± 3%) than that observed for WKY rats (data not shown). These findings confirm that nebivolol can stimulate NO release in the intact artery of both WKY rats and SHR in a stereoselective manner.
The effect of equimolar (10 μmol/L) concentration of nebivolol, its enantiomers, or atenolol peak concentration of NO generated from WKY rats and SHR mesenteric arteries are shown in Figure 3A. The effect of l-nebivolol on the concentration of NO released from the endothelium was the most significant (340 ± 20 nmol/L and 261 ± 17 nmol/L) for WKY rats and SHR respectively (Fig. 3A). NO release following atenolol stimulation was significantly lower than that observed for the nebivolol and its enantiomers and was limited to 58 ± 26 nmol/L and 25 ± 5 nmol/L in WKY rats and SHR, respectively (Fig. 3A).
The difference between WKY rats and SHR endothelial NO release after stimulation with nebivolol (racemate and both l- and d- forms) was much lower (≈15%) than that observed after stimulation with CI, Ach, or atenolol (≈40%). The maximal concentrations of ONOO− produced in the endothelium of WKY rats and SHR after stimulation with nebivolol (racemate), both l- and d-enantiomers, and atenolol are shown in Figure 3B. The ONOO− concentration produced by the endothelium of SHR is uniformly higher than that measured in WKY rats after stimulation with nebivolol racemate, nebivolol enantiomers, and atenolol. Figure 4 shows the ratios of NO concentration to ONOO− concentration. This ratio reflects on the balance between bioavailable NO and the level of nitroxidative stress (ONOO−) produced by the endothelium after stimulation with different eNOS agonists. The decrease in [NO]/[ONOO−] can be directly correlated with the increase in eNOS uncoupling. After stimulation with CI, ACh, nebivolol, or atenolol, the nanosensors measured simultaneously the availability of both of these molecules in close proximity (5 ± 2 μm) to the endothelial cell membrane. In WKY rats, the ratio of [NO]/[ONOO−] was similar after stimulation with CI or ACh (2.77 ± 0.05 and 2.79 ± 0.04 respectively; Fig. 4. In SHR, the [NO]/[ONOO−] ratio was much lower (1.14 ± 0.11 after stimulation with CI and 1.04 ± 0.43 after stimulation with ACh) than that observed for WKY rats. A dramatic increase in [NO]/[ONOO−] ratio to a level of 4.9 ± 0.03 for WKY rats and 3.09 ± 0.04 for SHR was measured after stimulation with nebivolol. This accounts for ≈65% increase in [NO]/[ONOO−] for WKY rats and ≈180% increase for SHR as compared with CI or ACh stimulation. This favorable ratio of [NO]/[ONOO−] indicate that nebivolol increased the bioavailability of NO and decreased nitroxidative stress and significantly inhibit the eNOS uncoupling, particularly in SHR. In contrast, the atenolol showed a low ratio of [NO]/[ONOO−] (1.05 ± 0.43 for WKY and 0.38 ± 0.05 for SHR), indicating a relatively low NO bioavailability and high nitroxidative stress.
Molecular Interactions of Nebivolol and Atenolol with Membrane Lipids
Small-angle x-ray diffraction approaches were used to directly determine the molecular distribution of nebivolol and atenolol in the membrane lipid bilayer, as previously described.27 At 20°C, the d-space value for the membrane, including surface hydration, was 55 Å, which was consistent with previous x-ray diffraction studies of both intact and reconstituted vascular smooth muscle cell plasma membrane and cardiac sarcolemma.28,29
Membrane samples prepared in the absence and presence of drug produced 4 strong, reproducible diffraction orders. Fourier analysis of the data produced a 1-dimensional electron density profile (Å vs electrons/Å3), which depicts the electron density associated with the phospholipid bilayer (Fig. 5A). The 2 maxima of electron density correspond to the center-of-mass positions of the phospholipid headgroups; the nadir of electron density is associated with the terminal methylene segments of the phospholipid acyl chain because of the high ratio of hydrogen to carbon and low molecular density.
The addition of atenolol and nebivolol at a low concentration (<5% by mass) produced distinct changes in the molecular structure of the phospholipid bilayer, consistent with their different membrane locations. The addition of nebivolol produced a pronounced increase in electron density 0-20 Å from the membrane bilayer center (Fig. 5B). A similar change in electron density was observed with l-nebivolol (Fig. 5C) and d-nebivolol (data not shown). By contrast, the addition of atenolol to these membranes under identical conditions did not produce a demonstrable change in hydrocarbon core electron density (Fig. 5D). These results indicate that atenolol remains at the hydrated surface of the membrane bilayer. The specific membrane locations indicate that the compounds exhibit affinity for different regions of the lipid bilayer (hydrophobic acyl chains, charged headgroup). In particular, nebivolol, a lipophilic compound, is most energetically stable within the membrane hydrocarbon core. By contrast, atenolol is hydrophilic and interacts only with the hydrated membrane surface of the membrane (Fig. 5D).
Comparative Effects of Nebivolol and Atenolol on Lipid Peroxidation
The comparative effects of nebivolol and atenolol on rates of lipid peroxidation were compared under identical conditions in vesicles enriched with polyunsaturated fatty acids (Fig. 6). The lipid peroxidation reaction occurred gradually over time in the absence of an initiator, such as a transitional metal (e.g., iron, copper). Samples treated with probucol and Trolox, a water-soluble analog of vitamin E, were included as antioxidant controls. At a low pharmacological concentration (100 nmol/L), nebivolol had an inhibitory effect on lipid peroxidation (36%, P < 0.01). At 500 nmol/L, the antioxidant activity of nebivolol increased to 66% (P < 0.01), as compared with controls. The synthetic and highly lipophilic antioxidant probucol also inhibited lipid peroxidation by 42% and 67% at 100 and 500 nmol/L, respectively. Although Trolox inhibited lipid peroxidation by 21% at 500 nmol/L, the effect was not statistically significant. These results indicated that nebivolol and probucol interfered with lipid peroxidation, with activity greater than that of Trolox. Atenolol, the other β-blocker tested, had no significant antioxidant effects at either 100 or 500 nmol/L.
The essential finding from this study is that nebivolol is a highly efficient stimulator of endothelial-dependent NO release in arteries isolated from SHR, a well-characterized genetic model of hypertension linked to abnormalities in eNOS function. The efficiency of nebivolol-stimulated production of NO is enhanced by its ability to inhibit eNOS uncoupling and to scavenge O2−. Both of these effects accounts for the reduction of ONOO− level, a main component of nitroxidative stress in the endothelium. The activity of nebivolol was also present with both enantiomers of nebivolol. The l-nebivolol enantiomer showed a more potent effect on NO release than d-nebivolol or nebivolol racemate. It is of particular interest that whereas d-nebivolol is the active enantiomer for β1-selective blockade, it was the weakest stimulant of NO release from arterial vessels. In comparative studies, the effect of nebivolol was not seen with atenolol, another β1-selective inhibitor. In fact, atenolol treatment was associated with further endothelial dysfunction in SHR vessels, as evidenced by a decrease in the ratio of NO to ONOO−. The deleterious effect of atenolol is attributed in part to its hydrophilic properties that lead to distinct membrane interactions. These experimental findings may provide insight into the lack of cardiovascular benefit that has been observed for atenolol in large randomized trials, despite effective blood pressure reduction.30 Thus, the endothelial-dependent actions of nebivolol are distinct from atenolol and not attributed to β1 selectivity but to direct effects on eNOS coupling/uncoupling mechanisms.
In the SHR, we demonstrated a marked decrease in NO bioavailability following stimulation with either a receptor-dependent (ACh) or receptor-independent (CI) agonist. Previous studies have demonstrated that the SHR model is characterized by an attenuation in levels of functional NO, despite increased eNOS enzyme activity.17 The basis for this paradox is attributed to excessive O2− generation that reacts rapidly with NO to form the deleterious product ONOO−.18 Thus, enhanced eNOS expression and enzymatic activity are compensatory mechanisms for the SHR animal, in response to reduced NO bioavailability. This hypothesis is supported by the observation of similar changes in eNOS expression and O2− generation in Sprague-Dawley rats made hypertensive by aortic banding.31 Under conditions of enhanced oxidative stress, increased expression of eNOS leads to yet higher levels of the uncoupled eNOS, resulting in still greater amounts of O2−. These findings are consistent with other studies linking endothelial dysfunction in hypertension to excessive O2− production through eNOS uncoupling and NAD(P)H oxidase activity.19,20 Thus, the beneficial activity of nebivolol is attributed to both inhibitions of eNOS uncoupling and endogenous antioxidant properties that lead to free radical scavenging.
An antioxidant action for nebivolol is highly supported by our finding of direct free radical scavenging activity in lipid vesicles enriched with polyunsaturated fatty acids. At pharmacological concentrations, nebivolol produced a dose-dependent reduction in the levels of lipid hydroperoxides. In the absence of any receptors or enzymatic modulators of O2− generation, we attribute the activity of nebivolol to its ability to act as a chain-breaking antioxidant through proton donation and electron stabilization mechanisms. This physicochemical model is further supported by the results of the x-ray diffraction analyses, which showed that nebivolol intercalates into the membrane hydrocarbon core. At this location in the membrane bilayer, nebivolol is able to participate in proton-donation and electron-stabilization mechanisms, thereby interfering with lipid radical formation and propagation. The activity of nebivolol was similar to that of probucol, a well-characterized agent with potent antioxidant actions linked to vascular benefits in various models of atherosclerosis.32,33 By contrast, atenolol did not partition into the membrane hydrocarbon core, which is consistent with differences in its chemical properties, including its greater hydrophilicity compared with nebivolol. Nebivolol has also been shown to interfere with enzymatic sources of oxygen radicals, including O2− generation, that can complement direct scavenging activity, as suggested from previous studies.15,34,35 Thus, although both atenolol and nebivolol are inhibitors of β1-receptors, differences in their physicochemical properties account for distinct effects on oxidative stress pathways and, ultimately, for the synthesis of bioavailable NO. These studies also show that a high NO production is not necessarily desirable and it may trigger high peroxynitrite production. Therefore, the ratio of NO/ONOO− is a more optimal indicator of potential therapeutic effect of NO-releasing drugs.
The results of these experiments further support the hypothesis that nebivolol has distinct and favorable effects on the kinetics of NO generation and metabolism, independent of β1-selective interactions. In addition to the antioxidant activity described in this study, there is evidence that nebivolol may stimulate NO release through the activation of mechanical channels, adenosine triphosphate reflux activation of P2Y-purinoceptors, intracellular calcium release, and eNOS activation.14 The contrasting action of nebivolol vs atenolol in SHR has also been observed in Dahl salt-sensitive rats, again indicating a direct vasodilatory mechanism for nebivolol independent of β1-blockade.36 A lack of vascular benefit with atenolol has also been observed in a small clinical investigation. In a crossover study, it was observed that treatment with an inhibitor of the renin-angiotensin system caused a marked improvement in endothelial-dependent vasodilatation in small resistance arteries obtained from patients who had been treated with atenolol for 1 year.37 The lack of benefit with atenolol was observed despite identical blood pressure reduction with the 2 treatments.
The activity of nebivolol with respect to endothelial NO release has important implications for its use in the treatment of cardiovascular disorders characterized by endothelial dysfunction including hypertension, heart failure, and coronary artery disease. The favorable effects of nebivolol on NO release from arterial endothelial cells would complement its ability to modulate the sympathetic nervous system in the treatment of cardiovascular disease. This has been demonstrated in comparative hemodynamic studies of hypertensive patients after 2 weeks of treatment. As compared with atenolol, nebivolol demonstrated direct vasodilatory properties as evidenced by reduced peripheral vascular resistance, improved cardiac output, and modest heart rate reduction.38
The results of this study provide further insights into mechanisms of hypertension and its treatment. SHR demonstrated marked reductions in endothelial-dependent NO release, as compared with normal animals. As a potent free radical scavenger, the ability of nebivolol to stimulate NO release is consistent with the hypothesis that a key contributor to hypertension, secondary to a loss of functional NO, is the excessive production of superoxide and peroxynitrite. Thus, the antioxidant activity of nebivolol, linked to its molecular membrane location, contributes to mechanisms of endothelial-dependent NO release, independent of β1-blockade. The adverse effects of atenolol on endothelial function indicate important differences in the vascular actions of lipophilic vs hydrophilic agents. Thus, nebivolol represents a new approach to the management of hypertension by mollifying the injury associated with endothelial dysfunction.
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