Oxidative stress has been suggested to be an etiological factor in cerebro- and cardiovascular disorders. We examined antioxidant activities of carvedilol, a [beta]- and [alpha]-adrenoreceptor blocker. Carvedilol suppressed lipid auto-oxidation and protein carbonyl formation in brain homogenate in a dose-dependent manner. Carvedilol also suppressed superoxide generation of human neutrophils. These properties of carvedilol should be suitable for treating hypertension resulting in cerebro- and cardiovascular diseases. [Neurol Res 2003; 25: 749-753]
Keywords: Carvedilol; antioxidant; lipid peroxidation; protein carbonyls; superoxide
Oxidative stress has been proposed as an important pathologic factor of ischemia-reperfusion and age-associated vascular pathologies such as arteriosclerosis1,2. In addition, oxidative stress is considered to play a cytotoxic role in age-related neurodegenerative disorders including Parkinson's and Alzheimer's diseases2-4. Neuronal cells are extremely sensitive to injury by reactive oxygen species that mediate cell death in various pathologic conditions. Therefore, agents with antioxidant activity as well as antihypertensive activity should be more beneficial in treating hypertension. Hypertension is the most critical risk factor of cerebrovascular diseases and is often associated with them.
Carvedilol, (+ or -)-1-(carbazol-4-yloxy)-3-[2-methoxyphenoxy] ethylamino-2-propanol, has been used clinically to treat hypertension for several years. The clinical effect is mainly derived from [beta]- and [alpha]-adrenoreceptor blockade. In addition, carvedilol has been shown to act as an antioxidant5-2. Several studies reported the antioxidative effects of carvedilol, using different systems for radical generation including auto-oxidation of dihydroxyfumaric acid in the presence or absence of iron ions7,8, ferrous iron with ascorbic acid9, stimulated macrophages5,6, and enzymatic systems9. However, the mechanism of this antioxidative effect is not completely clear.
To investigate whether carvedilol has neuroprotective ability, we examined its antioxidant properties by three independent approaches: 1. auto-oxidation of brain homogenate measured as thiobarbituric acid-reactive substances, 2. protein oxidation of brain homogenate monitored by protein carbonyl formation, and 3. superoxide-generation from neutrophils.
MATERIALS AND METHODS
Effects on lipid peroxidation
A whole swine brain was rapidly removed after slaughter. The cerebral cortex was collected and stored at -80[degrees]C until use.
The cerebral cortex was homogenized in modified Hank's balanced salt solution (mHBSS, Nissui, Japan) without phenol red and sodium bicarbonate, adjusted to pH 7.4 by addition of 280 mOsm disodium hydrogen phosphate. To induce auto-oxidation, the 5% homogenate with or without carvedilol was incubated at 37[degrees]C. Lipid peroxides produced by auto-oxidation were monitored by levels of thiobarbituric acid-reactive substances (TBARS).
After the incubation period, 500 [mu]l of the reaction mixture was added to 2 ml TBA reagent consisting of 15% trichloroacetic acid (Wako Pure Chemical, Osaka, Japan), 0.375% TBA, 0.25 M HCl dissolved in distilled water, 30 [mu]l butylated hydroxytoluene (BHT; 50 mM ethanol solution) and 470[mu]l distilled water, was heated in a 100[degrees]C water bath for 15 min. TBA and BHT were purchased from Tokyo Kasei (Japan) and Wako Pure Chemical (Osaka, Japan), respectively. After cooling in ice-cold water, the mixture was centrifuged at 1,250xg for 10 min. The absorbance of the supernatant at 535 nm was read against a blank which contained 2 ml of TBA reagent, 30 [mu]l BHT and 970 [mu]l of distilled water, using a spectrophotometer (U-2000, Hitachi, Tokyo, Japan). The concentration of TBARS was calculated using a molar absorption coefficient of 156,000 M cm^sup -1^.
Under the same conditions, anti-oxidant properties of carvedilol were compared with those of [alpha]-tocopherol (a kind gift of Eisai, Tokyo, Japan).
Effects on protein oxidation
After the whole brain was rapidly removed from a male Wistar rat, the cerebral cortex was collected and stored at -80[degrees]C until use. The cerebral cortex (50 mg) was homogenized in 0.55 ml of buffer in a glass homogenizer. The buffer consisted of 0.5 ml of homogenizing buffer (0.1% digitonin in 100 mM potassium dihydrogen orthophosphate buffer; PDO buffer, pH 7.4), 50 [mu]l of protease inhibitor cocktail (5 [mu]g ml^sup -1^ aprotinin, 5 [mu]g ml^sup -1^ leupeptin, 7 [mu]g ml^sup -1^ pepstatin A and 10 mM EDTA), and 1 [mu]l of phenylmethylsulphonyl fluoride (40 mg ml^sup -1^ dissolved in ethanol). Digitonin, aprotinin, leupeptin, pepstatin A, EDTA, and phenylmethylsulphonyl fluoride were purchased from Sigma Chemicals, St. Louis, MO, USA. The homogenized tissue was left at room temperature for 15 min and then centrifuged at 2,755xg for 10 min. The supernatant was removed and incubated at room temperature for 15 min with 1% streptomycin sulfate solution. Centrifugation at 2,755xg for 10 min removed any nucleic acids that might interfere with the carbonyl assay. The supernatant was used as the sample.
Protein oxidation induced by exposure to hydrogen peroxide
Bovine serum albumin (BSA; Sigma Chemicals) dissolved in PDO buffer (0.5 mg ml^sup -1^) and the homogenate of rat cerebral cortex were exposed to 0.2 mM hydrogen peroxide (H^sub 2^O^sub 2^; Kanto Chemical, Tokyo, Japan) in 100 mM PDO buffer, and then incubated for 20 min at 37[degrees]C. Carvedilol at various concentrations was added to the basal system before H^sub 2^O^sub 2^ exposure. To remove H^sub 2^O^sub 2^, manganese oxide was added. After centrifugation twice at 10,000xg for 5 min, the supernatant was used as the protein sample.
Derivatization of proteins with 2,4-dinitrophenylhydrazine (2,4-DNPH)
Using the OxyBlot(TM) Protein Oxidation Detection Kit (Intergen, Purchase, NY, USA) and according to its protocol, protein carbonyls were derivatized with 2,4-DNPH. Briefly, after denaturation of 5 [mu]l of protein samples by adding 5 [mu]l of 12% SDS, the samples were derivatized by adding 10 [mu]l of 1xDNPH solution and then incubated for 15 min at room temperature. Finally, 7.5 [mu]l of neutralization solution and 1.6 [mu]l of 2-mercaptoethanol were added. Derivatized samples were assayed within 24 h.
Detection of derivatized proteins by Western blot
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by standard procedures using the Mini Protean II system (Bio-Rad, Hercules, CA, USA). Samples treated with DNPH (derivatized BSA and brain homogenate) and molecular weight protein standards (OxyBlot(TM) Protein Oxidation Detection Kit; Intergen) were loaded on a 10% acrylamide resolving gel with a 4% acrylamide stacking gel. After electrophoresis, proteins were transferred to a PVDF membrane (Amersham Pharmacia, Buckingham, UK) using the Trans-Blot SD system (Bio-Rad) at 15 V for 20 min. The membranes were blocked for 1 h with 5% nonfat milk powder in PBS-T consisting of phosphate buffer saline supplemented with 0.1% Tween 20 (Wako Pure Chemical) and were then incubated overnight with rabbit anti-DNP antibody diluted with 1% nonfat milk powder in PBS-T (1 : 150). After washing with PBS-T (3x30 min), blots were incubated with a 1 : 300 dilution in PBS-T of secondary goat anti-rabbit antibody conjugated with horseradish peroxidase for 1 h. After washing (3x30 min with PBS-T), the blots were visualized using a chemiluminescence reagent, ECL-PLUS (Amersham). Finally, the membranes were exposed to Hyperfilm-ECL (Amersham).
Semi-quantitative analysis for protein carbonyls by densitometry with dot blot
Aliquots of 3 [mu]l of sample treated with DNPH were applied to a PVDF membrane. After the membranes were dried, they were blocked with 5% nonfat milk powder in PBS-T. The following procedures were the same as the Western blot assay mentioned above. Optical densities of the immunoblots or total protein carbonyl content were compared. The density of each band was measured using image processing software, NIH image (NIH, Bethesda, MD, USA).
Effects on superoxide production
Xantine oxidase (from buttermilk, grade III) and phorbolmyristate acetate (PMA) were purchased from Sigma Chemicals. PMA was dissolved in a small amount of dimethyl sulfoxide (DMSO; Sigma Chemicals), diluted with 50% DMSO and 50% mHBSS to the specified concentration, and then stored at -80[degrees]C. Hypoxantine was obtained from Wako. 2-Methyl-6- (p-methoxyphenyl)-3,7dihydroimidazo [1,2-a] pyrazin-3-one (MCLA) was purchased from Tokyo Kasei and prepared by the method described previously13.
Blood was drawn into a heparinized syringe (20 units ml^sup -1^) from healthy volunteers. Leukocytes were isolated by sedimentation in the presence of dextran followed by brief hypotonic lysis of contaminating erythrocytes13. The leukocytes, which contained 60%-80% neutrophils, were suspended in mHBSS and kept at 4[degrees]C for no longer than 3 h prior to use.
Superoxide production was determined by the MCLA dependent chemiluminescence method using a BLR 301 Luminescence Reader (Aloka, Tokyo, Japan)14. All experiments were carried out in the incubation chamber of the Luminescence Reader at 37[degrees]C in a total volume of 1 ml. The reaction mixture for the basal system consisted of neutrophils (5.0x10^sup 4^ cells ml^sup -1^) and 1.0 [mu]M MCLA in mHBSS with pre-incubation for 5 min at 37[degrees]C. The reaction was started by the addition of 0.1 [mu]g ml^sup -1^ of PMA. Superoxide production was expressed as photon counts per min. The maximum photon count was defined as the peak count14. The incubation period was 5 min. Carvedilol at various concentrations was added to the basal system before pre-incubation.
The hypoxanthine-xanthine oxidase system was used to test the scavenging effect of carvedilol on superoxides and the possible interaction with MCLA as described previously15. A system containing 1.0 [mu]M MCLA, 0.18 mg ml^sup -1^ of BSA, 45 [mu]M hypoxanthine and 0.1% N, N-dimethylformamide (Sigma Chemicals) in mHBSS was incubated with or without 100 [mu]M carvedilol at 37[degrees]C. Chemiluminescence produced in this system by addition of xanthine oxidase (28 units ml^sup -1^) was monitored.
Effects on lipid peroxidation
TBARS formation was increased with increasing incubation time (Figure 1). The addition of 25 [mu]M carvedilol significantly (about 50% inhibition) suppressed TBARS formation. Comparison between the two groups using two-factor analysis of variance (ANOVA) (one factor for the groups with or without carvedilol and another for the incubation time) revealed significant differences in the groups (p
Effects on protein oxidation
Carvedilol strongly inhibited protein carbonyl formation both in BSA and rat brain homogenate (Figure 3). Carvedilol (100-200 [mu]M) significantly suppressed protein carbonyl formation. The inhibitory effect was found to be dose-dependent (Figure 4). In the Western blot assay, carvedilol had different antioxidant effects on each protein band (Figure 3).
Effects on Superoxide production
Carvedilol suppressed the chemiluminescence of neutrophils stimulated by PMA and reduced the peak count in a dose-dependent manner (Figure 5). Complete inhibition was achieved by addition of 150 [mu]M carvedilol. The concentration required for 50% inhibition of the peak count was about 60 [mu]M.
In all experiments using the MCLA-dependent chemiluminescence method, the complete scavenging effect of 0.5 [mu]M SOD revealed that the chemiluminescence was derived from Superoxide15. In the hypoxanthinexanthine oxidase system, chemiluminescence produced in the presence of 100 [mu]M carvedilol was essentially the same as that shown in the system without carvedilol (data not shown). Therefore, carvedilol itself showed no detectable superoxide-scavenging activity and no interference with MCLA, but had an inhibitory effect on the superoxide-generating system of neutrophils.
We demonstrated that carvedilol has antioxidative ability by three independent approaches; lipid peroxidation, protein oxidation, and Superoxide production.
In the present study, carvedilol markedly inhibited lipid auto-oxidation of rat brain homogenates. This result agrees with those of previous studies5. Its inhibition of brain auto-oxidation is significantly more pronounced than that of [alpha]-tocopherol, a noble antioxidant breaking free-radical chain reactions in biological membranes. Carvedilol is a lipophilic agent which can be easily incorporated into membranes and is able to cross the blood-brain barrier. Although the mechanism of carvedilol inhibition of auto-oxidation of brain homogenate has not been elucidated, its lipid solubility is advantageous for antioxidant activities that inhibit membrane lipid peroxidation because initiation and propagation of radical chain reactions prefer lipophilic conditions. Carvedilol may act as an antioxidant breaking radical chain reactions by scavenging peroxyl and alkoxyl radicals, which are abundantly generated by iron-dependent decomposition of preformed lipid peroxides16. Our results suggest that this effect of carvedilol may protect biologic membranes from oxidative stress.
We also showed marked antioxidant activity of carvedilol in protein oxidation. Many studies suggest that oxidative stress is responsible for age associated pathologies such as arteriosclerosis and neurodegenerative disorders2-4. Oxidation of proteins modifies the side chains of methionine, histidine and tyrosine, forming cystein disulfide bonds17. Metal catalyzed oxidation of proteins introduces carbonyl groups (aldehydes and ketones) in lysine, arginine, proline or threonine residues in a site-specific manner17. Many investigators have demonstrated that hydroxyl radical causes oxidative modification of amino acid residues of proteins and that cross-linking and fragmentation of the proteins result in loss of function and increased susceptibility to proteases18-21. However, there have been no reports showing inhibitory effects of carvedilol on protein oxidation in the brain. We demonstrated this property for the first time. The inhibitory effect of carvedilol on protein oxidation is not clear, but it may inhibit production of the hydroxyl radical. Our findings suggest that carvedilol may protect against cellular and extracellular protein damage from oxidative stress and permit maintenance of physiological function.
We demonstrated that carvedilol did not scavenge Superoxide, but suppressed the superoxide-generating system. Since Superoxide is a potential precursor of the hydroxyl radical, which seems to be a more hazardous free radical, carvedilol may indirectly prevent hydroxyl radical formation by inhibition of Superoxide production from neutrophils. Recently, neutrophils have been suggested to play a role in ischemia/reperfusion injury. The removal of circulating neutrophils is reported to reduce ischemic brain edema22. Superoxide produced by neutrophils is one of the critical chemical mediators that alter vascular permeability and accelerate the development of cerebral edema22,23. Our findings suggest that carvedilol may have a protective effect on brain tissue during the acute phase of ischemia/reperfusion injury by suppression of superoxide production from neutrophils, thus reducing the size of infarction.
We demonstrated that carvedilol has antioxidative properties by three independent approaches; lipid peroxidation, protein oxidation, and superoxide production. This property was significantly stronger than that of [alpha]-tocopherol. This is the first report to demonstrate the inhibitory effect of carvedilol on protein oxidation in the brain. Carvedilol may act as a neuroprotective agent in cerebrovascular disorders.
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Yoko Suzuki, Makoto Tanaka, Makoto Sohmiya, Toshihiko Yoshida and Koichi Okamoto
Department of Neurology, Gunma University Graduate School of Medicine, Gunma, Japan
Correspondence and reprint requests to: Makoto Tanaka, Department of Neurology, Gunma University School of Medicine, 3-39-22 Showamachi, Maebashi, Gunma 371-8511, Japan. [email@example.com] Accepted for publication April 2003.
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