Antioxidant Effects of Calcium Antagonists Rat Myocardial
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223 Antioxidant Effects of Calcium Antagonists ['Ii] Rat Myocardial Membrane Lipid Peroxidation Hitoshi Sugawara, Katsuyuki Tobise, and Kenjiro Kikuchi We studied the antioxidant effects of nine calcium antagonists (nisoldipine, benidipine, nilvadipine, felo- dipine, nicardipine; nitrendipine, nifedipine, verapamil, and diltiazem) by means of rat myocardial membrane lipid peroxidation with a nonenzymatic active oxygen-generating system (DHF/FeC13-ADP). The order of antioxidant potency of these agents was nilvadipine > nisoldipine > felodipine > nicardipine > verapamil > benidipine. Their IC50 values (,uM) were 25.1, 28.2, 42.0, 150.0, 266.1, and 420.0, re- spectively. In contrast, nitrendipine, nifedipine, and diltiazem had little inhibitory effect on lipid peroxi- dation.These six calcium antagonists could be divided into four types on the basis of their antioxidant mechanisms. Nilvadipine, nisoldipine, and verapamil, which showed antioxidant effects both before and after the addition of active oxygen, and reduced the dihydroxyfumarate (DHF) auto-oxidation rate, were chain-breaking and preventive antioxidants. Felodipine, which showed antioxidant effects both before and after exposure to active oxygen and increased the DHF auto-oxidation rate, was only a chain-break- ing antioxidant. Nicardipine, which showed an antioxidant effect only before exposure to active oxygen and reduced the DHF auto-oxidation rate, was mainly a preventive antioxidant. Benidipine, which showed an antioxidant effect only before exposure to active oxygen and had no appreciable effect on the DHF auto-oxidation rate, could interrupt the chain reaction of lipid peroxidation at the initial step alone. Although these results suggest that the antioxidant properties of some calcium antagonists may be beneficial clinically in protecting against cellular damage caused by lipid peroxidation, further studies are required to establish the antioxidant effects of these agents in vivo. (Hypertens Res 1996; 19: 223-228) Key Words: antioxidant effect, calcium antagonist, dihydropyridine calcium antagonists, myocardial membrane lipid peroxidation Cellular damage due to lipid peroxidation associ- there are conflicting reports with regard to nifedi- ated with the production of active oxygen species pine, one of the most widely used dihydropyridine plays an important role in various pathologic states, calcium antagonists. Janero et al. found that nisoldi- such as ischemia-reperfusion injury (1), coronary pine was the most potent dihydropyridine antiox- arteriosclerosis (2), glomerular sclerosis (3), di- idant, whereas nifedipine had little effect as com- abetes mellitus (1), and the process of "normal" ag- pared with other dihydropyridine calcium antagon- ing (4). Calcium antagonists have been established ists in studies using a xanthine oxidase-dependent to be safe and effective in the management of active oxygen-generating system and liposomes de- essential hypertension and angina pectoris. Since rived from rat cardiac membrane phospholipids (6). treatment with anti-hypertensive and anti-anginal In contrast, Mak and Weglicki found that nifedipine agents is usually long term, additional protective had the highest antioxidant activity among all cal- effects against lipid peroxidation by oxygen radicals cium antagonists tested with an active oxygen- may be beneficial in preventing organ damage. generating system using dihydroxyfumarate (DHF) However, it is unclear whether the calcium antago- auto-oxidation and isolated sarcolemma of the nists have such antioxidant effects and, if so, what canine heart (7). The results of our previous report mechanisms are involved. (8) confirmed those of Janero et al. (6) and indi- Janero et al. first reported a direct effect of cal- cated that the membrane peroxidation reaction mix- cium antagonists on the lipid peroxidation of car- ture used by Mak and Weglicki (7) may be unsuit- diac membranes (5). They found that bepridil and able due to the generation of sucrose-derived prenylamine were the most potent antioxidants thiobarbituric acid-reactive substances (TBARS) among the calcium antagonists tested. However, rather than lipid-derived TBARS. From the First Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Hokkaido, Japan. Address for Reprints: Hitoshi Sugawara, M.D., Ph.D., Omiya Medical Center, Jichi Medical School, 1-847 Amanuma- cho, Omiya, Saitama 330, Japan. Received September 8, 1995; accepted in revised form July 15, 1996. 224 Hypertens Res Vol. 19, No. 4 (1996) To determine the antioxidant effects and mechan- pellet was resuspended in 10 mM Tris-HC1 buffer ( isms of antioxidant action of nine calcium antagon- pH 7.4) and adjusted to a protein concentration of ists (nisoldipine, benidipine, nilvadipine, felodipine, 1-2 mg/ml and stored at - 80°C. Protein concentra- nicardipine, nitrendipine, nifedipine, verapamil and tions were determined according to the method of diltiazem) we studied rat myocardial membrane Lowry et al. (10). lipid peroxidation by means of a nonenzymatic ac- tive oxygen-generating system. We were able to de- Active Oxygen-Generating System monstrate differences in antioxidant potency among A nonenzymatic active oxygen-generating system in- calcium antagonists and to classify the mechanisms volving the auto-oxidation of DHF/Fe3+ -ADP was of the antioxidant effects of these agents into four used as described by Mak et al. (11). After the types. auto-oxidation of DHF, the resultant superoxide an- ions produced hydroxyl radicals in a reaction cataly- zed by Fe3+-ADP and initiated a lipid peroxidation Methods chain reaction (12). Animals Seven-week-old male Sprague-Dawley rats were Experimental Protocols purchased from Charles River Laboratories, Inc. Experiment 1: Under amber lighting provided by a (Tokyo, Japan) and housed for one week at the sodium lamp, 100 g of myocardial membrane pro- animal laboratory of Asahikawa Medical College tein was incubated and shaken in 10 mM Tris-HC1 before use in this study. buffer (pH 7.4) for 30 min at 37°C in a warm water bath with each agent at a final concentration of 1 Materials mM. After adding 6.66 mM DHF, 1 mM ADP, and Dihydroxyfumarate (DHF) was purchased from 0.1 mM FeC13 (in a total volume of 0.5 ml), the Aldrich Chemical Co . (Milwaukee, WI). Adenosine mixtures were incubated and shaken in a warm wa- diphosphate (ADP), nifedipine, nicardipine, and ter bath at 37°C for a further 120 min, and the ex- verapamil were purchased from Sigma Chemical tent of lipid peroxidation was measured. As shown Co. (St. Louis, MO). Nisoldipine was provided by previously (8), the IC50 value was determined for Bayer Yakuhin Ltd. (Osaka, Japan), benidipine by all agents that inhibited lipid peroxide production Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan), significantly as compared with control. nilvadipine by Fujisawa Pharmaceutical Co., Ltd. Experiment 2: Subsequent experiments were per- (Osaka, Japan), felodipine by Hoechst Japan Ltd. ( formed with the agents that inhibited lipid peroxida- Tokyo, Japan), nitrendipine by Yoshitomi Phar- tion significantly as compared with control. Under maceutical Industry Ltd. (Osaka, Japan), and dil- similar conditions as experiment 1, 100 g of mem- tiazem by Tanabe Seiyaku Co., Ltd. (Osaka, brane protein in 10 mM Tris-HC1 buffer (pH 7.4) Japan). The other agents used were purchased from was preincubated and shaken in a warm water bath Nacali Tesque Inc. (Tokyo, Japan). Concentrated at 37°C for 30 min, and 6.66 mM DHF and 0.1 mM stock solutions of nisoldipine, benidipine, nilvadi- FeCl3-1 mM ADP were added. After 30 min, the pine, felodipine, nicardipine, nitrendipine, nifedi- test agent (1 mM) was added in a total volume of pine, verapamil, or diltiazem were diluted with 0.5 ml, the mixture was incubated and shaken in a ethanol before use, so that the final solvent concen- warm water bath at 37°C for a further 90 min, and tration in the reaction system did not affect lipid the extent of lipid peroxidation was measured. peroxidation. Water was purified with a Milli-Q Plus filter (Millipore, Tokyo, Japan) before use. Determination o f Lipid Peroxidation Lipid peroxide production was measured as thiobar- Preparation of Crude Myocardial Membranes bituric acid-reactive substances (TBARS) using the Eight-week-old rats were sacrificed by decapitation. improved thiobarbituric acid (TBA) test of Ogura et Their hearts were isolated and washed with phy- al. (13). Although the results of this test may not be siological saline. The aorta, atria, and fatty tissue related directly to actual lipid peroxide production were removed, and the ventricles were frozen in li- (14), we defined TBARS (nmol) as the malondial- quid nitrogen and stored at - 80°C. All subsequent dehyde (MDA) equivalent of lipid peroxide produc- procedures were conducted at 4°C. With the use of tion based on data obtained with a standard solu- the method of Wagner et al., as modified by Kane- tion of 1,1,3,3-tetraethoxypropane (9.487 nmol/ml). ko et al. (9), the crude myocardial membrane frac- After the last incubation in a warm water bath, tion was prepared from the heart tissue without uti- 10x1 of 2% (W/V) butylated hydroxytoluene (BHT) lizing chelating agents such as EDTA or a sucrose in methanol was immediately added to stop the gradient. Briefly, the myocardium was minced in 50 reaction. Then, 200pl of 10% (W/V) sodium mM Tris-HC1 buffer (pH 7.4), homogenized for 20 s dodecyl sulfate, and 3 ml of the TBA reagent were with a Polytron PTA 10S homogenizer (Brinkman added. The TBA reagent was a mixture of equal Inst., Inc.) on setting No.S, and then centrifuged at volumes of 0.67% (W/V) TBA aqueous solution 1,000 X g for 10 min. The supernatant obtained was and glacial acetic acid. Next, the mixture was heat- centrifuged at 48,000 X g for 25 min. The resultant ed for 30 min at 90°C and subsequently chilled in pellet was then resuspended in the same buffer and ice water for 10 min, after which 3 ml of chloroform was centrifuged again at 48,000 X g for 25 min. This was added while stirring.