[62] Antioxidant activity of calcium channel blocking drugs

620

ANTIOXlDANT CHARACTERIZATION AND ASSAY

[62]

plasmalogens by a plasmalogen-selective phospholipase A 2 is also possible. 64 In conclusion, biological systems involving oxidative stress need to be reexamined to establish whether oxidative decomposition of plasmalogens takes place under physiological conditions. 64 D. A. Ford, S. L. Hazen, J. E. Saffitz, and R. W. Gross, J. Clin. Invest. 88, 331 (1991).

[62] A n t i o x i d a n t A c t i v i t y o f C a l c i u m C h a n n e l Blocking Drugs

By I. TONG MAK and WILLIAM B. WEGLICKI Introduction In common with most cardiovascular agents, the clinically used calcium channel blockers (nicardipine, nifedipine, verapamil, diltiazem) are amphiphilic in nature. Thus, in addition to their specific binding to protein receptors, these agents may readily partition into the phospholipid domain of cardiovascular membranes to various degrees according to their lipophilicity. Efforts from our laboratory have focused on the effects of such agents on the sensitivities of cardiac membranes and vascular cells to free radical injury. 1-7 At the membrane level, we have chosen the highly purified sarcolemmal membranes of ventricular myocytes as model membranes. Compared to other subcellular membranes, the sarcolemmal membranes were much more sensitive to free radical-mediated damage, 8 probably owing to the highly enriched phospholipid content. 8-1° To assess the extent of membrane lipid peroxidation, we chose to use the thiobarbituric acid (TBA) method because of its sensitivity and convenience. 1 I. T. Mak and W. B. Weglicki, Circ. Res. 63, 262 (1988). 2 I. T. Mak and W. B. Weglicki, Circ. Res. 66, 1449 (1990). 3 I. T. Mak, C. M. Arroyo, and W. B. Weglicki, Circ. Res. 65, 1151 (1989). 4 W. B. Weglicki, I. T. Mak, and M. G. Simic, J. Mol. Cell. Cardiol. 22, 1199 (1990). 5 I. T. Mak, A. M. Freedman, B. F. Dickens, and W. B. Weglicki, Biochem. Pharmacol. 40, 2169 (1990). 6 I. T. Mak, P. Boehme, and W. B. Weglicki, Circ. Res. 70, 1099 (1992). 7 I. T. Mak, J. H. Kramer, and W. B. Weglicki, Coronary Artery Dis. 3, 1095 (1992). s j. H. Kramer, I. T. Mak, and W. B. Weglicki, Circ. Res. 55, 120 (1984). 9 W. B. Weglicki, K. Owens, F. F. Kennett, A. Kessner, L. Harris, R. M. Wise, and G. V. Vahoouny, J. Biol. Chem. 255, 3605 (1980). l0 W. B. Weglicki, J. H. Kramer, I. T. Mak, B. F. Dickens, and T. M. Phillips, in "Isolated Adult Cardiomyocytes" (H. Piper and G. Isenberg, eds.), Vol. 1, p. 1. CRC Press, Boca Raton, Florida, 1988.

METHODS IN ENZYMOLOGY,VOL. 234

Copyright© 1994by AcademicPress, Inc. All rightsof reproductionin any formreserved.

[62]

ANTIOXlDANT PROPERTIES OF CALCIUM BLOCKERS

621

Increasing evidence has accumulated to suggest that lipid peroxidative processes are involved in the pathogenesis of vascular diseases.ll,12 Among those processes, oxidative injury of endothelial cells may represent a critical event in propagating atherogenesis. 11 The endothelial cells are potential targets of reactive oxygen radicals released from activated blood cells (e.g., neutrophils, macrophages, platelets) and oxidizable drugs and chemicals. Therefore, at the cellular level, we have used cultured endothelial cells to assess the cytoprotective effects of the calcium channel blockers against free radical-induced loss of glutathione and increased membrane permeability. Both parameters were relatively sensitive to the oxidative stress generated from a chemical oxygen-radical system. Cardiac Sarcolemmal Membrane Model

Sarcolemmal Preparation. Sarcolemmal membranes are isolated from adult canine ventricular myocytes. The isolation procedure has been described in detail elsewhere, s-l° Briefly, adult canine myocytes are isolated from ventricular tissue by enzymatic digestion with 0.05% collagenase. Following disruption of the myocytes by nitrogen cavitation (1000 psi, 30 min), the sarcolemmal membranes are enriched by differential and sucrose gradient centrifugation. The sarcolemmal fractions, which band between 21 and 26% sucrose, are about 80-fold enriched in the specific activity of the marker enzyme Na ÷, K+-ATPase over that of the myocyte homogenate.9'l° Generation of Free Radicals. The chemical system we use to generate oxygen-radicals consists of dihydroxyfumarate (DHF) and FeCI3-ADP. 13 Oxidation of D H F (Sigma Chemical Co., St. Louis, MO) in solution generates sustained levels of superoxide anions14'15; the rate of production is further promoted by the presence of metal chelates such as Fe-ADP. Hydroxyl radicals (.OH) are generated according to the following chemical reactions: DHF + 02--> .DHF + 02 ~ •DHF + 02 ~ diketosuccinate + O2 ~ 202: + 2H ÷ ~ H202 + 02 Fe3+-ADP + 02 ~ ~ Fe2+-ADP + 02 Fe2+-ADP + H202 --> -OH + Fe3+-ADP + O H 11 B. Hennig and C. K. Chow, Free Radical Biol. Med. 4, 99 (1988). 12 B. Halliwell, Br. J. Exp. Pathol. 70, 737 (1989). 13 I. T. Mak, H. P. Misra, and W. B. Weglicki, J. Biol. Chem. 258, 13733 (1983). t4 S. A. Goscin and I. Fridovich, Arch. Biochem. Biophys. 153, 778 (1972). t5 B. Halliwell, Biochem. J. 163, 441 (1977).

622

ANTIOXIDANT CHARACTERIZATION AND ASSAY

[62]

Presumably, other iron-oxygen species (such as hypervalent iron complexes) may also be formed in Fenton-type reactionsl6; these species could be just as deleterious as .OH radicals. In our system, a concentrated solution of D H F is prepared in the incubation buffer; a brief period (<2 min) of heating in an 80° water bath is required to dissolve the D H F in solution. The dissolved D H F solution is placed on ice and adjusted to pH 7 by adding 1 N KOH. The F e - A D P chelate is prepared by mixing FeCI3 (1-2 mM) with a 10-fold higher concentration (10-20 mM) of ADP to achieve a final concentration ratio of F e 3+ to ADP of 1 : 10. Incubation and Measurement of Lipid Peroxidation. With the membrane system, we found that the TBA colorimetric method is quite satisfactory for assessing the membrane antioxidant activities of calcium blockers. Owing to the varying lipophilicity of the calcium channel blockers, stock solutions of the agents (1-20 mM) are prepared in absolute ethanol. Because of light sensitivity of the dihydropyridines, all experiments are conducted at minimal light. Sarcolemmal membranes ( - 5 0 /zg protein/0.50 ml) are mixed with 10/zl of each calcium-blocking drug and preincubated for 10 min, 37°, in a reaction buffer consisting of 120 mM KCI, 50 mM sucrose, and 10 mM potassium phosphate, pH 7.2; lipid peroxidation reactions are initiated by addition of freshly prepared F e - A D P (final concentrations: 0.025 mM FeCI 3 chelated by 0.25 mM ADP and DHF (0.83) mM). The rate of superoxide production is about 3 nmol/min/ml. At various times of incubation, the levels of TBA-reactive materials are determined by adding 0.5 ml of 0.5% TBA, 50 /.d of 10% trichloroacetis acid (TCA) and 10/xl of 1% butylated hydroxytoluene (BHT) to 0.5 ml of reaction mixture. The mixture is then vigorously vortexed and the color is developed by heating in a water bath at 80° for 30 min. After cooling on ice, 0.5 ml of 70% TCA is added to each sample which is then vortexed and centrifuged; the chromophore developed in the supernatant is measured at 532 nm. The TBA-reactive materials are estimated by using standards of malonaldehyde bis(dimethyl acetal) subjected to an identical heating procedure; the results are expressed as malondialdehyde (MDA) equivalents. The time course of lipid peroxidation in the sarcolemmal membranes, with or without added calcium blockers, is displayed in Fig. 1. The accumulation of reaction products in most samples appeared to be quasi-linear up to 20 min. Results in Fig. 1 indicate that 100/xM verapamil, nifedipine, 16 B. H. J. Bielski, Basic Life Sci. 49, 123 (1988).

[62]

ANTIOXIDANT PROPERTIES OF CALCIUM BLOCKERS A

623

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FIG. 1. Time course of free radical-mediated lipid peroxidation in isolated sarcolemmal membranes in the absence and presence of various calcium channel blockers (100/zM each). Values are means of 3-8 determinations. O, R' control; A, + diltiazem; T, + verapamil; 0 , + nifedipine; II, + nicardipine.

and nicardipine provided varying degrees of significant inhibition throughout 45 min of incubation. The same level of diltiazem provided a significant inhibition at 20 min but not at 45 min. These time course studies led us to choose 20 min as the incubation time to compare the inhibitory potency of the calcium blockers. As represented in Fig. 2, all four calcium blockers exhibited concentration-dependent inhibition of sarcolemmal lipid peroxidation. The order of potency was nicardipine > nifedipine > verapamil > diltiazem; their respective values of ECs0 (in micromolar) were estimated to be 22.6 for nicardipine, 38 for nifedipine, 210 for verapamil, and 850 for diltiazem. At least 5 min of preincubation of the agents and membranes is required prior to the free radical reaction, suggesting that membrane-drug interactions are necessary to effect the subsequent results. To evaluate the initial association of the drugs with the membranes, the following experiments are designed according to Scheme 1. Samples for set A are incubated normally as described. For the duplicate set (set B), the sarcolemmal membranes with or without (controls) drug treatment (100/zM each, 10 min, 37°) are transferred to the airfuge tubes and centrifuged at 150,000g

624

[62]

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for 10 min by using a Beckman Airfuge (Beckman Inst. Inc., Palo Alto, CA). The supernatants, containing some portion of drugs not associated with the membranes, are removed. The subsequent membranous pellets are resus¢-

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Log [uM] of Agents FIG. 2. Comparative inhibitory effects of the calcium channel blockers on sarcolemmal lipid peroxidation. The ECs0values (/zM)were as follows: (U) nicardipine, 22.6; (O) nifedipine, 38; (V) verapamil, 206; and (A) diltiazem, 850. (Results partially adapted from Mak and Weglicki,2 with permission.)

[62]

ANTIOXIDANT PROPERTIES OF CALCIUM BLOCKERS

625

TABLE I REQUIREMENT OF MEMBRANE PARTITIONING OF CALCIUM CHANNEL BLOCKERS FOR ANTIOXIDANT ACTIVITIES a M D A formation b

Conditions

Set A

Set B

Controls (R.) Agents (100 p.M) added: Nicardipine Nifedipine Verapamil Diltiazem

0.246

0.192

0.010 0.035 0.160 0.181

0.007 0.029 0.132 0.163

a Incubation conditions for set A were as described on p. 634. The conditions for set B were as described in Scheme 1; after the 10 min of membrane --- drug preincubation, the supernatants were removed by centrifugation and the membrane pellets were resuspended in fresh medium before addition of the free radical components. Other conditions were as described in Figs. 1 and 2. b (Absorbance at 532 nm)

pended in fresh buffer; the samples are then subjected to the free radical reaction for 20 min. Results summarized in Table I indicate that the calcium channel blockers in the samples of set B provided an extent of inhibition similar to those of set A. These results indicate that the calcium blockers mediate their antioxidant action in the lipid domain of the membranes. The overall results suggest that the calcium channel blockers provide lipophilic "chain-breaking" activity similar to that of a-tocopherol. Comments. The rate of DHF oxidation in solution can be followed by measuring the fall in absorbance at 300 nm.14 The corresponding rate of superoxide generation by the chemical oxygen radical system can be measured independently by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (75/xM) according to the method of Kellogg and Fridovich/7 In the presence of Fe-ADP, we did not find that the addition of SOD (10 /zg/ml) would inhibit the rate of DHF oxidation. With electron spin resonance (ESR) spectroscopy using 5,5-dimethyl-1pyrolidine N-oxide (DMPO) as the spin trap, the formation of hydroxyl radicals was confirmed and the steady-state level could be monitored. 3'18 17 E. W. Kellogg and I. Fridovich, J. Biol. Chem. 252, 6721 (1977). i8 C.M. Arroyo, I. T. Mak, and W. B. Weglicki, Free Radical Res. C o m m u n . 5, 369 (1989).

626

ANTIOXIDANT CHARACTERIZATION AND ASSAY

[62]

Under these conditions, none of the calcium blockers affected the rates of D H F oxidation and superoxide generation; none of the agents had any effect on the steady-state signal intensity of the D M P O - O H adducts. Despite some concerns about the specificity of the TBA method, ~9we found the method to be relatively reliable for the isolated membrane system; many of the intracellular components and metabolic systems that might influence the T B A - M D A chromophore formation were absent. To determine potential agent-mediated interference on the TBA reaction, experiments were performed in which the calcium blockers were added at the end of the 20-min, 37° incubation but before the 80 ° heating stage; the results indicated that none of the drugs interfered with the TBA assay. In a separate study, ~° we observed that the extent of free radical-induced sarcolemmal membrane MDA formation was accompanied by degradation of phospholipids.

Endothelial Cell Model

Cell Culture. Bovine aortic endothelial cells (GM 07372A) are obtained from the Coriell Institute for Medical Research (Camden, N J). The cells are cultured in Dulbecco's modified Eagle's medium supplemented with 15% calf serum. For subsequent subculturing, the cells are split 1 : 5 at confluence using trypsin-EDTA. Confluent plates are trypsinized by 0.05% trypsin in Hanks' balanced salt solution (HBSS) with 0.02% EDTA. The digestion is stopped by adding growth medium with serum. The cells are pelleted, washed twice at room temperature, and finally resuspended in the incubation buffer. Under these conditions, the isolated cells routinely displayed over 95% viability based on the trypan blue exclusion assay. Oxidative Incubation and Drug Treatment. Oxygen-radicals are generated from the D H F / F e - A D P system. The free radical generating system is prepared just before each experiment. Endothelial cells (1 x 106/ml) are resuspended gently in a buffer consisting of 10 mM glucose, 125 mM NaCI, 1.2 mM MgCI2, and 10 mM potassium phosphate, pH 7.2. Ten microliters of either calcium blocker (5-20 nmol) or BHT dissolved in ethanol is added to the 0.5 ml cell suspensions; 10 ttl of ethanol is added to the vehicle controls. All samples (with or without drug) are preincubated at 37° for 15 min in a shaking water bath. Finally, 25/xl of F e - A D P (final concentration of 50/.tM FeC13 chelated by 0.5 mM ADP) and 25 /zl of DHF (I .67 mM) are added to each sample to initiate the oxidative reaction. All experiments are performed in minimal light. The incubations are con19 D. R. Janero, Free RadicalBiol. Med. 9, 515 (1990).

[62]

ANTIOXIDANT PROPERTIES OF CALCIUM BLOCKERS

627

tinued up to 60 min; at various times, samples are assayed for thiols and viability. Assessments of Effects of Drugs on Losses of Cellular Glutathione and Viability. Because the oxygen radicals are generated in the extracellular space, presumably, the initial oxidative target is the plasmalemmal membrane. We therefore chose to use the increase in permeability to trypan blue as an index of membrane integrity. Cell aEiquots are mixed with an equal volume of 0.2% (w/v) trypan blue; the percentage of cells permeable to the dye is expressed as the percent loss of viability. The gEutathione system is considered critical in providing protection against oxidative stress in endothelial celEs.2° Cell aliquots for glutathione determinations are centrifuged at 200 g for 5 min; the cell pellets are resuspended in the incubation buffer containing 5% (w/v) 5-sulfosalicylic acid (SSA); cell disruption is achieved by sonication at 100 W (Fisher Sonic Dismembrator Model 300) for 45 sec. Total glutathione (GSH + ½ GSSG) in the cell supernatant fractions is determined by the enzymatic DTNB [5,5'-dithiobis(2-nitrobenzoic acid)]-GSSG reductase "cyclic method" originally described by Tietze 21 and modified by Anderson. 22 Briefly, pipette (i) 800/xE of working buffer (140 mM sodium phosphate, pH 7.5, 6.2 mM EDTA, with 245 ~g NADPH/ml added on the day of assay), (ii) 100/~1 of 6 mM DTNB solution, and (iii) 100 ~1 of sample of appropriate dilution into a cuvette. After mixing, the cuvettes are brought to 30° for 5 min; the assay is initiated by the final addition of 10/xl of GSSG reductase solution (133 U/mE). The rate of 5-thio-2-nitrobenzoic acid (TNB) formation, which is proportional to the total GSH content, is followed at 412 nm; the reaction is linear for at least 10 min. Standards of GSH in the range of 0. I-2 nmol containing the same amount of SSA as the samples are prepared similarly. For initial experiments, oxidized glutathione (GSSG), and protein thiols (protein SH) are determined. GSSG is measured by the above DTNB-GSSG reductase method with prior masking of GSH in the supernatant by 2% (v/v) 2-vinylpyridine22; the mixture is adjusted to pH 6-7 by adding 10/zE of triethanolamine per 200/zl sample volume. The GSSG standards are prepared in a solution containing 2-vinylpyridine and triethanolamine. Under alE conditions, we find that the level of GSSG is less than 5% of the total glutathione; therefore, for most studies only total

2o j. F. Jongkind, A. Verkerk, and R. G. A. Baggen, Free Radical Biol. Med. 7, 507 (1989). 21 F. Tietze, Anal. Biochern. 27, 502 (1969). 22 M. E. Anderson, in "Handbook of Methods for Oxygen Radical Research" (R. A. Greenwald, ed.), pp. 317-323. CRC Press, Boca Raton, FL, 1988.

628

[62]

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Time (Minutes) FIG. 3. Time course of losses caused by free radicals in total glutathione (V), protein thiols (©), and viability (&) in endothelial cells. (*p <~ 0.05, **p ~< 0.01 vs values at time zero.) (Reproduced with permission from Mak et al. 6)

glutathione is determined. Protein SH is measured according to the method of Di Monte e t al. 23 After disruption by sonication in the presence of SSA, the cell pellets (derived from 0.5 ml cell suspensions) are washed twice with 1 ml of 6% TCA and resuspended in 2 ml of 0.5 M Tris buffer, pH 7.6, containing 100 txM DTNB; after 20 min of incubation at 30°, the absorbance is measured at 412 nm. Total glutathione levels vary from 3.5 to 6 nmol/106 cells among preparations of endothelial cells. For the purpose of comparison, losses of total glutathione are expressed as percentages of controls at time zero. Figure 3 presents the time course for losses of endothelial glutathione, protein SH, and cellular viability in the presence of free radicals. On addition of the free radical system, total glutathione decreased rapidly for the initial 30 min but at a slower rate afterward; in close association, the loss of cell viability also occurred in a parallel manner. In contrast, the loss of protein SH was only moderate at 30 min of incubation. As summarized in Table II, pretreatment of the cells with each agent dose-dependently protected against the losses of GSH and viability at 30 min of incubation. The order of potency of cytoprotective effects is 23 D. Di Monte, D. Ross, G. Bellomo, L. Eklow, and S. Orrenius, Arch. Biochem. Biophys. 235, 334 (1984).

[62]

629

ANTIOXIDANT PROPERTIES OF CALCIUM BLOCKERS

TABLE II PROTECTIVE EFFECTS OF CALCIUMCHANNEL BLOCKERSAGAINSTFREE RADICAL-INDUCED LOSSES OF ENDOTHELIAL GLUTATHIONE AND VIABILITYa

Conditions Buffer controls R. (DHF + Fe-ADP) R. plus calcium blockers: Nicardipine Nifedipine Verapamil Diltiazem

a

Concentration (/xM)

20 5 20 5 20 5 20 5

GSH loss (%)

Cell death (%)

4 45

12 61

7 21 9 24 11 26 15 29

20 27 25 31 28 40 35 42

The cells were preincubated with each calcium blocker for 15 min before the addition of DHF plus F e - A D P (R.). After 30 min of incubation, the losses of glutathione and cell viability were determined. Values are means of 3-8 determinations. Results adapted from Mak e t a l . , 6 with permission.

nicardipine > nifedipine > verapamil > diltiazem; this appears to follow their membrane antiperoxidative activities.

Comparison of Pharmacologically Active and Inactive Enantiomers of Nicardipine. Of all the calcium blockers tested, nicardipine (racemic form) is the most effective cytoprotective agent. In an effort to further TABLE III ANTIOXIDANT PROPERTIES OF ISOMERS OF NICARDIPINE AND BUTYLATED HYDROXYTOLUENE IN ENDOTHELIAL CELLSa

Conditions Buffer control R. (DHF + Fe-ADP) R' plus agent: ( + )-Nicardipine ( - )-Nicardipine BHT

Concentration (~M)

20 5 20 5 5

GSH loss (%)

Cell death (%)

2 47

11 59

8 24 11 26 7

19 28 22 31 16

Incubation conditions were as described in Table II. Values are means of 4-9 determination. Results adapted from Mak et al., 7 with permission.

630

ANTIOXIDANT CHARACTERIZATION AND ASSAY

[62]

distinguish the nature of this cytoprotection, we have examined the effects of the pharmacologically active ( + ) and inactive ( - ) isomers of nicardipine. Owing to an asymmetric carbon at position 4 of the dihydropyridine ring, nicardipine has two optical isomers, namely, (+)- and (-)-nicardipine; the ( + ) isomer is about 5-fold more potent than the ( - ) isomer as a vasodilator or calcium channel blocker. 24 Both (+)- and (-)-nicardipine are supplied by Syntex Research Laboratory (Palo Alto, CA). When the membrane antioxidant activities of these two compounds are studied using the sarcolemmal membranes, both isomers display identical dose-dependent inhibitory activities against lipid peroxidation; the estimated ECs0 values are 20.6/xM for ( + )-nicardipine and 22.8/xM for ( - )-nicardipine. For comparison, the ECs0 value for BHT is 5.2/xM. Therefore the antioxidant potency of either nicardipine isomer was about one-fourth that of BHT. Using the endothelial cell system, the protection of the nicardipine isomers against free radical-induced losses of GSH and viability is evaluated. As summarized in Table III, both nicardipine isomers (5 and 20 /zM) provide comparable, concentration-dependent protection against the peroxidative depletion of GSH and loss of cellular viability. These results indicate that the cytoprotective effects of the agents are due to intrinsic chemical antioxidant activity rather than calcium channel blocking ability. Comments. The initial rate of endothelial GSH loss is proportional to the concentration of the free radicals generated; doubling the concentration of the free radical components leads to a 50% loss of GSH in 15 min of incubation. Linear regression analysis indicates that the loss of cellular viability is correlated significantly with the decrease of GSH (r = 0.89, p < 0.001). Lipid peroxidation at the cell level cannot be assessed accurately (compared to the isolated membranes); however, we believe that the loss of endothelial GSH is due to its oxidation by increased levels of lipid peroxides. The calcium blocker-mediated effects appear to be secondary to their inhibition of peroxide formation in the membranes. Our results indicate that the dihydropyridine calcium blockers exhibit the greatest antioxidant potency, which is partly due to their higher membrane partitioning and, perhaps, to their more active redox chemistry. It remains to be determined whether such antioxidant properties contribute to their established anti-atherogenic effects, z5 Acknowledgments This research was supported by National Institutes of Health Grants PO 1-HL-38079 and ROI-HL-36418. 24 K. Iwatsuli, F. Iijima,and S. Chiba, Clin. Exp. PharmacoL Physiol. 11, 1 (1984). 25 p. D. Henry, J. Cardiovasc. Pharmacol. 15~ $6 (1991).