262
Brief Communication
Protection by /3-Blocking Agents Against
Free Radical-Mediated Sarcolemmal
Lipid Peroxidation
I. Tong Mak and William B. Weglicki
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The effects of ^-blocking and class I antiarrhythmic agents on free radical-mediated sarcolemmal lipid peroxidation were examined. Highly purified canine myocytic sarcolemmal
membranes were pretreated with 10-800 /iM of selected ^-blocking (propranolol, pindolol,
metoprolol, atenolol, or sotalol) and class I (quinidine, lidocaine, procainamide, or diphenylhydantoin) antiarrhythmic agents at 37° C for 10 minutes. Subsequently, a superoxide radical
(derived from dihydroxyfumarate) driven, Fe3+-ADP catalyzed free radical generating system
was added and incubated for up to 45 minutes. Lipid peroxidation of sarcolemma was
determined by malondialdehyde formation. Pretreatment of the membranes with the five
/J-blockers resulted in various degrees (20-95%) of inhibition of sarcolemmal peroxidation in
a concentration- and time-dependent manner. All the class I agents were less effective (<20%
inhibition). The order of potency of the 0-blockers was propranolol>pindolol>metoprolol>
atenolol>sotalol and appeared to relate to their degree of lipophih'city. Propranol, the most
potent agent, achieved half-maximal inhibition of peroxidation at about 100 ftM and achieved
significance (p<0.01) at 20 juM. At pH 6.0, the efficacy of pindolol, metoprolol, atenolol, and
sotolol diminished by 30-50% compared to pH 7.2, but the potency of propranolol remained
unchanged. Since increased free radical production may occur during myocardial ischemia/reperfusion injury, the above findings suggest that the lipophilic 0-blockers may provide
additional antiperoxidative protection of ischemic tissue. (Circulation Research 1988;63:262-266)
D
uring cardiac ischemia, free oxygen radicals
have been reported to increase upon
reperfusion.'-4 The phospholipid-rich sarcolemma of ventricular myocytes may be a major
site of free radical attack. We recently reported that
isolated canine myocytic sarcolemmal membranes
were readily peroxidized by free oxygen radicals5;
in addition, the reactions were greatly influenced by
the presence of lipid amphiphiles.6
Most clinically used antiarrhythmic agents are
amphiphilic in nature7 and may readily partition into
the hydrophobic regions of the sarcolemmal membrane; we sought to determine whether the sensitivity of the membrane to free radical attack might
be affected. In the present study, we selected several /3-blockers and class I antiarrhythmic agents
From the Departments of Medicine and Physiology, Division
of Experimental Medicine, The George Washington University
Medical Center, Washington, DC 20037.
Supported by a Grant-in-Aid from the American Heart Association, Nation's Capitol Affiliate, and by National Institutes of
Health grants R0I-HL364I8 and P0I-HL380790.
Address for reprints: I. Tong Mak, PhD, Department of
Medicine, The George Washington University Medical Center,
2300 Eye Street, N.W., Washington, DC 20037.
Received August 17, 1987; accepted February 3, 1988.
and examined their dose-related effects on free
radical-mediated peroxidation in highly purified sarcolemmal membranes.
Materials and Methods
Materials
Dihydroxyfumarate (DHF), ADP, FeCl3 • 6H2O,
2-thiobarbituric acid, DL-propranolol, pindolol,
metoprolol-tartrate, atenolol, sotalol, quinidine, lidocaine, procainamide-HCI, and 5,5-diphenylhydantoin were purchased from Sigma Chemical,
St. Louis, Missouri; naphthalene and 1-methoxynaphthalene were from Fisher Scientific. Sarcolemmal membranes were prepared from adult canine
myocytes according to the procedure of Weglicki et
al.8 Briefly, the myocytes were isolated from ventricular tissues by digestion with 0.2% collagenase.
Freshly isolated myocytes were then disrupted by
nitrogen cavitation (500 PSI 30 minutes, 4° C), and
the sarcolemmal membranes were enriched by differential and sucrose density centrifugations. The
sarcolemmal fractions used for these studies were
enriched at least 50-fold in the specific activities of
the marker enzymes K+-dependent p-nitrophenyl
phosphatase and Na,K-ATPase.8
Mak and Weglicki
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Incubation Procedure and Measurement of Lipid
Peroxidation
For this study, free radicals were generated from
our established superoxide anion-driven, ironcatalyzed free radical system.5-6-9 Superoxide anions
were generated during the auto-oxidation of DHF;
iron was present as Fe3+-ADP chelate. Sarcolemmal membranes (75-100 fig protein/ml) were
preincubated with or without each antiarrhythmic
agent at specified concentrations for 10 minutes at
37° C in a reaction buffer consisting of 120 mM KCI,
50 mM sucrose, and 10 mM potassium phosphate,
pH 7.2 or 6.0. Other conditions are described in
"Results." Reactions were initiated by the final
additions of freshly prepared Fe3+-ADP (0.025 mM
FeCl3 chelated by 0.25 mM ADP) and 0.83 mM
DHF. The rates of membrane lipid peroxidation
were measured by the formation of malondialdehyde (MDA), which was determined by the thiobarbituric acid (TBA) method as described previously.9 To prevent nonspecific color formation due
to overheating,10 the temperature of the chromophore development step was maintained at 80° C
(for 30 minutes). Under these conditions, complete
development of the TBA-MDA value was achieved,
and no interference from the sucrose and iron in the
TABLE 1. Effects of ^-Blocking and Class I Antiarrhythmic Agents
on Sarcotanmal Lipid Peroxidation
Additions
Control system (Fe-ADP + DHF)
A. /3-Blocking agents
Propranolol
20/iM
200 MM
Pindolol
20fiM
200/iM
Metoprolol
20 jiM
200 MM
Atenolol
20 ^M
200 ^M
Sotalol
20 /iM
200/xM
B. Class 1 antiarrhythmi(: agents
Quinidine
20/iM
200/tM
Lidocaine
20 /iM
200 ^M
Procainamide
20 uM
200 fiM
Diphenylhydantoin
20/iM
200/tM
MDA Formation
(% of control system)
100
75.3T
34.5t
80.7*
54.0t
88.9
73.1*
84.3*
73.0t
85.7
74.7t
95.0
85.2*
%.3
95.3
97.0
%.5
100
98
Sarcolemmal membranes (75-100 jig protein/ml) were preincubated with each agent initially dissolved in 10 n\ ethanol/ml
reaction buffer for 10 minutes at 37° C before the additions of
Fe-ADP (0.025 mM FeCI,, 0.250 mM ADP) and DHF (0.83 mM)
in a medium of 0.120 M KCI, 0.05 M sucrose, and 0.010 M
potassium phosphate, pH 7.2. After 20 minutes of incubation,
samples were assayed for MDA formation and expressed as
percentage of control. Values are means of three to eight
separate determinations.
*p<0.05; tp<0.OI versus values for controls.
Antioridant Effects of ^-Blocking Agents
263
100
2
o
80
60
40
Q
20
10
20
45
Time at 37°C (ntin)
FIGURE I. Time course of free radical-mediated lipid
peroxidation in isolated sarcolemma in the absence (control) or presence of various fi-blockers (200 fiM each).
Values are mean±SD of three to eight separate determinations. O, control; • , propranolol; A, pindolol; •, metoprolol; A, atenolol; O, sotalol.
system was observed. None of the antiarrhythmic
agents was found to have an effect on the TBA
reaction. Protein determinations were performed
according to Lowry et al." Statistical differences
between mean values were determined by unpaired
Student's / test.
Results
With the chosen levels of the free radical generating components, the time course of lipid peroxidation in the control samples is presented in Figure
1. At 20 minutes, the level of MDA formed in the
control samples was 39.9±3.8 nmol/mg protein.
The effects of the five /3-blocking and four class I
antiarrhythmic agents on the induced sarcolemmal
lipid peroxidation are summarized in Table 1. The
four class I antiarrhythmic agents only showed
modest (<20%) effects. However, pretreatment of
the sarcolemmal membranes for at least 10 minutes
with the ^-blocking agents resulted in significant
(p^0.05 or less) protection against the membrane
peroxidation. Results from experiments in which
each 0-blocker (200 (JM) was added with no preincubation period showed inconsistent (propranolol
or pindolol) or no (metoprolol, atenolol, or sotalol)
protection against sarcolemmal peroxidation. Additional control experiments, in which various agents
were added at the end of the incubation, did not
show interference with the TBA assay. Separate
experiments were also performed indicating that the
rates of DHF auto-oxidation or superoxide radical
generation, measured by the method of Goscin and
Fridovich,12 were not affected by any of the /3blocking agents.
Time course studies showed that 200 /AM of
propranolol, pindolol, or metoprolol provided pro-
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Circulation Research
Vol 63, No I, July 1988
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tective effects throughout 45 minutes of incubation;
however, the same levels of atenolol or sotalol only
produced significant effects at 20 minutes but not at
45 minutes of incubation (Figure 1).
In the next series of experiments, the ability of
the five ^-blocking agents to inhibit lipid peroxidation in sarcolemma was compared under conditions
described in Table 1. As shown in Figure 2, all five
/3-blockers were able to inhibit peroxide formation
in a concentration-dependent manner; their relative
inhibitory potency was propranolol>pindolol>
metoprolol>atenolol>sotalol. Propranolol is a highly
potent and effective agent that produced complete
inhibition (>95%) at high concentrations and
achieved a significant effect at 20 /xM (24.7% inhibition, p<0.01). In data not shown, complete inhibition could not be achieved with the other four
/J-blockers at concentrations higher than 800 /AM.
Based on the data in Figure 2, the concentrations of
propranolol, pindolol, and metoprolol required to
inhibit MDA formation by 50% were calculated and
compared with their relative hydrophobicities, which
are expressed as log octanol/water partition coefficients (Table 2). Propranolol, the most effective
agent, is most hydrophobic, whereas atenolol and
sotalol are less hydrophobic and less effective.
Since the pH of ischemic cardiac tissue may fall
to as low as 5.8-6.0,13 the effect of acidotic pH on
/3-blocker-inhibited lipid peroxidation was studied
(Figure 3). Compared with pH 7.2, the level of
MDA formation in the control samples decreased
less than 10% (36.3±3.2 vs. 39.9±3.8 nmol/mg
protein). Another concentration-effect analysis indicates that the efficacy of propranolol was unchanged
by pH; its EC^ value was calculated to be 105 fiM.
The efficacy of pindolol (EC50, 458 fiM) decreased
TABLE 2. Inhibition of Sarcotetnmal Lipid Peroxidation: EC» of
Various 0-Blocfcers
Agent
Propranolol
Pindolol
Metoprolol
Atenolol
Sotalol
ECW (/iM)*
103
349
671
>l,000
>l,000
Log octanol/water
partition coefficientt
3.65
1.75
2.15
0.23
-0.79
•Concentration of agent required toreduceMDA formation by
50%. Data are estimated from results in Figure 1.
tAdapted from Cruickshank.14
slightly whereas those of metoprolol, atenolol, and
sotalol decreased to a larger extent (30-50%) at
high concentrations (Figure 3).
Discussion
As an index of membrane lipid peroxidative injury,
MDA formation assayed by the TBA method was
used in our study because of its sensitivity and
simplicity. Despite some controversy about the
specificity of the TBA-MDA methodology, the assay
remains a useful tool in monitoring relative lipid
peroxidation events in vitro. 1015 With an isolated
membrane system, many of the intracellular components and metabolic systems that may affect the
level of MDA formation or interfere with the TBAMDA assay1015 are absent; therefore, the TBA
assay can still be used as a reasonable indicator of
the relative extent of the membrane lipid peroxidation. In this study, since nonspecific assay interference from the agents has been ruled out, the data
are interpreted to indicate that /3-blockers possess
significant antiperoxidative potency against sarcolemmal lipid peroxidation. In addition, their order
FIGURE 2. Concentration-dependent protective
effect of propranolol (•), pindolol (A), metoprolol
(•), atenolol (A), and sotalol (O) on sarcolemmal
lipid peroxidation. After20 minutes of incubation,
samples were assayed for MDA formation and
expressed as percent relative to the controls. Values are mean±SD of three to eight separate
determinations.
100
10
20
40
100
Concentration
200
400
S00
Mak and Weglicki
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100
Concentration
200
400
goo
( ^ )
FIGURE 3. Effect of acidotic pH on sarcolemmal lipid
peroxidation in the presence of propranolol (•), pindolol
(A), metoprolol (•), atenolol (A), and sotalol (O). Samples
were incubated for 20 minutes at pH 6.0; other conditions
were as described in Figure 2.
of potency, with the exception of metoprolol,
appears to follow the degree of lipophilicity; propranolol, being most lipophilic, is the most potent
agent. Since a preincubation period is required, we
suggest that the observed effect does not result from
direct scavenging of free radicals generated in the
aqueous phase but rather from an interaction of the
/3-blockers with the sarcolemmal membrane lipids.
In addition to their receptor blocking activity,
/3-blockers such as propranolol and pindolol possess membrane stabilizing/local anesthetic properties.16 Since the concentrations required for the
/J-blockers to produce significant effects were well
above the range needed to block /3-adrenergic receptors, it is reasonable to speculate that all of the
effects observed were contributed by nonspecific
membrane effects such as membrane stabilizing
activity. However, both quinidine and lidocaine are
well-known local anesthetics and are relatively
lipophilic with their log octanol/water partition coefficient (log P) vaJues (1.56 and 1.81, respectively),
comparable to that of pindolol (1.75); yet neither
quinidine nor lidocaine provides major inhibition of
sarcolemmal peroxidation, whereas pindolol and
other /3-blockers do (Table 1).
As common structural features, all /3-blockers
consist of an aromatic moiety linked to the ethanolamine side chain by an oxymethylene bridge (except
sotalol).16 Presumably, much of the lipophilicity of
each agent is contributed by the aromatic ring
structure. Propranolol is more lipophilic because of
its naphthalene moiety (or a two-ring structure).
Antioxidant Effects of /3-Blocklng Agents
265
Interactions of propranolol with model membranes
and protein-depleted sarcoplasmic reticulum lipids,
as studied by neutron diffraction techniques, indicate that the naphthalene moiety of propranolol is
localized within the hydrocarbon center of the membrane bilayer.17 In additional experiments, sarcolemmal membranes were pretreated with the highly
lipophilic (log P>3.50) naphthalene (up to 400 fiM)
before exposure to free radicals; interestingly, no
protection (<10%) was observed. This result suggests that solvation of the lipophilic aromatic structure alone into the membranes does not provide
protection. However, a structurally related compound, 1-methoxynaphthalene at 200 ^.M, was found
to have protective activity approaching 50-60% of
that of propranolol at the same concentration. This
observation suggests that the naphthyoxy structure
may possess "true" antioxidant activity similar to
that of conventional chainbreaking antioxidants.18
Antioxidants of this class are usually phenols or
aromatic amines, and they owe their antioxidant
activity to their ability to trap peroxy radicals inside
the membrane.18 From a structural point of view, all
/3-blockers do have the aromatic resonance rings to
stabilize trapped radicals as most classic antioxidants have in common. However, such a property
for all the /3-blockers remains to be determined by
more direct experiments measuring the rate constant of the agents with peroxy radicals.18 Alternatively, since /J-blockers are cationic amphiphiles,
and since a variety of cationic amphiphiles have
been demonstrated to form complexes with
phospholipids,1920 interactions of the /3-blockers
with the sarcolemmal membrane might result in
drug-phospholipid complexes, which are less susceptible to free radical attack. The efficacy of each
agent may then depend on the degree of drug-lipid
complexing, which in turn depends on the lipophilicity of each agent. The present results neither
support nor exclude this possibility.
To our knowledge this study represents the first
report describing protective effects of these cardiovascular drugs against lipid peroxidation in cardiac
membranes, though anti-cancer agents such as
anthracenediones (which are compounds with polyaromatic rings) were reported to have inhibitory
effects on cardiac lipid peroxidation.21 Although
elucidation of the exact mechanisms by which /3blockers, especially propranolol, protect against
sarcolemmal lipid peroxidation requires further
study, the observed phenomenon may be of potential therapeutic significance. During cardiac ischemia, the level of free radicals, measured by electron spin resonance techniques, has been reported
to increase fourfold in the coronary venous effluent
of canine heart within 5 minutes after coronary
artery occlusion.2 Electron spin resonance studies
of free radical production in ischemic/reperfused rat
hearts in our laboratory clearly indicate that maximal oxygen free radical production is seen in the
effluent 3 minutes after the introduction of
266
Circulation Research
Vol 63, No 1, July 1988
reperfusion.22 Both studies suggest that excessive
free radicals are present in the extracellular compartment and may be able to attack the sarcolemmal
membranes of the cardiocytes. Since long term
administration of amphiphilic drugs such as propranolol may produce a significant accumulation in
myocardial tissue,23 it is reasonable to speculate
that the /3-blockers, in the lipid phase of the sarcolemmal membrane, may provide a protective effect
in vivo when free radicals are present.
Acknowledgments
The authors wish to thank Ms. Lisa Kopyta for
providing excellent technical assistance and Ms.
Miriam Cole and Ms. Suzanne Swim for assisting in
the preparation of the manuscript.
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KEY WORDS • lipid peroxidation • sarcolemma • class I
antiarrhythmic agents • ^-blockers • antioxidant activity
Protection by beta-blocking agents against free radical-mediated sarcolemmal lipid
peroxidation.
I T Mak and W B Weglicki
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Circ Res. 1988;63:262-266
doi: 10.1161/01.RES.63.1.262
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Copyright © 1988 American Heart Association, Inc. All rights reserved.
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