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Inhibition of epinephrine-induced myocardial necrosis in rats by administration of single doses of ethanol
Drug and Alcohol Dependence, 2 (1977) 397 - 407 @ Elsevier Sequoia S.A., Lausanne -Printed in the Netherlands
397
INHIBITION OF EPINEPHRINE-INDUCED MYOCARDIAL NECROSIS IN RATS BY ADMINISTRATION OF SINGLE DOSES OF ETHANOL
SAMUEL MALLOV and ROBERT F. GILMOUR Department of Pharmacology, State Symcuse, N.Y. 13210 (U.S.A.)
University
of New York, Upstate Medical Center,
Summary The oral administration of single doses (0.5 - 6.0 g/kg) of ethanol to rats, shortly before injecting them with a large dose (3.0 mg base/kg) of epinephrine subcutaneously, significantly reduced the severity of the myocardial damage produced by the epinephrine. The larger the dose of ethanol, the greater was the protective effect. The results were the same, regardless of the method used to determine the degree of cardiac injury. Experiments employing various agents to determine the mechanisms of ethanol action have tentatively suggested that a platelet de-aggregating or an osmotic effect of alcohol may be involved.
Introduction It has been known for many years that the presence of high levels of sympathomimetic amines, including catecholamines, in uivo in experimental animals [l, 21 and in man [3, 41 can cause’myocarditis and myocardial necrosis. We have been using the epinephrine-induced necrotic heart in rats as an experimental model for studies of the mechanism of this type of cardiac injury. The deleterious effects of acute and chronic ethanol ingestion on the heart are also well known [ 5,6]. It was therefore surprising to us when we observed, during investigation of the effects of certain drugs on the severity of myocardial necrosis produced by the administration of large doses of epinephrine, that ethanol exerted a protective effect. This was a serendipitous finding since the ethanol was merely added to the aqueous medium to increase the solubility of a drug (Anginin) that turned out to have no effect at all. We report here our observations regarding the protective effect of ethanol as well as the results of studies we have begun to elucidate the mechanism of this effect.
398
Methods Myocardial necrosis was produced in 300 - 350 g male Sprague-Dawley rats by injecting them once, subcutaneously (s.c.), with a large dose of epinephrine (3.0 mg base/kg) in saline solution. Control rats were injected with saline solution alone. Some of these rats were force-fed, by stomach tube, either 15 min or 2 h prior to the time of injection, 10% or 50% solutions of ethanol in water (v/v), while others were given equal volumes of water or an isocaloric solution of glucose in place of the ethanol. The doses of ethanol were 0.5, 3.0, or 6.0 g/kg. The rats were either in the fed or fasted state when force-fed the ethanol, water, or glucose. The severity of myocardial injury produced by the epinephrine was determined in three different ways: (a) by visual inspection and grading, (b) by determination of the quantities of certain enzymes released by the hearts in uitro, and (c) by determination of the cardiac uptake of technetium-99m stannous pyrophosphate (99mTc-PYP) in duo. Hearts were graded as to the severity of the necrosis produced, after visual inspection, as follows: grade 0, no visual necrosis; grade 1, necrosis limited to intraventricular septum (IVS) or apex; grade 2, necrosis in IVS or apex and also in base of left ventricle (BLV); grade 3, necrosis in IVS or apex, BLV and in base of right ventricle (BRV); grade 4, necrosis in IVS or apex, BLV, BRV and in free wall of left ventricle. Eighteen hours after the injection of epinephrine or saline, some of the rats were anesthetized by the intraperitoneal (i.p.) administration of pentobarbital. The hearts of these animals were removed, inspected and perfused via their aortas as Langendorff preparations, according to the technique previously described [ 71. The perfusion fluid, composed of oxygenated KrebRinger bicarbonate buffer at pH 7.4 containing 10 n-&f glucose, was not recirculated. The flow rate through each heart was maintained at 10.0 ml/ min. In the first experiments the perfusion fluid leaving each heart was collected in 5-min fractions for a period of 45 min, following an initial lomin period of blood wash-out and equilibration, and each fraction was then analyzed for enzyme activities. Lactate dehydrogenase (LDH) and glutamicoxaloacetic transaminase (GOT) activities were determined by the methods of Henry [8], creatine phosphokinase (CPK) by the method of Rosalki [9], and a-hydroxybutyrate dehydrogenase (HBDH) by the method of Ellis and Goldberg [lo] . The activities of the enzyme were expressed as milli-international units (mIU) per ml of perfusate. Other rats were injected intravenously (i.v.) with 25 &i of ggmTc-PYP 18 h after receiving epinephrine. The hearts of these animals were removed exactly 100 min later and their total radioactivities determined with the aid of a Beckman Biogamma II counter. ‘*‘Tc-PYP uptake was expressed as the ratio (% of total dose absorbed by the heart)/(% of body weight represented by the heart) in order to correct for variations in dose, heart and body weights [ 111. The measurement of the elevation of certain enzymes in serum to indicate the occurrence and to assess the severity of myocardial infarction is a
399
Fig. 1. Release of lactate dehydrogenase from isolated perfused hearts of saline- and epinephrine-treated rats into perfusion fluid. Epinephrine was injected S.C. in a dose of 3 mg/kg 18 h before the hearts were removed. Open circles, control rat hearts; closed circles, epinephrine-injected rat hearts.
s
301
@ GOT
E
2
1
‘; >
20
0
CPK
n
HBDH
LDH
t
INFARCT
GRADE
Fig. 2. Release of enzymes from isolated perfused hearts of control and epinephrine-treated rats into the perfusion fluid, in relation to the severity of cardiac injury as graded after visual inspection. Different doses of epinephrine were injected S.C. to produce different degrees of cardiac injury. Perfusates were collected for periods of 45 min. Numbers in parentheses refer to numbers of hearts. Vertical lines represent standard errors of the means.
well-known clinical as well as experimental technique [8, 12,131. Determination of these enzymes released from isolated perfused hearts has the added advantage of eliminating enzymes released from organs other than the heart. Uptake of s9mTc-PYP or related radioactive nuclides by the heart, in duo, has also been used both experimentally and clinically to assess the severity of myocardial infarction [ 14, 151. This sensitive method depends on the entrance into and binding of ssmTc-PYP by reversibly or irreversibly injured cells that surround areas of necrotic tissue.
INFARCT
GRADE
rats in Fig. 3. Cardiac uptake of 9s’nTc-PYP in v iuo of control and epinephrineinjected relation to the severity of cardiac injury as graded after visual inspection. Different doses of epinephrine were injected S.C. to produce different degrees of cardiac injury.
Results It was observed that isolated hearts from epinephrine-treated rats released significantly greater quantities of all four enzymes measured throughout the period of perfusion than did hearts from control rats. Figure 1 shows, for example, the results of experiments in which the activities of LDH released into the perfusion fluid from the hearts of control and epinephrine-treated rats were determined. When different doses of epinephrine were administered to rats to produce myocardial necrosis of different degrees of severity, it was found that there was a good correlation between the degree of severity of the lesion as graded after visual inspection and the release of each of the enzymes from the isolated perfused hearts into the perfusion fluid. This is shown in Fig. 2. Similarly, it was found that there was a good correlation between the visually determined severity of necrosis and the cardiac uptake of ss”Tc-PYP in uiuo, as shown in Fig. 3. Thus each of these methods was suitable for determination of the effects of drugs on the severity of epinephrine-induced cardiac damage. Effect
of ethanol
It was found that the oral administration of ethanol 15 min or 2 h before epinephrine injection inhibited the production of myocardial injury. The degree of protection increased as the dose of ethanol was raised from *Mean + s.e.m. **p values are given in those cases in which the myocardial necrotic grade is statistically significantly different from the necrotic grade in rats receiving only epinephrine. In all cases the dose of epinephrine administered was 3.0 mg base/kg. EtOH = ethanol, pyaa. = pyridylacetic acid, glut. = glucose, dipyr. = dipyridamole (Persantine), asp. = aspirin, Lprop. = L-propranolol, D-prop. = D-propranolol, and morph. = morphine.
401 TABLE
1
Effect of administering various agents on severity rats by epinephrine injection Agent
Dose
None (controls) Epin.
3 mg/kg,
Epin. + EtOH
0.5 g/kg, p.o.
Epin. + EtOH
6.0 g/kg, p.o.
Epin.
3 mg/kg,
S.C.
S.C.
3.0 g/kg, p.o.
Epin. + EtOH
6.0 g/kg, p.o.
Epin. + EtOH
0.5 g/kg, p.o.
Epin. + EtOH
3.0 g/kg, p.o.
Epin. + EtOH
6.0 g/kg, p.o.
Epin. + pyaa
100 mg/kg,
Epin. + glut.
33
fed
1.9 kO.2
18
fed
36
fasted
18
fasted
6 9
1.2 to.2
2h
0.3 to.1
2h
2.2 1.5 to.2
2h
0.3 kO.2
2h
1.3 to.2
15 min
0.6 + 0.2
CO.01
15 min
0.5 to.2
15 min 20 min
2.1 f 0.4
20 min
2.1 to.5
fasted fasted fasted fasted fasted fasted
equicaloric with 6.0 mg/kg ETOH
30.0 mglkg, p.o. 5.0 mg/kg, p.o.
Time of Admin. prior to epin.
fed
i.p.
Epin. + asp. + dipyr.
p**
0.0 io.0
10
30.0 mg/kg,
Infarct grade*
fed
5
Epin. + asp.
in
14
16
5.0 mg/kg,
produced
Nutr. state
9
Epin. + dipyr.
infarction
No. of rats
19
Epin. + EtOH
of myocardial
5
fasted
twice/day, 4 days
1.7 to.4
10
fasted
twice/day, 4 days
1.6 to.3
5
fasted
twice/day, 4 days
1.7 to.3
Epin. 10.0 mg/kg, + D-prop.
S.C. 6
fasted 20 min
2.0 to.4
Epin. 10.0 mg/kg, + L-prop.
S.C. 8
20 min
1.4 +O.l
20 min
2.0 +0.5
20 min
3.4 to.2
p.o. p.o.
fasted
Epin. 8 + Morph 2.0 mg/kg, S.C.
fasted
Epin. + morph
fasted
*‘**Please
13 5.0 mg/kg, see opposite
S.C. page for Table footnotes.
<0.05
402
Fig. 4. Effect of prior administration of ethanol to rats treated with epinephrine on release of enzymes from isolated perfused hearts of these animals. Ethanol was force-fed in single doses of 0.5 or 6.0 g/kg to fed rats 2 h before they were injected S.C. with 3 mg/kg epinephrine. Other rats were treated with epinephrine alone or given neither ethanol nor epinephrine. Numbers in parentheses refer to numbers of hearts. Vertical lines represent standard errors of the means.
0
CONTROL
EPI
EPI
EPI
EPI
E+t:O)
E,;60)
Efk.5,
2 hours
EPI Et(t3.0,
EPI Et&01
I5 minutes
Fig. 5. Effect of prior administration of ethanol to rats treated with epinephrine on cardiac uptake of “‘?‘c-PYP in Go. Ethanol was force-fed in single doses of 0.5, 3.0, or 6.0 g/kg to rats previously fasted for 22 h, either 15 min or 2 h before they were injected with 3 mg/kg epinephrine S.C. Other rats were treated with epinephrine alone or given neither ethanol nor epinephrine. Numbers in parentheses refer to numbers of hearts. Vertical lines represent standard errors of the means. *Statistically significantly different from epinephrine-treated rats, p < 0.05. **Significantly different, p < 0.01.
0.5 to 6.0 g/kg and protection occurred in both fasted and fed rats. The effects of administering doses of 0.5 and 6.0 g ethanol/kg to fed rats, 2 h prior to epinephrine injection, on the severity of cardiac lesions produced,
403
are shown in Table 1 and Fig. 4. Both doses of ethanol produced significant reduction in degree of injury as judged either by visual inspection of the necrosis or cardiac ““Tc-PYP uptake. The larger doses exerted the greater protective effect. Table 1 and Fig. 5 show that similar results were obtained when ethanol was administered in various doses, either 15 min or 2 h before epinephrine injection, to fasted rats. Reduction in severity of injury was significant, whether the alcohol was given 15 min or 2 h before the epinephrine. Protection was greater as the dose of ethanol was increased. Effects
of other agents
A number of hypotheses have been considered in the attempt to explain the protective effect of ethanol. We have begun to test these hypotheses by administering to rats certain agents whose pharmacological activities are known, and determining whether these agents exert any protective action against epinephrine-induced myocardial necrosis. When the protective effect of ethanol was first observed, small doses of the latter were being administered and the possibility that the plasma free fatty acid (FFA)-lowering effect of small or moderate doses of ethanol might be the basis of its protective action was considered. FFA has been reported to cause injury to isolated cells and heart and to uncouple oxidative phosphorylation in mitochondria. The catecholamines act to mobilize FFA from adipose tissues, thereby raising plasma FFA levels, and this results in increased cardiac extraction of FFA. We therefore administered pyridylacetic acid, an analogue of nicotinic acid that inhibits FFA mobilization and reduces plasma FFA levels for a number of hours [16]. The compound was injected i-p. in a dose of 100 mg/kg 20 min before epinephrine was administered. It did not decrease the cardiac damage, however. Alcohol, therefore, presumably is not protective by virtue of its ability to decrease plasma FFA. In addition, as previously reported by this laboratory [17], large doses of ethanol, which we have now found to be most protective in this situation, cause a rise in FFA concentrations. Protection due to a caloric effect of ethanol was ruled out since isocaloric quantities of glucose exerted no protective action. Haft and co-workers have reported that cardiac damage produced in dogs by prolonged epinephrine infusion is associated with and presumably caused by epinephrine-provoked platelet aggregation with consequent blockade of the circulation through small blood vessels of the heart [X3]. They also found that two drugs whose pharmacological activities include the inhibition of platelet aggregation, aspirin and dipyridamole (Persantine), inhibited epinephrine-produced cardiac damage [ 191. It has been reported that ethanol inhibits catecholamine-induced platelet aggregation in vitro [20]. We administered aspirin and dipyridamole separately and together, orally, to rats in doses of 30 and 5 mg/kg, respectively, twice a day for 4 days before injecting the animals with epinephrine. There was a tendency for aspirin to reduce the epinephrine-induced cardiac damage slightly, but
(5) (31) T
CONTROL
EPI
(4)
(81
EPI a PiAA
EPI a GLU
T
T
E,P’
E,Pl
E,pl
PER
AiP
ASP P:R
Fig. 6. Effects of administering 100 mg/kg pyridylacetic acid, 30 mg/kg dipyridamole (Persantine), 5 mg/kg aspirin, dipyridamole plus aspirin in the same doses, or glucose in amounts isocalorically equivalent to 6 g/kg ethanol to 22 h-fasted rats, before injecting them S.C. with 3 mg/kg epinephrine, on cardiac uptake of gg’nl”c-PYP. None of these agents significantly reduced the cardiac Tc uptake in the epinephrine-treated rats. Numbers in parentheses indicate numbers of rats. Vertical lines represent standard errors of the means.
neither agent produced a significant decrease in cardiac damage. Recently we have been administering larger doses of these agents. Better protective results have been achieved, but are complicated by the toxicity of these agents, especially aspirin, at higher doses. We are presently varying doses and dosing schedules of both agents in an attempt to remedy this problem. Table 1 and Fig. 6 show the lack of significant protective action of the foregoing agents. We next considered the possibility that the protective action of ethanol may be due to its reported ability to decrease cardiac contractility [21] . We administered L-propranolol, a beta adrenergic blocking agent, or D-propranolol, which is not such a blocker but which acts to reduce cardiac contractility [22], in doses of 10 mg/kg S.C. to rats 20 min before they were injected with epinephrine. It has been reported by other investigators that beta adrenergic blocking agents do reduce or prevent catecholamine-induced cardiac necrosis [23] . We confirmed this in our experiments with L-propranolol. D-propranolol, however, did not reduce the epinephrine-provoked cardiac damage which suggests that decreased contractility is not the basis of ethanol’s protective action. We also thought that administration of large doses of epinephrine might cause cardiac pain in our rats and that one consequence of such pain might be a central nervous system-mediated discharge of norepinephrine from sympathetic nerve endings in the heart which, in turn, could be involved in the production of the myocardial injury. It has been reported, for example, that some of the deleterious effects of coronary artery occlusion are prevented by the ligation of the cardiac sympathetic nerves [24]. We therefore
405
d-PROP
I-PROP
M&c
12.0)
MbR
15.01
Fig. 7. Effects of prior administration of L-propranolol (10 mg/kg SC.), D-propranolol (10 mg/kg s.c.) or morphine (2 or 5 mg/kg s.c.) to rats injected with 3 mg/kg epinephrine reduced and 5.0 mg/kg s.c., on cardiac uptake of “9~-PYP. L-prop ranolol significantly morphine significantly increased the Tc uptake.
considered the possibility that the analgesic effect of ethanol might be involved in its protective action. Morphine was administered S.C. to rats in doses of 2 or 5 mg/kg, 15 min before the epinephrine. These doses produced discernable analgesia but no obvious respiratory depression. Morphine, however, increased the degree of damage produced by epinephrine. Perhaps the release by morphine of catecholamines [25] or of histamine [26] may be involved in this potentiating effect. This matter is under investigation. Table I and Fig. 7 show the results of our experiments with propranolol and morphine. The question also arises as to whether ethanol reduces the circulating levels of administered epinephrine as a result of its diuretic effect or by otherwise shifting epinephrine from the circulation into another fluid compartment of the body. We have begun to make determinations of plasma epinephrine levels in rats after the administration of epinephrine alone or of epinephrine after ethanol. Thus far we have found no significant differences in the plasma epinephrine levels of ethanol-pre-treated rats and rats not given ethanol, 2 and 3 h after epinephrine administration, when plasma levels of the amine appear to be highest. Most recently we have been thinking about the possibility that ethanol may be effective by acting temporarily to increase plasma osmolality and thereby preventing the interstitial and intracellular edema often seen in the
406
heart after the administration of large doses of catecholamines [27]. Initial experiments with mannitol have indeed shown that this agent exerts a protective effect also. Studies with mannitol and other agents that alter osmotic pressures in the circulation are being made.
Discussion We have observed that ethanol, when administered orally in a single dose a short time before epinephrine is injected into rats, inhibits the production of cardiac injury that results from the presence of high levels of catecholamines in vivo. We do not know, at present, whether this protective action of ethanol extends to other catecholamines, to sympathomimetic agents in general, or to other species of animals. Most important, we do not yet know whether ethanol exerts a protective action against myocardial infarction produced by blockade of the coronary circulation. It would be most interesting to learn the mechanism by which ethanol exerts its protective action. Ethanol has numerous pharmacological effects, and any one of these may be the basis of its protective action. We have tentatively ruled out some possibilities and high-lighted others. Thus, two mechanisms appear promising: (a) a platelet aggregation-inhibiting effect and (b) an osmotic effect. The effects of ethanol on epinephrine-induced calcium accumulation in cardiac cells and on cyclic AMP levels in the heart are also well worth investigating.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
G. Rona et al., Arch. Path., 67 (1959) 443. I. Rosenblum, A. Wohl and A. A. Stein, Toxicoi. Appl. Pharmacol., 7 (1965) J. E. Szakacs and A. Cannon, Amer. J. Clin. Pathol., 30 (1958) 425. T. Winsor and H. J. Berger, Amer. Heart J., 89 (1975) 814. G. D. Talbott, Ann. New York Acad. Sci., 252 (1975) 237. T. R. Regan et al., Ann. New York Acad. Sci., 252 (1975) 250. S. Mallov, Biochem. Pharmacol., 25 (1976) 1645. R. J. Henry et aZ., Amer. J. Clin. Pathol., 34 (1960) 381. S. B. Rosalki, J. Lab. Clin. Med., 69 (1967) 696. G. Ellis and D. M. Goldberg, Amer. J. Clin. Pathol., 56 (1971) 627. Z. D. Grossman et al., J. Nucl. Med., 18 (1977) 51. F. Wroblewski and J. S. LaDue, Proc. Sot. Exp. Biol. Med., 90 (1955) 210. A. Karman, F. Wroblewski and J. S. LaDue, J. Clin. Invest., 34 (1954) 126. L. M. Buja et al., Circulation, 52 (1975) 596. B. L. Zaret et al., Circulation, 53 (1976) 422. J. N. Pereira, J. Lipid Res., 8 (1967) 239. S. Mallov, Quart. J. Studies Ale., 22 (1961) 250. J. I. Haft et al., Circulation, 46 (1972) 698. J. I. Haft et al., Amer. J. Cardiol., 30 (1972) 838. M. J. Haut and D. H. Lonan, Amer. J. Med., 56 (1974) 22. T. J. Regan et al., J. Clin. Invest., 45 (1966) 270. A. M. Barrett and V. A. Cullum, Brit. J. Pharmacol., 34 (1968) 43.
1.
407 23 L. Dorigotti et al., J. Pharm. Pharmac., 21 (1969) 188. 24 S. F. Schaal, A. G. Wallace and W. C. Sealy, Cardiovascular Res., 3 (1969) 241. 25 J. A. Richardson, E. F. Woods and A. K. Richardson, J. Pharmacol. Exp. Ther., (1958) 64A. 26 I. Klein and G. S. Levey, J. Clin. Invest., 50 (1971) 1012. 27 V. J. Ferrans et al., Ann. New York Acad. Sci., 156 (1969) 309.
122
Discussion FREUND - Have you had an opportunity ne injection? Does blood pressure increase MALLOV -We have not actually it should increase.
to measure appreciably
measured
FREUND - Do you feel that myocardial an increase in blood pressure?
blood
infarctions
blood pressure following epinephriwith the dose that you used?
pressure
in these experiments,
but yes,
are caused by vasoconstriction
MALLOV - Myocardial infarctions are a result of decreased On the other hand, the causes of the catecholamine-induced studying are unknown.
and
blood flow and hypoxia. necrosis such as we are
FREUND - I was going to suggest that you give phenobarbital or another sedative to quiet down the animal and lessen the overload to the heart caused by physical activity after epinephrine injection. MALLOV
-We
have done this and it does not seem to make any difference.
D. LESTER - It seems to me that there should be implications for treatment from your data. What, for example, would happen if you gave 0.5 g/kg every two hours over the period of the 18 hours of the development of the infarction? What would happen if you do not start giving alcohol prior to the epinephrine injection but begin afterwards? MALLOV - We thought about these questions but have not examined them yet; however, alcohol may have ameliorating effects on epinephrine-induced damage. SEIXAS - I think it should be emphasized that you used only one dose of ethanol. The effects of chronic and prolonged alcohol consumption can be quite different from the effect of a single acute dose. GOLDSTEIN
- Does alcohol
change
the coronary
MALLOVThis is another experiment. coronary dilators and see what appens.
blood
flow?
We are going to try nitroglycerine
and other
ROSS - At high concentrations of ethanol in vitro, it has been reported that there is an increase or inhibition of calcium influx. Both ethanol and propranolol may be simply having a membrane stabilization effect. MALLOV
- I agree,
GOLDSTEIN - If, as you suggested, infarctions are caused by hypoxia, you can look at the effects of ethanol on such a preparation by ligating the coronary artery. What would you predict would happen? MALLOV
- I hope that ethanol
would
have the same type of protective
effect.
397
INHIBITION OF EPINEPHRINE-INDUCED MYOCARDIAL NECROSIS IN RATS BY ADMINISTRATION OF SINGLE DOSES OF ETHANOL
SAMUEL MALLOV and ROBERT F. GILMOUR Department of Pharmacology, State Symcuse, N.Y. 13210 (U.S.A.)
University
of New York, Upstate Medical Center,
Summary The oral administration of single doses (0.5 - 6.0 g/kg) of ethanol to rats, shortly before injecting them with a large dose (3.0 mg base/kg) of epinephrine subcutaneously, significantly reduced the severity of the myocardial damage produced by the epinephrine. The larger the dose of ethanol, the greater was the protective effect. The results were the same, regardless of the method used to determine the degree of cardiac injury. Experiments employing various agents to determine the mechanisms of ethanol action have tentatively suggested that a platelet de-aggregating or an osmotic effect of alcohol may be involved.
Introduction It has been known for many years that the presence of high levels of sympathomimetic amines, including catecholamines, in uivo in experimental animals [l, 21 and in man [3, 41 can cause’myocarditis and myocardial necrosis. We have been using the epinephrine-induced necrotic heart in rats as an experimental model for studies of the mechanism of this type of cardiac injury. The deleterious effects of acute and chronic ethanol ingestion on the heart are also well known [ 5,6]. It was therefore surprising to us when we observed, during investigation of the effects of certain drugs on the severity of myocardial necrosis produced by the administration of large doses of epinephrine, that ethanol exerted a protective effect. This was a serendipitous finding since the ethanol was merely added to the aqueous medium to increase the solubility of a drug (Anginin) that turned out to have no effect at all. We report here our observations regarding the protective effect of ethanol as well as the results of studies we have begun to elucidate the mechanism of this effect.
398
Methods Myocardial necrosis was produced in 300 - 350 g male Sprague-Dawley rats by injecting them once, subcutaneously (s.c.), with a large dose of epinephrine (3.0 mg base/kg) in saline solution. Control rats were injected with saline solution alone. Some of these rats were force-fed, by stomach tube, either 15 min or 2 h prior to the time of injection, 10% or 50% solutions of ethanol in water (v/v), while others were given equal volumes of water or an isocaloric solution of glucose in place of the ethanol. The doses of ethanol were 0.5, 3.0, or 6.0 g/kg. The rats were either in the fed or fasted state when force-fed the ethanol, water, or glucose. The severity of myocardial injury produced by the epinephrine was determined in three different ways: (a) by visual inspection and grading, (b) by determination of the quantities of certain enzymes released by the hearts in uitro, and (c) by determination of the cardiac uptake of technetium-99m stannous pyrophosphate (99mTc-PYP) in duo. Hearts were graded as to the severity of the necrosis produced, after visual inspection, as follows: grade 0, no visual necrosis; grade 1, necrosis limited to intraventricular septum (IVS) or apex; grade 2, necrosis in IVS or apex and also in base of left ventricle (BLV); grade 3, necrosis in IVS or apex, BLV and in base of right ventricle (BRV); grade 4, necrosis in IVS or apex, BLV, BRV and in free wall of left ventricle. Eighteen hours after the injection of epinephrine or saline, some of the rats were anesthetized by the intraperitoneal (i.p.) administration of pentobarbital. The hearts of these animals were removed, inspected and perfused via their aortas as Langendorff preparations, according to the technique previously described [ 71. The perfusion fluid, composed of oxygenated KrebRinger bicarbonate buffer at pH 7.4 containing 10 n-&f glucose, was not recirculated. The flow rate through each heart was maintained at 10.0 ml/ min. In the first experiments the perfusion fluid leaving each heart was collected in 5-min fractions for a period of 45 min, following an initial lomin period of blood wash-out and equilibration, and each fraction was then analyzed for enzyme activities. Lactate dehydrogenase (LDH) and glutamicoxaloacetic transaminase (GOT) activities were determined by the methods of Henry [8], creatine phosphokinase (CPK) by the method of Rosalki [9], and a-hydroxybutyrate dehydrogenase (HBDH) by the method of Ellis and Goldberg [lo] . The activities of the enzyme were expressed as milli-international units (mIU) per ml of perfusate. Other rats were injected intravenously (i.v.) with 25 &i of ggmTc-PYP 18 h after receiving epinephrine. The hearts of these animals were removed exactly 100 min later and their total radioactivities determined with the aid of a Beckman Biogamma II counter. ‘*‘Tc-PYP uptake was expressed as the ratio (% of total dose absorbed by the heart)/(% of body weight represented by the heart) in order to correct for variations in dose, heart and body weights [ 111. The measurement of the elevation of certain enzymes in serum to indicate the occurrence and to assess the severity of myocardial infarction is a
399
Fig. 1. Release of lactate dehydrogenase from isolated perfused hearts of saline- and epinephrine-treated rats into perfusion fluid. Epinephrine was injected S.C. in a dose of 3 mg/kg 18 h before the hearts were removed. Open circles, control rat hearts; closed circles, epinephrine-injected rat hearts.
s
301
@ GOT
E
2
1
‘; >
20
0
CPK
n
HBDH
LDH
t
INFARCT
GRADE
Fig. 2. Release of enzymes from isolated perfused hearts of control and epinephrine-treated rats into the perfusion fluid, in relation to the severity of cardiac injury as graded after visual inspection. Different doses of epinephrine were injected S.C. to produce different degrees of cardiac injury. Perfusates were collected for periods of 45 min. Numbers in parentheses refer to numbers of hearts. Vertical lines represent standard errors of the means.
well-known clinical as well as experimental technique [8, 12,131. Determination of these enzymes released from isolated perfused hearts has the added advantage of eliminating enzymes released from organs other than the heart. Uptake of s9mTc-PYP or related radioactive nuclides by the heart, in duo, has also been used both experimentally and clinically to assess the severity of myocardial infarction [ 14, 151. This sensitive method depends on the entrance into and binding of ssmTc-PYP by reversibly or irreversibly injured cells that surround areas of necrotic tissue.
INFARCT
GRADE
rats in Fig. 3. Cardiac uptake of 9s’nTc-PYP in v iuo of control and epinephrineinjected relation to the severity of cardiac injury as graded after visual inspection. Different doses of epinephrine were injected S.C. to produce different degrees of cardiac injury.
Results It was observed that isolated hearts from epinephrine-treated rats released significantly greater quantities of all four enzymes measured throughout the period of perfusion than did hearts from control rats. Figure 1 shows, for example, the results of experiments in which the activities of LDH released into the perfusion fluid from the hearts of control and epinephrine-treated rats were determined. When different doses of epinephrine were administered to rats to produce myocardial necrosis of different degrees of severity, it was found that there was a good correlation between the degree of severity of the lesion as graded after visual inspection and the release of each of the enzymes from the isolated perfused hearts into the perfusion fluid. This is shown in Fig. 2. Similarly, it was found that there was a good correlation between the visually determined severity of necrosis and the cardiac uptake of ss”Tc-PYP in uiuo, as shown in Fig. 3. Thus each of these methods was suitable for determination of the effects of drugs on the severity of epinephrine-induced cardiac damage. Effect
of ethanol
It was found that the oral administration of ethanol 15 min or 2 h before epinephrine injection inhibited the production of myocardial injury. The degree of protection increased as the dose of ethanol was raised from *Mean + s.e.m. **p values are given in those cases in which the myocardial necrotic grade is statistically significantly different from the necrotic grade in rats receiving only epinephrine. In all cases the dose of epinephrine administered was 3.0 mg base/kg. EtOH = ethanol, pyaa. = pyridylacetic acid, glut. = glucose, dipyr. = dipyridamole (Persantine), asp. = aspirin, Lprop. = L-propranolol, D-prop. = D-propranolol, and morph. = morphine.
401 TABLE
1
Effect of administering various agents on severity rats by epinephrine injection Agent
Dose
None (controls) Epin.
3 mg/kg,
Epin. + EtOH
0.5 g/kg, p.o.
Epin. + EtOH
6.0 g/kg, p.o.
Epin.
3 mg/kg,
S.C.
S.C.
3.0 g/kg, p.o.
Epin. + EtOH
6.0 g/kg, p.o.
Epin. + EtOH
0.5 g/kg, p.o.
Epin. + EtOH
3.0 g/kg, p.o.
Epin. + EtOH
6.0 g/kg, p.o.
Epin. + pyaa
100 mg/kg,
Epin. + glut.
33
fed
1.9 kO.2
18
fed
36
fasted
18
fasted
6 9
1.2 to.2
2h
0.3 to.1
2h
2.2 1.5 to.2
2h
0.3 kO.2
2h
1.3 to.2
15 min
0.6 + 0.2
CO.01
15 min
0.5 to.2
15 min 20 min
2.1 f 0.4
20 min
2.1 to.5
fasted fasted fasted fasted fasted fasted
equicaloric with 6.0 mg/kg ETOH
30.0 mglkg, p.o. 5.0 mg/kg, p.o.
Time of Admin. prior to epin.
fed
i.p.
Epin. + asp. + dipyr.
p**
0.0 io.0
10
30.0 mg/kg,
Infarct grade*
fed
5
Epin. + asp.
in
14
16
5.0 mg/kg,
produced
Nutr. state
9
Epin. + dipyr.
infarction
No. of rats
19
Epin. + EtOH
of myocardial
5
fasted
twice/day, 4 days
1.7 to.4
10
fasted
twice/day, 4 days
1.6 to.3
5
fasted
twice/day, 4 days
1.7 to.3
Epin. 10.0 mg/kg, + D-prop.
S.C. 6
fasted 20 min
2.0 to.4
Epin. 10.0 mg/kg, + L-prop.
S.C. 8
20 min
1.4 +O.l
20 min
2.0 +0.5
20 min
3.4 to.2
p.o. p.o.
fasted
Epin. 8 + Morph 2.0 mg/kg, S.C.
fasted
Epin. + morph
fasted
*‘**Please
13 5.0 mg/kg, see opposite
S.C. page for Table footnotes.
<0.05
402
Fig. 4. Effect of prior administration of ethanol to rats treated with epinephrine on release of enzymes from isolated perfused hearts of these animals. Ethanol was force-fed in single doses of 0.5 or 6.0 g/kg to fed rats 2 h before they were injected S.C. with 3 mg/kg epinephrine. Other rats were treated with epinephrine alone or given neither ethanol nor epinephrine. Numbers in parentheses refer to numbers of hearts. Vertical lines represent standard errors of the means.
0
CONTROL
EPI
EPI
EPI
EPI
E+t:O)
E,;60)
Efk.5,
2 hours
EPI Et(t3.0,
EPI Et&01
I5 minutes
Fig. 5. Effect of prior administration of ethanol to rats treated with epinephrine on cardiac uptake of “‘?‘c-PYP in Go. Ethanol was force-fed in single doses of 0.5, 3.0, or 6.0 g/kg to rats previously fasted for 22 h, either 15 min or 2 h before they were injected with 3 mg/kg epinephrine S.C. Other rats were treated with epinephrine alone or given neither ethanol nor epinephrine. Numbers in parentheses refer to numbers of hearts. Vertical lines represent standard errors of the means. *Statistically significantly different from epinephrine-treated rats, p < 0.05. **Significantly different, p < 0.01.
0.5 to 6.0 g/kg and protection occurred in both fasted and fed rats. The effects of administering doses of 0.5 and 6.0 g ethanol/kg to fed rats, 2 h prior to epinephrine injection, on the severity of cardiac lesions produced,
403
are shown in Table 1 and Fig. 4. Both doses of ethanol produced significant reduction in degree of injury as judged either by visual inspection of the necrosis or cardiac ““Tc-PYP uptake. The larger doses exerted the greater protective effect. Table 1 and Fig. 5 show that similar results were obtained when ethanol was administered in various doses, either 15 min or 2 h before epinephrine injection, to fasted rats. Reduction in severity of injury was significant, whether the alcohol was given 15 min or 2 h before the epinephrine. Protection was greater as the dose of ethanol was increased. Effects
of other agents
A number of hypotheses have been considered in the attempt to explain the protective effect of ethanol. We have begun to test these hypotheses by administering to rats certain agents whose pharmacological activities are known, and determining whether these agents exert any protective action against epinephrine-induced myocardial necrosis. When the protective effect of ethanol was first observed, small doses of the latter were being administered and the possibility that the plasma free fatty acid (FFA)-lowering effect of small or moderate doses of ethanol might be the basis of its protective action was considered. FFA has been reported to cause injury to isolated cells and heart and to uncouple oxidative phosphorylation in mitochondria. The catecholamines act to mobilize FFA from adipose tissues, thereby raising plasma FFA levels, and this results in increased cardiac extraction of FFA. We therefore administered pyridylacetic acid, an analogue of nicotinic acid that inhibits FFA mobilization and reduces plasma FFA levels for a number of hours [16]. The compound was injected i-p. in a dose of 100 mg/kg 20 min before epinephrine was administered. It did not decrease the cardiac damage, however. Alcohol, therefore, presumably is not protective by virtue of its ability to decrease plasma FFA. In addition, as previously reported by this laboratory [17], large doses of ethanol, which we have now found to be most protective in this situation, cause a rise in FFA concentrations. Protection due to a caloric effect of ethanol was ruled out since isocaloric quantities of glucose exerted no protective action. Haft and co-workers have reported that cardiac damage produced in dogs by prolonged epinephrine infusion is associated with and presumably caused by epinephrine-provoked platelet aggregation with consequent blockade of the circulation through small blood vessels of the heart [X3]. They also found that two drugs whose pharmacological activities include the inhibition of platelet aggregation, aspirin and dipyridamole (Persantine), inhibited epinephrine-produced cardiac damage [ 191. It has been reported that ethanol inhibits catecholamine-induced platelet aggregation in vitro [20]. We administered aspirin and dipyridamole separately and together, orally, to rats in doses of 30 and 5 mg/kg, respectively, twice a day for 4 days before injecting the animals with epinephrine. There was a tendency for aspirin to reduce the epinephrine-induced cardiac damage slightly, but
(5) (31) T
CONTROL
EPI
(4)
(81
EPI a PiAA
EPI a GLU
T
T
E,P’
E,Pl
E,pl
PER
AiP
ASP P:R
Fig. 6. Effects of administering 100 mg/kg pyridylacetic acid, 30 mg/kg dipyridamole (Persantine), 5 mg/kg aspirin, dipyridamole plus aspirin in the same doses, or glucose in amounts isocalorically equivalent to 6 g/kg ethanol to 22 h-fasted rats, before injecting them S.C. with 3 mg/kg epinephrine, on cardiac uptake of gg’nl”c-PYP. None of these agents significantly reduced the cardiac Tc uptake in the epinephrine-treated rats. Numbers in parentheses indicate numbers of rats. Vertical lines represent standard errors of the means.
neither agent produced a significant decrease in cardiac damage. Recently we have been administering larger doses of these agents. Better protective results have been achieved, but are complicated by the toxicity of these agents, especially aspirin, at higher doses. We are presently varying doses and dosing schedules of both agents in an attempt to remedy this problem. Table 1 and Fig. 6 show the lack of significant protective action of the foregoing agents. We next considered the possibility that the protective action of ethanol may be due to its reported ability to decrease cardiac contractility [21] . We administered L-propranolol, a beta adrenergic blocking agent, or D-propranolol, which is not such a blocker but which acts to reduce cardiac contractility [22], in doses of 10 mg/kg S.C. to rats 20 min before they were injected with epinephrine. It has been reported by other investigators that beta adrenergic blocking agents do reduce or prevent catecholamine-induced cardiac necrosis [23] . We confirmed this in our experiments with L-propranolol. D-propranolol, however, did not reduce the epinephrine-provoked cardiac damage which suggests that decreased contractility is not the basis of ethanol’s protective action. We also thought that administration of large doses of epinephrine might cause cardiac pain in our rats and that one consequence of such pain might be a central nervous system-mediated discharge of norepinephrine from sympathetic nerve endings in the heart which, in turn, could be involved in the production of the myocardial injury. It has been reported, for example, that some of the deleterious effects of coronary artery occlusion are prevented by the ligation of the cardiac sympathetic nerves [24]. We therefore
405
d-PROP
I-PROP
M&c
12.0)
MbR
15.01
Fig. 7. Effects of prior administration of L-propranolol (10 mg/kg SC.), D-propranolol (10 mg/kg s.c.) or morphine (2 or 5 mg/kg s.c.) to rats injected with 3 mg/kg epinephrine reduced and 5.0 mg/kg s.c., on cardiac uptake of “9~-PYP. L-prop ranolol significantly morphine significantly increased the Tc uptake.
considered the possibility that the analgesic effect of ethanol might be involved in its protective action. Morphine was administered S.C. to rats in doses of 2 or 5 mg/kg, 15 min before the epinephrine. These doses produced discernable analgesia but no obvious respiratory depression. Morphine, however, increased the degree of damage produced by epinephrine. Perhaps the release by morphine of catecholamines [25] or of histamine [26] may be involved in this potentiating effect. This matter is under investigation. Table I and Fig. 7 show the results of our experiments with propranolol and morphine. The question also arises as to whether ethanol reduces the circulating levels of administered epinephrine as a result of its diuretic effect or by otherwise shifting epinephrine from the circulation into another fluid compartment of the body. We have begun to make determinations of plasma epinephrine levels in rats after the administration of epinephrine alone or of epinephrine after ethanol. Thus far we have found no significant differences in the plasma epinephrine levels of ethanol-pre-treated rats and rats not given ethanol, 2 and 3 h after epinephrine administration, when plasma levels of the amine appear to be highest. Most recently we have been thinking about the possibility that ethanol may be effective by acting temporarily to increase plasma osmolality and thereby preventing the interstitial and intracellular edema often seen in the
406
heart after the administration of large doses of catecholamines [27]. Initial experiments with mannitol have indeed shown that this agent exerts a protective effect also. Studies with mannitol and other agents that alter osmotic pressures in the circulation are being made.
Discussion We have observed that ethanol, when administered orally in a single dose a short time before epinephrine is injected into rats, inhibits the production of cardiac injury that results from the presence of high levels of catecholamines in vivo. We do not know, at present, whether this protective action of ethanol extends to other catecholamines, to sympathomimetic agents in general, or to other species of animals. Most important, we do not yet know whether ethanol exerts a protective action against myocardial infarction produced by blockade of the coronary circulation. It would be most interesting to learn the mechanism by which ethanol exerts its protective action. Ethanol has numerous pharmacological effects, and any one of these may be the basis of its protective action. We have tentatively ruled out some possibilities and high-lighted others. Thus, two mechanisms appear promising: (a) a platelet aggregation-inhibiting effect and (b) an osmotic effect. The effects of ethanol on epinephrine-induced calcium accumulation in cardiac cells and on cyclic AMP levels in the heart are also well worth investigating.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
G. Rona et al., Arch. Path., 67 (1959) 443. I. Rosenblum, A. Wohl and A. A. Stein, Toxicoi. Appl. Pharmacol., 7 (1965) J. E. Szakacs and A. Cannon, Amer. J. Clin. Pathol., 30 (1958) 425. T. Winsor and H. J. Berger, Amer. Heart J., 89 (1975) 814. G. D. Talbott, Ann. New York Acad. Sci., 252 (1975) 237. T. R. Regan et al., Ann. New York Acad. Sci., 252 (1975) 250. S. Mallov, Biochem. Pharmacol., 25 (1976) 1645. R. J. Henry et aZ., Amer. J. Clin. Pathol., 34 (1960) 381. S. B. Rosalki, J. Lab. Clin. Med., 69 (1967) 696. G. Ellis and D. M. Goldberg, Amer. J. Clin. Pathol., 56 (1971) 627. Z. D. Grossman et al., J. Nucl. Med., 18 (1977) 51. F. Wroblewski and J. S. LaDue, Proc. Sot. Exp. Biol. Med., 90 (1955) 210. A. Karman, F. Wroblewski and J. S. LaDue, J. Clin. Invest., 34 (1954) 126. L. M. Buja et al., Circulation, 52 (1975) 596. B. L. Zaret et al., Circulation, 53 (1976) 422. J. N. Pereira, J. Lipid Res., 8 (1967) 239. S. Mallov, Quart. J. Studies Ale., 22 (1961) 250. J. I. Haft et al., Circulation, 46 (1972) 698. J. I. Haft et al., Amer. J. Cardiol., 30 (1972) 838. M. J. Haut and D. H. Lonan, Amer. J. Med., 56 (1974) 22. T. J. Regan et al., J. Clin. Invest., 45 (1966) 270. A. M. Barrett and V. A. Cullum, Brit. J. Pharmacol., 34 (1968) 43.
1.
407 23 L. Dorigotti et al., J. Pharm. Pharmac., 21 (1969) 188. 24 S. F. Schaal, A. G. Wallace and W. C. Sealy, Cardiovascular Res., 3 (1969) 241. 25 J. A. Richardson, E. F. Woods and A. K. Richardson, J. Pharmacol. Exp. Ther., (1958) 64A. 26 I. Klein and G. S. Levey, J. Clin. Invest., 50 (1971) 1012. 27 V. J. Ferrans et al., Ann. New York Acad. Sci., 156 (1969) 309.
122
Discussion FREUND - Have you had an opportunity ne injection? Does blood pressure increase MALLOV -We have not actually it should increase.
to measure appreciably
measured
FREUND - Do you feel that myocardial an increase in blood pressure?
blood
infarctions
blood pressure following epinephriwith the dose that you used?
pressure
in these experiments,
but yes,
are caused by vasoconstriction
MALLOV - Myocardial infarctions are a result of decreased On the other hand, the causes of the catecholamine-induced studying are unknown.
and
blood flow and hypoxia. necrosis such as we are
FREUND - I was going to suggest that you give phenobarbital or another sedative to quiet down the animal and lessen the overload to the heart caused by physical activity after epinephrine injection. MALLOV
-We
have done this and it does not seem to make any difference.
D. LESTER - It seems to me that there should be implications for treatment from your data. What, for example, would happen if you gave 0.5 g/kg every two hours over the period of the 18 hours of the development of the infarction? What would happen if you do not start giving alcohol prior to the epinephrine injection but begin afterwards? MALLOV - We thought about these questions but have not examined them yet; however, alcohol may have ameliorating effects on epinephrine-induced damage. SEIXAS - I think it should be emphasized that you used only one dose of ethanol. The effects of chronic and prolonged alcohol consumption can be quite different from the effect of a single acute dose. GOLDSTEIN
- Does alcohol
change
the coronary
MALLOVThis is another experiment. coronary dilators and see what appens.
blood
flow?
We are going to try nitroglycerine
and other
ROSS - At high concentrations of ethanol in vitro, it has been reported that there is an increase or inhibition of calcium influx. Both ethanol and propranolol may be simply having a membrane stabilization effect. MALLOV
- I agree,
GOLDSTEIN - If, as you suggested, infarctions are caused by hypoxia, you can look at the effects of ethanol on such a preparation by ligating the coronary artery. What would you predict would happen? MALLOV
- I hope that ethanol
would
have the same type of protective
effect.