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Cardiovascular effects of intravenous acetaldehyde and propionaldehyde in the anesthetized rat
TOXICOLOGY
AND
APPLIED
PHARMACOLQGY
Cardiovascular Effects Propionaldehyde
2d,636-644
(1973)
of Intravenous Acetaldehyde in the Anesthetized Rat1
and
JOHN L. EGLE, JR., PATRICIA M. HUDGINS AND FONG M. LAI Department of Pharmacology, Medical College of Virginia, Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia 23219 Received June 9,1972
Cardiovascular Effects of Intravenous Acetaldehyde and Propionaldehyde in the Anesthetized Rat. EGLE, J. L., JR., HUDGINS, P. M. and LAI, F. M. (1973). Toxicol. Appl. Pharmacol. 24, 636-644. Acetaldehyde and propionaldehyde have both been previously shown to possesssympathomimeticactivityand to affect thecardiovascular system. The objectives of the present investigation were to study the dose-effect relationships of the two aldehydes on the cardiovascular system of the anesthetized rat and to establish the mechanism of the pressor and depressor effects of the compounds. Low iv doses of acetaldehyde and propionaldehyde produced a consistent dose-related rise in blood pressure.These responseswere slightly reduced by adrenalectomy and more strongly antagonized by pretreatment with reserpine or phentolamine. This indicates that the pressor effects of these compounds are primarily due to vasoconstriction mediated by norepinephrine released from sympathetic nerve endings in vascular smooth muscle. Higher doses of the two aldehydes produced a sharp fall in blood pressure and a severe bradycardia. Atropine reduced the hypotensive and cardioinhibitory effects of the aldehydes, suggesting that these actions are mediated by the vagus nerve. This was confirmed by experiments in vagotomized rats. After vagotomy, high doses of acetaldehyde and propionaldehyde produced an increase in blood pressure and a positive chronotropic response. The results of this study indicate that acetaldehyde and propionaldehyde exert two opposing actions on the cardiovasctilar system: (1) a sympathomimetic effect which predominates at doses below 20 mg/kg resulting in a rise in blood pressure; and (2) stimulation of higher centers resulting in bradycardia and hypotension with higher doses. Aliphatic aldehydes are a group of compounds with considerable pharmacologic activity. Attention has primarily focused on acetaldehyde since it was shown by Handovsky (1934) to produce a pressor effect when administered intravenously to anesthetized dogs. Other investigators have observed the pressor effect of acetaldehyde and have suggested that the compound releases catecholamines from the adrenal medulla and other tissues (Roman0 et uZ., 1954; Eade, 1959; Akabane et d., 1964; James and Bear, 1968; Schneider, 1971). Interest in the biologic effects of acetaldehyde has been largely due to the fact that it is formed when ethanol is metabolized, and also that it is present in the vapor phase of cigarette smoke (Newsome e2 al., 1965) and is a 1 This study was aided by a grant from the American Medical Association Education and Research Fonndation. A preliminary report was presented at the Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, Bnrlington, Vermont, August 26, 1971. Copyright 0 1973 by Academic Press, Inc. 636 All rights of reproduction in any form reserved.
CARDIOVASCULAR
EFFECTS
OF ALDEHYDES
631
factor in smoke-induced ciliastasis (Kensler and Battista, 1963). James et af. (1970) have suggested that acetaldehyde may play a role in the development of cardiovascular disease in both smokers and alcoholics. Propionaldehyde is also present in the gas phase of tobacco smoke (Newsome er al.. 1965) and there is evidence that it also affects the cardiovascular system. Reports dealing with propionaldehyde have been descriptive in nature (Skog, 1952; Romano et aI., 1954; Wingard et al., 1955). Evidence has been presented that indicated that propionaldehyde has sympathomimetic properties similar to those of acetaldehyde (Eade, 1959; James and Bear, 1968). The present study was undertaken to investigate the dose-effect relationships of acetaldehyde and propionaldehyde on the cardiovascular system of anesthetized rats. The doses used in this investigation are unquestionably well above the level of aldehyde which might be presented to the cardiovascular system as a result of cigarette or ethanol consumption. An attempt has been made to establish and compare the mechanism of pressor and depressor effects of both aldehydes. Attention was directed toward elucidating the mechanism of the depressor effects observed after intravenous doses of aldehydes ranging from 20 to 40 mg/kg. Evidence is presented which implicates a central effect of these compounds which is cardioinhibitory and mediated by the vagus nerves. METHODS
All experiments were performed with male Wistar rats (250-350 g) which were anesthetized with pentobarbital sodium (50 mg/kg ip). The right femoral artery was cannulated and mean blood pressure was recorded by means of a Statham blood pressure transducer. Heart rate was measured with a cardiotachometer by means of fine needle electrodes inserted through the skin of the rat. The left femoral vein was cannulated to permit intravenous administration of drugs. The volume of injections was within the range of 0.1 to 0.2 ml, followed by flush with 0.2 ml of saline. Animals received control saline injections which were found to have no effect on resting blood pressure and heart rate. Doses of acetaldehyde and propionaldehyde ranging from 1 to 5 mg/kg were administered at lo-min intervals; doses from 10 to 40 mg/kg were administered at 20-min intervals. Subsequent injections were not made until all parameters had returned to preinjection values. In a portion of the experiments, bilateral adrenalectomy or vagotomy in the neck region was carried out 30 min prior to the first injection of aldehyde. A Grass Model 7 polygraph was used to record responses. The following agents were used in an attempt to antagonize the actions of acetaldehyde and propionaldehyde: phentolamine (10 mg/kg), propranolol (1 mg/kg) and atropine (0.5 mg/kg). Blockade was assessed by injections of the appropriate agonist. These were norepinephrine (0.3 pg/kg), isoproterenol (0.01 pg/kg) and acetylcholine (0.5 pg/kg), respectively. One antagonist was administered to one animal. The time between antagonist administration and drug challenge was 5-10 mitt, except in the case of propranolol which was 20 min. Selective blockade was achieved at these doses and time periods. Experimental results have been expressed as a percentage of the initial resting blood pressure or heart rate in each case. This was considered necessary in order to make relative comparisons between the effects of acetaldehyde and propionaldehyde.
638
EGLE,
HUDGINS
AND
LA1
Significant variations were observed in initial blood pressure and heart rate after drug pretreatment or surgical procedures (Table 1). Statistical comparisons were made with Dunnett’s test (Steel and Torrie, 1960). TABLE INITIAL
RESTING
BLOOD
PRESSURE
Drug or procedure Control Adrenalectomy Adrenalectomy + reserpine Phentolamine Propranolol Atropine Vagotomy
AND RAT
1 HEART
RATE
IN THE ANESTHETIZED
Mean blood pressure (mm Hg * SE)
Heart rate (mine1 & SE)
101.8 * 3.2 (9)” 116.0 5 3.4 (8)
405 * 14.1 (9)
73.4 74.9 82.8 96.0
zt 2.1 It 3.4 dz 3.2 z!c3.5
(7) (10) (7) (9)
135.1 Zt 5.2 (7)
396 * 12.8 (8) 236 394 327 401
+ 14.1 =k 16.7 zt 12.4 It 13.4
(7) (10) (7) (9)
416 & 12.1 (7)
’ Numbers in parentheses refer to the number of animals used in each experiment.
RESULTS Blood Pressure Responses to Acetaldehyde and Propionaldehyde
Tables 2 and 3 summarize the results of experiments in which anesthetized rats received acetaldehyde or propionaldehyde in doses ranging from 5 to 40 mg/kg iv. Although minor differences exist between blood pressure responses to acetaldehyde and propionaldehyde, the similarities are more striking. After 5 mg/kg of the aldehydes, pressor responses predominated. Exceptions are noted after phentolamine and propranolol and subsequent acetaldehyde administration. Similarly, after IO mg/kg of the aldehydes, pressor responses predominated. Phentolamine administered before acetaldehyde resulted in an equal number of pressor and depressor responses. After 20 mg/kg of acetaldehyde, depressor responses predominated, except following atropine administration or bilateral vagotomy. Propionaldehyde (20 mg/kg] responses were more variable and appeared to be lessdependent upon endogenous catecholamines than acetaldehyde for its action. Pressor as well as depressor responses were observed in a significant number of rats after adrenalectomy, and reserpine pretreatment followed by adrenalectomy and propranolol. These procedures completely abolished the pressor activity of acetaldehyde. Profound depressor responses were observed after acetaldehyde (40 mg/kg) in all instances except after administration of atropine or bilateral vagotomy. Atropine appeared to partially antagonize the depressor response to acetaldehyde. A dose of 40 mg/kg of propionaldehyde administered to anesthetized rats resulted in profound depressor responses in 81 ‘A of control animals. Atropine administration partially antagonized the depressor effects of propionaldehyde, but bilateral vagotomy was more effective. After vagotomy, a marked rise in blood pressure was the predominant response. Propionaldehyde produced a slight depressor response in 27% of animals after this latter procedure and was more potent than acetaldehyde in producing this effect.
CARDIOVASCULAR
EFFECTS
OF ALDEHYDES
OF VARIOUS
3
8.4 & 4.8 (818) 6.8 It 1.0 (21/Z]) 12.2 k 2.4 (13/13)
PropranoIol
A tropine
Vagotomy
PRESSURE
20
TO INTRAVENOUS
21.6 xt 3.9 (14/14) -
8.3 + 1.8 (U/15) -
10.9 h 4.6 (919) -
6.3 zk 1.1 (13/26) 6.4 z!z1.7 (13/26)
9.8 ic 1.7 (5/16) 14.1 zt 2.4 (11/16)
18.2 k 3.5 (11/15) 24.5 dz 5.9 (4/15)
12.4 * 1.9 (18/21) 12.3 zt 8.3 (3/21)
22.1 Lt 4.9 (10/14) 6.7 * 1.5 (4/14)
17.2 zt 2.0 (16/21) 9.8 zt 2.6 (5/21)
14.3 Liz1.7 (319) 18.5 + 7.7 (6/9)
16.6 * 4; (17/17)
10.0 & 1.0 (5/16) 10.4 zt 1.5 (1 l/16)
12.2 +z 3.4 (12/15) 35.0 zt 15.5 (3/15)
23.6 xk 3.1 (9/20) 40.0 zt 8.1 (ll/ZO)
32.9 k 5.6 (8/l 1) 13.0 zt 4.9 (3/l I)
26.0 I!= 1.1 (3/11) 19.4 zt 8.3 (8/11)
55.7 zt 8.1 (7/7)
-
iG
z E 5
-
z z
8 5
60.4 & 4.1 (14/14) 40.8 zt 5.6 (14/14)
58.0 zt 3.7 (15/15)
23.0 + 8.9 (3/16) 63.9 zt 7.2 (13/16)
40
PROPIONALDEHYDE
Percent of change from resting blood pressure (mean & SE)
IO
(mg/kg)
RESPONSES
PropionaIdehyde
RAY
u Responses ate cakulated as percent of change from Iesting blood pressure. These values were taken from each experiment. Mean + SE of the resting blood pressures appear in Table 1. ’ Number in parentheses refers to the frequency of each response as a function of the number of observations.
4.3 zk 1.7 (26/26) -
Phentolamine
9.8 & 1.4 (12/12) -
10.5 zt 1.1 (17/17)b -
5
PROCEDURES UPON THE BLOOD IN THE ANESTHETIZED
Adrenalectomy
i
SURGICAL
1 I.6 A 2.2 (16/16) -
+ reserpine
AND
Direction of change
DRUGS
Adrenalectomy
Control
Drug or procedure
INFLUENCE
TABLE
CARDIOVASCULAREFFECTSOFALDEHYDES
Heart
641
Rate Responses to Acetaldehyde and Propkmaldehyde
Table 4 summarizes the pronounced effects of the aldehydes (40 mg/kg iv) on resting heart rate. Doses of aldehyde between 5 and 20 mg/kg did not alter heart rate significantly and changes which did occur were clearly secondary to blood pressure changes in most cases. However, after 40 mg/kg the blood pressure responses appeared to be secondary to heart rate changes. It is evident that adrenalectomy with or without reserpine pretreatment significantly enhanced the cardioinhibitory effects of acetaldehyde and propionaldehyde. TABLE 4 HEART
RATE
CHANGE IN RESPONSE TO 40 MC/KG PROPIONALDEHYDEINTHEANESTHETIZEDRAT
ACETALDEHYDE
AND
A B Acetaldehyde Propionaldehyde Percent of control heart rate Drug or procedure
:/; * SE ~-.-
Control 4 64.8 5 6.8 (15)” Adrenalectomy J 87.8 It 4.1 (9)* Adrenalectomy + reserpine 4 99.2 * 0.8 (10)’ Phentolamine (10 mg/kg) J 75.0 zk 2.1 (7) Propranolol(1 mg/kg) 4 86.2 k 5.8 (9T Atrophine (0.5 mg/kg) 4 17.6 + 6.6 (lo)< ; 11.3 * 5.4 (l2)C Vagotomy
‘!< -7 SE ;
71.2 pi 6.1 (16) 86.2 + 7.4 (16)
; 100.0 t 0.1 (13jC ; 45.7 Jo7.6 (14)’ , 60.1 I 7.6(7) ~ 21.6 -L 9.0 (I l)C : 6.6 +2.1 (IIF
n Numbers in parentheses refer to the number of animals used in each experiment. b Significantly different from control (p c 0.05). C Significantly different from control(p c 0.01).
Adrenergic antagonists, phentolamine and propranolol had little effect on the cardioinhibitory effect of acetaldehyde, but appeared to attenuate the effect of propionaldehyde. Atropine administration significantly altered the myocardial depressant effects whereas bilateral vagotomy completely reversed the cardioinhibitory effects of the aldehydes. DISCUSSION The pressor responses observed after intravenous administration of relatively low doses of acetaldehyde and propionaldehyde are consistent with earlier indication5 that the compounds possess sympathomimetic activity (Handovsky- 1934; Roman<) cr a/., 1954). In the present study a relationship between the dose of aldehyde and response was apparent up to a dose of 20 mg/kg. A dose of 5 mg/kg of either compound always produced a pressor response. At doses of IO mg/kg there was a fail in blood pressure in 14’;); of cases after propionaldehyde administration. An equal number (~1’ pressor and depressor effects occurred after 20 mg/kg of both aldehydes. However. when the dose was elevated to 40 mg/kg, a pronounced depressor effect was evident and ~1rise in pressure was seen in only a few cases after propionaldehyde. Alterations in the frequency and magnitude of these control responses by the pharmacologic and surgical
642
EGLE,
HUDGINS
AND
LA1
procedures employed provide considerable information regarding the mechanism involved. Adrenalectomy failed to alter the pressor response to the lower dose of either aldehyde. There was an increase in the frequency of depressor responses after 10 and 20 mg/kg, but pressor effects predominated except after acetaldehyde (20 mg/kg). These results tend to indicate that the adrenals are considerably less important as a source of catecholamines in the production of pressor responses to acetaldehyde and propionaldehyde than sympathetic nerve endings. When reserpine pretreatment preceded adrenalectomy, depressor responses were observed more frequently with both aldehydes at doses of 10 and 20 mg/kg. Depletion of catecholamines by reserpine significantly impaired the. ability of the aldehydes to produce a pressor effect. The c+adrenergic blocking agent phentolamine also strongly inhibited the pressor effects of the two aldehydes. These results indicate that the pressor effects of acetaldehyde and propionaldehyde are primarily due to vasoconstriction and mediated by norepinephrine released from sympathetic nerve endings in vascular smooth muscle. The severe bradycardia produced by 40 mg/kg of the aldehyde was enhanced by adrenalectomy and adrenalectomy after reserpine pretreatment (Table 4). This is in contrast to the findings of Moller (1971) obtained with isolated guinea pig hearts. This discrepancy suggests that the bradycardia is due to extracardiac effects of the aldehydes rather than a direct effect on the myocardium. Further experiments were conducted in order to establish the mechanism of the bradycardia and depressor effect of high doses of the aldehydes, Walsh et al. (1969) studied the effects of acetaldehyde (0.3-30 mh%)on isolated left guinea pig atria. Doses between 0.3 and 10 rnM produced a positive inotropic response while 10 rnM (at 3 min) and 30 rnM produced a negative inotropic response. Propranolol blocked all inotropic responses to acetaldehyde. Blockade of the positive inotropic response was ascribed to /?-receptor blockade, whereas the blockade of the negative inotropic response was ascribed to a secondary action of propranolol on the membrane. In the present study only chronotropic responses were measured; however, with 20-40 mg/kg acetaldehyde and propionaldehyde depressor and negative chronotropic responses predominated after propranolol administration. Thus, propranolol did not appear to prevent these responses to the aldehydes in anesthetized rats. This finding suggested that the aldehydes may be affecting higher centers and indirectly affecting the heart. Subsequent experiments were carried out to verify this hypothesis. However, it is also possible that a negative inotropic effect of these aldehydes on the heart could account for a portion of the observations. In the animals pretreated with atropine, about 80 % of the injections of acetaldehyde at 20 mg/kg produced a pressor response compared with none in the control rats. A depressor response was still observed after 40 mg/kg of the aldehydes, but the magnitude was sharply reduced. The bradycardia produced was significantly less than in control rats. Pressor responses to propionaldehyde at a dose of 20 mg/kg predominated in the atropine-treated rats. In the five cases in which there was a depressor effect, the mean was only 9.8 % of the resting blood pressure compared to 40 y0 under similar conditions in control rats. At 40 mg/kg the fall in blood pressure and heart rate was reduced from 63.9% and 71.2x, respectively, and in the control rats to 19.4% and
CARDIOVASCULAR
EFFECTS
OF ALDEHYDES
643
2l.4:! in atropine-pretreated rats. This reduction in the hypotensive and cardioinhibitory effects of the aldehydes by atropine strongly suggests that these actions are mediated by the vagus nerve. This hypothesis was also confirmed by experiments in vagotomized rats. After vagotomy acetaldehyde (40 mg/kg) produced an increase in blood pressure and a positive chronotropic response. Responses to propionaldehyde were similar, except that depressor responses were observed in a few cases. It appears that propionaldehyde may have more direct activity on a-adrenergic receptors than acetaldehyde. This is suggested by the fact that adrenergic blockade was somewhat more effective in preventing the pressor activity and negative chronotropic activity of this aldehyde. The results of this study indicate that acetaldehyde and propionaldehyde exert two opposing actions on the cardiovascular system: (1) a sympathomimetic effect which results primarily from release of norepinephrine and produces vasoconstriction and rise in blood pressure. In the intact animal a reflex bradycardia ensues. However, following vagotomy, the released catecholamines act unopposed on the heart resulting in a positive chronotropic effect. (2) Stimulation of higher centers results in bradycardia and hypotension secondary to this effect. This response is mediated by the vagus and diminished by atropine; it is abolished by vagotomy. In the normal rat the sympathomimetic effect predominates at lower doses ( l-10 mg/kg), and a pressor response is usually seen. At high doses (40 mg/kg) the vagal stimulatory component overpowers the sympathomimetic action and there are profound decreases in blood pressure and heart rate. At a dose of 20 mg/kg, there appears to be an equal chance of either a pressor or depressor effect occurring. Which of these is seen would apparently depend upon such factors as degree of sympathetic and vagal tone in the animal prior to injection. It is also obvious that impairing either of the systems involved by drugs or surgery results in a shift in responses seen at diflerent doses. For example, with peripheral adrenergic blockade by phenotolamine, vagal stimulatory effects on the circulation frequently predominate at lower doses (IO mg/kg). Conversely, sympathetic stimulation is observed at higher doses following atropine or vagotomy. Moderate doses of acetaldehyde appear to possess more indirect sympathomimetic activity than PropionaIdehyde, and the latter agent appears to possess more direct activity on adrenergic receptors. The evidence to support this conclusion was obtained in experiments in which selective adrenergic antagonists were used. The predominate effects observed after administration of either aldehyde were clearly the result of release of catecholamines from sympathetic nerve endings and the adrenal medulla. However, the former source of norepinephrine appears to be of greater importance in the production of the pressor response. The effect of the aldehydes on the myocardium appears to be less important than the indirect effects, and these are clearly mediated by the vagus nerve.
ACKNOWLEDGMENTS The authors are grateful to Mrs. Elizabeth Miller and Miss Jacqueline Beckner for excellent technical assistanceduring this study.
644
EGLE,
HUDGINS
AND
LA1
REFERENCES J., NAKANISHI, S., KOHEI, H., MATSUMURA, R. AND OGATA, H. (1964). Studies on sympathomimetic action of acetaldehyde. I. Experiments with blood pressure and nictitating membrane responses. Jap. J. Pharmacol. 14, 295-307. EADE, N. R. (1959). Mechanism of sympathomimetic action of aldehydes. J. Pharmacol. AKABANE,
Exp. Ther. 127,29-34.
H. (1934). An sujet des proprietes biologiques et pharmacodynamiques de l’acetaldehyde. C. R. Sot. Biol. 117,238-241. JAMES, T. N. AND BEAR, E. S. (1968). Cardiac effects of some simple aliphatic aldehydes. HANDOVSKY,
J. Pharmacol.
Exp. Ther. 163, 30&308.
T. N., BEAR, E. S., LANG, K. F., GREEN, E. W. AND WINKLER, H. H. (1970). Adrenergic mechanisms in the sinus node. Arch. Intern. Med. 125, 512-547. KENSLER, C. J. AND BATTISTA, S. P. (1963). Components of cigarette smoke with ciliary depressant activity. N. Engl. J. Med. 269, 1161-1166. MILLER, S. E. (1971). Studies on the mode of action of acetaldehyde on the isolated guinea-pig heart. Acta Pharmacol. Toxicol. 30, U9457. NEWSOME, J, R., NORMAN, V. AND KEITH, C. H. (1965). Vapor phase analysis of cigarette smoke. Tobacco Sci. 9, 102-l 10. ROMANO, C., MEYER, F. H. AND ANDERSON, H. H. (1954). Pharmacological relationship between aldehydes and arterenol. Arch. Znt. Pharmacodyn. 99, 378-390. SCHNEIDER, F. H. (1971). Acetaldehyde-induced catecholamine secretion from the cow adrenal medulla. J. Pharmacol. Exp, Ther. 177, 109-l 18. SKOG, E. (1952). Anesthetic and haemolytic action of lower aliphatic aldehydes and their effect on respiration and blood pressure. Acta Pharmacol. Toxicol. 8, 275-289. STEEL, R. D. G. AND TORRIE, J. H. (1960). Princr$ZesandProceduresofStatistics. McGraw-Hill, New York. WALSH, M. J., HOLLANDER, P. B. AND TRUITT, E. B., JR. (1969). Sympathomimetic effects of acetaldehyde on the electrical and contractile characteristics of isolated left atria of guinea pigs. J. Pharmacol Exp. Ther. 167, 173-186. WINGARD, C., HITCHCOCK, F. AND TEAGUE, R. S. (1955). A survey of aldehydes with respect to their action on blood pressure. Arch. Int. Pharmacodyn. 102, 65-84. JAMES,
AND
APPLIED
PHARMACOLQGY
Cardiovascular Effects Propionaldehyde
2d,636-644
(1973)
of Intravenous Acetaldehyde in the Anesthetized Rat1
and
JOHN L. EGLE, JR., PATRICIA M. HUDGINS AND FONG M. LAI Department of Pharmacology, Medical College of Virginia, Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia 23219 Received June 9,1972
Cardiovascular Effects of Intravenous Acetaldehyde and Propionaldehyde in the Anesthetized Rat. EGLE, J. L., JR., HUDGINS, P. M. and LAI, F. M. (1973). Toxicol. Appl. Pharmacol. 24, 636-644. Acetaldehyde and propionaldehyde have both been previously shown to possesssympathomimeticactivityand to affect thecardiovascular system. The objectives of the present investigation were to study the dose-effect relationships of the two aldehydes on the cardiovascular system of the anesthetized rat and to establish the mechanism of the pressor and depressor effects of the compounds. Low iv doses of acetaldehyde and propionaldehyde produced a consistent dose-related rise in blood pressure.These responseswere slightly reduced by adrenalectomy and more strongly antagonized by pretreatment with reserpine or phentolamine. This indicates that the pressor effects of these compounds are primarily due to vasoconstriction mediated by norepinephrine released from sympathetic nerve endings in vascular smooth muscle. Higher doses of the two aldehydes produced a sharp fall in blood pressure and a severe bradycardia. Atropine reduced the hypotensive and cardioinhibitory effects of the aldehydes, suggesting that these actions are mediated by the vagus nerve. This was confirmed by experiments in vagotomized rats. After vagotomy, high doses of acetaldehyde and propionaldehyde produced an increase in blood pressure and a positive chronotropic response. The results of this study indicate that acetaldehyde and propionaldehyde exert two opposing actions on the cardiovasctilar system: (1) a sympathomimetic effect which predominates at doses below 20 mg/kg resulting in a rise in blood pressure; and (2) stimulation of higher centers resulting in bradycardia and hypotension with higher doses. Aliphatic aldehydes are a group of compounds with considerable pharmacologic activity. Attention has primarily focused on acetaldehyde since it was shown by Handovsky (1934) to produce a pressor effect when administered intravenously to anesthetized dogs. Other investigators have observed the pressor effect of acetaldehyde and have suggested that the compound releases catecholamines from the adrenal medulla and other tissues (Roman0 et uZ., 1954; Eade, 1959; Akabane et d., 1964; James and Bear, 1968; Schneider, 1971). Interest in the biologic effects of acetaldehyde has been largely due to the fact that it is formed when ethanol is metabolized, and also that it is present in the vapor phase of cigarette smoke (Newsome e2 al., 1965) and is a 1 This study was aided by a grant from the American Medical Association Education and Research Fonndation. A preliminary report was presented at the Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, Bnrlington, Vermont, August 26, 1971. Copyright 0 1973 by Academic Press, Inc. 636 All rights of reproduction in any form reserved.
CARDIOVASCULAR
EFFECTS
OF ALDEHYDES
631
factor in smoke-induced ciliastasis (Kensler and Battista, 1963). James et af. (1970) have suggested that acetaldehyde may play a role in the development of cardiovascular disease in both smokers and alcoholics. Propionaldehyde is also present in the gas phase of tobacco smoke (Newsome er al.. 1965) and there is evidence that it also affects the cardiovascular system. Reports dealing with propionaldehyde have been descriptive in nature (Skog, 1952; Romano et aI., 1954; Wingard et al., 1955). Evidence has been presented that indicated that propionaldehyde has sympathomimetic properties similar to those of acetaldehyde (Eade, 1959; James and Bear, 1968). The present study was undertaken to investigate the dose-effect relationships of acetaldehyde and propionaldehyde on the cardiovascular system of anesthetized rats. The doses used in this investigation are unquestionably well above the level of aldehyde which might be presented to the cardiovascular system as a result of cigarette or ethanol consumption. An attempt has been made to establish and compare the mechanism of pressor and depressor effects of both aldehydes. Attention was directed toward elucidating the mechanism of the depressor effects observed after intravenous doses of aldehydes ranging from 20 to 40 mg/kg. Evidence is presented which implicates a central effect of these compounds which is cardioinhibitory and mediated by the vagus nerves. METHODS
All experiments were performed with male Wistar rats (250-350 g) which were anesthetized with pentobarbital sodium (50 mg/kg ip). The right femoral artery was cannulated and mean blood pressure was recorded by means of a Statham blood pressure transducer. Heart rate was measured with a cardiotachometer by means of fine needle electrodes inserted through the skin of the rat. The left femoral vein was cannulated to permit intravenous administration of drugs. The volume of injections was within the range of 0.1 to 0.2 ml, followed by flush with 0.2 ml of saline. Animals received control saline injections which were found to have no effect on resting blood pressure and heart rate. Doses of acetaldehyde and propionaldehyde ranging from 1 to 5 mg/kg were administered at lo-min intervals; doses from 10 to 40 mg/kg were administered at 20-min intervals. Subsequent injections were not made until all parameters had returned to preinjection values. In a portion of the experiments, bilateral adrenalectomy or vagotomy in the neck region was carried out 30 min prior to the first injection of aldehyde. A Grass Model 7 polygraph was used to record responses. The following agents were used in an attempt to antagonize the actions of acetaldehyde and propionaldehyde: phentolamine (10 mg/kg), propranolol (1 mg/kg) and atropine (0.5 mg/kg). Blockade was assessed by injections of the appropriate agonist. These were norepinephrine (0.3 pg/kg), isoproterenol (0.01 pg/kg) and acetylcholine (0.5 pg/kg), respectively. One antagonist was administered to one animal. The time between antagonist administration and drug challenge was 5-10 mitt, except in the case of propranolol which was 20 min. Selective blockade was achieved at these doses and time periods. Experimental results have been expressed as a percentage of the initial resting blood pressure or heart rate in each case. This was considered necessary in order to make relative comparisons between the effects of acetaldehyde and propionaldehyde.
638
EGLE,
HUDGINS
AND
LA1
Significant variations were observed in initial blood pressure and heart rate after drug pretreatment or surgical procedures (Table 1). Statistical comparisons were made with Dunnett’s test (Steel and Torrie, 1960). TABLE INITIAL
RESTING
BLOOD
PRESSURE
Drug or procedure Control Adrenalectomy Adrenalectomy + reserpine Phentolamine Propranolol Atropine Vagotomy
AND RAT
1 HEART
RATE
IN THE ANESTHETIZED
Mean blood pressure (mm Hg * SE)
Heart rate (mine1 & SE)
101.8 * 3.2 (9)” 116.0 5 3.4 (8)
405 * 14.1 (9)
73.4 74.9 82.8 96.0
zt 2.1 It 3.4 dz 3.2 z!c3.5
(7) (10) (7) (9)
135.1 Zt 5.2 (7)
396 * 12.8 (8) 236 394 327 401
+ 14.1 =k 16.7 zt 12.4 It 13.4
(7) (10) (7) (9)
416 & 12.1 (7)
’ Numbers in parentheses refer to the number of animals used in each experiment.
RESULTS Blood Pressure Responses to Acetaldehyde and Propionaldehyde
Tables 2 and 3 summarize the results of experiments in which anesthetized rats received acetaldehyde or propionaldehyde in doses ranging from 5 to 40 mg/kg iv. Although minor differences exist between blood pressure responses to acetaldehyde and propionaldehyde, the similarities are more striking. After 5 mg/kg of the aldehydes, pressor responses predominated. Exceptions are noted after phentolamine and propranolol and subsequent acetaldehyde administration. Similarly, after IO mg/kg of the aldehydes, pressor responses predominated. Phentolamine administered before acetaldehyde resulted in an equal number of pressor and depressor responses. After 20 mg/kg of acetaldehyde, depressor responses predominated, except following atropine administration or bilateral vagotomy. Propionaldehyde (20 mg/kg] responses were more variable and appeared to be lessdependent upon endogenous catecholamines than acetaldehyde for its action. Pressor as well as depressor responses were observed in a significant number of rats after adrenalectomy, and reserpine pretreatment followed by adrenalectomy and propranolol. These procedures completely abolished the pressor activity of acetaldehyde. Profound depressor responses were observed after acetaldehyde (40 mg/kg) in all instances except after administration of atropine or bilateral vagotomy. Atropine appeared to partially antagonize the depressor response to acetaldehyde. A dose of 40 mg/kg of propionaldehyde administered to anesthetized rats resulted in profound depressor responses in 81 ‘A of control animals. Atropine administration partially antagonized the depressor effects of propionaldehyde, but bilateral vagotomy was more effective. After vagotomy, a marked rise in blood pressure was the predominant response. Propionaldehyde produced a slight depressor response in 27% of animals after this latter procedure and was more potent than acetaldehyde in producing this effect.
CARDIOVASCULAR
EFFECTS
OF ALDEHYDES
OF VARIOUS
3
8.4 & 4.8 (818) 6.8 It 1.0 (21/Z]) 12.2 k 2.4 (13/13)
PropranoIol
A tropine
Vagotomy
PRESSURE
20
TO INTRAVENOUS
21.6 xt 3.9 (14/14) -
8.3 + 1.8 (U/15) -
10.9 h 4.6 (919) -
6.3 zk 1.1 (13/26) 6.4 z!z1.7 (13/26)
9.8 ic 1.7 (5/16) 14.1 zt 2.4 (11/16)
18.2 k 3.5 (11/15) 24.5 dz 5.9 (4/15)
12.4 * 1.9 (18/21) 12.3 zt 8.3 (3/21)
22.1 Lt 4.9 (10/14) 6.7 * 1.5 (4/14)
17.2 zt 2.0 (16/21) 9.8 zt 2.6 (5/21)
14.3 Liz1.7 (319) 18.5 + 7.7 (6/9)
16.6 * 4; (17/17)
10.0 & 1.0 (5/16) 10.4 zt 1.5 (1 l/16)
12.2 +z 3.4 (12/15) 35.0 zt 15.5 (3/15)
23.6 xk 3.1 (9/20) 40.0 zt 8.1 (ll/ZO)
32.9 k 5.6 (8/l 1) 13.0 zt 4.9 (3/l I)
26.0 I!= 1.1 (3/11) 19.4 zt 8.3 (8/11)
55.7 zt 8.1 (7/7)
-
iG
z E 5
-
z z
8 5
60.4 & 4.1 (14/14) 40.8 zt 5.6 (14/14)
58.0 zt 3.7 (15/15)
23.0 + 8.9 (3/16) 63.9 zt 7.2 (13/16)
40
PROPIONALDEHYDE
Percent of change from resting blood pressure (mean & SE)
IO
(mg/kg)
RESPONSES
PropionaIdehyde
RAY
u Responses ate cakulated as percent of change from Iesting blood pressure. These values were taken from each experiment. Mean + SE of the resting blood pressures appear in Table 1. ’ Number in parentheses refers to the frequency of each response as a function of the number of observations.
4.3 zk 1.7 (26/26) -
Phentolamine
9.8 & 1.4 (12/12) -
10.5 zt 1.1 (17/17)b -
5
PROCEDURES UPON THE BLOOD IN THE ANESTHETIZED
Adrenalectomy
i
SURGICAL
1 I.6 A 2.2 (16/16) -
+ reserpine
AND
Direction of change
DRUGS
Adrenalectomy
Control
Drug or procedure
INFLUENCE
TABLE
CARDIOVASCULAREFFECTSOFALDEHYDES
Heart
641
Rate Responses to Acetaldehyde and Propkmaldehyde
Table 4 summarizes the pronounced effects of the aldehydes (40 mg/kg iv) on resting heart rate. Doses of aldehyde between 5 and 20 mg/kg did not alter heart rate significantly and changes which did occur were clearly secondary to blood pressure changes in most cases. However, after 40 mg/kg the blood pressure responses appeared to be secondary to heart rate changes. It is evident that adrenalectomy with or without reserpine pretreatment significantly enhanced the cardioinhibitory effects of acetaldehyde and propionaldehyde. TABLE 4 HEART
RATE
CHANGE IN RESPONSE TO 40 MC/KG PROPIONALDEHYDEINTHEANESTHETIZEDRAT
ACETALDEHYDE
AND
A B Acetaldehyde Propionaldehyde Percent of control heart rate Drug or procedure
:/; * SE ~-.-
Control 4 64.8 5 6.8 (15)” Adrenalectomy J 87.8 It 4.1 (9)* Adrenalectomy + reserpine 4 99.2 * 0.8 (10)’ Phentolamine (10 mg/kg) J 75.0 zk 2.1 (7) Propranolol(1 mg/kg) 4 86.2 k 5.8 (9T Atrophine (0.5 mg/kg) 4 17.6 + 6.6 (lo)< ; 11.3 * 5.4 (l2)C Vagotomy
‘!< -7 SE ;
71.2 pi 6.1 (16) 86.2 + 7.4 (16)
; 100.0 t 0.1 (13jC ; 45.7 Jo7.6 (14)’ , 60.1 I 7.6(7) ~ 21.6 -L 9.0 (I l)C : 6.6 +2.1 (IIF
n Numbers in parentheses refer to the number of animals used in each experiment. b Significantly different from control (p c 0.05). C Significantly different from control(p c 0.01).
Adrenergic antagonists, phentolamine and propranolol had little effect on the cardioinhibitory effect of acetaldehyde, but appeared to attenuate the effect of propionaldehyde. Atropine administration significantly altered the myocardial depressant effects whereas bilateral vagotomy completely reversed the cardioinhibitory effects of the aldehydes. DISCUSSION The pressor responses observed after intravenous administration of relatively low doses of acetaldehyde and propionaldehyde are consistent with earlier indication5 that the compounds possess sympathomimetic activity (Handovsky- 1934; Roman<) cr a/., 1954). In the present study a relationship between the dose of aldehyde and response was apparent up to a dose of 20 mg/kg. A dose of 5 mg/kg of either compound always produced a pressor response. At doses of IO mg/kg there was a fail in blood pressure in 14’;); of cases after propionaldehyde administration. An equal number (~1’ pressor and depressor effects occurred after 20 mg/kg of both aldehydes. However. when the dose was elevated to 40 mg/kg, a pronounced depressor effect was evident and ~1rise in pressure was seen in only a few cases after propionaldehyde. Alterations in the frequency and magnitude of these control responses by the pharmacologic and surgical
642
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procedures employed provide considerable information regarding the mechanism involved. Adrenalectomy failed to alter the pressor response to the lower dose of either aldehyde. There was an increase in the frequency of depressor responses after 10 and 20 mg/kg, but pressor effects predominated except after acetaldehyde (20 mg/kg). These results tend to indicate that the adrenals are considerably less important as a source of catecholamines in the production of pressor responses to acetaldehyde and propionaldehyde than sympathetic nerve endings. When reserpine pretreatment preceded adrenalectomy, depressor responses were observed more frequently with both aldehydes at doses of 10 and 20 mg/kg. Depletion of catecholamines by reserpine significantly impaired the. ability of the aldehydes to produce a pressor effect. The c+adrenergic blocking agent phentolamine also strongly inhibited the pressor effects of the two aldehydes. These results indicate that the pressor effects of acetaldehyde and propionaldehyde are primarily due to vasoconstriction and mediated by norepinephrine released from sympathetic nerve endings in vascular smooth muscle. The severe bradycardia produced by 40 mg/kg of the aldehyde was enhanced by adrenalectomy and adrenalectomy after reserpine pretreatment (Table 4). This is in contrast to the findings of Moller (1971) obtained with isolated guinea pig hearts. This discrepancy suggests that the bradycardia is due to extracardiac effects of the aldehydes rather than a direct effect on the myocardium. Further experiments were conducted in order to establish the mechanism of the bradycardia and depressor effect of high doses of the aldehydes, Walsh et al. (1969) studied the effects of acetaldehyde (0.3-30 mh%)on isolated left guinea pig atria. Doses between 0.3 and 10 rnM produced a positive inotropic response while 10 rnM (at 3 min) and 30 rnM produced a negative inotropic response. Propranolol blocked all inotropic responses to acetaldehyde. Blockade of the positive inotropic response was ascribed to /?-receptor blockade, whereas the blockade of the negative inotropic response was ascribed to a secondary action of propranolol on the membrane. In the present study only chronotropic responses were measured; however, with 20-40 mg/kg acetaldehyde and propionaldehyde depressor and negative chronotropic responses predominated after propranolol administration. Thus, propranolol did not appear to prevent these responses to the aldehydes in anesthetized rats. This finding suggested that the aldehydes may be affecting higher centers and indirectly affecting the heart. Subsequent experiments were carried out to verify this hypothesis. However, it is also possible that a negative inotropic effect of these aldehydes on the heart could account for a portion of the observations. In the animals pretreated with atropine, about 80 % of the injections of acetaldehyde at 20 mg/kg produced a pressor response compared with none in the control rats. A depressor response was still observed after 40 mg/kg of the aldehydes, but the magnitude was sharply reduced. The bradycardia produced was significantly less than in control rats. Pressor responses to propionaldehyde at a dose of 20 mg/kg predominated in the atropine-treated rats. In the five cases in which there was a depressor effect, the mean was only 9.8 % of the resting blood pressure compared to 40 y0 under similar conditions in control rats. At 40 mg/kg the fall in blood pressure and heart rate was reduced from 63.9% and 71.2x, respectively, and in the control rats to 19.4% and
CARDIOVASCULAR
EFFECTS
OF ALDEHYDES
643
2l.4:! in atropine-pretreated rats. This reduction in the hypotensive and cardioinhibitory effects of the aldehydes by atropine strongly suggests that these actions are mediated by the vagus nerve. This hypothesis was also confirmed by experiments in vagotomized rats. After vagotomy acetaldehyde (40 mg/kg) produced an increase in blood pressure and a positive chronotropic response. Responses to propionaldehyde were similar, except that depressor responses were observed in a few cases. It appears that propionaldehyde may have more direct activity on a-adrenergic receptors than acetaldehyde. This is suggested by the fact that adrenergic blockade was somewhat more effective in preventing the pressor activity and negative chronotropic activity of this aldehyde. The results of this study indicate that acetaldehyde and propionaldehyde exert two opposing actions on the cardiovascular system: (1) a sympathomimetic effect which results primarily from release of norepinephrine and produces vasoconstriction and rise in blood pressure. In the intact animal a reflex bradycardia ensues. However, following vagotomy, the released catecholamines act unopposed on the heart resulting in a positive chronotropic effect. (2) Stimulation of higher centers results in bradycardia and hypotension secondary to this effect. This response is mediated by the vagus and diminished by atropine; it is abolished by vagotomy. In the normal rat the sympathomimetic effect predominates at lower doses ( l-10 mg/kg), and a pressor response is usually seen. At high doses (40 mg/kg) the vagal stimulatory component overpowers the sympathomimetic action and there are profound decreases in blood pressure and heart rate. At a dose of 20 mg/kg, there appears to be an equal chance of either a pressor or depressor effect occurring. Which of these is seen would apparently depend upon such factors as degree of sympathetic and vagal tone in the animal prior to injection. It is also obvious that impairing either of the systems involved by drugs or surgery results in a shift in responses seen at diflerent doses. For example, with peripheral adrenergic blockade by phenotolamine, vagal stimulatory effects on the circulation frequently predominate at lower doses (IO mg/kg). Conversely, sympathetic stimulation is observed at higher doses following atropine or vagotomy. Moderate doses of acetaldehyde appear to possess more indirect sympathomimetic activity than PropionaIdehyde, and the latter agent appears to possess more direct activity on adrenergic receptors. The evidence to support this conclusion was obtained in experiments in which selective adrenergic antagonists were used. The predominate effects observed after administration of either aldehyde were clearly the result of release of catecholamines from sympathetic nerve endings and the adrenal medulla. However, the former source of norepinephrine appears to be of greater importance in the production of the pressor response. The effect of the aldehydes on the myocardium appears to be less important than the indirect effects, and these are clearly mediated by the vagus nerve.
ACKNOWLEDGMENTS The authors are grateful to Mrs. Elizabeth Miller and Miss Jacqueline Beckner for excellent technical assistanceduring this study.
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