- Email: [email protected]
Influence of beta2-adrenoceptor stimulation and blockade on cardiac electrophysiologic properties and serum potassium concentration in the anesthetized dog
Influence of beta21 blockade on cardiac electrophysiok@c properties and in the anesthetized dog
nand
The influence of selective &-adrenoceptor stimuiation~and blockade on cardiac eiectrophysioiogic properties was studied in 18 anesthetized dogs. Seiecttve &adrenoceptor activation by saibutamoi faked to alter myocordiai excitebftfty but stgnifkantiy iowered serum potassium concentration. Excitabiiity, refractoriness, and ventricular fibriiiation threshold were also not changed after administration of 100 and 200 fig/kg of the selective &antagonist ICI 118,551. However, at a dose of !500 pg/kg, refractoriness was prolonged and ventricular fibriiiation threshold increased. These changes appear to be due to blockade of B,-adrenoceptors rather than to membrane stabilizing effects, since in catechoiamtne-depieted animais even the highest dose of ICI 118,551 did not alter myocardtai electrical properties. it is concluded that &adrenoceptors do not influence eiectrophysioiogic properties of the canine myocardium. (AM HEART J 1987;113:1066.)
Stefan H. Hohnloser, Boston, Mass.
M.D., Richard
L. Verrier, Ph.D., and Bernard
The effects of /I,-adrenoceptor blockade on the electrophysiologic properties of the heart have been examined in detai1.l There is growing evidence, however, that not only &-, but also &-adrenoceptors are present in various cardiac tissues.2-6 For example, Dukes and Vaughan Williams7 found that repolarization of Purkinje fibers and papillary muscle cells of the rabbit heart is accelerated when ,f3,-adrenoceptors are stimulated. In human left atrial and left ventricular tissue, the presence of @,-adrenoceptors was demonstrated by means of in vitro binding techniques.~ g To our knowledge, however, the role of B2-adrenoceptors in modulating cardiac electrical stability has not been characterized in the intact heart. We therefore examined the influence of selec-
From
the Cardiovascular
Laboratories,
Harvard
School
of Public
Health.
This study was supported by Grants HL-33567 and HL-35138 from the National Heart, Lung, and Blood Institute of the National Institutes of Health, United States Public Health Service, Bethesda, Md.. and by The Rappaport International Program in Cardiology. Dr. Hohnloser is a fellow of the Fritz Thyssen Foundation, Cologne, Federal Republic of Germany. Received Reprint Harvard 02115.
1066
for publication requests: School
Richard of Public
Apr.
7, 1986;
L. Verrier, Health,
accepted
Sept.
5, 1986.
Ph.D., Cardiovascular Laboratories, 665 Huntington Ave., Boston, MA
Lown, M.D.
tive @,-adrenoceptor stimulation and blockade on cardiac electrical stability and on serum potassium in anesthetized dogs. METtWiN Experimental preparation. The experiments were carried out in 18 healthy adult mongrel dogs of either sex, weighing 15 to 25 kg. The animalswere anesthetized with intravenous a-chloralose (100 mg/kg, 10% weight in volume), and additional dosesof 50 mg/kg were given as required to maintain a constant level of anesthesia.Ventilation was carried out by a cuffed endotracheal tube attached to a Harvard respirator, which was adjusted to keep pH, PO,, and pC0, within the physiologic range. A femoral artery and vein were cannulated with a polyethylene catheter for arterial blood sampling, blood pressure
monitoring,anddrugadministration. Cardiac electrical stimulation. Electrical stimulation was accomplishedthrough an intracavitary electrode system consisting of a transvenous bipolar pacing catheter (Medtronic 6601, Medtronic Inc., Minneapolis, Minn.) and an electrographic recording catheter (No. 4 French semifloating bipolar temporary pacing catheter; USCI, Division of CR. Bard, Billerica, Mace.). The catheters were bound to each other with silk ligatures and were positioned at the right ventricular apex by way of a jugular vein under Auoroscopic control. Heart rate was held constant (200 bpm) by ventricular pacing throughout the
Volume Number
113 5
testing procedure. The pacing stimulus was a rectangular pulse 2 msec in duration at twice mid-diastolic threshold. Test impulses were generated by a constant-current Grass S44 square wave pulse generator (Grass Instrument Company, Quincy, Mass.) in conjunction with an optically isolated constant-current source of our own manufacture. The constant-current source used voltage-controlled voltage-source circuitry to generate constant-current pulses directly proportional to the voltage input pulses. The output of the device has a voltage compliance of 300 V direct current. Current output of the optically isolated constant-current source can be verified by taking the voltage across a variety of dummy load resistors (1Ofi to lOfit) and employing Ohm’s law. Current output, under actual operating conditions, can be verified by taking the voltage across a small resistor (approximately l(M2) with an oscilloscope equipped with a differential input amplifier, and again with the use of Ohm’s law. The timing of the stimuli was controlled by a digital timer with a crystalcontrolled time base having an accuracy of *O.Ol% or better. Timing was synchronized with the pacemaker stimulus. The stimulation system is equipped with appropriate circuitry to inhibit the output of the pacemaker from 1 to 5 seconds after delivery of the test stimulus. The test stimuli were delivered after every tenth to fifteenth paced beat. Determination tive refractory
of mid-diastolic threshold and effecperiod. The excitability of the heart was
determined by setting a test stimulus of 0.1 mA intensity and 2 msecduration in mid-diastole and increasing stimulus intensity in 0.1 mA stepsuntil a propagated response was evoked. The minimal current required to elicit this responsedefined the diastolic threshold. The refractory period was determined by mapping the cardiac cycle at 2 msecintervals with stimulus intensities of 3,5, and 7 rnA.‘O The shortest R to stimulus interval that did not result in a propagated responsemarked the refractory period. Determination le and ventricular
and ventricular
Beta,-adrenoceptors
of thresholds for repetitive extrasystofibrillation. Constant-current stimuli of
5 msecduration were used to scanelectrical diastole in 5 msecintervals in order to determine ventricular fibrillation thresholds. The distal electrode wascathodal according to standard convention. Scanning beganat the boundary of the effective refractory period and terminated beyond the T wave. Current strength was increasedin 2 mA stepsuntil two or more extrasystoleswere evoked by a single stimulus. The lowest current at which this repetitive responsewas observed in two out of three trials was considered the threshold for repetitive extrasystole.” Scanning wasthen continued until ventricular fibrillation occurred, thus defining the fibrillation threshold. Defibrillation was promptly accomplishedwithin 5 secondsby a 200 to 300 Wsec discharge from a Lown cardioverter through a pair of copper plates previously fastened to the thorax. j3,-adrenoceptor blockade. To achieve selective & adrenoceptor blockade, ICI 118,551 (erythro-dl-1-[7methylindan-4-yloxyl]-3-isopropylaminobutan-2-o1) was administered in intravenous bolusesof 100, 200, and 500
MID-
DIASTOLIC
vulnerability
1067
THRESHOLD
70 60
P
% CHANGE
*
p< 0.05
REFRACTORINESS
% CHANGE
IO 6 6 4 2 0
Control
100 pg/kg
200
500
ICI 116551
1. Influence of increased doses of ICI 118,551 on electrophysiologic properties of the heart. The highest doseof the drug significantly increasedelectrical stability of the myocardium. Mean values from six dogs. Fig.
pg/kg in six dogs.ICI 118,551beginsto act after 3 minutes following intravenous administration and hasa half-life of 1.3to 2 hours. The effects of the drug on electrophysiologic properties of the heart were examined 30 to 60 minutes after injection. &-adrenoceptor stimulation. Selective stimulation of &adrenoceptors was carried out by intravenous administration of salbutamoP at a doseof 0.1 &kg in six dogs. This concentration has been demonstrated to be selective for &adrenoceptors. I3 Changesin serum potassium concentration and cardiac electrophysiologic properties were monitored for 1 hour. Thereafter, 1.0 pg/kg salbutamol was given and the measurementswere repeated. lsoproterenol administration. To examine a possible influence of &-adrenoceptors on heart rate modulation, the effects of ICI 118,551on isoproterenol-induced tachycardia were examined. The ,&adrenoceptor agonist was given at a doseof 1.0 pg/kg, and peak heart rate response was determined before and 15 minutes after injection of ICI 118,551. Catecholamine depletion. Endogenouscatecholamines were depleted by administration of 0.3 mg/kg reserpine intramuscularly 24 hours before performing the experiments.14This experimental seriesconsistedof six dogs. Measurement of serum potassium. Serum potassium concentration wasmonitored throughout the experiments. Blood sampleswere taken from the femoral vein. An ion-selective electrode (Orion Research 1020, Orion ResearchInc., Cambridge, Mass.) was used for the measurements,and its accuracy wasroutinely checked with a flame photometer (IL 143). Statistical analysis. Statistical comparisonswere made by analysis of variance and by Student’s t test for paired samples.Data were normalized to percent change from control and are reported as mean 2 standard error of mean. Differences were taken assignificant when p < 0.05 (two-tailed).
May 1987
1066
Hohnloser,
Verrier,
and Lown
American
Heart
Journal
l50pao.01 T
THRESHOLD (mAI
.iL 100.
25 HEART RATE INCREASE (bratshin)
2D
50.
-
pg/kg
ICI
118551
Fig. 2. Effects of ICI 118,551 on the thresholds for repetitive extrasystoles (shaded bars) and ventricular fibrillation. The ratio between these thresholds is diminished by the highest drug concentration from 0.64 to 0.52 (p < 0.02).
RESULTS Effects animals
of j3,-adrenoceptor
blockade
in normal
Influence on myocardial excitable properties. Cardiac excitability, refractoriness, and vulnerability to ventricular fibrillation (VF) were not altered following injection of 100 and 200 pg/kg of ICI 118,551. After administration of 500 pg/kg, the effective refractory period was prolonged from 134 +- 2.4 to 143 + 1.7 msec, and the excitability threshold was raised from 0.2 to 0.29 mA (p = 0.06) (Fig. 1). The threshold for ventricular fibrillation was increased from 21.0 + 2.8 mA to 28.3 + 3.3 mA (p < 0.01) (Fig. 2). The threshold for repetitive extrasystoles was not significantly influenced by the /3,-adrenoceptor blocking drug (13.5 & 1.5 mA before vs 14.4 + 2.0 mA after 500 &kg ICI 118,551). The ratio between the thresholds for repetitive extrasystoles and ventricular fibrillation declined from 0.64 +- 0.05 to 0.52 + 0.05 (p < 0.02). Mean arterial blood pressure was not significantly changed by ICI 118,551 administration. Epinephrine-induced changes in serum potassium. To assess the level of &adrenoceptor blockade, epinephrine (1.0 &kg/min) was infused for 15 minutes. This is known to cause a decrease in serum potassium concentration of about 25% to 30%) which is mediated by &adrenoceptors.13y 15-17In the present experiments, the mean serum potassium concentration was 3.4 + 0.07 mEq/L during control, whereas the lowest value following epinephrineinfusion was 3.37 rt 0.1 mEq/L. Thus, abolition of the hypokalemic response indicated elective blockade of the &adrenoceptors by ICI 118,551. Heart-rate response to isoproterenol. Isoproterenol-induced tachycardia was used to assess the effect of ICI 118,551 on P1-adrenoceptors. The increase in heart rate was significantly attenuated
CONTROL
100
zoo *g/kg
ICI
500 118551
Fig. 3. Effects of ICI 118,551on isoproterenol-induced tachycardia. Whereasadministration of 100and 200kg/kg attenuated the response,500 pg/kg of the drug decreased it significantly. Mean values of six dogs.
after administration (-36.0 + 9.3 bpm (Fig. 3).
of 200 pg/kg of ICI 118,551 compared to control; p < 0.05)
Effects of &adrenoceptor blockade in catecholamhw-depleted animais. To preclude the possibility
that the observed decrease in cardiac excitability after 500 pg/kg ICI 118,551 is produced by &adrenoceptor blockade rather than through nonspecific membrane stabilization, the experiments were repeated in catecholamine-depleted dogs. In these animals, mid-diastolic threshold, effective refractory period, and the thresholds for repetitive extrasystoles and ventricular fibrillation were not altered by ICI 118,551 in doses up to 500 pg/kg (Table I). As in normal animals, epinephrine administration failed to produce hypokalemia following use of the & blocking agent. Effects of &-adrenoceptor stimulation. &-adrenoceptor stimulation by salbutamol (0.1 pg/kg) caused a decline in serum potassium concentration from 3.37 + 0.07 to 3.08 +- 0.08 mEq/L 03 < 0.01) but was without effect on refractoriness (129 t- 2.4 vs 131 + 3.2 msec; NS) or the threshold for ventricular fibrillation (18.0 & 1.7 vs 17.3 +- 1.1 mft; NS). Administration of 1.0 &kg salbutamol shortened the refractory period from 129 * 2.5 to 122 + 1.7 msec 03 < 0.01) and lowered the threshold for repetitive extrasystoles from 11.3 f 1.5 to 7.0 +- 1.3 mA (p < 0.06) and the threshold for fibrillation from 18.0 + 1.7 to 10.8 & 1.0 mA (p < 0.01). With the hiiher dose of the drug, mean arterial blood pressure showed a slight decline that did not reach statistical significance. DlSClJSSfON
There is persuasive evidence indicating that many mammalian organs contain both &- and &-adrenoceptors. The relative proportion of these receptors
VOlume Number
113 5
varies among species and organs.2-7 For example, both types of /3-adrenoceptors have been identified in human atrial and ventricular tissues.6,g The present study examined the effects of &adrenoceptor stimulation and blockade on cardiac electrophysiologic properties and serum potassium. The recently developed agent ICI 118,551, which has been shown to block &adrenoceptors selectively in doses up to 100 to 200 pg/kg,ls,‘g was used for blockade and salbutamol was used for stimulation of @,-adrenoceptors. Injection of ICI 118,551 in doses considered to be selective for ,6,-adrenoceptors did not significantly influence excitability of the canine heart. This is in contrast to results obtained in isolated Purkinje fibers and papillary muscles, in which ICI 118,551 prevented the &adrenoceptormediated shortening in action potential duration.7 Possible explanations for this apparent discrepancy are that the response of adrenoceptors differs under in vitro and in vivo conditions, and that the relative distribution of &- and &adrenoceptors differs among various species. 3,5*20Thus, it is possible that the absence of electrophysiologic changes following administration of 100 and 200 pg/kg ICI 118,551 in dogs is due to the relatively low /32-adrenoceptor density in the canine myocardium.20 During p2adrenoceptor stimulation with salbutamol (0.1 kg/ kg), serum potassium concentration declined significantly without altering myocardial excitability. These results indicate that /3,-adrenoceptors are not involved in modifying cardiac electrophysiologic properties. When salbutamol was given at a concentration of 1.0 Ng/kg, vulnerability to ventricular fibrillation was markedly enhanced. This effect was comparable to the influence of epinephrine16 or norepinephrinezl and suggests additional &adrenoceptor stimulation. Since epinephrine administration has also been shown to cause a &adrenoceptor-mediated decline in serum potassium concentration of approximately 25% to 30%,13*15-17infusion of this catecholamine was used to assess effectiveness of &-adrenoceptor blockade. Administration of 100 pg/kg of ICI 118,551 completely prevented the epinephrineinduced hypokalemia. The changes in cardiac electrophysiologic properties observed after administration of 500 pg/kg of ICI 118,551 are most likely due to A-adrenoceptor blocking effects of the drug, which were of comparable magnitude to those observed with oxprenolol and propranolol. * Another possible explanation would be that the substance has some mild membrane stabilizing activity, thereby decreasing cardiac vulnerability. However, in the catecholaminedepleted animals, ICI 118,551 did not alter cardiac
Betapadrenoceptors
and ventricular
vulnerability
I. Effects of ICI 118,551on cardiac excitability reserpinized dogs Table
100 Control MDT
0.23 + 0.03
(mA)
ERP 139.0+ 1.6 (msec) RET 14.8 2 1.6
d&
1069
in six
500 &kg
P
0.20 + 0.02
0.20 + 0.02
NS
139.0+ 2.0
140.0+ 1.9
14.3 + 2.3
14.7 f 1.8
NS
22.7 f 1.9
24.0 TL 2.1
NS
NS
b-4
VFT
22.0 + 1.9
(-4) = mid-diastolic threshold; ERP = effective refractory period; RET = repetitive extrasystolic threshold; VFT = ventricular fibrillation threshold.
MDT
excitability. The fact that ICI 118,551 attenuated the isoproterenol-induced tachycardia suggests that the observed electrophysiologic effects are mediated by &-adrenoceptors and do not represent a membrane-stabilizing effect of the drug. Matta et al.” reported that the threshold for provoking repetitive extrasystoles bears a relatively fixed relationship to the fibrillation threshold, which is preserved under diverse experimental interventions, such as vagus nerve and stellate ganglion stimulation22 or acute myocardial ischemia.23 In the present study, ICI 118,551, in doses which caused /3,-adrenoceptor blockade, diminished the ratio between these thresholds from 0.64 to 0.52. It is conceivable that this is due to a nonspecific pharmacologic effect of the drug, since &adrenoceptor blockade with practolol has also been demonstrated not to interrupt this relationship.” In conclusion, neither stimulation nor blockade of &adrenoceptors alters myocardial excitability. This indicates that &adrenoceptors do not participate in the regulation of cardiac electrical stability in the canine heart. The decline in serum potassium concentration following salbutamol or epinephrine administration is caused by &-adrenoceptors and can therefore be blocked by the selective antagonist ICI 118,551. We express our appreciation to Imperial Chemical Industries PLC, for providing us with ICI 118,551, and to Glaxo Inc., for providing us with saibutamol. We thank Dr. J. D. Fitzgerald and Dr. D. E. Rileyfor adviceconcerning the pharmacokinetics of ICI 118,551, and Dr. S. Sykas for advice concerning salbutamol administration. We also express our appreciation to Dr. E. Raeder for careful review of this article. REFERENCES
1. Raeder EA, Verrier RL, Lown B. Intrinsic activity and the effects of beta-adrenergic vulnerability to ventricular fibrillation. 1983;1:1442-1446.
sympathomimetic blocking drugs on J Am Co11 Cardiol
1070
Hohnloser,
Verrier, and Lown
2. Hedberg A, Minneman KP, Molinoff PB. Differential distribution of beta,-and beta,-adrenergic receptors in cat and guinea-pig heart. J Pharmacol Exp Ther 1980,213:503-508. 3. Brodde OE, Leifert FJ, Krehl HJ. Coexistence of &- and &adrenoceptors in the rabbit heart: Quantitative analysis of the regional distribution by (-)-‘H-Dihydroalprenolol binding. J Cardiovasc Pharmacol 1982;4:34-43. 4. DiCarlo S, Hamra M, Stone HL. The role of beta,-adrenergic receptors on coronary vascular resistance during exercise. Fed Proc 1984;43:626. 5. Johansson L-H, Persson H. &adrenoceptors in guinea-pig atria. J Pharm Pharmacol 1983;35:804-807. 6. Hedberg A, Mattsson H. Beta adrenoceptor interaction of full and partial agonists in the cat heart and soleus muscle. J Pharmacol Exp Ther 1981;219:798-808. 7. Dukes ID, Vaughan Williams EM. Effects of selective a,-, a*, PI-, and &-adrenoceptor stimulation on potentials and contractions in the rabbit heart. J Physiol [Lond] 1984;355:523546. 8. Robberecht P, Delhaye M, Taton G, De Neef P, Waelbroeck M, De Smet JM, Leclerc JL, Chatelain P, Christophe J. The human heart beta-adrenergic receptors. I. Heterogeneity of the binding sites: Presence of 50% beta,- and 50% beta,adrenergic receptors. Mol Pharmacol 1983;24:169-173. 9. Waelbroeck M, Taton G, Delhaye M, Chatelain P, Camus JC, Pochet R, Leclerc JL, De Smet JM, Robberecht P, Christophe J. The human beta-adrenergic receptors. II. Coupling of beta,-adrenergic receptors with the adenylate cyclase system. Mol Pharmacol 1983;24:174-182. 10. Schwartz PJ, Verrier RL, Lown B. Effect of stellectomy and vagotomy on ventricular refractoriness in dogs. Circ Res 1977;40:536-540. 11. Matta RJ, Verrier RL, Lown B. Repetitive extrasystole as an index of vulnerability to ventricular fibrillation. Am J Physiol 1976;230:1469-1473. 12. Brittain RT, Farmer JB, Jack D, Martin LE, Simpson WT. oi-[(t-Butylamino)methyl]-4-hydroxy-m-xylene-cu’,ol’-diol (AH.3365): A selective @-adrenergic stimulant. Nature 1968;219:862-863.
American
May 1997 Heart Journal
13. Vincent HH, Boomsma F, Man in’t Veld AJ, Derkx FHM, Wenting GJ, Schalekamp MADH. Effects of selective and nonselective agonists on plasma potassium and norepinephrine. J Cardiovasc Pharmacol 1984;6:107-114. 14. Blinks JR, Waud DR. Effects of graded doses of reserpine on the response of myocardial contractility to sympathetic nerve stimulation. J Pharmacol Exp Ther 1961;131:205-210. 15. Clausen T, Flatman JA. Beta,-adrenoceptors mediate the stimulating effect of adrenaline on active electrogenic NaK-transport in rat soleus muscle. Br J Pharmacol 1980;68:749-755. 16. Hohnloser SH, Verrier RL, Lown B. Role of potassium in epinephrine-induced changes in myocardial electrical stability. Fed Proc 1985;44:1359. 17. Brown MJ, Brown DC, Murphy MB. Hypokalemia from beta,-receptor stimulation by circulating epinephrine. N Engl J Med 1983;309:1414-1419. 18. Bilski AJ, Halliday SE, Fitzgerald JD, Wale JL. The pharmacology of a &-selective adrenoceptor antagonist (ICI i18,551). J Cardiovasc Pharmacol 1983:5:430-437. 19. Harry JD, Young J, Stribling D. Effects of ICI 118551 (a &adrenoceptor blocker) on metabolic changes induced by isoprenaline and on tachycardia induced by exercise. Br J Pharmacol 1982;14:584-585. 20. Manalan AS, Besch HR Jr, Watanabe AM. Characterization of [‘H]( ~)carasolol binding to beta-adrenergic receptors: Application to study of beta-adrenergic receptor subtypes in canine ventricular myocardium and lung. Circ Res 1981;49:326-336. 21. Rabinowitz SH, Verrier RL, Lown B. Muscarinic effects of vagosympathetic trunk stimulation on the repetitive extrasystole (RE) threshold. Circulation 1976;53:622-627. 22. Lown B. Verrier RL. Neural activitv and ventricular fibrillation. N Engl J Med 1976;294:1165-i170. 23. Kowey PR, Verrier RL, Lown B. The repetitive extrasystole as an index of vulnerability to ventricular fibrillation during myocardial ischemia in the canine heart. AM HEART J 1983;106:1321-1325.
nand
The influence of selective &-adrenoceptor stimuiation~and blockade on cardiac eiectrophysioiogic properties was studied in 18 anesthetized dogs. Seiecttve &adrenoceptor activation by saibutamoi faked to alter myocordiai excitebftfty but stgnifkantiy iowered serum potassium concentration. Excitabiiity, refractoriness, and ventricular fibriiiation threshold were also not changed after administration of 100 and 200 fig/kg of the selective &antagonist ICI 118,551. However, at a dose of !500 pg/kg, refractoriness was prolonged and ventricular fibriiiation threshold increased. These changes appear to be due to blockade of B,-adrenoceptors rather than to membrane stabilizing effects, since in catechoiamtne-depieted animais even the highest dose of ICI 118,551 did not alter myocardtai electrical properties. it is concluded that &adrenoceptors do not influence eiectrophysioiogic properties of the canine myocardium. (AM HEART J 1987;113:1066.)
Stefan H. Hohnloser, Boston, Mass.
M.D., Richard
L. Verrier, Ph.D., and Bernard
The effects of /I,-adrenoceptor blockade on the electrophysiologic properties of the heart have been examined in detai1.l There is growing evidence, however, that not only &-, but also &-adrenoceptors are present in various cardiac tissues.2-6 For example, Dukes and Vaughan Williams7 found that repolarization of Purkinje fibers and papillary muscle cells of the rabbit heart is accelerated when ,f3,-adrenoceptors are stimulated. In human left atrial and left ventricular tissue, the presence of @,-adrenoceptors was demonstrated by means of in vitro binding techniques.~ g To our knowledge, however, the role of B2-adrenoceptors in modulating cardiac electrical stability has not been characterized in the intact heart. We therefore examined the influence of selec-
From
the Cardiovascular
Laboratories,
Harvard
School
of Public
Health.
This study was supported by Grants HL-33567 and HL-35138 from the National Heart, Lung, and Blood Institute of the National Institutes of Health, United States Public Health Service, Bethesda, Md.. and by The Rappaport International Program in Cardiology. Dr. Hohnloser is a fellow of the Fritz Thyssen Foundation, Cologne, Federal Republic of Germany. Received Reprint Harvard 02115.
1066
for publication requests: School
Richard of Public
Apr.
7, 1986;
L. Verrier, Health,
accepted
Sept.
5, 1986.
Ph.D., Cardiovascular Laboratories, 665 Huntington Ave., Boston, MA
Lown, M.D.
tive @,-adrenoceptor stimulation and blockade on cardiac electrical stability and on serum potassium in anesthetized dogs. METtWiN Experimental preparation. The experiments were carried out in 18 healthy adult mongrel dogs of either sex, weighing 15 to 25 kg. The animalswere anesthetized with intravenous a-chloralose (100 mg/kg, 10% weight in volume), and additional dosesof 50 mg/kg were given as required to maintain a constant level of anesthesia.Ventilation was carried out by a cuffed endotracheal tube attached to a Harvard respirator, which was adjusted to keep pH, PO,, and pC0, within the physiologic range. A femoral artery and vein were cannulated with a polyethylene catheter for arterial blood sampling, blood pressure
monitoring,anddrugadministration. Cardiac electrical stimulation. Electrical stimulation was accomplishedthrough an intracavitary electrode system consisting of a transvenous bipolar pacing catheter (Medtronic 6601, Medtronic Inc., Minneapolis, Minn.) and an electrographic recording catheter (No. 4 French semifloating bipolar temporary pacing catheter; USCI, Division of CR. Bard, Billerica, Mace.). The catheters were bound to each other with silk ligatures and were positioned at the right ventricular apex by way of a jugular vein under Auoroscopic control. Heart rate was held constant (200 bpm) by ventricular pacing throughout the
Volume Number
113 5
testing procedure. The pacing stimulus was a rectangular pulse 2 msec in duration at twice mid-diastolic threshold. Test impulses were generated by a constant-current Grass S44 square wave pulse generator (Grass Instrument Company, Quincy, Mass.) in conjunction with an optically isolated constant-current source of our own manufacture. The constant-current source used voltage-controlled voltage-source circuitry to generate constant-current pulses directly proportional to the voltage input pulses. The output of the device has a voltage compliance of 300 V direct current. Current output of the optically isolated constant-current source can be verified by taking the voltage across a variety of dummy load resistors (1Ofi to lOfit) and employing Ohm’s law. Current output, under actual operating conditions, can be verified by taking the voltage across a small resistor (approximately l(M2) with an oscilloscope equipped with a differential input amplifier, and again with the use of Ohm’s law. The timing of the stimuli was controlled by a digital timer with a crystalcontrolled time base having an accuracy of *O.Ol% or better. Timing was synchronized with the pacemaker stimulus. The stimulation system is equipped with appropriate circuitry to inhibit the output of the pacemaker from 1 to 5 seconds after delivery of the test stimulus. The test stimuli were delivered after every tenth to fifteenth paced beat. Determination tive refractory
of mid-diastolic threshold and effecperiod. The excitability of the heart was
determined by setting a test stimulus of 0.1 mA intensity and 2 msecduration in mid-diastole and increasing stimulus intensity in 0.1 mA stepsuntil a propagated response was evoked. The minimal current required to elicit this responsedefined the diastolic threshold. The refractory period was determined by mapping the cardiac cycle at 2 msecintervals with stimulus intensities of 3,5, and 7 rnA.‘O The shortest R to stimulus interval that did not result in a propagated responsemarked the refractory period. Determination le and ventricular
and ventricular
Beta,-adrenoceptors
of thresholds for repetitive extrasystofibrillation. Constant-current stimuli of
5 msecduration were used to scanelectrical diastole in 5 msecintervals in order to determine ventricular fibrillation thresholds. The distal electrode wascathodal according to standard convention. Scanning beganat the boundary of the effective refractory period and terminated beyond the T wave. Current strength was increasedin 2 mA stepsuntil two or more extrasystoleswere evoked by a single stimulus. The lowest current at which this repetitive responsewas observed in two out of three trials was considered the threshold for repetitive extrasystole.” Scanning wasthen continued until ventricular fibrillation occurred, thus defining the fibrillation threshold. Defibrillation was promptly accomplishedwithin 5 secondsby a 200 to 300 Wsec discharge from a Lown cardioverter through a pair of copper plates previously fastened to the thorax. j3,-adrenoceptor blockade. To achieve selective & adrenoceptor blockade, ICI 118,551 (erythro-dl-1-[7methylindan-4-yloxyl]-3-isopropylaminobutan-2-o1) was administered in intravenous bolusesof 100, 200, and 500
MID-
DIASTOLIC
vulnerability
1067
THRESHOLD
70 60
P
% CHANGE
*
p< 0.05
REFRACTORINESS
% CHANGE
IO 6 6 4 2 0
Control
100 pg/kg
200
500
ICI 116551
1. Influence of increased doses of ICI 118,551 on electrophysiologic properties of the heart. The highest doseof the drug significantly increasedelectrical stability of the myocardium. Mean values from six dogs. Fig.
pg/kg in six dogs.ICI 118,551beginsto act after 3 minutes following intravenous administration and hasa half-life of 1.3to 2 hours. The effects of the drug on electrophysiologic properties of the heart were examined 30 to 60 minutes after injection. &-adrenoceptor stimulation. Selective stimulation of &adrenoceptors was carried out by intravenous administration of salbutamoP at a doseof 0.1 &kg in six dogs. This concentration has been demonstrated to be selective for &adrenoceptors. I3 Changesin serum potassium concentration and cardiac electrophysiologic properties were monitored for 1 hour. Thereafter, 1.0 pg/kg salbutamol was given and the measurementswere repeated. lsoproterenol administration. To examine a possible influence of &-adrenoceptors on heart rate modulation, the effects of ICI 118,551on isoproterenol-induced tachycardia were examined. The ,&adrenoceptor agonist was given at a doseof 1.0 pg/kg, and peak heart rate response was determined before and 15 minutes after injection of ICI 118,551. Catecholamine depletion. Endogenouscatecholamines were depleted by administration of 0.3 mg/kg reserpine intramuscularly 24 hours before performing the experiments.14This experimental seriesconsistedof six dogs. Measurement of serum potassium. Serum potassium concentration wasmonitored throughout the experiments. Blood sampleswere taken from the femoral vein. An ion-selective electrode (Orion Research 1020, Orion ResearchInc., Cambridge, Mass.) was used for the measurements,and its accuracy wasroutinely checked with a flame photometer (IL 143). Statistical analysis. Statistical comparisonswere made by analysis of variance and by Student’s t test for paired samples.Data were normalized to percent change from control and are reported as mean 2 standard error of mean. Differences were taken assignificant when p < 0.05 (two-tailed).
May 1987
1066
Hohnloser,
Verrier,
and Lown
American
Heart
Journal
l50pao.01 T
THRESHOLD (mAI
.iL 100.
25 HEART RATE INCREASE (bratshin)
2D
50.
-
pg/kg
ICI
118551
Fig. 2. Effects of ICI 118,551 on the thresholds for repetitive extrasystoles (shaded bars) and ventricular fibrillation. The ratio between these thresholds is diminished by the highest drug concentration from 0.64 to 0.52 (p < 0.02).
RESULTS Effects animals
of j3,-adrenoceptor
blockade
in normal
Influence on myocardial excitable properties. Cardiac excitability, refractoriness, and vulnerability to ventricular fibrillation (VF) were not altered following injection of 100 and 200 pg/kg of ICI 118,551. After administration of 500 pg/kg, the effective refractory period was prolonged from 134 +- 2.4 to 143 + 1.7 msec, and the excitability threshold was raised from 0.2 to 0.29 mA (p = 0.06) (Fig. 1). The threshold for ventricular fibrillation was increased from 21.0 + 2.8 mA to 28.3 + 3.3 mA (p < 0.01) (Fig. 2). The threshold for repetitive extrasystoles was not significantly influenced by the /3,-adrenoceptor blocking drug (13.5 & 1.5 mA before vs 14.4 + 2.0 mA after 500 &kg ICI 118,551). The ratio between the thresholds for repetitive extrasystoles and ventricular fibrillation declined from 0.64 +- 0.05 to 0.52 + 0.05 (p < 0.02). Mean arterial blood pressure was not significantly changed by ICI 118,551 administration. Epinephrine-induced changes in serum potassium. To assess the level of &adrenoceptor blockade, epinephrine (1.0 &kg/min) was infused for 15 minutes. This is known to cause a decrease in serum potassium concentration of about 25% to 30%) which is mediated by &adrenoceptors.13y 15-17In the present experiments, the mean serum potassium concentration was 3.4 + 0.07 mEq/L during control, whereas the lowest value following epinephrineinfusion was 3.37 rt 0.1 mEq/L. Thus, abolition of the hypokalemic response indicated elective blockade of the &adrenoceptors by ICI 118,551. Heart-rate response to isoproterenol. Isoproterenol-induced tachycardia was used to assess the effect of ICI 118,551 on P1-adrenoceptors. The increase in heart rate was significantly attenuated
CONTROL
100
zoo *g/kg
ICI
500 118551
Fig. 3. Effects of ICI 118,551on isoproterenol-induced tachycardia. Whereasadministration of 100and 200kg/kg attenuated the response,500 pg/kg of the drug decreased it significantly. Mean values of six dogs.
after administration (-36.0 + 9.3 bpm (Fig. 3).
of 200 pg/kg of ICI 118,551 compared to control; p < 0.05)
Effects of &adrenoceptor blockade in catecholamhw-depleted animais. To preclude the possibility
that the observed decrease in cardiac excitability after 500 pg/kg ICI 118,551 is produced by &adrenoceptor blockade rather than through nonspecific membrane stabilization, the experiments were repeated in catecholamine-depleted dogs. In these animals, mid-diastolic threshold, effective refractory period, and the thresholds for repetitive extrasystoles and ventricular fibrillation were not altered by ICI 118,551 in doses up to 500 pg/kg (Table I). As in normal animals, epinephrine administration failed to produce hypokalemia following use of the & blocking agent. Effects of &-adrenoceptor stimulation. &-adrenoceptor stimulation by salbutamol (0.1 pg/kg) caused a decline in serum potassium concentration from 3.37 + 0.07 to 3.08 +- 0.08 mEq/L 03 < 0.01) but was without effect on refractoriness (129 t- 2.4 vs 131 + 3.2 msec; NS) or the threshold for ventricular fibrillation (18.0 & 1.7 vs 17.3 +- 1.1 mft; NS). Administration of 1.0 &kg salbutamol shortened the refractory period from 129 * 2.5 to 122 + 1.7 msec 03 < 0.01) and lowered the threshold for repetitive extrasystoles from 11.3 f 1.5 to 7.0 +- 1.3 mA (p < 0.06) and the threshold for fibrillation from 18.0 + 1.7 to 10.8 & 1.0 mA (p < 0.01). With the hiiher dose of the drug, mean arterial blood pressure showed a slight decline that did not reach statistical significance. DlSClJSSfON
There is persuasive evidence indicating that many mammalian organs contain both &- and &-adrenoceptors. The relative proportion of these receptors
VOlume Number
113 5
varies among species and organs.2-7 For example, both types of /3-adrenoceptors have been identified in human atrial and ventricular tissues.6,g The present study examined the effects of &adrenoceptor stimulation and blockade on cardiac electrophysiologic properties and serum potassium. The recently developed agent ICI 118,551, which has been shown to block &adrenoceptors selectively in doses up to 100 to 200 pg/kg,ls,‘g was used for blockade and salbutamol was used for stimulation of @,-adrenoceptors. Injection of ICI 118,551 in doses considered to be selective for ,6,-adrenoceptors did not significantly influence excitability of the canine heart. This is in contrast to results obtained in isolated Purkinje fibers and papillary muscles, in which ICI 118,551 prevented the &adrenoceptormediated shortening in action potential duration.7 Possible explanations for this apparent discrepancy are that the response of adrenoceptors differs under in vitro and in vivo conditions, and that the relative distribution of &- and &adrenoceptors differs among various species. 3,5*20Thus, it is possible that the absence of electrophysiologic changes following administration of 100 and 200 pg/kg ICI 118,551 in dogs is due to the relatively low /32-adrenoceptor density in the canine myocardium.20 During p2adrenoceptor stimulation with salbutamol (0.1 kg/ kg), serum potassium concentration declined significantly without altering myocardial excitability. These results indicate that /3,-adrenoceptors are not involved in modifying cardiac electrophysiologic properties. When salbutamol was given at a concentration of 1.0 Ng/kg, vulnerability to ventricular fibrillation was markedly enhanced. This effect was comparable to the influence of epinephrine16 or norepinephrinezl and suggests additional &adrenoceptor stimulation. Since epinephrine administration has also been shown to cause a &adrenoceptor-mediated decline in serum potassium concentration of approximately 25% to 30%,13*15-17infusion of this catecholamine was used to assess effectiveness of &-adrenoceptor blockade. Administration of 100 pg/kg of ICI 118,551 completely prevented the epinephrineinduced hypokalemia. The changes in cardiac electrophysiologic properties observed after administration of 500 pg/kg of ICI 118,551 are most likely due to A-adrenoceptor blocking effects of the drug, which were of comparable magnitude to those observed with oxprenolol and propranolol. * Another possible explanation would be that the substance has some mild membrane stabilizing activity, thereby decreasing cardiac vulnerability. However, in the catecholaminedepleted animals, ICI 118,551 did not alter cardiac
Betapadrenoceptors
and ventricular
vulnerability
I. Effects of ICI 118,551on cardiac excitability reserpinized dogs Table
100 Control MDT
0.23 + 0.03
(mA)
ERP 139.0+ 1.6 (msec) RET 14.8 2 1.6
d&
1069
in six
500 &kg
P
0.20 + 0.02
0.20 + 0.02
NS
139.0+ 2.0
140.0+ 1.9
14.3 + 2.3
14.7 f 1.8
NS
22.7 f 1.9
24.0 TL 2.1
NS
NS
b-4
VFT
22.0 + 1.9
(-4) = mid-diastolic threshold; ERP = effective refractory period; RET = repetitive extrasystolic threshold; VFT = ventricular fibrillation threshold.
MDT
excitability. The fact that ICI 118,551 attenuated the isoproterenol-induced tachycardia suggests that the observed electrophysiologic effects are mediated by &-adrenoceptors and do not represent a membrane-stabilizing effect of the drug. Matta et al.” reported that the threshold for provoking repetitive extrasystoles bears a relatively fixed relationship to the fibrillation threshold, which is preserved under diverse experimental interventions, such as vagus nerve and stellate ganglion stimulation22 or acute myocardial ischemia.23 In the present study, ICI 118,551, in doses which caused /3,-adrenoceptor blockade, diminished the ratio between these thresholds from 0.64 to 0.52. It is conceivable that this is due to a nonspecific pharmacologic effect of the drug, since &adrenoceptor blockade with practolol has also been demonstrated not to interrupt this relationship.” In conclusion, neither stimulation nor blockade of &adrenoceptors alters myocardial excitability. This indicates that &adrenoceptors do not participate in the regulation of cardiac electrical stability in the canine heart. The decline in serum potassium concentration following salbutamol or epinephrine administration is caused by &-adrenoceptors and can therefore be blocked by the selective antagonist ICI 118,551. We express our appreciation to Imperial Chemical Industries PLC, for providing us with ICI 118,551, and to Glaxo Inc., for providing us with saibutamol. We thank Dr. J. D. Fitzgerald and Dr. D. E. Rileyfor adviceconcerning the pharmacokinetics of ICI 118,551, and Dr. S. Sykas for advice concerning salbutamol administration. We also express our appreciation to Dr. E. Raeder for careful review of this article. REFERENCES
1. Raeder EA, Verrier RL, Lown B. Intrinsic activity and the effects of beta-adrenergic vulnerability to ventricular fibrillation. 1983;1:1442-1446.
sympathomimetic blocking drugs on J Am Co11 Cardiol
1070
Hohnloser,
Verrier, and Lown
2. Hedberg A, Minneman KP, Molinoff PB. Differential distribution of beta,-and beta,-adrenergic receptors in cat and guinea-pig heart. J Pharmacol Exp Ther 1980,213:503-508. 3. Brodde OE, Leifert FJ, Krehl HJ. Coexistence of &- and &adrenoceptors in the rabbit heart: Quantitative analysis of the regional distribution by (-)-‘H-Dihydroalprenolol binding. J Cardiovasc Pharmacol 1982;4:34-43. 4. DiCarlo S, Hamra M, Stone HL. The role of beta,-adrenergic receptors on coronary vascular resistance during exercise. Fed Proc 1984;43:626. 5. Johansson L-H, Persson H. &adrenoceptors in guinea-pig atria. J Pharm Pharmacol 1983;35:804-807. 6. Hedberg A, Mattsson H. Beta adrenoceptor interaction of full and partial agonists in the cat heart and soleus muscle. J Pharmacol Exp Ther 1981;219:798-808. 7. Dukes ID, Vaughan Williams EM. Effects of selective a,-, a*, PI-, and &-adrenoceptor stimulation on potentials and contractions in the rabbit heart. J Physiol [Lond] 1984;355:523546. 8. Robberecht P, Delhaye M, Taton G, De Neef P, Waelbroeck M, De Smet JM, Leclerc JL, Chatelain P, Christophe J. The human heart beta-adrenergic receptors. I. Heterogeneity of the binding sites: Presence of 50% beta,- and 50% beta,adrenergic receptors. Mol Pharmacol 1983;24:169-173. 9. Waelbroeck M, Taton G, Delhaye M, Chatelain P, Camus JC, Pochet R, Leclerc JL, De Smet JM, Robberecht P, Christophe J. The human beta-adrenergic receptors. II. Coupling of beta,-adrenergic receptors with the adenylate cyclase system. Mol Pharmacol 1983;24:174-182. 10. Schwartz PJ, Verrier RL, Lown B. Effect of stellectomy and vagotomy on ventricular refractoriness in dogs. Circ Res 1977;40:536-540. 11. Matta RJ, Verrier RL, Lown B. Repetitive extrasystole as an index of vulnerability to ventricular fibrillation. Am J Physiol 1976;230:1469-1473. 12. Brittain RT, Farmer JB, Jack D, Martin LE, Simpson WT. oi-[(t-Butylamino)methyl]-4-hydroxy-m-xylene-cu’,ol’-diol (AH.3365): A selective @-adrenergic stimulant. Nature 1968;219:862-863.
American
May 1997 Heart Journal
13. Vincent HH, Boomsma F, Man in’t Veld AJ, Derkx FHM, Wenting GJ, Schalekamp MADH. Effects of selective and nonselective agonists on plasma potassium and norepinephrine. J Cardiovasc Pharmacol 1984;6:107-114. 14. Blinks JR, Waud DR. Effects of graded doses of reserpine on the response of myocardial contractility to sympathetic nerve stimulation. J Pharmacol Exp Ther 1961;131:205-210. 15. Clausen T, Flatman JA. Beta,-adrenoceptors mediate the stimulating effect of adrenaline on active electrogenic NaK-transport in rat soleus muscle. Br J Pharmacol 1980;68:749-755. 16. Hohnloser SH, Verrier RL, Lown B. Role of potassium in epinephrine-induced changes in myocardial electrical stability. Fed Proc 1985;44:1359. 17. Brown MJ, Brown DC, Murphy MB. Hypokalemia from beta,-receptor stimulation by circulating epinephrine. N Engl J Med 1983;309:1414-1419. 18. Bilski AJ, Halliday SE, Fitzgerald JD, Wale JL. The pharmacology of a &-selective adrenoceptor antagonist (ICI i18,551). J Cardiovasc Pharmacol 1983:5:430-437. 19. Harry JD, Young J, Stribling D. Effects of ICI 118551 (a &adrenoceptor blocker) on metabolic changes induced by isoprenaline and on tachycardia induced by exercise. Br J Pharmacol 1982;14:584-585. 20. Manalan AS, Besch HR Jr, Watanabe AM. Characterization of [‘H]( ~)carasolol binding to beta-adrenergic receptors: Application to study of beta-adrenergic receptor subtypes in canine ventricular myocardium and lung. Circ Res 1981;49:326-336. 21. Rabinowitz SH, Verrier RL, Lown B. Muscarinic effects of vagosympathetic trunk stimulation on the repetitive extrasystole (RE) threshold. Circulation 1976;53:622-627. 22. Lown B. Verrier RL. Neural activitv and ventricular fibrillation. N Engl J Med 1976;294:1165-i170. 23. Kowey PR, Verrier RL, Lown B. The repetitive extrasystole as an index of vulnerability to ventricular fibrillation during myocardial ischemia in the canine heart. AM HEART J 1983;106:1321-1325.