An Official Journal of the American Heart

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1 Circulation Research An Official Journal of the American Heart DECEMBER VOL. XXVII Association 1970 NO. 6 Neuretransmitter Control off Sinoatrial Pacemaker Frequency in Isolated Rat Atria and in Intact Rabbits By Albeit S. Grodner, M.D., Hans-Gunther Lahrtz, M.D., Peter E. Pool, M.D., and Eugene Braunwald, M.D. ABSTRACT The sinoatrial node is under the control of both parasympathetic and sympathetic influences. To study the interaction between these opposing influences, the response of the frequency of contraction of a spontaneously contracting rat right atrium to combinations of the neurotransmitters, norepinephrine and acetylcholine was observed. Norepinephrine in concentrations of 1 X 1(HM, 1 X ICH'M, and 1 X 10-SM, which by themselves increased atrial frequency by averages of 30%, 51% and 82%, respectively, and acetylcholine in concentrations of 1 X 10-5 M, 1 X ICMM, and 1 X ICHM, which by themselves decreased atrial frequency by averages of 17%, 52%, and 97%, respectively, were added in all combinations to the atrial preparations. The response was not an algebraic sum of the effects of each of the agents; rather, the bradycrotic influence of acetylcholine predominated in all cases. This action of acetylcholine was blocked by atropine. To evaluate this interaction under more physiologic circumstances, the response of heart rate of anesthetized rabbits to isoproterenol, a potent beta-receptor stimulant, and to asphyxia, a stimulus which produces a marked parasympathetic discharge, was studied. Asphyxia alone induced a marked bradycardia that could be abolished by vagotomy. However, this bradycardia was not prevented by isoproterenol. It is concluded that the predominance of the parasympathetic nervous system on the sinoatrial frequency is a result of the interaction of the neurotransmitters, norepinephrine and acetylcholine on the sinoatrial node. In this interaction the presence of acetylcholine appears to prevent the action of norepinephrine. ADDITIONAL KEY WORDS sinoatrial node vagotomy Since alterations in the frequency of contraction constitute one of the most impor- From the Department of Medicine, University of California at San Diego, La Jolla, California This work was supported in part by U. S. Public Health Service Grants HE and HE from the National Heart and Lung Institute and from the Central Valley and Riverside Heart Associations; also by a National Institutes of Health Fellowship HE from the National Heart Institute. Received July 22, 1970; accepted for publication October 2, norepinephrine asphyxia isoproterenol acetylcholine tant means by which the heart adjusts to varying conditions, an understanding of the control of this variable is essential to a detailed understanding of circulatory regulation. The frequency of discharge of the sinoatrial node is determined by its intrinsic automaticity as well as by the action of the sympathetic and parasympathetic nervous systems on this automaticity. Thus, control of heart rate may involve variations in the CitculnHtm Ruearth, Vol. XXVII, December

2 868 GRODNER, LAHRTZ, POOL, BRAUNWALD activities of both divisions of the autonomic nervous system (1), and it is likely that both divisions may influence heart rate simultaneously (2). The interaction between these two opposing nervous influences on heart rate is of considerable importance but has not been studied in great depth. In 1875, Baxt (3) observed that there was greater slowing of heart rate if the vagus and accelerator nerves were stimulated simultaneously than if the vagus were stimulated alone. Hunt (4) then noted that the effect of simultaneous stimulation of the two nerves depended on the intensity of the stimuli to each and concluded that the resultant effect on rate was the arithmetic mean of the rate induced by the stimulation of each nerve alone. Rosenblueth and Simeone (5) studied the effect of the combined discharge of these nerves on heart rate. They noted that if the alteration of frequency were expressed as percent change of the control rate then the results were the same with or without simultaneous stimulation of the other nerve. Samaan (6) observed that the parasympathetic nervous system predominates over the sympathetic in the control of heart rate and concluded that moderate vagal stimulation may provoke a bradycardia which will mask the effect of strong cardioaccelerator stimulation. More recently, the findings of Warner and Russell (7), and of Levy and Zieske (8) have been consistent with those of Samaan. The purpose of this investigation was to shed additional light on the control of heart rate by describing the interaction of the two neurotransmitters, acetylcholine and norepinephrine, on the spontaneously beating rat right atrium in vitro. Additionally, the response of the atrial rate to the combination of a beta-receptor stimulant and an intense vagal discharge was determined in the intact animal to study the interaction of these two stimuli under more physiologic conditions. Methods and Procedures A. STUDIES ON ISOLATED ATRIA Male, Sprague-Dawley derived, albino rats (Simonson Labs, Gilroy, California) weighing 300 to 500 g were decapitated. The hearts were rapidly excised and placed in oxygenated, modified Krebs solution (9). The right atrium, including the area of the sinus node, was dissected free. One end of the atrium was attached to a Statham force-displacement transducer (G ) via a 5-0 noncapillary silk suture. The other end of the atrium was placed in a lucite clip in a myograph containing modified Krebs solution aerated with 95% O 2 and 5% CO 2. Temperature was maintained at 30 C. Contractions were recorded on a Sanborn A direct-writing recorder. Atrial contraction frequency was determined from 10-second samples of the recorded contractions or from the output of a model A cardiotachometer preamplifier. About 10% of the atria exhibited arrhythmias or did not contract spontaneously and were discarded. All others were permitted to equilibrate for 30 to 120 minutes until atrial contraction frequency was stable for 5 to 10 minutes. To equalize the effect of passive stretch on the frequency of contraction (10), each atrium was stretched so that it contracted at the peak of its length-active tension curve. Solutions of acetylcholine and norepinephrine were made up from the crystalline reagent (Sigma Chemical Co., St. Louis, Mo.) in lx 10-2 N HC1. Doseresponse curves were constructed for each neurotransmitter by determining the frequency 2 minutes after adding the solution to the muscle bath. Low, intermediate, and high concentrations of each agent that elicited changes in the atrial contraction frequency of similar magnitude but opposite direction were then selected. All possible combinations of these two neurotransmitters in the selected concentrations were then added to the bath and the changes in atrial contraction frequency were recorded 2 minutes later. The concentration of neurotransmitters was calculated as that which existed after dilution in the muscle bath. To obviate the influence of residual drug effect or deterioration of the atria, only one observation of the addition of the agent or agents studied was made in each preparation and the preparation was then discarded. It was noted that acetylcholine had a more rapid onset of action than norepinephrine, although the effect on atrial frequency was fully established at 2 minutes with both neurotransmitters. However, no difference in the resultant frequency was noted when norepinephrine was added to the bath 30 seconds before acetylcholine in two additional experiments. The frequency of contraction did not change when the diluent alone was added to the muscle bath in three experiments. B. STUDIES ON INTACT RABBITS The response of the heart rate to the combined effects of a high intensity parasympathetic Circulation Research, Vol. XXVII, December 1970

3 NEUROTRANSMITTER CONTROL OF HEART RATE 869 NE discharge and to isoproterenol, a beta-receptor stimulant, was studied in the intact animal. Male, New Zealand rabbits, weighing 2.8 to 4.6 kg, were anesthetized with pentobarbital, 100 to 150 mg, supplemented as needed during the experiment. Incisional sites were infiltrated with 2% lidocaine. Arterialpressure was measured through a catheter in the right femoral artery by a Statham P23D pressure transducer. The heart rate was obtained from a, cardiotachometer preamplifier driven by the EGG output. The trachea was cannulated and respiration was spontaneous. An increase in parasyrnpathetic activity was induced by producing asphyxia in 24 rabbits by clamping the tracheal cannula. A comparison was made between the atrial frequency observed during the control period and that observed 60 seconds following the induction of asphyxia. This time period was selected so that the results would not be influenced by direct anoxic effects on the sinus node. The animals were divided into the following groups: (I) Six animals were subjected to 60 seconds of asphyxia; (II) six animals were subjected to 60 seconds of asphyxia following bilateral cervical vagotomy; (III) six animals were given 10 /Mg isoproterenol intravenously and the heart rate was determined 30 seconds later; (IV) six animals were subjected to asphyxia and 10 /jlg isoproterenol was administered intravenously 30 seconds after its onset; and (V) six vagotomized rabbits were given 10 fig isoproterenol 30 seconds after the induction of asphyxia. The electrocardiogram was monitored during each intervention and in each experiment sinus rhythm was present. Results A. Effect of Acetyloholine am} Norepinephrine on Atrial Frequency Figure 1 shows the changes in atrial contraction frequency following the addition of the neurotransmitters both alone and in combination. 1X 10~ 7 M, 1 X IO^M and 1 X 10~5M norepinephrine increased the frequency of contraction by averages of 30 ± 8%, 51 ± 4% and 82 ± 15% of control frequency, respectively. Acetylcholine in concentrations of 1 X 10" 5 M, 1 X KHM and 1 x 10"3M decreased atrial frequency by averages of 17 ±3%, 52 ±15% and 97 ±2% of control, respectively. When the atria were exposed to combinations of the low concentrations of acetylcholine (1 X 10~ 5 M) and norepinephrine (1X10~ 7 M), there was a reduction of atrial frequency of the same magnitude as when this concentration of acetylcholine was added alone, i.e., frequency fell by an average of 21 ± 10%. When the intermediate concentration of norepinephrine (1X10~ M) was ACh , hill Ml 82 hull ijij: 1=> i? HUl -97 iiiij-iilfjjjijj ;s-*-r* ** : :*: ij-ji::: : ::;; -21 jjiiijjljf 58 ) : :i; 93/: Ill 1! mi p2 : Wr 38 1 ill Mi 111 II : lll.lll FIGURE 1 Diagramatic representation of the change of atrial contraction frequency following the addition of the agents used, expressed as percent change from the control frequency. Lined area indicates an increase in frequency, stippled area indicates a decrease w frequency and the clear area indicates essentially no change. Mean control rate 176 ±2 heats/min. NE = norepinephrine; ACh := acetylcholine. :j;i: i i=i co X; Circulation Research, Vol. XXVII, Decemb 1970

4 870 GRODNER, LAHRTZ, POOL, BRAUNWALD combined with the low concentration of acetylcholine (1X10~ 5 M), there was no significant change in atrial frequency. Had there been a simple algebraic sum of the effects of these concentrations of each transmitter, a substantial increase in frequency would have been expected; since when given individually, there was a 51 ± 4% increase due to norepinephrine and only a 17 ± 3% reduction due to acetylcholine. No concentration of norepinephrine, not even 1 X 10" 5 M, which by itself elevated heart rate by an average of 82 ± 15%, altered the response of atrial frequency in combination with the highest concentration of acetylcholine. In the presence of the lowest concentration of acetylcholine, only the highest concentration of norepinephrine (1 X 10~ 5 M) increased atrial frequency. However, this high concentration of norepinephrine failed to affect atrial frequency in the presence of 1X 1(HM acetylcholine. The dominance of acetylcholine is apparent. It was possible that the above results could be interpreted to indicate that norepinephrine was competitively blocked from its receptor by acetylcholine. To investigate this possibility, the combination of 1 X 10~ e M norepinephrine and 1 X ICHM acetylcholine was added to atria that had been exposed to 1X 10" 5 M atropine for 2 minutes. Atropine alone did not change the atrial contraction frequency. In the presence of atropine, acetylcholine had no observable effect, i.e., the addition of the combination of norepinephrine and acetylcholine produced a change in frequency of an average of 51 ± 8%, the same as if this concentration of norepinephrine were acting alone. If acetylcholine were blocking norepinephrine at its receptor site, blunting of the tachycardiac effect of norepinephrine would have been evident. This was not observed. B. Influence of a Circulating Beta-Receptor Stimulant on Heart Rate in the Presence of a Strong Parasympathetic Discliarge. The heart rate response of the rabbits to the interventions studied are shown in Figure 2. The mean control rate for the animals studied J AND VAGOTOMY ISOPRO- TERENOL AND ISOPRO- TERENOL VAGOTOMY AND ISOPRO- TERENOL " CHANGE IN HEART RATE beats/min ± SE FIGURE 2 The change in heart rate of rabbits in response to the interoentions studied. The direction of the arrows indicates the direction of the change from the control heart rate (heavy line) to the resultant rate (broken line). Circulation Research, Vol. XXVll, December 1970

5 NEUROTRANSMITTER CONTROL OF HEART RATE 871 was 282 beats/min. This rapid rate suggested that the anesthetic agent had reduced vagal inhibition of heart rate. Sixty seconds after the induction of asphyxia (group I) there was a marked bradycardia. After bilateral cervical vagotorny, the basal heart rate was similar to that observed in the control group with intact vagi; however, the bradycardia produced by asphyxia was abolished (group II). After the rapid intravenous administration of 10 fig of isoproterenol, cardiac frequency increased by an average of 65±8 beats/min (group III). A slightly greater increment in frequency could be obtained with 50 fig isoproterenol but this.dose was associated with a high incidence of arrhythmias. When 10 fig of isoproterenol was administered to rabbits 30 seconds after the induction of asphyxia (group IV), heart rate slowed to almost the same rate as during simple asphyxia, i.e., isoproterenol had little influence on the bradychrotic effect of asphyxia. However, when this same dose of isoproterenol was administered to vagotomized rabbits 30 seconds after the induction of asphyxia (group V), the increment in heart rate was not statistically different from that occurring with isoproterenol alone. Arterial pressure was consistently elevated during the first 60 seconds of asphyxia both in the intact and vagotomized animals. Discussion The results of the present study suggest that the parasympathetic neurotransrnitter, acetylcholine, is capable of suppressing the response of the sinoatrial node to the sympathetic transmitter, norepinephrine. In contrast, norepinephrine is much less able to inhibit the effects of acetylcholine. Thus, these results are consistent with and extend the findings of Samaan (6), Warner and Russell (7) and Levy and Zieske (8), who showed in intact animals that the effects of sympathetic activity on heart rate are reduced as vagal tone is increased. James (11) has noted that nerves within the sinus node do not terminate directly on the cell membrane of pacemaker cells and surmised from this observation that Circulation Research, Vol. XXVll, December 1970 the effects of the neurotransmitter substances begin after they diffuse across a finite space to reach the cell surface. This observation by James as well as the data presented in this study enable us to conclude that it is the action of the neurotransrriitters themselves, rather than preferential innervation by the vagus nerve fibers of the pacemaker cells, that is responsible for the dominance of the parasympathetic over the sympathetic nervous system on heart rate. The concentrations of acetylcholine over the range employed in this experiment were one hundred times the concentrations of norepinephrine, and this difference could be considered to have biased the results in favor of the parasympathetic neurotransmitter. This is unlikely, however, since concentrations of these agents used in this study, when given alone, caused similar although opposite changes in frequency of contraction. This seeming disparity may be due to the presence of relatively high cholinesterase activity in the sinus node (11), and it has recently been confirmed (12) that this activity may determine the concentration of acetylcholine required to produce a change in the rate of the spontaneously beating rat atrium. Roberts and Konjovic (12) noted that inhibition of cholinesterase with physostigmine increased the sensitivity of the sinoatrial node of the isolated rat atrium to acetylcholine by a factor of The concentrations of acetylcholine employed by these investigators in preparations untreated with the cholinesterase inhibitor were in the same range as those used in this study. A further explanation for the high concentrations of exogenous acetylcholine required to slow atrial frequency was also provided by Rosenblueth (13), who noted that whereas only a few pacemaker fibers of the sinus node need be accelerated by norepinephrine to produce an increment in the heart rate, it is necessary for all such elements to be affected by acetylcholine before a decrement in the heart rate would be observed. There was a substantial decrease in the heart rate of the intact animal induced by asphyxia, but no decrease occurred with

6 872 GRODNER, LAHRTZ, POOL, BRAUNWALD asphyxia after vagotomy. This observation is consistent with the conclusion of Litwin and Skolasinska (14) that asphyxic bradycardia results from increased vagal activity. Since vagotomy alone had little if any effect on heart rate, it was likely that the control heart rate of the rabbits studied was elevated due to inhibition of parasympathetic tone by the anesthetic agent. Nevertheless, a profound bradycardia occurred with the increase in parasympathetic outflow produced by asphyxia alone as well as with the addition of a potent beta-receptor stimulating agent, isoproterenol. These results confirm the predominance of parasympathetic activity on the control of the heart rate. The mechanism responsible for the dominance of acetylcholine over norepinephrine in the control of the heart rate is not known. One possible explanation is that acetylcholine possesses, in addition to its direct depressant effect on sinoatrial automaticity, the ability to block beta receptors and thereby inhibit the ability of norepinephrine or isoproterenol to augment atrial frequency. However, the findings in atria previously treated with atropine are not consistent with this possibility; blockade of the muscarinic action of acetylcholine by atropine eliminated completely the interference of the parasympathetic transmitter with the action of the sympathetic transmitter. It has been shown that acetylcholine is capable of blocking not only the chronotropic but other actions of catecholamines on the heart as well. For example, the positive inotropic response of ventricular muscle to epinephrine, norepinephrine and tyramine as well as sympathetic nerve stimulation may be prevented by acetylcholine (15, 16). Furchgott et al. (17) have noted that acetylcholine can counteract the effect of epinephrine on the action potential in the guinea pig atrium, and it has been demonstrated that the ability of catecholamines to enhance phosphorylase activity and glycogenolysis may be inhibited by acetylcholine (18). It has been suggested that both the metabolic and inotropic effects on the heart induced by catecholamines may be correlated with the activation of adenyl cyclase and the myocardial concentration of cyclic AMP (19), and it is possible that this "secondary messenger" could be involved in the differential effects of norepinephrine and acetylcholine on contraction frequency. This possibility is supported by the demonstration that carbamylcholine, a parasympathomimetic drug, inhibits the formation of cyclic AMP in broken-cell preparations from the dog heart (20). In addition, it has been reported that carbamylcholine decreases tension development, adenyl cyclase activity and cyclic AMP concentration in atria and that these responses may be blocked by atropine (21). The recent finding that the dibutyryl derivative of cyclic AMP can increase the contraction frequency of isolated, cultured rat heart cells (22) certainly supports the suggestion that the positive chronotropic action of catecholamines is mediated by the formation of intracellular cyclic AMP. On the basis of the investigations reviewed above, as well as the experiments reported herein, it may be postulated that acetylcholine may interfere with the ability of catecholamines to elevate intracellular cyclic AMP. It would appear that these effects of acetylcholine do not involve blockade of beta receptors; but since they can be blocked by atropine, they can be classified as muscarinic. Although firm evidence to support these hypotheses is not yet available, they do provide an explanation of the available data on the interaction of the sympathetic and parasympathetic neurotransmitters on the sinoatrial node, and support a framework around which future experiments can be planned. References 1. BERKOWITZ, W. D., SCHERLAG, B. J., STEIN, E., AND DAMATO, A. N.: Relative roles of sympathetic and parasympathetic nervous systems in the carotid sinus reflex in dogs. Circ Res 24: 447, GLICK, G., AND BRAUNWALD, E.: Relative roles of the sympathetic and parasympathetic nervous systems in the reflex control of heart rate. Circ Res 16: 363, BAXT, N.: Ueber die Stellung des n. vagus zum n. accelerans cordis. Ber Verhandl Saechs Akad Wiss Liepzig Math Phys H 27: 323, Circulation Research, Vol. XXVII, December 1970

7 NEUROTRANSMITTER CONTROL OF HEART RATE HUNT, R.: Experiments on the relation of the inhibitory to the accelerator nerves of the heart. J Exp Med 2: 151, ROSENBLUETH, A., AND SIMEONE, F. A.: Interrelations of vagal and accelerator effects on the cardiac rate. Amer J Physiol 110: 42, SAMAAN, A.: Antagonistic cardiac nerves and heart rate. J Physiol (London) 83: 332, WARNER, H. R., AND RUSSELL, R. O., JB.: Effect of combined sympathetic and vagal stimulation on heart rate in the dog. Circ Res 24: 567, LEVY, M. N., AND ZIESKE, H.: Autonomic control of cardiac pacemaker activity and atrioventricular transmission. J Appl Physiol 27: 465, POOL, P. E., AND SONNENBLICK, E. H.: Mechanochemistry of cardiac muscle: I. The isometric contraction. J Gen Physiol 50: 951, LANCE, G., LU, H. H., CHANC, A., AND BROOKS, C., McC.: Effect of stretch on isolated cat sinoatrial node. Amer J Physiol 211: 1192, JAMES, T. N.: Cardiac innervation: Anatomic and pharmacologic relations. Bull NY Acad Med 43: 1041, ROBERTS, C. M., AND KONJOVIC, J.: Differences in the chronotropic and inotropic response of the rat atrium to choline esters, cholinesterase inhibitors and certain blocking agents. J Pharmacol Exp Ther 169: 109, ROSENBLUETH, A.: Transmission of Nerve Impulses at Neuroeffector Junctions and Peripheral Synapses. New York, The Technology Press of Massachusetts Institute of Technology and John Wiley and Sons, 1950, p LITWIN, J., AND SKOLASINSKA, K.: On the mechanism for bradycardia induced by acute systemic anoxia in the dog. Pflueger Arch Ges Physiol 289: 109, HOLLENBERG, M., CARRTJEHE, S., AND BAHGER, A. C.: Biphasic action of acetylcholine on ventricular myocardium. Circ Res 16: 527, MEESTER, W. D., AND HARDMAN, H. F.: Blockade of the positive inotropic actions of epinephrine and theophyiine by acetyloholine. J Phannacol Exp Ther 158: 241, FUHCHGOTT, R. F., SLEATOR, W., JR., AND DE CUBAREFF, T.: Effects of acetylcholine and epinephrine on the contractile strength and action potential of electrically driven guinea pig atria. J Phannacol Exp Ther 158: 405, BLUKOO-ALLOTEY, J. A., VINCENT, N. H., AND ELLIS, S.: Interactions of acetylcholine and epinephrine on contractility, glycogen and phosphorylase activity of isolated mammalian hearts. J Pharmacol Exp Ther 170: 27, SUTHERLAND, E. W., ROBISON, G. A., AND BUTCHER, R. W.: Some aspects of the biological role of adenosine 3' 5'-monophosphate (cyclic AMP). Circulation 37: 279, MURAD, F., CM, Y. M., RALL, T. W., AND SUTHERLAND, E. W.: Adenyl cyclase: III. The effect of catecholamines and choline esters on the formation of adenosine 3' 5'-phosphate by preparations from cardiac muscle and liver. J Biol Chem 237: 1233, LARAIA, P. J., AND SONNENBLICK, E. H.: Adrenergic and cholinergic control of adenyl cyclase and cyclic AMP in the myocardium. Circulation 40 (suppl. 3): 129, KRAUSE, E. G., HALLE, W., KALLABIS, E., AND WOLLENBERGER, A.: Positive chronotropic response of cultured isolated rat heart cells to Nc, 2'-O-dibutyryl-3', 5'-adenosine monophosphate. J Molec Cell Cardiol 1: 1, Research, Vol. XXVII, Dec