Direct Positive Inotropic Effect of Acetylcholine on Myocardium

Transcription

1 Direct Positive Inotropic Effect of Acetylcholine on Myocardium EVIDENCE FOR MULTIPLE CHOLINERGIC RECEPTORS IN THE HEART By Robert A. Buccino, M.D., Edmund H. Sonnenblick, M.D., Theodore Cooper, M.D., Ph.D., and Eugene Braunwald, M.D. ABSTRACT The effects of acetylcholine and nicotine on isometric tension, in the presence and absence of atropine, hexamethonium, and endogenous norepinephrine stores, were examined in isolated cat atria and papillary muscles. Acetylcholine exerted a negative inotropic effect, competitively inhibited by atropine, in atrial and papillary muscle; in higher concentrations in papillary muscles, it produced a positive inotropic effect that was independent of cardiac norepinephrine stores and that was not blocked by atropine or hexamethonium. Nicotine, in addition to releasing norepinephrine, was found to exert, in higher concentrations, a direct positive inotropic effect, not antagonized by hexamethonium. These data are compatible with the existence of two distinct cholinergic receptors in the myocardium: a muscarinic receptor intimately associated with vagal nerve endings; and a spatially separate receptor, whose stimulation produces responses similar to those produced by nicotine. This hypothesis allows for the reconciliation of much apparently contradictory data concerning acetylcholine and parasympathetic control of the heart; it accounts for the opposite inotropic effects of acetylcholine on atria (negative) and ventricles (positive) and for the opposite effects of vagal nerve stimulation (negative) and exogenous acetylcholine (positive) on ventricular myocardium. ADDITIONAL KEY WORDS parasympathetic control of the heart neuroeffector junction anesthetized cats Although the effects of acetylcholine on atrial muscle are well known, its mechanism of action on ventricular myocardium is unsettled. Some investigators have concluded that vagal stimulation does not affect the contractility of the mammalian ventricle but alters cardiac function by depressing atrial contractility and rate (1). Others have reported From the Cardiology Branch, National Heart Institute, Bethesda, Maryland, and the Department of Surgery, St. Louis University School of Medicine, St. Louis, Missouri. This work was supported by grants from the John A. Hartford Foundation and by Grants HE and HE-K from the National Institutes of Health, U. S. Public Health Service. Dr. Cooper's present address is Department of Pharmacology, University of New Mexico School of Medicine, Albuquerque, New Mexico. Accepted for publication October 24, cardiac norepinephrine stores muscarinic nicotinic that vagal stimulation exerts a direct negative inotropic effect on the ventricles (2, 3). Acetylcholine, on the other hand, is said to produce a positive inotropic effect on ventricular myocardium, both in isolated tissues (4, 5) and the intact heart (5, 6, 7). In addition, a biphasic action, positive and negative, has been described for both acetylcholine (8, 9) and vagal stimulation (10). Burn and Rand, among others, have proposed that acetylcholine mediates release of norepinephrine from sympathetic neural stores (11, 12, 13), and it has been suggested that acetylcholine exerts its positive inotropic effect on the ventricle through this mechanism (14). The present study was undertaken to determine the relationship between cardiac norepinephrine stores and the response of mammalian myocardium to acetylcholine, and to CircuUtio* Rtsurcb, Vol. XIX, Dictmbi

2 1098 BUCCINO, SONNENBLICK, COOPER, BRAUNWALD explore the apparent contradiction between the effects of acetylcholine and vagal stimulation on ventricular function. The basic experimental plan was to compare the responses of isolated atria and papillary muscles obtained from normal hearts and those depleted of norepinephrine to a wide range of concentrations of acetylcholine and nicotine and to examine the effects of atropine and hexamethonium on these responses. Norepinephrine stores were depleted either by reserpine or by extrinsic cardiac denervation (15); the latter method avoids the administration of norepinephrine-depleting drugs which by themselves might alter the response to acetylcholine. Methods Atria and right ventricular papillary muscles were isolated from 17 normal cats, 15 cats that had undergone cardiac denervation 1 to 4 weeks previously, and 13 cats that had received reserpine intraperitoneally. All 13 cats given reserpine received 1.0 to 3.0 mg/kg per day for 2 to 3 days and 6 of these animals received, in addition, 0.02 to 0.10 mg/kg per day for 3 to 16 days. The heart was totally denervated by mediastinal neural ablation, as described previously (15). Right ventricular norepinephrine content was measured spectrophotofluorometrically by the trihydroxyindoleacetic method (16). It has previously been shown that papillary muscles obtained from hearts depleted of norepinephrine by denervation or reserpine treatment do not respond to tyramine and it has therefore been assumed that this tissue is depleted of norepinephrine along with the right ventricular myocardium (17). Cats were anesthetized with intraperitoneal sodium pentobarbital (25 mg/kg) and a right ventricular papillary muscle and both atria were removed rapidly and, as described in detail previously (18), suspended in a myograph containing oxygenated Krebs solution. The papillary muscle was held at its lower nontendinous end by a spring-loaded cl i p, forming the end of a rigid pin that was attached directly to a Statham (GI-4-250) force transducer. Isometric tension was corrected for cross-sectional area and expressed in grams per square millimeter. Whole atria were similarly attached but tension was expressed as percentage of control since atrial fibers are not parallel and thus do not allow for meaningful correction in terms of cross-sectional area. The papillary muscles and left atria were stimulated with square wave, direct current impulses of 9 msec duration and voltage 10 to 25% greater than threshold, delivered through field electrodes placed parallel to their long axes. Electro-release of norepinephrine in the papillary muscle has been shown to occur with massive field stimulation (19). We have observed that this requires strength of stimuli greatly in excess of those employed in this study. Right atria were allowed to contract spontaneously and left atria were stimulated at a constant frequency of 80/min. The bath temperature was 30 C and the frequency of contraction of the papillary muscles 12/min. Papillary muscles and atria contracted isometrically at the apex of their length-tension curves and the active tension, its first derivative, and the stimulus artifact were recorded. Prior to the addition of drugs, preparations were permitted to equilibrate for 45 min; performance remained stable for periods of at least 3 hours. Solutions of acetylcholine chloride were prepared prior to each experiment and cumulative dose response curves were obtained over the concentration range to 300 /j.g/m\. The concentration of atropine sulfate employed (1 //.g/ml) was the largest that exerted little or no effect on developed tension but still competitively inhibited the effects of acetylcholine. The effects of nicotine in concentrations of 10, 100, 300, and 1000 fmg/m], and the effects of various concentrations of hexamethonium, ranging from 1.0 to jug/ml, on the response to acetylcholine and to nicotine were also studied in 12 muscles from normal, 4 from denervated, and 10 from reserpine-treated animals. In all instances a muscle was utilized for studying the effects of a single drug, alone or in the presence of one concentration of antagonist. Results Cardiac Norepinephrine Concentrations Cardiac denervation and reserpine pretreatment essentially eliminated cardiac norepinephrine stores. In 11 normal animals, the right ventricular norepinephrine concentration averaged 2.13±0.30 (SEM) /xg/g; in the 15 denervated animals, it was reduced to 0.01 ± 0.01 yxg/g; and in the 13 reserpinetreated animals, to 0.03 ± 0.01 Responses to Acetylcholine Papillary muscles. Acetylcholine was added to the bath at 4-min intervals in increasing concentrations, from to 300 /ig/ml. The maximum response recorded usually occurred between 30 and 150 sec. In Figure 1 are shown the average dose response curves for all three groups of muscles. The predominant CircuUiion ResHTch, Vol. XIX, Dtctmbtr 1966

3 CHOLINERGIC RECEPTORS IN THE HEART Normal (10) D«n*rvat«d (10) Rewrpin* (6) A X -.10 ACETYLCHOLINE (jig/ml) FIGURE 1 Effect of acetylcholine, in the absence of other drugs, on isometric tension in ventricular myocardium (papillary muscle). Mean and standard error of data from papillary muscles of 10 normal (m), 10 deneroated (A) and 6 reserpine-treated (o) cats; effect expressed as change in absolute tension, corrected for cross-sectional area of individual muscles. Maximum response, which usually occurred between 30 and 150 sec, was recorded at each concentration. response was positive in all groups with higher concentrations (10.0 to 300 fig/m\) of acetylcholine (Fig. 2); a small negative response occurred in 4 of the 8 normal muscles in which the effects of low concentrations (0.001 to 0.1 fig/ml) were studied. In 2 of the 10 normal muscles, 1 of the 10 muscles obtained from denervated hearts, and 3 of the 6 muscles obtained from reserpine-treated animals, the sustained increase in tension observed with higher concentrations of acetylcholine was preceded by a transient negative response (Fig. 3). Although there was considerable variation in the response between muscles, the predominant effect of acetylcholine was clearly to stimulate contractility. This was mediated primarily by an increase in the rate of tension development, with little or no effect on time to peak tension (Table 1). Atria. Dose-response curves to acetylcholine were obtained in 8 spontaneously beating right atria and 9 electrically stimulated left atria. The atria were always responsive to considerably smaller concentrations of acetyl- CircnUlicm Rtiurcb, Vol. XIX, Dtctmbtr 1966 choline than were the papillary muscles, and the negative inotropic effect was the same for atria from normal and denervated animals (Fig. 4A and B). The magnitude of the negative inotropic effect was identical regardless of whether atrial frequency was maintained constant or allowed to decrease as acetylcholine was added. The negative chronotropic effects of acetylcholine were identical in right atria removed from normal and denervated cats (Fig. 4C). Effect of Atropine on the Response to Acetylcholine Atria. In the presence of atropine (1 ml), the dose response curve to acetylcholine was displaced to the right, as shown in Figure 5. In 2 of the 6 atria, in the presence of atropine, a slight increase in tension, 11? and 12% of control, occurred with 10 /Ag/ml of acetylcholine, a dose just below that producing the negative inotropic effect. Papillary muscles. In 5 normal muscles, 2 from denervated hearts, and 2 from reserpine-

4 1100 BUCCINO, SONNENBLICK, COOPER, BRAUNWALD Normal ACH 10 ng /ml 8. Op g /turf Denervoled < 001 Mg /i) NE ACH 30Mg/ml / nvri* 0' 0 Ss«\ - UMOJH: Reserpine 001 NE ACH l2.0r g / mm' 6.0- FIGURE 2 Positive inotropic response of papillary muscles to acetylcholine, in the absence of other drugs. Upper tracing, normal muscle with intact norepinephrine (NE) stores; middle tracing, muscle from a denervated heart in which right ventricular norepinephrine measured less tlian 0.5% of normal and lower tracing, muscle from a reserpine-treated animal in which right ventricular norepinephrine measured less than 0.5% of normal. g/mm1 I0CH treated animals, atropine did not prevent a positive response to acetylcholine; the response of normal muscles was actually enhanced, as shown in Figure 6, while norepinephrine depletion appeared to abolish this enhancement of the response to acetylcholine in the presence of atropine. The small depression of tension produced by low con- ACH 30/iq/ml FIGURE 3 Transient negative response of papillary muscle to acetylcholine, followed by a sustained positive response. Muscle obtained from a reserpine-treated cat; no other drugs present. CircuUtio* Ruttrcb, Vol. XIX, Dtcnubf 1966

5 CHOLINERGIC RECEPTORS IN THE HEART 1101 Papillary Muscle Response to Acetylcholine Normal (10) Denervated (10) Reserpine (6) Control Tension (g/mm«) Maximal response ± 0.5* 5.4 ± ± 0.6 # * TABLE 1 Time to peak tension (msec) Maximal Control response ± * 10* 13* Rste of tension development (g/mm'/sec) Maximal Control responi* f * t Control taken prior to addition of acetylcholine and maximal response as the difference at that concentration associated with maximum tension. Rate of tension development measured as maximum slope of the tension curve. = P<.01. t = P <.05. * = Not significant. -STIMULATED NormalM) Denervottdl5! B -SPONTANEOUS Normal (41 DmrmttdH) RATE -SPONTANEOUS Normal H) DenerrattdM 10 I0 2 If f3 Iff 2 Iff 1 I 10 I0 2 ACETYLCHOLINE (/ig/ml) Iff 3 Iff 2 Iff 1 I 10 I0 2 FIGURE 4 Effect of acetylcholine, in the absence of other drugs, on isometric tension and spontaneous rate in atrial myocardium. A, mean and standard error of observations on left atria, from 4 normal (u) and 5 denervated (A) cats, stimulated at a constant rate. B and C, mean and standard error of observations on right atria, from 4 normal (u) and 4 denervated (A) cats, allowed to contract spontaneously. Tension and rate expressed as percentage of control. centrations of acetylcholine and the initial depression produced by high concentrations were not seen in the presence of atropine. Effect of Hexametlionium on the Response of Papillary Muscles to Acetylcholine In 7 normal muscles and 3 of the 4 muscles from denervated hearts, the positive inotropic effect of acetylcholine was not blocked by concentrations of hexamethonium ranging from 1.0 to 100O.0 /xg/ml, as illustrated in Figure 7. Response of Papillary Muscles to Nicotine It is well established that nicotine, 10 fig/ ml, produces a positive inotropic effect in the normal papillary muscle and that this response CircuUlioH Reiurcb, Vol. XIX, Dectmbtr 1966 can be blocked by low concentrations of hexamethonium (10 /zg/ml) and prevented by norepinephrine depletion (5). We confirmed these effects in a total of 8 muscles. However, we found that nicotine in higher concentrations of 100, 300, and 1000 fig/m] exerted a distinct positive inotropic effect on 3 papillary muscles obtained from denervated hearts and 8 from reserpine-treated animals, as illustrated in Figure 8. This positive inotropic effect was not blocked by hexamethonium, 10 //.g/ml, in 3 muscles from denervated hearts and not by 1000 / A g/ml in 3 muscles from reserpine-treated animals. Discussion This study demonstrated that acetylcholine exerts a positive inotropic effect on ventricular

6 1102 BUCCINO, SONNENBLICK, COOPER, BRAUNWALD 00 - i ^ a a 5 \ \ \ 80 %0F CONTROL \ \ 20 Untreated (8) " u Atropin»-Ifig/ml(6) T \ x \ 1 n " IO H I 10 ACETYLCHOLINE (/ig/ml) K> 2 10" FIGURE 5 Effect of atropine, 1 fxs/ml, on the dose-response curve for acetylchoune in normal atrial myocardium. Mean and standard error of response to acetylchoune in 8 atria, 4 spontaneous and 4 stimulated, in the absence of atropine (m) and 6 spontaneous atria in the presence of atropine (a). Tension expressed as percentage of control. A ( g / mm 2 ) 1.50 r L20 - Untreated (10) o-o Atropine - l/i g/ml (5) 10"* I 10 ACETYLCHOLINE (/tg/ml) FIGURE 6 Effect of atropine, 1 fig/ml, on the dose response curve for acetylchoune in normal ventricular myocardium (papillary muscle). Mean and standard error of response to acetylchoune in 10 papillary muscles in the absence of atropine (»} and 5 muscles in the presence of atropine (a). Effect expressed as change in absolute tension, corrected for cross-sectional area of individual muscles. CircnUtion Rtstrcb, Vol. XIX, Dtctmbtr 1966

7 CHOLINERGIC RECEPTORS IN THE HEART 1103 g /nr O ON 9 / r FIGURE 7 Positive inotropic effect of acetylcholine on ventricular myocardium in the presence of hexamethonium. Upper tracing, normal papillary muscle, pretreated with 1000 fig/ml hexamethonium; and lower tracing, papillary muscle from a denervated heart (0.01 fmg/g NE) in the presence of 10 fig/ml hexamethonium. NICOTINE 10 ^q /ml I g NICOTINE 100 ^g/ml NICOTINE r I 1 05sec FIGURE 8 Positive inotropic effect of nicotine on ventricular myocardium (papillary muscle from a denervated heart, < 0.01 ag/g NE) in the absence of cardiac NE stores and in the absence of other drugs. Upper, middle, and lower tracings represent sequential additions of nicotine to a single papillary muscle from a denervated heart in which right ventricular NE measured less than 0.5% of normal. CircnUtum Rtstarcb, Vol. XIX, Dtumbtr 1966

8 1104 BUCCINO, SONNENBLICK, COOPER, BRAUNWALD myocardium even in the absence of cardiac norepinephrine stores and helps to define the complex nature of the autonomic neuroeffector junction in the heart. The hypothesis that acetylcholine exerts a stimulating effect on myocardium by releasing catecholamines was considered by Spadolini and Domini in 1940 (8) but rejected when this effect was not prevented by alpha-adrenergic sympatholytics. Hoffman and associates in 1945 (6), Middleton and co-workers in 1956 (4), and Lee and Shideman in 1959 (5), in studies on the intact canine heart and cat papillary muscles, added support to but provided no definitive proof for the concept that acetylcholine, in concentrations comparable to those employed in the present study, exerts its positive inotropic effect by releasing norepinephrine. Burn and Rand have developed the most comprehensive application of this hypothesis as a basis for autonomic nervous transmission in general. In studies carried out on the rabbit atrium (20), vessels of the rabbit ear (21), the piloerector muscle, nictitating membrane, and spleen of the cat (22, 23), and the isolated canine uterus (23), they found that die sympathomimetic effects of acetylcholine, observed in the presence of atropine, were not seen when animals were pretreated with reserpine; the sympathomimetic effects of acetylcholine were, therefore, thought to depend upon intact norepinephrine stores and acetylcholine was thought to play an intermediary role in the release of norepinephrine from sympathetic nerve endings. From the findings of the present study, it appears that the hypothesis that acetylcholine exerts its "sympathetic-like" effect by releasing norepinephrine (13), need not be invoked to explain the positive inotropic effect of acetylcholine, since the response is essentially identical in muscles from norepinephrinedepleted hearts and in normal muscles (Fig. 1). In addition, acetylcholine and norepinephrine appear to increase tension by different mechanisms; that is, acetylcholine increases the rate of tension development without substantially changing time to peak tension, in a manner similar to calcium, while norepinephrine also increases the rate of tension development but strikingly shortens the time to peak tension (18). It cannot be denied, however, that acetylcholine may result in norepinephrine release under certain conditions. Richardson and Woods reported that the positive inotropic effect of acetylcholine observed in isolated atropinized rabbit hearts was accompanied by the release of norepinephrine determined both chemically and by bioassay in the coronary effluent (7). This observation was recently confirmed by Angelakos and Bloomquist (24). The finding that acetylcholine exerts a positive inotropic effect in the absence of norepinephrine suggests that the quantity of norepinephrine released may be so small that it plays little if any role in the positive inotropic response. Alternatively, it is conceivable that the release of norepinephrine is consequent to the stimulatory effect of acetylcholine and not necessarily the cause of the positive inotropic effect. Such an interpretation is supported by the work of Hollenberg, Carriere, and Barger, who showed that the positive inotropic effect associated with the intracoronary infusion of acetylcholine is not blocked by pronethalol (9), and by the findings of La Farge and associates, who have reported that norepinephrine efflux from the left ventricle may be stimulated by an increase of peak systolic pressure (25). The positive inotropic effect of acetylcholine is not blocked by atropine, which characteristically inhibits the action of acetylcholine at muscarinic receptor sites, or by hexamethonium, which characteristically inhibits the effects of nicotine at receptor sites in autonomic ganglia. Middleton and co-workers (4) and Lee and Shideman (5) made similar observations with regard to atropine, but reported that hexamethonium blocked the positive inotropic effect of acetylcholine. However, it seems clear from the present observations that the positive inotropic effect of acetylcholine is not blocked by hexamethonium. Ten of 11 papillary muscles, from both normal and denervated hearts, responded normally to acetylcholine in the presence of a CircuUiion Ruttrcb, Vol. XIX, Dtctmbtr 1966

9 CHOLINERGIC RECEPTORS IN THE HEART 1105 VAGUS NERVE ATRIUM VENTRICLE RESPONSE wide range of concentrations of hexamethonium (Fig. 7), which had been shown to block the usual stimulant effect of low concentrations of nicotine (10 /tg/ml). A working hypothesis may be proposed to explain the results of the present experiments and to correlate these findings with those of previous investigators. There appear to be at least two distinct types of cholinergic receptor sites in the heart, as depicted in Figure 9. The first, type I, respond to small concentrations of acetylcholine, exert a negative inotropic effect, and may be termed muscarinic, since they are specifically blocked by atropine; the other, type II, respond to larger concentrations of acetylcholine, exert a positive inotropic effect, and are not blocked by atropine. Postulating that the type I cholinergic sites are intimately related to vagal nerve endings and that the type II sites are spatially separate from these endings allows the reconciliation of many apparently contradictory findings. These include the predominantly negative effect of acetylcholine on atria in contrast to the predominantly positive effect on the ventricles as well as the negative effect of vagal stimulation on the ventricles as opposed to the positive effect of exogenous acetylcholine. Since vagal nerve fibers are more abundant in the atria than in the ventricles, type I sites related to nerve endings might be expected to follow a similar distribution, thus accounting for the predominantly negative inotropic effect both of acetylcholine and of vagal nerve stimulation (26) on the atria. Type I sites in the ventricles are few, corresponding to the paucity of vagal nerve endings in these chambers (27). By responding to appropriately small concentrations of acetylcholine, these type I sites in the ventricle, however, would account for the slight negative phase of the acetylcholine dose-response curve of papillary muscles (Fig. 1) and the initial transient negative effect seen with larger concentrations (Fig. 3). This hypothesis is supported by the finding that atropine abolishes both of these negative effects. By being more accessible to acetylcholine released at vagal nerve endings, type I sites also account for the negative inotropic effect of vagal stimulation in the intact heart. The predominance of type II sites in the ventricle, unrelated to the vagus nerve, would permit the positive inotropic effect produced by larger concentrations of exogenous acetylcholine. Whether this positive response is a direct effect of acetylcholine on the myocardium or is mediated by release of some unknown substance, as suggested by Hollenberg et al. (9), is unresolved. However, both possibilities are consistent with the hypothesis outlined here, since stimulation of type II sites might result in a positive effect directly TO ACETYLCHOLINE TYPE I (-) NEGATIVE ATRIUM (VENTRICLE) SMALL DOSE (0.3 /*g/ml) BLOCKED BY ATROPINE (1 /ig/ml) VAGAL TYPE I (+) POSITIVE VENTRICLE LARGE DOSE (30 /ig/ml) NOT BLOCKED BY ATROPINE (1 /ig/ml) NON-VAGAL FIGURE 9 Schematic representation of the two types of chounergic receptor sites in the heart. See text for explanation. CircuUiion Rsittrch, Vol. XIX, Dictmitr 1966

10 1106 BUCCINO, SONNENBLICK, COOPER, BRAUNWALD or require a sequence of reactions involving other substances. If this positive inotropic effect of acetylcholine is exerted through one or more mediators, it is clear that norepinephrine is not the sole mediator. The nature of the proposed type II receptor site for acetylcholine and its physiologic role, if any, are not entirely clear. Relatively large concentrations of acetylcholine are required to demonstrate its positive inotropic effect; this may represent a nonspecific, membrane effect rather than interaction with a discrete receptor site. It is not a nicotinic receptor in the conventional sense, as defined at autonomic ganglia, since it is not blocked by hexamethonium (28). However, it is clear that the papillary muscle or, more likely, its adrenergic innervation apparatus, contains what may be considered true nicotinic receptors. Thus, the normal papillary muscle responds dramatically to small concentrations of nicotine (10 /ig/ml) and this response can be prevented by norepinephrine depletion or hexamethonium, as shown by previous workers (5) and confirmed in the present investigation. Since no autonomic ganglia have been I I Atroplne VAGAL SYMPATHETIC Propranolol identified histologically in the papillary muscle, it is likely that these receptors are located in sympathetic nerves themselves, at or near their endings. It was found in the present study that nicotine, in higher concentrations (100 /xg/ml or higher), is capable of producing a positive inotropic effect of its own, in the absence of norepinephrine or sympathetic nerves and in the presence of hexamethonium. This second receptor responsive to nicotine, appears to be distal to the nerve ending and on the heart muscle cell itself and may, in fact, be the type II cholinoceptive site discussed above. The implications of this hypothesis extend to the mechanism of parasympathetic nervous transmission and the nature of the neuroeffector junction in general. Muscarinic and nicotinic receptor sites have been demonstrated to exist in close proximity on both the pre- and postsynaptic fibers in autonomic ganglia (29) and in the central nervous system (30, 31). The findings of the present study are consistent with the view that nicotine also acts directly on the effector tissue and emphasize the need to reevaluate the ACh-I ACh -E N N-S Hexamethonium Agonist Acatylcholina 0.3 fig/ml Acetylcholine 30 (ig/ml Nicotine 10 pg/ml Nicotine 100 jig/ml Location Effector (at vagal ending) Effector (separate from vagus) Pro-synoptic sympathetic nding Effector Antagonist Atropine Yes No Hexomethonium No No Yes No FIGURE 10 Schematic summary of receptor sites at the myocardial neuroeffector junction. See text for explanation. ACh = neurotransmitter acetylcholine. NE = neuwtransmitter norepinephrine. ACh-I = muscarinic cholinergic receptor. B =z beta-adrenergic receptor site. N = classical nicotinic receptor. ACh-ll = nonmuscarinic cholinergic receptor, independent of norepinephrine and not blocked by hexamethonium. N-S = nicotine-sensitioe receptor on heart muscle which is independent of norepinephrine and not blocked by hexamethonium. Dashed lines indicate pharmacologic antagonism. CircuUtiom Rtsircb, Vol. XIX, Dtctmitr 1966

11 CHOLINERGIC RECEPTORS IN THE HEART 1107 classical concept that nicotinic cholinoceptive sites are restricted to autonomic ganglia and that muscarinic cholinoceptive sites are restricted to the effector tissue. Although the majority of ganglionic receptor sites may be nicotinic and the dominant fraction at the effector tissue may be muscarinic, it seems important to recognize that each level of the autonomic nervous system may respond to both muscarine and nicotine. The concept of various receptor sites at the myocardial neuroeffector junction that emerges from the results of the present study is depicted in Figure 10. Of the two cholinoceptive sites, the one termed acetylcholine, type I, appears to be the one of physiologic importance since it is associated with vagal nerve endings, is typically muscarinic in that it is blocked specifically by atropine, and produces the negative inotropic effect anticipated of a parasympathetic receptor. The nicotinic receptor appears to be located at the nerve ending rather than on the effector membrane, since it depends upon intact sympathetic nerves and norepinephrine stores. Since this receptor is blocked by hexamethonium, it behaves like a typical nicotinic receptor at autonomic ganglia. A second receptor responsive to nicotine, on the other hand, appears to be distal to the nerve ending and on the effector itself, since it can be demonstrated in papillary muscles from denervated and reserpine-treated animals. Thus, it differs from the classical nicotinic receptor in that it induces a positive inotropic effect independent of norepinephrine and is not blocked by hexamethonium. The finding that the acetylcholine receptor, type II, behaves in a manner similar to this receptor which is responsive to nicotine suggests that these two receptors may, in fact, be identical. References 1. SARNOFF, S. J., BROCKMAN, S. K., GILMORE, J. P., LINDEN, R. J., AND MITCHELL, J. H.: Regulation of ventricular contraction: Influence of cardiac sympathetic and vagal nerve stimulation on atrial and ventricular dynamics. Circulation Res. 8: 1108, DECEEST, H., LEVY, M. N., ANTJ ZIESKE, H.: Negative inotropic effect of the vagus nerves CircuUlitmRt ittrcb, Vol. XIX, Dtc-tmitr 1966 upon the canine ventricle. Science 144: 1223, DAGGETT, W. M., NUGENT, G. G., CARR, P. W., POWERS, P. C., HARADA, Y., AND COOPER, T.: Influence of efferent vagal stimulation on ventricular performance, O 2 consumption, and coronary blood flow. Federation PTOC. 25: 335, MIDDLETON, S., OBEHTI, C, PRACER, R., AND MIDDLETON, H. H.: Stimulating effect of acetylcholine on the papillary myocardium. Acta Physiol. Latinoam. 6: 82, LEE, W. C, AND SHTDEMAN, F. E.: Mechanism of the positive inotropic response to certain ganglionic stimulants. J. Pharmacol. Exptl. Therap. 126: 239, HOFFMANN, F., HOFFMANN, E. J., MIDDLETON, S., AND TAIESNIK, J.: The stimulating effect of acetylcholine on the mammalian heart and the liberation of an epinephrine-like substance by the isolated heart. Am. J. Physiol. 144: 189, RICHARDSON, J. A., AND WOODS, E. F.: Release of norepinephrine from the isolated heart. Proc. Soc. Exptl. Biol. Med. 100: 149, SPADOLTNI, I., AND DOMINI, G.: La duplice azione dell acetilcolina sul cuove isolato di cavia (Double action of acetylcholine on isolated heart of guinea pig). Arch. Fisiol. 40: 148, HOLLENBERG, M., CARRIERE, S., AND BARCER, A. C.: Biphasic action of acetylcholine on ventricular myocardium. Circulation Res. 16: 527, LEVY, M. N., NG, M., MARTIN, P., AND ZIESKE, H.: Sympathetic and parasympathetic interactions upon the left ventricle of the dog. Circulation Res. 19: 5, BURN, J. H., AND RAND, M. J.: Sympathetic postganglionic mechanism. Nature 184: 163, BURN, J. H., AND RAND, M. J.: Acetylcholine in adrenergic transmission. Ann. Rev. Pharmacol. 5: 163, BURN, J. H.: Adrenergic transmission. Pharmacol. Rev. 18: 459, FERRY, C. B.: Cholinergic link hypothesis in adrenergic neuroeffector transmission. Physiol. Rev. 46: 420, COOPER, T., GILBERT, J. W., BLOODWEIX, R. D., AND CROUT, J. R.: Chronic extrinsic cardiac denervation by regional neural ablation. Circulation Res. 9: 275, SPANN, J. F., JR., CHTDSEY, C. A., POOL, P. E., AND BRAUNWALD, E.: Mechanism of norepinephrine depletion in experimental heart failure produced by aortic constriction in the guinea pig. Circulation Res. 17: 312, SPANN, J. F., JR., SONNENBLJCT, E. H., COOPER,

12 1108 BUCCINO, SONNENBLICK, COOPER, BRAUNWALD T., CHIDSEY, C. A., WILLMAN, V. L., AND BRAUNWALB, E.: Cardiac norepinephrine stores and the contractile state of heart muscle. Circulation Res. 19: 317, SONNENBLICK, E. H.: Force-velocity relations in mammalian heart muscle. Am. J. Physiol. 202: 931, BLINKS, J. R.: Field stimulation as a means of effecting the graded release of autonomic transmitters in isolated heart muscle. J. Pharmacol. Exptl. Therap. 151: 221, BURN, J. H., AND RAND, M. J.: Action of nicotine on the heart. Brit. Med. J. 1: 137, BURN, J. H., AND RAND, M. J.: Noradrenaline in artery walls and its dispersal by reserpine. Brit. Med. J. 1: 903, BURN, J. H., LEACH, E. H., RAND, M. J., AND THOMPSON, J. W.: Peripheral effects of nicotine and acetylcholine resembling those of sympathetic stimulation. J. Physiol. (London) 148: 332, BURN, J. H., AND RAND, M. J.: Sympathetic postganglionic cholinergic fibers. Brit. J. Pharmacol. 15: 56, ANCELAKOS, E. T., AND BLOOMQUIST, E.: Release of norepinephrine from isolated hearts by acetylcholine. Arch. Intern. Physiol. 73: 397, LA FARCE, C. C, MONROE, R. G., GAMBLE, W. J., ROSENTHAL, A., AND HAMMOND, R. P.: Left ventricular pressures and cardiac norepinephrine efflux. Federation Proc. 25: 336, WILLIAMS, J. F., SONNENBLICK, E. H., AND BRAUNWALD, E.: Determinants of atrial contractile force in the intact heart. Am. J. Physiol. 209: 1061, JACOBOWTTZ, D., COOPER, T., AND BARKER, H. B.: Histochemical and chemical studies of the localization of adrenergic and cholinergic nerves in normal and denervated cat hearts. Federation Proc. 25: 383, VOLLE, R. L., AND KOELLE, G. B.: Ganglionic stimulating and blocking agents. In The Pharmacological Basis of Therapeutics, 3rd ed., edited by Goodman, L. S., and Gilman, A. New York, The Macmillan Company, 1965, Chap. 27, p VOLLE, R. L.: Pharmacology of the autonomic nervous system. Ann. Rev. Pharmacol. 3: 129, CURTIS, D. R., AND ECCLES, R. M.: Excitation of Renshaw cells by pharmacologic agents applied electrophoretically. J. Physiol. (London) 141: 435, KHIJEVIC, K., AND PHILLIS, J. W.: Pharmacologic properties of acetylcholine sensitive cells in the cerebral cortex. J. Physiol. (London) 166: 328, CircuUtiom Ruircb. Vol. XIX, Dtc*mbtr 1966