In vitro autoradiographic localization of binding to angiotensin receptors in the rat heart

International Elsevier

CARD10

Journal of Cardiology,

25

28 (1990) 25-33

01053

In vitro autoradiographic localization of binding to angiotensin receptors in the rat heart A.M. Allen, H. Yamada Department

and F.A.O. Mendelsohn

of Medicine, University of Melbourne, Austin Hospital, Heidelberg, (Received

14 August

1989; revision

accepted

18 December

Allen AM, Yamada H, Mendelsohn FAO. In vitro autoradiographic receptors in the rat heart. Int J Cardiol 1990;27:25-33.

Victoria, Australia

1989)

localization

of binding

to angiotensin

The distribution of binding to angiotensin II receptors in the rat heart was determined by quantitative in vitro autoradiography employing ‘251-[Sar1, De’] angiotensin II as the radioligand. Low density binding occurs in the myocardium of both the atriums and ventricles and in the media of the aorta, pulmonary arteries and superior caval vein. Dense punctate binding is found over parasympathetic nerve bundles and some cells of the intracardiac ganglions. Binding is very low in nerves which do not stain for acetylcholinesterase. A moderate to high density of binding sites occurs throughout the conduction system, including the sinus node (low), the atrioventricular node (high) and the atrioventricular bundle (moderate). An extremely high density of binding is observed over the remnant of the arterial duct. These findings demonstrate many sites at which angiotensin II could exert its cardiac actions, including anatomical evidence supporting an action on cardiac myocytes, terminals of the vagal nerves, parasympathetic ganglion cells, and the conduction system. Key words:

Angiotensin

II; Receptor;

Heart;

Autonomic

Introduction Angiotensin II has potent actions on both indirectly (through fluid and homeostasis and regulation of vascular directly [1,2]. The direct actions include

the heart, electrolyte tone) and a positive

Correspondence to: A.M. Allen, Dept. of Medicine, Austin Hospital, Heidelberg, Victoria 3084, Australia. This work was supported by the National Heart Foundation of Australia, the National Health & Medical Research Council of Australia, and the Austin Hospital Medical Research Foundation. A.M. Allen is a National Health & Medical Research Foundation Biomedical Research Postgraduate Scholar.

0167-5273/90/$03.50

0 1990 Elsevier Science

Publishers

nervous

system

inotropic effect [3,4], mediated via receptors observed on fractions of myocardial membranes [5,6] and, more recently, on cultured cardiac myocytes [7]. Angiotensin II also exerts chronotropic effects through postjunctional myocardial receptors [8,9], stimulation of cardiac sympathetic efferent activity [lo-131, prejunctional potentiation of sym-’ pathetic function [14], central inhibition of cardiac vagal efferent activity [15,16], and prejunctional inhibition of cardiac vagal efferent activity [17] at the cardiac ganglions [18]. Angiotensin II also has direct actions on the cells of the ventricular conduction tissues [ 191. We have observed transport of receptors for angiotensin II in the peripherally projecting vagus

B.V. (Biomedical

Division)

26

nerve [20]. This has also been demonstrated

in the dog [21]. One likely destination for these sites of binding is the heart, since a prejunctional effect of angiotensin II on cardiac vagal motor activity has been described [17]. Considering this anatomical evidence, the multiple effects of angiotensin II on cardiac function, and the clinical importance of agents which block the renin-angiotensin system in the treatment of hypertension and heart failure [22,23], we have mapped the distribution of binding sites for angiotensin II in the heart by in vitro autoradiography. The association of receptors for angiotensin II with cardiac muscle has been reported [5-71, and there are recent reports of binding sites in the conduction system of the rat heart [24]. The purpose of this paper is to map systematically the distribution of receptors for angiotensin II throughout the rat heart. Materials and Methods Tissue

Male Sprague Dawley rats (n = 5) were killed by decapitation and the heart removed. The heart was bisected between atriums and ventricles, filled with Optimal cutting temperature compound (Tissue Tek, Miles Inc., IN, U.S.A.), and frozen in isopentane on dry ice (- 40” C). Serial 10 pm or 20 pm sections were cut on a cryostat at - 20 o C, mounted onto gelatin-coated slides and dehydrated under reduced pressure at 4“ C before storage at -80°C. Sections were either submitted to in vitro autoradiography for localization of binding sites for angiotensin II, stained with haematoxylin and eosin or stained for localization of acetylcholinesterase. A further three rats were decapitated and the hearts dissected into blocks containing the major components of the conduction system. For the sinus node, the superior caval vein together with its junction with the right atria1 appendage were removed, frozen in isopentane and sectioned as described above. For the atrioventricular node, the heart was bisected below the atrioventricular junction and the atriums removed, leaving the inter-atria1 septum intact. This was then frozen and prepared as described above.

In vitro autoradiography

Binding sites were localized by in vitro autoradiography employing ‘*‘I-[Sar’, Ile*] angiotensin II as radioligand. The method has been described in detail elsewhere [25,26]. Sections were preincubated at room temperature in 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl, 5 mM Na,EDTA, 0.14 mM bacitracin and 0.2% bovine serum albumin, to remove endogenous ligand, prior to incubation for 1 hour at room temperature in fresh buffer containing ‘251[Sar’, Iles] angiotensin II (0.2 pCi/ml; - 130 PM). Following incubation, the slides were washed in cold Tris-buffered saline (pH 7.4) before drying and exposure to Agfa Scopix CR3B X-ray film (Agfa Gevaert, Belgium) for 3-5 weeks. Standards, prepared by applying known amounts of ‘251-radioactivity to 20 pm sections of 5 mm rat brain cores mounted on gelatin-coated slides, were exposed with the sections to enable quantitation. Non-specific binding was determined by the addition of 1 PM unlabelled angiotensin II amide (Hypertensin, Ciba, .NJ, U.S.A.) to the incubation. Acetylcholinesterase

histochemistry

Sections were incubated overnight (approximately 16 hours) in 50 mM sodium acetate solution (pH 5.0) containing 4 mM copper sulphate glycine, 0.12% S-acetylthiocholine iodide and 0.003% ethopropazine [27]. The slides were rinsed in tap water prior to developing in a 1% solution of sodium sulphide (pH 7.4) for 10 minutes. The sections were then rinsed, fixed in 4% buffered paraformaldehyde and mounted. Quantitation

Selected regions of the rat heart were quantitated using a microcomputer imaging device system (Imaging Research Inc. Ontario, Canada) operated by an IBM AT computer with a high resolution CCD camera (Sierra Scientific, CA, U.S.A.). The radioactive standards were fitted to a calibration curve by the computer to enable conversion of optical density to radioactivity per unit area

27

(dpm/mm’). Three serial 20 pm sections of each region from each of three rats were quantitated and are expressed as means f standard error of the mean.

lb, d), superior caval veins (Fig. 2a) and the large coronary arteries (Fig. lh), display moderate levels of receptor binding (Table 1). Autonomic

Results Myocardium Low levels of angiotensin II receptors occur over cardiac muscle throughout both the atriums and ventricles: Binding tends to be higher in the atriums than in the ventricles (Table 1; Figs. If, h and 2~). Dense punctate binding of angiotensin II is found over the epicardium of the atria1 muscle (Figs. lb, d and 2c, Table 1). The resolution obtained, however, did not allow further identification of these structures. Subjacent to the origin of the leaflets of the aortic valve, the density of binding is increased compared to the remaining aortic smooth muscle (Fig. If). Large vessels The medial walls of all the great vessels, including the aorta (Fig. lb, d), pulmonary arteries (Fig.

nervous system

Dense streaks of angiotensin II binding occur in the vagus and recurrent laryngeal nerves, and in the vagal branches to the heart (Figs. Id and 3e, Table 1). Low density binding is also observed in some cholinesterase-negative nerves, but this was not prominent. Light binding is associated with intracardiac ganglion cells, with high density punctate binding occurring over a small proportion ( < 5%) of the cells (Fig. 3h, Table 1). This dense binding encompasses approximately 3-5 cells within some sections of the ganglions. Punctate binding is observed over cells in all the ganglions described by Pardini and colleagues [28], including those situated around the aorta and superior caval vein, the interatrial septum (inferior to the bifurcation of the pulmonary arteries), in the wall of the right atrium and between the left atrium and bronchus. Conduction system

TABLE Regional

1 densities

of cardiac

Region

angiotensin

II receptor

binding

Mean + SEM (dpm/mm’)

Atria1 myocardium Epicardial puncta Ventricular myocardium Aorta and pulmonary arteries (media) Aorta and pulmonary arteries (adventitia) Myocardium adjacent to origin of valve leaflets Choline@ nerve bundles Intracardiac ganglions Atrioventricular node Sinus node Remnant of arterial duct

9.7+ 61.3+ 8.3f 24.6k

0.4 8.7 0.8 0.5

32.8+

3.5

42.5+

2.3

The conduction system of the rat heart contains light binding in the sinus node (not shown, Table 1) and moderate to high density binding in the atrioventricular node (Figs. lh, 3b, Table l), atrioventricular bundle, and left and right bundle branches (Fig. lh). Arterial duct

47.2* 5.0 74.8* 5.1 52.7+ 5.9 18.8+ 1.8 252 +32

Table of regional densities of angiotensin II receptor binding sites in the heart. Values (mean + SEM, dpm/mm’) were obtained from three sections from each of three rat hearts, with the exception of the sinoatrial node which was from two sections from each of two hearts only. Non-specific binding (3-5 dpm/mm’) has been subtracted.

The most striking receptor binding is observed in a region superior to the bifurcation of the pulmonary arteries, this area stretching to the descending aorta. This is the highest level of bind-’ ing in the entire rat heart and corresponds to the region of the remnant of the arterial duct (Fig. lb, Table 1). All animals displayed consistent binding of angiotensin II within the areas of the heart described. All binding described was displaced totally when incubated in the presence of 1 PM unlabelled angiotensin II (Fig. 2a-d), indicating

28

Fig. 1. Photomicrographs of coronal sections of rat heart (a, c, e, g) and autoradiographs demonstrating angiotensin II receptor binding sites (b, d, f, h). The stained sections are immediately adjacent to the sections used for autoradiographs and were stained with haematoxylin and eosin. AAo = ascending aorta; AoV = aortic valve; AVN = atrioventricular node; Br = bronchus; ca = coronary artery; DA = ductus arteriosus remnant: DAo = descending aorta; LA = left atrium; LV = left ventricle; N = nerve; Pa = pulmonary artery; PV = pulmonary vein; RA = right atrium; RV = right ventricle.

29

Fig. 2. Coronal sections of the rat heart demonstrating total (a, c) and non-specific (b, d) binding of ‘251-[Sar’, Iles] angiotensin II in two representative sections. Non-specific binding is performed in the presence of 1 pM unlabelled angiotensin II. 2a is superior to the section in la, whilst 2c is approximately at the level shown in lg. Abbreviations are as in Fig. 1; SVC = superior vena cava; Tr = trachea.

that non-specific binding of “‘1-[Sar’, Ile*] angiotensin to the rat heart is very low (Fig. 2). The autoradiographs shown in the figures thus represent specific binding.

Discussion This paper describes the distribution of binding to receptors for angiotensin II throughout the rat

Fig. 3. Photomicrographs of coronal sections of rat heart demonstrating the atrioventricular node (a-c), a cardiac nerve (N) (d-f ) and an intracardiac ganglion (G) (g-i). Sections a, d and g are stained with haematoxylin and eosin and are immediately adjacent (20 pm) to the autoradiographs representing angiotensin II receptor binding sites (b, e, f). Sections c, f and i are stained for acetylcholinestl erase and are 40-60 pm from the corresponding sections used for autoradiography. Consequently the acetylcholinesterase positive CellS shown in “i” do not correspond exactly to those containing angiotensin II receptors (h). However, the binding in “h” does 01rerlie nerve cell bodies stained with haematoxylin and eosin shown in “g” (see arrows). Abbreviations not included in Fig. 1 are: bv = blood vessel; G = intracardiac ganglion.

31

heart. Such receptors have been described in membranes from rat heart [5,6], and in cultured rat cardiomyocytes, where this peptides stimulates the frequency of contraction [7]. This study demonstrates that the density of receptors is relatively low in the cardiac muscle. Binding in the atriums tends to be higher than that in the ventricles. The highest density of binding in the atriums was found in dense punctate areas on the atria1 surface, associated with epicardium. As demonstrated previously for vascular smooth muscle elsewhere [29], we observe moderate levels of binding in the medial walls of the large cardiac vessels and some large coronary vessels. The highest density of binding in the rat heart is found overlying the arterial ligament (remnant of the arterial duct) between the junction of the pulmonary arteries and the descending aorta. The function of these receptors, if any, presents an intriguing problem in the adult heart. Pardini et al. [28] described the distribution of four intracardiac collections of ganglion cells which project to specific sites in the heart. They suggested each cell group would control parasympathetic nerve activity in different regions. We found sites of binding of receptors for angiotensin II over a small proportion of cells ( < 5%) in each of the intracardiac ganglions, thus making it impossible to ascribe an action of angiotensin II to regional parasympathetic nervous activity. Neurons rich in receptors for angiotensin II were found clumped together in the ganglions in groups of 3-5 cells. Previous investigators have suggested that angiotensin II modulates release of acetylcholine from vagal motor neurons at intracardiac ganglions [18]. This observation is strengthened by our results in both this and a previous study [20]. We demonstrate in this paper that receptors are found in high concentrations in acetylcholinesterasepositive nerve bundles, as well as over cells in the cardiac ganglions. Previously, we have shown that receptors for angiotensin II are transported distally in the vagal trunk [20]. Together, these results suggest that the receptors may be produced in the vagal motor neurones and transported in the vagus to the nerve terminals in the intracardiac gan-

glions to mediate inhibition of release of acetylcholine by angiotensin II. Vagal motor neurones projecting to the heart mostly originate in the nucleus ambiguus, with only a small percentage being found in the dorsal motor nucleus [30]. The nucleus ambiguus, however, is conspicuously devoid of binding sites for angiotensin II [31]. In contrast, the dorsal motor nucleus contains a high density of receptors [31]. Thus, the sparsity of projections of the dorsal motor nucleus to the heart may correlate with the small proportion of cells in the intracardiac ganglions which possess binding sites for angiotensin II. The distribution of 125I-[Sar’, Ile8] Ang II binding in the human heart has recently been described [32]. Neuronal binding in this species was associated with sympathetic, and not parasympathetic nerve bundles. This observation supports the demonstration that angiotensin II potentiates sympathetic nervous activity in the heart by a presynaptic mechanism [14]. However, we cannot detect significant levels of angiotensin II receptor binding in non-cholinesterase containing nerve bundles in rat atriums or ventricles. One possible explanation may be that the observed nerve bundles in the rat heart contain mixed populations of sympathetic and parasympathetic nerves. However, in preliminary experiments, removal of the middle and inferior cervical ganglia in the rat [33], which results in a 95% reduction of nonadrenaline content in the cardiac ventricles, has no detectable effect on the angiotensin II binding in the nerve bundles, intracardiac ganglions or conduction tissue in the rat atrium described here (unpublished observations). Previously reported [24], we have demonstrated angiotensin II receptors throughout the conduction system of the rat heart. Unfortunately, the resolution obtained did not allow us to determine whether these receptors were associated with the innervation of this region, or the cells of the conduction system. Angiotensin II induces tachycardia independently of release of catecholamine [9] and increases strength of contraction and potentiates the action potential of the peripheral fibres of the ventricular conduction system [19].

32

Further work is required to determine the direct interactions of angiotensin II with this conduction system. The distribution of angiotensin converting enzyme has recently been mapped in the rat heart by in vitro autoradiography [34]. This enzyme occurs in high concentrations in the valvar leaflets, the endothelial and adventitial layers of the great vessels, and in moderate concentrations in the myocardium. No angiotensin converting enzyme could be detected in either nervous tissue or the conduction system. Thus, localization of receptors for angiotensin II and angiotensin converting enzyme are not directly correlated. The distribution of angiotensin converting enzyme in sites exposed to high blood flows, nonetheless, suggests the intracardiac production of angiotensin II. This angiotensin II could then diffuse to its sites of action. It is also possible that angiotensin II may be present in components of the autonomic nervous system and thus be released to access the receptors observed in this study. Experiments localizing the distribution of angiotensin II in autonomic ganglia and nerves by immunohistochemistry would answer this question. Overall, this study supports a wide role for angiotensin II in the regulation of cardiac function, both directly on cardiac myocytes, and indirectly through modulation of cardiac autonomic activity.

Acknowledgement We thank Dr. T. Shimada, Ohita School, Japan for assistance in identifying nents of the conduction system.

Medical compo-

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