Effects of endurance training on cholinergic and adrenergic receptors of rat heart

J Mol Cell Cardiol 16, 395-403 (1984)

E f f e c t s o f E n d u r a n c e T r a i n i n g on C h o l i n e r g i c a n d A d r e n e r g i c Receptors of Rat Heart R. Sanders Williams,* t Thomas F. Schaible, Timothy Bishop and Miriam Morey Departments of Medicine and Physiology (Cardiovascular Division) Duke University Medical Center, Durham, NC 27710 and Departments of Medicine and Physiology (Cardiology Division) Albert Einstein College of Medicine, Montefiore Hospital and Medical Center, Bronx, N Y 10467, USA (Received 8 April 1983, accepted in revised form 8 August 1983) R. S. WILLIAMS,T. F. SCHAIBLE,T. BISHOPANDM. MOREY. Effects of Endurance Training on Cholinergic and Adrenergic Receptors of Rat Heart. Journal of Molecular and Cellular Cardiology (1984) 16, 395-403. To test the hypothesis that alterations in adrenergic or cholinergic receptors occur in response to physical training, and that changes in receptor properties could be mechanistically important in producting the altered cardiovascular physiology of the trained state, we studied the effects of endurance training by swimming upon beta adrenergic, alpha adrenergic, and muscarinic cholinergic receptors of rat heart. Because of previously reported sex-related differences in the cardiac adaptation to training, male and female rats were studied separately. Despite tile occurrence of demonstrable training bradycardia in males, and of cardiac hypertrophy in females, there were no discernible effects of the training program upon the properties of cardiac beta adrenergic receptors. However, hearts from swimmers of both sexes demonstrated fewer numbers of muscarinic cholinergic and alpha adrenergic receptors than sedentary controls, without differences in the receptor affinities for antagonist or agonist compounds. These findings are inconsistent with the hypothesis that altered cardiac sensitivity to neurotransmitters contributes directly to training bradycardia. KEy WORDS: Physical conditioning; Autonomic nervous system; Beta adrenergic receptors; Alpha adrenergic receptors.

Introduction The regulation of cellular receptors for hormones and neurotransmitters has been the subject of extensive recent investigation [9, 18, 27]. In some cases, observations regarding changes in hormone receptors produced by various stimuli, including exercise training, have been convincingly related-to physiologic or clinical phenomena, and have extended our understanding of such phenomena to the molecular level [1, 2, 13, 24, 26]. Because a number of cardiovascular adaptations to physical training involve changes in the autonomic or hor-

monal control of circulatory function [6, 29], we sought to test the hypothesis that these physiological adaptations are related to alterations of adrenergic or cholinergic receptors of the heart. A prominent characteristic that distinguishes physically active from sedentary animals or humans is a slower heart rate, both at rest and during submaximal exercise workloads. Largely on the basis of studies involving selective adrenergic or cholingergic antagonist compounds, most investigators have agreed that training bradycardia is produced by both enhanced cholinergic tone

* Dr Williams is the recipient of NHLBI Young Investigator's Grant Number HL 25146. This work is also supported by the Pepsico Foundation, the Henry J. Kaiser Family Foundation, and by USPHS Grant Numbers HL 16037 and HL 25392. ~"To whom corrrespondence should be addressed. 0022-2828/84/050395+09 $03.00/0

~? 1984 Academic Press Inc. (London) Limited

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(more important for resting bradycardia) and diminished sympathetic drive to the sinus node (relatively more important for slow heart rates during submaximal exercise) [11, 12]. However, the relative contribution of altered neural input to the sinus node compared with altered intrinsic responsiveness to catecholamines or to ace@choline in producing the observed changes in autonomic tone that are induced by habitual exercise has remained controversial [4, 16, 25, 29, 31-33,

36]. Previous investigations in our laboratory revealed no discernible effects of 8 weeks of a moderate swimming program (75 min, 5• upon receptor n u m b e r or upon antagonist affinity for beta adrenergic or muscarinic cholinergic receptors in membrane particulate fractions derived from rat heart [34]. To address the possibility that different results might be observed with a greater duration of training, and to study female as well as male animals, we performed the additional experiments described in this communication. In addition, we have expanded our previous analysis of receptor properties to include quantification ofagonist as well as antagonist affinities, and to investigate the effects of training u p o n alpha and as well as beta adrenergic receptors. Methods

Conditioning program Male CD strain rats (Charles River) or female Wistar rats ten weeks of age and weighing 150 to 200 g were randomly allocated into a swimming and a sedentary group. At the onset of the conditioning program males swam in groups of 20 to 25 in a plastic tank 107 • 76 cm filled to a depth of 50 cm with water adjusted to 34~ The duration of the swimming was increased by 10 min daily until they swam continuously for 90 min. They maintained this schedule 5 days/week for 14 weeks. Both swimmers and controls were housed two animals/cage, given free access to food and water, and were maintained on a 12 h light-dark cycle. To control for the effects of handling and immersion, but to avoid any significant exertion, sedentary controls were removed from their cages weekly and placed for several minutes in 4 to

5 cm water at 34~ Female rats were subjected to the swimming protocol (75 min twice daily, 5 days/week for 8 weeks) previous employed by Scheuer and colleagues in numerous investigations [20, 28]. Control female animals were handled frequently, as outlined previously. At the completion of the conditioning program, resting heart rates were determined in male rats from electrocardiograms recorded from subdermal electrodes with the animals at ambient activity in individual cages. Swimmers were tested 16 to 20 h following their most recent swimming session. Electrocardiograms obtained via an Elema Mingograf 8-channel recorder at a paper speed of 25 cm/s were recorded for 16 s on two occasions 5 to 15 min apart. Resting heart rate was calculated as the mean from the two determinations. Heart rates during staged submaximal exercise on a rodent treadmill were determined in five randomly selected males from each group. These animals were habituated to the apparatus by walking 10 min at 0.8 km/h and 0% grade daily for 5 days. We judged this to be an inadequate stimulus for inducing training effects in the sedentary controls, and the swimmers continued their customary swimming regimen. Heart rates were recorded during the last 30 s of 3-min stages of exercise at 0.8 km/h at 0%, 3%, 5%, and 8% grade. Heart rate was not determined in females, but this training regimen has been shown to produce a conditioning effect as evidenced by cardiac hypertrophy and enhanced contractile performance [28] in addition to biochemical adaptations of contractile proteins and sacroplasmic retriculum [20]. Male animals were killed 16 to 24 h following their last swimming session by inhalation of 100% CO 2 for 1 min followed by a blow to the head. Female animals were anesthetized with ether. The hearts from both groups were rapidly excised, rinsed free of blood in sucrose buffer (0.25 M sucrose, 5 mM Tris-HC1, 1 m~t Mg C12, pH 7.5) at 0~ and frozen with Wollenberger tongs chilled in liquid nitrogen.

Membrane preparation and radioligand bindings We prepared myocardial m e m b r a n e suspensions by one of two published methods [3,

Endurance Training and Cardiac Receptors

35]. These preparations consist of sarcolemmal membranes containing adrenergic and cholinergic receptors, but have not been purified and also contain fragments of Ttubules, mitochondria, and other intracellular membranes. In pilot binding experiments the two techniques produced identical results, and differed only in the yield of membrane protein derived from a given mass of heart tissue. Except where specified below, the entire hearts from male animals including the atria were utilized to yield myocardial membrane preparations. Only the ventricles from frmale animals were used for preparation of membranes. We utilized (-) [sH] dihydroalprenolol (DHA) (Amersham; specific activity 80 Ci/mmol) to identify beta adrenergic receptors in cardiac membranes. For labelling cardiac alpha adrenergic receptors and muscarinic cholinergic receptors we utilized [3H] prazosin (Pfizer; specific activity 33 Ci/mmol), and racemic (males) or the 150

isomer (females) of [3HI quinuclidinyl benzilate (QNB) (New England Nuclear; specific activity 40 Ci/mmol) respectively. We incubated rat myocardial membranes (0.9 to 1.4 mg protein) in an assay volume of 900/~1 containing 50 mM Tris-HC1, 16.7 mM MgC12, pH 7.4 for 20 rain at 25~ with either varying concentrations of radioligand (saturation curves; Fig. 1) or a fixed concentration of radioligand and varying concentrations of unlabelled competing ligands (competition curves; Fig. 2). Bound radioactivity was separated from free radioactivity and quantitated as previously described [35]. We defined non-specific binding as the residual binding in the presence of 10-SM (-t-) propranolol (DHA-heart), 10-SM phentolamine ([SH] prazosin heart) or 10-6M unlabelled (-) Q NB (QNB-heart). We quantitated protein by the method of Lowry et aL [19]. Data analysis We analyzed saturation curves, illustrated in Figure 1, by a non-linear least squares curve

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FIGURE 1. Binding of (-) [3H] dihydroalprenolol (DHA) to cardiac beta adrenergic receptors (BAR). Closed symbols depict binding to cardiac membranes from conditioned male animals whereas open symbols represent the results from sedentary controls. The circles depict total radioligand binding at each concentration of DHA whereas the squares depict binding in the presence of 10-SM (+) propranolol (non-specific binding). Data points are mean values from three experiments each analyzing pooled membranes from four hearts in each group. Solid lines (sedentary animals) or dashed lines (conditioned animals) represent the computer-derived best fit of the binding data within each group as defined by a generalized formula for complex ligand-receptor interactions as described in the test. The saturation curves derived from four experiments utilizing hearts from female animals were very similar.

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FIGURE 2. Competition by unlabelled oxotremorine with [SH] quinuclidinyl benzilate (QNB) (0.25 riM) for muscarinic cholinergic receptors in cardiac membranes from male rats. Closed squares depict results in cardiac mernbranes derived from conditioned animals and open circles depict data from sedentary controls. Solid lines (sedentary animals) and dashed lines (conditioned animals) represent the computer-derived best fit of the binding data within each group as defined by a fourparameter generalized model for dose-response relationships as described in the text. Data points are mean values from three experiments each analyzing pooled membranes from four hearts in each group. We observed no differences in agonist competition curves between membranes from swimmers and sedentary controls of either sex for any class of receptors (alpha adrenergic, beta adrenergic, muscarinic cholinergic).

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fitting procedure employing a generalized model for complex ligand-receptor interactions and performed using an iterative program in PL/1 on a PDP 11/45 computer. The details of this analytical approach have been previously published [17]. To determine the statistical significance of differences in receptor affinity (KD) or receptor n u m b e r (Bmax) observed in m e m b r a n e fractions from trained as opposed to sedentary animals, each curve was first analyzed independently to determine the parameter estimates that provided the best fit of the observed data. The curves were then analyzed simultaneously constraining parameter estimates (K D or Bmax) of curve A to be equivalent to the corresponding estimates of curve B. The goodness of fit obtained by analyzing the data first independently, and then with this constraint, was assessed by measuring the residual variance between the data and each of the fitted curves, and comparing these variances by an F ratio test. For example, the K D estimated for radioligand binding in one tissue homogenate (A) would be considered significantly different from the K D estimates in a different homogenate (B) when the residual variance of curves constraining K D (A) = K D (B) was increased significantly (by F test) from the residual variance observed when KD (A) and K D (B) were estimated independently. We analyzed competition curves illustrated in Figure 2 by a four-parameter generalized model for dose-response relationships [10] and compared the relevant parameter estimates (slope factor (Pseudo-Hill coefficient) and half-maximal inhibitory concentration (ECs0)) observed for membranes derived

from exercising and sedentary animals by the statistical methods described above. We compared heart rates, heart weights, and body weights by one-way analysis of variance. These variables are reported as mean _+ S.D. Parameter estimates from radioligand binding curves are reported as mean + S.E. Results We observed the expected sequelae of physical training upon resting heart rate, body weight, and heart weight. Trained male animals (n = 34) had lower resting heart rates (338 + 29 v. 378 + 30 beats/rain; P < 0.01), lower heart rates during submaximal exercise (Table 1), failed to gain weight as fast as sedentary controls (n = 36) (final weights 449_+46 v. 576_+55g; P < 0 . 0 0 0 1 ) , and demonstrated a 7% increase in heart wet weight (1.7_+ 0.2 v. 1.6-+ 0.2 g; P < 0.01). Female swimmers (n = 16) displayed a more striking 17% increase in heart wet weight compared with controls (n = 13) (0.74 + 0.05 v. 0.63 _+ 0.05 g; P < 0.001), but only slightly lower body weights (241 -+ 12 v. 251 _+ 11 g; P < 0.05). The results of the radioligand binding studies of m e m b r a n e preparations derived from the hearts of these animals are summarized in Table 2. Beta adrenergic receptors (Fig. 1) were unaltered either in terms of receptor n u m b e r (Bmax), antagonist affinity, agonist affinity, or the slope factors of the agonist displacement curves. Deviation of this latter value from 1.0 reflects the formation of the guanine-nucleotide-sensitive, highaffinity binding complex that has been shown

TABLE 1. Heart rates during treadmill exercise Grade at 0.5 mph (%) 3 Heart rate (beats/min) (mean _+S.D.) Swimmers (n = 5) Sedentary controls (n = 5) a p < 0.05

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430+ 25 a 478•

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444• 476•

443+21 a 490_+32

Endurance Training and Cardiac Receptors

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T A B L E 2. B i n d i n g p r o p e r t i e s o f c a r d i a c r e c e p t o r s f r o m e x e r c i s e d a n d s e d e n t a r y rats

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0.11+_0.11 5.4_+2.1 6.3 +_ 2.3

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M F Agonist slope factor M F

aAll values are m e a n + s.E. of pooled data from duplicate determinations from 3 to 4 separate experiments. bF ratio test on parameter estimates from pooled data. M, males; F, females; SED,control group; EX, s w i m m i n g group; ISO, (--) isoproterenol; EPI, (-) epinephrine; NE, (-) norepinephrine; Bm~,, n u m b e r of binding sites; KD, dissociation constant; EC 50, half-maximal inhibitory concentrations.

in model systems to be an obligato W step in the activation of beta adrenergic and muscarinic cholinergic responses by agonist compounds [17]. Muscarinic cholinergic receptors (Table 2, Figs 2 and 3) were reduced in number by about 15% in hearts from both male and female swimmers, without significant change in any of the other binding properties. The apparently higher affinity for O_,NBin females is probably due to our shift to the 1-isomer from the racemic form used in the earlier studies of male animals.

Alpha adrenergic receptors were reduced in number bv 23% in female swimmers and by 14% in males (Table 2 and Fig. 4). No other receptor properties were altered. These changes were statistically significant for females (P= 0.001), but fell just short of statistical significance in males (P = 0.064). (-) [H s] Dihydroalprenolol does not distinguish B 1 from B2 receptors. Because there are recent data to suggest that cardiac B 2 receptors, as well as the more familiar cardiac B 1 receptors, may influence the chronotropic response to catecholamines [5], we sought to

400

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address the possibility that exercise training p r o d u c e s a shift in the relative p r o p o r t i o n s o f B 1 and B 2 receptors, Such a change w o u l d be expected to alter the relative affinities of 1e p i n e p h r i n e and 1-norepinephrine. As Table 2 indicates, we could detect no significant changes in the half-maximal inhibitory concentrations for inhibition o f D H A b i n d i n g to cardiac BAR o f these two c o m p o u n d s . Since the possibility exists that exercise training alters beta adrenergic receptors of the sinoatrial node, b u t that these effects w o u l d not be discernible in m e m b r a n e preparations derived f r o m whole hearts, we assessed (-) [SH]-dihydroalprenolol b i n d i n g to m e m b r a n e preparations derived f r o m

FIGURE 4. (a) Binding of[3H] prazosin to cardiac alpha adrenergic receptors from male rats. Symbols are explained in the legend to Figure 1, with the exception that 10-5Munlabelled phentolamine was used to define non-specific binding, and that data points are mean values from duplicate determinations in four experiments, each using a single heart from each group. (b) Binding of [aH] prazosin to cardiac alpha receptors from female rats. Symbols are identical to 4(a).

atrial tissue only. Technical limitations prohibited direct analysis o f sinus n o d e tissue, b u t we reasoned that atrial specimens w o u l d be enriched in specialized p a c e m a k e r and c o n d u c t i o n tissue, and w o u l d perhaps allow detection o f changes limited to these calls as o p p o s e d to working myocytes. In three experiments utilizing p o o l e d atrial specimens f r o m 30 male swimmers and 30 controls, we observed no differences in beta adrenergic receptor n u m b e r 32 + 2 v. 31 ___4 f m o l / m g protein: P = 0.877), or in antagonist affinity (2.0 + 0.5 v. 1.6 + 0.4 nM; P = 0.498).

Discussion

O u r current data s u p p o r t the hypothesis that the bradycardia o f physical training is

Endurance Training and Cardiac Receptors

mediated to a greater degree by altered autonomic neural input to the heart than by altered characteristics of the myocardial receptors for acetylcholine or for catechotamines. Confirming our previous findings [34] as well as those recently reported by Moore et al. [22], we could discern no alterations in the properties of cardiac beta adrenergic receptors in trained rats. Unlike our previous report, we did observe a downregulation ofmuscarinic cholinergic receptor number in cardiac tissue after a longer duration (males) or intensity (females) of physical training. This change would be expected to produce diminished cholinergic sensitivity in trained hearts, and does not support the hypothesis that enhanced responsiveness to acetylcholine plays a role in training bradycardia. Likewise, the downregulation of cardiac alpha adrenergic receptors we observed cannot, on the basis of existing knowledge regarding the physiologic effects of cardiac alpha receptor stimulation [21], be mechanistically related to training bradycardia in a plausible fashion. It should be recognized that our data were obtained from animals subjected to swimming as the training stimulus, and it is conceivable that different responses could occur in response to other forms of exercise. Likewise, our data cannot exclude the possibility that receptor changes occur within the sinus node that are different from those occurring elsewhere in the heart. However, these current findings that cardiac beta adrenergic receptors are unchanged by exercise conditioning are consistent with our previous human studies in which no differences in the chronotropic responsiveness to isoproterenol were observed in elite marathon runners as compared with age-matched sedentary control subjects [33]. Furthermore, no rightward shift of the concentration-activity relationship expressing exercising heart rate as a function of plasma catecholamine levels has been observed in longitudinal studies of physical conditioning in humans [14]. Although techniques for directly quantitating autonomic nerve discharge rates during exercise in either animals or humans are unavailable, the fact that conditioned subjects have lower plasma catecholamine levels at rest and during submaximal exercise [7, 15] provides additional

401

evidence that sympathetic nerve activity is reduced in trained individuals. The hearts of animals participating in endurance training are exposed transiently to higher levels of catecholamines and to a withdrawal of cholinergic tone during their daily exercise periods, yet during most of the day encounter reduced adrenergic tone and enhanced cholinergic tone in the resting state [11, 12]. Therefore, the manner in which agonist-induced down-regulation of receptor number, a prominent feature of receptor regulation in model adrenergic and cholinergic systems [13, 18], could be involved in producing the results we observed is difficult to delineate. It seems plausible that down-regulation of muscarinic cholinergic receptors is attributable to resting vagotonia in trained animals, but that the degree of resting adrenergic withdrawal in swimmers is insufficient to produce a concomitant upregulation of beta adrenergic receptors. Alternatively, the kinetics of agonist-induced receptor regulation may differ between the cholinergic and adrenergic systems, such that down-regulation of adrenergic receptors during acute exercise counterbalanced (beta receptors) or exceeded (alpha receptors) upregulation due to reduced resting adrenergic tone at the time point at which we assessed receptor properties. Furthermore, numerous hormonal influences other than agonist exposure are known to modulate receptor number [9, 19, 27] and may, in an as yet undetermined fashion, affect the responses of cardiac receptors to physical training. The changes we observed in alpha adrenergic receptor number invite some interesting speculation on the basis of recent data suggesting an important role of cardiac alpha receptors in mediating ventricular arrhythmias during acute myocardial ischemia. Sheridan et al. [30] have reported that phentolamine but not propranolol prevents ventricular arrhythmias during acute coronary occlusion and reperfusion in dogs, and both these and other investigators have observed up-regulation of cardiac alpha adrenergic receptors during acute ischemia [8, 23]. If stimulation of cardiac alpha adrenergic receptors is an important determinant of arrhythmia risk during acute myocardial ischemia, a down-regulation of

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cardiac alpha receptors by exercise training c o u l d e x e r t a p r o t e c t i v e effect. A p o t e n t i a l relationship between exercise-induced downregulation of cardiac alpha-adrenergic receptors and reduced arrhythmogenesis during myocardia ischemia and reperfusion, t h o u g h h i g h l y s p e c u l a t i v e at this p o i n t , w o u l d a p p e a r to m e r i t f u r t h e r study.

Acknowledgments T h e a u t h o r s a r e g r a t e f u l to M r R o b e r t T e e r f o r his t e c h n i c a l assistance, to M r J a c k Kasell for his a s s i s t a n c e in t h e h e a r t rate r e c o r d i n g s , to D r A n d r e D e L e a n for statistical advice, a n d to M s J a n i c e J a m e s a n d Ms R i t a O d e n f o r t h e preparation of the manuscript.

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