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Repeated swimming stress and responsiveness of the isolated rat pacemaker to the chronotropic effect of noradrenaline and isoprenaline: Role of adrenal corticosteroids
Gen. Pharmac. Vol. 19, No. 4, pp. 553-557, 1988 Printed in Great Britain. All rights reserved
0306-3623/88 $3.00+ 0.00 Copyright © 1988 Pergamon Press plc
REPEATED SWIMMING STRESS A N D RESPONSIVENESS OF THE ISOLATED RAT PACEMAKER TO THE CHRONOTROPIC EFFECT OF N O R A D R E N A L I N E A N D ISOPRENALINE: ROLE OF A D R E N A L CORTICOSTEROIDS REGINA CI~L1A SPADARII and Sf~RGIO DE MORAES2. tDepartment of Physiology and Biophysics, Institute of Biology and 2Department of Pharmacology, Faculty of Medical Sciences, Universidade Estadual de Campinas, Campifias, Silo Paulo, Brazil (Received 29 September 1987)
Abstract--1. Repeated swimming stress (three daily sessions) resulted in an increased plasma corticosterone level and subsensitivity of the isolated rat pacemaker to noradrenaline and isoprenaline. 2. Repeated swimming stress was found to decrease the affinity of fll-adrenoreceptors for metoprolol. 3. Bilateral adrenalectomy performed 2 days before repeated swimming stress abolished the development of pacemaker subsensitivity to noradrenaline and isoprenaline and the decrease in fll-adrenoreceptors affinity for metoprolol. 4. It is concluded that adrenal corticosteroids, at least partially, mediate the swimming stress-induced subsensitivity of the isolated rat pacemaker to noradrenaline and isoprenaline. INTRODUCTION It is generally accepted that repeated stress causes subsensitivity of rat brain slices to the noradrenaline (NA)-cyclic A M P generating effect (Stone, 1979a,b; Stone and Platt, 1982; Stone et al., 1985). The mechanisms by which repeated stress alters brain cyclic A M P response have not been ascertained yet. It has been reported that repeated restraint stress produces a reduction of rat brain fl-adrenoceptors density (U'Prichard and Kvetnansk~, 1980) which mediates the cyclic A M P response to N A and isoprenaline (ISO) in rat brain tissue. The sensitivity of the brain /~-adrenoceptors-adenylate cyclase system to N A is increased after adrenalectomy (Mobley et al., 1983) or decreased after administration of A C T H (Kendall et al., 1982; Stone et al., 1986), suggesting that adrenal corticosteroids play a role in the modulation of the brain fl-adrenoceptor-adenylate cyclase system sensitivity to NA. Contrasting with brain slice studies, few attempts have been made to study the sensitivity to catecholamines in peripheral isolated fl-adrenoceptor-containing tissues, such as the rat pacemaker, during repeated stressful situations. Pacemakers isolated from cold-stressed rats showed subsensitivity to the chronotropic effect of N A probably caused by a conformational alteration of pacemaker fl~-adrenoceptors (Callia and De Moraes, 1984). Recently, it has been reported that repeated footshock stress induces subsensitivity to N A and supersensitivity to ISO (Bassani and De Moraes, 1987). Acute swimming stress increases the sensitivity to ISO leaving unaltered that to N A (Spadari et al., 1988). Taken together, these studies suggest, as previously proposed by Stone (1979a), that fl-adrenoceptor subsensitivity to N A is a mechanism related to adaptation to repeated stress both in brain and peripheral tissues. *To whom all correspondence should be addressed.
It is the aim of the present report to extend our previous findings on the effects of a single 50-min swimming session on the sensitivity of the isolated rat pacemaker to the chronotropic effect of N A and ISO (Spadari et aL, 1988). In the present experiments rats were submitted to three daily swimming sessions of 5, 15 and 30 min, respectively. Our intention was also to study the role played by adrenal steroids on the repeated swimming stress-induced subsensitivity to the chronotropic effect of N A and ISO. MATERIALS AND METHODS
Stress protocol Male Wistar rats (200-250 g) were used. The animals were housed individually in a well-ventilated room, in plastic cages with standard laboratory chow and tapwater freely available, during a week prior to the experiments. Forced swimming stress was obtained by placing each rat into a glass tank filled with lukewarm water (28-30°C) and measuring 70 cm length, 44 cm width and 50 cm depth. The animals were submitted to swimming during three subsequent days, the duration of each session being 5, 15 and 30 min, respectively. Immediately after the ending of the swimming sessions the animals were returned to their homecages. All swimming sessions were carried out between 10:00 and 11:00 a.m. Naive rats were used as control. Plasma corticosterone levels Immediately at the ending of the third swimming session, rats were given an i.p. injection of sodium pentobarbital (45 mg/kg). After 45 min an abdominal incision was made and blood (4-5 ml) withdrawn from the right renal vein, collected into heparinized vials and centrifuged (2300 g) at 5°C. Plasma corticosterone levels were fluorimetrically determined according to Mattingiy (1962) as reported elsewhere (Callia and De Moraes, 1984). Bilateral adrenalectomy Rats were anesthetized with ether and the adrenal glands removed via the dorsal route, 2 days before the first swimming session. The animals were post-operatively maintained on 0.9% NaC1. Completeness of adrenalectomy was
553
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REGINA Cf:LIA SPADARIand SI~RGIODE MORAES
verified after the third swimming session by determination of plasma corticosterone levels as described above. Naive sham-operated rats were used as control.
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Organ-bath studies 7) After the third swimming session, rats were killed by a ' blow to the back of the head. Hearts were immediately removed and atria were isolated and suspended in 20 ml tu organ-baths containing Krebs-Henseleit solution of the following mM composition: NaCI, 115.0; KC1, 4.6; IIJ CaC12.2H20, 2.5; KH2PO 4, 1.2; MgSO4.7H20, 2.5; NaHCO3, 25.0 and glucose II.0. To minimize oxidation of I O 10 8) the two catecholamines 0.I 1 mM of ascorbic acid was Eg included. Diastolic tension was enough to permit recording o O of the spontaneous beating (0.5 cm/beat). After the equiO libration period of 1 hr with changes of bathing medium at tD z 15min intervals, a stable resting rate was reached. Precautions were taken to avoid the influence of junctional Z catecholamine dissipating mechanisms on the pacemaker o (-~ t¢) sensitivity to the chronotropic effect of the catecbolamines. Tissues were incubated with phenoxybenzamine (10/tM) during 15 rain followed by a thorough washout period of 30 min in order to block ~t-adrenoceptors and extraneuronal uptake (O'Donnell and Wanstall, 1979). When NA was used Fig. I. Plasma corticosterone levels of rats submitted to the experiments were carried out in the presence of cocaine three daily swimming sessions of 5, 15 and 30 min duration. (10 #M) to impair the neuronal uptake process. To reduce The plasma level of the steroid was determined 45 min after the possible participation of endogenous NA, atria were in the ending of the third swimming session. Naive rats i.e. vitro chemically denervated using 6-hydroxydopamine animals not submitted to swimming were used as control. (Aprigliano and Hermsmeyer, 1976). The efficiency of such Adrenalectomy (ADX) was performed 2 days before the procedure has been previously assessed (Callia and De first swimming session. Vertical bars indicate SEM. NumMoraes, 1984). Full cumulative concentration-effect curves bers in parentheses indicate the number of experiments. to ISO or NA were determined. Only one *P < 0.05 in relation to the control group. Plasma corticosterone in the control group was 10.3 ___1.9 itg/100 ml of concentration-effect curve was obtained with each atrium. plasma. The agonist concentration producing an effect which is 50% of maximum (ECs0) was calculated and presented as mean negative logarithm (pD2). swimming session attempted to escape, showing signs pA 2 values of metoprolol, a selective fll-adrenoceptor of emotional distress. Curiously, removal of the antagonist was determined according to Arunlakshana and Schild (1959). Full cumulative concentration-effect curves adrenal glands did not completely eliminate the to NA were obtained in the absence and presence of plasma level of corticosterone. However, the plasma metoprolol (1 300nM). The antagonist equilibrium time level of the steroid did not increase after the third was 60 min. Dose ratios (DR) were calculated by dividing swimming session, when compared to the remaining the ECs0 of NA in the presence and absence of metoprolol. level of the steroid observed in adrenalectomized rats Schild plots were drawn and the slopes determined to not submitted to swimming. The data on the effects validate pA 2 values which were also calculated according to of repeated swimming stress on the plasma level of the formula (Mackay, 1978): corticosterone are shown in Fig. 1. pA 2 = log (DR - 1) - log [B] There were no differences in resting rates and the maximum responses to the chronotropic effects of where [B] corresponds to the molar concentration of N A and ISO in isolated pacemakers of control and metoprolol. swimming-stressed rats. Swimming during three daily Statistical analysis sessions of 5, 15 and 30 min resulted in rightward The Student t-test for unpaired samples was used for the displacements of the concentration--effect curve to difference between means. Analysis of variance was also N A and ISO. The subsensitivity of the pacemaker to used. Schild plots were drawn using linear regression calcu- N A was 2.5-fold at the p D 2 level (P < 0.05) and that lated by the least squares method. to ISO was 7.9-fold (P <0.01). These data are presented in Table 1 and illustrated by Figs 2 and 3. Bilateral adrenalectomy carried out 2 days before the RESULTS first swimming session abolished the effects of repeated swimming stress on the pacemaker sensitivity As previously reported (Spadari et al., 1988) rats to the neurotransmitter (Fig. 4) and to ISO (result not swam vigorously, attempted to escape and showed shown). defecation during the first swimming session. HowFigure 5 depicted pA 2 values of metoprolol using ever, during the second swimming session the animals N A as the agonist. Pacemakers isolated from swam calmly and quickly learnt how to prop themselves with their hindlegs keeping their heads above swimming-stressed rats showed a statistically significant increase in the pA2 value of metoprolol the water. The animals displayed this habituation (5.7-fold, P < 0.01) indicating a reduction in the behavior during all the third swimming session. Plasma corticosterone levels were increased after the fl~-adrenoceptors affinity for the antagonist. Howthird swimming session as compared to naive rats. ever, pacemakers isolated from previously bilaterally Bilaterally adrenalectomized rats did not show any adrenalectomized rats which were submitted to the sign of habituation to swimming and even in the third swimming sessions showed pA2 values of metoprolol
Stress effects on rat pacemaker #1 adrenoreceptor
555
Table 1. The effects of swimming stress on the chronotropic response of the isolated rat pacemaker to noradrenaline and isoprenaline. Adrenalectomy (ADX) was performed 2 days before the first swimming session. Shown are mean values with 95% confidence intervals Group
Noradrenaline pD~
Na
Control Swimming ADX
8 8 8
8.24(8.01-8.47) 7.83(7.50-8.10)* 8.15(7.71-8.39)
Isoprenaline pD 2
Ratio: N" -2.57 1.23
6 6 6
Ratio c
9.64(9.20-10.0) 8.74(8.48-9.0)** 9.30(9.08-9.45)
-7.94 2.18
aNumber of experiments. bNegative logarithm of molar concentration of the agnnist producing a response which is 50% of the maximum. CAntilogarithm of the difference between pD 2 values. *Significantly different from the control group (P < 0.05). **Significantly different from the control group (P < 0.01).
which did n o t differ from those o b t a i n e d in the control group. In all experimental groups, slopes of Schild plots were n o t statistically different from 1.0 (Table 2).
DISCUSSION
The present report shows t h a t three daily swimming sessions o f 5, 15 a n d 30 m i n respectively, caused subsensitivity of the isolated rat p a c e m a k e r to the
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Fig. 2. Mean concentration-effect curves for the cbronotropic effect of ISO in the isolated rat pacemaker. Rats were submitted to three daily swimming sessions of 5, 15 and 30 min duration (swimming). Naive rats i.e. rats not submitted to swimming were used as control. Vertical bars indicate SEM. Numbers in parentheses indicate the number of experiments. loo
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Fig. 4. Mean concentration-effect curves for the chronotropic effect of NA in the isolated rat pacemaker. Rats were submitted to bilateral adrenalectomy performed 2 days before the first swimming session and submitted to the three daily swimming sessions of 5, 15 and 30min duration (ADX-swimming). Naive sham-operated rats were used as control. Vertical bars indicate SEM. Numbers in parentheses indicate number of experiments. 3.0
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Fig. 3. Mean concentration-effect curves for the chronotropic effect of NA in the isolated rat pacemaker. Rats were submitted to three daily swimming sessions of 5, 15 and 30 min duration (swimming). Naive rats i.e. rats not submitted to swimming were used as control. Vertical bars indicate SEM. Number in parentheses indicate the number of experiments.
t 5
, 6
7
8
9
.eTOPROLO,[-Log.] Fig. 5. Schild plots for the antagonism of metoprolol vs the chronotropic effect of NA in the isolated rat pacemaker. Rats were submitted to three daily swimming sessions of 5, 15 and 30min (swimming) or submitted to bilateral adrenalectomy performed 2 days before the first swimming session and submitted to three daily swimming sessions of 5, 15 and 30 min duration (ADX-swimming). Naive shamoperated animals were used as control. Numbers in parentheses indicate the combined number of points and animals.
556
REGINA CI~LIASPADARIand SI~RGIODE MORAES
Table 2. The effects of swimming stress on pA 2 values of metoprolol in the isolated rat pacemaker. Noradrenaline was used as the agonist. Adrenalectomy (ADX) was performed 2 days before the first swimming session. Shown are mean values with 95% confidence intervals Group
Na
pA2b
Slope
Control Swimming ADX
9 16 13
8.28 (8.22-8.35) 7.52 (7.40-7.70)* 8.04 (7.95-8.14)
1.0 (0.80-1.20) 0.90 (0.60-1.20) 1.0 (0.70-1. I 0)
aNumber of experiments. bpA2 = log (DR - 1) - log [B] (Mackay, 1978). *Significantly different from the control group (P > 0.01).
chronotropic effect of ISO and NA. Previous studies have shown that a single swimming session of 50 min duration increased the pacemaker sensitivity to ISO leaving unaltered the sensitivity to NA (Spadari et al., 1988). The development of subsensitivity to the NA cyclic A M P generating effect in rat brain tissue after repeated stress is thought to be related to the mechanisms of cell adaptation (Stone, 1979a,b). Recently, we have reported that repeated footshock stress decreases the number of pacemaker fll-adrenoceptors but causes subsensitivity to the chronotropic effect of NA through an increased efficiency of the neuronal reuptake process (Bassani and De Moraes, 1987). It has been reported that repeated restraint stress decreases the fl-adrenoceptors density of rat heart (U'Prichard and Kvetnansk2~, 1980). However, a discrete reduction in the number of fl~-adrenoceptors would not be expected to shift to the right the concentration-effect curve to ISO and NA since as reported (Buckner et al., 1978) there is a large amount of pacemaker spare flt-adrenoceptors in rat heart. An increase in the efficiency of the pacemaker catecholamine-dissipating mechanisms during the repeated swimming stress has to be ruled out because all experiments were carried out in the presence of cocaine, after in vitro denervation with 6-OHDA and pretreatment of the tissue with phenoxybenzamine which blocks the extraneuronal uptake of ISO. The chronotropic response to catecholamines in rat heart appears to be mediated by a pharmacologically homogeneous flt-adrenoceptors population (Bryan et al., 1981; Callia and De Moraes, 1984; Juberg et al., 1985; O'Donnell and Wanstall, 1985). However, some binding studies claim the presence of a pharmacological relevant fl2-adrenoceptors subpopulation (Minneman and Molinoff, 1980; Molinoff et al., 1981). ISO is a non-selective fl-adrenoceptor agonist whereas NA is a selective fl~-adrenoceptor agonist. Consequently, repeated swimming stress-induced subsensitivity to the chronotropic effect of NA and ISO could be produced by alterations located at the pacemaker fl~-adrenoceptors and/or modifications of the cyclic A M P system to which flradrenoceptors are coupled. Repeated swimming stress decreases the affinity of the pacemaker fl~-adrenoceptors for metoprolol. Two important heuristic aspects of pA 2 must be considered now. Firstly, when the slopes of Schild plots are not statistically different from 1.0, identical receptor populations should give rise to the same pA 2 value for a given antagonist unless receptor conformational alterations have occurred. Secondly, in homogeneous receptor populations pA 2 values are independent of the agonist selectivity, receptor density and/or events
located beyond the receptor level (for a review see Kenakin, 1982, 1984). Hence, our results consistently show that repeated swimming stress induces a conformational alteration of the pacemaker fll-adrenoceptors which is, at least partially, responsible by the reduced sensitivity to ISO and NA. The present report also shows that the repeated swimming stress-induced subsensitivity to the chronotropic effect of ISO and NA observed in the isolated rat pacemaker is under the influence of the adrenal gland steroids. Adrenal cortical hormones have been shown to modulate fl-adrenoceptors function both in brain (Duman et al., 1984; Roberts and Bloom, 1981) and peripheral tissues (Davies and Lefkowitz, 1984). Recently, it has been suggested that glucocorticoids can decrease fl-adrenoceptors affinity in intact tissues (Insel, 1984). The plasma level of corticosterone, the major adrenal steroid in the rat, was increased after the third swimming session and removal of the adrenal glands 2 days before the swimming session abolished the development of swimming stress-induced pacemaker subsensitivity to the chronotropic effect of NA and ISO. Moreover, adrenalectomy prevented the reduction of the affinity of fl~-adrenoceptors for metoprolol suggesting that, at least partially, the corticosterone plasma level should play a role in the conformational alteration of the pacemaker flradrenoceptors. An argument against the proposed role of corticosterone is that adrenalectomy also prevents the well-known stressinduced increase in plasma catecholamines level, which could be responsible for desensitization of the pacemaker fl~-adrenoceptors. However, rat pacemaker fll-adrenoceptors down-regulated by exposure to ISO during 4 days showed reduced sensitivity to the chronotropic effect of ISO, but not a statistically significant reduction of the affinity for atenolol, a selective fll-adrenoceptor antagonist (Kenakin, 1984). Recently, it has been suggested that the administration of ACTH to rats has the same effect as repeated restraint stress, i.e. induces a reduction of the sensitivity of rat cerebral cortex to the NA cyclic generating effect suggesting that an increase in the plasma level of adrenal cortical hormones could mediate the stress effects (Stone et al., 1986). The molecular mechanisms involved with repeated stressinduced decrease of pacemaker fl~-adrenoceptors chronotropic function remain obscure. Corticosterone might alter flradrenoceptor synthesis and/or change the structure of fl~-adrenoceptors through alterations of the cell membrane organization. Nevertheless, the interactions between adrenal cortical hormones and peripheral fl~-adrenoceptors seem to be important during adaptation to repeated stress. SUMMARY
The effects of three daily swimming sessions on plasma corticosterone level and the fl~-adrenoceptor mediated chronotropic response to noradrenaline and isoprenaline were studied. Swimming stress induces a persistent increase in the plasma corticosterone level which was detected 45 min after the ending of the third swimming session. Repeated swimming stress was found to cause subsensitivity to
Stress effects on rat pacemaker fit adrenoreceptor the chronotropic effect of noradrenaline (2.5-fold at the p D 2 level, P < 0.05) and isoprenaline (7.9-fold, P <0.01). Determination of pA2 values of metoprolol indicated that swimming stress decreases the affinity of pacemaker fl~-adrenoceptors for the antagonist. Adrenalectomy carried out 2 days before the first swimming session was found to prevent the development of pacemaker subsensitivity to noradrenaline and isoprenaline and the decrease of flradrenoceptors affinity for metoprolol. It is concluded that swimming stress-induced pacemaker subsensitivity to the chronotropic effect of noradrenaline and isoprenaline is, at least partially, mediated by adrenal corticosteroids. Acknowledgements--The authors wish to thank Mrs So-
lange Aparecida dos Santos Basso for the preparation of the manuscript. This paper was submitted by R.C.S. in partial fulfilment of the requirements for the Ph.D. degree in Physiology at the Institute of Biomedical Sciences, University of Silo Paulo, Brazil. REFERENCES
Aprigliano O. and Hermsmeyer K. (1976) In vitro denervation of the portal vein and caudal artery of the rat. J. Pharmac. exp. Ther. 198, 568-577. Arunlakshana O. and Schild H. O. (1959) Some quantitative uses of drug antagonists. Br. J. Pharmac. Chemother. 14, 48-58. Bassani R. A. and De Moraes S. (1987) Subsensitivity to fl-adrenoceptor agonists in right atria isolated from footshock-stressed rats. Gen. Pharmac. 18(5), 473-477. Bryan L. J., Cole J. J., O'Donnell S. R. and Wanstall J. C. (1981) A study designed to explore the hypothesis that flcadrenoceptors are "innervated" receptors and fl2-adrenoceptors are "hormonal" receptors. J. Pharmac. exp. Ther. 216, 395-400. Buckner C. K., Torphy T. and Costa D. J. (1978) Studies on fl-adrenoceptor mediated changes in mechanical events and adenosine 3"-5' monophosphate levels in rat atria. Eur. J. Pharmac. 47, 259-271. Callia M. L. and De Moraes S. (1984) Heterogeneity of beta adrenoceptors in right atria isolated from cold-exposed rats. J. Pharmac. exp. Ther. 230, 450-454. Davies A. O. and Lefkowitz R. J. (1984) Regulation of fl-adrenergic receptors by steroid hormones. A. Rev. Physiol. 46, 119-130. Duman R. S., Andr6e T., Kendall D. A. and Enna S. J. (1984) Effect of adrenocorticotropin administration on fl-adrenergic receptor adaptations in rat brain cerebral cortex. J. Neurochem. 42, 33-37. Insel P. A. (1984) Identification and regulation of adrenergic receptors in target cells. Am. J. Physiol. 247, E53-E70. Juberg E. N., Minneman K. P. and Abel P. W. (1985) fir and fl2-adrenoceptor binding and functional responses in right and left atria of rat heart. Naunyn-Schmiedeberg's Arch. Pharmac. 330, 193-202. Kendall D. A., Duman R., Slopes J. and Enna S. J. (1982) Influence of adrenocortropic hormone and yohimbine on antidepressant-induced declines in rat brain neurotransmitter receptor binding and function. J. Pharmac. exp. Ther. 222, 566-571.
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Kenakin T. P. (1982) The Schild regression in the process of receptor classification. Can. J. Physiol. Pharmac. 60, 249-265. Kenakin T. P. (1984) The classification of drugs and drugreceptors in isolated tissues. Pharmac. Rev. 36, 165-222. Mackay D. (1978) How should values of pA 2 and affinity constants for pharmacological competitive antagonists be estimated? J. Pharm. Pharmac. 30, 312-313. Mattingiy D. A. (1962) A simple fluorimetric method for the estimation of free I l-hydroxycorticoids in human plasma. J. clin. Path. 15, 374-379. Minneman K. P. and Molinoff P. B. (1980) Classification and quantification of beta-adrenergic receptor subtypes. Biochem. Pharmac. 29, 1317-1323. Mobley P. L., Manier D. H. and Sulser F. (1983) Norepinephrine-sensitive adenylate cyclase system in rat brain: role of adrenal corticosteroids. J. Pharmac. exp. Ther. 226, 71-77. Molinoff P. B., Wolfe B. B. and Willand G. A. (1981) Quantitative analysis of drug-receptor interactions: I. Determination of the properties of receptor subtypes. Life Sci. 29, 427-443. O'Donnell S. R. and Wanstall J. C. (1979) The importance of choice of agonist in studies designed to predict fit: f12 adrenoceptor selectivity of antagonists from pA2 values of guinea-pig trachea and atria. Naunyn-Schmiedeberg's Arch. Pharmac. 308, 183-190. O'Donnell S. R. and Wanstall J. C. 0985) Responses to the beta2-selective agonist procaterol of vascular and atrial preparation with different beta-adrenoceptor populations. Br. J. Pharmac. 84, 227-235. Roberts D. C. and Bloom F. E. (1981) Adrenal steroidinduced changes in/Ladrenergic receptor binding in rat hippocampus. Eur. J. Pharmac. 74, 37-42. Spadari R. C., Bassani R. A. and De Moraes S. (1988) Supersensitivity to isoprenaline and epinephrine in right atria isolated from rats submitted to a single swimming session. Gen. Pharmac. 19(1), 129-135. Stone E. A. (1979a) Reduction by stress of norepinephrinestimulated accumulation of cyclic AMP in rat cerebral cortex. J. Neurochem. 32, 1335-1337. Stone E. A. (1979b) Subsensitivity to norepinephrine as a link between adaptation to stress and antidepressant therapy: an hypothesis. Res. Commun. Psychol. Psychiatry 4, 241-255. Stone E. A. and Platt J. E. 0982) Brain adrenergic receptors and resistance to stress. 8rain Res. 237, 405-414. Stone E. A., Slucky A. V., Platt J. E. and Trullas R. 0985) Reduction of the cyclic adenosine 3', 5'-monophosphate response to catecholamines in rat brain slices following repeated restraint stress. J. Pharmac. exp. Ther. 233, 382-388. Stone E. A., Platt J. E., Herrera A. S. and Kirk L. K. (1986) Effect of repeated restraint stress, desmethylimipramine or adrenocorticotropin on the alpha and beta adrenergic components of the cyclic AMP response to norepinephrine in rat brain slices. J. Pharmac. exp. Ther. 237, 702-707. U'Prichard D. C. and Kvetnansk~, R. (1980) Central and peripheral adrenergic receptors in acute and repeated immobilization stress. In Catecholamine and Stress: Recent Advances (Edited by Usden E., Kvetnansk~' R. and Koppin J. J.), pp. 229-232. Elsevier/North Holland, New York.
0306-3623/88 $3.00+ 0.00 Copyright © 1988 Pergamon Press plc
REPEATED SWIMMING STRESS A N D RESPONSIVENESS OF THE ISOLATED RAT PACEMAKER TO THE CHRONOTROPIC EFFECT OF N O R A D R E N A L I N E A N D ISOPRENALINE: ROLE OF A D R E N A L CORTICOSTEROIDS REGINA CI~L1A SPADARII and Sf~RGIO DE MORAES2. tDepartment of Physiology and Biophysics, Institute of Biology and 2Department of Pharmacology, Faculty of Medical Sciences, Universidade Estadual de Campinas, Campifias, Silo Paulo, Brazil (Received 29 September 1987)
Abstract--1. Repeated swimming stress (three daily sessions) resulted in an increased plasma corticosterone level and subsensitivity of the isolated rat pacemaker to noradrenaline and isoprenaline. 2. Repeated swimming stress was found to decrease the affinity of fll-adrenoreceptors for metoprolol. 3. Bilateral adrenalectomy performed 2 days before repeated swimming stress abolished the development of pacemaker subsensitivity to noradrenaline and isoprenaline and the decrease in fll-adrenoreceptors affinity for metoprolol. 4. It is concluded that adrenal corticosteroids, at least partially, mediate the swimming stress-induced subsensitivity of the isolated rat pacemaker to noradrenaline and isoprenaline. INTRODUCTION It is generally accepted that repeated stress causes subsensitivity of rat brain slices to the noradrenaline (NA)-cyclic A M P generating effect (Stone, 1979a,b; Stone and Platt, 1982; Stone et al., 1985). The mechanisms by which repeated stress alters brain cyclic A M P response have not been ascertained yet. It has been reported that repeated restraint stress produces a reduction of rat brain fl-adrenoceptors density (U'Prichard and Kvetnansk~, 1980) which mediates the cyclic A M P response to N A and isoprenaline (ISO) in rat brain tissue. The sensitivity of the brain /~-adrenoceptors-adenylate cyclase system to N A is increased after adrenalectomy (Mobley et al., 1983) or decreased after administration of A C T H (Kendall et al., 1982; Stone et al., 1986), suggesting that adrenal corticosteroids play a role in the modulation of the brain fl-adrenoceptor-adenylate cyclase system sensitivity to NA. Contrasting with brain slice studies, few attempts have been made to study the sensitivity to catecholamines in peripheral isolated fl-adrenoceptor-containing tissues, such as the rat pacemaker, during repeated stressful situations. Pacemakers isolated from cold-stressed rats showed subsensitivity to the chronotropic effect of N A probably caused by a conformational alteration of pacemaker fl~-adrenoceptors (Callia and De Moraes, 1984). Recently, it has been reported that repeated footshock stress induces subsensitivity to N A and supersensitivity to ISO (Bassani and De Moraes, 1987). Acute swimming stress increases the sensitivity to ISO leaving unaltered that to N A (Spadari et al., 1988). Taken together, these studies suggest, as previously proposed by Stone (1979a), that fl-adrenoceptor subsensitivity to N A is a mechanism related to adaptation to repeated stress both in brain and peripheral tissues. *To whom all correspondence should be addressed.
It is the aim of the present report to extend our previous findings on the effects of a single 50-min swimming session on the sensitivity of the isolated rat pacemaker to the chronotropic effect of N A and ISO (Spadari et aL, 1988). In the present experiments rats were submitted to three daily swimming sessions of 5, 15 and 30 min, respectively. Our intention was also to study the role played by adrenal steroids on the repeated swimming stress-induced subsensitivity to the chronotropic effect of N A and ISO. MATERIALS AND METHODS
Stress protocol Male Wistar rats (200-250 g) were used. The animals were housed individually in a well-ventilated room, in plastic cages with standard laboratory chow and tapwater freely available, during a week prior to the experiments. Forced swimming stress was obtained by placing each rat into a glass tank filled with lukewarm water (28-30°C) and measuring 70 cm length, 44 cm width and 50 cm depth. The animals were submitted to swimming during three subsequent days, the duration of each session being 5, 15 and 30 min, respectively. Immediately after the ending of the swimming sessions the animals were returned to their homecages. All swimming sessions were carried out between 10:00 and 11:00 a.m. Naive rats were used as control. Plasma corticosterone levels Immediately at the ending of the third swimming session, rats were given an i.p. injection of sodium pentobarbital (45 mg/kg). After 45 min an abdominal incision was made and blood (4-5 ml) withdrawn from the right renal vein, collected into heparinized vials and centrifuged (2300 g) at 5°C. Plasma corticosterone levels were fluorimetrically determined according to Mattingiy (1962) as reported elsewhere (Callia and De Moraes, 1984). Bilateral adrenalectomy Rats were anesthetized with ether and the adrenal glands removed via the dorsal route, 2 days before the first swimming session. The animals were post-operatively maintained on 0.9% NaC1. Completeness of adrenalectomy was
553
554
REGINA Cf:LIA SPADARIand SI~RGIODE MORAES
verified after the third swimming session by determination of plasma corticosterone levels as described above. Naive sham-operated rats were used as control.
2G
L
Organ-bath studies 7) After the third swimming session, rats were killed by a ' blow to the back of the head. Hearts were immediately removed and atria were isolated and suspended in 20 ml tu organ-baths containing Krebs-Henseleit solution of the following mM composition: NaCI, 115.0; KC1, 4.6; IIJ CaC12.2H20, 2.5; KH2PO 4, 1.2; MgSO4.7H20, 2.5; NaHCO3, 25.0 and glucose II.0. To minimize oxidation of I O 10 8) the two catecholamines 0.I 1 mM of ascorbic acid was Eg included. Diastolic tension was enough to permit recording o O of the spontaneous beating (0.5 cm/beat). After the equiO libration period of 1 hr with changes of bathing medium at tD z 15min intervals, a stable resting rate was reached. Precautions were taken to avoid the influence of junctional Z catecholamine dissipating mechanisms on the pacemaker o (-~ t¢) sensitivity to the chronotropic effect of the catecbolamines. Tissues were incubated with phenoxybenzamine (10/tM) during 15 rain followed by a thorough washout period of 30 min in order to block ~t-adrenoceptors and extraneuronal uptake (O'Donnell and Wanstall, 1979). When NA was used Fig. I. Plasma corticosterone levels of rats submitted to the experiments were carried out in the presence of cocaine three daily swimming sessions of 5, 15 and 30 min duration. (10 #M) to impair the neuronal uptake process. To reduce The plasma level of the steroid was determined 45 min after the possible participation of endogenous NA, atria were in the ending of the third swimming session. Naive rats i.e. vitro chemically denervated using 6-hydroxydopamine animals not submitted to swimming were used as control. (Aprigliano and Hermsmeyer, 1976). The efficiency of such Adrenalectomy (ADX) was performed 2 days before the procedure has been previously assessed (Callia and De first swimming session. Vertical bars indicate SEM. NumMoraes, 1984). Full cumulative concentration-effect curves bers in parentheses indicate the number of experiments. to ISO or NA were determined. Only one *P < 0.05 in relation to the control group. Plasma corticosterone in the control group was 10.3 ___1.9 itg/100 ml of concentration-effect curve was obtained with each atrium. plasma. The agonist concentration producing an effect which is 50% of maximum (ECs0) was calculated and presented as mean negative logarithm (pD2). swimming session attempted to escape, showing signs pA 2 values of metoprolol, a selective fll-adrenoceptor of emotional distress. Curiously, removal of the antagonist was determined according to Arunlakshana and Schild (1959). Full cumulative concentration-effect curves adrenal glands did not completely eliminate the to NA were obtained in the absence and presence of plasma level of corticosterone. However, the plasma metoprolol (1 300nM). The antagonist equilibrium time level of the steroid did not increase after the third was 60 min. Dose ratios (DR) were calculated by dividing swimming session, when compared to the remaining the ECs0 of NA in the presence and absence of metoprolol. level of the steroid observed in adrenalectomized rats Schild plots were drawn and the slopes determined to not submitted to swimming. The data on the effects validate pA 2 values which were also calculated according to of repeated swimming stress on the plasma level of the formula (Mackay, 1978): corticosterone are shown in Fig. 1. pA 2 = log (DR - 1) - log [B] There were no differences in resting rates and the maximum responses to the chronotropic effects of where [B] corresponds to the molar concentration of N A and ISO in isolated pacemakers of control and metoprolol. swimming-stressed rats. Swimming during three daily Statistical analysis sessions of 5, 15 and 30 min resulted in rightward The Student t-test for unpaired samples was used for the displacements of the concentration--effect curve to difference between means. Analysis of variance was also N A and ISO. The subsensitivity of the pacemaker to used. Schild plots were drawn using linear regression calcu- N A was 2.5-fold at the p D 2 level (P < 0.05) and that lated by the least squares method. to ISO was 7.9-fold (P <0.01). These data are presented in Table 1 and illustrated by Figs 2 and 3. Bilateral adrenalectomy carried out 2 days before the RESULTS first swimming session abolished the effects of repeated swimming stress on the pacemaker sensitivity As previously reported (Spadari et al., 1988) rats to the neurotransmitter (Fig. 4) and to ISO (result not swam vigorously, attempted to escape and showed shown). defecation during the first swimming session. HowFigure 5 depicted pA 2 values of metoprolol using ever, during the second swimming session the animals N A as the agonist. Pacemakers isolated from swam calmly and quickly learnt how to prop themselves with their hindlegs keeping their heads above swimming-stressed rats showed a statistically significant increase in the pA2 value of metoprolol the water. The animals displayed this habituation (5.7-fold, P < 0.01) indicating a reduction in the behavior during all the third swimming session. Plasma corticosterone levels were increased after the fl~-adrenoceptors affinity for the antagonist. Howthird swimming session as compared to naive rats. ever, pacemakers isolated from previously bilaterally Bilaterally adrenalectomized rats did not show any adrenalectomized rats which were submitted to the sign of habituation to swimming and even in the third swimming sessions showed pA2 values of metoprolol
Stress effects on rat pacemaker #1 adrenoreceptor
555
Table 1. The effects of swimming stress on the chronotropic response of the isolated rat pacemaker to noradrenaline and isoprenaline. Adrenalectomy (ADX) was performed 2 days before the first swimming session. Shown are mean values with 95% confidence intervals Group
Noradrenaline pD~
Na
Control Swimming ADX
8 8 8
8.24(8.01-8.47) 7.83(7.50-8.10)* 8.15(7.71-8.39)
Isoprenaline pD 2
Ratio: N" -2.57 1.23
6 6 6
Ratio c
9.64(9.20-10.0) 8.74(8.48-9.0)** 9.30(9.08-9.45)
-7.94 2.18
aNumber of experiments. bNegative logarithm of molar concentration of the agnnist producing a response which is 50% of the maximum. CAntilogarithm of the difference between pD 2 values. *Significantly different from the control group (P < 0.05). **Significantly different from the control group (P < 0.01).
which did n o t differ from those o b t a i n e d in the control group. In all experimental groups, slopes of Schild plots were n o t statistically different from 1.0 (Table 2).
DISCUSSION
The present report shows t h a t three daily swimming sessions o f 5, 15 a n d 30 m i n respectively, caused subsensitivity of the isolated rat p a c e m a k e r to the
tOO
100 8O
8O 6o
In
~60 40 40
x
TROL (6)
:/, o /
;
o iw,..,NO ~s) o
o-~ 0
1'2
i
i
i
i
i
11 10 9 8 7 ISOPRENAUNE [ - L o g
*
NTROL (6) 0 ADX SWimMiNG (S:
20
,
6 M']
5
1"1
Fig. 2. Mean concentration-effect curves for the cbronotropic effect of ISO in the isolated rat pacemaker. Rats were submitted to three daily swimming sessions of 5, 15 and 30 min duration (swimming). Naive rats i.e. rats not submitted to swimming were used as control. Vertical bars indicate SEM. Numbers in parentheses indicate the number of experiments. loo
1'0
9
8
"l
6
5
4.
Fig. 4. Mean concentration-effect curves for the chronotropic effect of NA in the isolated rat pacemaker. Rats were submitted to bilateral adrenalectomy performed 2 days before the first swimming session and submitted to the three daily swimming sessions of 5, 15 and 30min duration (ADX-swimming). Naive sham-operated rats were used as control. Vertical bars indicate SEM. Numbers in parentheses indicate number of experiments. 3.0
z 0Q.
so ~:' 2.0
60
lo ~e 20
.CO.T.O, (,) 0 SWI..INa(16)
~
) 0
o ,
\
~o ~ ai 7i ~ ~, NO,AO,, NA'.,N [ ,-o, , ]
,;
Fig. 3. Mean concentration-effect curves for the chronotropic effect of NA in the isolated rat pacemaker. Rats were submitted to three daily swimming sessions of 5, 15 and 30 min duration (swimming). Naive rats i.e. rats not submitted to swimming were used as control. Vertical bars indicate SEM. Number in parentheses indicate the number of experiments.
t 5
, 6
7
8
9
.eTOPROLO,[-Log.] Fig. 5. Schild plots for the antagonism of metoprolol vs the chronotropic effect of NA in the isolated rat pacemaker. Rats were submitted to three daily swimming sessions of 5, 15 and 30min (swimming) or submitted to bilateral adrenalectomy performed 2 days before the first swimming session and submitted to three daily swimming sessions of 5, 15 and 30 min duration (ADX-swimming). Naive shamoperated animals were used as control. Numbers in parentheses indicate the combined number of points and animals.
556
REGINA CI~LIASPADARIand SI~RGIODE MORAES
Table 2. The effects of swimming stress on pA 2 values of metoprolol in the isolated rat pacemaker. Noradrenaline was used as the agonist. Adrenalectomy (ADX) was performed 2 days before the first swimming session. Shown are mean values with 95% confidence intervals Group
Na
pA2b
Slope
Control Swimming ADX
9 16 13
8.28 (8.22-8.35) 7.52 (7.40-7.70)* 8.04 (7.95-8.14)
1.0 (0.80-1.20) 0.90 (0.60-1.20) 1.0 (0.70-1. I 0)
aNumber of experiments. bpA2 = log (DR - 1) - log [B] (Mackay, 1978). *Significantly different from the control group (P > 0.01).
chronotropic effect of ISO and NA. Previous studies have shown that a single swimming session of 50 min duration increased the pacemaker sensitivity to ISO leaving unaltered the sensitivity to NA (Spadari et al., 1988). The development of subsensitivity to the NA cyclic A M P generating effect in rat brain tissue after repeated stress is thought to be related to the mechanisms of cell adaptation (Stone, 1979a,b). Recently, we have reported that repeated footshock stress decreases the number of pacemaker fll-adrenoceptors but causes subsensitivity to the chronotropic effect of NA through an increased efficiency of the neuronal reuptake process (Bassani and De Moraes, 1987). It has been reported that repeated restraint stress decreases the fl-adrenoceptors density of rat heart (U'Prichard and Kvetnansk2~, 1980). However, a discrete reduction in the number of fl~-adrenoceptors would not be expected to shift to the right the concentration-effect curve to ISO and NA since as reported (Buckner et al., 1978) there is a large amount of pacemaker spare flt-adrenoceptors in rat heart. An increase in the efficiency of the pacemaker catecholamine-dissipating mechanisms during the repeated swimming stress has to be ruled out because all experiments were carried out in the presence of cocaine, after in vitro denervation with 6-OHDA and pretreatment of the tissue with phenoxybenzamine which blocks the extraneuronal uptake of ISO. The chronotropic response to catecholamines in rat heart appears to be mediated by a pharmacologically homogeneous flt-adrenoceptors population (Bryan et al., 1981; Callia and De Moraes, 1984; Juberg et al., 1985; O'Donnell and Wanstall, 1985). However, some binding studies claim the presence of a pharmacological relevant fl2-adrenoceptors subpopulation (Minneman and Molinoff, 1980; Molinoff et al., 1981). ISO is a non-selective fl-adrenoceptor agonist whereas NA is a selective fl~-adrenoceptor agonist. Consequently, repeated swimming stress-induced subsensitivity to the chronotropic effect of NA and ISO could be produced by alterations located at the pacemaker fl~-adrenoceptors and/or modifications of the cyclic A M P system to which flradrenoceptors are coupled. Repeated swimming stress decreases the affinity of the pacemaker fl~-adrenoceptors for metoprolol. Two important heuristic aspects of pA 2 must be considered now. Firstly, when the slopes of Schild plots are not statistically different from 1.0, identical receptor populations should give rise to the same pA 2 value for a given antagonist unless receptor conformational alterations have occurred. Secondly, in homogeneous receptor populations pA 2 values are independent of the agonist selectivity, receptor density and/or events
located beyond the receptor level (for a review see Kenakin, 1982, 1984). Hence, our results consistently show that repeated swimming stress induces a conformational alteration of the pacemaker fll-adrenoceptors which is, at least partially, responsible by the reduced sensitivity to ISO and NA. The present report also shows that the repeated swimming stress-induced subsensitivity to the chronotropic effect of ISO and NA observed in the isolated rat pacemaker is under the influence of the adrenal gland steroids. Adrenal cortical hormones have been shown to modulate fl-adrenoceptors function both in brain (Duman et al., 1984; Roberts and Bloom, 1981) and peripheral tissues (Davies and Lefkowitz, 1984). Recently, it has been suggested that glucocorticoids can decrease fl-adrenoceptors affinity in intact tissues (Insel, 1984). The plasma level of corticosterone, the major adrenal steroid in the rat, was increased after the third swimming session and removal of the adrenal glands 2 days before the swimming session abolished the development of swimming stress-induced pacemaker subsensitivity to the chronotropic effect of NA and ISO. Moreover, adrenalectomy prevented the reduction of the affinity of fl~-adrenoceptors for metoprolol suggesting that, at least partially, the corticosterone plasma level should play a role in the conformational alteration of the pacemaker flradrenoceptors. An argument against the proposed role of corticosterone is that adrenalectomy also prevents the well-known stressinduced increase in plasma catecholamines level, which could be responsible for desensitization of the pacemaker fl~-adrenoceptors. However, rat pacemaker fll-adrenoceptors down-regulated by exposure to ISO during 4 days showed reduced sensitivity to the chronotropic effect of ISO, but not a statistically significant reduction of the affinity for atenolol, a selective fll-adrenoceptor antagonist (Kenakin, 1984). Recently, it has been suggested that the administration of ACTH to rats has the same effect as repeated restraint stress, i.e. induces a reduction of the sensitivity of rat cerebral cortex to the NA cyclic generating effect suggesting that an increase in the plasma level of adrenal cortical hormones could mediate the stress effects (Stone et al., 1986). The molecular mechanisms involved with repeated stressinduced decrease of pacemaker fl~-adrenoceptors chronotropic function remain obscure. Corticosterone might alter flradrenoceptor synthesis and/or change the structure of fl~-adrenoceptors through alterations of the cell membrane organization. Nevertheless, the interactions between adrenal cortical hormones and peripheral fl~-adrenoceptors seem to be important during adaptation to repeated stress. SUMMARY
The effects of three daily swimming sessions on plasma corticosterone level and the fl~-adrenoceptor mediated chronotropic response to noradrenaline and isoprenaline were studied. Swimming stress induces a persistent increase in the plasma corticosterone level which was detected 45 min after the ending of the third swimming session. Repeated swimming stress was found to cause subsensitivity to
Stress effects on rat pacemaker fit adrenoreceptor the chronotropic effect of noradrenaline (2.5-fold at the p D 2 level, P < 0.05) and isoprenaline (7.9-fold, P <0.01). Determination of pA2 values of metoprolol indicated that swimming stress decreases the affinity of pacemaker fl~-adrenoceptors for the antagonist. Adrenalectomy carried out 2 days before the first swimming session was found to prevent the development of pacemaker subsensitivity to noradrenaline and isoprenaline and the decrease of flradrenoceptors affinity for metoprolol. It is concluded that swimming stress-induced pacemaker subsensitivity to the chronotropic effect of noradrenaline and isoprenaline is, at least partially, mediated by adrenal corticosteroids. Acknowledgements--The authors wish to thank Mrs So-
lange Aparecida dos Santos Basso for the preparation of the manuscript. This paper was submitted by R.C.S. in partial fulfilment of the requirements for the Ph.D. degree in Physiology at the Institute of Biomedical Sciences, University of Silo Paulo, Brazil. REFERENCES
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