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Sympathetic Nerve Stimulation Activates Both β1 and β2-Adrenoceptors of SA and AV Nodes in Anesthetized Dog Hearts
Sympathetic
Manabu
Nerve Stimulation Activates Both 81 Adrenoceptors of SA and AV Nodes in Anesthetized Dog Hearts Takei,
Yasuyuki
Lei-Ming
Ren,
Furukawa, Yasuyuki
Masahiro
Karasawa
Narita,
Makoto
and Shigetoshi
and 82
Murakami,
Chiba *
Department of Pharmacology, Shinshu University School of Medicine, Matsumoto 390, Japan Received
November
1, 1991
Accepted
January
16, 1992
ABSTRACT We investigated blocking effects of the selective 8 1-adrenoceptor blocker atenolol (0.1 100 ,ug/kg, i.v.), the selective fl2-adrenoceptor blocker ICI 118,551 (1-1000 ,ug/kg, i.v.) and the com bination of the two drugs on positive chronotropic and dromotropic responses to norepinephrine (NE) released by stimulation of the sympathetic nerves in anesthetized, neurally decentralized, open-chest dogs after atropine was given. Stimulation of the intracardiac sympathetic nerves to the SA nodal re gion or to the AV nodal region selectively increased heart rate or decreased AV conduction time, re spectively. ICI 118,551 inhibited the chronotropic or dromotropic response to each stimulation in a dose-dependent manner, but its inhibition of the dromotropic response was less than that of the chro notropic response. Atenolol similarly inhibited either the positive chronotropic or dromotropic response to each stimulation in a dose-related manner. The combination of atenolol and ICI 118,551 attenuated the responses to each stimulation more than atenolol alone. These data indicate that sympathetic nerve stimulation activates both 81 and 82-adrenoceptors of the SA and AV nodes and that the proportion of ,62-adrenoceptor-mediated effects on the AV node is less than that on the SA node. These results suggest that neurally released NE in part controls physiological functional cardiac responses mediated through 82-adrenoceptors, in addition to the responses predominantly mediated through ~' 1-adreno ceptors. Keywords:
Atenolol,
Heart
rate,
ICI 118,551,,8-Adrenoceptors,
Norepinephrine (NE) released by activation of sym pathetic nerve fibers to the heart produces positive chronotropic, dromotropic and isotropic responses mediated through ,6-adrenoceptors (1). NE is pos tulated as a selective 8 1-adrenoceptor agonist to / adrenoceptors (2 5), although it has been suggested that circulating catecholamines cause a positive chrono tropic effect mediated by ,E2-adrenoceptors in the ex ercising dog (6). Many previous studies examined the role of 8-adreno ceptor subtypes on the cardiac functions by exoge nously administered catecholamines or selective 81 and /.92-agonistsin isolated heart tissues (3, 4, 7). By radioli gand binding studies, the amounts of 81 and /32-adreno ceptors have been determined in the right atrium and
Sympathetic
nerve
stimulation
left ventricle of cat, guinea pig and dog hearts (8, 9), and the proportion of 82-adrenoceptors in the right atrium is higher than that in the left ventricle. Similar to the proportion of 82-adrenoceptors, the 82 adrenoceptor-mediated effects on the SA node are sub stantially greater than those on atrial muscle, whereas only slight effects are observed on ventricular muscle (7). An autoradiographic study using X-ray film indi cated that the AV node also contains both 81 and 82 adrenoceptors (10, 11). Thus, it is important to under stand whether ,62-adrenoceptors in the AV node medi ate a functional effect like they do in the SA node. In the present study, therefore, we investigated whether neurally released NE induced positive dromo tropic and chronotropic effects functionally mediated through 82-adrenoceptors in the dog heart in situ, and if this is so, whether there were differential 92
adrenoceptor-mediated chronotropic these aims,
changes
responses to we investigated
91-adrenoceptor 92-adrenoceptor
antagonist antagonist
chronotropic response tracardiac sympathetic gion and the positive tion of the discrete
in
dromotropic
and
released NE. To achieve the effects of the selective atenolol and the selective ICI 118,551 on the positive
to stimulation of the discrete nerve fibers to the SA nodal dromotropic intracardiac
fibers to the AV nodal region
in re
response to stimula sympathetic nerve
in anesthetized
dog heart.
MATERIALS AND METHODS Preparations Surgical and experimental procedures were approved by the Shinshu University Animal Welfare Committee. We used 23 mongrel dogs, weighing from 10 to 30 kg. Each dog was anesthetized with sodium pentobarbital (30 mg/kg, i.v.). A tracheal cannula was inserted and intermittent positive-pressure ventilation was started. The chest was opened transversely at the fifth intercos tal space. Each cervical vagus nerve was crushed with a tight ligature and each stellate ganglion was ligated tightly at its junction with the ansa subclavia. These maneuvers have been shown to remove virtually all tonic neural activity to the heart (12). A bipolar electrode was placed on the base of the epicardial surface of the right atrial appendage to re cord the electrical activity. A bipolar recording elec trode was also placed on the epicardial surface of the ventricle. We refer to the deflection recorded during depolarization of the right auricle as "A" and the de flection recorded during depolarization of the right ven tricle as "V". The heart rate (HR) and atrioventricular conduction time (AVCT, AV interval) were measured and displayed on a thermo-writing rectigraph (Nihon Kohden WT685T). Systemic arterial pressure was also recorded via the femoral artery. The left femoral vein was also cannulated for physiological saline infusion to adjust spontaneous fluid losses and drug injection. When the surgical procedure was ended, the sterno tomy was covered by a vinyl sheet and, by adjusting the distance of the table lamp to the cardiac surface, epi cardial temperature was maintained. Two bipolar plate silver electrodes, 2 mm inter electrode distance, were used to stimulate the intra cardiac sympathetic nerve fibers (13). One bipolar elec trode was placed on the fatty tissue overlying the right side of the left atrial junctions of the right pulmonary veins, and this was used to stimulate the intracardiac sympathetic nerve fibers to the SA nodal region; we re fer to this as "SAS stimulation". The second electrode was placed on the fatty tissue at the junction of the in
ferior vena cava and left atrium. It was used to stimu late the intracardiac sympathetic nerve fibers to the AV nodal region; we refer to that as "AVS stimulation". When we studied the cardiac responses to sympathe tic nerve stimulation, a dog was given atropine sulfate (0.2 mg/kg, i.v.), and 0.1 or 0.2 mg/kg, i.v. of atropine was given each hour thereafter to block the responses mediated by the muscarinic receptors. We used repeti tive bursts of stimuli (Nihon Kohden SEN 7103) with a 5 mA pulse amplitude and 1 to 2 msec pulse duration. The pulse amplitude and duration were adjusted so that the stimuli were above threshold for neighboring nerve fibers but below threshold for the cardiac cells (13). The interpulse interval within each burst was 10 msec. A brief burst of stimuli was delivered in each cardiac cycle. Each stimulus burst contained from 1 to 5 pulses. Stimulation was triggered by the atrial depolarization ("A" wave); the time from the beginning of the "A" wave to the beginning of the stimulus burst was 10 msec. Thus, a burst with 5 pulses terminated within 60 msec from the beginning of atrial depolarization. To avoid excitation of these structures, we used brief bursts of stimuli delivered at preselected times each car diac cycle, to coincide with the absolute refractory period. Each 30-sec train of stimuli to the nerves was followed by a recovery period of at least 4 min. Experimental protocols Four groups of experiments were carried out. In the first group with 4 dogs, we repeated SAS and AVS sti mulations eight times to confirm that the response to each stimulation would be maintained at a similar level during a series of experiments. The stimulation inter vals of each SAS or AVS stimulation were 10 min. In the second group of experiments, the effects of ICI 118,551 (n = 8) on the positive chronotropic response to SAS stimulation and the positive dromotropic re sponse to AVS stimulation were studied. ICI 118,551 at doses of 1 to 1000pg/kg, i.v. was given into the femor al vein. In the third group with 6 dogs, we studied the effects of atenolol on the positive chronotropic response and the positive dromotropic response to each stimula tion. We also injected atenolol at doses of 0.1 to 100 ,ug/kg, i.v. into the femoral vein. We measured the HR and AVCT before and more than 5 min after admin istration of ICI 118,551 or atenolol. In the fourth group with 5 dogs, we compared the effect of atenolol (10 ,ug/kg, i.v.) alone and the combination of atenolol (10 ,ug/kg, i.v.) and ICI 118,551 (30,ug/kg, i.v.) on the positive chronotropic response to SAS stimulation or the dromotropic response to AVS stimulation. We meas ured the HR and AVCT before and more than 5 min after atenolol alone or the combination of atenolol and
ICI 118,551. The order of treatment with atenolol alone or the combination of atenolol and ICI 118,551 was randomized, and sufficient recovery time was allowed between two treatments. Drugs The drugs used in the present experiments were ate nolol (Sigma, St. Louis, MO, USA), atropine sulfate (Wako Pure Chemical Co., Osaka, Japan), ICI 118,551 (erythro [ ± ]-1-[7-methylindan-4-yloxy]-3-isopropyl-amino butan-2-ol) (generously donated by Imperial Chemical Industries Co., Macclesfield, England), and propranolol hydrochloride (Sigma, St. Louis, MO, USA). Each drug was dissolved in physiological saline before start ing the experiments. Statistical analysis All data were expressed as a percentage of change in the respective control value of each stimulation obtained just before treatment with /3-adrenoceptor antagonists. These data were expressed as means ± S.E. Fifty percent inhibition doses (ID50) were deter mined for each dose-inhibition curve. An analysis of variance was used for the statistical analysis of multiple comparisons of data and Student's t-test for paired and unpaired samples was used for comparisons between the two groups. P values of less than 0.05 were consi dered to indicate a statistically significant difference.
RESULTS The changes in HR and AVCT induced by SAS or AVS stimulation Stimulation of the intracardiac sympathetic nerve fibers to the SA nodal region (SAS stimulation) consis tently increased HR and variably changed AVCT in an anesthetized, neurally decentralized open-chest dog af ter atropine was given (Fig. 1A, control). The changes in AVCT induced by SAS stimulation were not due to direct effects of stimulation, but caused by a shift of the pacemaking site within the sino-atrial pacemaker com plex (13). Stimulation of the intracardiac sympathetic nerve fibers to the AV nodal region (AVS stimulation) consistently decreased AVCT, but did not affect the HR (Fig. 1B, control). In the first group, the first SAS stimulation increased HR by 31 ± 7 beats/min (n = 4) from the prestimulation control level. The first AVS sti mulation decreased AVCT by 21 ± 3 msec from the basal level. Changes in HR and AVCT induced by re petition of each stimulation did not significantly change during a series of experiments. The 8th SAS and AVS stimulations changed HR and AVCT to 93 ± 3% and 95 ± 3% of the changes in HR and AVCT to each first stimulation, respectively. Therefore, the changes in HR and AVCT in response to each stimulation before treat ment with /8-adrenoceptor antagonists were regarded as control values.
Fig. 1. Effects of ICI 118,551 and propranolol on the positive chronotropic (HR) and dromotropic (AVCT) responses to A) stimulation of the intracardiac sympathetic nerve fibers to the SA nodal region (SAS stimulation) and B) stimulation of the intracardiac sympathetic nerve fibers to the AV nodal region (AVS stimulation) in a neurally decentralized, open chest, anesthetized dog.
Blocking effects of ICI 118,551 The basal HR and AVCT in 8 anesthetized dogs for a second ICI 118,551-treated group were 106 ± 5 beats/min, and 133 ± 7 msec, respectively. These values were not significantly different from those after treatment with ICI 118,551. SAS stimulation increased HR by 38 ± 4 beats/min from the prestimulation con trol value. AVS stimulation decreased AVCT by 34 ± 3 msec from the basal level. As shown in Fig. 1A, ICI 118,551 at doses of 1 to 1000,ug/kg, i.v. inhibited the increases in HR induced by SAS stimulation dose-dependently in an anesthe tized, open-chest dog treated with atropine. ICI 118,551 (100 and 1000 ,ug/kg, i.v.) also attenuated the shorten ing of the AVCT induced by AVS stimulation, although ICI 118,551 at doses of 1 to 10,ug/kg, i.v. lit tle affected the effects of AVS stimulation (Fig. 1B). The positive chronotropic response to SAS stimulation and the positive dromotropic response to AVS stimula tion were not completely blocked by ICI 118,551 at a dose of 1000,ug/kg, i.v. Propranolol at the dose of 1 mg/kg, i.v. abolished the residual positive responses to SAS or AVS stimulation (Fig. 1, A and B). The composite data from 8 experiments are shown in Fig. 2. ICI 118,551 at doses of 1 to 1000,ug/kg, i.v. sig nificantly (P < 0.05) inhibited the positive chronotropic response to SAS stimulation in a dose-dependent man ner. On the other hand, the positive dromotropic re sponse to AVS stimulation was significantly (P < 0.05) attenuated by ICI 118,551 at doses of 30,ug/kg, i.v.
and more (100 and 1000,ug/kg, i.v.) dose-dependently. The inhibition of ICI 118,551 at doses of 3 to 1000 ,ug/kg, i.v. on the positive dromotropic response to AVS stimulation was less than that on the positive chronotropic response to SAS stimulation at each dose (Fig. 2). Blocking effects of atenolol The basal HR and AVCT in 6 anesthetized dogs for an atenolol-treated group were 111 ± 10 beats/min and 136 ± 7 msec, respectively. These values were not sig nificantly different from those after treatment with ate nolol. SAS stimulation increased HR by 32 ± 3 beats/min from the prestimulation control value. AVS stimulation decreased AVCT by 35 ± 3 msec from the basal level. These data were not significantly different from the data of the ICI 118,551-treated group. Atenolol significantly (P < 0.05) inhibited both the positive chronotropic response to SAS stimulation and the positive dromotropic response to AVS stimulation at 0.3,ug/kg, i.v. and more (1 to 100,ug/kg, i.v.) in a dose-dependent manner (Fig. 3). The inhibition of ate nolol (0.1 to 100,ug/kg, i.v.) on the positive chronotro pic response to SAS stimulation was not significantly different from that on the positive dromotropic re sponse to AVS stimulation at each dose (Fig. 3). The IC50 values of atenolol for the chronotropic and dro motropic responses to each stimulation were 6.9 ± 2.8 ,ug/kg, i.v. and 12.9 ± 6.1,ug/kg, i.v., respectively. These values were not significantly different.
Fig. 2. The effects of ICI 118,551 at doses of 1 to 1000 ,ug/kg, i.v. on the positive chronotropic response to stimulation of the intracardiac sympathetic nerve fibers to the SA nodal region (0) and on the positive dromotropic response to stimula tion of the intracardiac sympathetic nerve fibers to the AV nodal region (•) in 8 anesthetized dogs . Vertical bars show S.E. *P < 0.05, **P < 0.01 compared to control values.
Blocking effects of the combination of atenolol and ICI 118,551 SAS stimulation increased HR by 35 ± 4 beats/min from the prestimulation control value in the atenolol group and by 30 ± 5 beats/min in the combination of atenolol and ICI 118,551 group. These data were not significantly different. AVS stimulation decreased AVCT by 24 ± 3 msec from the basal level in the ate nolol group and by 22 ± 3 msec in the combination of atenolol and ICI 118,551 group. These data were not significantly different.
The blocking effect of the combination of atenolol (10,ug/kg, i.v.) and ICI 118,551 (30,ug/kg, i.v.) on the positive chronotropic response to SAS stimulation was significantly (P < 0.05) greater than that of atenolol (10 ug/kg, i.v.) alone (Fig. 4A). The blocking effect of the combination of atenolol and ICI 118,551 on the positive dromotropic response to AVS stimulation was not signi ficantly different from that of atenolol, but the com bination treatment (41 ± 8%) tended to inhibit the re sponse more than atenolol alone (53 ± 6%) (Fig. 4B).
Fig. 3. The effects of atenolol at doses of 0.1 to 100 ug/kg, i.v. on the positive chronotropic response to stimulation of the intracardiac sympathetic nerve fibers to the SA nodal region ((--)) and on the positive dromotropic response to stimula tion of the intracardiac sympathetic nerve fibers to the AV nodal region (•) in 6 anesthetized dogs. Vertical bars show S.E. *P < 0.05, **P < 0.01 compared to control values.
Fig. 4. The effects of atenolol (AT, 10 ug/kg, i.v.) and the combination atenolol (10 ug/kg, i.v.) and ICI 118,551 (30 pg/kg, i.v.) (AT + ICI) on the positive chronotropic response to SAS stimulation (panel A) or the dromotropic response to AVS stimulation (panel B). Vertical bars show S.E. * P < 0.05, compared with atenolol alone and the combination of two drugs.
DISCUSSION In the present study, we demonstrated that positive dromotropic and chronotropic responses to sympathetic nerve stimulation were induced by activation of both 1 and N 92-adrenoceptors in anesthetized dog hearts and the proportion of the 82-adrenoceptor-mediated effect for SA nodal pacemaker activity was greater than that for AV conductivity, although the cardiac re sponses to sympathetic stimulation were predominantly mediated through 81-adrenoceptors. After atropine was given to the anesthetized dog, sti mulation of the intracardiac sympathetic nerve fibers to the SA nodal region (SAS stimulation) increased HR and variably changed AVCT. The changes in AVCT were caused by a shift of the pacemaker site within the sino-atrial pacemaker complex (13, 14). Stimulation of the intracardiac sympathetic nerve fibers to the AV nodal region (AVS stimulation) decreased AVCT, but it did not alter the HR. Thus, AVS stimulation permit ted us to examine the effects of sympathetic stimulation on the AVCT without concomitant secondary influ ences of the changes in HR, pacemaker location, and conduction pathway (15 -17). SAS stimulation increased HR and the positive chro notropic response was dose-dependently inhibited by the selective 82-adrenoceptor antagonist ICI 118,551 in the dose range of 1 to 1000,ug/kg, i.v. ICI 118,551 blocks ,82-adrenoceptors selectively in doses of up to 100,ug/kg, i.v. in anesthetized dogs (5, 18). Atenolol also inhibited the positive chronotropic response to SAS stimulation (Fig. 3). The effect of the combination of atenolol (10,ug/kg, i.v.) and ICI 118,551 (30 pg/kg, i.v.) on the positive chronotropic response to SAS sti mulation was significantly greater than that of atenolol (10pg/kg, i.v.) (Fig. 4A). Atenolol is a selective ," 1 adrenoceptor antagonist and its selectivity ratio for 81 adrenoceptors versus /32-adrenoceptors is approximately 6 to 20 (3, 5). Thus, our results indicate that the posi tive chronotropic response to sympathetic nerve sti mulation is in part mediated by ,82-adrenoceptors in addition to ,81-adrenoceptors in anesthetized dog hearts and suggest that neurally released NE in part controls the functional chronotropic response mediated through /32-adrenoceptors. These results were inconsistent with a previous report by Bilski et al. (5). They showed that the increases in HR due to sympathetic stimulation were not significantly inhibited by ICI 118,551 in anes thetized dogs. Although we do not know the exact reasons of the discrepancy between our results and theirs, it is postulated that 1) we stimulated the discrete intracardiac sympathetic nerves to the SA nodal region, and 2) they used only four dogs to composite data.
NE is postulated as a selective N 1-adrenoceptor ago nist for /8-adrenoceptors (2-5). It was found that the affinity value of the N 1-adrenoceptor subtype for NE was 15 times greater than that of the ,82-adrenoceptor subtype in the isolated dog atrial tissue and the potency of NE in occupying 81 and ,82-adrenoceptors was simi lar to its potency in stimulating f1 and /82-subtype mediated increases in adenylate cyclase activity (2). Thus, those studies support our results that the positive chronotropic response to sympathetic stimulation is in part mediated by ,82-adrenoceptors in the dog heart in situ. AVS stimulation decreased AVCT in anesthetized dogs with atropine. Atenolol inhibited the positive dro motropic response to AVS stimulation in a dose-depend ent manner, whereas ICI 118,551 at doses of 30 and 100 ,ug/kg, i.v. attenuated the positive dromotropic re sponse to AVS stimulation slightly but significantly. Although ICI 118,551 blocks ,82-adrenoceptors selec tively in doses of up to 100,ug/kg, i.v. in anesthetized dogs (5, 18), in the present study, ICI 118,551 at a dose of 100,ug/kg, Lv. inhibited the positive chronotropic response to sympathetic stimulation by almost 50% (Fig. 2). Thus, it is likely that that dose of ICI 118,551 inhibited partly the positive dromotropic responses to neurally released NE mediated by /81-adrenoceptors be cause the ,82/,81-adrenoceptor ratio was 30%/70% in the dog atrium (2). ,82-Adrenoceptors in the AV node of the mammalian hearts have been shown by auto radiography (11, 19). Motomura and Hashimoto (20) reported briefly that ICI 118,551 inhibited the positive dromotropic effect of a selective ,82-adrenoceptor ago nist, procaterol, in the canine isolated, blood-perfused AV node preparation. Therefore, our results suggest that sympathetic nerve stimulation induces a little posi tive dromotropic effect mediated through 82-adreno ceptors in addition to 81-adrenoceptors in anesthetized dogs. Presynaptic 82-adrenoceptors have been postulated to facilitate NE release from nerve endings (21, 22). If ICI 118,551 blocked only presynaptic ,82-adrenoceptors, ICI 118,551 would inhibit the positive chronotropic and dromotropic responses to each stimulation similarly. However, the ratio of /32-adrenoceptor-mediated dro motropic response to NE was less than that of the ,82 adrenoceptor-mediated chronotropic response to NE in our experiments. In guinea pig atria, neurally released epinephrine but not neurally released NE facilitated the stimulation induced release of NE (22). Therefore, it is likely that the ,82-adrenoceptors of the postsynaptic site are inhibited by ICI 118,551 in our experiments, although it is difficult to exclude completely that pre synaptic ,82-adrenoceptors are in part involved.
It has been demonstrated that stimulation of the /32 adrenoceptors by selective adrenoceptor ,82-agonists, procaterol and salbutamol, produces a positive chrono tropic effect in anesthetized dogs (23) and humans (24). However, it does not mean such stimulation of the re ceptors to be physiological relevant (24). The effects of atenolol and propranolol were studied on the hemody namic responses induced by exercise in dogs and sug gested that ,82-adrenoceptors in the SA node might be stimulated by circulating catecholamines and contri buted greatly to sympathetic modulation of HR during heavy exercise in dogs (6). It was shown that failing hu man ventricular myocardium contained a relative high proportion of X82-adrenoceptors, due to selective down regulation of ,81-adrenoceptors using a radioligand binding study (25). Recently, it was found that selective /31-adrenoceptor blockade enhanced positive inotropic responses to catecholamines as well as salbutamol medi ated through 82-adrenoceptors in the human atrium (4). In the present study, we demonstrated that the positive chronotropic and dromotropic responses to sympathetic stimulation were mediated through 82 adrenoceptors in addition to predominant ,81-adreno ceptors in the anesthetized dog. Therefore, we suggest that neurally released NE controls functional cardiac re sponses mediated through ,82-adrenoceptors, especially during 81-adrenoceptor blockade and pathophysiologi cal conditions. Acknowledgment We supplies
thank
Imperial
of ICI
Chemical
Industries
Co.,
Ltd.
for
generous
118,551.
REFERENCES
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,8-adrenoceptor in the regulation of noradrenaline release by nerve stimulation through a positive feed-back mechanism. Br. J. Pharmacol. 53, 43-50 (1975) 22 Majewski, H., McCulloch, M.W., Rand, M.J. and Story, D.F.: Adrenaline activation of prejunctional ,9-adrenoceptors in guinea-pig atria. Br. J. Pharmacol. 71, 435-444 (1980) 23 Yabuuchi, Y., Yamashita, S. and Tei, S.-S.: Pharmacological studies of OPC-2009, a newly synthesized selective beta adreno ceptor stimulant, in the bronchomoter and cardiovascular sys tem of the anesthetized dog. J. Pharmacol. Exp. Ther. 202, 326 336 (1977)
24
25
Hall, J.A., Petch, M.C. and Brown, M.J.: Intracoronary
in
jection of salbutamol demonstrate the presence of functional ,92-adrenoceptors in the human heart. Circ. Res. 65, 546-553 (1989) Bristow, M.R., Ginsburg, R., Umans, V., Fowler, M., Minobe, W., Rasmussen, R. et al.: ,B1 and l2-Adrenergic-re ceptor subpopulations in nonfailing and failing human ven tricular myocardium: Coupling of both receptor subtypes to muscle contraction and selective 81-receptor down-regulation in heart failure. Circ. Res. 59, 297-309 (1986)
Manabu
Nerve Stimulation Activates Both 81 Adrenoceptors of SA and AV Nodes in Anesthetized Dog Hearts Takei,
Yasuyuki
Lei-Ming
Ren,
Furukawa, Yasuyuki
Masahiro
Karasawa
Narita,
Makoto
and Shigetoshi
and 82
Murakami,
Chiba *
Department of Pharmacology, Shinshu University School of Medicine, Matsumoto 390, Japan Received
November
1, 1991
Accepted
January
16, 1992
ABSTRACT We investigated blocking effects of the selective 8 1-adrenoceptor blocker atenolol (0.1 100 ,ug/kg, i.v.), the selective fl2-adrenoceptor blocker ICI 118,551 (1-1000 ,ug/kg, i.v.) and the com bination of the two drugs on positive chronotropic and dromotropic responses to norepinephrine (NE) released by stimulation of the sympathetic nerves in anesthetized, neurally decentralized, open-chest dogs after atropine was given. Stimulation of the intracardiac sympathetic nerves to the SA nodal re gion or to the AV nodal region selectively increased heart rate or decreased AV conduction time, re spectively. ICI 118,551 inhibited the chronotropic or dromotropic response to each stimulation in a dose-dependent manner, but its inhibition of the dromotropic response was less than that of the chro notropic response. Atenolol similarly inhibited either the positive chronotropic or dromotropic response to each stimulation in a dose-related manner. The combination of atenolol and ICI 118,551 attenuated the responses to each stimulation more than atenolol alone. These data indicate that sympathetic nerve stimulation activates both 81 and 82-adrenoceptors of the SA and AV nodes and that the proportion of ,62-adrenoceptor-mediated effects on the AV node is less than that on the SA node. These results suggest that neurally released NE in part controls physiological functional cardiac responses mediated through 82-adrenoceptors, in addition to the responses predominantly mediated through ~' 1-adreno ceptors. Keywords:
Atenolol,
Heart
rate,
ICI 118,551,,8-Adrenoceptors,
Norepinephrine (NE) released by activation of sym pathetic nerve fibers to the heart produces positive chronotropic, dromotropic and isotropic responses mediated through ,6-adrenoceptors (1). NE is pos tulated as a selective 8 1-adrenoceptor agonist to / adrenoceptors (2 5), although it has been suggested that circulating catecholamines cause a positive chrono tropic effect mediated by ,E2-adrenoceptors in the ex ercising dog (6). Many previous studies examined the role of 8-adreno ceptor subtypes on the cardiac functions by exoge nously administered catecholamines or selective 81 and /.92-agonistsin isolated heart tissues (3, 4, 7). By radioli gand binding studies, the amounts of 81 and /32-adreno ceptors have been determined in the right atrium and
Sympathetic
nerve
stimulation
left ventricle of cat, guinea pig and dog hearts (8, 9), and the proportion of 82-adrenoceptors in the right atrium is higher than that in the left ventricle. Similar to the proportion of 82-adrenoceptors, the 82 adrenoceptor-mediated effects on the SA node are sub stantially greater than those on atrial muscle, whereas only slight effects are observed on ventricular muscle (7). An autoradiographic study using X-ray film indi cated that the AV node also contains both 81 and 82 adrenoceptors (10, 11). Thus, it is important to under stand whether ,62-adrenoceptors in the AV node medi ate a functional effect like they do in the SA node. In the present study, therefore, we investigated whether neurally released NE induced positive dromo tropic and chronotropic effects functionally mediated through 82-adrenoceptors in the dog heart in situ, and if this is so, whether there were differential 92
adrenoceptor-mediated chronotropic these aims,
changes
responses to we investigated
91-adrenoceptor 92-adrenoceptor
antagonist antagonist
chronotropic response tracardiac sympathetic gion and the positive tion of the discrete
in
dromotropic
and
released NE. To achieve the effects of the selective atenolol and the selective ICI 118,551 on the positive
to stimulation of the discrete nerve fibers to the SA nodal dromotropic intracardiac
fibers to the AV nodal region
in re
response to stimula sympathetic nerve
in anesthetized
dog heart.
MATERIALS AND METHODS Preparations Surgical and experimental procedures were approved by the Shinshu University Animal Welfare Committee. We used 23 mongrel dogs, weighing from 10 to 30 kg. Each dog was anesthetized with sodium pentobarbital (30 mg/kg, i.v.). A tracheal cannula was inserted and intermittent positive-pressure ventilation was started. The chest was opened transversely at the fifth intercos tal space. Each cervical vagus nerve was crushed with a tight ligature and each stellate ganglion was ligated tightly at its junction with the ansa subclavia. These maneuvers have been shown to remove virtually all tonic neural activity to the heart (12). A bipolar electrode was placed on the base of the epicardial surface of the right atrial appendage to re cord the electrical activity. A bipolar recording elec trode was also placed on the epicardial surface of the ventricle. We refer to the deflection recorded during depolarization of the right auricle as "A" and the de flection recorded during depolarization of the right ven tricle as "V". The heart rate (HR) and atrioventricular conduction time (AVCT, AV interval) were measured and displayed on a thermo-writing rectigraph (Nihon Kohden WT685T). Systemic arterial pressure was also recorded via the femoral artery. The left femoral vein was also cannulated for physiological saline infusion to adjust spontaneous fluid losses and drug injection. When the surgical procedure was ended, the sterno tomy was covered by a vinyl sheet and, by adjusting the distance of the table lamp to the cardiac surface, epi cardial temperature was maintained. Two bipolar plate silver electrodes, 2 mm inter electrode distance, were used to stimulate the intra cardiac sympathetic nerve fibers (13). One bipolar elec trode was placed on the fatty tissue overlying the right side of the left atrial junctions of the right pulmonary veins, and this was used to stimulate the intracardiac sympathetic nerve fibers to the SA nodal region; we re fer to this as "SAS stimulation". The second electrode was placed on the fatty tissue at the junction of the in
ferior vena cava and left atrium. It was used to stimu late the intracardiac sympathetic nerve fibers to the AV nodal region; we refer to that as "AVS stimulation". When we studied the cardiac responses to sympathe tic nerve stimulation, a dog was given atropine sulfate (0.2 mg/kg, i.v.), and 0.1 or 0.2 mg/kg, i.v. of atropine was given each hour thereafter to block the responses mediated by the muscarinic receptors. We used repeti tive bursts of stimuli (Nihon Kohden SEN 7103) with a 5 mA pulse amplitude and 1 to 2 msec pulse duration. The pulse amplitude and duration were adjusted so that the stimuli were above threshold for neighboring nerve fibers but below threshold for the cardiac cells (13). The interpulse interval within each burst was 10 msec. A brief burst of stimuli was delivered in each cardiac cycle. Each stimulus burst contained from 1 to 5 pulses. Stimulation was triggered by the atrial depolarization ("A" wave); the time from the beginning of the "A" wave to the beginning of the stimulus burst was 10 msec. Thus, a burst with 5 pulses terminated within 60 msec from the beginning of atrial depolarization. To avoid excitation of these structures, we used brief bursts of stimuli delivered at preselected times each car diac cycle, to coincide with the absolute refractory period. Each 30-sec train of stimuli to the nerves was followed by a recovery period of at least 4 min. Experimental protocols Four groups of experiments were carried out. In the first group with 4 dogs, we repeated SAS and AVS sti mulations eight times to confirm that the response to each stimulation would be maintained at a similar level during a series of experiments. The stimulation inter vals of each SAS or AVS stimulation were 10 min. In the second group of experiments, the effects of ICI 118,551 (n = 8) on the positive chronotropic response to SAS stimulation and the positive dromotropic re sponse to AVS stimulation were studied. ICI 118,551 at doses of 1 to 1000pg/kg, i.v. was given into the femor al vein. In the third group with 6 dogs, we studied the effects of atenolol on the positive chronotropic response and the positive dromotropic response to each stimula tion. We also injected atenolol at doses of 0.1 to 100 ,ug/kg, i.v. into the femoral vein. We measured the HR and AVCT before and more than 5 min after admin istration of ICI 118,551 or atenolol. In the fourth group with 5 dogs, we compared the effect of atenolol (10 ,ug/kg, i.v.) alone and the combination of atenolol (10 ,ug/kg, i.v.) and ICI 118,551 (30,ug/kg, i.v.) on the positive chronotropic response to SAS stimulation or the dromotropic response to AVS stimulation. We meas ured the HR and AVCT before and more than 5 min after atenolol alone or the combination of atenolol and
ICI 118,551. The order of treatment with atenolol alone or the combination of atenolol and ICI 118,551 was randomized, and sufficient recovery time was allowed between two treatments. Drugs The drugs used in the present experiments were ate nolol (Sigma, St. Louis, MO, USA), atropine sulfate (Wako Pure Chemical Co., Osaka, Japan), ICI 118,551 (erythro [ ± ]-1-[7-methylindan-4-yloxy]-3-isopropyl-amino butan-2-ol) (generously donated by Imperial Chemical Industries Co., Macclesfield, England), and propranolol hydrochloride (Sigma, St. Louis, MO, USA). Each drug was dissolved in physiological saline before start ing the experiments. Statistical analysis All data were expressed as a percentage of change in the respective control value of each stimulation obtained just before treatment with /3-adrenoceptor antagonists. These data were expressed as means ± S.E. Fifty percent inhibition doses (ID50) were deter mined for each dose-inhibition curve. An analysis of variance was used for the statistical analysis of multiple comparisons of data and Student's t-test for paired and unpaired samples was used for comparisons between the two groups. P values of less than 0.05 were consi dered to indicate a statistically significant difference.
RESULTS The changes in HR and AVCT induced by SAS or AVS stimulation Stimulation of the intracardiac sympathetic nerve fibers to the SA nodal region (SAS stimulation) consis tently increased HR and variably changed AVCT in an anesthetized, neurally decentralized open-chest dog af ter atropine was given (Fig. 1A, control). The changes in AVCT induced by SAS stimulation were not due to direct effects of stimulation, but caused by a shift of the pacemaking site within the sino-atrial pacemaker com plex (13). Stimulation of the intracardiac sympathetic nerve fibers to the AV nodal region (AVS stimulation) consistently decreased AVCT, but did not affect the HR (Fig. 1B, control). In the first group, the first SAS stimulation increased HR by 31 ± 7 beats/min (n = 4) from the prestimulation control level. The first AVS sti mulation decreased AVCT by 21 ± 3 msec from the basal level. Changes in HR and AVCT induced by re petition of each stimulation did not significantly change during a series of experiments. The 8th SAS and AVS stimulations changed HR and AVCT to 93 ± 3% and 95 ± 3% of the changes in HR and AVCT to each first stimulation, respectively. Therefore, the changes in HR and AVCT in response to each stimulation before treat ment with /8-adrenoceptor antagonists were regarded as control values.
Fig. 1. Effects of ICI 118,551 and propranolol on the positive chronotropic (HR) and dromotropic (AVCT) responses to A) stimulation of the intracardiac sympathetic nerve fibers to the SA nodal region (SAS stimulation) and B) stimulation of the intracardiac sympathetic nerve fibers to the AV nodal region (AVS stimulation) in a neurally decentralized, open chest, anesthetized dog.
Blocking effects of ICI 118,551 The basal HR and AVCT in 8 anesthetized dogs for a second ICI 118,551-treated group were 106 ± 5 beats/min, and 133 ± 7 msec, respectively. These values were not significantly different from those after treatment with ICI 118,551. SAS stimulation increased HR by 38 ± 4 beats/min from the prestimulation con trol value. AVS stimulation decreased AVCT by 34 ± 3 msec from the basal level. As shown in Fig. 1A, ICI 118,551 at doses of 1 to 1000,ug/kg, i.v. inhibited the increases in HR induced by SAS stimulation dose-dependently in an anesthe tized, open-chest dog treated with atropine. ICI 118,551 (100 and 1000 ,ug/kg, i.v.) also attenuated the shorten ing of the AVCT induced by AVS stimulation, although ICI 118,551 at doses of 1 to 10,ug/kg, i.v. lit tle affected the effects of AVS stimulation (Fig. 1B). The positive chronotropic response to SAS stimulation and the positive dromotropic response to AVS stimula tion were not completely blocked by ICI 118,551 at a dose of 1000,ug/kg, i.v. Propranolol at the dose of 1 mg/kg, i.v. abolished the residual positive responses to SAS or AVS stimulation (Fig. 1, A and B). The composite data from 8 experiments are shown in Fig. 2. ICI 118,551 at doses of 1 to 1000,ug/kg, i.v. sig nificantly (P < 0.05) inhibited the positive chronotropic response to SAS stimulation in a dose-dependent man ner. On the other hand, the positive dromotropic re sponse to AVS stimulation was significantly (P < 0.05) attenuated by ICI 118,551 at doses of 30,ug/kg, i.v.
and more (100 and 1000,ug/kg, i.v.) dose-dependently. The inhibition of ICI 118,551 at doses of 3 to 1000 ,ug/kg, i.v. on the positive dromotropic response to AVS stimulation was less than that on the positive chronotropic response to SAS stimulation at each dose (Fig. 2). Blocking effects of atenolol The basal HR and AVCT in 6 anesthetized dogs for an atenolol-treated group were 111 ± 10 beats/min and 136 ± 7 msec, respectively. These values were not sig nificantly different from those after treatment with ate nolol. SAS stimulation increased HR by 32 ± 3 beats/min from the prestimulation control value. AVS stimulation decreased AVCT by 35 ± 3 msec from the basal level. These data were not significantly different from the data of the ICI 118,551-treated group. Atenolol significantly (P < 0.05) inhibited both the positive chronotropic response to SAS stimulation and the positive dromotropic response to AVS stimulation at 0.3,ug/kg, i.v. and more (1 to 100,ug/kg, i.v.) in a dose-dependent manner (Fig. 3). The inhibition of ate nolol (0.1 to 100,ug/kg, i.v.) on the positive chronotro pic response to SAS stimulation was not significantly different from that on the positive dromotropic re sponse to AVS stimulation at each dose (Fig. 3). The IC50 values of atenolol for the chronotropic and dro motropic responses to each stimulation were 6.9 ± 2.8 ,ug/kg, i.v. and 12.9 ± 6.1,ug/kg, i.v., respectively. These values were not significantly different.
Fig. 2. The effects of ICI 118,551 at doses of 1 to 1000 ,ug/kg, i.v. on the positive chronotropic response to stimulation of the intracardiac sympathetic nerve fibers to the SA nodal region (0) and on the positive dromotropic response to stimula tion of the intracardiac sympathetic nerve fibers to the AV nodal region (•) in 8 anesthetized dogs . Vertical bars show S.E. *P < 0.05, **P < 0.01 compared to control values.
Blocking effects of the combination of atenolol and ICI 118,551 SAS stimulation increased HR by 35 ± 4 beats/min from the prestimulation control value in the atenolol group and by 30 ± 5 beats/min in the combination of atenolol and ICI 118,551 group. These data were not significantly different. AVS stimulation decreased AVCT by 24 ± 3 msec from the basal level in the ate nolol group and by 22 ± 3 msec in the combination of atenolol and ICI 118,551 group. These data were not significantly different.
The blocking effect of the combination of atenolol (10,ug/kg, i.v.) and ICI 118,551 (30,ug/kg, i.v.) on the positive chronotropic response to SAS stimulation was significantly (P < 0.05) greater than that of atenolol (10 ug/kg, i.v.) alone (Fig. 4A). The blocking effect of the combination of atenolol and ICI 118,551 on the positive dromotropic response to AVS stimulation was not signi ficantly different from that of atenolol, but the com bination treatment (41 ± 8%) tended to inhibit the re sponse more than atenolol alone (53 ± 6%) (Fig. 4B).
Fig. 3. The effects of atenolol at doses of 0.1 to 100 ug/kg, i.v. on the positive chronotropic response to stimulation of the intracardiac sympathetic nerve fibers to the SA nodal region ((--)) and on the positive dromotropic response to stimula tion of the intracardiac sympathetic nerve fibers to the AV nodal region (•) in 6 anesthetized dogs. Vertical bars show S.E. *P < 0.05, **P < 0.01 compared to control values.
Fig. 4. The effects of atenolol (AT, 10 ug/kg, i.v.) and the combination atenolol (10 ug/kg, i.v.) and ICI 118,551 (30 pg/kg, i.v.) (AT + ICI) on the positive chronotropic response to SAS stimulation (panel A) or the dromotropic response to AVS stimulation (panel B). Vertical bars show S.E. * P < 0.05, compared with atenolol alone and the combination of two drugs.
DISCUSSION In the present study, we demonstrated that positive dromotropic and chronotropic responses to sympathetic nerve stimulation were induced by activation of both 1 and N 92-adrenoceptors in anesthetized dog hearts and the proportion of the 82-adrenoceptor-mediated effect for SA nodal pacemaker activity was greater than that for AV conductivity, although the cardiac re sponses to sympathetic stimulation were predominantly mediated through 81-adrenoceptors. After atropine was given to the anesthetized dog, sti mulation of the intracardiac sympathetic nerve fibers to the SA nodal region (SAS stimulation) increased HR and variably changed AVCT. The changes in AVCT were caused by a shift of the pacemaker site within the sino-atrial pacemaker complex (13, 14). Stimulation of the intracardiac sympathetic nerve fibers to the AV nodal region (AVS stimulation) decreased AVCT, but it did not alter the HR. Thus, AVS stimulation permit ted us to examine the effects of sympathetic stimulation on the AVCT without concomitant secondary influ ences of the changes in HR, pacemaker location, and conduction pathway (15 -17). SAS stimulation increased HR and the positive chro notropic response was dose-dependently inhibited by the selective 82-adrenoceptor antagonist ICI 118,551 in the dose range of 1 to 1000,ug/kg, i.v. ICI 118,551 blocks ,82-adrenoceptors selectively in doses of up to 100,ug/kg, i.v. in anesthetized dogs (5, 18). Atenolol also inhibited the positive chronotropic response to SAS stimulation (Fig. 3). The effect of the combination of atenolol (10,ug/kg, i.v.) and ICI 118,551 (30 pg/kg, i.v.) on the positive chronotropic response to SAS sti mulation was significantly greater than that of atenolol (10pg/kg, i.v.) (Fig. 4A). Atenolol is a selective ," 1 adrenoceptor antagonist and its selectivity ratio for 81 adrenoceptors versus /32-adrenoceptors is approximately 6 to 20 (3, 5). Thus, our results indicate that the posi tive chronotropic response to sympathetic nerve sti mulation is in part mediated by ,82-adrenoceptors in addition to ,81-adrenoceptors in anesthetized dog hearts and suggest that neurally released NE in part controls the functional chronotropic response mediated through /32-adrenoceptors. These results were inconsistent with a previous report by Bilski et al. (5). They showed that the increases in HR due to sympathetic stimulation were not significantly inhibited by ICI 118,551 in anes thetized dogs. Although we do not know the exact reasons of the discrepancy between our results and theirs, it is postulated that 1) we stimulated the discrete intracardiac sympathetic nerves to the SA nodal region, and 2) they used only four dogs to composite data.
NE is postulated as a selective N 1-adrenoceptor ago nist for /8-adrenoceptors (2-5). It was found that the affinity value of the N 1-adrenoceptor subtype for NE was 15 times greater than that of the ,82-adrenoceptor subtype in the isolated dog atrial tissue and the potency of NE in occupying 81 and ,82-adrenoceptors was simi lar to its potency in stimulating f1 and /82-subtype mediated increases in adenylate cyclase activity (2). Thus, those studies support our results that the positive chronotropic response to sympathetic stimulation is in part mediated by ,82-adrenoceptors in the dog heart in situ. AVS stimulation decreased AVCT in anesthetized dogs with atropine. Atenolol inhibited the positive dro motropic response to AVS stimulation in a dose-depend ent manner, whereas ICI 118,551 at doses of 30 and 100 ,ug/kg, i.v. attenuated the positive dromotropic re sponse to AVS stimulation slightly but significantly. Although ICI 118,551 blocks ,82-adrenoceptors selec tively in doses of up to 100,ug/kg, i.v. in anesthetized dogs (5, 18), in the present study, ICI 118,551 at a dose of 100,ug/kg, Lv. inhibited the positive chronotropic response to sympathetic stimulation by almost 50% (Fig. 2). Thus, it is likely that that dose of ICI 118,551 inhibited partly the positive dromotropic responses to neurally released NE mediated by /81-adrenoceptors be cause the ,82/,81-adrenoceptor ratio was 30%/70% in the dog atrium (2). ,82-Adrenoceptors in the AV node of the mammalian hearts have been shown by auto radiography (11, 19). Motomura and Hashimoto (20) reported briefly that ICI 118,551 inhibited the positive dromotropic effect of a selective ,82-adrenoceptor ago nist, procaterol, in the canine isolated, blood-perfused AV node preparation. Therefore, our results suggest that sympathetic nerve stimulation induces a little posi tive dromotropic effect mediated through 82-adreno ceptors in addition to 81-adrenoceptors in anesthetized dogs. Presynaptic 82-adrenoceptors have been postulated to facilitate NE release from nerve endings (21, 22). If ICI 118,551 blocked only presynaptic ,82-adrenoceptors, ICI 118,551 would inhibit the positive chronotropic and dromotropic responses to each stimulation similarly. However, the ratio of /32-adrenoceptor-mediated dro motropic response to NE was less than that of the ,82 adrenoceptor-mediated chronotropic response to NE in our experiments. In guinea pig atria, neurally released epinephrine but not neurally released NE facilitated the stimulation induced release of NE (22). Therefore, it is likely that the ,82-adrenoceptors of the postsynaptic site are inhibited by ICI 118,551 in our experiments, although it is difficult to exclude completely that pre synaptic ,82-adrenoceptors are in part involved.
It has been demonstrated that stimulation of the /32 adrenoceptors by selective adrenoceptor ,82-agonists, procaterol and salbutamol, produces a positive chrono tropic effect in anesthetized dogs (23) and humans (24). However, it does not mean such stimulation of the re ceptors to be physiological relevant (24). The effects of atenolol and propranolol were studied on the hemody namic responses induced by exercise in dogs and sug gested that ,82-adrenoceptors in the SA node might be stimulated by circulating catecholamines and contri buted greatly to sympathetic modulation of HR during heavy exercise in dogs (6). It was shown that failing hu man ventricular myocardium contained a relative high proportion of X82-adrenoceptors, due to selective down regulation of ,81-adrenoceptors using a radioligand binding study (25). Recently, it was found that selective /31-adrenoceptor blockade enhanced positive inotropic responses to catecholamines as well as salbutamol medi ated through 82-adrenoceptors in the human atrium (4). In the present study, we demonstrated that the positive chronotropic and dromotropic responses to sympathetic stimulation were mediated through 82 adrenoceptors in addition to predominant ,81-adreno ceptors in the anesthetized dog. Therefore, we suggest that neurally released NE controls functional cardiac re sponses mediated through ,82-adrenoceptors, especially during 81-adrenoceptor blockade and pathophysiologi cal conditions. Acknowledgment We supplies
thank
Imperial
of ICI
Chemical
Industries
Co.,
Ltd.
for
generous
118,551.
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24
25
Hall, J.A., Petch, M.C. and Brown, M.J.: Intracoronary
in
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