Pharmacological Analysis of Positive Chrono and Inotropic Responses to Denopamine (TA-064) in Dog Cross-Circulated Atrial and Ventricular Preparations KunioAKAHANE, Yasuyuki FURUKAWA, Yasuyuki KARASAWA, Lei-MingREN and ShigetoshiCHIBA* Departmentof Pharmacology,Shinshu University School of Medicine, Matsumoto390, Japan AcceptedSeptember 21, 1989

Abstract-Positive chrono and inotropic responses to denopamine (TA-064, (-) (R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino] ethanol), a new and orally active cardiotonic agent, were investigated in the canine isolated right atrial or left ventricular preparation which was cross-circulated with blood from another support dog. Denopamine dose-dependently increased the sinus rate, right atrial and left ventricular contractile force. Denopamine was one to two orders of mag nitude less potent than isoproterenol. The positive chrono and inotropic effects of denopamine in isolated, blood-perfused right atria were dose-dependently in hibited by treatment with propranolol and atenolol. The effects of denopamine were only slightly attenuated by ICI 118,551 in doses which completely suppressed the positive chrono and inotropic effects of procaterol. The increases in sinus rate and atrial contractility induced by denopamine were partially but significantly attenuated by treatment with imipramine in a dose which suppressed the effects of tyramine and potentiated the effects of norepinephrine. These results indicate that denopamine is a highly selective beta-1 adrenoceptor agonist in isolated, blood perfused dog heart preparations, and they also suggest a mild catecholamine releasing activity through tyramine-like action in isolated right atria.

Congestive heart failure is characterized by Recently, orally active positive Inotropic com a state of insufficient cardiac output to fulfill pounds have been developed which have the metabolic requirements of the body. One beta -sympathom1metic (1-4) or phosphodies of the therapies for congestive heart failure is terase inhibiting action (5-9), and these agents augmenting the cardiac pumping function are structurally unrelated to cardiac gly (positive Inotropic action). Cardiac gly cosides. Denopamine, a newly synthesized cosides are the drugs used for producing phenylethanolamine derivative, is the former Positive inotropic effects, but their effec type of cardiotonic agent. Ikeo et al. (10) have tiveness and safety remain controversial be reported that, administered parenterally or cause of their toxicity. Some catecholamines orally, denopamine induced a significant such as dopamine and dobutamine, which dose-dependent increase in contractile force have a strong positive inotropic effect, are and dp/dtmax in the left ventricle with a little clinically available only by intravenous ad change in heart rate in anesthetized and con ministrations. Therefore, potent positive Ino scious dogs. The cardiotonic effects of tropic drugs which are orally active and pos denopamine with a weak arrhythmogenic ac sess a wide margin of safety are of great tivity were also demonstrated in guinea pigs, interest in the treatment of heart failure. rabbits, cats, monkeys, rats and pigs (11-14). Kino et al. (15) have revealed that the in

To whom correspondence should be addressed. travenous infusion of denopamine produced a marked increase in dp/dtmax in the left ventri pump (Harvard Apparatus model 1210). A cle without significant effects on heart rate in pneumatic resistance was placed in parallel both normal and diseased human hearts. The with the perfusion system so that the per mechanism of the cardiotonic activity of fusion pressure could be maintained at 100 denopamine mainly involves its ability to mmHg. The blood flow rate to the isolated selectively stimulate cardiac beta-1 receptors atrium was 6 to 11 ml/min. The venous ef (16-18). However, many of the studies were fluent from the preparation was led to a col performed by in vivo experiments or by the lecting funnel, from which it was returned Langendorff's method using small animals continuously to the support dog via the ex such as guinea pigs or rabbits in which beta ternal jugular vein. The ventricular margin of 2 adrenoceptors have a minor role for in the atrium was attached to a rigid stainless creasing heart rate or cardiac contractility steel bar, and the preparation was placed in a (19-22). Recently, we have reported the glass container which was kept at a constant existence of positive chrono and inotropic temperature of 37'C by means of a heating responses mediated by beta-2 adrenoceptors bath circulator (Haake FE 2). The upper part in addition to predominant beta-1 adreno of the atrium was connected to a force-dis ceptors in isolated, blood-perfused right atrial placement transducer (Nihon Kohden AP and left ventricular preparations of the dog 620 G) by a silk thread. The atrial muscle was (23). Therefore, this study was designed to usually stretched to a resting tension of 2 g. examine the direct cardiac effects of denop The isometric tension was recorded on a amine on isolated, b!ood-perfused right atrial thermo-writing rectigraph (Nihon Kohden and left ventricular preparations of the dog WT 685T). A pair of silver electrodes was which are free from extracardiac modifica brought into contact with the epicardial tions such as autonomic nervous reflexes. surface of the isolated atrium to record the atrial electrogram. The tachometer was driven Materials and Methods by the atrial electrogram. Preparation of the isolated, blood-perfused The left ventricular muscle along the right atrial or left ventricular muscle: Experi anterior descending branch of the left coro ments were carried out on mongrel dogs of nary artery was excised, and the anterior either sex anesthetized with sodium pento descending artery was cannulated. The left barbital (30 mg/kg, i.v.). Isolated right atria or ventricular preparation was perfused with the left ventricles were obtained from 28 recipient heparinized blood from the support dog using dogs weighing 7 to 16 kg, and each prepara the same perfusion system for the right atrial tion was perfused with arterial blood from a preparation. A pair of bipolar silver electrodes second support dog. The details of these prep was sewn on the ventricular free wall, and the arations have been described in Previous preparation was driven by an electrical stimu papers (24-26). Sodium heparin (500 USP lator (Nihon Kohden SEN 7103) at a fre units/kg, i.v.) was administered to each dog quency of 2 Hz with square wave pulses of 2 at the beginning of the perfusion, and 200 msec duration and twice the threshold voltage USP units/kg were given each hour thereafter. (usually 4 V). The left ventricular tension After heparin (200 USP units/kg, i.v.) was development was measured isometrically by a administered, the right atrium or the left force-displacement transducer through a fine ventricle was excised and immersed in cold thread connected to a ventricular surface. The Ringer's solution. The wet weight of the resting tension of the ventricular muscle was isolated right atrial and left ventricular prep 2 g. arations varied from 8 to 16 g and 10 to 18 g, The femoral arterial blood pressure and respectively. heart rate derived from the ECG lead II of the In the right atrial preparation, the sinus support dog and the blood flow rate to a prep node artery was cannulated via the right aration were simultaneously recorded. coronary artery, and it was perfused with Drugs: The drugs used in the experiments heparinized blood led from the carotid artery were denopamine (TA-064, (-)-(R)-1-(p of the support dog with the aid of a peristaltic hydroxyphenyl) -2 [(3,4-dimethoxypheneth yl) amino]ethanol, generously donated by usually assessed 1-1.5 min after treatment Tanabe Seiyaku Co., Ltd., Osaka, Japan), / with an antagonist. isoproterenol hydrochloride (Nikken Kagaku), Statistical analysis: All changes in positive d,/-norepinephrine hydrochloride (NE, San chrono and inotropic responses induced by kyo), procaterol hydrochloride (Otsuka), each dose of substance were expressed as tyramine hydrochloride (Wako Pure Chemi percent changes from their predrug levels. cal), propranolol hydrochloride (Sigma), Values presented are means±S.E.M. Data, atenolol (Sigma), ICI 118,551 (generously which were obtained as the maximum re donated by Imperial Chemical Industries, sponses to each drug, were analyzed by Macclesfield, England) and imipramine Student's t-test for paired data. A P value less hydrochloride (Fujisawa). All drugs were than 0.05 was considered as statistically dissolved in physiological saline before the significant. start of the experiment. The volume of the Results drug solution injected into the sinus node artery of the isolated right atrium or the an Effects of denopamine, isoproterenol, NE terior descending branch of the isolated left and procaterol on sinus rate and tension ventricle was 0.01-0.03 ml over a period of 4 development in isolated right atria: When sec. The dose of a cardiotonic agent which denopamine was injected into the sinus node evoked about 50-60% increase in atrial con artery of the spontaneously beating dog right tracti!e force with an obvious chronotropic atrium, the drug dose-dependently increased response was roughly selected for the control the sinus rate and atrial contractile force, and response, i.e., denopamine at 1 or 3 nmol, EN the positive chrono and inotropic responses at 0.1 or 0.1 nmol, procaterol at 1 or 3 nmol to denopamine at high doses lasted more than and tyramine at 10 or 30 nmol, respectively. 10 min (Fig. 1). Dose-response curves of Since the effect of each antagonist appeared denopamine, isoproterenol, NE and pro in less than 1 min and continued for approxi caterol for the sinus rate and atrial tension mately 20-30 min, the positive chrono and development are shown in Fig. 2. The thresh inotropic responses to each agonist were old doses for increasing the sinus rate and

Fig. 1. Typical tracings of positive chrono and inotropic responses to 0.3. 3 and 30 nmol of denopamine in an isolated dog right atrium (A) and positive inotropic responses to 1, 10 and 100 nmol of denopamine in an isolated dog left ventricle (B). atrial tension were 0.001 nmol (isopro nopamine was about 300 times less potent terenol), 0.01 nmol (NE), 0.03 nmol (pro than isoproterenol for increasing sinus rate, caterol) and 0.1 nmol (denopamine). The whereas denopamine was only 30 times less rank order of potency for the positive chrono potent than isoproterenol for increasing atrial and inotropic responses were isoproterenol> contractility (Table 1). Although a selective NE>denopamine (Fig. 2, Table 1). De beta-2 agonist, procaterol, increased the sinus rate more than atrial contractility, the maximal responses were much smaller than those produced by isoproterenol. Effects of denopamine, isoproterenol and procaterol on tension development in isolated left ventricles: When denopamine was ad ministered into the cannulated anterior de scending branch of the left coronary artery of the electrically driven left ventricle, positive inotropic effects were induced in a dose related manner. Typical tracings of increasing doses of denopamine are shown in Fig. 1 (lower panel). Summarized data of dose responses to denopamine, isoproterenol and procaterol are shown in Fig. 3. The threshold doses for inducing the positive inotropic responses were 0.001 nmol (isoproterenol), 0.03 nmol (procaterol) and 0.1 nmol (de nopamine), respectively. The effective dose for increasing ventricular contractility by 100% (ED, 00) of denopamine was 49 nmol and was about 200 times larger than that of isoprotere nol (Table 1). Effects of propranolol on positive chrono Fig. 2. Dose-response curves of isoproterenol (/, and inotropic responses induced by de n=10), norepinephrine (NE, E], n=6), denopamine nopamine and NE: After treatment with a non (0, n=6) and procaterol (0, n=9) for sinus rate selective beta-blocker, propranolol (0.1-10 (upper panel) and tension development (lower nmol), the positive chrono and inotropic re panel) of isolated right atria. Data are expressed as sponses to both denopamine (1 or 3 nmol) percentage changes of each basal value. Basal sinus and NE (0.1 or 0.3 nmol) were dose-de rate and atrial contractile force were 110.4±5.5 beats/min and 2.0±0.3 g, respectively. Points pendently suppressed in the isolated, cross represent mean values, and vertical bars show S.E.M. circulated dog right atrium. Summarized data

Table 1. ED values of denopamine, isoproterenol and NE for sinus rate, atrial and ventricular developed tension in isolated, blood-perfused canine right atrium and left ventricle

Data were obtained from 6-9 isolated right atria and 4-6 isolated left ventricles. Each va!ue is presented as a mean±S.E.M. Basal sinus rate, atrial contractile force and ventricular contractile force were 110.4±5.5 beats/min, 2.0±0.3 g and 5.0±0.5 g, respectively. SR, sinus rate; AT, atrial tension development; VT, ventricular tension development; NE, norepinephrine. of the effects of propranolol are shown in Fig. tagonist, atenolol (0.1-10 nmol), dose de 4. pendently inhibited the increases in sinus Effects of atenolol on positive chrono and rate and atrial contractility elicited by denop inotropic responses induced by denopamine, amine and NE, whereas atenolol only slightly NE and procaterol: A selective beta-1 an attenuated the positive chrono and inotropic

Fig. 3. Dose-response curves of isoproterenol (/, n=6), denopamine (0, n=4) and procaterol (0, n=6) for tension development of isolated left ventricles. Data are expressed as percentage changes of each basal value. Basal ventricular contractile force was 5.0±0.5 g. Points represent mean values, and vertical bars show S.E.M.

Fig. 4. Effects of treatment with propranolol (0.1-10 nmol) on increases in sinus rate (upper panel) and atria) tension development (lower panel) induced by denopamine (1 or 3 nmol, open columns) and norepinephrine (NE, 0.1 or 0.3 nmo!, hatched columns) in 5 isolated, blood-perfused right atria. Data are expressed as percentage changes of each basal value. Vertical bars show S.E.M. *P<0.05, **P< 0.01, compared with the respective control (C). responses to procaterol in the dog right atrium were 63.8±5.9 and 55.9±7.9%, respectively; (Fig. 5). Mean percentage inhibitions in the and those in the NE-induced responses by the denopamine-induced positive chrono and same dose of atenolol were 79.5±1.6 and inotropic responses by 10 nmol of atenolol 79.4±3.6%, respectively.

Fig. 5. Effects of treatment with atenolol (0.1-10 nmol) on increases in sinus rate (upper panel) and atrial tension development (lower panel) induced by denopamine (1 or 3 nmol, open columns), nor epinephrine (NE, 0.1 or 0.3 nmol, hatched columns) and procaterol (1 or 3 nmol, dotted columns) in 5-10 isolated, blood-perfused right atria. Data are expressed as percentage changes of each basal value. Vertical bars show S.E.M. *P<0.05, **P<0.01, compared with the respective control (C).

Fig. 6. Effects of treatment with ICI 118,551 (0.1-10 nmol) on increases in sinus rate (upper panel) and atrial tension development (lower panel) induced by denoparnine (1 or 3 nmol, open columns), norepinephrine (NE, 0.1 or 0.3 nmol, hatched columns) and procaterol (1 or 3 nmol, dotted columns) in 5-9 isolated, blood-perfused right atria. Data are expressed as percentage changes of each basal value. Vertical bars show S.E.M. *P<0.05, **P<0.01, compared with the respective control (C). Effects of ICI 118,551 on positive chrono of 300 nmol significantly inhibited the positive and inotropic responses evoked by denop chrono and inotropic responses to tyramine amine, NE and procaterol: A selective beta-2 (10 or 30 nmol) and potentiated the effects of antagonist, ICI 118,551 (0.1-10 nmol), dose NE (0.1 or 0.3 nmol) in 7-8 isolated, blood dependently inhibited the increases in sinus perfused dog right atria. After treatment with rate and atrial contractile force elicited by the same dose of imipramine, the denop procaterol. ICI 118,551 did not significantly amine-induced increases in sinus rate and affect the responses to N E, and the antagonist atrial contractile force were slightly but signifi at lower doses also did not significantly cantly depressed (Fig. 7). Mean percentage modify the positive chrono and inotropic re inhibitions in the positive chrono and inotro sponses to denopamine. However, ICI pic responses to tyramine by imipramine were 118,551 at 10 nmol significantly (P<0.05) 68.417.4 and 69.4±5.1%, respectively; and attenuated the positive chronotropic response those in the denopamine-induced responses to denopamine, and it tended to decrease the were 48.0+6.6 and 30.5±10.7%, respectively. positive inotropic response to denopamine (0.05metoprolol, and sinus rate in isolated, blood-perfused dog the effect was completely inhibited by pro right atria (23). Denopamine increased atrial pranolol. Nagao et al. (12) showed that the contractility more than the sinus rate. Further small vasodilating effect of denopamine on more, the positive chrono and inotropic the femoral artery of the anesthetized dog was effects induced by denopamine in isolated shifted after treatment with propranolol. It right atria were dose-dependently inhibited has been suggested that beta-adrenergic by treatment with a non-selective beta receptors in renal, mesenteric and femoral blocker, propranolol, which inhibited the NE arteries were of the beta-2 subtype (42). induced responses. These responses were These results may suggest that denopamine also blocked by a selective beta-1 antagonist, also possesses a weak beta-2 adrenoceptor atenolol, in a dose range which hardly affected stimulating property. the positive responses to procaterol. The Nagao et al. (12) have reported that in the positive chrono and inotropic responses to reserpinized dog, denopamine exerted in NE which is known to have a much higher creases in cardiac contractile force, heart rate selectivity for beta-1 adrenoceptors than and blood pressure similar to those in the non beta-2 adrenoceptors (20, 29-31) were in reserpinized normal dog, suggesting that the hibited by 10 nmol of atenolol more strongly cardiotonic actions of denopamine were not than those to denopamine (Fig. 5). Although due to the release of endogenous catechol Lands et al. (32, 33) first subclassified beta amines. Kino et al. (15) have also reported adrenoceptors into beta-1 and beta-2 sub that the positive inotropic effects of denop types with absolute organ-specific distribu amine were not mediated by the release of the tion such as beta-1 receptors in the heart and intrinsic catecholamines by measuring the plasma catecholamine levels. However, in tonic activity of MDL 17,043. J. Cardiovasc. this study, the positive chrono and inotropic Pharmacol. 4, 509-514 (1982) responses to denopamine was significantly 6 Endoh, M., Yanagisawa, T., Morita, T. and attenuated by treatment with imipramine. The Taira, N.: Differential effects of sulmazole (AR-L same dose of imipramine significantly de 115 BS) on contractile force and cyclic AMP levels in canine ventricular muscle: Comparison pressed the tyramine-induced responses and significantly potentiated the responses to NE, with MDL 17,043. J. Pharmacol. Exp. Ther. 234, indicating the dose of imipramine acted as an 267-273 (1985) 7 Alousi, A.A. and Johnson, D.C.: Pharmacology uptake blocker of endogenously released or of the bipyridines: amrinone and milrinone. externally administered NE. 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