In recent years, a new mechanism underly ing the positive inotropic action has been described for some new cardiotonic agents such as sulmazole (1), pimobendan (2), DPI 201-106 (3), APP 201-533 (4), MCI-154 (6-[4-(4'-pyridyl)aminophenyl]-4.5-dihydro 3(2H)-pyridazinone hydrochloride) (5, 6) and adibendan (7). These drugs increase Ca2+ sensitivity of the cardiac contractile protein system, and this effect is proposed to participate in the cardiotonic action of the agents. Among the aforementioned cardiotonic agents, only MCI-154 has been reported to enhance the interaction between action and myosin (5, 6). In the present study, we com pared the effects of MCI-154 on the con

Japan. J. Pharmacol.50, 411-419 (1989) 411

Comparison of the Effects of MCI-154, a New Cardiotonic Agent, and Some Cat+-Sensitizing Agents on the Response of the Contractile System to Ca 2+ in Skinned Cardiac Muscle

Yoshimi KITADA, Miyuki MORITA and Akihiro NARIMATSU PharmaceuticalsLaboratory, Life Science ResearchSector, Research Center, MitsubishiKasei Corporation, Yokohama 227, Japan Accepted April20, 1989

Abstract-Several cardiotonic agents (MCI-154, sulmazole, pimobendan and adibendan) were examined for their ability to influence Ca2+-activated tension development and MgATP-activated tension development in the absence of free Ca2+ (rigor tension), using the chemically skinned fiber from guinea pig papillary muscles. MCI-154, sulmazole, pimobendan and adibendan all increased the tension development induced by pCa (-log[Ca2+]M) 5.8 in a concentration dependent manner (10-6 to 10-4 M). The order of the potency was as follows: MCI-1 54>pimobendan>adibendan>sulmazole. MCI-154 enhanced the maximum tension developed at pCa 4.4 but sulmazole, pimobendan and adibendan did not enhance it. MCI-154, but not sulmazole, pimobendan and adibendan, enhanced the tension development induced by pMgATP (-log[MgATP]M) 6.0 in the absence of free Ca2+. MCI-154, sulmazole, pimobendan and adibendan concentration dependently (10-7 to 10-4 M) increased the force of contraction in isolated guinea pig papillary muscles. The order of the potency was as follows: MCI-154> adibendan>pimobendan>sulmazole. These results demonstrated that the Ca2+ sensitizing action on the contractile system may be involved in the positive inotropic action of MCI-154, sulmazole, pimobendan and adibendan, and that MCI-154 is the most potent among these drugs. Furthermore, sulmazole, pimobendan and adibendan did not enhance the interaction of actin and myosin, suggesting that the mechanism of actions of these drugs are qualitatively different from that of MCI-154.

tractile apparatus with those of other Ca2+ sensitizing agents, sulmazole, pimobendan and adibendan, using chemically skinned fiber from guinea pig papillary muscle.

Materials and Methods Chemically skinned fiber: Papillary muscles in the right ventricles of male guinea pigs (weight, 250-350 g) were used in the present experiments. Muscles were dissected in a Ringer's solution (see below). Then the ex ternal solution was replaced by a relaxing solution (see below). A small bundle with a diameter of about 0.1 to 0.15 mm and length of about 1 to 1.5 mm was dissected. To obtain the skinned fiber, the small bundles were treated with the relaxing solution con 412 Y. Kitada, M. Morita & A . Narimatsu

taining 250 ug/ml of saponin for 30 min (8, were obtained by replacing EGTA with EDTA. 9). This concentration of saponin affects not The strongly chelates both Mg2+ and Cat+. only the surface membrane but also the The apparent stability constants at pH 7.0 sarcoplasmic reticulum membrane (8, 9). were also taken from Martell and Smith (13): The composition of the Ringer's solution Ca-EDTA, 2.4X10-7 M, Mg-EDTA, 2.3X10-5 was as follows: 150 mM NaCI, 2 mM KCI, M. The solutions contained 3 mM ATP (total) 5 mM 4-(2-hydroxyethyl)-1 -piperazine and a fixed amount (5 mM) of free EDTA.The ethanesulfonic acid, 2 mM CaCI2 and 5.5 mM concentrations of total Mg and added Mg glucose, pH 7.4. For the skinned fiber experi EDTA necessary to obtain the desired pMg ments, appropriate amounts of stock solu ATP and pMg were calculated as the de tions, 1 M K-methanesulfonate (Ms), 0.1 M scribed above. CaMs2, 0.1 M MgMs2, 0.05 M ATP-Na2 Saponin was dissolved in the relaxing (KOH), 0.1 M ethylene glycol bis ((9 amino solution shortly before each experiment. ethylether N, N'-tetraacet1c acid) (EGTA) Isolated guinea pig papillary muscles: Male (KOH) and 0.2 M piperazine-N,N'-bis (2 guinea pigs, weighing 250-350 g, were killed ethanesulfonic acid) (PIPES) (KOH) were by a sharp blow on the skull. Right ventricular mixed and diluted to make solutions. The total papillary muscles were rapidly excised from concentrations of the different chemicals the isolated hearts and mounted in an organ needed to produce the desired free concen bath (30 ml). The bath solution was a Krebs trations of Cat+, Mg2+ and MgATP2 were Henseleit solution of the following composi calculated on a computer by an interactive tion: 118 mM NaCI, 24 mM NaHCO3, 4.8 mM procedure to solve the binding equations that KCI, 1.2 mM KH2PO4, 2.5 mM CaCI2, 1.2 mM use the stability constants described by Smith MgSO4 and 11 mM glucose. The solution was and Martell (10, 11). The following relaxing bubbled with 95% 02-5% CO2 at a temper solution was used: 129 mM KMs, 5.1 mM ature of 32 °C. Muscles were stretched to a MgMs2, 4.2 mM ATP-Na2, 2 mM EGTA and resting tension of about 0.5 g and stimulated 20 mM PIPES, brought to pH 7.0 with KOH. with pulses of a voltage about 20% above the The concentration of EGTA was altered when threshold at a frequency of 1 Hz. During the necessary. In Cat+-containing solutions, 10 equilibration period of 1 hr, the resting tension mM EGTA was used and specified amounts of the muscle was adjusted so as to give the of CaMs2 were added. The force-producing maximal contractile force. Force of contrac capacity of the muscle preparation was in tion was recorded on a thermal pen writing vestigated at varying free Ca2+ concentrations, oscillograph (NEC Sanei Instruments, Ltd., a MgATP2 concentration of 3.5 mM and a Tokyo, Japan, Recti-Horiz-8) by the use of free Mg2+ concentration of 1.5 mM. In all of force displacement transducers (Nihon the above alterations from the normal relaxing Kohden FD Pick up, TB-612T, Tokyo, Japan). solution, the ionic strength was kept constant In order to eliminate the interindividual (0.2 M) by adjusting the concentration of variability in the maximum inotropic response KMs. to cardiotonic drugs used in the present ex Very low MgATP2 was needed in some periments, isoproterenol was used as a experiments (Mg-ATP activation-tension), standard. The maximal response to isopro the achievement of which with the previously terenol was determined at the end of each ex described buffer system would have required periment after washout of all other drugs. The total ATP in the micromolar range. The concentration of isoproterenol was increased myoplasmic ATP would have been uncertain (usually up to 10-5 M) until the maximum re because of the hydrolysis of ATP within the sponse was obtained. The inotropic effect of skinned fiber. This hydolysis could have drugs used in the present study is expressed resulted in a gradient between the concen as the percentage of the maximal response to trations of ATP in the perfusion medium and in isoproterenol determined in the same muscle. the center of the skinned fiber (12). To avoid Drugs: All the chemicals used were reagent this problem, total ATP below 1.0 mM was not grade. Saponin was obtained from NBC used. Instead, low free Mg2+ and MgATP2 (Cleveland, Ohio, U.S.A.). MCI-154, sul Cardiotonic Agents and Cal+ Sensitivity 413

mazole, pimobendan and adibendan were tension development by 10-4 M MCI-154 synthetized at our Research Center. MCI-154, amounted to 116±25% (n=8) (Fig. 2). sulmazole and adibendan were dissolved in Pimobendan, adibendan and sulmazole also distilled water, and pimobendan was dis increased the pCa 5.8-induced tension de solved in dimethylsulfoxide. In the skinned velopement in a concentration-dependent fiber experiments, the volume of drug solution manner (10-6 to 10-4 M) (Figs. 1 and 2). The added to the 495 /,,I bath was 5 /cl. In the maximal increases in the tension development isolated cardiac muscle experiments, the after pimobendan, adibendan and sulmazole volume added to the 30 ml bath was 300 /tl. amounted to 43:L1% (n=7), 37.7±7% (n=7) and 2014% (n=7), respectively. When com Results pared in the concentrations producing 20% Skinned fiber experiments: Skinned fiber increase in the tension development at pCa from the guinea pig papillary muscle was pre 5.8, MCI-154 was about 10, 15 and 85 times contracted with pCa 5.8, which was a sub more potent than pimobendan, adibendan maximal activation ranging from 30 to 35% of and sulmazole, respectively. the maximal response to Cat'. After addition As shown in Fig. 3, only MCI-154 en of MCI-154, a concentration-dependent in hanced the maximum tension developed at crease in tension development was observed pCa 4.4 (a concentration of Ca2+ showing a in the concentration range of 10-6 to 10-4 M saturating effect). The effect of MCI -154 was (Figs. 1 and 2). The maximal increases in concentration-dependent (10-6 to 10-4 M)

Fig. 1. Typical recordings of the effects of MCI-1 54 (A), sulmazole (B), adibendan (C) and pimobendan (D) on the tension development induced by pCa 5.8 in the skinned fibers from the guinea pig papillary muscles. The fibers were treated with 250 ug/ml of saponin for 30 min before the experiments. 414 Y. Kitada, M. Morita & A. Narimatsu

Fig. 2. Concentration-dependent effects of MCI-1 54 (A), sulmazole (B), adibendan (C) and pimobendan (D) on the submaximal tension development of the skinned fibers from guinea pig papillary muscles. The submaximal contraction was induced by pCa 5.8. The fibers were treated with 250 ,g/ml of saponin for 30 min before the experiments. Data are expressed as % of the pre-drug value. Means±S.E. are given; n=6-8 for MCI-1 54, 7 for sulmazole, adibendan and pimobendan.

(Fig. 3). On the other hand, sulmazole, tigated in the present experiments. MCI-154 pimobendan and adibendan had no effect on enhanced the MgATP-induced tension de the tension development induced by pCa 4.4 velopment in a concentration -dependent (Fig. 3). manner (10-6 to 10-4 M) (Figs. 4 and 5). On We have previously described that in the the other hand, sulmazole, pimobendan and absence of free Cat', a bell-shaped curve of adibendan did not affect the MgATP-induced tension as a function of pMgATP was ob tension development at all (Fig. 4). tained in the skinned cardiac fiber from guinea Isolated cardiac muscle preparations: The pig papillary muscles (6). The maximum concentration-response curve for the positive tension activated by MgATP (pMgATP=6.0) inotropic effect of MCI-154 in isolated guinea was 42.0±1.8% (mean±S.E.) of the maximal pig papillary muscles is shown in Fig. 6. The tension activated by pCa 4.4 that was ob force of contraction was increased by MCI tained at 3.5 mM MgATP. Because the maxi 154 in a concentration-dependent manner mum tension was observed at [MgATP2-] (10-7 to 10-4 M). For comparison, the con 10-6 M, the effects of cardiotonic agents on tractile responses to pimobendan, adibendan the tension development induced by pMgATP and sulmazole are also presented in Fig. 6. 6.0 in the absence of free Ca2+ was inves When compared in the concentrations in Cardiotonic Agents and Ca2+ Sensitivity 415

Fig. 3. Effects of MCI-154, sulmazole, adibendan and pimobendan on the maximal Ca2+-activated tension development of the skinned fibers from guinea pig papillary muscles. The maximal con traction was induced by pCa 4.4. The fibers were treated with 250 Ug/ml of saponin for 30 min before the experiments. Data are expressed as % of the predrug value. Means±S.E. are given; n=5-9 for MCI 154, 6 for sulmazole, 7 for adibendan and pimobendan.

Fig. 4. Typical recording traces of the effects of (`,SCI-154 (A), sulmazole (B), adibendan (B) and pimobendan (B) on the tension development induced by pMgATP 6.0 in the absence of free Ca 2+ ion. The fibers were treated with 250 pg/ml of saponin for 30 min before the experiments.

In the isolated guinea pig papillary muscles, MCI-154 increased the force of contraction in a concentration-dependent manner. The effect of MCI-154 was much more potent than those of pimobendan, adibendan and sulmazole. This result is consistent with that obtained in the skinned fiber experiments. MCI-154 increased the developed tension in the isolated guinea pig papillary muscles in the same concentration range (10-6 to 10-4 M), causing the enhancement of the Ca2+ activated force in the skinned cardiac muscles. Similar results were obtained for other Ca2+ sensitizing agents used in the present study. Most of the new cardiotonic agents, in cluding sulmazole, pimobendan and adiben dan used in the present study, as well as amrinone, milrinone, fenoximone, piroximone and CI-914, inhibit cyclic AMP phosphodies terase and thereby increase intracellular cyclic AMP in the cardiac muscle; and this be lieved to be responsible for their positive inotropic effects (7, 17-22). These drugs in creased the levels of cyclic AMP in the mam

416 Y. Kitada. M. Morita & A. Narimatsu

sulmazole has been found to increase Ca2+ binding to troponin (14), the Ca2+ sensitizing effect of this drug and probably also pimo bendan and adibendan may be mainly due to an increased affinity of troponin C for Ca2} in the presence of the drugs. MCI-154 also stimulates the Ca2+ binding to troponin purified from the canine ventricular muscles and increases the activity of the myofibrillar and reconstituted actomyosin ATPases (15). Therefore the increase in response of the contractile system to Ca2+ induced by MCI 1 54 could be due to both an increase in the affinity of troponin C for Ca2+ and a facilitation of the actin-myosin interaction. This indicates that the effects of MCI-154 on the skinned Fig. 5. Concentration-dependent effects of MCI fiber preparations are qualitatively different 154 on the MgATP (10-6 M)-induced tension from those of sulmazole, pimobendan and development of the skinned fibers from guinea pig adibendan. The present findings are in agree papillary muscles. The fibers were treated with 250 ment with those of Perreault et al., (16), who ;ug/ml of saponin for 30 min before the experiments. Data are expressed as % of the pre-drug value. revealed that MCI-154 (10-6 to 10-4 M) Means+S.E. are given, n=5. enhanced both the sensitivity of the myofila ment to Ca2+ and pCa 4.5-induced maximum developed tension in chemically skinned ducing the positive inotropic response cor fibers from ferret and human hearts. There responding to about 20% of the maximum fore, the enhancement by MCI-154 of the response to isoproterenol, MCI-154 was responsiveness of the contractile system to about 15, 50 and 100 times more potent than Ca2+ seems to be independent of the species. adibendan, pimobendan and sulmazole, respectively.

Discussion In the present study, we have found that MCI-154 markedly enhanced the Ca2+-ac tivated contraction of the skinned cardiac muscle fibers in comparison with sulmazole, pimobendan and adibendan. In this respect, MCI-154 was about 10, 15 and 85 times more potent than pimobendan, adibendan and sulmazole, respectively, which have been re ported to produce an increase in Ca2+ sensi tivity of the myocardial contractile apparatus (1, 2, 7). The present study has confirmed that MCI-1 54 is the only positive intoropic agent which can enhance the interaction between actin and myosin, because this drug enhanced the maximum tension developed at a Ca2+ ion concentration giving a saturating effect (pCa 4.4) and augmented the tension development induced by MgATP in the absence of free Ca2+ (6). Unlike MCI-154, sulmazole, pimobendan and adibendan had no such effects. Since Cardiotonic Agents and Ca2+ Sensitivity 417

Fig. 6. Effects of MCI-154 (A), sulmazole (B), adibendan (C) and pimobendan (D) on the force of contraction in isolated guinea pig papillary muscles (stimulation-frequency 1 Hz, 32°C). Basal force of contraction and maximal response to isoproterenol in each group were as follows: 0.35±0.5 g and 1.27±0.16 g in muscles to which MCI-154 was administered, 0.41 ±0.10 g and 1.05±0.10 g in muscles to which sulmazole was administered, 0.35±0.10 g and 1.13±0.18 g in muscles to which adibendan was administered, and 0.53±0.10 g and 1.85±0.52 g in muscles to which pimobendan was administered. Means±S.E. are given; n=7 for MCI-154, n=6 for sulmazole, adibendan and pimobendan. malian hearts at a concentration (<10-4) positive inotropic drugs (24). MCI-154 scare lower than that needed for Cat+-sensitization. cely increases cyclic AMP levels in the canine In contrast, the positive inotropic effect of ventricular muscles in concentrations pro MCI-154 was not significantly affected by ducing definite positive inotropic effects carbachol (23), which has been reported to (10-7 to 10-4 M) and increase it only at higher inhibit the cyclic AMP-mediated action of concentrations (>10-4 M) (23). The positive

ing and coronary dilatory properties. Naunyn Schmiedebergs Arch. Pharmacol. 329, 316-325 (1985) 4 Salzmann, R., Bormann, G., Herzig, J.W., Markstein, R. and Scho!tysik, G.: Pharmaco logical actions of APP 201-533, a novel cardio tonic agent. J. Cardiovasc. Pharmacol. 7, 588- 596 (1985) 5 Kitada, Y., Narimatsu, A., Matsumura, N. and Endo, M.: Contractile proteins: a possible targets for the cardiotonic action of MCI-154, a novel cardiotonic agent? Eur. J. Pharmacol. 134, 229 231 (1987) 6 Kitada, Y., Narimatsu, A., Matsumura, N. and Endo, M.: Increase in Ca2+ sensitivity of the contractile system by MCI-154, a novel cardio tonic agent, in chemically skinned fibers from the guinea pig papillary muscles. J. Pharmacol. Exp. Ther. 243, 633-638 (1987) 7 Muler-Beckmann, B., Freund, P., Honerjager, P., Kling, L. and Ruegg, J.C.: In vitro investigations a new positive inotropic and vasodilating agent (BM 14,478) that increases myocardial cyclic AMP content and myofibrillar calcium sensitivity. J. Cardiovasc. Pharmacol. 11, 8-16 (1988) 8 Endo, M. and Kitazawa, T.: E-C coupling studies on skinned cardiac fibers. In Biophysical Aspects of Cardiac Muscle, Edited by Morad, M., p. 307 327, Academic Press, New York (1978) 9 Endo, M. and lino, M.: Specific perforation of muscle cell membranes with preserved SR functions by saponin treatement J. Muscle Res. Cell Motil. 1, 89-100 (1980) 10 Smith, R.M. and Martell, A.E.: Critical Stability Constants. Vol. 2, Plenum Press, New York (1975) 11 Smith, R.M. and Martell, A.E.: Critical Stability Constants. Vol. 3, Plenum Press, New York (1976)

418 Y. Kitada, M. Morita & A. Narimatsu inotropic effect of MCI-154 is not modified by force, stiffness and unloaded shortening velocity a and Q-adrenergic, muscarinic and his (Vmax) in isolated contractile structures from tamine (H1 and H2) receptors or channel an mammalian heart muscle. Arzneimittelforschung tagonists (25-27). Unlike cardiac glycosides, 31, 188-191 (1981 ) MCI-154 does not inhibit Na+,K+-ATPase 2 Ruegg, J.C., Pfitzer, G., Eubler, D. and Zeugner, C.: Effect on contractility of skinned fibers from (25). Taken together, these results suggest that the increased Ca2+ sensitivity of the mammalian heart and smooth muscle by a new benzimidazole derivative, 4,5-dihydro-6-[2-(4 myocardial contractile system may be re methoxy-phenyl)-1 H-benzimidazole-5-yl]-5 sponsible for the cyclic AMP-independent methyl-3(2H)-pyridazinone. Arzneimittelforsch action of MCI-154. However, other mecha ung 34, 1736-1738 (1984) nisms that increase intracellular free Ca2+ have 3 Scholtysik, G., Salzmann, R., Berthold, R., not been excluded, and such a mechanism Herzig, J.W., Quast, U. and Markstein, R.: DPI can be expected (28, 29), since the com 201-106, a novel cardioactive agent. Combi pound has been shown to inhibit the cyclic nation of c-AMP-independent positive inotropic, AMP phosphodiesterase (23). negative chronotropic, action potential prolong It has been well-established that classical positive inotropic agents such as cardiac glycosides and catecholamines act through a final common pathway, i.e., an increase in the amount of Ca2+ avaiable for activation of the contractile system, and therefore all bear a potential risk of Ca2+ overload induced by drugs. The Ca2+ overload and accompanying cardiac necrosis may cause the reduction in survival time in chronic heart failure. There fore, the clinical treatment for heart failure with positive inotropic agents should be most safe if cardiotonics that do not increase the intracellular Ca2+ concentration could be used. Interestingly, Colucci et al. (30) have also suggested that the ideal positive inotro pic agent may be one whose sole action is to increase the sensitivity of the contractile system to Ca2+ ions. In this context, the in troduction of a Ca2 sensitizing agent such as MCI-154 may stimulate the development of newer cardiotonic agents. In conclusion, the present study has shown that the Ca2+-sensitizing action on the con tractile system may be involved in the positive inotropic effects of MCI-154, sulmazole, pimobendan and adibendan, and MCI-154 is the most potent among these drugs. Furthermore, sulmazole, pimobendan and adibendan did not enhance the interaction between actin and myosin, suggesting that the mechanism of action of these drugs are qualitatively different from that of MCI-154. References 1 Herzig,J.W., Feile, K. and Ruegg,J.C.: Activating effect of AR-L 115 BS on the Ca'-sensitive Cardiotonic Agents and Cal+ Sensitivity 419

12 Reuben, J.P., Brabdt, P.W., Berman, M. and the mechanism of action of Cl-914, a new Grundfest, H.: Regulation of tension in the cardiotonic agent. Eur. J. Pharmacol. 119, 205 skinned crayfish muscle fiber. J. Gen. Physiol. 215 (1985) 57, 385-407 (1971 ) 23 Kitada, Y., Narimatsu, A., Suzuki, R., Endoh, M. 13 Martell, A.E. and Smith, R.M.: Critical Stability and Taira, N.: Does the positive inotropic action Constants. Vol. 1, Plenum Press, New York of a novel cardiotonic agent, MCI-154 involve (1974) mechanisms other than cyclic AMP? J. Phar 14 Solaro, R.J. and Ruegg, J.C.: Stimulation of Ca2+ macol. Exp. Ther. 243, 639-645 (1987) binding and ATPase activity of dog cardiac 24 Endoh, M.: Correlation of cyclic AMP and cyclic myofibrils by AR-L 115 BS, a novel cardiotonic GMP levels with changes in contractile force of agent. Circ. Res. 51, 290-294 (1982) dog ventricular myocardium during cholinergic 15 Kitada, Y., Kobayashi, M., Narimatsu, A. and antagonism of positive inotropic actions of Ohizumi, Y.: Potent stimulation of myofilament histamine, glucagon, theophylline and papa force and adenosine triphosphatase activity of verine. Japan. J. Pharmacol. 29, 855-864 (1979) canine cardiac muscle through a direct enhance 25 Narimatsu, A., Kitada, Y., Satoh, N., Suzuki, R., ment of troponin C Ca 2+ binding by MCI-154, a Kobayashi, M. and Okushima, H.: Cardiovascular novel cardiotonic agent. J. Pharmacol. Exp. Ther. profile of MCI-154, a novel and potent cardio (1989) (in press) tonic agent with vasodilator effect. Japan. J. 16 Perreault, C., Hajjar, R.J. and Morgan, J.P.: Pharmacol. 40, Supp. 234P (1986) Effect of MCI-154 on Ca 2+-activation in skinned 26 Narimatsu, A., Kitada, Y., Satoh, N., Suzuki, R. human and ferret cardiac muscle. J. Am. Coll. Kobayashi, M. and Okushima, H.: Cardio Cardiol. 11, Supp. 2, 139A (1988) vascular pharmacology of MCI-154, a novel and 17 Honerjager, P., Schafer-Korting, M. and Reiter, potent cardiotonic compound with vasodilator M.: Involvement of cyclic AMP in the direct property: comparison with other cardiotonics. inotropic action of arrinone. Naunyn Schmiede Fed. Proc. 45, 810 (1986) bergs Arch. Pharmacol. 318, 112-120 (1981) 27 Narimatsu, A., Kitada, Y., Satoh, N., Suzuki, R. 18 Honerjager, P., Heiss, A., Schafer-Korting, M., and Okushima, H.: Cardiovascular phar Schonsteiner, G. and Reiter, M.: UD-CG 115 macology of 6-[4-(4'-pyridyl)amino phenyl] a cardiotonic pyridazinone which elevates cyclic 4,5-dihydro-3(2H)-pyridazinone hydrochloride, AMP and prolongs the action potential in guinea a novel and potent cardiotonic agent with pig papillary muscle. Naunyn Schmiedebergs vasodilator properties. Arzneimittelforschung 37, Arch. Pharmacol. 325, 258-269 (1984) 398-406 (1987) 19 Endoh, M., Yamashita, S. and Taira, N.: Positive 28 Endoh, M.: Characteristics of regulation of intra inotropic effect of amrinone in relation to cyclic cellular calcium mobilization and sensitivity by nucleotide metabolism in the canine ventricular jf-adrenocetor and muscarinic agonists, and a muscle. J. Pharmacol. Exp. Ther. 221, 775-783 novel inotropic agent, MCI-154 in canine ven (1982) tricular myocardium. In Recent Advances in Ca 20 Endoh, M., Yanagisawa, T., Morita, T. and Taira, Channels and Ca-Antagonists, Edited by Yamada, N.: Differential effects of sulmazole (AR-L 115 K., Shibata, S., Katz, A.M. and Fabiato, A., BS) on contractile force and cyclic AMP levels Pergamon Press, Elmford (1989) (in press) in canine ventricular muscle: Comparison with 29 Warren, S.E., Kihara, Y., Pesaturo, J., Gwathmey, MDL 17,043. J. Pharmacol. Exp. Ther. 234, 267 J.K., Phillips, P. and Morgan, J.P.: Inotropic and 273 (1985) lusitropic effects of MCI-154 (6-4[4-pyridyl) 21 Endoh, M., Yanagisawa, T., Taira, N. and aminophenyl]-4,5-dihydro-3(2H)-pyridazinone) Blinks, J.R.: Effects of new inotropic agents on on human myocardium. J. Mol. Cell. Cardiol. cyclic nucleotide metabolism and calcium (1989) (in press) transients in dog ventricular muscle. Circulation 30 Colucci, W.S., Wright, R.F. and Braunwarld, E.: 73, Supp. III, 117-133 (1986) New positive inotropic agents in the treatment of 22 Weishaar, R., Quade, M., Schenden, J.A., Boyd, congestive heart failure. N. Engl. J. Med. 314, D. and Evans, D.B.: Studies aimed elucidating 349-358 (1986)