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The cardioactive properties of SC4453, a digoxin analogue with a C17β-pyridazine ring
European Journal o f Pharmacology, 60 (1979) 337--344
337
© Elsevier/North-Holland Biomedical Press
THE CARDIOACTIVE PROPERTIES OF 8C4453, A DIGOXIN ANALOGUE WITH A C17~-PYRIDAZINE RING THI~OPHILE G O D F R A I N D and JOSIANE GHYSEL-BURTON
Laboratoire de Pharmacodynamie Ggndrale et de Pharmacologie, Universitd Catholique de Louvain, Av. E. Mounier 73, UCL 7350, 1200 BruxeUes, Belgium Received 14 May 1979, revised MS received 4 September 1979, accepted 2 October 1979
T. G O D F R A I N D and J. GHYSEL-BURTON, The cardioactive properties o f SC4453, a digoxin analogue with a Cl 7~-pyridazine ring, European J. Pharmacol. 60 (1979) 337--344. SC4453 is a digoxin analogue with a pyridazine instead of a lactone ring in C175. SC4453 was compared with digoxin with respect to effect on contractility and on activity of the sodium pump in guinea-pig isolated left atria stimulated at 3.3 Hz. The two glycosides stimulated the sodium pump at low concentrations and inhibited at high concentrations. At the time to peak inotropic effect, for a similar inhibition of the Na pump, the increase in systolic tension was higher with SC4453 than with digoxin, whereas the increase in diastolic tension was similar for both. These observations confirm that the inhibition of the Na pump is not the only mechanism responsible for the positive inotropic effect of cardiac glycosides. Digoxin
C17~-Pyridazine
Na pump
Myocardial contractility
1. Introduction The lactone ring plays an important role in the biological action of cardiac glycosides. Its saturation yielding dihydroderivatives reduces their biological potency and, as observed with ouabain, suppresses their ability to stimulate b u t not to inhibit the sodium pump (GhyselBurton and Godfraind, 1976, 1979). However, in several natural and synthetic c o m p o u n d s with cardioactive action, the substituant in C17~ is n o t a lactone ring (Thomas et al., 1974). SC4453 is a derivative of digoxin in which the lactone ring in C17~ has been replaced b y a pyridazine ring (fig. 1). In another report from this laboratory, it was shown that SC4453 inhibited (Na ÷ +K÷)ATPase within the same range of concentration as digoxin (Godfraind and Tona Lutete, 1979). The purpose of the present experiment was to compare, in isolated guinea-pig atria, the actions of SC4453 and digoxin on the activity
o f the sodium pump and on contractility. The results show that SC4453 has cardioactive properties similar to those of digoxin and that, for a similar inhibition of the sodium pump, it evoked a higher positive inotropic effect than digoxin.
.0 0 0 OH
CH 3
OH
SC ~453
CH3
9H
CH3 oH
OH
CH 3
OH
DIGOXIN Fig. 1. Structure of the drugs.
338
T. G O D F R A I N D , J. G H Y S E L - B U R T O N
2. Materials and methods
2.1. Preparation Albino guinea pigs weighing approximatively 400 g were killed by a blow on the head, exsanguinated and the hearts rapidly removed. The atria were dissected out in physiological solution. The left atria were suspended in an organ bath between platinum electrodes, under a resting tension of 500 mg. The atria were stimulated with rectangular pulses of 10 msec (strength at least twice threshold) at a rate of 3.3 Hz.
2.2. Physiological solution The composition of the Tyrode solution was (mM): NaC1 137, KC1 2.7 or 6, CaC12 1.82 MgCl~ 0.105, NaH2PO4 0.417, NaHCO3 11.9 glucose 5.5. It was equilibrated at 30°C with a mixture of 95% 02--5% COs.
2.3. Determination of 14C inulin space The procedure was similar to that followed with smooth muscle (Godfraind, 1976).
2.4. Ionic content determinations At the end of the incubation, the atria were removed from the organ bath and each preparation was blotted on filter paper and pressed three times with a roller weighing 350 g. After weighing, the atrium was placed in a quartz crucible and left overnight at 100°C then weighed. To remove organic material, it was left at 500°C for 18 h. The residue was dissolved in 1 ml HC1 (1 N) and assayed by atomic absorption as described previously for another tissue (Godfraind, 1976). Results have been expressed in term of intracellular water according to the formula C i ~- ( C w - - Co ×
ECS)/((H20)T -- ECS) mmol/1
fibre water where Ci is the cellular concentration, C T the
total concentration of the preparation expressed as mmol/kg wet wt, Co the cation concentration in the perfusion fluid (mmol/1), ECS (inulin space) and (H20)T (water content of the atria) were expressed as 1/kg wet wt. (H20)T and Ci were not affected by the 3-4 h incubation under the conditions reported in this paper. Once the equilibrium effect of the glycoside has been reached the changes in cellular K and Na content, expressed in percent of the maximal change with the most effective drug, are likely to reflect the percentage change in the rate of the Na pump. An estimate of the EDs0 for cardenolide inhibition of the Na pump based on changes in Nai might be biased if the incubation time is not sufficient for the effect to reach equilibrium.
2.5. Uptake of 42K At the end of a 45 min or 3 h preincubation period in the presence or in the absence of the glycosides, 42K was added to the solution (between 185 and 370 Bq/ml), 10 min later the atria were washed for 5 sec in a non-radioactive solution. The atria were blotted and weighed as described for the ionic determination. Each atrium was dissolved in 0.2 ml of a solution composed of equal parts of perchloric acid (37°C, w/v) and H202 (30 vol). This solution was heated at 75°C for 30 min, cooled and 5 ml of Aqualuma (Lumac) then added. The radioactivity of the samples was counted in a liquid scintillation counter (Packard Tricard 3375). The efficiency was the same for both tissue and solution. The uptakes were expressed as: mmol/kg wet wt = = (c/min in muscle)/(wet wt kg)) × × (mmol/1 medium)/(c/min/1 medium) The action of the glycoside was expressed as a percentage of the ouabain-sensitive K uptake. Ouabain-sensitive K uptake is defined as the difference between the 4~K uptake in the
CARDIOACTIVE PROPERTIES OF SC4453 AND DIGOXIN absence and that in the presence of ouabain 10 -4 M. Statistical analysis was d o n e with the net 42K uptake data and n o t with values related to ouabain-sensitive 42K uptake. In the controls, 42K uptake was equal to 6.18 +- 0.11 (n = 41) m m o l / k g wet wt for the 10 min in radioactive solution, the uptake in t he presence o f ouabain 1 0 - 4 M was reduced t o 4.58 + 0.08 (n = 24) m m ol / kg wet wt.
2.6. Recording o f the contractions and experimental protocol Recordings o f th e contractile activity were made with an isometric level using t w o strain gauges as part o f a balanced bridge, the outp u t o f which was fed into a p o t e n t i o m e t r i c recorder. T h e dissection was p e r f o r m e d in KC1 2.7 mM, the atria were stimulated for 30 min in this solution which was t he r eaf t e r exchanged for the physiological solution containing KC1 6 m M . The systolic tension was sensitive to the change in KC1. A time of 30 min was sufficient to reach the new level of contractility which, with KC1 6 mM, was constant for a f u r th er 90 min observation period. Expressed in g/100 mg, the systolic tension was equal to 1.13 + 0.02 (n = 193) for KC1 2.7 mM and to 0.75 + 0.05 (n -- 109) for KC1 6 mM. Changes in systolic tension evoked by digoxin and SC4453 were expressed in percent of the tension developed before t he addition o f the drug.
2. 7. Statistical methods Whenever possible, values are presented as means-+ S.E. o f mean. The significance o f differences between means was checked with Student's t-test.
2.8. Drugs Digoxin and SC4453 were gift of Simes (Milano, Italy). The stock solution (10 -2 M) was kept in ethanol-water ( 7 : 3 ) . The dilutions were prepared with t he physiological
339
solution. 4:KCI was obtained from Amersham (U.K.).
3. Results
3.1. Ionic content of atria incubated in the presence of digoxin and SC4453 The Na, K, Ca and Mg cont ent s were determined in guinea-pig atria incubated for 3 h in a physiological solution containing KC1 6 mM and various concentrations of digoxin o f SC4453. This 3 h incubation time was f o u n d t o be required for t he ouabain effect to reach equilibrium (Godfraind and Ghysel-Burton, 1977). The action of digoxin and SC4453 on the Na and K c o n t e n t was qualitatively different according to the drug c o n c e n t r a t i o n used. A r e d u c t i o n of Nai c o n t e n t and an increase in Ki were evoked b y digoxin 5 × 10 -'o mol/1 ( 0 . 0 1 < P < 0.02) and SC4453 10 -9 mol/1 ( 0 . 0 2 < P < 0.05), whereas digoxin 10 -9 mol/1, SC4453 3 × 10 -9 mol/1 or higher concentrations of the two glycosides evoked a gain of Nai and a loss of Ki. Maximum changes in m onoval ent cation c o n t e n t were evoked with 10 -4 mol/1 of either of the com pounds. The dose of digoxin and SC4453 producing a 50% increase in Nai c o n t e n t was f o u n d to be 3.7 X 10 -4 M (fig. 2). The gain in Na~ was compensated by an equivalent loss of Ki. F u r t h e r m o r e , the atria treated with the glycosides at concent rat i ons higher than 10 -s M had a higher c o n t e n t o f Ca and a reduced c o n t e n t of Mg.
3.2. Action o f digoxin and SC4453 on 42K uptake The actions of t he t w o glycosides on 42K uptake were measured after a 45 min incubation. This time was sufficient to obtain the m a x i m u m inotropic effect (see below). Fig. 3 shows that the high doses inhibited 42K uptake whereas t he lowest doses increased this uptake, an observation consistent with
T. G O D F R A I N D , J. G H Y S E L - B U R T O N
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Glycoside (M)
Fig. 2. Cellular s o d i u m c o n t e n t (retool/1 fibre w a t e r ) o f s t i m u l a t e d left guinea-pig atria i n c u b a t e d for 3 hrs in physiological s o l u t i o n at 30°C in t h e p r e s e n c e o f d i g o x i n (A) a n d S C 4 4 5 3 (o), A b s c i s s a : glycoside conc e n t r a t i o n (M). O r d i n a t e : Na i c o n t e n t of atria. Each p o i n t is t h e m e a n o f at least 4 e x p e r i m e n t s . T h e limits o f S.E. are s h o w n w h e n t h e y e x c e e d e d t h e d i a m e t e r s o f t h e s y m b o l . Differences b e t w e e n t r e a t e d a n d u n t r e a t e d : * P < 0 . 0 5 ; ** P < 0.01, for c o n c e n t r a t i o n s higher than 10 -7 M P < 0 . 0 1 .
previous reports (Godfraind and Lesne, 1972; Godfraind and Ghysel-Burton, 1977; GhyselBurton and Godfraind, 1976 and 1979). The doses producing a 50% inhibition o f 42K uptake were 4.2 X 10 -6 M and t o 2.4 × 10-6 M for SC4453 and digoxin respectively. When 42K uptake was measured after 3 h incubation in the presence o f 10 -8 M digoxin, inhibition o f the ouabain-sensitive uptake was observed instead o f the stimulation evoked after 45 min (fig. 3). This was consistent with the Nai increase and the K i decrease observed after 3 h incubation in the presence o f 10 -8 M of the glycoside. Other experiments, also shown in fig. 3, were carried out in order to measure the inhibition o f 42K uptake after a 3 h incubation in the presence o f digoxin. The concentration producing an inhibition o f 50% of the m a x i m u m u p tak e was 2 × 10 -4 M, a value close to the one estimated after a 45 min incubation.
0
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Glycoside (M)
Fig. 3. Dose-response r e ] a t i o n o f the e f f e c t o f digoxin a n d S C 4 4 5 3 o n 42K u p t a k e o f s t i m u l a t e d left guineapig atria at 30°C in t h e p r e s e n c e of KC1 6 raM. Abscissa: glycoside c o n c e n t r a t i o n (M). O r d i n a t e : o u a b a i n - s e n s i t i v e 42K u p t a k e as a p e r c e n t a g e o f t h e c o n t r o l m e a s u r e d a f t e r 45 rain p r e i n c u b a t i o n w i t h d i g o x i n (A) a n d S C 4 4 5 3 (o) a n d a f t e r 3 h w i t h digoxin (v). Each p o i n t is t h e m e a n o f at least 4 d e t e r m i n a tions. T h e d i f f e r e n c e s b e t w e e n t r e a t e d a n d u n t r e a t e d were a n a l y s e d w i t h r e s p e c t t o n e t 42K u p t a k e . * P < 0 . 0 5 ; ** P < 0.01.
3.3. The action o f cardioactive steroids on the contractility o f isolated guinea-pig atria The contractility of electrically driven left guinea-pig atria was m o n i t o r e d in a physiological solution containing KC1 6 mM and various concentrations o f digoxin and SC4453 ranging from 5 X 10 -1° to 10 -4 mol/1. The threshold effect was found with digoxin and SC4453 5 × 10 -1° mol/1 which evoked an 8% increase in systolic tension (fig. 4). The onset of this action was observed 16 min after the addition o f digoxin and 27 min after t he addition of SC4453. The peak effect was reached after 40 min for digoxin and 46 min for SC4453. When the drug concentration was increased, the time t o onset and t he time to peak decreased in a dose-dependent manner. The magnitude of the inotropic
341
C A R D I O A C T I V E P R O P E R T I E S O F SC4453 A N D D I G O X I N 150.
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Fig. 4. Dose-response relation of the effect of digoxin and SC4453 o n the peak increase in systolic tension of stimulated left guinea-pig atria at 30°C in the presence of KC1 6 mM. Abscissa: glycoside concentration. Ordinate: peak increase in systolic tension in % of initial tension for digoxin (z~) and SC4453 (©). Each point is the m e a n of at least 3 experiments. The limits of S.E. are s h o w n w h e n t h e y exceeded the diameter of the symbol.
effect was related to t he c o n c e n t r a t i o n of the glycosides. However, for glycoside concentrations higher than 10 -6 mol/1, the increase in systolic tension was n o t sustained as it had been for the lower concentrations; the systolic tension decreased once the diastolic tension increased. The increase in diastolic tension and its time to onset were d e p e n d e n t on the glycoside c o n c e n t r a t i o n (fig. 5). Fig. 4 illustrates the relation between digoxin and SC4453 concentrations and the peak increase in systolic tension measured in t h e presence o f KC1 6 mM. T he glycoside concentration producing an increase in systolic tension equal to 50% of t he m a x i m u m observed was 10 -6 mol/1 for SC4453 and digoxin. When the increase in diastolic tension measured af 4 5 m i n or 3 h was plotted as a fu n c t i o n of glycoside c o n c e n t r a t i o n the doseeffect curves were not superimposable as for the increase in systolic tension. Digoxin appeared to evoke an increase in diastolic tension at lower concentrations than SC4453 ( P < 0.05 for concentrations lower than 3 × 10 -6 M, fig. 5).
1.0.
~
'0- 5
io-' °~Io-6
~0-s
~'o'~
Glycoside {M)
Fig. 5. The relation between the increase in diastolic tension and the glycoside concentration. Abscissa: glycoside concentration. Ordinate: increase in diastolic tension (g/100 mg atria) a after 45 min, b after 3 h in the presence of SC4453 (©) and digoxin (A).
The m a x i m u m increase in tension was the same for t he t w o glycosides. Fig. 5. illustrates t he increase in diastolic tension evoked b y digoxin and SC4453. The glycoside concentration producing an increase in diastolic tension equal to 50% of the m a x i m u m observed after 3 h r s , was 2 × 10-6mol/1 for digoxin and 4 X 10 -6 mol/1 for SC4453. The relation bet w een t he inhibition of 42K uptake and t he increase in systolic tension was studied after a 45 min incubation in t he presence o f digoxin and SC4453 at concentrations lower t han those producing an increase in diastolic tension and whose inotropic action was sustained. A positive inotropic effect was observed in the absence o f p u m p inhibition (fig. 6a). F u r t h e r m o r e , for a similar inhibition of the pump, the increase in systolic tension evoked by SC4453 was higher than t he one evoked by digoxin ( P < 0.001). In order to find w h e t h e r this difference could be due to the release of catecholamines, t he inotropic effect of SC4453 was measured in t he presence of propranolol 5 × 10 -6 M. The time to peak and the increase in systolic tension evoked by SC4453 3 X 10 -7 M were n o t modified by the/]-blocker. For the t w o glycosides used the relationship bet w een the
342
T. GODFRAIND, J. GHYSEL-BURTON
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f s'o
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Fig. 6. The relation between the inhibition of 42K uptake and the increase in systolic tension with SC4453 (o) and digoxin (A). Abscissa: inhibition of the ouabain-sensitive 42 K uptake evoked by SC4453 (©) a, at concentrations 10 -6 M, 1.5 x 10 -6 M, 2 x 10 -6 M; b, at concentrations 3 x 10 -6 M, 4.5 x 10 -6 M, 10 -s M and evoked by digoxin (A) a, at concentrations 3 x 10 -TM, 7 x 10 -TM, 10 -6M; b, at concentrations 10 -6 M, 1.5 X 10 -6 M, 3 x 10 -6 M, 10 -s M, after 45 min incubation. Ordinate: a, increase in systolic tension in percent of the tension recorded before the addition of the glycosides. Each value is the mean of at least 3 determinations; b, increase in diastolic tension (g/100 mg atria).
increase in diastolic t e n s i o n a n d t h e i n h i b i t i o n o f 42K u p t a k e m e a s u r e d a f t e r 45 m i n was v e r y similar (fig. 6b). This indicates t h a t changes in diastolic t e n s i o n c o r r e l a t e d b e t t e r w i t h inhibit i o n o f t h e Na p u m p t h a n w i t h changes in systolic t e n s i o n .
4. Discussion T h e p r e s e n t results s h o w t h a t r e p l a c e m e n t o f t h e l a c t o n e ring o f d i g o x i n b y a p y r i d a z i n e ring did n o t abolish t h e c a r d i o a c t i v e actions as S C 4 4 5 3 s h o w e d p r o p e r t i e s similar to t h e parent compound. T h e i n t e r a c t i o n w i t h t h e s o d i u m p u m p was qualitatively similar f o r t h e t w o glycosides. As a l r e a d y r e p o r t e d f o r o u a b a i n a n d d i g i t o x i n ( G o d f r a i n d a n d Lesne, 1 9 7 2 ; G o d f r a i n d , 1 9 7 5 ; G o d f r a i n d a n d G h y s e l - B u r t o n , 1 9 7 7 ; GhyselB u r t o n and G o d f r a i n d , 1 9 7 6 , 1 9 7 9 ) l o w doses
o f digoxin and o f S C 4 4 5 3 s t i m u l a t e d the Na p u m p . This e f f e c t was s h o w n b y t h e e n h a n c e m e n t o f 42K u p t a k e m e a s u r e d a f t e r a 45 m i n i n c u b a t i o n a n d suggested b y t h e r e d u c t i o n o f Na i c o n t e n t a f t e r a 3 h i n c u b a t i o n . T h e p r e s e n t results s h o w t h a t w h e n t h e d u r a t i o n o f t h e i n c u b a t i o n in t h e p r e s e n c e o f t h e glycoside was p r o l o n g e d , t h e s t i m u l a t i o n o f t h e p u m p w a n e d a n d was t r a n s i e n t f o r conc e n t r a t i o n s such as 10 -s M and 10 -7 M. Such an o b s e r v a t i o n was r e p o r t e d s o m e years ago b y Klaus et al. ( 1 9 6 2 ) f o r digitoxigenin. I n d e e d , a c o m p a r i s o n o f figs. 2 and 3 s h o w s that the concentrations which stimulated 42K u p t a k e a f t e r a 45 m i n i n c u b a t i o n , r e d u c e d t h e Nai c o n t e n t o f t h e atria i n c u b a t e d for 3 hrs. This o b s e r v a t i o n is in a g r e e m e n t w i t h e l e c t r o p h y s i o l o g i c a l studies o f C o h e n et al. (1976) and with the report by Deitmer and Ellis ( 1 9 7 8 ) o f a t r a n s i e n t c h a n g e in intracellular s o d i u m activity e v o k e d b y cardiac glycosides in Purkinje fibers. It d o e s n o t s e e m t h a t a change in a f f i n i t y o f t h e i n h i b i t o r y sites m i g h t a c c o u n t f o r this, as t h e EDs0 for inhibit i o n o f t h e Na p u m p m e a s u r e d a f t e r 45 m i n or 3 h i n c u b a t i o n did n o t d i f f e r m u c h . In previous r e p o r t s ( G h y s e l - B u r t o n a n d G o d frain, 1 9 7 6 , 1 9 7 9 ) , we have s h o w n t h a t t h e s t i m u l a t o r y a n d i n h i b i t o r y sites o f t h e N a p u m p sensitive to o u a b a i n b e h a v e d d i f f e r e n t l y when the composition of the perfusing mediu m was c h a n g e d . In view o f t h e o b s e r v a t i o n t h a t t h e b i n d i n g o f o u a b a i n to s t i m u l a t o r y sites o c c u r r e d w i t h a Hill c o e f f i c i e n t higher t h a n 1, t h e s e sites c o u l d s h o w c o o p e r a t i v e properties. Therefore, a prolonged incubation w i t h t h e glycosides m i g h t p r o d u c e an allosteric change o f t h e s t i m u l a t o r y sites leading to a reduction of their pumping activity. Further e x p e r i m e n t s will be n e c e s s a r y in o r d e r to clarify this p o i n t . It has b e e n p r o p o s e d t h a t t h e positive inot r o p i c e f f e c t o f cardiac glycosides was related to t h e i n h i b i t i o n o f t h e s o d i u m p u m p (Langer, 1 9 6 8 ; A k e r a et al., 1 9 7 0 ; Besch et al., 1 9 7 0 ) , which might interfere with the tr a nsme mbr a ne Na-Ca e x c h a n g e . This h y p o t h e s i s is still a m a t t e r o f c o n t r o v e r s y (Lee and Klaus, 1 9 7 1 ;
CARDIOACTIVE PROPERTIES OF SC4453 AND DIGOXIN Godfraind, 1975; Brody and Akera, 1977; B e n t f e l d e t al., 1 9 7 7 ; O k i t a , 1 9 7 7 ) . K u e t al. (1974) have shown that the positive inotropic e f f e c t o f d i g i t o x i n in g u i n e a - p i g a t r i a w a s c o r r e l a t e d w i t h t h e i n h i b i t i o n o f S6Rb u p t a k e c o n s i d e r e d as a n i n d e x o f N a p u m p a c t i v i t y . The present findings confirm these observations and provide additional information about the relation between the inhibition of t h e N a p u m p a n d t h e i n o t r o p i c e f f e c t as t w o different glycosides were compared. When the activity of the sodium pump was measured at the peak of the inotropic effect, it was observed that, for the same inhibition of the pump, the inotropic effect evoked by SC4453 was twice that evoked by digoxin. Furthermore, a positive inotropic effect was produced by glycoside concentrations which stimulated the sodium pump. This confirms our previous observations (Godfraind and Ghysel-Burton, 1978; Ghysel-Burton and Godfraind, 1979) t h a t t h e i n h i b i t i o n o f t h e s o d i u m p u m p is n o t the only mechanism responsible for the positive inotropic effect of cardiac glycosides.
Acknowledgements This work was supported by the Fonds National de la Recherche Scientifique M6dicale and the Fonds de D~veloppement Scientifique de l'Universit~ Catholique de Louvain. We wish to thank Mrs G. Leonardy, Mr. A. Goffin and Mr. P. Simon for their technical assistance.
References Akera, T., F.S. Larson and T.M. Brody, 1970, Correlation of cardiac sodium and potassium activated adenosine triphosphatase activity with ouabaininduced inotropic stimulation, J. Pharmacol. Exp. Therap. 173,145. Bentfeld, M., H. Ltillman, T. Peters and D. Proppe, 1977, Interdependence of ion transport and the action ouabain in heart muscle, Brit. J. Pharmacol. 61, 19. Besch, J.R., J.C. Allen, G. Glick and A. Schwartz, 1970, Correlation between the positive inotropic action of ouabain and its effects on subcellular
343
enzyme systems from canine myocardium, J. Pharmacol. Exp. Therap. 14, 1. Brody, T.M. and T. Akera, 1977, Relations among Na ÷, K÷-ATPase activity, sodium pump activity, transmembrane sodium movement and cardiac contractility, Federation Proc. 36, 2219. Cohen, I., J. Daut and D. Noble, 1976, An analysis of the actions of low concentrations of ouabain on membrane currents in Purkinje fibres, J. Physiol. (London) 260, 55. Deitmer, J.M. and D. Ellis, 1978, The intracellular sodium activity of cardiac Purkinje fibres during inhibition and reactivation of the Na-K pump, J. Physiol. (London) 284,241. Ghysel-Burton, J. and T. Godfraind, 1976, Importance of the lactone ring for the action of therapeutic doses of ouabain in guinea-pig atria, J. Physiol. (London) 266, 75P. Ghysel-Burton, J. and T. Godfraind, 1979, Stimulation and inhibition of the sodium pump by cardioactive steroids in relation to their binding sites and their inotropic effect in guinea-pig isolated atria, Brit. J. Pharmacol. 66,175. Godfraind, T., 1975, Cardiac glycosides receptors in the heart, Biochem. Pharmacol. 24,823. Godfraind, T., 1976, Calcium exchange in vascular smooth muscle action of noradrenaline and lanthanum, J. Physiol. (London) 260, 21. Godfraind, T. and J. Ghysel-Burton, 1977, Binding sites related to ouabain-induced stimulation or inhibition of the sodium pump, Nature, (London) 265,165. Godfraind, T. and J. Ghysel-Burton, 1978, The inotropic effect of ouabain, ouabagenin and dihydro° ouabain in guinea-pig atria, Arch. Intern. Pharmacodyn. Therap. 232,339. Godfraind, T. and M. Lesne, 1972, The uptake of cardiac glycosides in relation to their actions in isolated cardiac muscle, Brit. J. Pharmacol. 46, 488. Godfraind, T. and N. Tona Lutete, 1979, Inhibition by digoxin and SC4453 of the (Na ÷ + K+)-ATPase isolated from human heart guinea-pig heart and guinea-pig brain, European J. Pharmacol. 60, 329. Klaus, W., G. Kuschinsky and H. Liillmann, 1962, Uber den Zusammenhang zwischen positiv inotroper Wirkung yon Digitoxigenin, Kalium flux und intracellul~/ren Ionenkonzentrationen im Herzmuskel, Naunyn-Schmiedeb. Arch. Exp. Pathol. Pharmakol. 242,480. Ku, D., T. Akera, C.L. Pew and T.M. Brody, 1974, Cardiac glycosides: correlations among Na ÷, K ÷ATPase, sodium pump and contractility in the guinea-pig heart, Naunyn-Schmiedeb. Arch. Pharmacol. 285, 185. Langer, G.A., 1968, Ion fluxes in cardiac excitation
344 and contraction and their relation to myocardial contractility, Physiol. Rev. 48,708. Lee, K.S. and W. Klaus, 1971, The subcellular basis for the mechanism of inotropic action of cardiac glycosides, Pharmacol. Rev. 23,193. Okita, G.T., 1977, Dissociation of Na ÷, K÷-ATPase
T. GODFRAIND, J. GHYSEL-BURTON inhibition from digitalis inotropy, Federation Proc. 36, 2225. Thomas, R., J. Boutagy and A. Gelbart, 1974, Synthesis and biological activity of semi-synthetic digitalis analogs, J. Pharm. Sci. 63, 1649.
337
© Elsevier/North-Holland Biomedical Press
THE CARDIOACTIVE PROPERTIES OF 8C4453, A DIGOXIN ANALOGUE WITH A C17~-PYRIDAZINE RING THI~OPHILE G O D F R A I N D and JOSIANE GHYSEL-BURTON
Laboratoire de Pharmacodynamie Ggndrale et de Pharmacologie, Universitd Catholique de Louvain, Av. E. Mounier 73, UCL 7350, 1200 BruxeUes, Belgium Received 14 May 1979, revised MS received 4 September 1979, accepted 2 October 1979
T. G O D F R A I N D and J. GHYSEL-BURTON, The cardioactive properties o f SC4453, a digoxin analogue with a Cl 7~-pyridazine ring, European J. Pharmacol. 60 (1979) 337--344. SC4453 is a digoxin analogue with a pyridazine instead of a lactone ring in C175. SC4453 was compared with digoxin with respect to effect on contractility and on activity of the sodium pump in guinea-pig isolated left atria stimulated at 3.3 Hz. The two glycosides stimulated the sodium pump at low concentrations and inhibited at high concentrations. At the time to peak inotropic effect, for a similar inhibition of the Na pump, the increase in systolic tension was higher with SC4453 than with digoxin, whereas the increase in diastolic tension was similar for both. These observations confirm that the inhibition of the Na pump is not the only mechanism responsible for the positive inotropic effect of cardiac glycosides. Digoxin
C17~-Pyridazine
Na pump
Myocardial contractility
1. Introduction The lactone ring plays an important role in the biological action of cardiac glycosides. Its saturation yielding dihydroderivatives reduces their biological potency and, as observed with ouabain, suppresses their ability to stimulate b u t not to inhibit the sodium pump (GhyselBurton and Godfraind, 1976, 1979). However, in several natural and synthetic c o m p o u n d s with cardioactive action, the substituant in C17~ is n o t a lactone ring (Thomas et al., 1974). SC4453 is a derivative of digoxin in which the lactone ring in C17~ has been replaced b y a pyridazine ring (fig. 1). In another report from this laboratory, it was shown that SC4453 inhibited (Na ÷ +K÷)ATPase within the same range of concentration as digoxin (Godfraind and Tona Lutete, 1979). The purpose of the present experiment was to compare, in isolated guinea-pig atria, the actions of SC4453 and digoxin on the activity
o f the sodium pump and on contractility. The results show that SC4453 has cardioactive properties similar to those of digoxin and that, for a similar inhibition of the sodium pump, it evoked a higher positive inotropic effect than digoxin.
.0 0 0 OH
CH 3
OH
SC ~453
CH3
9H
CH3 oH
OH
CH 3
OH
DIGOXIN Fig. 1. Structure of the drugs.
338
T. G O D F R A I N D , J. G H Y S E L - B U R T O N
2. Materials and methods
2.1. Preparation Albino guinea pigs weighing approximatively 400 g were killed by a blow on the head, exsanguinated and the hearts rapidly removed. The atria were dissected out in physiological solution. The left atria were suspended in an organ bath between platinum electrodes, under a resting tension of 500 mg. The atria were stimulated with rectangular pulses of 10 msec (strength at least twice threshold) at a rate of 3.3 Hz.
2.2. Physiological solution The composition of the Tyrode solution was (mM): NaC1 137, KC1 2.7 or 6, CaC12 1.82 MgCl~ 0.105, NaH2PO4 0.417, NaHCO3 11.9 glucose 5.5. It was equilibrated at 30°C with a mixture of 95% 02--5% COs.
2.3. Determination of 14C inulin space The procedure was similar to that followed with smooth muscle (Godfraind, 1976).
2.4. Ionic content determinations At the end of the incubation, the atria were removed from the organ bath and each preparation was blotted on filter paper and pressed three times with a roller weighing 350 g. After weighing, the atrium was placed in a quartz crucible and left overnight at 100°C then weighed. To remove organic material, it was left at 500°C for 18 h. The residue was dissolved in 1 ml HC1 (1 N) and assayed by atomic absorption as described previously for another tissue (Godfraind, 1976). Results have been expressed in term of intracellular water according to the formula C i ~- ( C w - - Co ×
ECS)/((H20)T -- ECS) mmol/1
fibre water where Ci is the cellular concentration, C T the
total concentration of the preparation expressed as mmol/kg wet wt, Co the cation concentration in the perfusion fluid (mmol/1), ECS (inulin space) and (H20)T (water content of the atria) were expressed as 1/kg wet wt. (H20)T and Ci were not affected by the 3-4 h incubation under the conditions reported in this paper. Once the equilibrium effect of the glycoside has been reached the changes in cellular K and Na content, expressed in percent of the maximal change with the most effective drug, are likely to reflect the percentage change in the rate of the Na pump. An estimate of the EDs0 for cardenolide inhibition of the Na pump based on changes in Nai might be biased if the incubation time is not sufficient for the effect to reach equilibrium.
2.5. Uptake of 42K At the end of a 45 min or 3 h preincubation period in the presence or in the absence of the glycosides, 42K was added to the solution (between 185 and 370 Bq/ml), 10 min later the atria were washed for 5 sec in a non-radioactive solution. The atria were blotted and weighed as described for the ionic determination. Each atrium was dissolved in 0.2 ml of a solution composed of equal parts of perchloric acid (37°C, w/v) and H202 (30 vol). This solution was heated at 75°C for 30 min, cooled and 5 ml of Aqualuma (Lumac) then added. The radioactivity of the samples was counted in a liquid scintillation counter (Packard Tricard 3375). The efficiency was the same for both tissue and solution. The uptakes were expressed as: mmol/kg wet wt = = (c/min in muscle)/(wet wt kg)) × × (mmol/1 medium)/(c/min/1 medium) The action of the glycoside was expressed as a percentage of the ouabain-sensitive K uptake. Ouabain-sensitive K uptake is defined as the difference between the 4~K uptake in the
CARDIOACTIVE PROPERTIES OF SC4453 AND DIGOXIN absence and that in the presence of ouabain 10 -4 M. Statistical analysis was d o n e with the net 42K uptake data and n o t with values related to ouabain-sensitive 42K uptake. In the controls, 42K uptake was equal to 6.18 +- 0.11 (n = 41) m m o l / k g wet wt for the 10 min in radioactive solution, the uptake in t he presence o f ouabain 1 0 - 4 M was reduced t o 4.58 + 0.08 (n = 24) m m ol / kg wet wt.
2.6. Recording o f the contractions and experimental protocol Recordings o f th e contractile activity were made with an isometric level using t w o strain gauges as part o f a balanced bridge, the outp u t o f which was fed into a p o t e n t i o m e t r i c recorder. T h e dissection was p e r f o r m e d in KC1 2.7 mM, the atria were stimulated for 30 min in this solution which was t he r eaf t e r exchanged for the physiological solution containing KC1 6 m M . The systolic tension was sensitive to the change in KC1. A time of 30 min was sufficient to reach the new level of contractility which, with KC1 6 mM, was constant for a f u r th er 90 min observation period. Expressed in g/100 mg, the systolic tension was equal to 1.13 + 0.02 (n = 193) for KC1 2.7 mM and to 0.75 + 0.05 (n -- 109) for KC1 6 mM. Changes in systolic tension evoked by digoxin and SC4453 were expressed in percent of the tension developed before t he addition o f the drug.
2. 7. Statistical methods Whenever possible, values are presented as means-+ S.E. o f mean. The significance o f differences between means was checked with Student's t-test.
2.8. Drugs Digoxin and SC4453 were gift of Simes (Milano, Italy). The stock solution (10 -2 M) was kept in ethanol-water ( 7 : 3 ) . The dilutions were prepared with t he physiological
339
solution. 4:KCI was obtained from Amersham (U.K.).
3. Results
3.1. Ionic content of atria incubated in the presence of digoxin and SC4453 The Na, K, Ca and Mg cont ent s were determined in guinea-pig atria incubated for 3 h in a physiological solution containing KC1 6 mM and various concentrations of digoxin o f SC4453. This 3 h incubation time was f o u n d t o be required for t he ouabain effect to reach equilibrium (Godfraind and Ghysel-Burton, 1977). The action of digoxin and SC4453 on the Na and K c o n t e n t was qualitatively different according to the drug c o n c e n t r a t i o n used. A r e d u c t i o n of Nai c o n t e n t and an increase in Ki were evoked b y digoxin 5 × 10 -'o mol/1 ( 0 . 0 1 < P < 0.02) and SC4453 10 -9 mol/1 ( 0 . 0 2 < P < 0.05), whereas digoxin 10 -9 mol/1, SC4453 3 × 10 -9 mol/1 or higher concentrations of the two glycosides evoked a gain of Nai and a loss of Ki. Maximum changes in m onoval ent cation c o n t e n t were evoked with 10 -4 mol/1 of either of the com pounds. The dose of digoxin and SC4453 producing a 50% increase in Nai c o n t e n t was f o u n d to be 3.7 X 10 -4 M (fig. 2). The gain in Na~ was compensated by an equivalent loss of Ki. F u r t h e r m o r e , the atria treated with the glycosides at concent rat i ons higher than 10 -s M had a higher c o n t e n t o f Ca and a reduced c o n t e n t of Mg.
3.2. Action o f digoxin and SC4453 on 42K uptake The actions of t he t w o glycosides on 42K uptake were measured after a 45 min incubation. This time was sufficient to obtain the m a x i m u m inotropic effect (see below). Fig. 3 shows that the high doses inhibited 42K uptake whereas t he lowest doses increased this uptake, an observation consistent with
T. G O D F R A I N D , J. G H Y S E L - B U R T O N
340 200.
100. o
E
=
10C
~_ 0 ~10 10
,.
10 9
,.
10 8
,.
10 7
,_
10 6
..
10 5
]04,
Glycoside (M)
Fig. 2. Cellular s o d i u m c o n t e n t (retool/1 fibre w a t e r ) o f s t i m u l a t e d left guinea-pig atria i n c u b a t e d for 3 hrs in physiological s o l u t i o n at 30°C in t h e p r e s e n c e o f d i g o x i n (A) a n d S C 4 4 5 3 (o), A b s c i s s a : glycoside conc e n t r a t i o n (M). O r d i n a t e : Na i c o n t e n t of atria. Each p o i n t is t h e m e a n o f at least 4 e x p e r i m e n t s . T h e limits o f S.E. are s h o w n w h e n t h e y e x c e e d e d t h e d i a m e t e r s o f t h e s y m b o l . Differences b e t w e e n t r e a t e d a n d u n t r e a t e d : * P < 0 . 0 5 ; ** P < 0.01, for c o n c e n t r a t i o n s higher than 10 -7 M P < 0 . 0 1 .
previous reports (Godfraind and Lesne, 1972; Godfraind and Ghysel-Burton, 1977; GhyselBurton and Godfraind, 1976 and 1979). The doses producing a 50% inhibition o f 42K uptake were 4.2 X 10 -6 M and t o 2.4 × 10-6 M for SC4453 and digoxin respectively. When 42K uptake was measured after 3 h incubation in the presence o f 10 -8 M digoxin, inhibition o f the ouabain-sensitive uptake was observed instead o f the stimulation evoked after 45 min (fig. 3). This was consistent with the Nai increase and the K i decrease observed after 3 h incubation in the presence o f 10 -8 M of the glycoside. Other experiments, also shown in fig. 3, were carried out in order to measure the inhibition o f 42K uptake after a 3 h incubation in the presence o f digoxin. The concentration producing an inhibition o f 50% of the m a x i m u m u p tak e was 2 × 10 -4 M, a value close to the one estimated after a 45 min incubation.
0
0
10-9
1'0-8
~0-7
1'0-6
10-5
Glycoside (M)
Fig. 3. Dose-response r e ] a t i o n o f the e f f e c t o f digoxin a n d S C 4 4 5 3 o n 42K u p t a k e o f s t i m u l a t e d left guineapig atria at 30°C in t h e p r e s e n c e of KC1 6 raM. Abscissa: glycoside c o n c e n t r a t i o n (M). O r d i n a t e : o u a b a i n - s e n s i t i v e 42K u p t a k e as a p e r c e n t a g e o f t h e c o n t r o l m e a s u r e d a f t e r 45 rain p r e i n c u b a t i o n w i t h d i g o x i n (A) a n d S C 4 4 5 3 (o) a n d a f t e r 3 h w i t h digoxin (v). Each p o i n t is t h e m e a n o f at least 4 d e t e r m i n a tions. T h e d i f f e r e n c e s b e t w e e n t r e a t e d a n d u n t r e a t e d were a n a l y s e d w i t h r e s p e c t t o n e t 42K u p t a k e . * P < 0 . 0 5 ; ** P < 0.01.
3.3. The action o f cardioactive steroids on the contractility o f isolated guinea-pig atria The contractility of electrically driven left guinea-pig atria was m o n i t o r e d in a physiological solution containing KC1 6 mM and various concentrations o f digoxin and SC4453 ranging from 5 X 10 -1° to 10 -4 mol/1. The threshold effect was found with digoxin and SC4453 5 × 10 -1° mol/1 which evoked an 8% increase in systolic tension (fig. 4). The onset of this action was observed 16 min after the addition o f digoxin and 27 min after t he addition of SC4453. The peak effect was reached after 40 min for digoxin and 46 min for SC4453. When the drug concentration was increased, the time t o onset and t he time to peak decreased in a dose-dependent manner. The magnitude of the inotropic
341
C A R D I O A C T I V E P R O P E R T I E S O F SC4453 A N D D I G O X I N 150.
1.s
,~ I
:~~oo.
£i
.,.,
o c -
u~
A/f
SO.
u
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1.0
o
c
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;P
z~_~o---~~ ° 0
I o-IO
i'o-9
io-8
i'o-7
~o-6
io -s
io"
Glycoside (M)
Fig. 4. Dose-response relation of the effect of digoxin and SC4453 o n the peak increase in systolic tension of stimulated left guinea-pig atria at 30°C in the presence of KC1 6 mM. Abscissa: glycoside concentration. Ordinate: peak increase in systolic tension in % of initial tension for digoxin (z~) and SC4453 (©). Each point is the m e a n of at least 3 experiments. The limits of S.E. are s h o w n w h e n t h e y exceeded the diameter of the symbol.
effect was related to t he c o n c e n t r a t i o n of the glycosides. However, for glycoside concentrations higher than 10 -6 mol/1, the increase in systolic tension was n o t sustained as it had been for the lower concentrations; the systolic tension decreased once the diastolic tension increased. The increase in diastolic tension and its time to onset were d e p e n d e n t on the glycoside c o n c e n t r a t i o n (fig. 5). Fig. 4 illustrates the relation between digoxin and SC4453 concentrations and the peak increase in systolic tension measured in t h e presence o f KC1 6 mM. T he glycoside concentration producing an increase in systolic tension equal to 50% of t he m a x i m u m observed was 10 -6 mol/1 for SC4453 and digoxin. When the increase in diastolic tension measured af 4 5 m i n or 3 h was plotted as a fu n c t i o n of glycoside c o n c e n t r a t i o n the doseeffect curves were not superimposable as for the increase in systolic tension. Digoxin appeared to evoke an increase in diastolic tension at lower concentrations than SC4453 ( P < 0.05 for concentrations lower than 3 × 10 -6 M, fig. 5).
1.0.
~
'0- 5
io-' °~Io-6
~0-s
~'o'~
Glycoside {M)
Fig. 5. The relation between the increase in diastolic tension and the glycoside concentration. Abscissa: glycoside concentration. Ordinate: increase in diastolic tension (g/100 mg atria) a after 45 min, b after 3 h in the presence of SC4453 (©) and digoxin (A).
The m a x i m u m increase in tension was the same for t he t w o glycosides. Fig. 5. illustrates t he increase in diastolic tension evoked b y digoxin and SC4453. The glycoside concentration producing an increase in diastolic tension equal to 50% of the m a x i m u m observed after 3 h r s , was 2 × 10-6mol/1 for digoxin and 4 X 10 -6 mol/1 for SC4453. The relation bet w een t he inhibition of 42K uptake and t he increase in systolic tension was studied after a 45 min incubation in t he presence o f digoxin and SC4453 at concentrations lower t han those producing an increase in diastolic tension and whose inotropic action was sustained. A positive inotropic effect was observed in the absence o f p u m p inhibition (fig. 6a). F u r t h e r m o r e , for a similar inhibition of the pump, the increase in systolic tension evoked by SC4453 was higher than t he one evoked by digoxin ( P < 0.001). In order to find w h e t h e r this difference could be due to the release of catecholamines, t he inotropic effect of SC4453 was measured in t he presence of propranolol 5 × 10 -6 M. The time to peak and the increase in systolic tension evoked by SC4453 3 X 10 -7 M were n o t modified by the/]-blocker. For the t w o glycosides used the relationship bet w een the
342
T. GODFRAIND, J. GHYSEL-BURTON
1 ja,
= o
o~ 1 . S
o~
u = o
50
1.0
._a
c
0
0
is
s0
0
Inhibition of /~2K uptake
f s'o
too
(%)
Fig. 6. The relation between the inhibition of 42K uptake and the increase in systolic tension with SC4453 (o) and digoxin (A). Abscissa: inhibition of the ouabain-sensitive 42 K uptake evoked by SC4453 (©) a, at concentrations 10 -6 M, 1.5 x 10 -6 M, 2 x 10 -6 M; b, at concentrations 3 x 10 -6 M, 4.5 x 10 -6 M, 10 -s M and evoked by digoxin (A) a, at concentrations 3 x 10 -TM, 7 x 10 -TM, 10 -6M; b, at concentrations 10 -6 M, 1.5 X 10 -6 M, 3 x 10 -6 M, 10 -s M, after 45 min incubation. Ordinate: a, increase in systolic tension in percent of the tension recorded before the addition of the glycosides. Each value is the mean of at least 3 determinations; b, increase in diastolic tension (g/100 mg atria).
increase in diastolic t e n s i o n a n d t h e i n h i b i t i o n o f 42K u p t a k e m e a s u r e d a f t e r 45 m i n was v e r y similar (fig. 6b). This indicates t h a t changes in diastolic t e n s i o n c o r r e l a t e d b e t t e r w i t h inhibit i o n o f t h e Na p u m p t h a n w i t h changes in systolic t e n s i o n .
4. Discussion T h e p r e s e n t results s h o w t h a t r e p l a c e m e n t o f t h e l a c t o n e ring o f d i g o x i n b y a p y r i d a z i n e ring did n o t abolish t h e c a r d i o a c t i v e actions as S C 4 4 5 3 s h o w e d p r o p e r t i e s similar to t h e parent compound. T h e i n t e r a c t i o n w i t h t h e s o d i u m p u m p was qualitatively similar f o r t h e t w o glycosides. As a l r e a d y r e p o r t e d f o r o u a b a i n a n d d i g i t o x i n ( G o d f r a i n d a n d Lesne, 1 9 7 2 ; G o d f r a i n d , 1 9 7 5 ; G o d f r a i n d a n d G h y s e l - B u r t o n , 1 9 7 7 ; GhyselB u r t o n and G o d f r a i n d , 1 9 7 6 , 1 9 7 9 ) l o w doses
o f digoxin and o f S C 4 4 5 3 s t i m u l a t e d the Na p u m p . This e f f e c t was s h o w n b y t h e e n h a n c e m e n t o f 42K u p t a k e m e a s u r e d a f t e r a 45 m i n i n c u b a t i o n a n d suggested b y t h e r e d u c t i o n o f Na i c o n t e n t a f t e r a 3 h i n c u b a t i o n . T h e p r e s e n t results s h o w t h a t w h e n t h e d u r a t i o n o f t h e i n c u b a t i o n in t h e p r e s e n c e o f t h e glycoside was p r o l o n g e d , t h e s t i m u l a t i o n o f t h e p u m p w a n e d a n d was t r a n s i e n t f o r conc e n t r a t i o n s such as 10 -s M and 10 -7 M. Such an o b s e r v a t i o n was r e p o r t e d s o m e years ago b y Klaus et al. ( 1 9 6 2 ) f o r digitoxigenin. I n d e e d , a c o m p a r i s o n o f figs. 2 and 3 s h o w s that the concentrations which stimulated 42K u p t a k e a f t e r a 45 m i n i n c u b a t i o n , r e d u c e d t h e Nai c o n t e n t o f t h e atria i n c u b a t e d for 3 hrs. This o b s e r v a t i o n is in a g r e e m e n t w i t h e l e c t r o p h y s i o l o g i c a l studies o f C o h e n et al. (1976) and with the report by Deitmer and Ellis ( 1 9 7 8 ) o f a t r a n s i e n t c h a n g e in intracellular s o d i u m activity e v o k e d b y cardiac glycosides in Purkinje fibers. It d o e s n o t s e e m t h a t a change in a f f i n i t y o f t h e i n h i b i t o r y sites m i g h t a c c o u n t f o r this, as t h e EDs0 for inhibit i o n o f t h e Na p u m p m e a s u r e d a f t e r 45 m i n or 3 h i n c u b a t i o n did n o t d i f f e r m u c h . In previous r e p o r t s ( G h y s e l - B u r t o n a n d G o d frain, 1 9 7 6 , 1 9 7 9 ) , we have s h o w n t h a t t h e s t i m u l a t o r y a n d i n h i b i t o r y sites o f t h e N a p u m p sensitive to o u a b a i n b e h a v e d d i f f e r e n t l y when the composition of the perfusing mediu m was c h a n g e d . In view o f t h e o b s e r v a t i o n t h a t t h e b i n d i n g o f o u a b a i n to s t i m u l a t o r y sites o c c u r r e d w i t h a Hill c o e f f i c i e n t higher t h a n 1, t h e s e sites c o u l d s h o w c o o p e r a t i v e properties. Therefore, a prolonged incubation w i t h t h e glycosides m i g h t p r o d u c e an allosteric change o f t h e s t i m u l a t o r y sites leading to a reduction of their pumping activity. Further e x p e r i m e n t s will be n e c e s s a r y in o r d e r to clarify this p o i n t . It has b e e n p r o p o s e d t h a t t h e positive inot r o p i c e f f e c t o f cardiac glycosides was related to t h e i n h i b i t i o n o f t h e s o d i u m p u m p (Langer, 1 9 6 8 ; A k e r a et al., 1 9 7 0 ; Besch et al., 1 9 7 0 ) , which might interfere with the tr a nsme mbr a ne Na-Ca e x c h a n g e . This h y p o t h e s i s is still a m a t t e r o f c o n t r o v e r s y (Lee and Klaus, 1 9 7 1 ;
CARDIOACTIVE PROPERTIES OF SC4453 AND DIGOXIN Godfraind, 1975; Brody and Akera, 1977; B e n t f e l d e t al., 1 9 7 7 ; O k i t a , 1 9 7 7 ) . K u e t al. (1974) have shown that the positive inotropic e f f e c t o f d i g i t o x i n in g u i n e a - p i g a t r i a w a s c o r r e l a t e d w i t h t h e i n h i b i t i o n o f S6Rb u p t a k e c o n s i d e r e d as a n i n d e x o f N a p u m p a c t i v i t y . The present findings confirm these observations and provide additional information about the relation between the inhibition of t h e N a p u m p a n d t h e i n o t r o p i c e f f e c t as t w o different glycosides were compared. When the activity of the sodium pump was measured at the peak of the inotropic effect, it was observed that, for the same inhibition of the pump, the inotropic effect evoked by SC4453 was twice that evoked by digoxin. Furthermore, a positive inotropic effect was produced by glycoside concentrations which stimulated the sodium pump. This confirms our previous observations (Godfraind and Ghysel-Burton, 1978; Ghysel-Burton and Godfraind, 1979) t h a t t h e i n h i b i t i o n o f t h e s o d i u m p u m p is n o t the only mechanism responsible for the positive inotropic effect of cardiac glycosides.
Acknowledgements This work was supported by the Fonds National de la Recherche Scientifique M6dicale and the Fonds de D~veloppement Scientifique de l'Universit~ Catholique de Louvain. We wish to thank Mrs G. Leonardy, Mr. A. Goffin and Mr. P. Simon for their technical assistance.
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