- Email: [email protected]
Divergent influences of Ca2+ on the action of several cardiotonic steroids in isolated heart muscle preparations
EUROPEAN JOURNAL OF PHARMACOLOGY 26 (1974) 331-337. NORTH-HOLLAND PUBLISHING COMPANY
DIVERGENT CARDIOTONIC
INFLUENCES STEROIDS
O F Ca 2+ O N T H E A C T I O N O F S E V E R A L
IN ISOLATED
HEART MUSCLE PREPARATIONS
Rudolf G. ALKEN, Uwe FRICKE and Wolfgang KLAUS Department of Pharmacology, Medical School of Hannover, 3000 Hannover-Kleefeld, Karl-Wiechert-Allee 9, W.-Germany
Received 14 August 1973
Accepted 17 December 1973
R.G. ALKEN, U. FRICKE and W. KLAUS, Divergent influences of Ca2+on the action of several cardiotonic steroids in isolated heart muscle preparations, European J. Pharmacol. 26 (1974) 331-337. In recent years, calcium-digitalis synergism has been challenged on the basis of some divergent observations in in vivo experiments on different species. The purpose of the present in vitro studies on isolated guinea pig papillary muscles was to eliminate extracardlac interferences and to test the possible calcium-digitalis interrelationship at the myocardial level. For evaluation of the positive inotropic and toxic actions the dose-response curves of several cardiotonic steroids were studied at different extracellular calcium concentrations. With ouabain, digitoxin and strophanthidin-3-acetate a continuous decrease of the EDso and an enhanced toxicity was observed when the calcium concentration was raised from 0.45 to 7.2 mM. In contrast, with digoxin and strophanthidin-3-bromoacetate, an increase of the EDso and a reduced toxicity was obtained in the calcium concentration range from 0.45 to 3.6 mM. These results do not support the concept of a calcium-digitalis synergism as a general rule for all cardenolides. Several possible explanations at the cellular level are discussed.
Cardiotonic steriods
Inotropic action
1. Introduction In experiments on isolated frog hearts, Werschinin (1910), Von Konschegg (1913), and Loewi (1917, 1918) found calcium ions and digitalis to have a synergistic effect on contractility. Edens and Huber (1916) and Billigheimer (1924) observed similar effects in man. Gold and Edwards (1928) confirmed this synergism in experiments on dogs: they found an increase in toxicity of digitalis with enhanced serum calcium levels induced by simultaneous infusion of calcium chloride or chronic treatment with parathyroid hormone. Several cases of death caused b y digitalis in man with increased serum calcium levels were reported in 1 9 2 5 - 1 9 3 6 and further supported the concept of a calcium-digitalis synergism (for ref. see Klaus, 1964). In experiments on mice, however, Koch (1952) reported for the first time differences between some cardiac glycosides with regard to this calcium-digi-
Papillary muscle
Toxity
Ca2+-dependence
talis interdependence: thus the lethal dose of digitoxin was decreased b y increasing the serum calcium level whereas the lethal doses of k-strophanthin and convallatoxin were increased by elevated serum calcium concentrations. F6rster and Lindenau (1963) obtained similar results in experiments on guinea pigs: thus, the toxicity of digitoxin was increased b y simultaneous infusion o f calcium chloride whereas the toxicity of digoxin and convallatoxin was decreased under the same conditions; the toxicities of k-strophanthin and ouabain were not influenced. More recently, Natorp and Schultz ( 1 9 6 8 ) s t r e n g t h ened these observations in a more detailed study o f the calcium-digitalis interaction on guinea pigs. All these results, however, were obtained in experiments on whole animals; thereby, extracardiac interferences between calcium and digitalis (e,g. protein binding, distribution, metabolism, etc,) cannot be excluded from being the basis of these divergent observations. For further elucidation of this interrelation,
332
R.G. Alken et al., Ca ~+ on the action o f cardiotonic steroids
the influence of varying the extracellular calcium concentration on the inotropic and toxic effects of various cardenolides was tested in experiments on isolated papillary muscles. The following substances were used: digitoxin, digoxin and ouabain (= the cardiac glycosides showing different calcium digitalis interactions in the above experiments), and besides these strophanthidin-3-acetate (= acetylstrophanthidin) and strophanthidin-3-bromoacetate. The latter was of special interest, because in other experiments this cardenolide showed unexpected behaviour with respect to the well known K+-digitalis antagonism (Fricke and Klaus, 1971a). Part of the results has been reported previously (Alken et al., 1972).
due to the cumulative application of the cardenolides. The EDs o values (= concentration for half maximum response) were obtained by probit analysis (Goldstein et al., 1968) of the individual dose-response curves. All these data were analysed by standard statistical methods (mean values and S.E.M., Student's t-test, Tukey test, U-test and analysis of variance respectively). Ouabain, digitoxin and digoxin were supplied by Merck AG, Darmstadt, Germany. Strophanthidin-3acetate (Fricke et al., in preparation) and strophanthidin-3-bromoacetate (Fricke and Klaus, 1971b) were synthesized in our laboratory.
3. Results 2. Materials and methods The inotropic action of the 5 cardenolides studied was measured on electrically stimulated (Grass SM-6 stimulator: square waves, 60/min, 3 msec, 50 V) papillary muscles isolated from fight ventricles of guinea pigs (300-400 g). The contractile force was measured isometrically by a Grass FT 03 transducer and registered on a Grass Model 7 Polygraph (GRASS Instr. Co., Mass. USA). The papillary muscles (length: 3 - 4 mm, diameter < 1 mm, preload: 0.5 p) were allowed to equilibrate for a period of 3 0 - 4 5 min in a muscle bath cGntaining 50 ml, then for 5 min in an organ bath containing 5 ml of modified Tyrode solution with varying Ca2+concentrations (table 1), maintained at 30°C, and gassed with 95% 0 2 - 5 % CO2. After this period, the cardenolide to be tested was added cumulatively to the incubation medium at 30 min intervals, until toxic effects (contracture or oscillations (Reiter, 1962) appeared. The cardenolides were dissolved in propanediol-l,2 (stock solutions of ouabain, digoxin, digitoxin 10-4 M, strophanthidin-3acetate and strophanthidin-3-bromoacetate 10-3 M), which has been reported to have no effect on contractility under the above conditions (Fricke and Klaus, 1971b). The changes in contractile force induced by the cardenolides were expressed both in percentages of the preceding control values and in absolute terms (pond/g dry weight). The toxicity was quantified by measuring the time to the appearance of toxic signs. This time is equivalent to a particular concentration
The influence of extracellular Ca 2+concentrations between 0.45 mM and 7.2 mM on the contractile force of isolated papillary muscles is shown in table 1. In the range studied, a nearly linear relationship between the contractile force and the log dose of the external Ca z+ concentration was observed. Regarding the dose-response curves of the cardenolides digitoxin, digoxin, ouabain, strophanthidin-3acetate and strophanthidin-3-bromoacetate in the presence of the five Ca 2÷ concentrations studied, the greatest increase in contractile force, expressed as a percentage of the control value, was found at the lowest Ca 2+ concentration tested (0.45 raM). Depending on the cardenolide, the contractile force could be increased by 214 -+ 45% to 573 + 50%. This increase was only 10.6 -+ 3.1% to 40.1 -+ 8.2% with 7.2 mM extracellular Ca ~÷. This dependence of the relative change in contractile force by the cardenolides on the extracellular Ca 2+ concentration may be attributed to the different control values under the respective conditions (table 1). Indeed, a comparison of the a b s o l u t e changes in contractile force confirmed this assumption: there was no consistent dependence of the maximum inotropic potency of any particular cardenolide on the various Ca 2÷ concentrations. Therefore, only two typical dose-response curves - digitoxin (fig. 1), digoxin (fig. 2) - are shown which are representative for the different calcium cardenolide interactions described below. In figs. 3 and 4, the variation of the responsiveness
R . G . A l k e n et al., Ca 2 + o n t h e a c t i o n o f e a r d i o t o n i c steroids
333
Table 1 Contractile force (pond/g dry weight) of guinea pig papillary muscles at increasing extracellular Ca 2+ concentrations (means ± S.E.M. of 22 37 experiments) and composition of the modified Tyrode solutions as determined by tlame photometry (means ± S.E.M., n = 2 -8). In addition, these solutions contained: Mg 2+ 1.05, C1- 143.2- 156.4, HCO3- 11.9, H2PO 4- 0.42, glucose 10.0 raM. The pH was 7.15 ± 0.02. Intended Ca 2+ variations in t h e T y r o d e s o l u t i o n s
Concentrations measured (raM)
Contractile f o r c e
(mM)
.............................................. Ca 2+ Na +
0.45 0.90
0 . 3 8 -+ 0 . 0 0 0 . 8 6 -+ 0 . 0 3
1.80
1.82 ± 0.04
3.60 7.20
3.68 ± 0.09 7.02 ± 0.07
it)/g d r y w e i g h t ) K+
1 4 5 . 5 +- 0.5 148.3 +- 0.7 146.8 -+ 1.6 1 4 9 . 3 +_ 4.1
147.8 +- 1.2
5.22 5.41 5.42 5.33 5.32
± 0.00 +- 0 . 0 5 ± 0.05 ± 0.10 ± 0.09
1
®
Oi~itoxin
%
T
240 -
.--
•
A
-=
A ~-
± 3.0 ± 13.0 ± 34.0 +- 4 2 . 0 ± 34.0
the EDso of ouabain (4.73 ± 0.27 × 10 -7 M -~ 2.71 ± 0.72 X 10 -7 M, p < 0.01), digitoxin (11.33 +- 1.25 × 10-TM --> 4.43 _+ 1.65 i0 -7 M, p < 0.005), and strophanthidin-3-acetate (11.08 ± 1.78 X 10 -6 M 5.94 -+ 1.54 X 10 -6 M, p < 0 . 0 1 ) were gradually decreased. In contrast, the EDso of digoxin was shifted
of the heart muscle preparations to the above cardenolides with increasing Ca 2+ concentrations is presented. The dependence of the dose for half-maximum effect ( E D s o ) of the different cardenolides on the external Ca 2÷ concentration is shown in fig. 3. By increasing the Ca 2+ concentration from 0.45 to 7.2 mM
®
30.0 109.0 226.0 385.0 498.0
0.45 m M
rnM Ca2*
-A
t 8 3.6
mM Cae" m M Ca 2 .
~
72
r a M Ca 2°
.
P g DW
Ca 2°
09
-800
ch
¢.
8 -600 R
ll ////// iIf~ i
160"
/
rb
I/
8
-400
~c~ 1
-
_
80" ,
l
:[/./
#
-200
-0
O-
1.10 x I0"/M Fig. I. Influence of different extracellular Ca 2+ concentrations on the dose-response curve of digitoxin on isolated papillary muscles. (A) Percentage increase in contractile force. (B) Absolute values of the initial contractile force and of the cardenolide induced changes (pond/g dry weight). Means and S.E.M. of 4--7 experiments.
I
I
®
%
o---o =--, ~.....~ a- -u
350"
250ii /1
Q
O.~S mM 0.9 mM 1.8 mM 3.6 mM
I
®
Oi~oxin
p
Ca2° Ca 2° Co z" Ca z°
-900
7,2 mM Co 2"
I
-600
16¢ /¢ ¢/
"
//
.,''1 ,T ,,,I 1//
80-
_,
/
i... t
. . . .
-300
I
i
.~...........
~o
,b ;o .~-~M ~u
-o
~ ;
,~
2. . , . 6. .~ . ~
,o-~o~,o".'
Fig. 2. Influence of different extracellular Ca 2+ concentrations on the dose-response curve of digoxin on isolated papillary muscles. (A) Percentage increase in contractile force. (B) Absolute values of the initial contractile force and of the cardenolide induced changes (pond/g dry weight). Means and S.E.M. of 4 - 6 experiments.
Concert. mltic~
['l
I ED50
Tk~# Toxicity
["~1
8 x lO'S .
190
6xtO "5 .
160
StrophcMtddin. 3Bromoc~etate
[,,o~# J; x ~ "s [-[.130
I
StrophantNdin- 3Bromoocetate
2 x I0"6
Stlophanthidin- 3 Acetate
a x ~ "e ] 2~ 6x10"6 1
2.0"
100
Digoxin
tgO
4 x tO'6 T t60
1.0,
~
~
Oigitoxin
2x~'6t t.?O
Digoxin 0.5,
StrophanthMin-3Acetate
Oigitoxin
2xXT6I
Ouaboin
I xtO4 t ~0 !
Ouaboin
# x tO"? ~ t30 0,1
o.'~s
d9
~8
Ls
712
.v-,.
i:~
Fig. 3. Ca 2+ dependence of the ED50 values (concentrations for half-maximum response in M) of the caxdenolides tested. Data derived by probit analysis from the respective individual experiments. Means and $.E.M. of 4 - 8 experiments.
o.,
,n
316
~b
. ~-'. ke']
Fig. 4. Ca 2+ dependence of the 'time to toxicity' (min) of the cardenolide tested. In addition, the corresponding concentrations (M) are given (for details see text). Data derived from the respective individual experiments. Means and S.E.M. of 4 - 8 experiments.
R.G. A l k e n et al., Ca 2+ on the action o f cardiotonic steroids
from 13.42 -+ 1.06 X IO-TM to 19.08 -+ 1.65 × 10-7 M (p < 0.005) by increasing the Ca 2÷ concentration from 0.45 to 1.8 mM. A further increase to 7.2 mM Ca 2÷, however, decreased the EDso (7.34 -+ 1.27 × 10 -7 M).
With strophanthidin-3-bromoacetate, the same initial shift of the EDso from 1 3 . 2 2 -+ 1.28 X 10 -6 M to 19.32 -+ 1.50 × 10 -6 M (p < 0.05) in the Ca ~÷ concentration range from 0.45 to 1.8 mM could be shown, whilst a further increase to 7.2 mM Ca 2+ again decreased the EDs0 (10.17 -+ 1.07 × 10 -6 M). Similar results were obtained when the time course of the onset of the toxic effects produced by these cardenolides was analysed at different Ca 2÷ concentrations (fig. 4). Since the time schedule for the application of the drugs was identical under all conditions (30 min intervals), the appearance of toxic effects could be expressed in minutes calculated from the beginning of the experiment. This procedure allows more distinct evaluation of the toxicity than the consideration of the cardenolide concentrations only, because of the rather large concentration intervals. By increasing the Ca 2÷ concentration from 0.45 to 7.2 mM a continuous decrease of the 'time to toxicity' could be observed with ouabain (186.7 -+ 5.6 min 137.5 + 4.8 min, p < 0.0005), digitoxin (176.5 -+ 3.0 min ~ 135.0 + 2.9 min, p < 0.0025), and strophanthidin-3-acetate (158.0 --- 5.2 min ~ 127.0 -+ 7.0 min, p < 0.0005). In contrast, with digoxin the 'time to toxicity' was augrnented from 194.0 -+ 3.0 min to 222.5 -+ 4.5 min (p < 0.0025) by increasing the Ca 2÷ concentration from 0.45 to 3.6 mM. However, a further increase to 7.2 mM Ca 2÷ decreased again the 'time to toxicity' (193.5 -+ 3.0 min). Similar results were obtained in the experiments with strophanthidin-3-bromoacetate: increasing the Ca 2÷ concentration from 0.45 to 1.8 mM caused a prolongation of the 'time to toxicity' from 148.2 -+ 5.0 min to 185.2 -+ 7.2 min (p < 0.0025), whereas a further increase of the Ca 2÷ concentration to 7.2 mM reduced the 'time to toxicity' (140.0 +- 8.0 min).
4. Discussion
Analysis of the inotropic action of the cardenolldes tested at different extracellular Ca 2÷ concentrations indicates lack of a pronounced relationship of
335
the absolute changes in contractility: though the initial values of the contractile force differ greatly with varying calcium concentrations, the maximum increase in contractile force produced by a particular cardenolide is almost identical at all calcium concentrations tested. This is in accordance with the assumption that the inotropic action of cardiotonic steroids is mediated by a cellular calcium fraction (Lee and Klaus, 1971), and is not related to myocardial calcium uptake processes which are known to be dependent on extracellular calcium concentrations (Scholz, 1970). Furthermore, our results allow a differentiation of two distinct groups of cardenolides with respect to the calcium dependence of their pharmacological actions on isolated papillary muscles of guinea pig hearts. (1) With ouabain, digitoxin and strophanthidin-3acetate, calcium-digitalis synergism (see introduction) could be demonstrated. In these experiments a significant shift of the EDs0 to lower cardenolide concentrations was found by increasing the extracellular calcium concentration from 0.45 to 7.2 mM (fig. 3). The time to the appearance of toxicity was also significantly reduced in these experiments (fig. 4). These observations are in agreement with the results obtained previously in other experiments on isolated heart muscle preparations (e.g. Hoditz, 1963; Heeg and Udo, 1964). (2) In contrast, with two of the studied cardenolides, digoxin and strophanthidin-3-bromoacetate, opposite results were obtained over a limited extracellular calcium range. The EDs 0's of strophanthidin-3bromoacetate and of digoxin were shifted significantly to higher concentrations by increasing the calcium concentration to 1.8 mM (fig. 3). Similar results were obtained when studying the 'time to toxicity' (fig. 4). Thus, these latter observations do not support the commonly accepted concept of a calcium-digitalis synergism, but even indicate antagonistic actions between calcium and digitalis - at least for some cardenolides within a certain calcium concentration range. These results obtained under in vitro conditions are in agreement with the previously reported differences in respective in vivo studies by Koch (1952), Forster and Lindenau (1963), and Natorp and Schultz (1968). In addition, our observations exclude
336
R.G. Alken et al., Ca 2+ on the action o f cardiotonic steroids
an extracardiac mechanism from being responsible for the differences in the calcium-digitalis interdependence, and clearly indicate a divergent interaction on the myocardial level. At present, it is not possible to give a final explanation for this phenomenon. Perhaps the differences in the calcium-digitalis interaction of the studied cardenolides might be related to the diverse influence of various cardiotonic steroids on the shape of the action potential as demonstrated by Ire et al. (1970): some (digitoxin, proscillaridin) caused a dose-dependent prolongation of the plateau period, others (ouabain, convaUatoxol) induced such an effect only in a low concentration range and produced a reduction with higher concentrations whereas digoxin and dihydro-ouabain did not influence this parameter. However, there is no further information on the calcium dependence of this phenomenon. Alternatively, calcium could influence the uptake of different cardenolides by the myocardium in a different manner. Our preliminary hypothesis of this kind of interaction is illustrated in fig. 5. It is based on the assumption that two different processes are involved in the reaction of digitalis (D) with the heart muscle cell. One of them is a Ca 2÷ sensitive 'specific' binding process (membrane ATPase?) which is supposed to be predominant in the interaction with some cardenolides, whereas the other one is a 'non-specific' mechanism based on the physico-chemical nature of these drugs. The extent to which each of these mechanisms is involved in the digitalis reaction with the cell may be different for different cardenolides. Both these processes determine the amount of digitalis
Fig. 5. Scheme of the supposed ealcium-digitafis interactions at the cellular level. ECS = extracellular space, D -- digitalis. For details see text.
which is effective in the 'mediator system' of the heart muscle cell with respect to the modulation of the intracellular calcium concentration (Lee and Klaus, 1971). The limited range of Ca 2÷ antagonism to the digoxin and strophanthidin-3-bromoacetate actions in the experiments demonstrated above could then be explained by the assumption that the calcium-dependent inhibition of the cardenolide reaction with the myocardial cell is overcome by the direct actions of Ca 2÷ on the 'effector system' at higher extracellular calcium concentrations.
References Alken, R.G., U. Frieke and W. Klaus, 1972, Differences in the [Ca] -dependence of the cardenolide action in isolated heart muscle preparations, Naunyn-Schmiedeb. Arch. Pharmacol. 274, R 12. Billigheimer, E., 1924, Vergleichende Untersuchungen fiber die Wirkung des Calcium und der Digitalis, Z. Kiln. Med. 100,411. Edens, E. und J.E. Huber, 1916, Uber Digitalisbigemie, Deutsch. Arch. Klin. Med. 118,476. F6ster,, W, und M. Lindenau, 1963, Untersuchungen zur Frage einer synergistisehen Wtrkung yon Ca ~ und Digitalisk6rpern, Klin. Wschr. 41,339. Fricke, U. and W. Klaus, 1971a, Comparative studies of the influence of various K ÷-concentrations " on the action of k-strophanthidin, digitoxin and strophanthidin-3-bromoacetate on papillary muscle and on membrane-ATPase of guinea-pig hearts, European J. Pharmacol. 15, 1. Fricke, U. und W. Klaus, 1971b, 0ber die Wirkung yon Strophanthidin-3-bromacetat am Papillarmuskel des Meerschweinchens, Naunyn-Schmiedeb. Arch. Pharmakol. 268, 192. Gold, H. and D.J. Edwards, 1928, The effect of ouabain on the heart in the presence of hypercalcemia, Amer. Heart J. 3,45. Goldstein, A., L. Aronow and S.M. Kalman, 1968, Principles of Drug Action (Hoeber Medical Division, Harper and Row, New York-Evanston-London) p. 343. Heeg, E. und E. Udo, 1964, Der Einfluss der extracellul~'ren Kalium-, Calcium- und Natriumkonzentrarion auf die 'therapeutische' und toxische Wirkung des Digitoxigenins, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 247, 333. Hoditz, H., 1963, Quantitative Untersuchungen am Meerschweinchenvorhof fiber die Abh~'ngigkeit der Digitoxigeninwirkung yon der extracellul//ren Calciumkonzentration, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 244,531. Ire, M., P.B. Hollander, B.H. Marks and S. Dutta, 1970, The effects of six cardiac glycosides on the transmembrane
R.G. A l k e n et al., Ca 2+ on the action o f cardiotonic steroids
potential and contractile characteristics of the right ventricle of guinea pigs, J. Pharmacol. Exptl. Therap. 172, 188. Klaus, W., 1964, Neuere Aspekte fiber den Wirkungsmechanismus der Herzglykoside, Z. Naturwiss.-Med. Grundlagenforsch. 2, 43. Koch, F.E., 1952, ExperimenteUe Untersuchungen tiber das herzwirksame Prinzip der ConvaUaria majalis, Madaus Jahresbericht 5, 9. Lee, K.S. and W. Klaus, 1971, The subceUular basis for the mechanism of inotropic action of cardiac glycosides, Pharmacol. Rev. 23,193. Loewi, O., 1917, Uber den Zusammenhang zwischen Digitalis- und Kalziumwirkung, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 82,131. Loewi, O., 1918, t0ber den Zusammenhang zwischen Digitalis- und Kalziumwirkung, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 83,336.
337
Natorp, W. und U. Schultz, 1968, [3ber den Einfluss einer Calcium chlorid-Infusion auf die Toxizitiit einiger Herzglykoside, Arzneim. Forsch. 18, 1609. Reiter, M., 1962, Die Entstehung yon 'Nachkontraktionen' im Herzmuskel unter Einwirkung yon Calcium und yon Digitalisglykosiden in Abh~nglgkeit yon der Reizfrequenz, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 242, 497. Scholz, H., 1970, Elektrophysiologische und IsotopenVersuche zur positiv inotropen Wirkung von Theophyllin, Adrenalin und Digitoxigenin an Warmbltiterherzen, Habilitationsschrift, Mainz. Von Konschegg, A., 1913, 0her die Beziehung zwischen Herzmittel und physiologischer Kationenwirkung, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 71,251. Werschinin, N., 1910, Uber die systolische und diastolische Herzwirkung des g-Strophanthins, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 63,386.
DIVERGENT CARDIOTONIC
INFLUENCES STEROIDS
O F Ca 2+ O N T H E A C T I O N O F S E V E R A L
IN ISOLATED
HEART MUSCLE PREPARATIONS
Rudolf G. ALKEN, Uwe FRICKE and Wolfgang KLAUS Department of Pharmacology, Medical School of Hannover, 3000 Hannover-Kleefeld, Karl-Wiechert-Allee 9, W.-Germany
Received 14 August 1973
Accepted 17 December 1973
R.G. ALKEN, U. FRICKE and W. KLAUS, Divergent influences of Ca2+on the action of several cardiotonic steroids in isolated heart muscle preparations, European J. Pharmacol. 26 (1974) 331-337. In recent years, calcium-digitalis synergism has been challenged on the basis of some divergent observations in in vivo experiments on different species. The purpose of the present in vitro studies on isolated guinea pig papillary muscles was to eliminate extracardlac interferences and to test the possible calcium-digitalis interrelationship at the myocardial level. For evaluation of the positive inotropic and toxic actions the dose-response curves of several cardiotonic steroids were studied at different extracellular calcium concentrations. With ouabain, digitoxin and strophanthidin-3-acetate a continuous decrease of the EDso and an enhanced toxicity was observed when the calcium concentration was raised from 0.45 to 7.2 mM. In contrast, with digoxin and strophanthidin-3-bromoacetate, an increase of the EDso and a reduced toxicity was obtained in the calcium concentration range from 0.45 to 3.6 mM. These results do not support the concept of a calcium-digitalis synergism as a general rule for all cardenolides. Several possible explanations at the cellular level are discussed.
Cardiotonic steriods
Inotropic action
1. Introduction In experiments on isolated frog hearts, Werschinin (1910), Von Konschegg (1913), and Loewi (1917, 1918) found calcium ions and digitalis to have a synergistic effect on contractility. Edens and Huber (1916) and Billigheimer (1924) observed similar effects in man. Gold and Edwards (1928) confirmed this synergism in experiments on dogs: they found an increase in toxicity of digitalis with enhanced serum calcium levels induced by simultaneous infusion of calcium chloride or chronic treatment with parathyroid hormone. Several cases of death caused b y digitalis in man with increased serum calcium levels were reported in 1 9 2 5 - 1 9 3 6 and further supported the concept of a calcium-digitalis synergism (for ref. see Klaus, 1964). In experiments on mice, however, Koch (1952) reported for the first time differences between some cardiac glycosides with regard to this calcium-digi-
Papillary muscle
Toxity
Ca2+-dependence
talis interdependence: thus the lethal dose of digitoxin was decreased b y increasing the serum calcium level whereas the lethal doses of k-strophanthin and convallatoxin were increased by elevated serum calcium concentrations. F6rster and Lindenau (1963) obtained similar results in experiments on guinea pigs: thus, the toxicity of digitoxin was increased b y simultaneous infusion o f calcium chloride whereas the toxicity of digoxin and convallatoxin was decreased under the same conditions; the toxicities of k-strophanthin and ouabain were not influenced. More recently, Natorp and Schultz ( 1 9 6 8 ) s t r e n g t h ened these observations in a more detailed study o f the calcium-digitalis interaction on guinea pigs. All these results, however, were obtained in experiments on whole animals; thereby, extracardiac interferences between calcium and digitalis (e,g. protein binding, distribution, metabolism, etc,) cannot be excluded from being the basis of these divergent observations. For further elucidation of this interrelation,
332
R.G. Alken et al., Ca ~+ on the action o f cardiotonic steroids
the influence of varying the extracellular calcium concentration on the inotropic and toxic effects of various cardenolides was tested in experiments on isolated papillary muscles. The following substances were used: digitoxin, digoxin and ouabain (= the cardiac glycosides showing different calcium digitalis interactions in the above experiments), and besides these strophanthidin-3-acetate (= acetylstrophanthidin) and strophanthidin-3-bromoacetate. The latter was of special interest, because in other experiments this cardenolide showed unexpected behaviour with respect to the well known K+-digitalis antagonism (Fricke and Klaus, 1971a). Part of the results has been reported previously (Alken et al., 1972).
due to the cumulative application of the cardenolides. The EDs o values (= concentration for half maximum response) were obtained by probit analysis (Goldstein et al., 1968) of the individual dose-response curves. All these data were analysed by standard statistical methods (mean values and S.E.M., Student's t-test, Tukey test, U-test and analysis of variance respectively). Ouabain, digitoxin and digoxin were supplied by Merck AG, Darmstadt, Germany. Strophanthidin-3acetate (Fricke et al., in preparation) and strophanthidin-3-bromoacetate (Fricke and Klaus, 1971b) were synthesized in our laboratory.
3. Results 2. Materials and methods The inotropic action of the 5 cardenolides studied was measured on electrically stimulated (Grass SM-6 stimulator: square waves, 60/min, 3 msec, 50 V) papillary muscles isolated from fight ventricles of guinea pigs (300-400 g). The contractile force was measured isometrically by a Grass FT 03 transducer and registered on a Grass Model 7 Polygraph (GRASS Instr. Co., Mass. USA). The papillary muscles (length: 3 - 4 mm, diameter < 1 mm, preload: 0.5 p) were allowed to equilibrate for a period of 3 0 - 4 5 min in a muscle bath cGntaining 50 ml, then for 5 min in an organ bath containing 5 ml of modified Tyrode solution with varying Ca2+concentrations (table 1), maintained at 30°C, and gassed with 95% 0 2 - 5 % CO2. After this period, the cardenolide to be tested was added cumulatively to the incubation medium at 30 min intervals, until toxic effects (contracture or oscillations (Reiter, 1962) appeared. The cardenolides were dissolved in propanediol-l,2 (stock solutions of ouabain, digoxin, digitoxin 10-4 M, strophanthidin-3acetate and strophanthidin-3-bromoacetate 10-3 M), which has been reported to have no effect on contractility under the above conditions (Fricke and Klaus, 1971b). The changes in contractile force induced by the cardenolides were expressed both in percentages of the preceding control values and in absolute terms (pond/g dry weight). The toxicity was quantified by measuring the time to the appearance of toxic signs. This time is equivalent to a particular concentration
The influence of extracellular Ca 2+concentrations between 0.45 mM and 7.2 mM on the contractile force of isolated papillary muscles is shown in table 1. In the range studied, a nearly linear relationship between the contractile force and the log dose of the external Ca z+ concentration was observed. Regarding the dose-response curves of the cardenolides digitoxin, digoxin, ouabain, strophanthidin-3acetate and strophanthidin-3-bromoacetate in the presence of the five Ca 2÷ concentrations studied, the greatest increase in contractile force, expressed as a percentage of the control value, was found at the lowest Ca 2+ concentration tested (0.45 raM). Depending on the cardenolide, the contractile force could be increased by 214 -+ 45% to 573 + 50%. This increase was only 10.6 -+ 3.1% to 40.1 -+ 8.2% with 7.2 mM extracellular Ca ~÷. This dependence of the relative change in contractile force by the cardenolides on the extracellular Ca 2+ concentration may be attributed to the different control values under the respective conditions (table 1). Indeed, a comparison of the a b s o l u t e changes in contractile force confirmed this assumption: there was no consistent dependence of the maximum inotropic potency of any particular cardenolide on the various Ca 2÷ concentrations. Therefore, only two typical dose-response curves - digitoxin (fig. 1), digoxin (fig. 2) - are shown which are representative for the different calcium cardenolide interactions described below. In figs. 3 and 4, the variation of the responsiveness
R . G . A l k e n et al., Ca 2 + o n t h e a c t i o n o f e a r d i o t o n i c steroids
333
Table 1 Contractile force (pond/g dry weight) of guinea pig papillary muscles at increasing extracellular Ca 2+ concentrations (means ± S.E.M. of 22 37 experiments) and composition of the modified Tyrode solutions as determined by tlame photometry (means ± S.E.M., n = 2 -8). In addition, these solutions contained: Mg 2+ 1.05, C1- 143.2- 156.4, HCO3- 11.9, H2PO 4- 0.42, glucose 10.0 raM. The pH was 7.15 ± 0.02. Intended Ca 2+ variations in t h e T y r o d e s o l u t i o n s
Concentrations measured (raM)
Contractile f o r c e
(mM)
.............................................. Ca 2+ Na +
0.45 0.90
0 . 3 8 -+ 0 . 0 0 0 . 8 6 -+ 0 . 0 3
1.80
1.82 ± 0.04
3.60 7.20
3.68 ± 0.09 7.02 ± 0.07
it)/g d r y w e i g h t ) K+
1 4 5 . 5 +- 0.5 148.3 +- 0.7 146.8 -+ 1.6 1 4 9 . 3 +_ 4.1
147.8 +- 1.2
5.22 5.41 5.42 5.33 5.32
± 0.00 +- 0 . 0 5 ± 0.05 ± 0.10 ± 0.09
1
®
Oi~itoxin
%
T
240 -
.--
•
A
-=
A ~-
± 3.0 ± 13.0 ± 34.0 +- 4 2 . 0 ± 34.0
the EDso of ouabain (4.73 ± 0.27 × 10 -7 M -~ 2.71 ± 0.72 X 10 -7 M, p < 0.01), digitoxin (11.33 +- 1.25 × 10-TM --> 4.43 _+ 1.65 i0 -7 M, p < 0.005), and strophanthidin-3-acetate (11.08 ± 1.78 X 10 -6 M 5.94 -+ 1.54 X 10 -6 M, p < 0 . 0 1 ) were gradually decreased. In contrast, the EDso of digoxin was shifted
of the heart muscle preparations to the above cardenolides with increasing Ca 2+ concentrations is presented. The dependence of the dose for half-maximum effect ( E D s o ) of the different cardenolides on the external Ca 2÷ concentration is shown in fig. 3. By increasing the Ca 2+ concentration from 0.45 to 7.2 mM
®
30.0 109.0 226.0 385.0 498.0
0.45 m M
rnM Ca2*
-A
t 8 3.6
mM Cae" m M Ca 2 .
~
72
r a M Ca 2°
.
P g DW
Ca 2°
09
-800
ch
¢.
8 -600 R
ll ////// iIf~ i
160"
/
rb
I/
8
-400
~c~ 1
-
_
80" ,
l
:[/./
#
-200
-0
O-
1.10 x I0"/M Fig. I. Influence of different extracellular Ca 2+ concentrations on the dose-response curve of digitoxin on isolated papillary muscles. (A) Percentage increase in contractile force. (B) Absolute values of the initial contractile force and of the cardenolide induced changes (pond/g dry weight). Means and S.E.M. of 4--7 experiments.
I
I
®
%
o---o =--, ~.....~ a- -u
350"
250ii /1
Q
O.~S mM 0.9 mM 1.8 mM 3.6 mM
I
®
Oi~oxin
p
Ca2° Ca 2° Co z" Ca z°
-900
7,2 mM Co 2"
I
-600
16¢ /¢ ¢/
"
//
.,''1 ,T ,,,I 1//
80-
_,
/
i... t
. . . .
-300
I
i
.~...........
~o
,b ;o .~-~M ~u
-o
~ ;
,~
2. . , . 6. .~ . ~
,o-~o~,o".'
Fig. 2. Influence of different extracellular Ca 2+ concentrations on the dose-response curve of digoxin on isolated papillary muscles. (A) Percentage increase in contractile force. (B) Absolute values of the initial contractile force and of the cardenolide induced changes (pond/g dry weight). Means and S.E.M. of 4 - 6 experiments.
Concert. mltic~
['l
I ED50
Tk~# Toxicity
["~1
8 x lO'S .
190
6xtO "5 .
160
StrophcMtddin. 3Bromoc~etate
[,,o~# J; x ~ "s [-[.130
I
StrophantNdin- 3Bromoocetate
2 x I0"6
Stlophanthidin- 3 Acetate
a x ~ "e ] 2~ 6x10"6 1
2.0"
100
Digoxin
tgO
4 x tO'6 T t60
1.0,
~
~
Oigitoxin
2x~'6t t.?O
Digoxin 0.5,
StrophanthMin-3Acetate
Oigitoxin
2xXT6I
Ouaboin
I xtO4 t ~0 !
Ouaboin
# x tO"? ~ t30 0,1
o.'~s
d9
~8
Ls
712
.v-,.
i:~
Fig. 3. Ca 2+ dependence of the ED50 values (concentrations for half-maximum response in M) of the caxdenolides tested. Data derived by probit analysis from the respective individual experiments. Means and $.E.M. of 4 - 8 experiments.
o.,
,n
316
~b
. ~-'. ke']
Fig. 4. Ca 2+ dependence of the 'time to toxicity' (min) of the cardenolide tested. In addition, the corresponding concentrations (M) are given (for details see text). Data derived from the respective individual experiments. Means and S.E.M. of 4 - 8 experiments.
R.G. A l k e n et al., Ca 2+ on the action o f cardiotonic steroids
from 13.42 -+ 1.06 X IO-TM to 19.08 -+ 1.65 × 10-7 M (p < 0.005) by increasing the Ca 2÷ concentration from 0.45 to 1.8 mM. A further increase to 7.2 mM Ca 2÷, however, decreased the EDso (7.34 -+ 1.27 × 10 -7 M).
With strophanthidin-3-bromoacetate, the same initial shift of the EDso from 1 3 . 2 2 -+ 1.28 X 10 -6 M to 19.32 -+ 1.50 × 10 -6 M (p < 0.05) in the Ca ~÷ concentration range from 0.45 to 1.8 mM could be shown, whilst a further increase to 7.2 mM Ca 2+ again decreased the EDs0 (10.17 -+ 1.07 × 10 -6 M). Similar results were obtained when the time course of the onset of the toxic effects produced by these cardenolides was analysed at different Ca 2÷ concentrations (fig. 4). Since the time schedule for the application of the drugs was identical under all conditions (30 min intervals), the appearance of toxic effects could be expressed in minutes calculated from the beginning of the experiment. This procedure allows more distinct evaluation of the toxicity than the consideration of the cardenolide concentrations only, because of the rather large concentration intervals. By increasing the Ca 2÷ concentration from 0.45 to 7.2 mM a continuous decrease of the 'time to toxicity' could be observed with ouabain (186.7 -+ 5.6 min 137.5 + 4.8 min, p < 0.0005), digitoxin (176.5 -+ 3.0 min ~ 135.0 + 2.9 min, p < 0.0025), and strophanthidin-3-acetate (158.0 --- 5.2 min ~ 127.0 -+ 7.0 min, p < 0.0005). In contrast, with digoxin the 'time to toxicity' was augrnented from 194.0 -+ 3.0 min to 222.5 -+ 4.5 min (p < 0.0025) by increasing the Ca 2÷ concentration from 0.45 to 3.6 mM. However, a further increase to 7.2 mM Ca 2÷ decreased again the 'time to toxicity' (193.5 -+ 3.0 min). Similar results were obtained in the experiments with strophanthidin-3-bromoacetate: increasing the Ca 2÷ concentration from 0.45 to 1.8 mM caused a prolongation of the 'time to toxicity' from 148.2 -+ 5.0 min to 185.2 -+ 7.2 min (p < 0.0025), whereas a further increase of the Ca 2÷ concentration to 7.2 mM reduced the 'time to toxicity' (140.0 +- 8.0 min).
4. Discussion
Analysis of the inotropic action of the cardenolldes tested at different extracellular Ca 2÷ concentrations indicates lack of a pronounced relationship of
335
the absolute changes in contractility: though the initial values of the contractile force differ greatly with varying calcium concentrations, the maximum increase in contractile force produced by a particular cardenolide is almost identical at all calcium concentrations tested. This is in accordance with the assumption that the inotropic action of cardiotonic steroids is mediated by a cellular calcium fraction (Lee and Klaus, 1971), and is not related to myocardial calcium uptake processes which are known to be dependent on extracellular calcium concentrations (Scholz, 1970). Furthermore, our results allow a differentiation of two distinct groups of cardenolides with respect to the calcium dependence of their pharmacological actions on isolated papillary muscles of guinea pig hearts. (1) With ouabain, digitoxin and strophanthidin-3acetate, calcium-digitalis synergism (see introduction) could be demonstrated. In these experiments a significant shift of the EDs0 to lower cardenolide concentrations was found by increasing the extracellular calcium concentration from 0.45 to 7.2 mM (fig. 3). The time to the appearance of toxicity was also significantly reduced in these experiments (fig. 4). These observations are in agreement with the results obtained previously in other experiments on isolated heart muscle preparations (e.g. Hoditz, 1963; Heeg and Udo, 1964). (2) In contrast, with two of the studied cardenolides, digoxin and strophanthidin-3-bromoacetate, opposite results were obtained over a limited extracellular calcium range. The EDs 0's of strophanthidin-3bromoacetate and of digoxin were shifted significantly to higher concentrations by increasing the calcium concentration to 1.8 mM (fig. 3). Similar results were obtained when studying the 'time to toxicity' (fig. 4). Thus, these latter observations do not support the commonly accepted concept of a calcium-digitalis synergism, but even indicate antagonistic actions between calcium and digitalis - at least for some cardenolides within a certain calcium concentration range. These results obtained under in vitro conditions are in agreement with the previously reported differences in respective in vivo studies by Koch (1952), Forster and Lindenau (1963), and Natorp and Schultz (1968). In addition, our observations exclude
336
R.G. Alken et al., Ca 2+ on the action o f cardiotonic steroids
an extracardiac mechanism from being responsible for the differences in the calcium-digitalis interdependence, and clearly indicate a divergent interaction on the myocardial level. At present, it is not possible to give a final explanation for this phenomenon. Perhaps the differences in the calcium-digitalis interaction of the studied cardenolides might be related to the diverse influence of various cardiotonic steroids on the shape of the action potential as demonstrated by Ire et al. (1970): some (digitoxin, proscillaridin) caused a dose-dependent prolongation of the plateau period, others (ouabain, convaUatoxol) induced such an effect only in a low concentration range and produced a reduction with higher concentrations whereas digoxin and dihydro-ouabain did not influence this parameter. However, there is no further information on the calcium dependence of this phenomenon. Alternatively, calcium could influence the uptake of different cardenolides by the myocardium in a different manner. Our preliminary hypothesis of this kind of interaction is illustrated in fig. 5. It is based on the assumption that two different processes are involved in the reaction of digitalis (D) with the heart muscle cell. One of them is a Ca 2÷ sensitive 'specific' binding process (membrane ATPase?) which is supposed to be predominant in the interaction with some cardenolides, whereas the other one is a 'non-specific' mechanism based on the physico-chemical nature of these drugs. The extent to which each of these mechanisms is involved in the digitalis reaction with the cell may be different for different cardenolides. Both these processes determine the amount of digitalis
Fig. 5. Scheme of the supposed ealcium-digitafis interactions at the cellular level. ECS = extracellular space, D -- digitalis. For details see text.
which is effective in the 'mediator system' of the heart muscle cell with respect to the modulation of the intracellular calcium concentration (Lee and Klaus, 1971). The limited range of Ca 2÷ antagonism to the digoxin and strophanthidin-3-bromoacetate actions in the experiments demonstrated above could then be explained by the assumption that the calcium-dependent inhibition of the cardenolide reaction with the myocardial cell is overcome by the direct actions of Ca 2÷ on the 'effector system' at higher extracellular calcium concentrations.
References Alken, R.G., U. Frieke and W. Klaus, 1972, Differences in the [Ca] -dependence of the cardenolide action in isolated heart muscle preparations, Naunyn-Schmiedeb. Arch. Pharmacol. 274, R 12. Billigheimer, E., 1924, Vergleichende Untersuchungen fiber die Wirkung des Calcium und der Digitalis, Z. Kiln. Med. 100,411. Edens, E. und J.E. Huber, 1916, Uber Digitalisbigemie, Deutsch. Arch. Klin. Med. 118,476. F6ster,, W, und M. Lindenau, 1963, Untersuchungen zur Frage einer synergistisehen Wtrkung yon Ca ~ und Digitalisk6rpern, Klin. Wschr. 41,339. Fricke, U. and W. Klaus, 1971a, Comparative studies of the influence of various K ÷-concentrations " on the action of k-strophanthidin, digitoxin and strophanthidin-3-bromoacetate on papillary muscle and on membrane-ATPase of guinea-pig hearts, European J. Pharmacol. 15, 1. Fricke, U. und W. Klaus, 1971b, 0ber die Wirkung yon Strophanthidin-3-bromacetat am Papillarmuskel des Meerschweinchens, Naunyn-Schmiedeb. Arch. Pharmakol. 268, 192. Gold, H. and D.J. Edwards, 1928, The effect of ouabain on the heart in the presence of hypercalcemia, Amer. Heart J. 3,45. Goldstein, A., L. Aronow and S.M. Kalman, 1968, Principles of Drug Action (Hoeber Medical Division, Harper and Row, New York-Evanston-London) p. 343. Heeg, E. und E. Udo, 1964, Der Einfluss der extracellul~'ren Kalium-, Calcium- und Natriumkonzentrarion auf die 'therapeutische' und toxische Wirkung des Digitoxigenins, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 247, 333. Hoditz, H., 1963, Quantitative Untersuchungen am Meerschweinchenvorhof fiber die Abh~'ngigkeit der Digitoxigeninwirkung yon der extracellul//ren Calciumkonzentration, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 244,531. Ire, M., P.B. Hollander, B.H. Marks and S. Dutta, 1970, The effects of six cardiac glycosides on the transmembrane
R.G. A l k e n et al., Ca 2+ on the action o f cardiotonic steroids
potential and contractile characteristics of the right ventricle of guinea pigs, J. Pharmacol. Exptl. Therap. 172, 188. Klaus, W., 1964, Neuere Aspekte fiber den Wirkungsmechanismus der Herzglykoside, Z. Naturwiss.-Med. Grundlagenforsch. 2, 43. Koch, F.E., 1952, ExperimenteUe Untersuchungen tiber das herzwirksame Prinzip der ConvaUaria majalis, Madaus Jahresbericht 5, 9. Lee, K.S. and W. Klaus, 1971, The subceUular basis for the mechanism of inotropic action of cardiac glycosides, Pharmacol. Rev. 23,193. Loewi, O., 1917, Uber den Zusammenhang zwischen Digitalis- und Kalziumwirkung, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 82,131. Loewi, O., 1918, t0ber den Zusammenhang zwischen Digitalis- und Kalziumwirkung, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 83,336.
337
Natorp, W. und U. Schultz, 1968, [3ber den Einfluss einer Calcium chlorid-Infusion auf die Toxizitiit einiger Herzglykoside, Arzneim. Forsch. 18, 1609. Reiter, M., 1962, Die Entstehung yon 'Nachkontraktionen' im Herzmuskel unter Einwirkung yon Calcium und yon Digitalisglykosiden in Abh~nglgkeit yon der Reizfrequenz, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 242, 497. Scholz, H., 1970, Elektrophysiologische und IsotopenVersuche zur positiv inotropen Wirkung von Theophyllin, Adrenalin und Digitoxigenin an Warmbltiterherzen, Habilitationsschrift, Mainz. Von Konschegg, A., 1913, 0her die Beziehung zwischen Herzmittel und physiologischer Kationenwirkung, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 71,251. Werschinin, N., 1910, Uber die systolische und diastolische Herzwirkung des g-Strophanthins, Naunyn-Schmiedeb. Arch. Exptl. Pathol. Pharmakol. 63,386.