Lack of interaction of spironolactone with ouabain in guinea pig isolated heart muscle preparations

European Journal of Pharmacology, 49 (1978) 363--371

363

© Elsevier/North-Holland Biomedical Press

LACK OF INTERACTION OF SPIRONOLACTONE WITH OUABAIN IN GUINEA PIG ISOLATED HEART MUSCLE PREPARATIONS * UWE FRICKE

Pharmakologisches Institut der Universit~it zu K~In, Gleueler Str. 24, D-5000 K~In 41, Germany Received 31 October 1977, revised MS received 10 February 1978, accepted 20 February 1978 U. FRICKE, Lack of interaction of spironolactone with ouabain in guinea pig isolated heart muscle preparations, European J. Pharmacol. 49 (1978) 363--371. The effect of spironolactone on ouabaln action was studied in experiments on guinea pig isolated heart muscle preparations. There was no effect of spironolactone on the ouabain-induced positive inotropic or toxic effects in isolated papillary muscles. This was accompanied by a lack of spironolactone effect on myocardial ouabain uptake. Only rather high spironolactone concentrations (1 × 10 -4 M) led to a reduced cardiac ouabain uptake (about 10%). The subcellular distribution pattern of ouabain, however, remained unchanged under these conditions. Studies on ouabaln binding to a cardiac NaK-ATPase supported the conclusion that a direct interaction of spironolactone and cardiac glycosides at the myocardium could be ruled out. Subcellular distribution Myocardial uptake

Inotropic effects Spironolactone

NaK-ATPase Interaction

1. Introduction Spironolactone and its water-soluble derivative potassium canrenoate are often used clinically in the treatment of edema. Early in 1969, Selye et al. (1969a, b) reported an antagonistic effect of spironolactone on the toxicity of cardiac glycosides in rats. Yeh et al. (1972, 1974) in a more detailed study on dogs demonstrated a potent antiarrhythmic activity of potassium canrenoate in digitalis toxicity. As the positive inotropic action of these drugs was not affected at all, these authors assumed that the canrenoate molecule might be a specific antagonist to the electrophysiological toxicity of cardiac glycosides. Similar experiments on isolated heart muscle preparations (Baskin et al., 1973; Coraboeuf and Deroubaix, 1974) partly confirmed these observations. A direct interaction at the Na÷--K+-ATPase level, however, could obviously be ruled out (Baskin et al., 1973).

* The study was supported by grants of the Deutsche Forschungsgemeinschaft (FR 407/1 + 3).

Toxicity

Ouabaln

Binding

More recently, Kuhlmann et al. (1977) studied the pharmacokinetic interaction of potassium canrenoate and cardiac glycosides in dogs and reported an increase in myocardial accumulation of digoxin and ouabain. The unexpected results of this study were explained on the basis of an increased coronary blood flow (Baskin et al., 1973; Coraboeuf and Deroubaix, 1974), of improved hemodynarnic effects (KStter et al., 1977) and a reduced renal clearance of cardiac glycosides (Steiness, 1974) induces by spironolactone. Therefore, to exclude the effects above described, the present study dealing with the interaction of spironolactone and ouabain was performed on isolated heart muscle preparations. The influence of spironolactone on the inotropic and toxic action of ouabain was tested with guinea pig isolated papillary muscles. Furthermore, the interaction of spironolactone with myocardial ouabain uptake and subcellular distribution was studied on isolated perfused guinea pig hearts. Finally, the influence of spironolactone was tested on ouabain binding to a microsomal Na÷--K÷-ATPase (NaK-ATPase) of guinea pig hearts.

364 2. Materials and methods

2.1. Methods 2.1.1. Inotropic action The inotropic action was evaluated on electrically stimulated (GRASS SM-6 stimulator: square waves, 60/min, 3 msec, 50 V) papillary muscles isolated from right ventricles o f guinea pig hearts (guinea pigs o f either sex, 200-250 g). Contractile force was measured isometrically b y means of a force~lisplacement transducer and recorded after amplification on a Grass Model 7 Polygraph (Grass Instr. Co., Mass. U.S.A.). The papillary muscles (resting tension: 0.5 p; length: 3--4 mm; diameter <~ 1 mm) were allowed to stabilize at 30°C for 35 min in an organ bath containing 5 ml of a modified Tyrode solution (mM) (150.2 + 0.4 Na ÷, 5.44+ 0.02 K ÷, 0.91 + 0.02 Ca 2÷ (n = 25), 1.05 Mg 2+, 144 Cl-, 11.9 HCO3-, 0.42 H2PO4- and 10.0 glucose; the Na ÷ and K ÷ concentration were determined b y flame p h o t o m e t r y and the Ca 2+ concentration b y titration with Calcein as an indicator). The T y r o d e solution was equilibrated with carbogen (95% O2--5% CO2) resulting in a pH o f 7.28 + 0.03 (n = 25). To test the influence o f spironolactone (stock solution: 1 × 10 -2 M in propanediol-l,2; this solvent has been reported to have no effect on contractile force under the above conditions, Fricke and Klaus, 1971), on contractile force this drug was added cumulatively to the organ bath at 30 min intervals. In another series of experiments the effect of spironolactone (concentrations as indicated) on ouabain actions was tested, After the stabilization period of 35 min (see above) a pre-incubation period (30 rain) in the presence or in the absence (control) o f spironolactone was allowed and a cumulative dose--response curve of ouabain (30 min intervals) was then done under the same conditons as used for the pre-incubation period. The experiments were terminated when toxic effects appeared (arrhythmias, contracture). All time intervals were chosen so as to allow a steady state of drug action.

u. FRICKE

2.1.2. Myocardial uptake and subcellular distribution Isolated hearts of guinea pigs (300--400 g) were perfused via the aorta at a constant flow (8.5 + 0.01 ml/min, corresponding to a mean perfusion pressure of 50.2 + 2.8 mm Hg, n = 12), b y means of a peristaltic pump (Fricke and Klaus, 1975). The perfusion medium used was a modified Tyrode solution containing (mM) 149.1 + 0.5 Na ÷, 5.47 + 0.02 K ÷, 0.90 + 0.01 Ca 2÷ (n = 12; other electrolytes and glucose as indicated above) maintained at 37°C and equilibrated with carbogen (95% 02--5% CO2) resulting in a pH of 7.40 + 0.02 in = 12). The contractile force of the electrically stimulated hearts (Stimulator S, Hugo Sachs Elektronik, Hugstetten, F.R.G.: square waves, 180/min, 6 msec, 2 × threshold voltage) was measured b y means of a pressure transducer (Bell & Howell, Type 4-327) connected with a fluid-filled Latex balloon (0.25 ml) placed into the left ventricle. The force of contraction was recorded after amplification (Datatran Amplifier, Bell & Howell, G.B.) on a recording oscillograph (Bell & Howell, T y p e 5-124 FB, G.B.). The perfusion pressure was measured b y means o f a second pressure transducer (see above) connected to the aortic cannula. After a 30 min stabilization period with drug-free T y r o d e solution the hearts were perfused for another 30 min with a Tyrode solution o f the same composition in the absence (control) or in the presence of either 1 × 10 -s or 1 X 10 -4 M spironolactone (stock solution: 2 × 10 -2 M in ethanol 90%). At the end of this pre-incubation period the perfusion medium was changed (for additional 30 min) to a T y r o d e solution containing in addition 1 X 10 -7 M 3H-ouabain. Estimation o f the extracellular space was performed with 14Csucrose (5 rag/l), which was added during the drug perfusion period (30 min). At the end o f each drug perfusion period the hearts were detached from the cannula, both atria and the aorta were removed and the ventricles weighed immediately after

365

SPIRONOLACTONE INTERACTION WITH OUABAIN gentle blotting. The ventricles were homogenized with 9 volumes of an isotonic sucrose solution (250 mM sucrose, 5 mM imidazole HC1, pH 7.4) and fractionally centrifuged to prepare nuclear membrane (600 × g for 10 min), mitochondrial (5000 × g for 10 rain) and microsomal fractions (68000 × g for 30 min) as described earlier (Fricke et al., 1975). Aliquots of the Tyrode solution, of the initial homogenate and of each fraction were then mixed with 10 ml of Tritosol, a Triton-X100 containing scintillation mixture, which can be used for aqueous and particulate samples (Fricke, 1975) and counted in a liquid scintillation counter (Mark II, Nuclear Chicago, U.S.A.). Quenching was corrected for with the external standard ratio method. After individual correction for the extracellular space (as determined by the 14C-sucrose contamination) the amount of 3H-ouabain in the homogenate and in each fraction was calculated on the basis of g wet weight and mg protein, respectively. The influence of homogenization, fractionation etc., on the subcellular distribution of 3H-ouabain was tested in another series of experiments. For this purpose hearts were perfused for 30 min with Tyrode solution and minced in a micro-mincer (Braun, Melsungen, F.R.G.). Before homogenizing and fractionating -- as outlined above -- 3Houabain was added in an amount approximately equal to that present in the homogenates of the drug-perfused hearts ("artificial", i.e. function independent distribution).

2.1.3. 3H-ouabain binding A microsomal NaK-ATPase from guinea pig hearts was freshly prepared before use as described recently (Fricke and Klaus, 1977). For the binding experiments, polyethylene tubes (Sarstedt, Niimbrecht, F.R.G.) were used with incubations at 37°C. The incubation medium contained the enzyme preparation (about 0.3 mg of protein per 4.0 ml), 5raM MgCI2, 140mM NaC1, 4raM Na2EDTA, 100 mM imidazole HC1 (uH 7.4) and

either no or I X I0 -s M or I X 10 -4 M spironolactone in the presence or absence of 2 m M Na2-ATP. After a preincubation time of 10 rain the reaction was started by the addition of 3H-ouabain in various concentrations (5 X 10 -9 M to 1 X 10 -6 M, final concentration). The reaction was stopped after 30 min by immersion in an ice bath (5 rain). The mixture was then centrifuged at 40,000 g for 30 rain at 0°C (Beckman Spinco L2 65B). The resulting sediment and an aliquot of the original incubation medium were each mixed with 10 ml of Tritosol scintillation cocktail (see above) and counted in a liquid scintillation counter (Mark If, Nuclear Chicago, U.S.A.). Correction for quenching was performed by the external standard ratio method. A T P dependent 3H~uabain binding ("specific binding") was calculated by subtracting the phosphate ligand independent 3H-ouabain binding from the 3H-ouabain binding in the presence of ATP. The SH-ouabain binding is expressed as pmol ouabain per m g protein. Protein determinations were carried out according to the method of Lowry et al. (1951) with Labtrol (Merz & Dade, Miinchen, F.R.G.) as a standard. All data were analyzed by standard statistical methods (means and S.E.M., Student's t-test).

2.2. Materials SH-ouabain (specific activity; 12 Ci/mmole) and 14C-sucrose (434 mCi/mmole) were obtained from NEN Chemicals (Dreieichenhain, F.R.G.). Radiochemical purity was checked by thin layer chromatography, Spironolactone was purchased from Calbiochem (Frankfurt, F.R.G.) and ATP-Na2 from Serva (Heidelberg, F.R.G.). All other reagents were supplied by Merck (Darmstadt, F.R.G.). 3. Results

3.1. Inotropic action Spironolactone induced negligible or no effects on the contractile force of isolated

366

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OUABAIN Fig. I. Influence of increasing concentrations of spironolactone (indicated in the fig.) on ouahain-induced contractile force (expressed as miIlipond, rap) in guinea pig heart isolated papillary muscles. C = pre-drug control

value; P = contractile force after addition of spironolactone as indicated. Means of 5--7 experiments are given.

papillary muscles from guinea pig heart up to a concentration o f I X 10 -6 M. Higher concentrations up to 1 × 10-4M resulted in a dose-dependent negative inotropic action. Maximum reduction in contractile force at I × 10 -4 M spironolactone was about 25% of the control value. This effect on contractility corresponded well with those effects obtained in the experiments on isolated perfused guinea pig hearts: with 1 X 10-SM spironolactone there was a negative inotropic effect of about 30% and with a concentration of 1 × 10 -4 M in the perfusion medium contractile force was reduced by about 50%. However, there was no influence of spironolactone on ouabain-induced positive inotropic and toxic effects in guinea pig heart isolated papillary muscles in the range of I X 10 -8 up to 1 × 10 -4 M spironolactone (fig. 1).

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Fig. 2. Myocardial uptake (pmoles/g wet weight) of 3H-ouabain in isolated perfused guinea pig hearts under control conditions (open column) and under the influence o f 1 x 10 -s M (hatched column) or 1 x 10 -4 M (dotted column) spironolactone. *P < 0.05. Means and S.E.M. o f 4 experiments each are shown. Ordinate: ouabain concentration (pmol/g wet weight).

SPIRONOLACTONE

INTERACTION

WITH OUABAIN

367

TABLE 1 M y o c a r d i a l u p t a k e a n d s u b c e l l u l a r d i s t r i b u t i o n o f a H - o u a b a i n (1 × 10 -7 M ) in i s o l a t e d p e r f u s e d g u i n e a pig h e a r t u n d e r c o n t r o l c o n d i t i o n s a n d u n d e r t h e i n f l u e n c e o f 1 X 10 - s o r 1 × 10 -# M s p i r o n o l a c t o n e . ( A ) O u a b a i n c o n c e n t r a t i o n in p m o l e s / g w e t w e i g h t ( c o l u m n I); (B) o u a b a i n c o n c e n t r a t i o n in p m o l e s / m g p r o t e i n ( c o l u m n I). C o l u m n II s h o w s t h e r a t i o o f t h e d i s t r i b u t i o n o f o u a b a i n in t h e s u b c e l l u l a r f r a c t i o n s o f p e r f u s e d t o n o n - p e r f u s e d h e a r t s (values > 1.0 i n d i c a t e a " s p e c i f i c " , i.e. f u n c t i o n - d e p e n d e n t b i n d i n g ) . M e a n s a n d S.E.M. o f 4 e x p e r i m e n t s e a c h are p r e s e n t e d . Fraction

Control

Spironolactone 1 × 10 -s M

I

II

I

1 x 10 -4 M II

I

II

(A) Homogenate Nuclear membrane Mitochondrial Microsomal Supernatant

298.3 61.1

_+ 12.7 + 2.4

-8.7

23.8 55.1 132.2

-+ 3.5 _+ 4.9 + 13.5

3.6 +_ 0.3 16.9 + 1.0 0 . 5 2 + 0.02

_+ 0.6

283.1 63.5

+ 7.4 + 2.9

-8.3 -+ 0.6

257.0 48.3

_+ 10.8 + 3.7

18.7 49.2 119.5

+ 1.6 _+ 1.2 _+ 5.6

3.3 -+ 0.1 17.9 + 1.0 0 . 5 3 -+ 0 . 0 3

17.8 48.1 111.9

+ 1.2 + 0.3 + 11.3

-9.8

_+ 0.4

3.1 _+ 0.2 16.4 _+ 0.4 0.51 -+ 0.01

(B) Homogenate Nuclear membrane Mitochondrial Microsomal Supernatant

2.95 + 0.21 1.43 _+ 0 . 1 0

-5.7

1.50 _+ 0.19 6.44 + 0.64 5.50 -+ 0 . 5 6

2.6 + 0.3 10.8 + 0.5 0 . 4 3 + 0.01

-+ 0.3

3.2. Myocardial uptake and subcellular distribution The myocardial uptake of ouabain and the interaction of spironolactone (1 × 10 -s and 1 × 10-4 M) with ouabain in isolated perfused guinea pig hearts is shown in fig. 2. Whereas 1 X 10-SM spironolactone did not influence myocardial ouabain uptake, the 10-fold higher concentration resulted in a small b u t significant (P < 0.05) reduction of cardiac ouabain uptake (see table 1). The extraceUular space (as indicated b y the 1"C-sucrose contamination) in these studies was in the range of 47.5 + 1.9% (n = 4) under control conditions and was slightly increased under the influence o f 1 × 10 -s M spironolactone (50.5 + 0.5%, n = 4). With the 10-fold higher spironolactone concentration a significantly (P < 0.05) higher sucrose space was found (53.4 + 1.6%, n = 4).

2.76 -+ 0 . 0 5 1.49 +_ 0.05

-5.5

1.24 + 0 . 0 8 5.96 + 0.53 5.25 + 0.19

2.3 + 0.1 10.3 -+ 1.0 0.45 + 0.04

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0.16 0.05

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0.05 0.44 0.56

2.3 + 0.1 10.6 -+0.6 0 . 4 2 + 0.03

+ 0.0

The subcellular distribution of ouabain under control conditions and under the influence of spironolactone is specified in table 1 (column I) on the basis of g wet weight and mg protein, respectively. Considering the subcellular distribution of ouabain in nonperfused hearts under the same conditions (see above), thus indicating an "artificial", function-independent distribution o f the cardiac glycoside, the ratio of the distribution in drug-perfused hearts to that in non-perfused hearts reflects the function dependent uptake of ouabain. These ratios are given in table 1 (column II). From these values a specific accumulation of ouabain can be derived for the nuclear membrane fraction and specially for the microsomal fraction. Spironolactone in the concentration range studied, however, did not change the subcellular distribution pattern of ouabain.

368

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Fig. 3. ATP-dependent binding of 3H-ouabain (pmoles/mg protein) to a microsomal NaK-ATPase of guinea pig heart under control conditions and under the influence of increasing concentrations of spironolactone as indicated. Means of 3--4 experiments each are presented.

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Fig. 4. Influence of increasing concentrations of spironolactone (as indicated) on the time course of the ATPdependent 3H-ouabain binding (pmoles/mg protein, ordinate) to a microsomal NaK-ATPase of guinea pig heart. 3H-ouabain concentration in the incubation medium: 5 X 10 -s M. Means of 3--4 experiments each are given.

Neither ATP~lependent nor ATP-independ e n t b i n d i n g o f o u a b a i n (see M e t h o d s ) w e r e influenced when the spironohctone concent r a t i o n was i n c r e a s e d f r o m 1 × 10 -8 t o 1 × 10 -4 M u n d e r s t e a d y s t a t e c o n d i t i o n s (fig. 3). I n a d d i t i o n , t h e r e was n o i n f l u e n c e o n t h e time-course of ouabain binding to NaK-ATPase u n d e r t h e s e c o n d i t i o n s (fig. 4). O n t h e basis o f t h e s e results t h e m a x i m u m b i n d i n g c a p a c i t i e s (Bma=) a n d t h e r e s p e c t i v e a p p a r e n t d i s s o c i a t i o n c o n s t a n t s (KD) w e r e determined by means of a Scatchard plot ( 1 9 4 9 ) . T h e s e d a t a are listed in t a b l e 2.

S P I R O N O L A C T O N E I N T E R A C T I O N WITH O U A B A I N TABLE 2 M a x i m u m binding capacities (Bmax) and apparent dissociation constants (K D) for 3H-ouabain u n d e r control c o n d i t i o n s and under 'the i n f l u e n c e of increasing c o n c e n t r a t i o n s of spironolact0ne. Analysis according to Scatchard (1949) o f t h e experimental data presented in fig. 3. Means and S.E.M. o f the indicated n u m b e r o f e x p e r i m e n t s are shown. Conc. [M]

B

-1 × 1 X 1 × 1× 1X

14.88 14.56 14.44 14.99 14.65 14.47

10-8 10 -7 10 -6 10 -s 10 -4

~ pm_ol 1 maXLmg ProtJ + 0.15' + 0.80 + 1.02 -+ 1.47 _+ 1.06 _+ 0.74

K D [X10 -7 M]

n

4.0 4.1 3.9 3.9 4.1 4.2

4 3 3 4 4 4

+ 0.1 -+ 0.2 + 0.2 + 0.4 + 0.2 _+ 0.1

4. Discussion Quite different results concerning the inotropic action of spironolactone or its watersoluble derivative potassium (or sodium) canrenoate can be found in the literature. Reduced contractility due to these drugs -- as in the present study -- has been reported in atrial preparations of rabbits (Briggs and Holland, 1959), guinea pigs (Bacciarelli, 1967) and rats (Coraboeuf and Deroubaix, 1974) as well as in isolated hearts of guinea pigs (Baskin et al., 1973) and rats (Coraboeuf and Deroubaix, 1974). Furthermore, a negative inotropic action has been observed in papillary muscles of the cat heart (Lucchesi and Haley, 1973). These effects on contractile force of isolated heart muscle preparations, however, could not be confirmed in experiments on dogs in vivo (Yeh et al., 1972). These authors did not find any change in contractility after a bolus injection of 0.5 mmoles of potassium canrenoate. On the other hand there are reports of dose
369

and Schiiren, 1972; SchrSder et al., 1972). These rather divergent effects of spironolactone on myocardial contractility, might be based on pronounced species differences and on the fact that, in the in vivo experiments, hemodynamic effects may be responsible for the improved contraction of the heart. Taking into account the reported protective effect of spironolactone on cardiac glycosideinduced toxicity (see Introduction), it was rather surprising that, in the present study, no such effects could be detected over a wide range of spironolactone concentrations (see fig. 1). However, looking more closely at the above mentioned studies it is obvious that these beneficial effects of spironolactone are mainly found in the intact animal (Selye et al., 1969a, b; Yeh et al., 1972; Yeh and Sung, 1972; Yeh et al., 1974), or in man (Varenne et al., 1967; Smolensky et al., 1969). On the contrary, there are negligible or even no effects in isolated heart muscle preparations (Bacciarelli, 1967; Baskin et al., 1973; Lucchesi and Haley, 1973). The failure of spironolactone to alter digitalis tolerance has also been reported in one clinical study (Kr{imet et al., 1973). These observations are in good agreement with the results regarding the interaction of spironolactone with the myocardial ouabain uptake obtained in the present study (see fig. 2, table 1). Only at rather high spironolactone concentrations (1 × 10-4 M) was there a slight reduction {about 10%) in myocardial uptake which, however, might not account for the above mentioned protective action of spironolactone on digitalis toxicity. Moreover, the subcellular distribution pattern of ouabain was not altered under the influence of spironolactone (see table 1), thereby ruling out the possibility of an interaction with cardiac glycosides in intracellular structures, e.g. the microsomal fraction, supposedly a predominant site of action of cardiac glycosides (for ref. see Lee and Klaus, 1971). These results could be further confirmed by the studies on a microsomal NaKATPase preparation (see figs. 3, 4; table 2). As

370

already demonstrated by Baskin et al. (1973) there was no change in ouabain binding under the influence of spironolactone. Thus, the protective effect of this drug on digitalis toxicity resp. an antiarrhythmic action of spironolactone (if there is such an action), cannot be related to a direct interaction at the myocardium. There are to be considered, however, in vivo (1) a beneficial effect of spironolactone induced hyperkalemia on digitalis tolerance, though this mechanism obviously could be ruled out in experiments on rats (Selye et al., 1969a, b) and mice (Buck and Lage, 1971) as well as in studies on man (Kr'dmer et al., 1973), and (2) an induction of hepatic microsomal enzyme activity and an increased biliary excretion of cardiac glycosides (Solymoss et al., 1969; Leber et al., 1971; Castle and Lage, 1972, 1973; Abshagen, 1973; Wirth and FrSlich, 1974; Klaasen, 1974). The latter point, how. ever, seems to be of minor importance in man (Wirth et al., 1976; Abshagen et al., 1976).

References Abshagen, U., 1973, Effects of pretreatment with spironolactone on pharmacokinetics of 4"'methyldigoxin in rats, Naunyn-Schmiedeb. Arch. Pharmacol. 278, 91. Abshagen, U., H. Rennekamp and J. Kuhlmann, 1976, Effects of pretreatment with spironolactone on pharmacokinetics of 4"t-methyldigoxin in man, Naunyn Schmiedeb. Arch. Pharmacol. 292, 87. Bacciarelli, C., 1967, L'effetto della digoxina, del dessossicorticosterone e dello spironolattone sul miocardio di mammifero, Arch. Intern. Pharmacodyn. 166, 150. Baskin, S.I., T. Akera, C.R. Puckett, S.L. Brody and T.M. Brody, 1973, Effect of potassium canrenoate on cardiac function and ( N a ÷ + K÷)-activated ATPase, Proc. Soc. Exp. Biol. Med. 143,495. Briggs, A.H. and W.C. Holland, 1959, Effect of steroids, acetylcholine, quinidine, and ionic environment on refractory period and K transport in isolated rabbit atria, Federation Proc. 18, 371. Buck, S.H. and G.L. Lage, 1971, Possible mechanism of the prevention of digitoxin toxicity by spironolactone in the mouse, Arch. Intern. Pharmacodyn. Therap. 189, 192.

U. FRICKE Castle, M.C. and G.L. Lage, 1972, Effect of pretreatment with spironolactone, phenobarbital or ~-diethylaminoethyl diphenylpropylacetate (SKF 525-A) on tritium levels in blood, heart and liver of rats at various times after administration of [3H]-digitoxin, Biochem. Pharmacol. 21, 1449. Castle, M.C. and G.L. Lage, 1973, Enhanced biliary excretion of digitoxin following spironolactone as it relates to the prevention of digitoxin toxicity, Res. Commun. Chem. Pathol. Pharmacol. 5, 99. Coraboeuf, E. and E. Deroubaix, 1974, Effect of a spironolactone derivative, sodium canrenoate, on mechanical and electrical activities of isolated rat myocardium, J. Pharmacol. Exptl. Therap. 191, 128. Fricke, U., 1975, Tritosol: A new scintillation cocktail based on Triton X-100, Anal. Biochem. 63,555. Fricke, U., U. Hollborn and W. Klaus, 1975, Inotropic action, myocardial uptake and subcellular distribution of ouabain, digoxin and digitoxin in isolated rat hearts, Naunyn Schmiedeb. Arch. Pharmacol. 288,195. Fricke, U. and W. Klaus, 1971, ~-ber die Wirkung yon Strophathidin-3-bromacetat am Papillarmuskel des Meerschweinchens, Naunyn Schmiedeb. Arch. Pharmakol. 268,192. Fricke, U. and W. Klaus, 1975, Dependence of the cardiac uptake of digitalis glycosides on the extracellular calcium concentration in isolated guinea pig hearts, Europ. J. Pharmacol. 30, 182. Fricke, U. and W. Klaus, 1977, Evidence for two different Na÷-dependent 3H-ouabain binding sites of a Na*-K*-ATPase of guinea pig hearts, Brit. J. Pharmacol. 61,423. Hiittemann, U. and K.P. Schiiren, 1972, Die Wirkung yon Aldactone auf Atmung und Lungenkreislauf beim chronischen Cot pulmonale, Klin. Wschr. 50, 953. Klaassen, C.D., 1974, Effect of microsomal enzyme inducers on the biliary excretion of cardiac glycosides, J. Pharmacol. Exptl. Therap. 191,201. KStter, V., E. Von Leitner, J. Kuhlmann and R. SchrSder, 1977, Vergleich der Auswirkung yon Digoxin und Canreoat-Kalium (Aldactone pro injectione) auf die H~imodynamik und InfarktgrSsse yon Hunden mit grossen Myokardinfarkten, Herz/Kreisl. 9,208. Kr~/mer, K.-D., P. Ghabussi and H. Hochrein, 1973, Klinische Priifung der subtoxischen Vollwirk- und Erhaltungsdosis yon Digoxin unter dem Einfluss von Spironolaktone. Untersuchungen an dekompensierten Herzkranken, Arzneim. Forach. 23,508. Kuhlmann, J., V. K6tter, H.F. V6hringer, N. Rietbrock and R. Schr~der, 1977, Influence of canrenoate-K and cardiac glycosides on their tissue distribution and elimination, Arzneim. Forsch. 27, 1505.

SPIRONOLACTONE INTERACTION WITH OUABAIN Leber, H.W., P. Rawer and G. Schiitterle, 1971, Beeinflussung mikrosomaler Enzyme der Rattenleber dutch Spironolacton, Etacryns~ute und Futosemid, Klin. Wschr. 49,116. Lee, K.S. and W. Klaus, 1971, The subcellular basis for the mechanism of inotropic action of cardiac glycosides, Pharmacol. Rev. 23,193. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193,265. Lucchesi, B.R. and N.R. Haley, 1973, Failure of potassium canrenoate to alter experimentally induced digitalis arrhythmias, European J. Pharmacol. 22,256. Scatchard, G., 1949, The attractions of proteins for small molecules and ions, Ann. N.Y. Acad. Sci. 51, 660. SchrSder, R., K.P. Schiiren, G. Biamino, V. Meyer and W. Sadie, 1971, Positiv-inotrope Herzwirkung yon Aldadien-Kalium (Aldactone pro injectione), Klin. Wschr. 49, 1093. Schr6der, R., B. Ramdohr, K. Hiittemann and K.P. Schiiren, 1972, Direkte positiv-inotrope Herzwirkung yon Aldactone (Spironolacton, Canrenoat-Kalium), Deutsch. Med. Wschr. 97, 1535. Selye, H., I. M~cs and T. Tamuta, 1969, Effect of spironoiactone and norbolethone on the toxicity of digitalis compounds in the rat, Brit. J. Pharmacol. 37,485. Selye, H., M. Krajny and L. Savoie, 1969, Digitoxin poisoning: Prevention by spironolactone, Science 164,842. Smolensky, V.S., I.S. Kun, V.S. Khudzhamirova, M.B. Gelfond and Y.S. Topschiashvili, 1969, Elimination of digitalis arrhythmia with concurrent antagonists of aldosterone and cocarboxilase, Ter. Arch. (Moskva) 41, 19. Solymoss, B., H.G. Classen and S. Varga, 1969, Increased hepatic microsomal activity induced by spironolactone, Proc. Soc. Exp. Biol. Med. 132, 940. Steiness, E., 1974, Renal tubular secretion of digoxin, Circulation 50,103.

371 Strauer, B.E., 1973, The influence of the aldosteroneantagonist spironolactone on myocardial contractility, Arch. Intern. Pharmacodyn. Therap. 201, 59. Strauer, B.E., H. Avenhaus, M. Nose, 1972, Evidence for positive inotropic effect of aldadiene (-K, -Na) on the isolated ventricular myocardium, Klin. Wschr. 50,387. Tanz, R.D., 1962, Studies on me inotropic action of aldosterone on isolated cardiac tissue preparations; including the effects of pH, ouabain and SC8109, J. Pharrnacol. Exptl. Therap. 135, 71. Tanz, R.D. and C.F. Kerby, 1961, The inotropic action of certain steroids upon isolated cardiac tissue; with comments on steroidal cardiotonic structure-activity relationships, J. Pharmacol. Exptl. Therap. 131, 56. Varenne, A., P. Andr~-Robaut, J.-B. Guiran, 1967, l~tude de l'action clinique d'une spiroiactone sur l'excitabilit~ musculaire et l'~lectrocardiogramme, G.M. de France 12, 5869. Wirth, K.E. and J.C. Fr~lich, 1974, Effect of spironolactone on excretion of 3H-digoxin and its metabolites in rats, European J. Pharmacol. 29, 43. Wirth, K.E., J.C. Fr~lich, J.W. Hollifield, F.C. Falkner, B.S. Sweetman and J.A. Oates, 1976, M#tabolism of digitoxin in man and its modification by spironolactone, European J. Clin. Pharmacol. 9,345. Yeh, B.K. and P.K. Sung, 1972, Prevention and abolition of ouabain-induced ventricuhtr tachycardia by potassium canrenoate in dogs, European J. Pharmacol. 19,151. Yeh, B.K., P.K. Sung and R. Lazzara, 1974, The specificity of potassium canrenoate, a steroidal antidysrhythmic drug, Clin. Pharmacol. Therap. 16, 1014. Yeh, B.K., P.K. Sung and A.K. Saha, 1972, Use of potassium canrenoate to suppress ouabain-induced ventricular arrhythmias in dog, Circulation Res. 31,915.