European Journal of Pharmacology, 57 (1979) 343--351 © Elsevier/North-Holland Biomedical Press
343
DIFFERENTIAL EFFECT OF POTASSIUM ON THE ACTION OF DIGOXIN AND DIGOXIGENIN IN GUINEA-PIG HEART * TAI AKERA, SALLY A. WIEST and THEODORE M. BRODY
Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824, U.S.A. Received 20 February 1979, accepted 14 May 1979
T. AKERA, S.A. WIEST and T.M. BRODY, Differential effect of potassium on the action of digoxin and digoxigenin in guinea-pig heart, European J. Pharmacol. 57 (1979) 343--351. The effect of potassium on the binding of digoxin or digoxigenin to isolated Na ÷, K÷-ATPase was compared with that of potassium on the positive inotropic action of the agents in guinea-pig hearts. The binding of digoxigenin to the enzyme in vitro was reduced to a greater extent by potassium than was the binding of digoxin. The digoxigenin-induced increase in the force of contraction of left atrial preparations estimated at steady state was reduced at higher potassium concentrations. Potassium had a lesser effect when digoxin was used as the inotropic agent. In contrast, potassium reduced the rate of development and also the rate of loss of the positive inotropic action of digoxin observed with left atrial and Langendorff preparations, respectively, to a greater extent than those of digoxigenin. The loss of the positive inotropic effect was more rapid with digoxigenin than with digoxin at each KCI concentration. These data support the contention that the extent of the interaction of digitalis with Na*,K÷-ATPase determines the degree of the positive inotropic effect. Potassium effects on digitalis action Digoxigenin
Na÷,K÷-ATPase
1. Introduction Potassium affects the binding of cardiac glycosides to, and subsequent release from, Na÷,K÷-ATPase (Matsui and Schwartz, 1968; Akera and Brody, 1971). The potassiuminduced inhibition of the glycoside binding results from a reduction in the concentration of phosphoenzyme which preferentially binds cardiac glycosides (Matsui and Schwartz, 1968; Post et al., 1969; Barnett, 1970). Our previous studies in rat brain enzyme preparations (Akera et al., 1974, 1976, 1978; Choi and Akera, 1977) suggest that potassium inhibits the release of the glycoside from the enzyme by increasing the effectiveness of a * This work was supported by a U.S. Public Health Service Grant, HL-16052 and by the Michigan Heart Association.
Inotropic action
Digoxin
lipid barrier which regulates the release of bound glycosides. For the glycoside, potassium markedly delays the binding velocity, but has a relatively minor effect on the equilibrium concentration of bound drug, because both forward and backward reaction velocities are reduced. In contrast, potassium markedly reduces the equilibrium concentration of the bound aglycone, because the backward reaction velocity is reduced to a lesser extent than the forward reaction velocity for these more lipid soluble compounds (Akera et al., 1978). These differences in the effects of potassium on the interaction between the glycoside or aglycone and Na÷,K÷-ATPase provide us with an opportunity to determine whether the concentration of the digitalis-enzyme complex formed at a given time is related to the degree of the drug-induced increase in the
344
force of cardiac contraction at that time. Thus, in the present study, the effects of potassium on the interaction between digoxin or digoxigenin and cardiac Na ÷,K*-ATPase were compared with those of potassium on the positive inotropic action of these agents.
2. Materials and methods Guinea pigs of either sex weighing 300-500 g were stunned by a sharp blow to the head and their hearts were rapidly removed. Homogenates, left atrial preparations, or Langendorff preparations were prepared immediately. [3H]-Labelled ouabin (generally labelled; specific activity, 19 Ci/mmole) was purchased from Amersham/Searle Corporation, Arlington Heights, Illinois. Ouabain octahydrate, Tris-ATP, digoxin and EDTA (free acid) were purchased from Sigma Chemical Company, St. Louis, Missouri. Digoxigenin was purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. 2.1. Fractional occupancy studies
Fractional occupancy of the steroid binding sites on Na÷,K÷-ATPase by either digoxin or digoxigenin in the presence of various concentrations of KC1 was estimated from the initial velocity of saturable [3H]ouabain binding using ventricular homogenates. Homogenate preparations were used because purification procedures might conceivably alter the properties of Na÷,K÷-ATPase with respect to the glycoside interaction (Choi and Akera, 1978). Approximately 0.8 g of ventricular muscle was minced with scissors and homogenized in 10 volumes of an ice-cold solution containing 0.25 M sucrose, 5 mM histidine-HC1 and 1 mM EDTA (pH adjusted to 7.0 with Tris base} using a Douce ball-type homogenizer. After obtaining a smooth homogenate, a 2.5 ml-aliquot was added to an additional 4 ml of the above solution and homogenized again. A 0.1-ml aliquot of the above homogenate
T. A K E R A E T AL.
was incubated at 37°C for 1 5 m i n with digoxin or digoxigenin in the presence of 30 mM NaC1, 5 mM MgC12, 5 mM Tris-ATP, 50 mM Tris-HC1 buffer (pH 7.5) and an indicated concentration of KC1 in a total volume of 1.0 ml. After a 15-min incubation, 0.025 ml of [3H]ouabain solution (carrier-free, 2.8 pM) was added yielding a final labelled ouabain concentration of 68 nM. The [3H]ouabain binding reaction was terminated 1.5 min later by the addition of 5 ml of an icecold solution containing 15 mM KCI and 1 mM nonlabelled ouabain. KC1 was added to stabilize the [3H]ouabain--enzyme complex (Akera and Brody, 1971). The mixture was immediately placed on a nitrocellulose filter (Millipore Corporation, Bedford, Massachusetts; type AA; pore size, 0.8 pm) and filtered under vacuum. The filter was rinsed twice with 15 ml of an ice-cold solution containing 15 mM KC1 and 1 mM nonlabelled otmbain. The radioactivity trapped on the filter (bound ouabain) was assayed using liquid scintillation counting after dissolving the filter in 1.0 ml of ethylene glycol monomethyl ether (Piersolve®, Pierce Chemical Company, Rockford, Illinois). Counting efficiency (approximately 27%) was monitored using external standard channel ratio method. Digoxin and digoxigenin were dissolved in 5 v/v % ethanol. The final concentration of ethanol in the incubation mixture was 0.5 v/v %. The same concentration of ethanol was added to the control incubation mixture. This concentration of ethanol failed to significantly affect the saturable [SH] ouabain binding. Fractional occupancy of the steroid binding sites on Na÷,K÷-ATPase in cardiac homogenates by either digoxin or digoxigenin was calculated from the reduction in saturable [SH]ouabain binding in the presence of these agents compared to that in their absence. Our previous study (Ku et al., 1974) indicates that the 1.5-min values of [3H]ouabain binding to cardiac homogenates can be used instead of the initial binding velocity
POTASSIUM AND DIGITALIS ACTION for this purpose. Nonspecific binding of [3H]ouabain was assayed concurrently by adding 1 mM nonlabelled ouabain to the incubation mixture. This value was subtracted from the total [3H]ouabain binding to calculate saturable [~H]oubain binding. The protein concentration of homogenates was determined by the method of Lowry et al. (1951).
2.2. Development of the positive inotropic effect Development of the positive inotropic effect of digoxin or digoxigenin was studied in electrically stimulated left atrial preparations of guinea-pig hearts. Left atria were vertically suspended in a modified Krebs-Henseleit solution containing l l 8 m M NaC1, 27.2mM NaHCO3, 1.0mM KH2PO4, 1.2 mM MgSO4, 1.8 mM CaC12, 11.1 mM glucose and either 2.5, 4.8 or 8.5 mM KC1 (final concentration of K*; 3.5, 5.8 or 9.5 mEq/liter) and aerated with a 95% 02/5% CO2 gas mixture at 30°C yielding the pH value of 7.4. The atria were stimulated with platinum field-stimulation electrodes at 1 Hz with square wave pulses of 3 msec duration at a voltage 15% above threshold. Nearly isometric force of contraction was recorded with a force
343
DIFFERENTIAL EFFECT OF POTASSIUM ON THE ACTION OF DIGOXIN AND DIGOXIGENIN IN GUINEA-PIG HEART * TAI AKERA, SALLY A. WIEST and THEODORE M. BRODY
Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824, U.S.A. Received 20 February 1979, accepted 14 May 1979
T. AKERA, S.A. WIEST and T.M. BRODY, Differential effect of potassium on the action of digoxin and digoxigenin in guinea-pig heart, European J. Pharmacol. 57 (1979) 343--351. The effect of potassium on the binding of digoxin or digoxigenin to isolated Na ÷, K÷-ATPase was compared with that of potassium on the positive inotropic action of the agents in guinea-pig hearts. The binding of digoxigenin to the enzyme in vitro was reduced to a greater extent by potassium than was the binding of digoxin. The digoxigenin-induced increase in the force of contraction of left atrial preparations estimated at steady state was reduced at higher potassium concentrations. Potassium had a lesser effect when digoxin was used as the inotropic agent. In contrast, potassium reduced the rate of development and also the rate of loss of the positive inotropic action of digoxin observed with left atrial and Langendorff preparations, respectively, to a greater extent than those of digoxigenin. The loss of the positive inotropic effect was more rapid with digoxigenin than with digoxin at each KCI concentration. These data support the contention that the extent of the interaction of digitalis with Na*,K÷-ATPase determines the degree of the positive inotropic effect. Potassium effects on digitalis action Digoxigenin
Na÷,K÷-ATPase
1. Introduction Potassium affects the binding of cardiac glycosides to, and subsequent release from, Na÷,K÷-ATPase (Matsui and Schwartz, 1968; Akera and Brody, 1971). The potassiuminduced inhibition of the glycoside binding results from a reduction in the concentration of phosphoenzyme which preferentially binds cardiac glycosides (Matsui and Schwartz, 1968; Post et al., 1969; Barnett, 1970). Our previous studies in rat brain enzyme preparations (Akera et al., 1974, 1976, 1978; Choi and Akera, 1977) suggest that potassium inhibits the release of the glycoside from the enzyme by increasing the effectiveness of a * This work was supported by a U.S. Public Health Service Grant, HL-16052 and by the Michigan Heart Association.
Inotropic action
Digoxin
lipid barrier which regulates the release of bound glycosides. For the glycoside, potassium markedly delays the binding velocity, but has a relatively minor effect on the equilibrium concentration of bound drug, because both forward and backward reaction velocities are reduced. In contrast, potassium markedly reduces the equilibrium concentration of the bound aglycone, because the backward reaction velocity is reduced to a lesser extent than the forward reaction velocity for these more lipid soluble compounds (Akera et al., 1978). These differences in the effects of potassium on the interaction between the glycoside or aglycone and Na÷,K÷-ATPase provide us with an opportunity to determine whether the concentration of the digitalis-enzyme complex formed at a given time is related to the degree of the drug-induced increase in the
344
force of cardiac contraction at that time. Thus, in the present study, the effects of potassium on the interaction between digoxin or digoxigenin and cardiac Na ÷,K*-ATPase were compared with those of potassium on the positive inotropic action of these agents.
2. Materials and methods Guinea pigs of either sex weighing 300-500 g were stunned by a sharp blow to the head and their hearts were rapidly removed. Homogenates, left atrial preparations, or Langendorff preparations were prepared immediately. [3H]-Labelled ouabin (generally labelled; specific activity, 19 Ci/mmole) was purchased from Amersham/Searle Corporation, Arlington Heights, Illinois. Ouabain octahydrate, Tris-ATP, digoxin and EDTA (free acid) were purchased from Sigma Chemical Company, St. Louis, Missouri. Digoxigenin was purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. 2.1. Fractional occupancy studies
Fractional occupancy of the steroid binding sites on Na÷,K÷-ATPase by either digoxin or digoxigenin in the presence of various concentrations of KC1 was estimated from the initial velocity of saturable [3H]ouabain binding using ventricular homogenates. Homogenate preparations were used because purification procedures might conceivably alter the properties of Na÷,K÷-ATPase with respect to the glycoside interaction (Choi and Akera, 1978). Approximately 0.8 g of ventricular muscle was minced with scissors and homogenized in 10 volumes of an ice-cold solution containing 0.25 M sucrose, 5 mM histidine-HC1 and 1 mM EDTA (pH adjusted to 7.0 with Tris base} using a Douce ball-type homogenizer. After obtaining a smooth homogenate, a 2.5 ml-aliquot was added to an additional 4 ml of the above solution and homogenized again. A 0.1-ml aliquot of the above homogenate
T. A K E R A E T AL.
was incubated at 37°C for 1 5 m i n with digoxin or digoxigenin in the presence of 30 mM NaC1, 5 mM MgC12, 5 mM Tris-ATP, 50 mM Tris-HC1 buffer (pH 7.5) and an indicated concentration of KC1 in a total volume of 1.0 ml. After a 15-min incubation, 0.025 ml of [3H]ouabain solution (carrier-free, 2.8 pM) was added yielding a final labelled ouabain concentration of 68 nM. The [3H]ouabain binding reaction was terminated 1.5 min later by the addition of 5 ml of an icecold solution containing 15 mM KCI and 1 mM nonlabelled ouabain. KC1 was added to stabilize the [3H]ouabain--enzyme complex (Akera and Brody, 1971). The mixture was immediately placed on a nitrocellulose filter (Millipore Corporation, Bedford, Massachusetts; type AA; pore size, 0.8 pm) and filtered under vacuum. The filter was rinsed twice with 15 ml of an ice-cold solution containing 15 mM KC1 and 1 mM nonlabelled otmbain. The radioactivity trapped on the filter (bound ouabain) was assayed using liquid scintillation counting after dissolving the filter in 1.0 ml of ethylene glycol monomethyl ether (Piersolve®, Pierce Chemical Company, Rockford, Illinois). Counting efficiency (approximately 27%) was monitored using external standard channel ratio method. Digoxin and digoxigenin were dissolved in 5 v/v % ethanol. The final concentration of ethanol in the incubation mixture was 0.5 v/v %. The same concentration of ethanol was added to the control incubation mixture. This concentration of ethanol failed to significantly affect the saturable [SH] ouabain binding. Fractional occupancy of the steroid binding sites on Na÷,K÷-ATPase in cardiac homogenates by either digoxin or digoxigenin was calculated from the reduction in saturable [SH]ouabain binding in the presence of these agents compared to that in their absence. Our previous study (Ku et al., 1974) indicates that the 1.5-min values of [3H]ouabain binding to cardiac homogenates can be used instead of the initial binding velocity
POTASSIUM AND DIGITALIS ACTION for this purpose. Nonspecific binding of [3H]ouabain was assayed concurrently by adding 1 mM nonlabelled ouabain to the incubation mixture. This value was subtracted from the total [3H]ouabain binding to calculate saturable [~H]oubain binding. The protein concentration of homogenates was determined by the method of Lowry et al. (1951).
2.2. Development of the positive inotropic effect Development of the positive inotropic effect of digoxin or digoxigenin was studied in electrically stimulated left atrial preparations of guinea-pig hearts. Left atria were vertically suspended in a modified Krebs-Henseleit solution containing l l 8 m M NaC1, 27.2mM NaHCO3, 1.0mM KH2PO4, 1.2 mM MgSO4, 1.8 mM CaC12, 11.1 mM glucose and either 2.5, 4.8 or 8.5 mM KC1 (final concentration of K*; 3.5, 5.8 or 9.5 mEq/liter) and aerated with a 95% 02/5% CO2 gas mixture at 30°C yielding the pH value of 7.4. The atria were stimulated with platinum field-stimulation electrodes at 1 Hz with square wave pulses of 3 msec duration at a voltage 15% above threshold. Nearly isometric force of contraction was recorded with a force
