Differential effect of R 56865 on ouabain binding to isolated sarcolemma and intact atrial tissue of guinea-pig.

1. R 56865 (N-[1-[4-(4-fluorophenoxy)-butyl]-4-piperidinyl]-N-methyl- 2-benzothiazolamine) is a compound known to antagonize cardiac glycoside intoxication. Therefore, the effect of the compound on ouabain binding to intact cardiac tissue as well as cardiac membrane preparations was investigated. 2. The binding of ouabain to highly purified sarcolemmal membranes was not influenced by R 56865 1 x 10(-6) mol l-1 (ouabain: KD = 1.3 x 10(-7) mol l-1, Bmax = 160 pmol mg-1; ouabain + R 56865: KD = 1.4 x 10(-7) mol l-1, Bmax = 168 pmol mg-1). 3. In contrast to the results in purified membranes, the binding of ouabain (10(-8) mol l-1 to 5 x 10(-7) mol l-1) to intact atria was significantly reduced. 4. Ouabain, 5 x 10(-7) mol l-1, led to a transient positive inotropic effect of about 220% followed by a developing negative inotropic effect after 3 h. R 56865, 10(-7) mol l-1, led to a maximal positive inotropic effect of about 290% also followed by a delayed decline of contractile force. A tenfold higher concentration of R 56865 led to sustained positive inotropic effect of about 250% in the same time interval. 5. The different effects of R 56865 on ouabain binding in subcellular preparations and intact tissue do not support the view that R 56865 interferes directly with the action of ouabain on Na/K-ATPase. An indirect effect, which may be mediated by a lowered intracellular sodium load is discussed.     

Consequences of specific [3H]ouabain binding to guinea pig left atria and cardiac cell membranes

An analysis of [3H]ouabain binding to electrically stimulated, contracting guinea pig left atria gave the following results. (1) A non-linear Scatchard plot with at least two binding sites: a high-affinity site (KD 1.1 X 10(-6) M) with about 430 receptors/micron2 related to positive inotropy, and a low-affinity site (KD' 2.1 X 10(-4) M) with about 18,000 receptors/micron2, possibly related to (Na+ + K+)ATPase inhibition. A crude left atrial homogenate gave about 530 receptors/micron2. (2) Half-maximal positive inotropic effects occurred at about 4 X 10(-7) M. (3) 86Rb+-uptake was significantly increased at all inotropic ouabain concentrations (10(-7) - 10(-6) M). Toxic concentrations (above 2 X 10(-6) M) inhibited 86Rb+-uptake (half-maximal inhibition at about 5 X 10(-6) M). [3H]Ouabain binding to partly purified guinea pig cardiac cell membranes showed: (a) linear Scatchard plots for (Mg2+, Pi)- and (Na+, ATP, Mg2+)-supported binding (KD 1.18 X 10(-7) M and 1.49 X 10(-7) M, respectively); (b) non-linear Scatchard plots for (Tyrode + ATP)-supported binding (KD 4.7 X 10(-7) M; KD' 6 X 10(-6) M); and (c) half-maximal [3H]ouabain binding occurred at a lower concentration (about 3.2 X 10(-7) M) than half-maximal inhibition of (Na+ + K+)ATPase activity (about 7.2 X 10(-7) M). Thus, we conclude that there may be more than one type of ouabain binding site in guinea pig left atria, and that measurable inhibition of (Na+ + K+)ATPase is not necessarily related to positive inotropy in the guinea pig.     

Lack of correlation between [3H]ouabain binding and Na-K ATPase inhibition in rat aorta

The binding of [3H]ouabain to intact strips of rat aorta was compared with the ability of ouabain to inhibit the uptake of 86Rb by the same preparation. When a cold temperature wash was used to process tissues after binding of [3H]ouabain, a class of relatively high affinity binding sites was found (KD = 1.2 X 10(-7) M). Binding was saturable and sensitive to both ATP depletion and elevated potassium. Elevation of cytoplasmic Ca2+ levels by phenylephrine or c-AMP levels by theophylline and terbutaline had no influence on [3H]ouabain binding. Ouabain inhibition of 86Rb uptake progressed to 60% of the total 86Rb uptake at 2 X 10(-3) from a threshold of about 10(-5) M. Half-maximal inhibition by ouabain occurred at a concentration of 10(-4) M. The disparity between [3H]ouabain binding and inhibition of 86Rb uptake indicates that the high affinity binding site in the rat does not contribute to inhibition of Na-K ATPase function.     

Inotropic effects and Na+,K+-ATPase inhibition of ouabain in isolated guinea-pig atria and diaphragm

Effects of ouabain on force of contraction were compared in electrically driven isolated tissue preparations of guinea-pig left atria and diaphragm. A distinct and steady positive inotropic effect of ouabain was observed in atrial preparations, whereas in diaphragm preparations, ouabain produced only a slight and transient positive inotropic effect, followed by the negative inotropic phase. The transient positive inotropic effect of ouabain was observed even in the absence of extracellular calcium, but was markedly dependent on the extracellular sodium concentration. In vitro [3H]ouabain binding studies revealed that the affinity of Na+,K+-ATPase for ouabain was about eight times higher and tissue concentration of the enzyme was significantly lower in diaphragm than in cardiac tissue. The Ki value for ouabain inhibition of the cardiac Na+,K+-ATPase was also approximately ten times higher than for the diaphragm enzyme. Ouabain-sensitive 86Rb uptake, an estimate of sodium pump activity, was inhibited by ouabain at a time when it produced its transient positive inotropic effect in diaphragm preparations. These results indicate that the lack of a distinct and steady positive inotropic effect of ouabain in diaphragm was due neither to the difference in the ouabain-Na+,K+-ATPase interaction between diaphragm and cardiac tissues nor the failure of sodium pump inhibition by ouabain in diaphragm.     

Effect of neuraminidase treatment on the inotropic response to ouabain, isoproterenol and calcium in the guinea pig heart

To determine the role of the glycocalyx sialic acids residues in excitation-contraction coupling and the inotropic response to cardiotonic agents, we studied the effect of neuraminidase treatment on the response to ouabain, isoproterenol, calcium and reduced extracellular sodium in Langendorff preparations of adult guinea pig hearts. Neuraminidase treatment (0.01 unit/ml, 1 h) reduced the magnitude of the positive inotropic response to 2.5 X 10(-7) M ouabain and the maximum response to 5 X 10(-7) M ouabain by about 46% and 30%, respectively, but did not prevent ouabain toxicity. Neuraminidase treatment did not affect the contractility produced by calcium concentration alterations up to 5 mM calcium or the positive inotropic effect produced by lowering external sodium to as low as 80 mM. The inotropic response to as high as 10(-8) M isoproterenol was also not affected. The contractility response developed to calcium concentrations greater than 5 mM and to 5 X 10(-8) M isoproterenol were significantly reduced (P less than 0.05) by neuraminidase treatment. The content of sialic acids in neuraminidase-treated hearts used in the above concentration-response studies of ouabain, isoproterenol, calcium, and sodium was reduced by 70.7%, 66.1%, 65.6% and 66.2%, respectively. Neuraminidase treatment had no effect on basal (Na+ - K+)ATPase and Mg2+ - ATPase activities of (Na+ - K+)ATPase-containing membrane preparations of the guinea pig left ventricle. Neuraminidase treatment neither influenced the sensitivity of the enzyme (Na+ - K+)ATPase to ouabain inhibition nor did it affect the characteristics of [3H]ouabain binding to the preparation. These results suggest that the sialic acids of the glycocalyx in the guinea pig left ventricle play an important role in part of the inotropic response to subtoxic concentrations of ouabain.     

Action of ouabain on rat heart: comparison with its effect on guinea-pig heart.

The inotropic dose-response curve of ouabain in rat cardiac ventricular strips exceeded a concentration range of two decades (1 X 10(-7) M to 3 X 10(-5) M) displaying an intermediate plateau phase. In guinea-pig ventricular strips the inotropic ouabain concentrations spanned only one decade (1 X 10(-7) M-1 X 10(-6) M). Ouabain-intoxication in guinea-pig ventricular strips occurring at 3 X 10(-6) M consisted of arrhythmia and contracture, while in rat ventricular strips at the toxic concentration of 1 X 10(-4) M only a progressive increase in diastolic tension was observed. By means of atomic absorption spectroscopy the ouabain-induced loss of cellular potassium and gain of sodium in rat ventricular strips was detected only at concentrations of ouabain higher than 10(-4) M. Ouabain reduced the activity of Na/K-ATPase prepared from rat and guinea-pig cardiac ventricles to half of its maximum at 6.5 X 10(-5) M in rat and 1.0 X 10(-6) M in guinea-pig, rat heart Na/K-ATPase thus being about 60 fold less sensitive towards ouabain. Specific [3H]-ouabain binding to membrane suspensions prepared from rat and guinea-pig ventricles was characterized by a similar affinity in rat (KD = 4 X 10(-8) M) and guinea-pig (KD = 13 X 10(-8) M). The number of ouabain binding sites in rat membranes was only about 10% of the number found in guinea-pig membranes. In rat the presence of additional ouabain-binding with low affinity and high capacity seemed possible, but could not be verified for methodological reasons. In the light of the biochemical results and binding data, the wider range of ouabain concentration exerting a positive inotropic effect in the rat may be attributed to the existence in the latter of two populations of receptors with different affinities for ouabain and different capacities. In contrast, in the guinea-pig, there is a single population. Nevertheless it is probable that all the receptors in both species are part of the Na/K-ATPase complex and mediate a positive inotropic effect after ouabain-binding in an identical manner.     

Comparison of ouabain receptors in sheep myocardium and Purkinje fibres

The conducting system of the heart has been reported to be more sensitive to the toxic effects of digitalis than the working myocardium. To investigate the molecular basis of these observations, we have characterized the ouabain receptor in Purkinje fibres and ventricular muscle of the digitalis-sensitive sheep heart using cell membrane preparations, crude homogenates and contracting heart tissues. [3H]-Ouabain binding has the following characteristics: in sheep left ventricular cell membranes, specific binding was of high affinity (KD 1.9 X 10(-9) M at 37 degrees); was co-incident with an inhibition of (Na+ + K+)-ATPase activity; and was inhibited by K+ and unlabelled cardiotonic steroids; in crude homogenates, the maximal binding capacity but not the affinity for ouabain varied in different parts of the sheep heart with Purkinje fibres containing markedly fewer binding sites (0.33 X 10(14)/g wet weight; left ventricle, 1.3 X 10(14)/g wet weight) and in isolated, contracting Purkinje fibres and right ventricular moderator band strips, concentration-response curves for [3H]-ouabain binding, increase in force of contraction and inhibition of [86Rb+]-uptake were co-incident. In both contracting tissues, a ouabain concentration of 3 X 10(-7) M occupied about 50% of the specific binding sites, gave the maximal inotropic effect without toxicity and inhibited [86Rb+]-uptake by about 50%. The maximal binding capacity was lower in contracting Purkinje fibres (2 X 10(14) binding sites/g wet weight) than in contracting moderator band strips (3.9 X 10(14) binding sites/g wet weight). The maximal inotropic effects were reached slightly faster in Purkinje fibres but toxicity also occurred faster in these fibers. We conclude that the specific ouabain binding site is the receptor mediating positive inotropy and inhibition of (Na+ + K+)-ATPase in the sheep heart. Further, this receptor is identical in both the conducting system and working myocardium but the conducting system contains many fewer receptors. This change in receptor number, rather than affinity, may underlie the increased ouabain toxicity observed in Purkinje fibres.     

Erythrosin B inhibits high affinity ouabain binding in guinea-pig heart Na+-K+-ATPase without influence on cardiac glycoside induced contractility.

Binding of [3H]-ouabain to guinea-pig heart membranes enriched in Na+-K+-ATPase revealed two different cardiac glycoside binding sites. High affinity binding was obtained at a KD = 2.2 X 10(-7) mol 1(-1) (Bmax = 16.8 pmol ouabain mg-1 protein) whereas low affinity ouabain binding occurred at a KD much greater than 10(-6) mol 1(-1). To discover whether the two ouabain binding sites are functional in guinea-pig heart muscle, erythrosin B, an inhibitor of the high affinity ouabain binding in rat brain tissue, was tested in guinea-pig isolated heart muscle preparations. Erythrosin B proved to be a potent inhibitor of the Mg2+ (Na+)-dependent-, as well as Na+-K+-activated ATPase (ID50 = 9 X 10(-6) mol 1(-1). Contractility of guinea-pig isolated papillary muscles, however, was not influenced by erythrosin B in concentrations up to 1 X 10(-5) mol 1(-1). Only very high concentrations (4 X 10(-4) mol 1(-1) resulted in a slightly negative inotropic effect (about 20%). Erythrosin B dose-dependently inhibited [3H]-ouabain binding to the Na+-K+-ATPase (KD = - 3.6 X 10(-6) mol 1(-1). In a concentration of 1 X 10(-5) mol 1(-1) the dye abolish high affinity [3H]-ouabain binding without affecting the low affinity binding sites. In contrast, in guinea-pig isolated atria, no functional antagonism between erythrosin B (5 X 10(-5) mol 1(-1) and ouabain was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac glycoside receptors in cultured heart cells--II. Characterization of a high affinity and a low affinity binding site in heart muscle cells from neonatal rats

The binding of [3H]ouabain has been studied in (Na+ + K+)-ATPase enriched cardiac cell membranes, as well as in cardiac muscle and non-muscle cells in culture--all obtained from hearts of neonatal rats. The binding has been correlated with ouabain-induced inhibition of (Na+ + K+)-ATPase (cardiac cell membranes) and the inhibition of active (86Rb+ + K+)-influx (cardiac muscle and non-muscle cells in culture). Furthermore, the effect of ouabain on the amplitude of cell-wall motion and contraction velocity has been studied in electrically driven cardiac muscle cells. In muscle and non-muscle cells, two classes of ouabain binding sites have been identified. In rat heart muscle cells, the high affinity binding site has a dissociation constant (KD) of 3.2 X 10(-8) M and a binding capacity (B) of 0.2 pmole/mg protein (80,000 sites/cell); the values for the low affinity binding site are: KD = 7.1 X 10(-6) M; B = 2.6 pmole/mg protein (10(6) sites/cell). The binding to both types of binding sites is depressed by K+ and abolished after heat denaturation of the cells. The kinetics of [3H]ouabain binding to rat heart muscle cells (association and dissociation rate constants, K+- and temperature-dependence of association and dissociation processes) have been characterized. In rat heart muscle and non-muscle cells, the binding of [3H]ouabain to the low affinity site results in inhibition of the (86Rb+ + K+)-influx (EC50 = 1.3 and 1.5 X 10(-5) M ouabain), a decrease in cell-K+ (EC50 = 1.9 and 1.4 X 10(-5) M) and an increase in cell-Na+ (10(-5)-10(-4) M). The ouabain-induced positive inotropic effect (increase in amplitude of cell-wall motion, increase in contraction velocity) in cardiac muscle cells is observed only at ouabain concentrations greater than or equal to 5 X 10(-6) M, and it is therefore probably attributed to occupation of the low affinity binding site. Coupling of occupation of the low affinity site by ouabain with drug-induced inhibition of the sodium pump and with drug-induced positive inotropic action is further substantiated by kinetic measurements. In contrast, occupation of the high affinity binding site does not produce any measurable inhibition of the sodium pump activity or positive inotropy.(ABSTRACT TRUNCATED AT 400 WORDS)     

The inotropic effect of ouabain and its antagonism by dihydroouabain in rat isolated atria and ventricles in relation to specific binding sites.

The inotropic effect of ouabain has been studied in rat ventricles and atria. The concentration-effect curve of ouabain may be fitted by a model assuming the existence of two saturable components. The component with the higher sensitivity to ouabain accounted for 30% of the maximal increase in systolic tension in ventricles and for only 5% in atria. Increase in diastolic tension was only apparent at ouabain concentrations required to observe the low sensitivity component. [3H]-ouabain binding has been examined in microsomes prepared from atria and ventricles. High and low affinity binding sites have been observed. The ratio of high and low affinity ouabain binding sites was 4 fold lower in microsomes from rat atria than from rat ventricles. This could account for the difference in the response of these two tissues to the inotropic action of ouabain. In ventricular strips the high sensitivity component was much less apparent in the presence of dihydroouabain than with ouabain. When ventricular strips were preincubated in the presence of dihydroouabain 3 microM, the increase in systolic tension evoked by ouabain 1 microM was significantly reduced. Cumulative concentration-effect curve studies showed dihydroouabain antagonism to the high sensitivity component.     

Enhanced ouabain sensitivity of the heart and myocardial sodium pump in aged rats

Tolerance to arrhythmogenic actions of digitalis decreases with advanced age. To determine if aged myocardium has reduced glycoside tolerance, ouabain sensitivity and associated biochemical changes were examined in young (3-month), adult (8-month) and aged (26-month old) Fisher 344 rats, because age-related changes in physiology and biochemistry are well characterized in this strain of rats. Although rats are uniquely tolerant to digitalis, basic mechanisms leading to inotropic and toxic actions of digitalis are not different from other species. In aged anesthetized rats, intravenous infusion of ouabain produced arrhythmias and cardiac arrest at lower doses. Aged myocardium had lower Na,K-ATPase activity and fewer high affinity [3H]ouabain binding sites. Affinity of the ouabain binding sites on Na,K-ATPase was not altered. Despite these findings, sodium pump activity, estimated from the ouabain-sensitive 86Rb+ uptake by ventricular muscle slices, was higher in the aged myocardium when the uptake was observed in the absence of ouabain and was more sensitive to inhibitory action of the glycoside. Differences in Na,K-ATPase and sodium pump data may be explained if sodium leak influx is increased in aged myocardium. Fewer sodium pumping sites and enhanced ouabain sensitivity of the sodium pump seem to be responsible for the reduced tolerance of aged heart to digitalis-induced toxicity.     

The effects of strychnine on the regulation of voltage-dependent calcium channels by dihydropyridines in brain and heart.

The effects of strychnine (STR) were investigated on K(+)-stimulated 45Ca2(+)-uptake into mouse brain neurons, the contractile activity of spontaneously beating rat atria and on [3H]nitrendipine and [3H]BAY K 8644 binding to dihydropyridine calcium channel antagonist and agonist binding sites on brain and cardiac membranes. STR (10(-6)-10(-4) M) had no effect on neuronal 45Ca2(+)-uptake. When combined at equimolar concentrations (10(-5) M), STR and nifedipine produced a potent (nM) inhibition (40%) of neuronal 45Ca2(+)-uptake. In the spontaneously beating rat atria, STR produced a dose-dependent (10(-7)-3 x 10(-4) M) decrease in chronotropy but did not affect inotropy. STR (10(-4) M) completely inhibited the positive chronotropic, but did not affect the positive inotropic effects of (-)-S-BAY K 8644. [3H]Nitrendipine and [3H]BAY K 8644 binding to brain and cardiac membranes was enhanced by STR in a concentration-dependent manner (EC50 8 X 10(-6) M). Scatchard analysis revealed that STR increased the affinity (decreased the Kd) of [3H]BAY K 8644 to a greater degree than that of [3H]nitrendipine for dihydropyridine binding sites. STR decreased the Kd of [3H]nitrendipine binding by increasing and decreasing the microassociation and microdissociation constants respectively. STR enhanced [3H]nitrendipine binding to the same extent in the cerebral cortex, striatum, hippocampus, cerebellum, brainstem and spinal cord. The enhancement of [3H]nitrendipine binding in brain was completely inhibited by Ca2+ and partially inhibited by Na+ in a concentration-dependent manner. Glycine (10(-2) M) did not affect the STR enhancement of [3H]nitrendipine binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiotoxic and inotropic effects of ouabain on atria isolated from rabbits of different ages.

1 In sino-atrial node-right atrial preparations and left atrial preparations obtained from rabbits of different ages (2 to 360 days old), cardiotoxic and inotropic effects of ouabain were compared. Tachyarrhythmias were produced in immature and mature rabbit atria exposed for 6 to 8 min to ouabain (5 x 109(-6) M). The total number of contractions before the arrhythmias developed varied inversely with age (2 to 90 days old). Percentage reductions in the resting membrane potential, overshoot and maximum rate of rise induced by ouabain were greater in mature rabbit atria than in immature rabbit atria. 2 Reduction of Ca2+ in the bathing media suppressed the development of tachyarrhythmias. 3 In electrically-driven left atria isolated from immature and mature rabbits, similar frequency-contractile force curves were obtained, maximum contractions being attained at a frequency of 120/min. Ouabain at 2 x 10(-7) M shifted the curve upward and at 10(-6) M greatly potentiated the contractile force developed at low frequencies (6 to 30/min). Increase in contractions induced by ouabain relative to pre-drug concentrations at a frequency of 60/min were greater in immature rabbit atria (15 days old) but the positive inotropic effect of 2 x 10(-7) M ouabain relative to the effect seen at 10(-6) M was appreciably less. 4 It may be concluded that atria isolated from immature rabbits tolerate higher concentrations of ouabain than those isolated from mature rabbits.     

Inotropic action,myocardial uptake and subcellular distribution of ouabain,digoxin and digitoxin in isolated rat hearts

In experiments on isolated, electrically driven (240/min) rat hearts, perfused via the aorta at a constant flow (3.8 ml/min), the pharmacologically effective concentration range, the myocardial uptake and the subcellular distribution of three cardiac glycosides (digitoxin, digoxin, ouabain) were determined. The following results were obtained: 1. The effective range varied depending on the cardiac glycoside tested: With digoxin and ouabain very similar results were found- the positive inotropic concentration ranges being within 8x10(-6)M and 6x10(-5)M, the maximum positive inotropic effects attainable being about 100% and the concentration for half maximum effects (ED-50) being 2.4x10(-5)M and 2.3x10(-5)M, respectively. With digitoxin the inotropic concentration range was found to be within 3.6x10(-6)M and 2.4x10(-5)M with a maximum inotropic effect attainable of about 50% only and an ED-50 of 9.5x10(-6)M. The analysis of the time course of the inotropic action revealed extremely short half times for all cardiac glycosides studied (between 48 and 54 sec). 2. The myocardial uptake correlated with the physicochemical behaviour of the three cardiac glycosides studied and was found-depending on the perfusion time (5 to 60 min)-to be in the range of 23 and 36 (ouabain), 66 and 98 (digoxin) and 169 and 264 (digitoxin) nmoles/g wet weight. The respective computed half times for these uptake processes were 2.5 min (digoxin, ouabain) and 3.4 min )digitoxin). 3. Regarding the subcellular distribution an accumulation exceeding an "unspecific" binding (non-perfused hearts) was found mainly in the nuclear-membrane fraction. On the basis of these results (very short half times of either the pharmacological action and the cardiac uptake) the site of action of cardiac glycosides in the rat heart is supposed to be located at the surface membrane of the heart muscle cells. Furthermore, the above results are discussed with respect to those obtained in digitalis-sensitive species.     

Sodium pump activity and contractile effect of ouabain in human placental veins.

The aim of the present study was to determine the number and affinity of [3H]ouabain binding sites, the sodium pump activity and the mechanisms involved in the contractile effects of ouabain in human placental veins. Scatchard analysis suggested the existence of a single population of binding sites with a KD of 196.7 nM and a Bmax of 1606 fmol/mg protein. The sodium pump activity was determined from the 86Rb+ uptake, which was reduced concentration dependently by ouabain (10(-8)-10(-4) M), and from the K+ (7.5 mM)-induced relaxation in veins preincubated in a K(+)-free medium and precontracted with PGF2 alpha (10(-6) M), which was also blocked by the glycoside (10(-6) M). Ouabain (10(-7)-10(-4) M) induced concentration-dependent contractions, which were not modified by either nifedipine or Bay K 8644 (10(-7) and 10(-6) M). Ca2+ omission from the medium or amiloride (10(-4) M) inhibited these contractions, whereas monensin (10(-6) M) potentiated them. These data indicate that human placental veins possess sodium pump activity and that its inhibition by ouabain induces potent contractions mainly mediated by Ca2+ entry through the Na(+)-Ca2+ exchange system.     

Studies on the mechanism of the positive inotropic effect of ATX II (Anemonia sulcata) on isolated guinea pig atria

The basic polypeptide ATX II (MW 4,770) isolated from the sea anemone Anemonia sulcata evokes a pronounced and dose-dependent positive inotropic effect in different mammalian heart preparations. The mechanism of this effect is so far unknown. (a) Investigations on isolated guinea pig atria indicate that changes of the steady state cellular Na, K and Ca concentrations cannot account for the positive inotropic effect. (b) An increase of the surface pressure of phospholipid monolayers was observed only at cardiotoxic ATX II concentrations. However, the 45Ca binding to phosphatidylserine, as the essential Ca-binding phospholipid, was not changed even at cardiotoxic ATX II concentrations. (c) Neither the enzymatic activity nor the ouabain inhibition kinetic of an isolated Na/K-ATPase preparation was affected by ATX II. (d) In intact electrically stimulated (1 Hz) guinea pig atria the binding of [3H]ouabain increases by about 50% at a positive inotropic ATX II concentration. The results suggest that the positive inotropic effect of ATX II is not caused by an unspecific membrane damaging action or by a direct interaction with the Na/K-ATPase. The increased binding of [3H]ouabain to intact heart muscles indirectly reflects an increased pump activity of the Na/K-ATPase, which is caused by an elevated Na transient due to the electrophysiologically well-established mechanism of the ATX II action on fast Na channel, i.e., delayed inactivation of the fast Na flux. However, the exact mechanism of the ATX II induced positive inotropic effect remains unknown.     

Digitalis structure-activity relationship analyses: Conclusions from indirect binding studies with cardiac (Na+ + K+)-ATPase

We have performed direct and indirect binding studies with [³H]ouabain or [³H]digitoxin on beef or guinea pig cardiac (Na⁺ + K⁺)-ATPase to measure the potencies of a broad range of cardiotonic steroids for structure-activity relationship (SAR) studies for comparison with previously determined positive inotropic potencies. The positive inotropic potencies of twelve compounds on contracting guinea pig left atria correlated well with the equilibrium dissociation constants (K_D values) from the inhibition of [³H]ouabain binding to guinea pig cardiac (Na⁺ + K⁺)-ATPase (r = 0.98 for seven 5β-compounds, r = 0.95 for five 5α-compounds). Further we calculated K_D values from the inhibition of [³H]ouabain binding data for a total of 33 digitalis derivatives on the digitalis-sensitive beef cardiac (Na⁺ + K⁺)-ATPase. For the 27 compounds tested on both beef cardiac (Na⁺ + K⁺)-ATPase and guinea pig left atria, the potencies showed a significant correlation (r = 0.92 for 22 5β-compounds, r = 0.96 for five 5/ga-compounds. For seven compounds, K_D values were measured on beef cardiac (Na⁺ + K⁺)-ATPase using inhibition of binding of [³H]digitoxin. These values correlated well (r = 0.99) with the K_D values from the [³H]ouabain studies.These results show that: (1) The significant correlation observed between K_D values on guinea pig cardiac (Na⁺ + K⁺)-ATPase and positive inotropic potency in guinea pig left atria is further evidence that the pharmacological receptor for inotropy is part of the enzyme, (2) Inhibition of the binding of [³H]ouabain or [³H]digitoxin can be used to determine the relative potencies of unlabelled digitalis derivatives. The similar relative potencies on beef and guinea pig cardiac (Na⁺ + K⁺)-ATPase of a broad range of digitalis derivatives indicate that the binding site is similar for both species; and (3) SAR studies indicate that functional groups on these steroids have the same influence on potency on either the positive inotropy or cardiac (Na⁺ + K⁺)-ATPase studies.     

Effects of androgens and antiandrogens on the inotropism induced by ouabain and isoproterenol on the left atrium of the rat in vitro.

1. The effect of androgens 5 beta- and 5 alpha-dihydrotestosterone (DHT, 10(-9) M), and the antiandrogens cyproterone acetate (CPA, 10(-8)-10(-6) M), chlormadinone acetate (CMA, 10(-8)-10(-6) M), medroxyprogesterone acetate (MPA, 10(-8)-10(-6) M), spironolactone (SPI, 10(-5) M), flutamide (F, 10(-5) M) and cimetidine (C, 10(-5) M), on inotropic positive effect induced by ouabain (10(-8)-10(-5) M) and isoproterenol (10(-8)-10(-6) M), on electrically stimulated left atria of rat, has been assayed. 2. Ouabain (10(-6) M) did not modify the inotropic effect of isoproterenol (10(-8)-10(-6) M). 3. The androgens 5 beta- and 5 alpha-DHT (10(-9) M) and the antiandrogens SPI (10(-6) M), F (10(-5) M) and C (10(-5) M) inhibit the inotropic effect of ouabain and isoproterenol on electrically stimulated left atria of the rat. 4. The antiandrogens CPA, MPA and CMA to 10(-7) M, inhibit the inotropic effect of ouabain. The CPA (10(-8)-10(-6) M) inhibit, in a dose-dependent way the positive inotropic effect of isoproterenol. MPA and CMA (10(-8)-10(-6) M) also inhibit the inotropic effect isoproterenol but the inhibitory effect is greater with 10(-8) M than 10(-6) M of both drugs. 5. Taken together, our results suggest that steroidal hormones could modulate the cardiac contractility through interference with Na-pump in a non-digitalic site and/or with intracellular mediators in left atrium.     

Sodium load and high affinity ouabain binding in rat and guinea-pig cardiac tissue.

An estimation of the actual Na/K-ATPase transport activity in intact cardiac cells was made by measuring the binding of [3H]-ouabain to rat and guinea-pig ventricular strips. At the low [3H]-ouabain concentration of 1 nM equilibrium binding was hardly obtained after an incubation time of five hours. Different procedures known to alter the sodium load of the cardiac preparations influenced [3H]-ouabain binding: the sodium ionophore monensin enhanced [3H]-ouabain binding, the local anaesthetic dibucaine and a reduction of external sodium ion concentration diminished [3H]-ouabain binding; [3H]-ouabain binding was similarly affected by these procedures in the rat and guinea-pig. Since [3H]-ouabain binding occurred predominantly at the high-affinity binding sites of rat myocardium under the applied experimental conditions, it was concluded that these binding sites represent Na/K-ATPase molecules involved in sodium ion transport.     

Role of Na-H exchange in the inotropic action of Bay K 8644 and of ouabain in guinea-pig isolated atria.

1. The inotropic effects of two concentrations of ouabain and of Bay K 8644 have been studied in isolated left atria of the guinea-pig in physiological solutions at pH lowered from 7.4 to 6.0 and in the presence of ethylisopropylamiloride (EIPA) an inhibitor of Na+/H+ exchange. The low concentration of ouabain (300 nM) was chosen to saturate the high affinity binding sites (it occupied about 7% of the low affinity sites). The high concentration of ouabain saturated both high and low binding sites. Bay K 8644 evoked a positive inotropic effect of a magnitude similar to ouabain (300 nM). 2. When comparing the positive inotropic effects of equi-effective concentrations of ouabain (300 nM) and of Bay K 8644 (100 nM), it was observed that extracellular acidification specifically depressed the inotropic effect of ouabain 300 nM; the positive inotropic effect of the high concentration of ouabain (3 microM) was barely affected by extracellular acidification. 3. EIPA 10 microM depressed the positive inotropic effect of ouabain 300 nM, but did not affect the peak response to Bay K 8644. The depressant action of EIPA on the positive inotropic effect of ouabain was concentration-dependent and was much more obvious on the effect of ouabain 300 nm than on ouabain 3 microM. 4. An increase in diastolic tension was evoked by 3 microM but not by 300 nM ouabain. This increase in tone was reduced dose-dependently by EIPA (10-30 microM). It was also significantly reduced when the extracellular pH was equal to 6.4 or 6.0. 5. Ouabain (300 nM) evoked a gain in tissue Na and an equivalent loss in tissue K.(ABSTRACT TRUNCATED AT 250 WORDS)