Thies Peters

INTERDEPENDENCE OF ION TRANSPORT AND THE ACTION OF OUABAIN IN HEART MUSCLE

1 The influence of ouabain (0.4 μM) on contractile force and cellular Na and K concentrations was investigated in isolated left atria of the guinea‐pig at rest and at different beat frequencies. Simultaneously the binding of ouabain to the tissue was determined. 2 Strict dependence of rates of onset of positive iontropic action and of binding of ouabain on beat frequency are limited to conditions where no alterations of cellular Na and K concentrations occur. A correlation was observed between sodium flux per unit time and the development of positive inotropism and binding to the receptors of ouabain. 3 Ouabain exerts its positive inotropic effect without affecting the intracellular Na and K concentrations in spite of the fact that under these conditions even the majority of binding sites, i.e. Na‐K‐adenosine triphosphatases (Na‐K‐ATPases), are occupied by the drug. The positive inotropic effect may be explained by a ouabain‐induced conformational alteration of the Na‐K‐ATPase which leads to structural alterations of the plasmalemma connected with an increased availability of coupling calcium. 4 Increasing the frequency of stimulation over a critical value, which appears to be determined by an overloading of the Na pump, induces a decrease in contractile force, cellular accumulation of Na and loss of K, and eventually contracture. 5 The rate of binding of ouabain appears to depend on the actual concentration of particular conformations of the Na‐K‐ATPase with high affinity for ouabain. These conformations transiently occur during a pumping cycle and their concentration may therefore be dependent on the frequency of cycling which in turn is determined by the frequency of contraction. 6 Ouabain can easily be washed out from the tissue irrespective of the condition of the muscle. If, however, the intracellular Na and K homeostasis is impaired, the inhibition of the pump persists even if ouabain is released from the binding sites upon wash‐out. It is suggested that the inhibition of the pump is maintained by an increased intracellular Ca ion concentration and a depletion of ATP. 7 A kinetic model is proposed for the interaction between cardiac glycosides and the Na‐K‐ATPase in intact heart muscle cells.

PLASMALEMMAL CALCIUM IN CARDIAC EXCITATION-CONTRACTION COUPLING

1. Mammalian heart muscle is extremely sensitive to the external calcium concentration. It reacts to alterations of the external calcium concentration with an immediate adaptation of contractile force.

Evidence for a dissociation between positive inotropic effect and inhibition of the Na+N+-ATPase by ouabain, cassaine and their alkylating derivatives

An attempt was made to gain further information on the correlation postulated between NaK-ATPase activity and the contractile state of heart muscle. The efficiency of a new alkylating agent, cassic acid mustard (CAM) on contractile force and on the NaK-ATPase was compared with cassaine (C) and ouabain (S). The ED50 valuess obtained in isolated guinea-pig atria were (× 10−7 M): C 0.9, CAM 2.7 and S 1.1; the ID50 values resulting from ATPase inhibition experiments were (× 10−6 M): C 1.0, CAM 24, and S 0.25. Low concentrations of all three drugs stimulated the ATPase activity up to 20%. The time course of stimulation is characterized by a half-life of 5 min at a concentration of 1 × 10−0 M ouabain. A considerable discrepancy exists between the time courses of onset of the positive inotropic effect and the development of enzyme inhibition. This is most obvious in the case of ouabain: a drug concentration of 1 × 10−7 M produces a positive inotropic equilibrium within 30 min, whereas the inhibition of the enzyme still continues after a period of more than 60 min. A positive inotropic effect of 80% can be washed out a half lives of 8.3 min for CAM, 2.8 min for C and 4.4 min for S. In contrast to this ready reversibility of the positive inotropic effect, the enzyme inhibition by S is not reversible and persists for more than 30 min upon dilution of the drug-enzyme complex. The data obtained are consistent with the interpretation, that the Na+K+− ATPase is not the mediator of the positive inotropic action of digitalis and related compounds. It is much more tempting to correlate ATPase inhibition with the toxic effects of these compounds.

The significance of a fast exchanging superficial calcium fraction for the regulation of contractile force in heart muscle

Abstract The force of contraction of isolated guinea-pig atria decreased with time during 5 h of incubation in Tyrode solution containing 0.9 m m -CaCl2. The relative effect of ouabain strongly depended on the duration of incubation (“age”) of the tissue. Freshly dissected atria showed a 12% increase in contractile force, whereas aged atria (5 h of incubation) produced a 240% increase in tension in the presence of ouabain (1.5 × 10−7 m ). In freshly dissected atrial preparations, after an equilibration period of 30 mins, ouabain had no influence on Ca metabolism (total tissue Ca, rate of exchange, and size of exchangeable fraction). After aging of the tissue the rate of Ca exchange was significantly decreased, and ouabain considerably accelerated the Ca exchange rate. The presence of pentobarbital also slowed the Ca exchange rate. Ouabain was able completely to restore the rate of Ca exchange to control valves. Analysis of the Ca exchange curves yielded a fast and a slowly exchanging Ca compartment. The size of the fast exchanging fraction was significantly reduced by aging and by the treatment with pentobarbital. Under these conditions ouabain enhanced the size of this particular Ca compartment. It is suggested that the fast exchanging Ca compartment is superficially located and is responsible for rapid changes in contractile force. This Ca fraction may provide a Ca concentration of about 1.5 × 10−5 m in the intracellular aqueous space, which is generally believed to be sufficient for the activation of contraction.

Absorption rates of some cardiac glycosides and portal blood flow

Abstract In guinea pigs, 3 H-labelled cardiac glycosides were infused intraduodenally and the absorption rates estimated. The relative rates for digitoxin, digoxin and ouabain amounted to 100:43:2.5. The absorption rates were highly dependent on intestinal blood flow. At a blood flow of 20 ml/min, the maximum absorption rate for digitoxin was calculated to be 65% and for digoxin 36% of the dose administered per hr. For ouabain this value could not be determined due to poor absorption. Within 1 hr, about 30% of the absorbed digitoxin was eliminated with the bile but only 1% with the urine. The corresponding figures for digoxin were 15% and 1.5% respectively. Metabolites were present in gut content after administration of digitoxin and digoxin although the bile was quantitatively collected. This finding suggests a participation of intestinal tissue in the metabolic degradation of digitalis glycosides.

The cardioactivity of digitoxin metabolites

Abstract Using isolated atria, the positive inotropic action of digitoxin and digoxin was compared quantitatively with the cardiac action of metabolites found in guinea pigs. The bis- and monodigitoxoside of digitoxigenin and the monodigitoxoside of digoxigenin showed the strongest inotropic effect with respect to the affinity as well as the maximum response. The corresponding conjugation products did not alter the contraction amplitude.

Influence of ouabain and dihydroouabain on the circular dichroism of cardiac plasmalemmal microsomes

Summary1.The influence of ouabain on the tertiary structure of cardiac plasmalemmal proteins was investigated by means of circular dichroism measurement. Purified plasmalemmal microsomes were obtained by sucrose gradient centrifugation. The CD-spectra of the membranal proteins were shifted to the red and the amplitudes were smaller than those of the same proteins after solubilization.2.Ouabain induced an increase of the ellipticity bands at 210 and 222 nm of about 50% above the level yielded with microsomes after sonication. At 222 nm ouabain exhibited the half maximum effect at a concentration of 5×10−9 M. The effect could, however, only be exerted if the inside of the microsomes was exposed to ouabain by sonication, thus reflecting the inside-out nature of the plasmalemmal microsomes.3.The high specificity of the ouabain effect was underlined by the following experiments: a) Dihydroouabain, a much less cardioactive derivative of ouabain proved to be ineffective in corresponding concentrations, b) ouabain had no influence upon the CD spectrum of microsomes derived from cardiac sarcoplasmic reticulum, c) a detergent-like action of ouabain underlying the observed effect can be excluded since highly active tensides, i.e. desoxycholate and dodecylsulfate, only influence the CD spectra at concentrations exceeding 10−3 M, d) electronmicrographs of microsomes exposed to ouabain demonstrated no alteration of either the appearance or size of the microsomes.4.The magnitude of the observed ouabain effect indicates that a large portion of the membrane-bound proteins is involved. The number of binding sites and their isolated structural alteration induced by ouabain are not sufficient to account quantitatively for the enhanced amplitudes of the CD spectra. This suggests that ouabain evokes structural changes of membrane proteins different from actual binding sites. It seems, therefor highly improbable that changes of the Na-K-ATPase present in the plasmalemmal microsomes are responsible for the observed effect.

STUDIES ON THE KINETICS OF [3 H]-OUABAIN UPTAKE AND EXCHANGE IN THE ISOLATED PAPILLARY MUSCLE OF THE GUINEA-PIG

1 The uptake, wash‐out and exchange of [3 H]‐ouabain was studied in isolated, resting, and electrically stimulated papillary muscles of the guinea‐pig. 2 At the equilibrium level of uptake, a different tissue/medium ratio was obtained for each of the concentrations used, i.e. 3.4, 1.8 and 0.82 for 1 × 10−7 M, 7.7 × 10−7 M, and 5 × 10−6 M ouabain, respectively. Equilibrium was reached more rapidly at high concentrations of ouabain. 3 The maximum number of binding sites for ouabain was estimated to be 1 × 1015 binding sites/g wet weight. 4 No difference in [3 H]‐ouabain uptake could be detected between resting and electrically stimulated papillary muscles. 5 The kinetics of the ouabain uptake, wash‐out and exchange are discussed. The results suggest that there is a saturable compartment in papillary muscle which can best be demonstrated if low concentrations of ouabain are used. Because of its small size, the saturable compartment submerges in the process of the filling of the extracellular space at high concentrations (5 × 10−6 M).

Influence of pH on the action of parasympathomimetic drugs

Abstract 1. 1. The influence of p H on the activity of tertiary parasympathomimetic drugs was investigated using guinea pig isolated atria and ileum segments. Carbachol and arecaidine methyl ester methiodide, which are both quaternary cholinomimetics, were used as reference compounds. 2. 2. The responses of the organs used in our experiments were unchanged within the p H ranges 6.2–8.4 (ileum) and 6.3–8.8 (atria). The p K values of the tertiary parasympathomimetic drugs investigated also lay within these ranges. 3. 3. The tertiary parasympathomimetic compounds had agonist activity only in the protonated form. Therefore, the apparent p D 2 value (at any particular p H) and the ‘true’ p D 2 value (when the degree of protonation is maximal) should be distinguished. 4. 4. The slope of the concentration-response curves was reduced for both tertiary and quaternary parasympathomimetic drugs as the OH − ion concentration was increased. 5. 5. In contrast to the behaviour of the quaternary compounds, the intrinsic activity of the tertiary parasympathomimetic drugs increased when the OH − concentration was raised. The intrinsic activity of the tertiary compounds may be even higher than that of the quaternized drugs. 6. 6. The p H value of the medium not only determined the degree of protonation of the tertiary agonists, but also influenced the slope of the concentration-response curves and the intrinsic activity.

Kinetic events determining the effects of cardiac glycosides

Abstract Drugs pose kinetic problems on three different levels: (1) pharmacokinetics (dealing with drug disposition), (2) receptor kinetics, and (3) transformation kinetics (describing the transformation of the drug-receptor interaction into effects). The events occurring on these three levels have a highly complex interdependence. Receptor and transformation kinetics of naturally occurring cardiac glycosides and their semisynthetic congeners deserve more attention in view of their therapeutic importance.