W. Klaus

ber die Wirkung von Digitoxigenin auf den cellulren Calcium-Umsatz im Herzmuskelgewebe

In neueren Untersuchungen fiber den Wirkungsmechanismus der Herzglykoside konnte durch Messung der transmembranen K-Bewegungen und der intracellul~ren Ionenkonzentrationen im Herzmuskelgewebe ausgeschlossen werden, dab eine Hemmung des aktiven Ionentransportes bzw. eine Abnahme der intracellul~ren K-Konzentrat ion in einem kausalen Zusammenhang zur positiv inotropen Wirkung dieser Substanzen steht (KLAuS, KUSCttl-NSKY u. Lt~LLMANN 1962a, b). Ein solcher Meehanismus war auf Grund zahlreicher Befunde an kardialem und extrakardialem Gewebe lange Zeit fiir wahrscheinlich gehalten worden (Literatur siehe bei HAJDU U. LEOTARD 1959 und KLAUS, Kvscn-msxY u. LffLLMAN~ 1962a), er scheint jedoch nur fiir das Erscheinungsbild der Glykosidintoxikation yon Bedeutung zu sein. Aus Messungen der intracelluli~ren Ca-Konzentration im Herzmuskelgewebe unter dem Einflul~ versehiedener Herzglykosidkonzentrationen (LEE, Yu, LEE u. Btm~STEn~ 1961; KLAVS, KUSCm-NSK¥ u. LfTLLMA~ 1962a) ergaben sich jedoch tLinweise auf einen m6glichen Angritfspunkt dieser Substanzen am cellul~tren Ca-Stoffwechsel. Ubereinstimmend wurde n/~mlieh eine geringe Verminderung des intraeellul~ren Ca-Gehal~es bei Anwendung therapeu~ischer Glykosidkonzentrationen und eine leichte Zunahme bei Verwendung toxiseher Dosen gefunden. Dieses Verhalten der intracellul/iren Ca-Konzentration kSnnte in einen engeren Zusammenhang zum eigentliehen ~Virkungsmeehanismus der Herzglykoside gebraeht werden als die Kund NaVerschiebungen bei Verabreiehung toxiseher Dosen, da bereits seit den Untersuehungen yon Rn~GwR (1882/83) die grundlegende Bedeutung der Ca-Ionen ffir die Kontraktionskraft des Herzmuskels bekannt ist und aul~erdem durch KONSCHECG (1913), CLARK (1915) und LOEW~ (1918) bereits auf die Abhi~ngigkeit der Glykosidwirkung yon der Ca-Konzentration hingewiesen wurde. Obwohl diese synergistische Weehselwirkung

ber den Zusammenhang zwischen positiv inotroper Wirkung von Digitoxigenin, Kaliumflux und intracellulren Ionenkonzentrationen im Herzmuskel

ZusammenfassungDer wirkungsmechanismus der Herzglykoside wird von einer Reihe von Untersuchern über eine Hemmung des aktiven Ionentransportes, bzw. eine Veränderung der intracellulären Kalium- und Natriumkonzentration, erklärt. Hierdurch würden Bedingungen geschaffen, die einen positiv inotropen Effekt zur Folge hätten.Zur Prüfung dieser Vorstellung wurde an isolierten, elektrisch gereizten Meerschweinchenvorhöfen die Wirkung verschiedener Digitoxigeninkonzentrationen auf das Mechanogramm, den K-Influx und -Efflux, die intracellulären K-, Na-, Ca-, Cl-Konzentrationen und den Wassergehalt untersucht. Es wurden vorwiegend zwei Digitoxigeninkonzentrationen geprüft: 3 · 10−7 g/ml, nur positiv inotrop wirkend (= „therapeutische“ Konzentration) und 10−6 g/ml, kontrakturerzeugend (= „toxische“ Konzentration).Die Kontrollvorhöfe zeigten während der zweistündigen Versuchsperiode keine Veränderungen der intracellulären K-, Na- und Ca-Konzentrationen. Ebenso blieb der K-Influx konstant, während der K-Efflux allmählich abnahm. Die Cl-Konzentration stieg leicht an, der Wassergehalt blieb unverändert.Die therapeutische Digitoxigeninkonzentration hatte keinen Einfluß auf den K-Efflux, der K-Influx wurde vorübergehend sogar gesteigert. Eine Veränderung der intracellulären K-Konzentration ließ sich jedoch nicht feststellen. Die intracelluläre Na- und Ca-Konzentration wurde deutlich erniedrigt.Die toxische Digitoxigeninkonzentration hemmte den K-Influx beträchtlich und steigerte den K-Efflux auf etwa das Doppelte. Dadurch wurde der intracelluläre K-Gehalt stark vermindert, während der Na-Gehalt entsprechend anstieg. Der Ca-Gehalt schien leicht gesteigert zu sein. Vorübergehend wurden während der positiv inotropen Phase der toxischen Digitoxigeninkonzentration gleiche Effekte auf die transmembranen K-Bewegungen und die intracellulären Ionenkonzentrationen beobachtet wie bei der therapeutischen Konzentration während der gesamten Einwirkungszeit (gesteigerter K-Influx, verminderte Na- und Ca-Konzentration). Die Cl-Konzentration und der Wassergehalt wurden durch die verschiedenen Digitoxigeninkonzentrationen nicht beeinflußt.Aus den Befunden wird geschlossen, daß die positiv inotrope Wirkung des Digitoxigenins nicht auf eine Hemmung des aktiven Ionentransportes bzw. eine Verminderung des intracellulären K-Gehaltes zurückzuführen ist. Diese Effekte charakterisieren vielmehr nur die toxischen Erscheinungen bei anwendung dieser Substanz. Zur Klärung der unter dem Einfluß therapeutischer Digitoxigeninkonzentrationen erfolgten Verminderung des intracellulären Na- und Ca-Gehaltes sind weitere Untersuchungen erforderlich.SummaryOne of the common conceptions concerning the mode of action of the heart glycosides considers that these substances act through inhibition of the active ion transport, producing a decrease in intracellular potassium and an increase in intracellular sodium concentrations. It is assumed that, thereby, conditions are created which improve the contraction of the heart muscle.This theory was tested by measuring the action of a “therapeutic” concentration (3 · 10−7 g/ml, producing only a positive inotropic effect) and a “toxic” concentration (10−6 g/ml, producing contracture) of digitoxigenin on isolated electrically stimulated guinea pig auricles. The mechanical activity, K influx and efflux, intracellular K, Na, Ca and Cl concentrations and the water content were determined.During control experiments, the intracellular K, Na and Ca concentrations remained constant. The K influx was unchanged, while the K efflux gradually decreased. The Cl concentration increased slightly, and the water content was unaltered.Therapeutic digitoxigenin concentrations had no effect on the K efflux. The K influx was initially increased, but an increase in the intracellular K concentration could not be measured. The intracellular Na and Ca concentrations decreased markedly.Toxic digitoxigenin concentrations produced substantial inhibition in K influx, and a twofold increase in K efflux. The intracellular K concentration was thereby reduced, while the Na concentration increased correspondingly. The Ca concentration seemed to be increased. During the initial positive inotropic phase of the toxic digitoxigenin effect changes in transmembrane K movements and intracellular ion concentrations identical to those occuring after therapeutic concentrations were observed (increased K influx, decreased Na and Ca concentrations). The Cl concentration and the water content were not influenced by the variou digitoxigenin concentrations.The results presented here indicate that the therapeutic, positive inotropic effect of digitoxigenin cannot be due to an alteration in the intracellular K concentration or to an influence on transmembrane K exchange. Such changes occur only after application of toxic digitoxigenin concentrations, and are most probably caused by inhibition of active ion transport and an increase in membrane permeability. The signifigance of the decrease in Na and Ca concentrations after thereapeutic digitoxigenin doses can only be determined after closer investigation with measurement of the Na and Ca fluxes under these conditions.

Fluorometrische bestimmung von mikromengen calcium in muskelgewebe

A method for fluorometric estimation of micro-amounts of calcium (about 10 nEq./sample) in biological materials using calcein as indicator. Its applicability to heart muscle specimens (6–100 mg wet weight) and its limitation by interfering substances are described.

Über die Wirkung von Prostaglandin E1 auf den Ca-Haushalt isolierter Meerschweinchenherzen

The stimulatory effect of PGE1 on different functions of isolated guinea-pig hearts (Langendorff method, Tyrode solution) was coupled with an increase in the rate of45Ca uptake from the perfusion medium. The total myocardial Ca content and the amount of exchangeable cellular Ca were not affected. This action of PGE1 on the myocardial Ca metabolism seems to be related to the positive inotropic action of PGE1 and can most probably be explained by an increase in the membrane permeability to Ca ions (similar to the action of epinephrine).

Über die Wirkung von Strophanthidin-3-bromacetat am Papillarmuskel des Meerschweinchens

SummaryIn experiments on isolated, electrically stimulated papillary muscles or auricles of guinea pig hearts some characteristics of the inotropic action of strophanthidin-3-bromaocetate (SBA), a derivative of k-strophanthidin which is supposed to be an irreversible inhibitor of the (Na+-K+)-activated ATPase, were studied and compared with the behaviour of k-strophanthidin and digitoxin.The dose-response curves were shifted to higher concentrations in the following order: digitoxin < k-strophanthidin < SBA, (ED50: 3.2×10−7M; 3.5×10−6M; 9.1×10−6M), but with each of these substances the same maximum effect could be obtained.To test the reversibility of the observed pharmacological effects the time course of the changes in contractility was followed in washout experiments. Digitoxin showed the slowest rate of decline (t 1/2≈30 min), k-strophanthidin the most rapid (t 1/2≈3.5 min), and SBA had an intermediate position due to a biphasic washout curve which showed that 70% of the inotropic effect was eliminated with a halftime of about 2 min (similar to k-strophanthidin) and 30% with a halftime of about 20 min (similar to digitoxin).A similar behaviour was observed in the rate of development of the positive inotropic effects. The halftime of this process was around 1 min with k-strophanthidin, and 5 min with digitoxin, whereas the effect of SBA showed again a biphasic time course: 30% of the positive inotropic effect developed with a t1/2 of about 3 min and 70% with at1/2 of about 20 min.Due to the reversibility of the pharmacological action of SBA it has been concluded that (a) either the inotropic action of this substance is not mediated by an inhibition of the (Na+-K+)-activated ATPase or (b) that this enzyme is not inhibited irreversibly by SBA under the prevailing conditions. The biphasic time course of the SBA action is discussed tentatively on the assumption of two specific receptors with different affinities for this drug.

Kinetische Analyse der Calcium-Kompartimente im Meerschweinchenherzen unter Kontrollbedingungen und Strophanthineinwirkung

SummaryA quantitative analysis of myocardial Ca-metabolism was carried out on isolated, isovolumetric (10 ml/min) perfused guinea pig hearts by combined determinations of the total Ca-content and kinetics of 45Ca-efflux (collecting period 60 min) (Fig. 1). The kinetics of 45Ca-uptake was estimated by extrapolating 45Ca-efflux curves of heart muscles isotopically loaded for different times (2, 5, 10, 30 60 min) to the end of the loading-period (Fig. 2).From the specific activity it was possible to express the absolute Ca-content of the extrapolated compartments in μM Ca/g w.w. The amount of activity in cannules and coronary vessels was estimated by dextranblue (MW about 2 millions).The results indicate the presence of three kinetically defined phases (K1, K2, K3) of calcium movements in guinea pig heart muscle. The content of the Ca compartments under equilibrium conditions (60 min 45Ca-loading period) was found to be: K1=0.29±0.05, K2=0.56±0.09, K3=0.30±0.04 μmoles/g w.w.The half-time of Ca movement in these compartments for 45Ca efflux is for K1 15.5±1.0 sec, for K2 2.2±0.1 min, for K3 17.9±0.9 min, and for 45Ca uptake K1<2, K23.9±1.01.1, K39.2±1.82.5 min.Total Ca content has been found to be 1.67±0.06 μmoles/g w.w. Under control conditions about 65% of total heart muscle Ca was exchangeable. Under the influence of ouabain (1.5·10−7 M) Ca of the heart muscle exchanged to about 100%. This was caused by an increase in Ca content of compartment K3 to 0.46±0.06 μmoles/g w.w. and a significant decrease in total Ca-content to 1.32±0.07 μmoles/g w.w. The rate of 45Ca-exchange was not influenced by ouabain. The value of Ca-turnover for Ca-availability in contraction cycle and the mechanism of ouabain action are discussed.

Die Haftung verschiedener Cardenolide am Papillarmuskel und einer mikrosomalen ATPase des Meerschweinchenherzens

SummaryIn experiments on isolated electrically stimulated guinea pig papillary muscles and on isolated cardiac Na+-K+-activated ATPase preparations the action and the reversibility of action of 3 different cardenolides-digitoxin, k-strophanthidin and strophanthidin-3-bromoacetate (SBA) (supposed to be an irreversible inhibitor of the transport ATPase)-were studied.The equieffective concentrations for maximum positive inotropic effects (around 90%) were 2×10−6, 2×10−5 and 4×10−5 M, respectively. In washout experiments the positive inotropic action of all these substances was found to be completely reversible: the rates of decline of the positive inotropic effects were about 2.7%/min with digitoxin, 24%/min with strophanthidin and 22%/min respectivety 5.7%/0/min (two components) with SBA.The equieffective concentrations for maximum inhibition (90–95%) of the Na+-K+-activated ATPase by digitoxin, strophanthidin and SBA were 10−4, 2×10−4 and 10−4 M respectively. In washout experiments (repeated centrifugations) different degrees of reversibility of these inhibitory effects were observed depending upon the experimental conditions. Preincubation of the enzyme with the cardenolides in theabsence of Na+, Mg2+ and ATP resulted in a persisting inhibition of the Na+-K+-ATPase of 14% with digitoxin, 10% with k-strophanthidin and- significantly higher (p < 0.05)-33% with SBA. Corresponding experiments with preincubation of the enzyme in thepresence of Na+, Mg2+ and ATP, however, demonstrated a full reversibility of the inhibitory action of all these substances.These results are in contrast, in certain respects, with those obtained in previous experiments on brain ATPase.It is concluded that SBA is able to inhibit irreversibly only the non-phosphorylated form of the cardiac Na+-K+-activated ATPase, whereas the phosphorylated intermediate of this enzyme seems to be protected against the irreversible inhibition by this substance. Assuming that the latter state of the enzyme is predominant in the intact heart muscle cell, a complete reversibility of the pharmacological action of SBA would be expected if the inotropic effect is mediated by an inhibition of the enzyme. Our results are compatible with this hypothesis.

Calcium als intracelluläre Überträgersubstanz und die mögliche Bedeutung dieses Mechanismus für pharmakologische Wirkungen

ZusammenfassungAuf Grund zahlreicher neuerer Befunde über die Bedeutung von Ca-Ionen für die funktionellen Reaktionen von Muskel-, Nerven- und Drüsengeweben wurde die Vorstellung entwickelt, daß die intracelluläre Ca-Freisetzung bzw. der transmembrane Ca-Einstrom beim Erregungsablauf als wirksames Prinzip des physiologischen Transformationsvorganges von Erregung in spezifische Zellfunktionen zu betrachten ist. Dieser Mechanismus der intracellulären Erregungsübertragung durch Ca-Ionen („elektro-funktionelle Kopplung“) kann zum Angriffspunkt von Pharmaka werden. Die Wirkung der Herzglykoside auf die Kontraktionskraft des Herzmuskels scheint auf eine primäre Beeinflussung des Gleichgewichtes zwischen den verschiedenen cellulären Ca-Fraktionen zurückzuführen zu sein. Unter Berücksichtigung neuerer Befunde der Muskelphysiologie und -morphologie wurde danach ein Schema über den primären Angriffspunkt der Herzglykoside auf cellulärer Basis entworfen.

Über die Abhängigkeit der Strophanthinwirkung auf den myokardialen Sauerstoffverbrauch vom Funktionszustand des Herzens

SummaryOn the basis of some divergent observations concerning the action of cardiac glycosides on the myocardial oxygen consumption, it was suggested that the direction and the magnitude of this effect might be dependent on the experimental conditions and on the functional state of the hearts. Further characterization of this dependence was expected from measurements of the oxygen consumption of isolated guinea pig hearts working under different, well defined hemodynamic conditions. The hearts were perfused with Tyrode solution (37°C, carbogen saturated) and the mean aortic pressure (30 mm Hg) and the stimulation frequency (180/min) were kept constant.In one series of experiments the venous filling pressure was fixed at 10 cm H2O and the end-diastolic pressure, the cardiac output, the coronary flow, and the oxygen consumption were measured. Ouabain (5×10−8g/ml) caused an, increase in cardiac output and a decrease in end-diastolic pressure, but did not significantly influence the coronary flow and the oxygen consumption. This result, however, is not conclusive because of the simultaneous and opposite change of two parameters (cardiac output, end-diastolic pressure) known to be involved in the regulation of the myocardial oxygen consumption.In another series of experiments the cardiac output was kept constant (30 ml/min), in addition to frequency and mean aortic pressure, and the changes in the aforementioned parameters were studied. Under these conditions the external cardiac work remained unchanged, only the end-diastolic pressure varied depending on the contractile ability of the particular heart muscle preparation. In both groups the oxygen consumption was increased with increasing end-diastolic pressure but the values for the ouabain-treated preparations were consistently higher than the corresponding control values at the same end-diastolic pressure. The maximum rate of rise in pressure development (dp/dt) showed an analogue dependence on the enddiastolic pressure. A combined analysis of all these data indicated that the myocardial oxygen consumption under all conditions studied was determined only by the magnitude of the maximum rate of rise of pressure development.Because of the dependence of this parameter on the end-diastolic pressure (besides the direct influence of ouabain) it is conceivable that the effect of ouabain on the myocardial oxygen consumption is dependent on the hemodynamic conditions that is on the degree of sufficiency. Therefore, different effects of ouabain on myocardial oxygen consumption (increase, reduction, no change) may result at different functional states of the myocardium. The possible involvement of other factors determining the myocardial oxygen consumption under the influence of cardiac glycosides is briefly discussed.

Die Reversibilität der Wirkung von Digitoxin, Strophanthidin und Strophanthidin-3-bromazetat am Papillarmuskel und einer mikrosomalen Na+-K+-aktivierbaren ATPase des Meerschweinchens

The reversibility of the inhibitory action of strophanthidin, strophanthidin-3-bromoacetate, and digitoxin on the Na+-K+-ATPase activity (microsomal fraction, guinea-pig brain) was compared with the rate of decline of their positive inotropic effects (papillary muscle, guinea-pig heart) after exposure to drug-free solution. The actions of strophanthidin and digitoxin were easily reduced on both systems. Strophanthidin-3-bromoacetate, however, was found to have an irreversible blocking effect on the transport ATPase, whereas its inotropic action could be rapidly abolished.