H. Scholz

Stimulatory effects of DB-c-AMP and adrenaline on myocardial contraction and 45Ca exchange. Experiments at reduced calcium concentration and low frequencies of stimulation

SummaryThe effects of adrenaline (2.2×10−6 M) and cyclic N6-2′-O-dibutyryl-adenosine-3′,5′-monophosphate (DB-c-AMP; 10−3 M) on mechanical performance, 45Ca uptake and total tissue calcium concentration were investigated in electrically stimulated left auricles isolated from female rats weighing 180–220 g. The experiments were performed at reduced [Ca]e of 0.45 mM and at various frequencies of stimulation (0–120 beats/min). In the first series of experiments 45Ca incubation time was 5 min. Under these conditions DB-c-AMP as well as adrenaline enhanced contractile force to 300–450% of the control values at all frequencies tested (Fig.1). This increase in contractile force was accompanied by a significant enhancement in 45Ca exchange (Fig.2) while the total tissue calcium concentration remained unchanged (Table 1). In resting auricles 45Ca exchange was not altered under the influence of both drugs.At long periods of 45Ca exposure (40–90 min) both drugs augmented contractile force in a way similar to that of the first series of experiments (Fig.4) but no influence of DB-c-AMP or adrenaline on 45Ca exchange could be detected (Fig.3).It is concluded that DB-c-AMP can mimic the wellknown effects of adrenaline on myocardial calcium movements. Under the assumption that DB-c-AMP is representative for c-AMP, the results thus provide experimental support for the view that the positive inotropic effect of adrenaline is mediated by primary changes in the intracellular level of c-AMP which secondarily might enhance calcium fluxes across the cardiac cell membrane.

Possible role of cyclic AMP in the relaxation process of mammalian heart: effects of dibutyryl cyclic AMP and theophylline on potassium contractures in cat papillary muscles.

SummaryThe effect of dibutyryl cyclic AMP (DB-c-AMP; 3×10−4–3×10−3 M) on electrically induced twitch and high potassium (142.4 mM KCl)-induced contracture tension was studied in papillary muscles from normal and reserpinized cats ([Ca]0 1.8 mM; 25°C; pH 7.4). In both groups of preparations, the increase in twitch tension evoked by DB-c-AMP was accompanied by an abbreviation of the time to peak force and of relaxation time. In the same preparations, the high potassium contracture was markedly depressed by DB-c-AMP in a concentration-dependent manner. Similar results were obtained with the N6-monobutyryl derivative of cyclic AMP.The relaxing effects of the cyclic nucleotides on KCl contractures did not appear to be due to possible non-cyclic breakdown products: adenosine; 5′-AMP and sodium butyrate did not attenuate contracture tension at concentrations up to 3×10−3 M. The same applies to ATP and non-cyclic N6-2′-O-3′-O-tributyryl-adenosine-monophosphate. Theophylline (10−2 M) was found to prolong the relaxation time of the twitch and to enhance the high KCl contracture.It is concluded that cyclic AMP may be capable of modulating the relaxation process of mammalian heart and that not only the positive inotropic but also the relaxant effects of catecholamines on myocardium described before may be mediated by the cyclic AMP system. The relaxant effects of cyclic AMP derivatives on intact myocardial preparations are attributed to a stimulation by cyclic AMP of the calcium transport of the sarcoplasmic reticulum (SR) and are interpreted to be a corollary to the effects of cyclic AMP previously obtained on isolated SR preparations.

Influence of cyclization and acyl substitution on the inotropic effects of adenine nucleotides

SummaryThis study was designed to further elucidate relevance and mechanism of the positive inotropic action of cyclic N6-2′-O-dibutyryl-AMP (DB-c-AMP). For this purpose the effects of cyclic N6-monobutyryl-AMP (N6-MB-c-AMP), noncyclic N6-2′-O-3′-O-tributyryl-5′-AMP (TB-AMP), c-AMP, adenosine and various adenine nucleotides (ATP, ADP, AMP) on myocardial contractile force (CF) were investigated and compared to that of DB-c-AMP. The experiments were performed on isolated, electrically driven (frequency 2 Hz) rat left auricles, i.e. on a preparation in which DB-c-AMP consistently produced positive inotropic effects. The following results were obtained:1.N6-MB-c-AMP (2×10−4–5×10−3 M) produced a concentration-dependent positive inotropic effect (Fig. 2). This effect began 5.5 to 1.8 min after addition of the drug (Table 2), developed gradually (Fig.2), and was maximal within 44.5 to 9.5 min (Table 2). It was preceded by a transitory decrease in CF which was also concentration-dependent (Fig.2, Table 1).2.The inotropic effects of cyclic N6-MB-AMP and cyclic DB-AMP were qualitatively similar. Quantitatively, the positive inotropic effect of N6-MB-c-AMP developed more slowly (Table 2) and was smaller in magnitude than that of DB-c-AMP (Fig.2). The initial negative inotropic effect, however, was more pronounced (Table 1) and longer in duration (Table 2) with N6-MB-c-AMP than with DB-c-AMP.3.The time course of the reversal of the positive inotropic effects during washout in drug-free solution was practically not different with both drugs (Fig.3). Predrug levels were reached within 50–60 min.4.No positive inotropic effects were found with the non-cyclic but butyryl substituted adenine nucleotide, TB-AMP. In contrast, this compound (10−7 to 10−3 M) produced a concentration-dependent decrease in CF (Fig.4).5.c-AMP, ATP, ADP, AMP and adenosine depressed CF. No secondary positive inotropic effects could be detected within 60 min (Fig.5; Fig.6). From the failure of non-cyclic TB-AMP to increase contractile force it is concluded that the positive inotropic effects seen with cyclic DB-AMP and cyclic N6-MB-AMP are due to both acyl substitution and the intact cyclophosphate structure. In so far we probably have ruled out one of the objections to the view that the positive inotropic responses to acyl substituted derivatives of c-AMP are “representative” for c-AMP itself. Since the effects of cyclic DB-AMP and cyclic N6-MB-AMP were qualitatively similar and since the time course of the loss of the positive inotropic effects during washout were practically not differen with both drugs, one may further conclude that within the cell both drugs are likely to act via the same compound. This agent could be c-AMP. However, cyclic N6-MB-AMP as the active intracellular compound cannot be ruled out.

Dibutyryl cyclic AMP and adrenaline increase contractile force and 45Ca uptake in mammalian cardiac muscle

SummaryThe effects of dibutyryl cyclic AMP (DB-AMP; 10−3M) and adrenaline (2.2×10−6 M) on contractile force, 45Ca uptake, and total myocardial Ca concentration were investigated in electrically driven left auricles isolated from rat hearts. The experiments were performed at an extracellular Ca concentration of 0.45 mM and at low frequency of stimulation (15 beats/min). 45Ca exposure was 5 min. Under the conditions used, both drugs increased contractile force and enhanced 45Ca uptake (expressed as relative specific activity) by about 30% (DB-AMP) and 40% (adrenaline), respectively. Thus, the results provide evidence that the effects of adrenaline on 45Ca uptake in mammalian cardiac muscle can be mimicked by DB-AMP.

Über den Einfluß von Mangan-Ionen auf die positiv inotrope Wirkung von Adrenalin, Theophyllin und Digitoxigenin an isolierten Meerschweinchenvorhöfen

SummaryThe influence of bivalent manganese ions (Mn++) on the positive inotropic effect of adrenaline, theophylline, and digitoxigenin was studied in isolated, electrically driven left guinea-pig auricles in phosphate-free Tyrodes solutions with different extracellular calcium concentrations ([Ca]e; 0.45; 1.8; 7.2 mM).Mn++ (0.1–50 mM) exerted a dose-dependent negative inotropic effect which was dependent on [Ca]e: Raising [Ca]e decreased the inhibitory action of Mn++. The negative inotropic effect of Mn++ was exclusively due to a decrease in the rate of tension development; the time to peak tension and the duration of contraction remained unchanged.In a solution containing 0.45 mM Ca, pretreatment with 0.1 mM Mn+ significantly diminished the positive inotropic effect of adrenaline (10−9-10−5 g/ml) and theophylline (5×10−6-10−3 g/ml), but did not influence the effect of digitoxigenin (2×10−7-2×10−6 g/ml). The depression of the positive inotropic effect of adrenaline and theophylline with higher concentrations of Mn+ (0.35–2.25 mM, producing a negative inotropic effect of about 50%) was influenced by the [Ca]e. The effect of Mn+ was most evident at 0.45 mM Ca, less pronounced (but significant) at 1.8 mM Ca and was not observed at 7.2 mM Ca. With the same concentrations of Mn+, however, the positive inotropic effect of digitoxigenin was only slightly decreased at 0.45 mM Ca, was not changed at 1.8 mM Ca and was increased at 7.2 mM Ca.As Mn+ selectively blocks the inward movement of Ca ions across the membrane of the myocardial cell during depolarisation, it is tentatively concluded from these experiments that the positive inotropic effect of adrenaline and theophylline may be due at least partially to an increase of the Ca influx during the excitation process, whereas the positive inotropic effect of digitoxigenin seems to be independent of this mechanism.

Effect of DB-c-AMP on mechanical characteristics of ventricular and atrial preparations of several mammalian species

SummaryConflicting results exist about the influence of cyclic N6-2′-O-dibutyryl-AMP (DB-c-AMP) on myocardial contractile force. The present study was designed to examine whether the positive inotropic action of DB-c-AMP is restricted to certain model preparations or whether it can be assumed to represent a more general effect of the drug. Therefore, the effects of DB-c-AMP on myocardial force and on various parameters of the isometric contraction curve were examined in isolated electrically driven (0.5–2Hz) ventricular and atrial preparations of several mammalian species (cat, rabbit, calf, sheep, rat and guinea-pig). The following results were obtained: 1.At concentrations above 3×10−4M, DB-c-AMP exerted concentrationdependent positive inotropic effects in all ventricular preparations studied. These effects were accompanied by increases in the rates of force development and relaxation and by decreases in time to peak force and relaxation time.2.Positive inotropic responses to DB-c-AMP were also obtained in atrial preparations of cats, rabbits, calves, sheep and rats. In guinea-pig auricles, similar effects were seen when the preparations were treated with the phosphodiesterase inhibitor papaverine. The results suggest that the positive inotropic action of DB-c-AMP is not restricted to certain model preparations and can be obtained in all cases under suitable experimental conditions.

Ca-abhängige Membranpotentialänderungen am Herzen und ihre Bedeutung für die elektro-mechanische Kopplung. Versuche mit Tetrodotoxin in Na-haltigen Lösungen

Summary1.Tetrodotoxin (TTX), at a concentration of less than 10−6 g/ml, had no effect on membrane potential and contraction of isolated, thin ventricular trabeculae of sheep and calf hearts. 10−6 to 2 × 10−5 g/ml TTX decreased the rate of rise, over-shoot, and duration (phase of 90% repolarisation) of the action potential and the amplitude of contraction, without change in the resting potential and the plateau (20% repolarisation phase) of the action potential. Excitation block regularly occurred only with 10−5 to 2×10−5 g/ml TTX.2.In a solution containing Na and TTX (5×10−6-2×10−5 g/ml) graded depolarisation was possible if the preparations were stimulated by square wave pulses of 500 msec duration across a sucrose bridge. In Ca-containing solutions the time-course of the electrotonic potentials showed two steps. The second step of depolarisation (SSD) began when the membrane potential reached−60 to−50 mV (threshold), and tension was initiated at the same level. Contractions reached their steady-state values only after about 5 depolarisations of the same size. There was no difference in the amplitude of contractions (steady-state level) elicited in Tyrode and in Tyrode + TTX.3.SSD and contraction were dependent on the [Ca]e. Rate of rise (V/sec) and amplitude (mV) of SSD and tension increased with increasing [Ca]e. In Ca-free solutions the electrotonic potentials reached their steady-state levels in one step. No SSD and tension were observed in the absence of Ca, even with reversal of membrane potential.4.In Tyrode + TTX the SSD was identical with the changes in membrane potential which could be observed in Na-free solution without TTX, but in Na-free solutions, contractions already reached their maximum during the first SSD.5.SSD in Na-free solution was not blocked by TTX. The results indicate that the second step of depolarization in Na-free as well as in Na-containing solution + TTX is due to a Ca inward current. It is tentatively concluded that not only in Na-free but also in Na-containing solution Ca ions carry charge across the membrane of cardiac muscle during depolarisation and that this Ca inward current is an important factor in excitation-contraction coupling.

Über den Mechanismus der positiv inotropen Wirkung von Theophyllin am Warmblüterherzen

SummaryIsometric tension was measured in isolated electrically driven left guinea-pig atria (frequency 3 Hz) before and 5 min after addition of theophylline (T).In normal Tyrode solution (1.8 mM Ca++), T produced a concentration-dependent positive inotropic effect, threshold and peak concentrations being 10−5 and 5×10−4 g/ml, respectively. Higher, i.e. toxic concentrations caused contractures and a decrease in contractile force (Figs. 1 and 3).The positive inotropic effect of T began immediately after addition of the drug and was maximal after about 2 min (Fig. 2). It was exclusively due to an increase in the rate of tension development; the time to peak tension and the duration of contraction remained unchanged (Tab. 2; Fig. 4). Toxic concentrations, however, resulted in a decrease in the rate of tension development and an increase in time to peak tension and duration of contraction (Tab. 2, Fig. 4).The action of T was dependent on the [Ca]e (Figs. 5 and 6). An increment in myocardial contractility was seen only at normal and reduced (0.45; 0.9 mM) [Ca]e. Quantitatively, there was no difference in the absolute effect of T (measured asΔg/g dry weight) at 0.45; 0.9 and 1.8 mM Ca++, but when plotted relatively (as % increase in force) the action of T was greater at 0.45 and 0.9 mM than at 1.8 mM Ca++. At 3.6 and 7.2 mM Ca++, low concentrations of T had no effect, but high concentrations diminished contractile force and caused contractures. In Ca++ free solution neither contractions nor potassium contractures were obtained within 15 min after addition of 5 · 10−4 g/ml T (Fig. 7).Reducing the [K]e from 5.4 to 4.05 mM did not increase the positive inotropic effect of T. Likewise, the action of T was not diminished at an increased [K]e of 8.1 mM (Fig. 10).Reduction of the [Na]e from 149.2 to 74.6 mM (Fig. 8) had no influence on the contractile response to T, provided that the [Ca]e/[Na]e2 ratio was kept constant.It could be excluded that the positive inotropic effect of T was mediated by a release of endogenous catecholamines, as it was found not to be reduced in reserpinized atria (Tab. 4).In conclusion, the experiments provide further evidence that the positive inotropic effect of T in therapeutic concentrations qualitatively ressembles that of adrenaline, which increases Ca++ influx during the excitation process. Differences in the actions of both drugs, however, are observed with toxic concentrations, especially in that T causes contractures, which do not occur with adrenaline even in supramaximal doses.

Über die positiv inotrope Wirkung von Dibutyryl-3′,5′-AMP an isolierten Rattenvorhöfen

Summary1.In isolated electrically driven (frequency 2 Hz) rat left atria the N6-2′-O-dibutyryl derivative of cyclic 3′,5′-AMP (DB-AMP; monosodium salt) produced a concentration-dependent and reversible positive inotropic effect. In normal Tyrode solution (1.8 mM Ca++, the threshold concentration was 5×10−5 M; the peak concentration (10−3 M) increased contractile force to 203% of control values. Negative inotropic responses were not observed in these experiments even with supramaximal DB-AMP concentrations (Fig. 1).2.The positive inotropic effect of DB-AMP developed gradualy (Fig. 2). It began 0.3–7.7 min after addition of the drug and was maximal within 3.5–28.3 min (Tab. 2). Once established, the effect was prolonged and decreased only slightly within 60 min (Fig. 2).3.The positive inotropic effect of DB-AMP was due to an increase in the rate of tension development. The time to peak tension and the duration of contraction remained unchanged (Tab. 3).4.At high concentrations (10−3–5×10−3M) DB-AMP caused contractures in some experiments (Tab. 1).5.The time course and the magnitude of the inotropic effects of DB-AMP were influenced by the [Ca]e. The increment in myocardial contractility caused by 10−3M DB-AMP developed faster at high than at low [Ca]e (Tab. 4; Fig. 5). On the other hand, the positive inotropic effect of 10−3M DB-AMP was most evident at normal (1.8 mM) and reduced (0.45; 0.9 mM) [Ca]e, less pronounced at 3.6 mM Ca++ and was insignificant at 7.2 mM Ca++ (Fig.3).6.Sodium butyrate (10−4–10−2M) did not alter the contractile behaviour of isolated rat atria.7.In isolated electrically driven (frequency 3 Hz) left guinea-pig atria no positive inotropic effects could be demonstrated with DB-AMP at concentrations up to 10−3M. It is concluded that the positive inotropic action of DB-AMP—at least in isolated rat atria—may resemble that of adrenaline or theophylline in some points, e.g. with regard to its dependence on the [Ca]e. But as no positive inotropic effect could be observed in guinea-pig atria and as the mechanism by which DB-AMP augments contractile force remains obscure, the results are not thought to necessarily support the view that the effects of adrenaline or theophylline on contractile behaviour of mammalian cardiac muscle occur via cyclic AMP.

Effect of theophylline on calcium exchangeability in ventricular myocardium.

SummaryThe effects of theophylline on contractile force and myocardial calcium exchangeability were studied in isolated, electrically driven Langendorff perfused guinea-pig hearts. Following a 30-min exposure to 45Ca, total cellular calcium and 45Ca activity were measured in right ventricular samples. “Non-toxic” theophylline concentrations (5×10−5–10−3 g/ml) which augmented contractile force without producing arrhythmias or contractures had no effect on total tissue calcium and did not alter the size of the fraction of cellular calcium exchangeable under steadystate conditions. A “toxic” concentration of theophylline (2×10−3 g/ml) induced contractures and increased the amount of exchangeable cellular calcium. The latter effect was due to an increase in total calcium; the unlabelled cellular calcium fraction remained unchanged under the influence of all theophylline concentrations studied. The results suggest that theophylline increases the steady-state calcium exchangeability in ventricular myocardium only when the total calcium concentration is also increased.