Depressant effects of chloroquine on the isolated guinea-pig heart.

Some anti-malarials have deleterious effects upon the heart. The actions of two of these, chloroquine and quinacrine, were compared on isolated guinea-pig atria and Langendorff preparations to assess their effects on several calcium pools. Both compounds decreased developed tension in a concentration (chloroquine 10-100 microM; quinacrine 3-100 microM) and time-dependent manner, with quinacrine being twice as potent as potent as chloroquine. Ventricular muscle was much more sensitive to chloroquine than was atrial muscle. Concentrations of chloroquine, comparable to that found in the serum of patients ingesting toxic doses, caused significant inhibition of developed tension. The effects of chloroquine were almost completely reversed on washout; quinacrine, however, was less readily reversible. Chloroquine also had a direct negative chronotropic effect, substantially reduced force-frequency relationships and developed tension in partially depolarized atrial preparations; while post-rest contraction and the positive inotropic effect of ouabain were unaffected. Increases in extracellular calcium antagonized the negative inotropic effect. Quinacrine had a marked effect on post-rest contraction and attenuated the positive inotropic action of ouabain. It is concluded that the action of chloroquine may involve a superficial calcium pool, while quinacrine may act upon several calcium pools.

Isolation and enrichment of Ca2+-tolerant myocytes for biochemical experiments from guinea-pig heart.

Isolated myocytes for biochemical experiments must be homogeneous and highly enriched in viable cells. For cardiac myocytes, isolation of Ca2+-tolerant cells in high yield and with good viability has been possible from rat. This paper describes myocyte isolation and enrichment procedures which are effective for several species including guinea-pig and rat. New methods for selection of collagenase and viable cells are presented. Using Ca2+-tolerant myocytes obtained from guinea-pig heart and enriched in viable cells, dihydropyridine binding sites are shown to be accessible only in depolarized cells.

Cardiodepressant actions of the orally active male contraceptive, gossypol, in guinea-pig heart muscle.

We have previously shown that gossypol has direct actions on isolated atrial muscle preparations. The possible mechanisms responsible for the transient positive and sustained negative inotropic effects of gossypol were examined under conditions that modify Ca2+ pools involved in contractile activation. In Langendorff preparations obtained from guinea-pig or rat heart, gossypol produced marked negative inotropic and arrhythmogenic effects but failed to produce a positive inotropic effect. Langendorff preparations were significantly more sensitive than atrial muscle preparations. In atrial muscle preparations, the negative inotropic effect of gossypol was not specific to utilization of superficial or intracellular Ca2+ pools; force-staircase phenomenon observed between 0.5 and 3 Hz, contractions elicited by slow action potentials in partially depolarized muscle, the inotropic effect of extracellular Ca2+ and potentiated post-rest contractions were all suppressed by gossypol to the similar extent. Low external Na+ concentrations abolished the positive inotropic effect of gossypol without affecting the negative inotropic effect. A low extracellular Ca2+ concentration enhanced the transient positive inotropic effect and delayed development of the negative inotropic effect. Simultaneous reduction of extracellular Na+ and Ca2+ concentrations abolished the positive inotropic effect and enhanced the negative inotropic effect. Gossypol inhibited ATP-dependent Ca2+ uptake by sarcolemmal vesicles obtained from dog heart. These results indicate that the actions of gossypol on cardiac muscle is not specific to utilization of either the superficial or intracellular Ca2+ pools involved in contractile activation.

The effect of stimulation frequency and calcium concentration on maintenance of developed tension in isolated heart muscle preparations.

The ability of isolated cardiac muscle preparations to maintain force of contraction was assessed at different extracellular Ca2+ concentrations and at various stimulation frequencies. Preparations were incubated in Krebs-Henseleit buffer at 32 degrees C and electrically stimulated at frequencies between 0.25 and 3.0 Hz. Extra-cellular Ca2+ was maintained at given concentrations between 0.8 and 2.4 mM, a range that included the plasma-ionized Ca2+ levels determined for rat and guinea pig. Developed tension of atrial or papillary muscle preparations of guinea pig heart was more stable at higher stimulation frequencies. Rat atria and ventricular strips were more stable at 0.25 than at 0.5 or 1.0 Hz, and the stability increased at 2.0 and 3.0 Hz. Guinea pig atrial muscle was maintained for a longer time at higher media Ca2+ concentrations; however, no such effect was observed with rat atrial tissue. Thus, the decrementing at developed tension in isolated cardiac muscle preparations is dependent on stimulation frequency and Ca2+ concentrations and can be retarded by selection of appropriate incubation conditions.

High affinity and low affinity ouabain binding sites in the rat heart.

Ventricular muscle of rat heart has two classes of receptors which are responsible for the positive inotropic effect of ouabain. Low affinity receptors are apparently related to Na+, K+-ATPase. To determine if high affinity receptors are also sarcolemmal Na+, K+-ATPase of muscle cells, their characteristics were examined. Binding of [3H]ouabain to the high affinity binding site required ATP in the presence of Mg2+ and Na+, was stimulated by Na+ in the presence of Mg2+ and ATP, and was inhibited by K+. Digoxin, digitoxin and cassaine all inhibited [3H]ouabain binding to the high affinity site. Cassaine was about an order of magnitude less potent than the glycosides. These results indicate similarities in high affinity ouabain binding sites in ventricular muscle of rat heart and Na+, K+-ATPase obtained from other sources. Destruction of sympathetic nerve terminals with 6-hydroxydopamine failed to affect the high affinity ouabain binding sites indicating that high affinity sites do not represent the Na+, K+-ATPase in sympathetic nerve terminals. Labeling of Na+, K+-ATPase from [gamma-32P]ATP indicates that high affinity ouabain binding sites account for 25% of the total enzyme molecules present in ventricular muscle of rat heart.

13-Propylberberine reduces response of guinea-pig myocardium to inotropic interventions including changes in extracellular Ca2+.

Berberine has been shown to increase developed tension in cardiac muscle but its derivatives have been reported to inhibit the catalytic subunit of adenylate cyclase. In the present study, the cardiac actions of the most potent derivative, 13-propylberberine, were examined. It produced a transient increase followed by a sustained decrease in developed tension in paced left atrial muscle preparations isolated from guinea-pig heart. In the presence of 13-propylberberine, isoproterenol caused only a transient increase in developed tension; marked desensitization to the positive inotropic effect of isoproterenol occurred within 20 min. After washout of isoproterenol and an additional 15-min incubation in the presence of 13-propylberberine, the muscle lost its sensitivity to isoproterenol. Moreover, the positive inotropic effect of ouabain or effects of decrease or increase in extracellular Ca2+ concentration on the force of muscle contraction were markedly attenuated. Isoproterenol-induced elevation of tissue cyclic AMP concentration was inhibited by 13-propylberberine; however, 13-propylberberine did not alter the basal cyclic AMP concentration and its effects on inotropic actions of ouabain or extracellular Ca2+ appear unrelated to tissue cyclic AMP concentration.

Lack of pharmacodynamic interactions between quinidine and digoxin in isolated atrial muscle of guinea pig heart.

Quinidine has been reported to have no effect on the positive inotropic action of digoxin observed in isolated cardiac muscle preparations. This is surprising because quinidine has been shown to reduce Na+ influx in cardiac muscle. The conditions which increase Na+ influx stimulate the glycoside binding to Na+- and K+-activated Mg++-dependent ATP phosphohydrolase (Na+,K+-ATPase), and therefore quinidine may be expected to have an opposite effect. Thus, the effects of quinidine on cardiac muscle and its possible interactions with digoxin were re-evaluated using electrically paced left atrial muscle preparations of guinea pig heart. Quinidine caused a frequency- and concentration-dependent decrease in maximal upstroke velocity and amplitude of the action potential without altering resting membrane potential. In addition, quinidine prolonged action potential duration markedly in a frequency-dependent manner. Despite action potential prolongation, the alkaloid reduced net Na+ influx as determined by a decrease in steady-state ouabain-sensitive 86Rb+ uptake. Under these conditions, however, quinidine failed to reduce the rate of onset or the maximal positive inotropic effect of digoxin; or did it reduce digoxin binding to Na+,K+- ATPase in beating atrial muscle preparations. Benzocaine, which reduced net Na+ influx without increasing the action potential duration, also failed to affect the peak inotropic effect of digoxin or the glycoside binding. Quinidine had no direct effects on glycoside binding to isolated cardiac Na+,K+-ATPase. Moreover, [3H]ouabain binding to isolated enzyme was relatively insensitive to changes in Na+ concentrations between 1 and 8 mM although binding was stimulated clearly by Na+ above 8 mM. These results indicate that quinidine, at therapeutic concentrations, does not interact pharmacodynamically with digoxin in isolated cardiac muscle.

Effects of BAY K-8644 on inotropic and arrhythmogenic actions of digoxin.

Myocardial intracellular 'Ca2+ overload' may be involved in the direct arrhythmogenic actions of cardiotonic steroids. This proposal was examined by determining if the sensitivity of guinea-pig atrial muscle to digoxin-induced arrhythmias was affected by BAY K-8644, a 1,4-dihydropyridine derivative which promotes Ca2+ influx via slow channels. BAY K-8644 significantly reduced both the time required for a given concentration of digoxin to produce arrhythmias and the amount of digoxin bound to atrial muscle at the onset of arrhythmias. In addition, BAY K-8644 increased the maximum developed tension observed in the presence of digoxin before the onset of arrhythmias. Similar results were obtained with increasing concentrations of buffer Ca2+. In contrast, A23187, a Ca2+ ionophore, enhanced the sensitivity to digoxin-induced arrhythmias without affecting maximum developed tension. These results suggest that increases in intracellular Ca2+ enhance cardiac sensitivity to digoxin-induced arrhythmias and that the arrhythmogenic action may involve Ca2+ overload at a pool other than that which activates contractile proteins.

Amiloride: effects on myocardial force of contraction, sodium pump and Na+/Ca2+ exchange.

The direct effects of amiloride on myocardial contractility were examined in electrically stimulated left atrial muscle of guinea-pig heart. Amiloride (0.3 to 1.5 mM) produced a positive inotropic effect which, at higher concentrations, was followed by a decline in developed tension. These effects were not accompanied by contracture or arrhythmia and were not affected by a combination of phentolamine, nadolol, cimetidine, tripelennamine and atropine. The above concentrations of amiloride prolonged the action potential duration during the development of the positive inotropic effect; however, no further change in the action potential duration was observed during the decline in developed tension caused by high concentrations of amiloride. Myocardial membrane Na,K-ATPase, ouabain-sensitive 86Rb+ uptake and Na+-dependent Ca2+ efflux from sarcolemmal membrane vesicles were all inhibited by amiloride. The positive inotropic effect of the agent is reduced and the negative inotropic action is enhanced in low Na+ solutions, i.e., under conditions likely to favor Ca2+ influx via Na+/Ca2+ exchange. These results suggest that amiloride, under the present conditions, has a complex interaction with cardiac muscle fibers. Amiloride may produce its inotropic effects in guinea-pig atrial muscle by several mechanisms including sodium pump inhibition, Na+/Ca2+ exchange inhibition, prolongation of the action potential duration, and/or actions such as Na+/H+ exchange inhibition which were not directly addressed in this study.

Suppression of positive inotropic and toxic effects of cardiac glycosides by amiloride.

Effects of amiloride on the inotropic and toxic actions of cardiac glycosides were examined using left atrial muscle isolated from guinea pig heart. Preincubation of atrial muscle with amiloride significantly decreased the maximum positive inotropic effect of dihydrodigoxin but failed to reduce that of isoproterenol. Amiloride prevented the contracture and significantly reduced the incidence of arrhythmias induced by 2 microM digoxin. Similar experiments examining 5 microM digoxin-induced arrhythmias showed that amiloride increased both the time required to produce arrhythmias and the fractional occupancy of sarcolemmal Na,K-ATPase by digoxin at the onset of arrhythmias. The antagonism of cardiac glycoside actions was best observed during the decline in developed tension elicited by amiloride subsequent to its initial positive inotropic effect. Amiloride had no effect on binding site concentration for ATP-dependent [3H]ouabain binding but decreased affinity of the binding sites for ouabain in membrane preparations obtained from guinea pig heart. Furthermore, amiloride inhibited Na,K-ATPase activity and increased the IC50 value for ouabain inhibition of the enzyme. These results indicate that amiloride antagonizes the positive inotropic and toxic effects of cardiac glycosides. Possible mechanisms for the antagonism include inhibition of sarcolemmal Na+/Ca2+ or Na+/H+ exchange.

Ascorbic acid: an endogenous inhibitor of isolated Na+,K+-ATPase.

During attempts to isolate and identify an endogenous ligand for the glycoside binding sites on Na+,K+-ATPase, bovine adrenal glands were found to contain a potent inhibitor of isolated Na+,K+-ATPase. The inhibitory principle was extracted from adrenal cortex, following homogenization in NaHCO3 solution and separation on a Sephadex G-10 column. The active principle was recovered from a fraction which eluted from the column after the 3H2O peak. The extract inhibited isolated Na+,K+-ATPase and the specific [3H]ouabain binding reaction. Sensitivity of the enzyme to the inhibitory action of the extract was species and tissue dependent; however, the pattern and the magnitude of the sensitivity were different from those of the digitalis glycosides. Moreover, the inhibitory principle failed to inhibit sodium pump activity, estimated from ouabain inhibitable 86Rb+ uptake by guinea pig brain slices. The activity of the extract to inhibit isolated Na+,K+-ATPase was stable under acidic condition but was lost rapidly at neutral pH, and could be eliminated by EDTA. In an acidic medium, the inhibitory principle had an absorption maximum at 244 nm which shifted to 264 nm and decayed rapidly at neutral pH. By using mass spectrometry, the principle was identified to be ascorbic acid, which has been shown previously to inhibit isolated Na+,K+-ATPase under appropriate conditions. Because ascorbic acid was incapable of inhibiting the sodium pump in intact cells, this inhibitor of the isolated enzyme does not appear to be the endogenous ligand which regulates sodium pump activity in vivo.

Effects of K+ on the interaction between cardiac glycosides and Na,K-ATPase.

Inhibition of Na,K-ATPase by cardiac glycosides is at least partially antagonized by K+. The kinetics of the antagonism, however, appear complicated because K+ is capable of reducing both association and dissociation rate constants for the glycoside-enzyme interaction. In order to better understand the effect of K+, inhibition of partially purified Na,K-ATPase obtained from rat brain, guinea-pig heart and rat heart by ouabain, digoxin, digoxigenin, dihydrodigoxin and cassaine were compared in the presence of 1, 3 or 10 mM K+. Higher concentrations of K+ caused a parallel shift to the right in the concentration-inhibition curves for these compounds. For ouabain or digoxin, the extent of the shift was minimal with rat brain enzyme, intermediate with guinea-pig heart enzyme and more substantial with rat heart enzyme. For digoxigenin, dihydrodigoxin or cassaine, the extent of the shift was substantial in all enzyme preparations. These results could not be explained from either the affinity of the enzyme for the compound or its lipid solubility alone. The concentrations of these compounds required to cause a 50 percent inhibition of enzyme activity were markedly different with rat brain enzyme, but relatively similar with rat heart enzyme. The effects of K+, which depend on the source of the enzyme and chemical structures of the compounds, have to be considered in studies on comparative effects of various compounds on Na,K-ATPase, [3H]ouabain binding, sodium pumping and the force of myocardial contraction.

The dual effect of sodium ion on the digitalis-sodium pump interaction.

Digitalis glycosides are specific inhibitors of the sodium pump. While their effects on isolated Na,K-ATPase, an enzymatic representation of the sodium pump, can be quantified easily and precisely, estimation of their effects on the sodium pump and evaluation of the physiological significance of sodium pump inhibition are complicated by several factors. In isolated Na,K-ATPase, the specific binding of cardiac glycosides observed in the presence of Mg2+ and ATP is stimulated by Na+. In intact myocardial cells, conditions which enhance Na+ influx, and hence the amount of Na+ to be transported by the sodium pump, such as stimulation at high frequencies, presence of monensin (a sodium ionophore), batrachotoxin or grayanotoxin I, enhance the glycoside binding to the sodium pump. In left atrial muscle preparations isolated from the guinea-pig heart and stimulated at 0.5 Hz, binding of ouabain to glycoside binding sites on Na,K-ATPase was eliminated by lowering the extracellular Na+ concentration from 145 to 27 mM, a condition reported to lower the intracellular Na+ concentration by more than 60%. When ouabain exposure of atrial muscle preparations was restricted to the quiescent period, glycoside binding to the sodium pump was minimal. Monensin, however, caused ouabain to bind to the sodium pump in quiescent preparations. These results indicate that glycoside binding to the sodium pump is enhanced by intracellular Na+. In addition to enhancing the glycoside binding to the sodium pump, an elevation of intracellular Na+ reduces the reserve capacity of the sodium pump and therefore increases the sensitivity of the myocardium to sodium pump inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

The inotropic effects of a synthetic pyrethroid decamethrin on isolated guinea pig atrial muscle.

Pyrethroid insecticides have been reported to increase transmembrane sodium influx and inhibit ion-dependent ATPases in insect, squid, and toad nerve tissues. Since changes in sarcolemmal ion fluxes and inhibition of membrane-bound ATPases can alter myocardial contractility, the effects of a potent synthetic pyrethroid, decamethrin, on mammalian myocardium were examined using isolated left atrial muscle of the guinea-pig heart electrically stimulated at 1.5 Hz. Decamethrin (0.1-10 microM) increased the force of isometric contraction in a dose-dependent manner without affecting the resting tension. Propranolol (5 microM) reduced the magnitude of the pyrethroid's inotropic effect; however, a significant inotropic effect was still observed with 1 and 10 microM decamethrin in the presence of this concentration of propranolol. Similar results were noted in the presence of 500 microM procainamide and in left atrial muscle obtained from reserpine-treated animals. The results suggest that decamethrin increased the force of myocardial contraction by two mechanisms; indirectly by releasing catecholamines from sympathetic nerve terminals and directly by an action on the myocardium. Inotropic concentrations of the pyrethroid did not inhibit partially purified rat brain or guinea pig heart Na+, K+-ATPase. Moreover, twitch tension recordings of isometric contractions showed that, in the presence of propranolol, decamethrin increased tension development without changing either the time to peak tension or the duration of contraction. Tetrodotoxin almost completely abolished the positive inotropic effect of decamethrin. Thus, the positive inotropic effect of decamethrin is apparently due to an increase in transmembrane sodium influx which causes catecholamine release from the sympathetic nerve terminals and also directly enhances muscle contraction.

Negative chronotropic and positive inotropic actions of phencyclidine on isolated atrial muscle in guinea pigs and rats.

Chronotropic and inotropic actions of phencyclidine were studied in spontaneously beating right atrial muscle and electrically paced left atrial muscle preparations isolated from guinea-pig or rat hearts. In right atrial muscle preparations, phencyclidine (10-100 microM) decreased the frequency of spontaneous beating. Guinea-pig and rat heart preparations had similar sensitivities to this action of phencyclidine. The negative chronotropic effect was not altered by atropine. A high concentration of naloxone failed to affect the chronotropic effect of phencyclidine in guinea-pig muscle, but significantly reduced the effect in rat heart muscle preparations. Phencyclidine (1-100 microM) caused positive inotropic effects in both guinea-pig and rat heart left atrial muscle electrically stimulated at 1.5 Hz; rat heart preparations had a higher sensitivity to the positive inotropic action of phencyclidine. The positive inotropic effect was reduced by verapamil, nifedipine and relatively high concentrations of diltiazem, but was not affected by propranolol, phentolamine, tripelennamine, atropine or ryanodine, indicating that the effect is not mediated by adrenergic, histaminergic or cholinergic systems or does not involve ryanodine-sensitive calcium pools. Inactivation of the fast sodium channels by partial membrane depolarization, and subsequent restoration of the contraction by raising the extracellular Ca++ concentration, did not abolish the positive inotropic action of phencyclidine. These results suggest that the negative chronotropic effect of phencyclidine is not mediated by a stimulation of the muscarinic receptor. The positive inotropic effects of phencyclidine seem to result from an increase in Ca++ influx through the slow channels of the cardiac cell membrane.

Effects of progesterone derivatives on sodium pump activity and force of myocardial contraction in isolated guinea pig heart.

A progesterone derivative, chlormadinone acetate, has been reported to inhibit isolated Na,K-ATPase, to increase intracellular sodium, to decrease the force of myocardial contraction and to antagonize the positive inotropic action of ouabain. Because several inhibitors of isolated Na,K-ATPase fail to interact with the sodium pump in intact cells, two progesterone derivatives, chlormadinone acetate and megestrol acetate were examined for their effects on Na,K-ATPase, sodium pump activity and the force of contraction in isolated guinea pig atria. Both megestrol acetate and chlormadinone acetate inhibited specific (ATP-dependent) 3H-ouabain binding to Na,K-ATPase isolated from guinea pig brain or heart, with a corresponding inhibition of enzyme activity. The effects on Na,K-ATPase obtained from brain were more pronounced than that obtained from heart. The inhibitory effect of megestrol acetate was partially antagonized by K+. In electrically stimulated left atrial muscle preparations, neither compound affected ouabain-sensitive 42K+ uptake. Additionally, these compounds failed to affect the force of myocardial contraction or to influence the positive inotropic action of ouabain. These data fail to support the contention that inhibition of the sarcolemmal sodium pump is unrelated to the ouabain-induced increase in the force of myocardial contraction.

How increased sodium influx enhances digoxin-induced arrhythmias in guinea-pig atrial muscle.

The hypothesis that digitalis-induced arrhythmias occur when Na,K-ATPase inhibition exhausts the sodium pump reserve capacity, producing an accumulation of intracellular Na+, was tested by reducing the reserve capacity in isolated left atrial muscle of guinea pig heart and estimating specific digoxin binding and Na,K-ATPase activity in atrial muscle homogenized at the onset of digoxin-induced arrhythmias. Reductions in reserve capacity were produced by either increasing the stimulation frequency of the atrial muscle or adding a sodium ionophore, monensin, to the media bathing the tissue. As stimulation frequency was increased, both the time required to produce arrhythmias with a given concentration of digoxin and the amount of digoxin bound to sarcolemmal Na,K-ATPase at the onset of arrhythmias were reduced. Similarly, monensin treatment produced reductions in the time to arrhythmia and in digoxin binding and Na,K-ATPase inhibition observed at the onset of arrhythmias. These results support the above proposal suggesting that a decrease in reserve capacity of the sodium pump enhances cardiac sensitivity to digitalis-induced arrhythmias.