Kyosuke Temma

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.

Sodium pump inhibition by sulfhydryl inhibitors and myocardial contractility.

SummaryIn order to determine whether the positive inotropic action of cardiac glycosides is related to the inhibition of sodium pump activity, the actions of known inhibitors:N-ethylmaleimide,p-chloromercuribenzoate (PCMB),p-chloromercuribenzene sulfonic acid (PCMBS) and digitoxin, were studied in isolated guinea pig hearts. In electrically driven left atrial preparations, all these agents increased isometric contractile force. The inotropic effect ofN-ethylmaleimide was only partially reduced in the presence of propranolol or by reserpine pretreatment, whereas the inotropic effects of PCMB and PCMBS were unaffected by reserpine pretreatment. (±)-Propranolol markedly delayed the development of the inotropic action of PCMB and PCMBS without affecting the magnitude of the peak inotropic response. Similar effect was observed with (+)-propranolol indicating that the delayed development of the inotropic action is probably due to the action of propranolol unrelated to β-adrenergic blockade. Among sulfhydryl blocking agents, the development of the inotropic effects was fastest withN-ethylmaleimide and slowest with PCMBS. Effects of these sulfhydryl inhibitors on resting potential and action potential were unrelated to the inotropic action. These agents caused a decrease in ouabain-sensitive86Rb uptake, an estimate of sodium pump activity, in sodium-loaded ventricular slices obtained from drug-perfused Langendorff preparations. Quantitative comparisons between the degree of inotropic response and sodium pump inhibition caused by these agents were not possible because of the difference in experimental conditions. These results, however, are consistent with the hypothesis that Na+, K+-ATPase inhibitors cause positive inotropic effects associated with sodium pump inhibition.

Reduction of the equilibrium binding of cardiac glycosides and related compounds to Na+, K+-ATPase as a possible mechanism for the potassium-induced reversal of their toxicity

SummaryThe influence of potassium ions on the equilibrium state of the binding of cardiac glycosides and their derivatives to partially purified dog heart and rat brain enzyme preparations was studied in vitro. The addition of potassium to the incubation mixture containing enzyme preparation, 3H-ouabain, Na+, Mg2+ and ATP, at the time when the binding reaction is close to equilibrium, caused an immediate reduction of the bound drug concentration; the concentration apparently shifting toward a lower equilibrium state. The degree of the potassium-induced reduction in bound drug concentration was dependent on the potassium concentration and on the chemical structure of the compound. The binding of aglycones, pentacetyl-gitoxin and cassaine was affected to a greater extent than that of the glycosides. These data suggest that one of the mechanisms by which potassium antagonizes the toxic actions of digitalis on the heart is to reduce the drug binding to cardiac Na+, K+-ATPase.

Aging and digitalis sensitivity of cardiac muscle in rats

The cause of the reduced tolerance of aged rats to the arrhythmogenic effect of ouabain was studied in left atrial and left ventricular muscle preparations obtained from 3-, 8- or 26-month old Fischer 344 rats. Under 1.5-Hz stimulation, the concentration-response curves for the positive inotropic effect of ouabain showed a tendency to shift to the left with age. The toxic effects occurred at lower ouabain or digoxin concentrations in the ventricular muscle obtained from senescent rats especially when the muscle was stimulated at a higher frequency (4 Hz), although differences in tolerance to the toxic effect of ouabain were not apparent in atrial muscle preparations stimulated at 1.5 Hz. No age-related difference in intracellular sodium or potassium concentration was observed in perfused ventricular muscle preparations paced at 1.5 Hz. There was no significant age effect on Na+-induced activation of Na+,K+-ATPase. In aged myocardium, however, the upstroke velocity of the action potential was reduced especially under high frequency stimulations, and the ability to follow high frequency stimulation was compromised. These results suggest that reserve capacity of the sodium pump is reduced in aged ventricular muscle, thereby elevating its sensitivity to the cardiac glycoside.

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.

Inotropic effects of digitoxin in isolated guinea-pig heart under conditions which alter contraction☆

In order to examine which calcium pool(s) might contribute to the therapeutic action of digitalis, the positive inotropic effect of digitoxin was quantified under conditions in which different calcium pools were either predominant or suppressed. Isolated left atrial muscle of guinea-pig heart was stimulated at 0.5 Hz at 30 degrees C. After a brief rest period, the first contraction (post-rest contraction) observed when electrical stimulation was resumed was markedly greater than the contraction observed under 0.5 Hz stimulation. Post-rest contraction was apparently dependent on the rest period and related to a ryanodine-sensitive, verapamil-insensitive calcium pool. Post-rest contraction was moderately enhanced by 0.2 microM digitoxin, either in the absence or presence of verapamil. A step-wise increase in the frequency of stimulation following the rest period caused a typical staircase phenomenon, which was markedly suppressed by verapamil but not by ryanodine. Digitoxin markedly augmented the staircase in the absence or presence of ryanodine. Paired-pulse stimulation markedly increased the developed tension, which was slightly reduced by verapamil but not by ryanodine. Digitoxin substantially increased the developed tension evoked by paired-pulse stimulation. In left atrial preparations of rat heart, an increase in stimulation frequency decreased the force of contraction; however, paired-pulse stimulation increased the force, indicating that the enhancement of developed tension by an increase in stimulation frequency and that by paired-pulse stimulation have different mechanisms. Thus, the positive inotropic action of digitoxin does not appear to be restricted to a specific calcium pool; however, the inotropic effect was greater under the conditions in which superficial calcium pool plays a predominant role.

Positive inotropic and toxic effects of brevetoxin-B on rat and guinea pig heart☆

Brevetoxin-B (GbTX-B), a cyclic polyether purified from the marine dinoflagellate Gymnodinium breve, produced positive inotropic and arrhythmogenic effects on isolated rat and guinea pig cardiac preparations at concentrations between 1.25 X 10(-8) and 1.87 X 10(-7) M. The toxin (10(-7) M) transiently increased left ventricular +dP/dt, hydraulic work, and oxygen consumption of paced working rat hearts, then reduced these variables during continuous exposure. Brevetoxin-B exerted a much smaller positive inotropic effect on working guinea pig hearts, but produced a marked and sustained inotropic effect on guinea pig left atria. The toxin also produced arrhythmias in rat and guinea pig hearts, characterized by ventricular tachycardia and A-V blockade. Sympatholytic procedures (beta blockade or reserpine pretreatment) partially blocked the positive inotropic effects, and eliminated the ventricular tachycardia, but not the A-V blockade. Tetrodotoxin markedly inhibited the positive inotropic effect of GbTX-B. Brevetoxin-B did not inhibit guinea pig cardiac Na,K-ATPase activities. The results show that GbTX-B is a potent cardiotoxin and suggest that GbTX-B exerts positive inotropic and arrhythmogenic effects by increasing sarcolemmal sodium permeability, and by releasing catecholamines from sympathetic nerve endings.

Inability of Na+, K+-ATPase inhibitor to cause hypertension in sodium-loaded or deoxycorticosterone-treated one kidney rats

The role of an endogenous inhibitor of Na+,K+-ATPase in hypertension observed in one-kidney NaCl-loaded rats treated with deoxycorticosterone (DOC) was examined. Ouabain or digitoxin, an exogenous inhibitor of Na+,K+-ATPase, failed to cause hypertension in one-kidney NaCl-loaded rats without DOC treatment or one-kidney DOC-treated rats without NaCl loading. Moreover, neither ouabain nor digitoxin acted additively with a putative endogenous inhibitor of Na+,K+-ATPase to augment hypertension observed in one-kidney NaCl-loaded rats treated with DOC. The results do not support the hypothesis that an endogenous inhibitor of Na+,K+-ATPase plays an important role in the development or maintenance of hypertension in this animal model.

Temperature-dependent development of ouabain action in isolated guinea pig heart: Differences in sodium influx?

At 30 degrees C the development of the positive inotropic action of 0.3 microM ouabain observed in isolated left atrial muscle preparations of guinea-pig heart was significantly slower and more dependent on the frequency of electrical stimulation than it was at 37 degrees C. The hypothesis was tested that such differences are due to a variation in the rate of leak sodium influx, and ensuing differences in glycoside binding to sarcolemmal Na,K-ATPase. Monensin or grayanotoxin, agents which have been shown to increase sodium influx, caused faster development of the inotropic action of ouabain at 30 degrees C when muscle preparations were stimulated at 0.5 or 1 Hz but their effect was smaller at 2 or 3 Hz stimulation. The contracture caused by toxic concentrations of ouabain in quiescent preparations developed faster at the higher temperature. Ouabain binding to sarcolemmal Na,K-ATPase which occurred during exposure of atrial muscle preparations to the glycoside at 25 degrees C for 30 min was less than the corresponding value observed after a similar exposure at 37 degrees C; however, electrical stimulation increased glycoside binding to a greater extent at the lower temperature. The ouabain-sensitive 42K+ uptake was lower in quiescent preparations at 30 degrees C than at 37 degrees C. Moreover, the enhancement of the ouabain-sensitive 42K+ uptake caused by electrical stimulation was greater at 30 degrees C than at 37 degrees C. These results are consistent with the hypothesis that, at lower temperatures, the rate of sodium influx is low and is the factor determining the rate of development of the inotropic action of ouabain. At a higher temperature, a relatively high sodium leak influx seems to make the onset of ouabain action less dependent on the frequency of stimulation.

Intracellular Sodium Enhancement of Ouabain Binding to Na,K-ATPase and the Development of Glycoside Actions

Publisher Summary This chapter discusses the intracellular sodium enhancement of ouabain binding to Na, K-ATPase and the development of glycoside. The binding of cardiac glycosides to Na, K-ATPase, observed in vitro in the presence of Mg 2+ and ATP, is enhanced by Na + . Because Na + activates the enzyme at the cytoplasmic side of the cell membrane, it seems to be intracellular Na + (Na + i ) that stimulates the glycoside binding. Bodemann and Hoffman, however, reported that Na + i fails to stimulate ouabain binding in resealed erythrocyte ghosts under certain ionic conditions. In myocardium, the positive inotropic and toxic actions of the glycosides develop faster when the muscle is electrically stimulated at higher frequencies. Because stimulation, and resulting membrane depolarization, increases the amount of Na + i available to Na,K-ATPase or the sodium pump, these finding favors the concept that Na + i stimulates glycoside binding. Na + i available to the sodium pump is the determinant of the specific 86 Rb + - or 42 K + -uptake in cardiac muscle preparations that are not preloaded with Na + . The specific uptake, therefore, may represent the rate of Na + influx that is equivalent to Na + efflux in steady-state preparations.