Control of brain slice respiration by (Na+ + K+)-activated adenosine triphosphate and the effects of enzyme inhibitors.

The involvement of membrane (Na+ + K+)-ATPase (Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, E.C. 3.6.1.3) in the oxygen consumption of rat brain cortical slices was studied in order to determine whether (Na+ + K+)-ATPase activity in intact cells can be estimated from oxygen consumption. The stimulation of brain slice respiration with K+ required the simultaneous presence of Na+. Ouabain, a specific inhibitor of (Na+ + K+)-ATPase, significantly inhibited the (Na+ + K+)-stimulation of respiration. These observations suggest that the (Na+ + K+)-stimulation of brain slice respiration is related to ADP production as a result of (Na+ + K+)-ATPase activity. However, ouabain also inhibited non-K+ -stimulated respiration. Additionally, ouabain markedly reduced the stimulation of respiration by 2,4-dinitrophenol in a high (Na+ + K+)-medium. Thus, ouabain depresses brain slice respiration by reducing the availability of ADP through (Na+ + K+)-ATPase inhibition and acts additionally by increasing the intracellular Na+ concentration. These studies indicate that the use of ouabain results in an over-estimation of the respiration related to (Na+ + K+)-ATPase activity. This fraction of the respiration can be estimated more precisely from the difference between slice respiration in high Na+ and K+ media and that in choline, K+ media. Studies were performed with two (Na+ + K+)-ATPase inhibitors to determine whether administration of these agents to intact rats would produce changes in brain respiration and (Na+ + K+)-ATPase activity. The intraperitoneal injection of digitoxin in rats caused an inhibition of brain (Na+ + K+)-ATPase and related respiration, but chlorpromazine failed to alter either (Na+ + K+)-ATPase activity or related respiration.

Gel electrophoretic identity of the (Na+ + Mg-2+)- and (Na+ + Ca-2+)-stimulated phosphorylations of rat brain ATPase.

The classical E2-P intermediate of (Na+ + K+)-ATPase dephosphorylates readily in the presence of K+ and is not affected by the addition of ADP. To determine the significance in the reaction cycle of (Na+ + K+)-ATPase of kinetically atypical phosphorylations of rat brain (Na+ + K+)-ATPase we compared these phosphorylated components with the classical E2-P intermediate of this enzyme by gel electrophoresis. When rat brain (Na+ + K+)-ATPase was phosphorylated in the presence of high concentrations of Na+ a proportion of the phosphorylated material formed was sensitive to ADP but resistant to K+. Similarly, if phosphorylation was carried out in the presence of Na+ and Ca-2+ up to 300 pmol/mg protein of a K+ -resistant, ADP-sensitive material were formed. If phosphorylation was from [gamma-32-P]CTP up to 800 pmol-32-P/mg protein of an ADP-resistant, K+ -sensitive phosphorylated material were formed. On gel electrophoresis these phosphorylated materials co-migrated with authentic Na+ -stimulated, K+ -sensitive, E2-P-phosphorylated intermediate of (Na+ + K+)-ATPase, supporting suggestions that they represent phosphorylated intermediates in the reaction sequence of this enzyme.

Sodium influx rate and ouabain-sensitive rubidium uptake in isolated guinea pig atria.

1. Ouabain-sensitive 86Rb+ uptake by tissue preparations has been used as an estimate of Na+ pump activity. This uptake, however, may be a measure of the Na+ influx rate, rather than capacity of the Na+ pump, since intracellular Na+ concentration is a determinant of the active Na+/Rb+ exchange reaction under certain conditions. This aspect was examined by studying the effect of altered Na+ influx rate on ouabain-sensitive 86Rb+ uptake in atrial preparations of guinea pig hearts. 2. Electrical stimulation markedly enhanced ouabain-sensitive 86Rb+ uptake without affecting nonspecific, ouabain-insensitive uptake. Paired-pulse stimulation studies indicate that the stimulation-induced enhancement of 86Rb+ uptake is due to membrane depolarizations, and hence related to the rate of Na+ influx. 3. Alterations in the extracellular Ca2+ concentration failed to affect the 86Rb+ uptake indicating that the force of contraction does not influence 86Rb+ uptake. 4. Reduced Na+ influx by low extracellular Na+ concentration decreased 86Rb+ uptake, and an increased Na+ influx by a Na+-specific ionophore, monensin, enhanced 86Rb+ uptake in quiescent atria. 5. Grayanotoxins, agents that increase transmembrane Na+ influx, and high concentrations of monensin appear to have inhibitory effects on ouabain-sensitive 86Rb+ uptake in electrically stimulated and in quiescent atria. 6. Electrical stimulation or monensin enhanced ouabain binding to (Na+ + K+)-ATPase and also increased the potency of ouabain to inhibit 86Rb+ uptake indicating that the intracellular Na+ available to the Na+ pump is increased under these conditions. 7. The ouabain-sensitive 86Rb+ uptake in electrically stimulated atria was less sensitive to alterations in the extracellular Na+ concentration, temperature and monensin than that in quiescent atria. 8. These results indicate that the rate of Na+ influx is the primary determinant of ouabain-sensitive 86Rb+ uptake in isolated atria. Electrical stimulation most effectively increases the Na+ available to the Na+ pump system. The ouabain-sensitive 86Rb+ uptake by atrial preparations under electrical stimulation at a relatively high frequency seems to represent the maximal capacity of the Na+ pump in this tissue.

Effects of alkali metal cations on phospho-enzyme levels and [3H] ouabain binding to (Na+ + K+)-ATPase.

The effects of several alkali metal cations on the relationship between steady state phospho-enzyme levels and initial velocity and equilibrium levels of [3H]-ouabain binding to (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3.) were examined. Only Na+ increased both phospho-enzyme and [3H] ouabain binding levels above those observed in the presence of Mg2+ alone. While Na+ stimulated phosphorylation with an apparent Km of about 1 mM, its stimulation of [3H] ouabain binding was biphasic, the lower Km for stimulation corresponding to the Km for formation of phospho-enzyme. Among the other alkali metal cations, potassium, rubidium and lithium were at least eight times more effect in reducing phospho-enzyme levels than in reducing [3H] ouabain binding. This discrepancy is not due to the stability of the enzyme-ouabain complex, nor to any action on the rates of formation or dissociation of the enzyme-ouabain complex. The data thus suggest that [3H] ouabain interacts with the K+, Rb+ or Li+ -enzyme complexes. For Li+, this hypothesis is further supported by the observation that Li+ can cirectly increase the equilibrium level of [3H] ouabain binding to this enzyme under certain conditions.

Is ouabain-sensitive rubidium or potassium uptake a measure of sodium pump activity in isolated cardiac muscle?

(1) The significance of the specific (ouabain-sensitive) 86Rb+ or 42K+ uptake by cardiac muscle preparations which are not 'sodium-loaded' was studied. (2) In left atrial preparations of guinea-pig heart, resting 86Rb+ uptake was relatively low. It was markedly increased by electrical stimulation. This stimulated uptake was further enhanced by isoproterenol and inhibited by verapamil. (3) In rat atria, the resting 86Rb+ uptake was somewhat higher than in guinea-pig atria, and the increase in uptake caused by electrical stimulation was smaller. In guinea-pig right ventricular papillary muscle, the resting uptake was highest among those tissues studied, and the response to electrical stimulation was smallest. In the latter tissue, verapamil produced only a minimal inhibition of the specific 86Rb+ uptake. (4) The effect of the frequency of electrical stimulation of 86Rb+ uptake paralleled its influence on the force of contraction, suggesting the involvement of intracellular sodium in both events. (5) In both left atrial and right papillary muscle preparations of guinea-pig heart, specific 42K+ uptake observed with 5.8 mM K+ was relatively high, and was increased only slightly by electrical stimulation. This electrical stimulation, however, increased ouabain-induced inhibition of 42K+ uptake, suggesting that the stimulation increases the amount of Na+ available to the sodium pump. (6) When the K+ concentration was 1 mM, the resting 42K+ uptake was low, and could be enhanced by electrical stimulation. (7) Thus, in cardiac muscle preparations which are not sodium loaded, the specific 86Rb+ or 42K+ uptake can be used to estimate the rate of sodium influx, which is equivalent to the rate of sodium efflux under steady-state conditions, provided that neither Rb+ nor K+ is in excess compared to the Na+ available to the pump. If Rb+ or K+ is in excess, its specific uptake may not reflect changes in transmembrane Na+ movement.

Saturable adenosine 5'-triphosphate-independent binding of [3H]-ouabain to brain and cardiac tissue in vitro.

1. Several investigators have proposed that membrane Na+, K+-adenosine 5'-triphosphatase (Na+, K+-ATPase) is a mechanism for the transmembrane transport of cardiac glycosides, rather than the receptor for pharmacological actions of these agents. This implies that the glycosides bind to an intracellular constituent (receptor) other than Na+, K+-ATPase. 2. In search for such a receptor site, saturable ATP-independent [3H]-ouabain binding was studied in rat brain and dog and guinea-pig heart homogenates. The binding of the glucoside to this site results in a relatively unstable complex which is stabilized by K+ to a lesser extent than is the complex formed with the ATP-dependent binding to Na+, K+-ATPase. 3. The ATP-independent ouabain binding sites are more abundant in rat brain tissue than in cardiac tissue, and have a lower ouabain affinity compared to the binding sites on Na+, K+-ATPase. 4. These results do not support the contention that there are intracellular inotropic receptors for digitalis.

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.

Prednisolone-3, 20-bisguanylhydrazone: Na+, K+-ATPase inhibition and positive inotropic action.

The relationship between two known actions of prednisolone-3, 20-bisguanylhydrazone (PBGH); Na+, K+-ATPase inhibition and positive inotropic effects, was investigated. In electrically driven left atrial preparations of guinea pig heart, the positive inotropic action of PBGH was not affected by beta-adrenergic or histamine antagonists. Pretreatment of animals with reserpine also failed to influence the positive inotropic action of PBGH. Inotropic concentrations of PBGH inhibited Na+, K+-ATPase and the ATP-dependent binding of (3/)-ouabain to Na+, K+- ATPase preparations in vitro. Additionally, ouabain-sensitive 86Rb uptake, an estimate of sodium pump activity was inhibited when sodium-loaded ventricular slices were obtained from Langendorff preparations at the peak inotropic response to PBGH. Guinea pig heart was highly sensitive to PBGH to the positive inotropic action, the inhibition of Na+, K+ -ATPase and (3H)-ouabain binding, whereas rat, rabbit and dog heart were markedly less sensitive. These findings suggest that the mechanism of the positive inotropic action of PBGH resembles that of ouabain and probably involves Na+, K+ -ATPase inhibition, although the mode of interaction of these steroids with Na+, K+ -ATPase may be different.

Lack of effect on brain stem and cerebral cortex Na+, K+-ATPase during heart block produced by chronic digoxin treatment.

The autonomic nervous system has been shown to play an important role in digitalis toxicity. In order to determine whether the central nervous system could be digitalis' site of action, the effect of chronic treatment with toxic doses of digitalis on brain Na+,K+-ATPase was studied in the dog. After four weeks of digoxin treatment, Na+,K+-ATPase activity of the brainstem or cerebral cortex was unaffected at the time when digitalis toxicity (heart block) was apparent. ATP-dependent (3H)-ouabain binding to these tissues was also unaffected indicating that a significant occupancy of brain Na+,K+-ATPase by digoxin did not occur during chronic drug treatment. In contrast, cardiac Na+,K+-ATPase was markedly inhibited with the concomitant binding of digoxin to the enzyme. Since Na+,K+-ATPase is the most digitalis sensitive system identified to date, it appears that digoxin does not affect neuronal function directly.

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.

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.

Mechanism of grayanotoxin III-induced afterpotentials in feline cardiac Purkinje fibers.

Grayanotoxins are known to produce cardiac tachyarrhythmias. It is unknown, however, which of three mechanisms, i.e., reentry, induction of automatic activity or induction of triggered activity, accounts for grayanotoxin-induced arrhythmias. To distinguish between these possible mechanisms, effects of grayanotoxin III (GTX III) on the electrical activity of isolated feline cardiac Purkinje fibers were examined under control conditions in which reentry was not likely to occur (perfusion with an oxygenated physiological saline solution at 30 degrees C and pH 7.4) and under several other conditions known to either enhance or block triggered activity. GTX III alone (1 microM) produced either 1-2 low amplitude or repetitive, suprathreshold after potentials within 15 min of administration. At the onset of GTX III-induced afterpotentials, the configuration of electrically driven action potentials was only slightly altered. Increasing stimulation frequency, raising extracellular calcium concentration or lowering extracellular potassium concentration, each of which augment triggered activity, enhanced the production of GTX III-induced afterpotentials. Furthermore, verapamil, raising extracellular potassium concentration or phenytoin, each of which block triggered activity, suppressed afterpotentials elicited by GTX III. These results indicate that the mechanism underlying grayanotoxin-induced arrhythmias is the production of triggered activity in the form of oscillatory afterpotentials.

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.

Differential effect of potassium on the action of digoxin and digoxigenin in guinea-pig heart.

The effect of potassium on the binding of digoxin or digoxigenin to isolated Na+, K+-ATPase was compared with that of potassium on the positive inotropic action of the agents in guinea-pig hearts. The binding of digoxigenin to the enzyme in vitro was reduced to a greater extent by potassium than was the binding of digotoxin. The digoxigenin-induced increase in the force of contraction of left atrial preparations estimated at steady state was reduced at higher potassium concentrations. Potassium had a lesser effect when digoxin was used as the inotropic agent. In contrast, potassium concentrations. Potassium had a lesser effect when digoxin was used as the ininotropic agent. In contrast, potassium reduced the rate of development and also the rate of loss of the positive inotropic action of digoxin observed with left atrial and Langendorff preparations, respectively, to a greater extent than those of digoxigenin. The loss of the positive inotropic effect was more rapid with digoxigenin than with digoxin at each KCl concentration. These data support the contention that the extent of the interaction of digitalis with Na+,K+-ATPase determines the degree of the positive inotropic effect.

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.

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.

Studies on the stable inhibition of Na+ + K+-ATPase by cassaine.

Exposure of rat brain Na+ + K+-ATPase (ATP phosphohydrolase E.C. 3.6.1.3) to concentrations of cassaine greater than 1 x 10(-4) M resulted in a poorly reversible inhibition of this enzyme. Inhibition did not require the presence of ATP and developed rapidly, but the final amount of inhibition observed was independent of time. The amount of inhibition observed at a given concentration of cassaine was reduced by increasing the concentration of membranes in the system. The inhibition of Na+ + K+-ATPase activity was associated with equivalent inhibition of the phosphorylation and (3H)-ouabain binding reactions of this enzyme, while the uninhibited enzyme was apparently kinetically normal. Concentrations of cassaine which produced this stable inhibition of Na+ + K+-ATPase had no effect on the Mg2+-activated ATPase or the NADH cytochrome-c-reductase activities of crude rat brain microsomal preparations. Cassaine inhibited the cholinesterase activity of rat brain microsomes with a Ki of about 5 x 10(-5) M, but his inhibition was fully reversible. The poorly reversible inhibitory actions of cassaine, thus, appeared specific for Na+ + K+-ATPase. Because this stable pattern of inhibition of the Na+ + K+-ATPase by cassaine required drug concentrations at least one hundred-fold greater than those which produce positive inotropic effects, it appears unlikely that this pattern of Na+ + K+-ATPase inhibition is involved in the cardiotonic actions of this drug.

Cardiotonic site directed irreversible inhibition of Na+ + K+-ATPase by 3-azidoacetylstrophanthidin, a photochemical analogue of strophanthidin.

A photochemical analogue of strophanthidin, 3-azidoacetylstrophanthidin (AAS) was synthesized and tested as a cardiotonic steroid (CS) site directed photoaffinity label for Na+ + K+-ATPase (ATP phosphohydrolase, E.C. 3.6.1.3). AAS-inhibited rat brain ATPase with an I50 of about 1 x 10(-6) M readily displaced 3H-ouabain from its specific binding sites on this enzyme and produced a positive inotropic effect in guinea pig atrial strips. In the absence of UV light its interaction with the CS binding sites of Na+ + K+-ATPase appeared reversible. In the presence of UV light and acetylphosphate, AAS produced about 15% irreversible inhibition of Na+ + K+-ATPase, compared with about 5% irreversible inhibition in the absence of either UV light or acetyl phosphate. Since acetylphosphate supports specific glucoside binding at the CS binding sites of Na+ + K+-ATPase these data are consistent with the concept that AAS is a cardiotonic steroid site directed photoactivatable inhibitor of Na+ + K+-ATPase.

Effects of monovalent cations on (Na+ + K+)-ATPase in rat brain slices.

The influence of monovalent cations on membrane (Na + K+)-ATPase was estimated in vitro in intact cells from the oxygen consumption of rat brain cortical slices. High concentrations of K+, Rb+ or Cs+ stimulated the respiration in the presence of Na+. This stimulation was antagonized by ouabain in a concentration- and time-dependent manner. Additionally, only combinations of monovalent cations, that stimulate (Na+ + K+)-ATPase, increased oxygen consumption, indicating that the stimulated portion of respiration is realted to the (Na+ + K+)-ATPase activity. Low concentrations of Rb+ and Cs+, however, failed to affect oxygen consumption. Li+ slightly and transiently stimulated oxygen uptake at low concentrations and inhibited it at higher concentrations. Low concentrations of Tl+ also stimulated respiration in a K+-free medium. However, the inhibitory effects of Tl+ were predominant at higher concentrations or in the presence of K+. Thus, monovalent cations can alter (Na+ + K+)-ATPase activity. While Rb+ and Li+ produce opposite effects on this enzyme system under certain conditions, these actions do not seem to be related to the antidepressant action of Rb+ and the antimanic action of Li+.

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.