Enhancement of cardiac actions of ouabain and its binding to Na+, K+-adenosine triphosphatase by increased sodium influx in isolated guinea-pig heart

The rate of development of the positive inotropic action of ouabain is enhanced when the heart is stimulated at higher frequencies. A hypothesis that this enhancement is due to a stimulation of the glycoside binding to sarcolemmal Na+,K+-adenosine triphosphatase (ATPase) caused by an increase in intracellular Na+ available to the sodium pump was tested in isolated left atrial muscle preparations of guinea-pig heart, incubated at 30 degrees C and electrically stimulated at 0.5, 1 or 2 Hz. The rate of development of the positive inotropic action of ouabain was dependent on the frequency of stimulation. Each preparation was homogenized at a predetermined time and the fractional occupancy of Na+,K+-ATPase by ouabain was estimated from the decrease in the initial velocity of ATP-dependent [3H]ouabain binding reaction. A parallel relationship was observed between effects of stimulation frequency of the positive inotropic action and those on the occupancy of Na+,K+-ATPase by ouabain. In quiescent preparations, a sodium ionophore, monensin, enhanced the development of contracture caused by a toxic concentration of ouabain and also the glycoside binding to Na+,K+-ATPase. Similar effects on the ouabain-induced contracture and on the glycoside binding were observed with either grayanotoxin I or batrachotoxin, agents known to increase sodium influx, when muscle preparations were exposed to these agents under 1.5 Hz stimulation and were subsequently tested for the actions of ouabain in quiescence. When the exposure to ouabain and either grayanotoxin I or batrachotoxin was restricted to quiescent period, the development of ouabain-induced contracture and glycoside binding to Na+,K+-ATPase were not significantly altered. Monensin, grayanotoxin I or batrachotoxin failed to significantly affect [3H]ouabain binding to muscle homogenates when added to the medium for the labeled glycoside binding assay. These results indicate that intracellular sodium ions promote the ouabain binding to Na+,K+-ATPase and thereby enhance the development of glycoside actions in the isolated atrial muscle of guinea-pig heart. The "beat-dependent" onset of the glycoside action is at least partially explained from the effect of membrane depolarization to increase Na+ available to the sodium pump and to enhance the glycoside binding.     

Cardiac Na+, K+-adenosine triphosphatase inhibition by ouabain and myocardial sodium: a computer simulation.

The major evidence against the hypothesis that Na+, K+-adenosine triphosphatase (Na+, K+-ATPase) inhibition is the mechanism of the positive inotropic action of digitalis is that the myocardial sodium content does not increase at the time of the inotropic response. In order to understand the relationship between sodium pump inhibition and myocardial sodium content, a computer simulation of the intracellular sodium concentration ([Na+]i) during a cycle of myocardial function was performed. The model for the computer simulation is a small compartment adjacent to the inner surface of the sarcolemma. The change in [Na+]i in this compartment is determined by the rate of sodium influx (published data utilized) and the rate of active sodium transport was estimated from the activities of partially purified dog heart Na+, K+-ATPase preparations assayed with various concentrations of sodium and ouabain. The initial rapid sodium influx results in maximal sodium pump activation, but the pump activity decreases with time as the [Na+]i decreases. Thus, the sodium pump functions at a rate close to its maximal velocity during the initial phase of each cycle but at reduced rates during the later phase. Inhibition of Na+, K+-ATPase by ouabain decreases the maximal velocity during the intiial phase of each cycle but at reduced rates during the later phase. Inhibition of Na+, K+-ATPase by ouabain decreases the maximal velocity of the sodium pump but increases the time in each cycle at which the sodium pump operates at its highest possible rate under these conditions, i.e., a rate close to the inhibited maximal velocity. A 40% inhibition of Na+, K+-ATPase activity, caused by inotropic concentrations of ouabain, increases the peak [Na+]i but fails to cause intracellular sodium accumulation since [Na+]i approaches control levels before the beginning of the next cardiac cycle. With greater enzyme inhibition, caused by arrhythmic concentrations of ouabain, [Na+]i fails to return to the precycle level and thus each subsequent cycle causes a progressive accumulation of myocardial sodium. Computer simulation predicts that a positive inotropic concentration of ouabain causes a myocardial sodium accumulation at a high heart rate but not at a lower heart rate. This was confirmed by experiments with Langendorff preparations of guinea-pig hearts. It is concluded that a moderate sodium pump inhibition by inotropic concentrations of ouabain enhances the intracellular sodium transient (a transient increase in intracellular sodium concentration associated with each membrane excitation) but does not cause a significant myocardial sodium accumulation at normal heart rates. A progressive myocardial sodium accumulation occurs only when the degree of Na+, K+-ATPase inhibition exceeds a critical magnitude.     

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.     

Species differences in sodium-potassium adenosine triphosphatase activity in the smooth muscle of the guinea-pig and rat vas deferens.

The acitvities of sodium-potassium-activated adenosine triphosphatase (Na+,K+-activated ATPase) and ouabain-inhibited, sodium-potassium-activated adensoine triphosphatase (Na+,K+-ATPase) in subcellular fractions of guinea-pig and rat vasa deferentia were compared to determine whether the ineffectiveness of ouabain and reduced extracellular potassium in the rat vas deferens observed in the preceding paper occurs because of a relatively low level of Na+,K+-ATPase and/or an insensitivity to ouabain. The results indicate that the specific and total activities of Na+,K+-activated ATPase and Na+,K+-ATPase (i.e., the transport enzyme) in the individual subcellular fractions and in the tissue were higher in the vas deferens of the rat than in the guinea pig. The percentage of inhibition of Na+,K+-activated activity by ouabain (8 x 10(-5) M) varied in the subcellular fractions; it was higher in the guinea-pig (range 31--87%) than in the rat (nonsignificant effect to 40%). A greater percentage of total Na+K+- activated ATPase activity was inhibited in the vas deferens of the guinea pig (56%) than the rat (30%). Differences in the effects of lowered extracellular potassium concentration or ouabain on resting membrane potential (preceding paper) are apparently unrelated to the amount of transport enzyme in the vasa deferentia or the two species, or to its relative sensitivity to ouabain.     

A polypeptide (AP-A) from sea anemone (Anthopleura xanthogrammica) with potent positive inotropic action.

Anthopleurin-A (AP-A), a polypeptide with MW ca. 5500 (53 amino acids), isolated from the sea anemone, Anthopleura xanthogrammica (Brandt), elicited a potent positive inotropic effect but without an accompanying chronotropic effect on the isolated cardiac muscles of rat, rabbit, guinea pig and cat. Similarly in dogs and cats in situ, i.p. injections of AP-A increased the contractile force without effect on heart rate or blood pressure. The cardiotonic potency for AP-A was equivalent to that of isoproterenol but much greater than that for ouabain or glucagon on the isolated cardiac muscle. AP-A increased the contractile force (cardiac output) and decreased atrial pressure in dog heart during pentobarbital-induced failure. This inotropic effect was not inhibited by propranolol pretreatment. The Ca++ requirement to restore the contractile force was less in AP-A-treated than in ouabain or isoproterenol-treated tissues. After AP-A treatment, the cardiac contractility was more resistant to hypoxia and to low or high temperature stress than ouabain-treated or control preparations. AP-A at 5 10(-9) M increased the duration of the action potential, its mean rate of rise and conduction in the guinea-pig atria and ventricles. At the maximum effective concentration, AP-A did not inhibit Na+, K+-activated adenosine triphosphatase, phosphodiesterase (high Km and low Km) and cyclic 3',5'-adenosine monophosphate content of guinea-pig heart. AP-A (5 X 10(-8) to 5 X 10(-7) M) neither contracted nor relaxed the isolated vascular smooth muscle. The results suggest that AP-A may be useful in the clinical management of cardiac failure and as an experimental tool to study the pharmacology and physiology of cardiac muscle.     

Extracellular magnesium and cardiotonic steroid toxicity in isolated myocardial preparations

This study examined effects of extracellular magnesium (Mg++0) on the positive inotropic and toxic actions of cardiotonic steroids in cardiac muscle isolated from guinea pig heart. Increasing concentrations of Mg++0 produced a negative inotropic effect in electrically paced, left atrial muscle and decreased the sensitivity to arrhythmogenic actions of digoxin without affecting the maximum developed tension observed before dysrhythmic activity. Other signs of toxicity such as contracture were less sensitive to the antagonistic effects of Mg++0. Estimates of fractional occupancy suggested that the increased tolerance to digoxin-induced arrhythmias was mediated by an altered responsiveness to given levels of receptor binding. Experiments in partially purified membrane preparations demonstrated that elevations in Mg++ increased affinity for [3H]ouabain without affecting binding site density. Na+,K+-adenosine triphosphatase activity in these membrane preparations was also enhanced by Mg++; however, increases in buffer Mg++ concentration had no effect on the Na+-pump in intact tissue. In summary, these results indicate that elevations in Mg++0 act directly on myocardium to diminish the sensitivity to cardiotonic steroid-induced arrhythmias. Furthermore, data suggest that this antagonistic action of Mg++0 is not mediated by alterations in receptor binding or Na+-pump reserve capacity.     

Digitalis-induced mechanical toxicity: protection by slow Ca++ channel blockers

In the in vitro perfusion of the isolated heart, toxic doses of cardiac glycosides produce an inotropic response which is followed by a decline in contractile force and an increase in the resting tension. Several reports in the literature indicate that the subsequent decline in contractile force may be related to cardiac cellular Ca++ overload. The purpose of the present study was to determine if the slow Ca++ channel blockers such as verapamil and nifedipine, which block Ca++ influx through voltage-dependent gated channels, can reduce or prevent the digitalis-induced decline in contractile force (mechanical toxicity). Langendorff preparations of isolated perfused guinea pig heart were used for the present study. The data obtained demonstrate that 1 to 2 microM ouabain in the perfusion medium produced mechanical toxicity in the hearts after an initial inotropic response. Verapamil or nifedipine, when combined with ouabain in the perfusion medium, increased the magnitude of the inotropic response and delayed or abolished the mechanical toxicity in a dose-dependent manner. No changes in the sarcolemmal Na+,K+-adenosine triphosphatase or ouabain binding were observed in the presence of verapamil or nifedipine. The data suggest that simultaneous use of verapamil or nifedipine may protect against digitalis-induced mechanical toxicity.     

Kinetics of ouabain binding and changes in cellular sodium content, 42K+ transport and contractile state during ouabain exposure in cultured chick heart cells

To define further the mechanism of positive inotropic action of cardiac glycosides, the temporal relationships among ouabain binding, sodium pump inhibition and positive inotropy were examined using cultured chick embryo ventricular cells. In K+-free medium, specific [3H]ouabain binding to intact cells followed pseudo first-order kinetics with saturation of binding sites occurring at 1 microM ouabain. The KD values calculated from the association and dissociation rate constants were 1.4 and 4.9 X 10(-7) M, respectively, in K+-free and 4 mM K+ medium. The Scatchard plot of binding in K+-free medium was linear, consistent with the presence of a single class of binding sites (KD = 1.3 X 10(-7) M). In 4 mM K+, 0.1 microM ouabain occupied 10% of the total binding sites and failed to produce an inotropic effect, inhibit 42K+ uptake or alter [Na+]i. Exposure of cells to 1 microM ouabain caused a significant increase in contractile state after 30 sec, reaching a plateau after 7 min with 50 +/- 6% augmentation of the amplitude of cell motion; the 42K+ uptake rate was concurrently inhibited by 36% accompanied by a 35% increase in [Na+]i and occupation of 38% of total ouabain binding sites. The initial rate of 42K+ uptake in cells loaded with Na+ by incubation in K+-free medium was 4 times greater than that observed without Na+ loading. These results indicate that more than 10% of sodium pump sites must be inhibited to produce an appreciable change in the rate of monovalent cation transport, [Na+]i or contractile state, due to the reserve capacity of uninhibited sodium pumps.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Na(+), K(+)-ATPase in morphine-induced antinociception

We evaluated the modulation by Na+,K+-ATPase inhibitors of morphine-induced antinociception in the tail-flick test and [3H]naloxone binding to forebrain membranes. The antinociception induced by morphine (1-32 mg/kg, s.c.) in mice was dose-dependently antagonized by ouabain (1-10 ng/mouse, i.c.v.), which produced a significant shift to the right of the morphine dose-response curve. The i.c.v. administration of three Na+,K+-ATPase inhibitors (ouabain at 0.1-100, digoxin at 1-1000, and digitoxin at 10-10000 ng/mouse) dose-dependently antagonized the antinociceptive effect of morphine (4 mg/kg, s.c.) in mice, with the following order of potency: ouabain > digoxin > digitoxin. This effect cannot be explained by any interaction at opioid receptors, since none of these Na+,K+-ATPase inhibitors displaced [3H]naloxone from its binding sites, whereas naloxone did so in a concentration-dependent manner. The antinociception induced by morphine (5 mg/kg, s.c.) in rats was antagonized by the i.c.v. administration of ouabain at 10 ng/rat, whereas it was not significantly modified by intrathecally administered ouabain (10 and 100 ng/rat). These results suggest that the activation of Na+,K+-ATPase plays a role in the supraspinal, but not spinal, antinociceptive effect of morphine.     

Effects of ouabain on contractions induced by manganese ions in C2+a-free, isotonic solutions with varying concentrations of K+ in guinea-pig taenia coli

The action of ouabain, a cell membrane Na+, K+-ATPase blocker, on contractions induced by manganese ions (Mn2+) in Ca2+-free, isotonic solutions with varying concentrations of K+ in the external medium were investigated in order to evaluate the underlying role of external Na+ in Mn2+-induced contractions in isolated taenia coli of the guinea-pig. Mn2+ at 5 mM induced greater contractions as external isotonic K+ concentrations progressively increased from 10 to 100 mM. Ouabain (2 x 10(-4) M) completely inhibited tension development stimulated by 5 mM Mn2+ in isotonic, 30 mM K+ (96 mM Na+) medium. Whereas, the tension inhibitory effects of ouabain became progressively weaker as isotonic, external K+ concentrations increased to 60 mM, which successively decreased external Na+ concentrations. Eventually, ouabain failed to affect contractions stimulated by Mn2+ in isotonic, 126 mM K+, Na+-deficient medium. Ouabain caused progressively greater increase in cellular Na+ concentrations as the Na+ concentrations increased in the isotonic, K+ medium. While, pyruvate, which penetrates cell independently of external Na+, reversed the inhibition of tension by ouabain in isotonic, 30 mM K+, Na+-sufficient (96 mM) medium containing 5 mM Mn2+. These results suggested that Mn2+ induced the contraction, which was maintained by glucose transport depending on external Na+, in the case of Na+-sufficient medium in K+-depolarized taenia coli. However, it induced the contraction independent of external Na+, in the case of Na+-deficient, K+ medium. Ouabain might exhibit greater inhibition of the contraction induced by Mn2+ as the decrease in the Na+ gradient across the cell membranes continues.     

Water depletion, not oral sodium loading, increases levels of sodium, potassium-dependent adenosine triphosphatase inhibitors in rat plasma

In order to define a physiological role for circulating inhibitors of sodium, potassium-dependent adenosine triphosphatase (Na+,K+-ATPase), plasma was obtained from control, water deplete, water repleted, sodium deplete and sodium loaded rats. The effect of this plasma on Na+,K+-ATPase activity, and its transport equivalent 86Rb uptake, was measured in separated guinea pig renal cortical tubules. Plasma from water deplete rats had a raised plasma osmolality and sodium concentration and a significant inhibitory effect on Na+,K+-ATPase (14%) and 86Rb uptake (24%) compared with control or water repleted rats. Inhibition of Na+,K+-ATPase and 86Rb transport was not seen with plasma from rats after dietary sodium loading (urine sodium 5.2 +/- 0.9 mmol/day) compared with low sodium diet controls (urine sodium 0.41 +/- 0.08 mmol/day). Des-amino arginine vasopressin in vivo produced no inhibition of Na+,K+-ATPase or Rb transport. These studies suggest, that in terms of common homoeostatic insults, circulating inhibitors of Na+,K+-ATPase are more responsive to water depletion than to oral sodium loading. The inhibitors may fulfil a physiological role in increasing sodium excretion to maintain osmolality after dehydration.     

Cellular potentials, electrogenic sodium pumping and sensitivity in guinea-pig atria

Intracellular recording techniques in guinea-pig atrial pacemaker and nonpacemaker cells were used to investigate 1) the role of membrane potential changes in postjunctional supersensitivity, 2) the electrogenicity of the Na+,K+ pump and 3) the role of electrogenic pumping in sensitivity of the atria to agonists. In nonpacemaker cells, ouabain (10(-6) M) had no effect on resting membrane potential (left atria) or maximum diastolic potential (right atria). However, ouabain effectively suppressed the transient hyperpolarization that followed cessation of electrical stimulation. In pacemaker cells, ouabain and chronic treatment with reserpine (0.1 mg/kg/day) produced quite different patterns of changes in intracellular potentials. Chronic treatment with reserpine induced chronotropic supersensitivity to isoproterenol but not to histamine. Ouabain did not alter the chronotropic sensitivity to either agonist. The effects of isoproterenol and histamine on intracellular potentials in pacemaker cells were investigated in the presence and absence of ouabain and in control atria vs. atria from guinea pigs chronically pretreated with reserpine. Analysis of the data indicated that 1) electrophysiological measurements do not provide a discernible explanation for chronotropic supersensitivity, 2) the Na+ pump has the capacity for electrogenic pumping under conditions of Na+ loading, but demonstrates little indication of electrogenicity under basal conditions and 3) chronic treatment with reserpine does suppress the Na+,K+ pump in some areas of the right atrium, but this activity probably does not contribute to chronotropic supersensitivity. Other possible mechanisms of postjunctional supersensitivity in atria are discussed.     

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.     

The effects of grayanotoxin I and alpha-dihydrograyanotoxin II on guinea-pig myocardium.

We have demonstrated recently that grayanotoxin I (GTX I) produces a positive inotropic effect in isolated guinea-pig atria. In order to determine whether this effect of GTX I is related to the reported action of this compound to increase the sodium permeability of cytoplasmic membranes, the effect of GTX I and alpha-dihydrograyanotoxin II (alpha-2H-GTX II) on electrical and mechanical properties and transmembrane cation movements were studied in guinea-pig myocardium. In electrically driven guinea-pig left atrial preparations, both grayanotoxins produced a slight depolarization and appear to decrease the upstroke velocity of the action potential, with a concomitant increase in isometric contractile force in the presence or absence of propranolol. Pretreatment with propranolol shifted the dose-response curves for the inotropic effect of both grayanotoxins slightly to the right. The magnitudes of changes in the electrical and mechanical properties induced by GTX I and alpha-2H-GTX II were similar. The rate of development and subsequent washout of the positive inotropic effects, however, was faster with alpha-2H-GTX II than with GTX I, consistent with a previous report that the action of alpha-2H-GTX II to increase membrane sodium permeability develops more rapidly than that of GTX I. At higher concentrations, both grayanotoxins produced arrhythmias. Arrhythmias induced by GTX I were characterized by extrasystoles whereas those induced by alpha-2H-GTX II were characterized by initial extrasystoles followed by a failure of the atria to follow electrical stimulation. Positive inotropic and arrhythmic effects of both grayanotoxins were reversible after the washout of the drug. Both types of arrhythmias produced by either GTX I or alpha-2H-GTX II were reversed by tetrodotoxin, an agent which has been demonstrated to antagonize the action of the grayanotoxins to increase membrane sodium permeability. Although both grayanotoxins had no marked effect on partially purified Na+, K+-adenosine triphosphatase, they produced dose-dependent increases in ouabain-sensitive 86Rb uptake of ventricular slices under conditions in which the intracellular sodium concentration determines the rate of active monovalent cation transport by the Na+, K+-adenosine triphosphatase system. These data suggest that the positive inotropic effects of grayanotoxins are due to an increased membrane sodium permeability and are consistent with a hypothesis that alterations in transmembrane sodium movements result in an altered myocardial contractility.     

Increased renal tubular sodium pump and Na+,K+-adenosine triphosphatase in streptozotocin-diabetic rats

The effects of chronic glucose osmotic diuresis on renal tubular sodium pump and Na+,K+-adenosine triphosphatase (ATPase) activities were studied in chronic streptozotocin-induced diabetic rats. Four to seven weeks after streptozotocin (60 mg/kg i.p.) injection, specific renal Na+,K+-ATPase activity showed a 34.8% increase as compared to the saline-citrate treated controls, whereas the nonspecific Mg++-ATPase was not altered. The concentration of Na+,K+-ATPase, estimated from the maximum [3H]ouabain binding site concentration, also showed a significant increase in the chronic streptozotocin-diabetic rats. To determine further the specificity of this increase in Na+,K+-ATPase, the activity of the sodium pump, estimated from ouabain-sensitive 86Rb uptake, was measured in nonenzymatically isolated renal tubules. Again, a significant (+106.4%) increase in the renal tubular sodium pump activity was observed in the streptozotocin-diabetic rats, whereas the nonspecific, ouabain-insensitive 86Rb uptake was not altered. Neither was there any difference in 86Rb uptake by the isolated renal glomeruli. Thus, it appears that chronic streptozotocin-induced diabetes in rats is associated with a significant increase in renal tubular sodium pump and Na+,K+-ATPase. The latter effects may represent an important physiologic adaptation of the kidneys to maintain electrolyte homeostasis in diabetes.     

Comparison of the effects of aminosugar cardiac glycosides with ouabain and digoxin on Na+, K+ -adenosine triphosphatase and cardiac contractile force.

Two aminosugar cardiac glycosides, 3-beta-O-(4-amino-4,6-dideoxy-beta-D-galactopyranosyl) digitoxigenin (ASI-222) and its 4-aminoglucose analog (ASI-254) have been shown in our laboratory to have a greater therapeutic index than ouabain (O) or digoxin (D). We have now compared the ability of ASI-222, its nonamino galactose analog (ASI-253), ASI-254, ouabain and digoxin to inhibit swine brain Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and to increase contractile force of isolated, driven rabbit atria. As inhibitors of Na+,K+ -ATPase, both ASI-222 and ASI-254 were found to be about 10 times more potent than ASI-253, O or D (I50:ASI-222, 1.3 X 10(-7) M; ASI-254, 1.4 X 10(-7) M; ASI-253, 1.15 X 10(-6) M; D, 1.6 X 10(-6) M; O, 1.75 X 10(-6) 7). Moreover the potency of these glycosides in inhibiting Na+, K+ -ATPase correlates closely with the ability of these same glycosides to increase contractile force. The concentration needed to obtain 50% of the maximum increase in contractile force was 9.7 X 10(-8) M for ASI-254, 1.5 X 10(-7) M for ASI-222, 8.8 X 10(-7) M for ASI-253 8.4 X 10(-7) M for O and 1.2 X 10(-6) M for D. Since ASI-253, a nonaminogalactose analog of ASI-222, exhibits a potency in both of our test systems which is similar to the other neutral sugar cardenolides, our data also indicate that the presence of an aminosugar group at position 4 of a sugar in a cardiac glycoside confers greater potency.     

Cytochemical localization of Na+, K+-ATPase in the rat hepatocyte.

The enzyme Na+,5+-ATPase was cytochemically localized in the rat hepatocyte by a modification of the Ernst potassium-dependent nitrophenyl phosphatase technique. Measurement of nitrophenol release from 50-micrometer liver slices confirmed the presence of ouabain-inhibitable nitrophenyl phosphatase activity that increased over the 30-min incubation period. Electron micrographs demonstrated that sinusoidal and lateral membrane reaction product deposition was K+-dependent, Mg++-dependent, inhibited by ouabain but not by alkaline phosphatase inhibitors, and was localized to the cytoplasmic side of the membrane. In contrast, canalicular reaction product was K+-independent, Mg++-dependent, inhibited by alkaline phosphatase inhibitors but not by ouabain, and was localized to the luminal side of the membrane. These findings indicate that Na+,K+-ATPase is localized to the sinusoidal and lateral portions of the rat hepatocyte plasma membrane and is not detectable on the bile canaliculus where alkaline phosphatase is confined. This basolateral localization of Na+,K+-ATPase is similar to that found in epithelia where secretion is also directed across the apical membrane.     

Studies on the mechanism of inhibition of the red cell metabolism by cardiac glycosides.

The purpose of this work was to test the previously suggested hypothesis that the inhibitory effect of ouabain on lactate production in human red cells is due to an interaction between phosphoglycerate kinase and (Na+ + k+)-activated adenosine triphosphatase (Na+,K+ATPase). An antibody to red cell phosphoglycerate kingase caused complete inhibition of the purified enzyme, whereas a portion of the phophoglycerate kinase activity of the red cell membranes was resistant to the antibody. When increasing amounts of the purified enzyme were added to the membranes, the antibody-resistant portion of the activity increased. The effects of the antibody and ouabain on lactate production from fructose-6,6-diphosphate in red cell hemolysates were studied. Ouabain, at a maximally effective concentration, produced about 30% inhibition of lactate formation. This value was doubled in the presence of the antibody. Red cell membranes, and rat brain Na+,K+-ATPase, did not catalyze the hydrolysis of 1,3-diphosphoglycerate. Ouabain did not affect the reactions of the Rapport-Luebering pathway of the red cells. These findings provide further support for the view that in red cells a membrane pool of phosphoglycerate kinase is oriented in the vicinity of Na+,K+-ATPase in a way that the product of each enzyme may be used as the immediate substrate of the other and that ouabain inhibits glycolysis by removing the regulatory effect of Na+,K+-ATPase on that portion of glycolysis which is channeled through this pool of phosphoglycerate kinase.     

(Na+, K+)-ATPase regulation and NaCl intake: effects on circulating inhibitor and sensitivity to noradrenaline

These experiments examined the effects of a high NaCl diet on (Na+,K+)-ATPase in kidney, heart and cerebral cortex, on the level of circulating inhibitor of (Na+,K+)-ATPase in plasma, and on stimulation of (Na+,K+)-ATPase by treatment with dextro (d)-amphetamine. High salt diet increased indices of (Na+,K+)-ATPase activity (K+-activated p-nitrophenyl-phosphatase activity and ouabain binding) in kidney medulla, prevented stimulation by amphetamine in cerebral cortex and reduced amphetamine stimulation in heart. High NaCl feeding increased the plasma level of circulating inhibitor of (Na+,K+)-ATPase. Amphetamine alone had no effect on inhibitor level but amphetamine administration reduced the increase in inhibitor with high NaCl feeding.     

Vascular and cardiac effects of a new dihydropyridine derivative, YC-170: a comparison with Bay K 8644

The pharmacological effects of YC-170, a new dihydropyridine derivative, were studied in the rabbit aortic strips and guinea pig cardiac preparations and compared with those of Bay K 8644. In the rabbit aortic strips, YC-170 produced contraction in normal physiological saline solution ([K+]0 = 5.9 mM) in a concentration-dependent manner. Increasing the [K+]0 of the medium to 15 mM enhanced the contractile response. The maximum contraction produced by YC-170 at [K+]0 of 15 mM was comparable to that by Bay K 8644. However, YC-170 induced relaxation when the strip was contracted by 60 mM K+. In guinea pig left atrium, YC-170 produced a positive inotropic effect in a concentration-dependent manner, but its extent was far less than that of Bay K 8644. Like Bay K 8644, however, YC-170 increased the time to peak tension and relaxation time of the isometric tension, and prolonged the action potential duration. YC-170 failed to produce a positive inotropic action in the papillary muscle in which Bay K 8644 was a potent positive inotropic agent. In spontaneously beating right atria, YC-170 caused a negative chronotropic effect, whereas Bay K 8644 a positive one. The positive inotropic and vasoconstrictor effects of YC-170 were antagonized competitively by a Ca++ antagonist nicardipine. When the left atria were depolarized with high-K+ medium, the positive inotropic effect of YC-170 was attenuated progressively with increasing [K+]0 and at 13.2 mM K+ a negative inotropic effect was induced by YC-170.(ABSTRACT TRUNCATED AT 250 WORDS)