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.     

Comparative species studies on the effect of monovalent cations and ouabain on cardiac Na+, K+-adenosine triphosphatase and contractile force.

The effects of ouabain, Rb+ and Tl+ on Na+, K+-adenosine triphosphatase (Na+,K+-ATPase; Mg++-dependent, Na+,K+-activated ATP phosphohydrolase, EC 3.6.1.3) and contractile force were compared in guinea-pig and rat hearts. Although ouabain produced a dose-dependent positive inotropic effect in rat as well as in guinea-pig atrial preparations, concentrations of ouabain needed to produce comparable positive inotropic effects were more than an order of magnitude higher in rats than in guinea pigs. Additionally, the time to reach the plateau of the inotropic response was significantly shorter in rat than in guinea-pig atrial preparations. Concentrations of ouabain needed to produce comparable inhibition of cardiac Na+, K+-ATPase in vitro observed with partially purified cardiac enzyme preparations were also more than an order to magnitude higher in rats than in guinea pigs.     

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.     

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.     

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.     

Effects of grayanotoxin I on cardiac Na + K + -adenosine triphosphatase activity, transmembrane potential and myocardial contractile force.

The relationship between altered transmembrane sodium movements and myocardial contractility was studied by opposing the action of the sodium pump with grayanotoxin I (GTX), an agent previously shown to increase resting sodium influx. GTX failed to affect Na+,K+-adenosine triphosphatase activity in vitro in concentrations as high as 0.1 mM. In electrically driven left atrial preparations of guinea-pig hearts, 1 mugM GTX produced a slight depolarization and appeared to decrease the upstroke velocity of the action potential, GTX (0.1-1 mugM) also produced a positive inotropic effect which developed over a 20-minute period. At higher concentrations, GTX produced arrhythmias. These effects of GTX were also observed in the presence of 10 mugM propranolol. Positive inotropic and arrhythmic effects of GTX were reversible after washout of the drug. These effects of GTX were also reversed by tetrodotoxin, an agent which has been shown to counteract the effect of GTX on sodium permeability. These data are consistent with a hypothesis that altered transmembrane sodium movement effects myocardial contractility.     

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.     

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)     

(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.     

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.     

On the mechanism of the positive inotropic action of the alpha adrenoceptor agonist, phenylephrine, in isolated rat left atria.

Alpha adrenoceptor agonists have been reported to increase contractile force and to stimulate Na+/H+ exchange in the heart. We studied the influence of hexamethylamiloride (HMA), a selective inhibitor of Na+/H+ exchange, on the positive inotropic action of phenylephrine in isolated, paced rat left atria (3 microM propranolol). HMA (10 microM) blocked the ouabain-induced contracture, an event dependent on Na+ uptake via the Na+/H+ exchanger. The same concentration of HMA prevented 50% of the positive inotropic effect of phenylephrine (10 microM), but had no effect on base-line developed force. HMA reduced the maximal effect (234 +/- 19 vs. 117 +/- 20% increase of base-line), but not the EC50 (4.4 +/- 1.0 vs. 3.6 +/- 2 microM) of phenylephrine. Phenylephrine (100 microM) caused both a leftward and upward shift of the Ca++ concentration-effect curve, but only a leftward shift, in the additional presence of HMA (3 microM). It is known that lithium, but not choline, will exchange for H+ via the Na+/H+ exchanger: phenylephrine's (100 microM) positive inotropic effect in choline-substituted solutions averaged 37% of that in lithium-substituted solutions. The positive inotropic effect of phenylephrine was amplified by ouabain (200 microM). These results are consistent with the hypothesis that alpha adrenoceptor agonists produce their positive inotropic effects, in part, via stimulation of Na+/H+ exchange. Such stimulation could cause an intracellular alkalinization and (in the presence of ouabain), elevated intracellular Na+.     

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.     

Digitalis toxicity: lack of marked effect on brain na+,k+-adenosine triphosphatase in the cat.

Effect of digitalis on central sympathetic neurons have been proposed to alter sympathetic influences on the heart and to contribute to the induction of arrhythmias. Recently, however, we have presented evidence which indicates that the involvement of a direct central action of digitalis is negligible in the alteration of sympathetic nerve activity after i.v. administration of the drug. Thus, a group of experiments were designed to determine if central drug concentrations or biochemical events in the brain would suggest a central action of the drug. Tritiated digoxin (20 microng/kg) was injected i.v. into cats every 15 minutes until ventricular fibrillation occurred. The concentrations of digoxin in cerebrospinal fluid and serum increased linearly with time as the cumulative dose of digoxin was increased. At the mean arrhythmic dose, 140 microng/kg, cerebrospinal fluid contained approximately 10 nM digoxin whereas free digoxin concentration in serum was approximately 30 nM and total digoxin concentration in serum was approximately 120 nM. Since inhibition of Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) is often associated with the pharmacological effects of digitalis, effects of nanomolar concentrations of digoxin on Na+,K+-ATPase activity were determined in vitro. The concentration of digoxin faund in cerebrospinal fluid at arrhythmia inhibited Na+,K+-ATPase only slightly (5-10%). Activity of Na+,K+-ATP-ase was also examined in brains of cats which had died in ventricular arrhythmias due to treatment with lethal dose of digitoxin. After ventricular fibrillation, the cat brains were removed and Na+,K+-ATPase activity and ouabain binding were determined in eight areas. No reduction in Na+,K+-ATPase activity or [3H]ouabain binding was observed in any area. Thus, it appeared that toxic doses of digitalis did not cause sail to provide evidence for central effects of toxic doses of digoxin or digitoxin.     

Effect of changes in dietary sodium on active electrolyte transport by erythrocytes at different stages of human pregnancy

1. Active electrolyte transport was examined in erythrocytes from women in the second and third trimesters of pregnancy and post partum, and compared with that in ovulating women. 2. There was a significant reduction in intracellular sodium ([Na]i) and increase in intracellular potassium ([K]i) in pregnancy with a return towards normal values in the post-partum period. 3. Maximum specific ouabain binding [number of Na+,K+-adenosine triphosphatase (Na+, K+-ATPase) units] was increased by 70% in pregnancy and returned slowly towards normal values post partum. 4. Na+,K+-ATPase activity as determined by ouabain-sensitive 86Rb influx in artificial media was also increased in pregnancy by 13%. It returned towards normal post partum. 5. The increases in Na+,K+-ATPase in pregnancy were not closely related to the concomitant increases in aldosterone or cholesterol nor to reticulocytosis and were not affected by 7 days of high (greater than 250 mmol/day) or low (less than 50 mmol/day) sodium intake.     

Ischemia-induced alterations in myocardial (Na+ + K+)-ATPase and cardiac glycoside binding.

The effects of ischemia on the canine myocardial (Na+ + K+)-ATPase complex were examined in terms of alterations in cardiac glycoside binding and enzymatic activity. Ability of the myocardial cell to bind tritiated ouabain in vivo was assessed after 1, 2, and 6 h of coronary occlusion followed by 45 min of reperfusion, and correlated with measurements of in vitro (Na+ + K+)-ATPase activity and in vitro [3H]ouabain binding after similar periods of ischemia. Regional blood flow alterations during occlusion and reperfusion were simultaneously determined utilizing 15 mum radioactive microspheres to determine the degree to which altered binding of ouabain might be flow related. Anterior wall infarction was produced in 34 dogs by snaring of confluent branches of the left coronary system. Epicardial electrograms delineated ischemic and border zone areas. Coronary reperfusion after 2 and 6 h of occlusion was associated with impaired reflow of blood and markedly impaired uptake of [3H]ouabain in ischemic myocardium. In both groups, in vivo [3H]ouabain binding by ischemic tissue was reduced out of proportion to the reduction in flow. Despite near-complete restoration of flow in seven dogs occluded for 1 h and reperfused, [3H]ouabain remained significantly reduced to 58 +/- 9% of nonischemic uptake in subendocardial layers of the central zone of ischemia. Thus, when coronary flow was restored to areas of myocardium rendered acutely ischemia for 1 or more hours, ischemic zones demonstrated progressively diminished ability to bind ouabain. To determine whether ischemia-induced alteration in myocardial (Na+ + K+)-ATPase might underlie these changes, (Na+ + K+)-ATPase activity and [3H]ouabain binding were measured in microsomal fractions from ischemic myocardium after 1, 2, and 6 h of coronary occlusion. In animals occluded for 6 h, (Na+ + K+)-ATPase activity was significantly reduced by 40% in epicardial and by 35% in endocardial layers compared with nonischemic myocardium. Comparable reductions in in vitro [3H]ouabain binding were also demonstrated. Reperfusion for 45 min after occlusion for 6 h resulted in no significant restoration of enzyme activity when compared to the nonreperfused animals. In six animals occluded for 2 h, a time at which myocardial creatine phosphokinase activity remains unchanged, (Na+ + K+)-ATPase activity was reduced by 25% compared with nonischemic enzyme activity. In five dogs occluded for 1 h, (Na+ + K+)-ATPase activity in ischemic myocardium was unchanged from control levels. We conclude that reduced regional myocardial blood flow, local alterations in cellular milieu, and altered glycoside-binding properties of (Na+ + K+)-ATPase all participate in the reduction of cardiac glycoside binding observed after reperfusion of ischemic myocardium. In addition, after 2 or more hours of severe ischemia, myocardial (Na+ + K+)-ATPase catalytic activity is significantly reduced despite incubation in the presence of optimal substrate concentrations.     

Potassium, Na+-K+ pump inhibitor and low-renin hypertension

A local increase in extracellular potassium concentration [K+]o, up to about 8 mEq/liter, by topical application or intra-arterial infusion of iso-osmotic solutions of K+ salts, causes arteriolar dilation and decreased resistance to blood flow in systemic vascular beds. A local decrease in [K+]o over physiologic ranges induces arteriolar constriction and increased resistance to blood flow. K+ vasodilation is accompanied by hyperpolarization of the smooth muscle cell, whereas the vasoconstriction is accompanied by depolarization. All of these responses can be blocked by ouabain, a potent Na+,K+-ATPase inhibitor. Thus it is thought that K+ vasodilation results from stimulation of the electrogenic Na+-K+ pump, and that the constriction results from its inhibition. Acute generalized inhibition of the Na+,K+-ATPase and Na+-K+ pump (hypokalemia, strophanthidin, methylguanidine, vanadate) in the anesthetized dog can raise blood pressure. In experiments in animals, myocardial Na+,K+-ATPase and vascular Na+-K+ pump activities were decreased in low-renin hypertension, and vascular Na+-K+ pump activity was decreased following acute volume expansion, changes associated with bioassay evidence of a Na+-K+ pump inhibitor in the plasma. The inhibitor appears to arise in, or to be influenced by the area of the anteroventral third ventricle of the brain. It induces electrogenic depolarization of vascular smooth muscle cells and may inhibit norepinephrine uptake by adrenergic nerve terminals. Potassium and a circulating endogenous Na+,K+-ATPase inhibitor of unknown molecular structure may partly regulate the mechanical activity of cardiovascular muscle and participate in the genesis of certain forms of hypertension. Potassium may be of value in the prevention and therapy of hypertension, partly by virtue of its vasodilator activity.     

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 rubidium on contractility and sodium pump activity in guinea-pig ventricle

Inhibition of the Na+-K+ active transport system has been postulated to be one mechanism through which myocardial contractility is increased. Rubidium is one substance which has been shown to increase the contractility of guinea-pig atria and inhibit the activity of the isolated Na+,K+-adenosine triphosphatase of guinea-pig ventricle. A reexamination of these results confirmed the positive inotropic effect of rubidium on guinea-pig atria and demonstrated that this effect on contractility is accompanied by a decrease in both resting potential and action potential duration. However, it was also found that rubidium produced a transient negative inotropic effect in guinea-pig ventricle. The latter response was closely paralleled by a transient shortening of action potential duration. A concentration of rubidium maximally effective in decreasing contractility (2.0 mM) had no effect on the slow response action potential or contraction. RbCl (0.1 mM) had no effect on cyclic adenosine 3':5'-monophosphate levels of the ventricle or atrium. RbCl did inhibit active transport in the ventricle, as evidenced by a significant reduction in the electrogenic contribution on the active transport system to the maximal diastolic membrane potential during high-frequency drive. These results demonstrate that RbCl has different effects on the contractility of atrial and ventricular muscle. They also suggest that inhibition of the sodium pump is not necessarily accompanied by an increased force of myocardial contraction.     

Ion flux and Na+,K+-ATPase activity of erythrocytes and leucocytes in thyroid disease

In hyperthyroidism, erythrocytes show decreased Na+,K+-ATPase activity, decreased [3H]ouabain binding capacity (an index of the number of sodium pumps) and decreased active sodium and potassium flux rates, with a high intracellular sodium concentration. As erythrocytes are non-nucleated and atypical cells, we have studied electrolyte status in thyroid disease using mixed leucocytes as well; the results obtained differed from those in erythrocytes. When compared with findings in healthy subjects, leucocyte Na+,K+-ATPase activity, [3H]-ouabain binding capacity, total and active rubidium (used instead of potassium) influx were all significantly increased in untreated hyperthyroidism and decreased in untreated hypothyroidism. In hyperthyroidism, there was also a decrease in plasma potassium, an increase in sodium efflux rate and efflux rate constant, but no significant changes in cell sodium and potassium concentrations. All these changes returned to normal in successfully treated patients. There was a significant correlation between these abnormalities of electrolyte status and thyroid disease status (as serum thyroid stimulating hormone and free thyroxine).     

Effect of canrenone on the disturbances of cation handling induced by ouabain in macrophages and vascular smooth muscle cells

Ouabain induced rapid and profound modifications of Na+ and K+ contents in mouse macrophages and cultured vascular smooth muscle cells. In mouse macrophages, we found a one-to-one net Na+ gain and K+ depletion, with a maximal initial rate of 30 to 35 mmol (l X cells X hr)-1 and with an IC50 of about 100 microM. The one-to-one exchange results from at least two additive effects: inhibition of the Na+,K+-pump and stimulation of a furosemide-sensitive, outward Na+,K+-cotransport by the increase in internal Na+ content. The latter effect helps the cell to maintain a normal cell volume in spite of the large changes in internal cation content. In cultured vascular smooth muscle cells from rat aorta, ouabain provoked net Na+ gain and stimulated a quinidine-sensitive, K+-efflux. This likely reflects the opening of Ca+-dependent, K+-channels in response to an increase in cytosolic-free Ca+ content. Canrenone, an antihypertensive drug, has been shown to behave like a partial agonist at the digitalisreceptor site of the Na+,K+-pump. We observed here in mouse macrophages and cultured vascular smooth muscle cells that: canrenone alone (or at low ouabain concentrations) induces slight Na+ gain and K+ depletion; canrenone partially counterbalances the very rapid cell Na+ gain (and K+ depletion) provoked by high ouabain concentrations, and canrenone reverses the secondary effects of ouabain on the Na+,K+-cotransport system and Ca+-dependent, K+-channels. It appears therefore that canrenone may partially reverse the disturbances of cation handling induced by high concentrations of ouabain in macrophages and vascular smooth muscle cells.