Captopril inhibits ouabain-sensitive Na+/K+-ATPase

Captopril has been reported to inhibit ouabain-sensitive Na+/K+-ATPase activity in erythrocyte membrane fragments. We investigated the effect of captopril on two physiological measures of Na+/K+ pump activity: 22Na+ efflux from human erythrocytes and K+-induced relaxation of rat tail artery segments. Captopril inhibited 22Na+ efflux from erythrocytes in a concentration-dependent fashion, with 50% inhibition of total 22Na+ efflux at a concentration of 4.8 X 10(-3) M. The inhibition produced by captopril (5 X 10(-3) M) and ouabain (10(-4) M) was not greater than that produced by ouabain alone (65.3 vs. 66.9%, respectively), and captopril inhibited 50% of ouabain-sensitive 22Na+ efflux at a concentration of 2.0 X 10(-3) M. Inhibition by captopril of ouabain-sensitive 22Na efflux was not explained by changes in intracellular sodium concentration, inhibition of angiotensin-converting enzyme or a sulfhydryl effect. Utilizing rat tail arteries pre-contracted with norepinephrine (NE) or serotonin (5HT) in K+-free solutions, we demonstrated dose-related inhibition of K+-induced relaxation by captopril (10(-6) to 10(-4) M). Concentrations above 10(-4) M did not significantly inhibit K+-induced relaxation but did decrease contractile responses to NE, although not to 5HT. Inhibition of K+-induced relaxation by captopril was not affected by saralasin, teprotide or indomethacin. We conclude that captopril can inhibit membrane Na+/K+-ATPase in intact red blood cells and vascular smooth muscle cells. The mechanism of pump suppression is uncertain, but inhibition of ATPase should be considered when high concentrations of captopril are employed in physiological studies.     

Characterization of fatty acid interaction with ouabain and vanadate binding to (Na+ + K+)-activated ATPase

The candidateship of unsaturated fatty acids as endogenous ouabain-like factors was studied. Binding of the artificial ligand vanadate at the intracellular phosphorylation epitope of membrane-bound Na+/K+-ATPase was unaffected by linoleic and arachidonic acid. In the (Mg2+ + Pi)-facilitated system for ouabain binding they were characterized as noncompetitive inhibitors of cardiac glycoside binding, however. The ouabain binding capacity as well as the affinity decreased and the ouabain dissociation rate was accelerated by fatty acids. In the presence of vanadate for facilitation of ouabain binding an increase in ouabain affinity was seen. It is concluded that elementary criteria for the characterization of unsaturated fatty acids as ouabain-like factors are not fulfilled. The ratio between E2-subconformations of Na+/K+-ATPase with different ouabain affinities may be changed by incorporation of fatty acids in the lipid membrane.     

Ca2+ transport by intact synaptosomes: the voltage-dependent Ca2+ channel and a re-evaluation of the role of sodium/calcium exchange

The verapamil-sensitive Ca2+ channel in the synaptosomal plasma membrane is investigated. Verapamil is without effect on Ca2+ uptake or steady-state content in synaptosomes with a polarized plasma membrane, but completely inhibits the additional Ca2+ uptake following plasma-membrane depolarization by high [K+], by veratridine plus ouabain or by high concentrations of the permeant cation tetraphenylphosphonium. Verapamil-insensitive Ca2+ influx and steady-state content are identical in polarized and depolarized synaptosomes, even though the Na+ electrochemical potential is greatly decreased in the latter, indicating that Na+/Ca2+ exchange is not a significant mechanism for Ca2+ efflux under these conditions. A transient Na+-dependent Ca2+ efflux can only be observed on addition of Na+ to Na+-depleted depolarized synaptosomes. While 0.2 mM verapamil decreases the ate of 86Rb+ efflux and 22Na+ entry during depolarization induced by veratridine plus ouabain, the final steady-state Na+ accumulation is not inhibited. Ca2+ efflux from synaptosomes following mitochondrial depolarization does not occur by a verapamil-sensitive pathway.     

No evidence for a role in signal-transduction of Na+/K+-ATPase interaction with putative endogenous ouabain.

A cascade of events (signal-transduction), mainly seen in rat cardiac myocytes and renal cells, is thought to occur after ouabain interaction with a minor fraction of Na+/K+-ATPase. A higher intracellular Na+ concentration followed sodium pump inhibition by ouabain with a subsequent gradual increase or oscillations in intracellular Ca2+ concentration. Whether this increase in intracellular Ca2+ concentration is part of the cascade, a result of the cascade or a totally independent phenomenon are conflicting interpretations that are discussed. At best, however, the cascade is initiated by ouabain concentrations several orders of magnitude higher than the measured plasma concentrations of putative endogenous ouabain. The experimentally high ouabain concentration may be critical for another reason. Most tissues contain various isoforms of the catalytic alpha-peptide of Na+/K+-ATPase with an individual sublocalization and, in rats, with different ouabain-sensitivity. The almost ouabain-insensitive alpha1-isoform of Na+/K+-ATPase is essentially unaffected by the high ouabain concentration, whereas ouabain-sensitive alpha-isoforms, possibly confined to membrane structures near cytosolic microdomains and Na+/Ca2+ exchangers, may be totally blocked. Classifying endogenous ouabain as a physiological inducer of the signaling system on this background seems hazardous.     

Metabolic changes after prior treatment with ethanol. Evidence against in involvement of the Na+ + K+-activated ATPase in the increase in ethanol metabolism.

In perfused rat liver, the inhibition of ethanol uptake by ouabain does not follow the rapid inhibition of the Na+ K+- activated ATPase as assessed by changes in perfusate [K+] (half-time, t 1/2 = 2--3 min), but correlated rather with the slow inhibition of oxygen uptake (maximal inhibition = 40% in 20 min). The data indicate that ouabain exerts its effect on ethanol metabolism via the following sequence of events; inhibition of the sodium pump is followed gradually by a perturbation of the intracellular cation milieu; this leads to an inhibition of the mitochondrial respiratory chain, resulting in diminished rate of NADH oxidation, which in turn causes in inhibition of ethanol metabolism.     

Facilitation of ouabain binding to (Na+ + K+)-ATPase by vanadate at in vivo concentrations.

In the presence of Mg2+ vanadate was shown to facilitate ouabain binding to (Na+ + K+)-ATPase in much the same way as Pi does. Thus the hypothesis that vanadate interacts with the phosphate site of the enzyme seems to be supported by ouabain binding experiments. At given ouabain concentrations maximum binding is achieved at microM concentrations of vanadate whereas mM concentrations of Pi are needed. Na+ as well as K+ counteract ouabain binding but some cardiac glycoside binding is still possible at in vivo concentrations of these cations. A minor contamination of the enzyme preparations with vanadate could explain the in vitro binding of ouabain that can be obtained with Mg2+ and in the absence of Pi.     

K+ transport in resting rat hind-limb skeletal muscle in response to paraxanthine, a caffeine metabolite

This study tested the hypothesis that paraxanthine, a caffeine metabolite, stimulates skeletal muscle potassium (K+) transport by an increase in Na+ -K+ ATPase activity. The unidirectional transport of K+ into muscle (J(in)K) was studied using a perfused rat hind limb technique. Using 12 hind limbs, we examined the response to 20 min of paraxanthine perfusion (0.1 mM), followed by 20 min perfusion with 0.1 mM paraxanthine and 5 mM ouabain (n = 5) to irreversibly inhibit Na+ -K+ ATPase activity. Paraxanthine stimulated J(in)K by 23+/-5% within 20 min. Ouabain abolished the paraxanthine-induced stimulation of J(in)K, suggesting the increase in K+ uptake was due to activation of the Na+ -K+ ATPase. To confirm the role of the Na+ -K+ ATPase, 14 hind limbs were perfused for 20 min with 5 mM ouabain prior to 20 min perfusion with 0.1 mM paraxanthine and 5 mM ouabain (n = 6). Ouabain alone resulted in a 41+/-7% decrease in J(in)K within 15 min. Inhibition of ouabain-sensitive J(in)K prevented the paraxanthine-induced increase in J(in)K. Hind limbs (n = 3) were also perfused with 0.1 mM paraxanthine for 60 min to examine the response to longer duration paraxanthine perfusion. The paraxanthine-induced increase in J(in)K continued for the entire 60 min. In another series, hind limbs were perfused with 0.01 (n = 9), 0.1 (n = 9), or 0.5 (n = 6) mM paraxanthine for 15 min. There was no concentration-dependent relationship between J(in)K and paraxanthine concentration, and 0.01, 0.1, and 0.5 mM paraxanthine increased J(in)K similarly (25+/-5, 22+/-4, and 27+/-6%, respectively). The effect of paraxanthine on J(in)K could not be reversed by subsequent perfusion with paraxanthine-free perfusate. Caffeine (0.05-1.0 mM) had no effect on K+ transport. It is concluded that paraxanthine increases J(in)K in resting skeletal muscle by stimulating ouabain-sensitive Na+ -K+ ATPase activity.     

Transport functions of the liver. Lack of correlation between hepatocellular ouabain uptake and binding to (Na+ + K+)-ATPase

Ouabain uptake was studied on isolated rat hepatocytes. Hepatocellular uptake of the glycoside is saturable (Km = 348 mumol/l, Vmax = 1.4 nmol/mg cell protein per min), energy dependent and accumulative. Concentrative ouabain uptake is not present on permeable hepatocytes, Ehrlich ascites tumor cells and AS-30D ascites hepatoma cells. There is no correlation between ouabain binding to rat liver (Na+ + K+)ATPase and ouabain uptake into isolated rat hepatocytes. While ouabain uptake is competitively inhibited by cevadine, binding to (Na+ + K+)-ATPase is not affected by the alkaloid. Although the affinities of digitoxin and ouabain to (Na+ + K+)-ATPase are similar, digitoxin is 10000-times more potent in inhibiting [3H]ouabain uptake as compared to ouabain. That binding to (Na+ + K+)-ATPase appears to be no precondition for ouabain uptake was also found in experiments with plasmamembranes derived from Ehrlich ascites tumor cells and AS-30D hepatoma cells. While tumor cell (Na+ + K+)-ATPase is ouabain sensitive, the intact cells are transport deficient. Hepatic ouabain uptake might be related to bile acid transport. Several inhibitors of the bile acid uptake system also inhibit ouabain uptake.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

Postnatal changes in Na,K-ATPase isoform expression in rat cardiac ventricle. Conservation of biphasic ouabain affinity.

The cardiac glycoside sensitivity of the rat heart changes during postnatal maturation and in response to certain pathological conditions. The Na,K-ATPase is thought to be the receptor for cardiac glycosides, and there are three isozymes of its catalytic (alpha) subunit with different cardiac glycoside affinities: alpha 1 (low affinity) and alpha 2 and alpha 3 (high affinity). We examined the developmental expression of the alpha subunit isozymes in rat ventricular membrane preparations by immunoblotting with isozyme-specific antibodies. The alpha 1 isozyme was present throughout all stages of maturation. A developmental switch from alpha 3 to alpha 2 occurred between 14 and 21 days after birth. Measurements of [3H]ouabain binding and inhibition of Na,K-ATPase activity indicated that alpha 2 and alpha 3 should make equivalent contributions to ion pump capacity; in both neonatal natal and adult preparations, ouabain interacted with a single class of high-affinity binding sites (KD = 15 or 40 nM, respectively; Bmax = 4-5 pmol/mg protein), and at low concentrations produced a similar degree of Na,K-ATPase inhibition (25%). The results indicate that the developmental difference in cardiac glycoside sensitivity cannot be explained by quantitative differences in the proportion of high-affinity isozymes of the Na,K-ATPase. The switch from alpha 3 to alpha 2 coincides with other major changes in cardiac electrophysiology and calcium metabolism.     

Na+/Ca2+ exchange in plasma membrane vesicles from a glucose-responsive insulinoma.

Plasma membrane vesicles from a glucose-responsive insulinoma exhibited properties consistent with the presence of a membrane Na+/Ca2+ exchange. The exchange was rapid, reversible, and was dependent on the external Ca2+ concentration (Km = 4.1 +/- 1.1 microM). External Na+ inhibited the uptake in a dose-dependent manner (IC50 = 15 mM). Dissipation of the Na+ gradient by 10 microM monensin decreased Na+/Ca2+ exchange from 0.74 +/- 0.17 nmoles/mg protein/s to 0.11 +/- 0.05 nmoles/mg protein/s. Exchange was not influenced by veratridine, tetrodotoxin and ouabain, or by modifiers of cAMP. No effect was seen using the calcium channel blockers, nitrendipine or nifedipine. Glucose had no direct effect on Na+/Ca2+ exchange, while glyceraldehyde, glyceraldehyde-3-phosphate and dihydroxyacetone inhibited the exchange. Na+ induced efflux of calcium was seen in Ca2+ loaded vesicles and was half maximal at [Na+] of 11.1 +/- 0.75 mM. Ca2+ efflux was dependent on [Na+], with a Hill coefficient of 2.7 +/- 0.07 indicating that activation of Ca2+ release involves a minimum of three sites. The electrogenicity of this exchange was demonstrated using the lipophilic cation tetraphenylphosphonium [( 3H]-TPP), a membrane potential sensitive probe. [3H]-TPP uptake increased transiently during Na+/Ca2+ exchange indicating that the exchange generated a membrane potential. These results show that Na+/Ca2+ exchange operates in the beta cell and may be an important regulator of intracellular free Ca2+ concentrations.     

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.     

Properties of (Na+ plus K+)-activated ATPase in rat liver plasma membranes enriched with bile canaliculi.

Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124-132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na+ plus K+)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na+ plus K+)-activated ATPase activity was documented by demonstrating specific cation requirements for Na+ and K+, while the divalent cation, Ca(2+), and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na+ plus K+)-activated ATPase activity averaged 10.07 plus or minus 2.80 mumol Pi/mg protein per h compared to 50.03 plus or minus 11.41 for Mg(2+)-activated ATPase and 58.66 plus or minus 10.07 for 5'-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na+ plus K+)-activated ATPase without any effect on Mg(2+)-activated ATPase. Both (Na+ plus K+)-activated ATPase and Mg(2+)-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na+ plus K+)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na+ plus K+)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis.     

Involvement of Na+/K+-ATPase in hydrogen peroxide-induced hypertrophy in cardiac myocytes

We have shown that increased production of reactive oxygen species (ROS) was required for ouabain-induced hypertrophy in cultured cardiac myocytes. In the present study we assessed whether long-term exposure of myocytes to nontoxic ROS stress alone is sufficient to induce hypertrophy. A moderate amount of H2O2 was continuously generated in culture media by glucose oxidase. This resulted in a steady increase in intracellular ROS in cultured cardiac myocytes for at least 12 h. Such sustained, but not transient, increase in intracellular ROS at a level comparable to that induced by ouabain was sufficient to stimulate protein synthesis, increase cell size, and change the expression of several hypertrophic marker genes. Like ouabain, glucose oxidase increased intracellular Ca2+ and activated extracellular signal-regulated kinases 1 and 2 (ERK1/2). These effects of glucose oxidase were additive to ouabain-induced cellular changes. Furthermore, glucose oxidase stimulated endocytosis of the plasma membrane Na+/K+-ATPase, resulting in significant inhibition of sodium pump activity. While inhibition of ERK1/2 abolished glucose oxidase-induced increases in protein synthesis, chelating intracellular Ca2+ by BAPTA-AM showed no effect. These results, taken together with our prior observations, suggest that ROS may cross talk with Na+/K+-ATPase, leading to the activation of hypertrophic pathways in cardiac myocytes.     

Membrane (Na+ + K+)-ATPase of canine brain, heart and kidney. Tissue-dependent differences in kinetic properties and the influence of purification procedures.

Effects of commonly used purification procedures on the yield and specific activity of (Na+ + K+)-ATPase (Mg2+-dependent, Na+ + K+-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP-dependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na+ + K+)-ATPase and the ratio between (Na+ + K+)-ATPase and Mg2+-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na+ + K+)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na+ + K+)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzymes preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain.enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purfication procedure. These results indicate that the presentyl used purification procedures may alter the properties of membrane (Na+ + K+)-ATPase and affect the interaction between cardiac glycosides and the enzyme. The effect of a given treatment depends on the source of the enzyme. For the in vitro studies involving purified (Na+ + K+)-ATPase preparations, the influence of the methods used to obtain the enzyme preparation should be carefully evaluated.     

Single Na+ channels activated by veratridine and batrachotoxin

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.     

Characterization of the electrogenic sodium channel from rat brain membranes using neurotoxin-dependent 22Na uptake

The sodium channel was studied in osmotically-sensitive membrane preparations from rat brain and in innervated and chronically denervated rat soleus and extensor digitorum longus muscles. These experiments were undertaken in order to define a set of parameters for sodium channel function at the subcellular level to be used as a measure of retention of channel integrity upon subsequent isolation of the channel. Various neurotoxins and drugs were employed to control the permeability of the brain membranes to 22Na and the sodium-conductance properties of the muscles. Batrachotoxin (ED50 = 0.2 micrometer), veratridine (ED50 = 1 micrometer), or grayanotoxin I (ED50 = 30 micrometers) stimulated 22Na uptake in brain membranes is inhibited in an apparently uncompetitive manner by the sodium channel blocking agents tetrodotoxin and saxitoxin in a simple competitive manner by Ca2+ and in a partial or allosteric competitive manner by lidocaine and procaine. This 22Na uptake assay, which can be equated to a measure of equilibrium toxin binding, shows dependence on the concentration of the membranes and is sensitive to pH, temperature, ionic strength, and the ionic composition of the media. Parallel biophysical studies on sodium channels in rat muscle show that the properties of the sodium channel are similarly affected by these agents.     

Ouabain-receptor interactions in (Na+ + K+)-ATPase preparations. A contribution to the problem of nonlinear Scatchard plots.

Specific [3H]ouabain binding to rat and guinea pig skeletal muscle (musculus soleus) was studied using a rapid centrifugation and a filtration method. Both assays gave identical results: the incubation of the cell membranes in 50 mM imidazole/HCl buffer pH 7.25 or 7.4 MgCl2, Pi caused a time dependent loss of (Na+ +K+)-ATPase activity indicating an alteration of the membrane preparation. Ouabain binding properties were changed concomitantly. If ouabain binding was allowed to proceed until equilibrium was reached (3 min in rat and 10 min in guinea pig) at 37 degrees C the data plotted according to Scatchard followed a straight line. The dissociation constants of the ouabain-receptor-complexes of the rat cell membrane preparation as calculated from the slope of the plot (KD = 134 nM) and from the ratio of the dissociation and association rate constants (KD = 175 nM) agreed within experimental error with that determined by Clausen and Hansen [(1974) Biochim. Biophys. Acta 345, 387-404] in intact soleus muscles (KD = 210 nM). If ouabain binding was allowed to proceed for a longer period, however, nonlinear Scatchard plots resulted with an identical maximal number of binding sites but inconstant and decreased affinity for the cardiac glycoside. Experimental evidence is presented that nonlinear Scatchard plots often obtained in hormone (drug)-receptor binding experiments may (among other things) be the result of damaged cell membrane particles in vitro.     

Regulation of the (Na+ + K+)-ATPase in cultured chick skeletal muscle. Modulation of expression by the demand for ion transport

The levels of (Na+ + K+)-ATPase expression during muscle development and in response to modulation of demand for ion transport were studied in chick skeletal muscle cells in culture. The number of (Na+ + K+)-ATPase molecules on the myogenic cell surface, quantified with 125I-labeled monoclonal antibodies, increased 20-fold during muscle differentiation, with a substantial increase in (Na+ + K+)-ATPase molecules/unit area of membrane. The demand for sodium ion transport by the (Na+ + K+)-ATPase was modulated by activating voltage-sensitive sodium channels with veratridine or exposing cultures to low [K+]o (0.5 mM). Exposure to veratridine (10 microM) resulted in a 60-100% increase in cell surface and a smaller increase in intracellular (Na+ + K+)-ATPase over a 24-36-h period. Neither high [K+]o (50 mM) nor Ca2+ ionophore A23187 (1 microM) produced any such change, suggesting that neither membrane depolarization nor elevated cytosolic calcium was mediating the effect of veratridine. Veratridine stimulated up-regulation was specific for the (Na+ + K+)-ATPase, blocked by tetrodotoxin, and completely reversible. The kinetics of the reversal (down-regulation) process were much faster (t1/2 = 3 h) than those of up-regulation (t1/2 = 18 h). Up-regulation of the (Na+ + K+)-ATPase by veratridine occurred by a combination of two mechanisms: the first an early phase involving a stimulated biosynthesis of the (Na+ + K+)-ATPase and a later phase in which the biosynthetic rate returned to approximately control levels while the degradation rate slowed (t1/2 control = 31 h, t1/2 veratridine = 64 h).     

Signal-transducing function of Na+-K+-ATPase is essential for ouabain's effect on [Ca2+]i in rat cardiac myocytes

We showed before that Na+-K+-ATPase is also a signal transducer in neonatal rat cardiac myocytes. Binding of ouabain to the enzyme activates multiple signal pathways that regulate cell growth. The aims of this work were to extend such studies to adult cardiac myocytes and to determine whether the signal-transducing function of Na+/K+-ATPase regulates the well-known effects of ouabain on intracellular Ca2+ concentration ([Ca2+]i). In adult myocytes, ouabain activated protein tyrosine phosphorylation and p42/44 mitogen-activated protein kinases (MAPKs), increased production of reactive oxygen species (ROS), and raised both systolic and diastolic [Ca2+]i. Pretreatment of myocytes with several Src kinase inhibitors, or overexpression of a dominant negative Ras, antagonized ouabain-induced activation of MAPKs and increases in [Ca2+]i. Treatment with PD-98059 (a MAPK kinase inhibitor) or overexpression of a dominant negative MAPK kinase 1 also ablated the effect of ouabain on MAPKs and [Ca2+]i. N-acetyl-cysteine, which blocks the effect of ouabain on ROS, did not prevent the ouabain-induced rise in [Ca2+]i. Clearly, the activation of the Ras/MAPK cascade, but not ROS generation, is necessary for ouabain-induced increases in [Ca2+]i in rat cardiac myocytes.