Na+, K(+)-ATPase, endogenous cardiotonic steroids and their transducing role

Na+, K(+)-ATPase--a protein complex of plasmatic membrane, which performs the dual function: firstly, it supports the Na+ and K+ homeostasis, and also transmembrane potential gradient, secondly, it serves as the transducer of signals and as the regulator of the expression of many key genes. Endogenous cardiotonic steroids, which are synthesized in the adrenal glands and hypothalamus, serve as the signal molecules. New concepts about the mechanisms of the realization of the Na+, K(+)-ATPase signal function and their connection with cellular functions, apoptosis, and with pathologies of cardiovascular system and water-salt homeostasis are described in the survey.     

Isolation and identification of hemin as an endogenous Na+/K(+)-ATPase inhibitor from porcine blood cells.

A substance which is a potent inhibitor of Na+/K(+)-ATPase activity and competitively displaces [3H]ouabain binding to this enzyme was isolated from porcine blood cells. From its chemical and physiochemical properties, this activity was identified as hemin (chloroprotohemin IX). Hemin showed a dose dependent curve for Na+/K(+)-ATPase inhibitory activity similar to that of ouabain and displaced [3H]ouabain binding as potent as 1/100 of ouabain itself.     

Novel peptide inhibitor (SPAI) of Na+, K+-ATPase from porcine intestine

Three unique inhibitors (SPAI-1, -+2, and -3) were first purified from porcine duodenal extract based on the Na+, K+-ATPase inhibitory activity. These peptide inhibitors had four disulfide bridges in common. The sequencing results of their S-carboxymethyl derivatives, lysilendopeptidase fragments, and chymotryptic peptides disclosed their entire primary structures. Both SPAI-2 and -3 consisted of 61 amino acids, respectively, and had almost the same sequences except for two amino acid substitutions, while SPAI-1 was found to lack the N-terminal twelve amino acid sequence of SPAI-2. The kinetics study revealed that SPAIs inhibited Na+, K+-ATPase by the competitive mode against Na+ and were uncompetitive with K+.     

Dissociation of Na+,K+-ATPase inhibition from digitalis inotropy.

Recent findings from our laboratory as well as those of other laboratories do not support the postulation that the mechanism of the positive inotropic action of digitalis is due to inhibition of NA,K-ATPase. Using short-acting digitalis steroids and drug washout experiments, in isolated myocardial preparations, it has been demonstrated that Na,K-ATPase isolated from such preparations is still significantly inhibited, whereas the positive inotropic effect is no longer present. Also, based on kinetic measurements the two exponential rate constants observed for drug half-life, a rapid and slow phase, were found to be associated, respectively, with the very short inotropic half-life and the very long enzyme inhibition half-life. In addition, a dissociation of the transient inotropic effects of digitalis was observed from the long lasting cardiotoxic effects of digitalis during drug washout. Moreover, a temporal correlation was noted between the persistent inhibitory effects of digitalis on Na,K-ATPase and the persistent cardiotoxic effects of digitalis. Therefore, it is concluded that inhibition of Na,K-ATPase is not responsible for the positive inotropic action of digitalis, but may be the mechanism, at least in part, for certain cardiotoxic effects of digitalis.     

A marine algal Na(+)-activated ATPase possesses an immunologically identical epitope to Na+,K(+)-ATPase.

Immunological homology was investigated between Heterosigma akashiwo (a marine algae) Na(+)-activated ATPase and animal Na+,K(+)-ATPase. The former polypeptide [(1989) Plant Cell Physiol. 30, 923-928] reacted with anti-serum raised against the amino-terminal half of the pig kidney Na+,K(+)-ATPase alpha subunit. It is suggested that the Na+,K(+)-ATPase epitope within the amino-terminal region is conserved in the plant Na(+)-activated ATPase, and the region containing the epitope may be important for Na ion transport.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase.

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

Na+,K(+)-ATPase activity in human colorectal carcinoma

Na+,K(+)-ATPase activities in macroscopically unchanged mucosa (conditionally normal tissue) and human colorectal carcinoma (mainly low-grade and moderately differentiated adenocarcinomas) have been investigated. Microsomal fractions are similar by dimensions of the membrane fragments detected by photon correlation spectroscopy analysis. The activation optima under digitonin pretreatment of the membrane fractions differ significantly for Na+,K(+)-ATPase and concomitant Mg(2+)-ATPase activity, but are the same in conditionally normal and cancerous tissues. This allows to detect correctly total levels of the Na+,K(+)-ATPase activity in the detergent-pretreated preparations. The moderate decrease of the Na+,K(+)-ATPase activity is revealed in carcinomas. It is concluded that a decrease of activity of the ouabain-sensitive human Na+,K(+)-ATPase is characteristic of colorectal carcinoma.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

Hydrolysis by acylphosphatase of erythrocyte membrane Na+, K(+)-ATPase phosphorylated intermediate.

Acylphosphatase, purified from human erythrocytes, actively hydrolyzes the phosphoenzyme intermediate of human red blood cell membrane Na+, K(+)-ATPase. This effect occurred with acylphosphatase amounts (up to 10 units/mg membrane protein) that fall within the physiological range. Acylphosphatase addition to erythrocyte membranes resulted in a significant increase in the rate of Na+, K(+)-dependent ATP hydrolysis. Maximal stimulation, observed with 10 units/mg membrane protein, was of about 80% over basal value. The same acylphosphatase amount enhanced of about 40% the rate of ATP driven Na+ transport into inside out red cell membrane vesicles. Taken together these findings suggest a potential role of acylphosphatase in the control of the activity of erythrocyte membrane Na,K pump.     

Digitalis sensitivity of Na+,K(+)-ATPase, myocytes and the heart.

Cardiac Na+,K(+)-ATPase, the receptor molecule for digitalis glycosides, have isoforms with different intrinsic affinities for the glycosides. Expression of these isoforms are under developmental and hormonal regulation. Switching in isoforms to those with lower intrinsic affinity may decrease digitalis sensitivity of the heart. In addition to the intrinsic affinity of the cardiac Na+,K(+)-ATPase for the glycoside, increases in the rate of Na+ influx and decreases in extracellular K+ concentrations increase glycoside sensitivity of the heart and also reduces the margin of safety by reducing reserve capacity of the sodium pump. Reserve capacity of the sodium pump is also reduced by pathological conditions or aging, resulting in reduced margin of safety for the glycoside. Events that follow sodium pump inhibition also affect sensitivity of the heart to digitalis toxicity. These are hypercalcemia and magnesium depletion. It is now feasible to predict digitalis sensitivity of the heart, not empirically but based on the understanding of the mechanisms responsible for the positive inotropic and toxic actions of the glycoside.     

Demonstration of an endogenous activator for the Na+, K+-ATPase system

A heat-labile, non-dialysable and protease-sensitive endogenous activator (NaAF) capable of stimulating the Na⁺, K⁺-ATPase system has been demonstrated. The activator (NaAF) activity was partially enriched (about 10 fold) by dialysis (30 kDa cutoff) under negative pressure and pH 4.8 precipitation. The NaAF has been found to occur in the cytosolic fractions of tissues such as the kidney and brain from two different species (rabbit and pig) tested so far. Also, the factor from one tissue stimulates with equal efficacy the Na⁺, K⁺-ATPase systems of other tissues regardless of the species; thus demonstrating universal nature of the activator. Some degree of cross-reactivity was noted between the activating effects of this activator (for the Na⁺,K⁺-ATPase) and that for the H⁺,K⁺-ATPase recently described (J. Biol. Chem. 262:5664–5670, 1987). The purified NaAF obtained from sephacryl S-300 column chromatography activates the pure renal medullary Na⁺,K⁺-ATPase in a dose dependent manner.A preliminary account of this work was published in Fed. Proc. 46(4): 4466, 1987     

Brain Na+,K(+)-ATPase inhibition induced by arginine administration is prevented by vitamins E and C

Hyperargininemia is a metabolic disorder caused by deficiency of arginase activity resulting in tissue accumulation of arginine and neurological dysfunction. We have previously demonstrated that arginine induces oxidative stress and decreases Na⁺,K⁺-ATPase in rat midbrain. In the present study we investigated the action of vitamins E and C on the inhibition of Na⁺,K⁺-ATPase provoked by arginine in the midbrain of 60-day-old rats. Animals were pretreated for 1 week with daily IP administration of saline (control) or vitamins E (40 mg/kg) and C (100 mg/kg). Twelve h after the last injection, animals received one injection of arginine (0.8 mol/g of body weight) or saline. Chemiluminescence was significantly increased, whereas total antioxidant capacity and Na⁺,K⁺-ATPase activity were significantly decreased. Furthermore, treatment with vitamins E and C prevented these effects. If these effects also occur in the human condition, it is possible that antioxidant administration might slow the progression of neurodegeneration in this disorder.     

Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump.

The giant-patch technique was used to study the Na+,K(+)-ATPase in excised patches from rat or guinea pig ventricular myocytes. Na+,K(+)-pump currents showed a saturable ATP dependence with aK(m) of approximately 150 microM at 24 degrees C. The pump current can be completely abolished by ortho-vanadate. Dissociation of vanadate from the enzyme in the absence of extracellular Na+ was slow, with a Koff of 3.10(-4) S-1 (K1 approximately 0.5 microM, at 24 degrees C). Stationary currents were markedly dependent on intracellular pH, with a maximum at pH 7.9. Temperature-dependence measurements of the stationary pump current yielded an activation energy of approximately 100 kJ mol-1. Partial reactions in the transport cycle were investigated by generating ATP concentration jumps through photolytic release of ATP from caged ATP at pH 7.4 and 6.3. Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slower decay to a stationary current. It was concluded that the fast rate constant of approximately 200 s-1 at 24 degrees C (pH 6.3) reflects a step rate-limiting the electrogenic Na+ release. Simulating the data with a simple three-state model enabled us to estimate the turnover rate under saturating substrate concentrations, yielding rates (at pH 7.4) of approximately 60 s-1 and 200 s-1 at 24 degrees C and 36 degrees C, respectively.     

Vascular smooth muscle expresses a truncated Na+, K(+)-ATPase alpha-1 subunit isoform.

By regulating transmembrane Na+ and K+ concentrations and membrane potential, the Na+,K(+)-ATPase plays an important role in regulating cardiac, skeletal, and smooth muscle function. A high degree of amino acid sequence and structural identity characterizes the three Mr 100,000 Na+,K(+)-ATPase alpha subunit isoforms expressed in cardiac and skeletal muscle. Strikingly, vascular smooth muscle utilizes alternative RNA processing of the alpha-1 gene to express a structurally distinct Mr approximately 65,000 isoform, alpha 1-T (truncated). Analysis of both its mRNA and protein structure reveals that alpha-1-T represents a major, evolutionarily conserved, truncated Na+,K(+)-ATPase isoform expressed in vascular smooth muscle. This demonstrates an unexpected complexity in the regulation of vascular smooth muscle Na+,K(+)-ATPase gene expression and suggests that a structurally novel, truncated alpha subunit may play a role in vascular smooth muscle active ion transport.     

Fluorescence competition assay for the assessment of ATP binding to an isolated domain of Na+, K(+)-ATPase

An equation allowing estimation of the dissociation constant for binding of a non-fluorescent ligand to the enzyme is presented that is based on the competitive replacement of the ligand by its fluorescent analog. We derived an explicit formula for the probe fluorescence intensity, which is suitable for nonlinear least-squares analysis. We used this formula to evaluate the binding of ATP to the large cytoplasmic loop of Na+,K(+)-ATPase. The estimated value of KD (6.2+/- 0.7 mM) is comparable with the results from other laboratories for similar constructs obtained by a different method.     

Na+/K(+)-ATPase regulation by neurotransmitters.

A long period of experimental work has led to the conclusion that Na+/K(+)-ATPase is the enzymatic version of the Na+/K+ pump. This enzymatic system is in charge of various important cell functions. Among them cationic equilibrium and recovering of resting membrane potential in neurons is relevant. A tetrameric ensemble of peptides conform the system known as alpha and beta subunits. The alpha subunit is subdivided in alpha 1, alpha 2 and alpha 3, according to different location and properties. Regulatory factors intrinsic to the Na+/K(+)-ATPase system are: ATP, Na+ and Mg2+ concentrations inside the cell, and K+ outside. The enzyme activity is also regulated by extrinsic factors like some hormones (insulin and thyroxine). Induction of gene expression or post-translational modifications of the preexisting pool of the enzyme are the basic mechanisms of regulation proposed. Other extrinsic factors that seem to regulate the enzyme activity are some neurotransmitters. Among them the most extensively studied are catecholamines, mainly norepinephrine (NE) and lately serotonin (5-HT). The mechanism suggested for NE activation of the enzyme seems to involve specific receptors or a non-specific chelating action related to the catechol group that would relieve the inhibition by divalent cations. Another possibility is that NE removes an endogenous inhibitory factor present in the cytoplasm. The Na+/K(+)-ATPase is activated also by 5-HT. In vivo pharmacological and nutriological manipulations of brain 5-HT are accompanied by parallel responses of Na+/K(+)-ATPase activity. Serotonin agonists do activate the enzyme and antagonists neutralize the activation. In vitro there is a different dose dependent activation, according to the brain region. The mechanism involved seems to implicate a specific receptor system. Serotonin-Na+/K(+)-ATPase interaction in the rat brain is probably of functional relevance because it disappears in amygdaloid kindling. Also it seems to influence the ionic regulation of the pigment transport mechanism in crayfish photoreceptors. In relation to other neurotransmitters, a weak response to histamine was observed with acetylcholine, GABA and glutamic acid, the results were negative.     

Stimulation of the Na+,K(+)-ATPase activity of K562 human erythroleukemia cells by triiodothyronine.

The effect of triiodothyronine (T3) on Na+,K(+)-ATPase activity of K562 human erythroleukemic cell was studied to understand why the erythrocyte sodium pump activity is decreased in hyperthyroidism. Na+,K(+)-ATPase activity of K562 cell lysates was assayed by measuring the release of inorganic phosphate (Pi) from ATP. Na+,K(+)-ATPase activity of K562 cell grown in the presence of T3 for 48 hours was significantly higher than that of control (0.98 +/- 0.05 mumol Pi h-1 mg protein-1 vs 0.82 +/- 0.10 mumol Pi h-1 mg protein-1, p < 0.05). The Na+,K(+)-ATPase activity could be stimulated in a time- and concentration-dependent manner; maximum stimulatory effect of T3 was seen at a concentration of 10(-7) mol/L. When an inducer [cytosine-beta-D-arabino-furanoside (ARA-C)] was added to the culture medium, the K562 cells showed signs of differentiation and synthesised haemoglobin. At the same time, the Na+,K(+)-ATPase activity remained high. We conclude that T3 stimulates Na+,K(+)-ATPase activity of K562 cells and in the presence of T3 during differentiation, the enzyme activity remains high.     

Interaction of sodium and potassium ions with Na+,K(+)-ATPase. IV. Affinity change for K+ and Na+ of Na+,K(+)-ATPase in the cycle of the ATP hydrolysis reaction

The Kd for ouabain-sensitive K+ or Rb+ binding to Na+,K(+)-ATPase was determined by the centrifugation method with radioactive K+ and Rb+ in the presence of various combinations of Na+, ATP, adenylylimidodiphosphate (AMPPNP), adenylyl-(beta,gamma-methylene)diphosphonate (AMPPCP), Pi, and Mg2+. From the results of the K+ binding experiments, Kd for Na+ was estimated by using an equation describing the competitive inhibition between the K+ and Na+ binding. 1) The Kd for K+ binding was 1.9 microM when no ligand was present. Addition of 2 mM Mg2+ increased the Kd to 15-17 microM. In the presence of 2 mM Mg2+, addition of 3 mM AMPPCP with or without 3 mM Na+ increased the Kd to 1,000 or 26 microM, respectively. These Kds correspond to those for K+ of Na.E1.AMPPCPMg or E1.AMPPCPMg, respectively. 2) Addition of 4 mM ATP with or without 3 mM Na+ decreased the Kd from 15-17 microM to 5 or 0.8 microM, respectively. Because the phosphorylated intermediate was observed but ATPase activity was scarcely observed in the K+ binding medium containing 3 mM ATP and 2 mM Mg2+ in the absence of Na+ as well as in the presence of Na+ at 0 degrees C, it is suggested that K+ binds to E2-P.Mg under these ligand conditions. 3) The Kd for Na+ of the enzyme in the presence of 3 mM AMPPCP or 4 mM ATP with Mg2+ was estimated to be 80 or 570 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)