Incorporation of Na,K-ATPase into human erythrocyte membranes using liposomes

A procedure for incorporation of isolated cattle brain Na,K-ATPase into erythrocyte membranes by proteoliposomes has been elaborated. The Na,K-ATPase activity of proteoliposome-treated human erythrocytes containing incorporated Na,K-ATPase does not exceed that of control erythrocytes. In the erythrocyte membrane the incorporated enzyme exists in a functionally active state and retains the vector properties of the Na+-pump. Exogenous ATP stimulates 22Na influx and 86Rb efflux in and from the erythrocytes.     

Na,K-ATPase is a target of cigarette smoke and reduced expression predicts poor patient outcome of smokers with lung cancer

Diminished Na,K-ATPase expression has been reported in several carcinomas and has been linked to tumor progression. However, few studies have determined whether Na,K-ATPase function and expression are altered in lung malignancies. Because cigarette smoke (CS) is a major factor underlying lung carcinogenesis and progression, we investigated whether CS affects Na,K-ATPase activity and expression in lung cell lines. Cells exposed to CS in vitro showed a reduction of Na,K-ATPase activity. We detected the presence of reactive oxygen species (ROS) in cells exposed to CS before Na,K-ATPase inhibition, and neutralization of ROS restored Na,K-ATPase activity. We further determined whether Na,K-ATPase expression correlated with increasing grades of lung adenocarcinoma and survival of patients with smoking history. Immunohistochemical analysis of lung adenocarcinoma tissues revealed reduced Na,K-ATPase expression with increasing tumor grade. Using tissue microarray containing lung adenocarcinomas of patients with known smoking status, we found that high expression of Na,K-ATPase correlated with better survival. For the first time, these data demonstrate that CS is associated with loss of Na,K-ATPase function and expression in lung carcinogenesis, which might contribute to disease progression.     

The Na,K-ATPase of nervous tissue

The Na,K-ATPase has been only partially purified from nervous tissue, yet it is clear that two forms (and +) of the catalytic subunit are present. is a component subunit of the glial Na,K-ATPase, which has a relatively low affinity for binding cardiac glycosides and + has been identified as a subunit of the Na,K-ATPase which has relatively high affinity for cardiac glycosides. The + form may also be sensitive to indirect modulation by neurotransmitters or hormones. The ratio of + / changes in the nervous system during development, and + appears to be the predominant species in adult neurones. Changes in Na,K-ATPase activity have been associated with several abnormalities in the nervous system, including epilepsy and altered nerve conduction velocity, but a causal relationship has not been definitively established. Although the Na,K-ATPase has a pivotal role in Na⁺ and K⁺ transport in the nervous system, a special role for the glial Na,K-ATPase in clearing extracellular K⁺ remains controversial.     

Ouabain-sensitive H,K-ATPase functions as Na,K-ATPase in apical membranes of rat distal colon

Na,K-ATPase activity has been identified in the apical membrane of rat distal colon, whereas ouabain-sensitive and ouabain-insensitive H,K-ATPase activities are localized solely to apical membranes. This study was designed to determine whether apical membrane Na,K-ATPase represented contamination of basolateral membranes or an alternate mode of H,K-ATPase expression. An antibody directed against the H, K-ATPase alpha subunit (HKcalpha) inhibited apical Na,K-ATPase activity by 92% but did not alter basolateral membrane Na,K-ATPase activity. Two distinct H,K-ATPase isoforms exist; one of which, the ouabain-insensitive HKcalpha, has been cloned. Because dietary sodium depletion markedly increases ouabain-insensitive active potassium absorption and HKcalpha mRNA and protein expression, Na, K-ATPase and H,K-ATPase activities and protein expression were determined in apical membranes from control and sodium-depleted rats. Sodium depletion substantially increased ouabain-insensitive H, K-ATPase activity and HKcalpha protein expression by 109-250% but increased ouabain-sensitive Na,K-ATPase and H,K-ATPase activities by only 30% and 42%, respectively. These studies suggest that apical membrane Na,K-ATPase activity is an alternate mode of ouabain-sensitive H,K-ATPase and does not solely represent basolateral membrane contamination.     

A kinetic analysis of the action of a synaptosomal factor on Na, K-ATPase

A synaptosomal factor stimulated by neurotransmitters activates the Na, K-ATPase system effecting the phosphorylating intermediates moving the Na, K-ATPase system in the mode of simultaneous transport of Na+ and K+. This conclusion has been made during the analysis of kinetics of the effect of MgATP complex, free Mg2+ ions and ATP on Na, K-ATPase activity. Unlike the EGTA, the factor under study does not change the number of essential activators (ions of Na+ and K+) of the Na, K-ATPase system at the equimolar ATP and Mg2+ correlation.     

Na,K-ATPase beta-subunit is required for epithelial polarization, suppression of invasion, and cell motility

The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an alpha- and beta-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin-mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and beta(1)-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase beta(1)-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin-mediated cell-cell adhesion requires the Na,K-ATPase beta-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the beta(1)-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells.     

Regulation of the number of k-binding sites of Na,K-ATPase by adenosine triphosphate

A possible existence of two functional states of Na,K-ATPase with different electrogenic coefficients has been experimentally proved. Regulation of electrogenicity is achieved by alteration in the number of K+ transport sites. A transition of Na,K-ATPase from one functional state to the other has been shown to occur during the binding of ATP free ions.     

Extracellular signal-regulated kinase (ERK) participates in the hypercapnia-induced Na,K-ATPase downregulation

Hypercapnia has been shown to impair alveolar fluid reabsorption (AFR) by decreasing Na,K-ATPase activity. Extracellular signal-regulated kinase pathway (ERK) is activated under conditions of cellular stress and has been known to regulate the Na,K-ATPase. Here, we show that hypercapnia leads to ERK activation in a time-dependent manner in alveolar epithelial cells (AEC). Inhibition of ERK by U0126 or siRNA prevented both the hypercapnia-induced Na,K-ATPase endocytosis and impairment of AFR. Moreover, ERK inhibition prevented AMPK activation, a known modulator of hypercapnia-induced Na,K-ATPase endocytosis. Accordingly, these data suggest that hypercapnia-induced Na,K-ATPase endocytosis is dependent on ERK activation in AEC and that ERK plays an important role in hypercapnia-induced impairment of AFR in rat lungs.     

The effect of carnosine on the activity of Na,K,ATPase: prospective uses in clinical cardiology]

The effects of carnosine on erythrocyte membrane Na,K-ATPase and isolated enzyme in vitro as well as on membrane Na,K-ATPase activity and lipid peroxidation (LPO) in chronic heart failure (CHF) and acute myocardial infarction (AMI) have been studied. CHF and AMI have been shown to be associated with significant inhibition of the erythrocyte membrane Na,K-ATPase activity and LPO activation. Marked activation of erythrocyte membrane Na,K-ATPase by carnosine in comparison with the isolated enzyme has been established. The ability of carnosine to induce Na,K-ATPase activation and prevent membrane depolarization indicates that the dipeptide may be a useful tool in the pathogenetic therapy of CFH and AMI.     

Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K(+) and Na(+) affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481 ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K(+) affinity of Na,K-ATPase alpha1/beta1 and alpha2/beta1 isozymes but increases their apparent Na(+) affinity, dependent on phospholemman phosphorylation at Ser(68). Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of alpha1/beta1 isozymes nor the K(+) affinity of alpha1/beta1 and alpha2/beta1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of alpha2/beta1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase alpha1/beta and alpha2/beta isozymes and increases their apparent Na(+) affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase alpha2/beta but not of alpha1/beta isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.     

Intracellular Na+ controls cell surface expression of Na,K-ATPase via a cAMP-independent PKA pathway in mammalian kidney collecting duct cells

In the mammalian kidney the fine control of Na+ reabsorption takes place in collecting duct principal cells where basolateral Na,K-ATPase provides the driving force for vectorial Na+ transport. In the cortical collecting duct (CCD), a rise in intracellular Na+ concentration ([Na+]i) was shown to increase Na,K-ATPase activity and the number of ouabain binding sites, but the mechanism responsible for this event has not yet been elucidated. A rise in [Na+]i caused by incubation with the Na+ ionophore nystatin, increased Na,K-ATPase activity and cell surface expression to the same extent in isolated rat CCD. In cultured mouse mpkCCDcl4 collecting duct cells, increasing [Na+]i either by cell membrane permeabilization with amphotericin B or nystatin, or by incubating cells in a K(+)-free medium, also increased Na,K-ATPase cell surface expression. The [Na+]i-dependent increase in Na,K-ATPase cell-surface expression was prevented by PKA inhibitors H89 and PKI. Moreover, the effects of [Na+]i and cAMP were not additive. However, [Na+]i-dependent activation of PKA was not associated with an increase in cellular cAMP but was prevented by inhibiting the proteasome. These findings suggest that Na,K-ATPase may be recruited to the cell membrane following an increase in [Na+]i through cAMP-independent PKA activation that is itself dependent on proteasomal activity.     

The molecular mechanism of the Na, K-ATPase system

Some new properties of Na,K-ATPase system have been revealed using the kinetic analysis of the complex enzymic systems. The fundamental mechanism of Na,K-ATPase functioning has been interpreted and the minimum model including all known working modes of the enzyme under different conditions has been built. The existence of new unknown modes and properties of Na,K-ATPase is predicted and confirmed by different authors.     

Human myocardial Na,K-ATPase concentration in heart failure

The Na,K-ATPase is of major importance for active ion transport across the sarcolemma and thus for electrical as well as contractile function of the myocardium. Furthermore, it is receptor for digitalis glycosides. In human studies of the regulatory aspects of myocardial Na,K-ATPase concentration a major problem has been to obtain tissue samples. Methodological accomplishments in quantification of myocardial Na,K-ATPase using vanadate facilitated ³H-ouabain binding to intact samples have, however, made it possible to obtain reliable measurements on human myocardial necropsies obtained at autopsy as well as on biopsies of a wet weight of only 1–2 mg obtained during heart catheterisation. However, access to the ultimately, normal, vital myocardial tissue has come from the heart transplantation programs, through which myocardial samples from cardiovascular healthy organ donors have become available. In the present paper we evaluate the various values reported for normal human myocardial Na,K-ATPase concentration, its regulation in heart disease and the association with digitalization. Normal myocardial Na,K-ATPase concentration level is found to be 700 pmol/g wet weight. No major variations were found between or within the walls of the heart ventricles. During the first few years of life a marked decrease in myocardial Na,K-ATPase concentration is followed by a stable level obtained in early adulthood and normally maintained throughout life. In patients with enlarged cardiac x-ray silhouette a significant positive, linear correlation between left ventricular ejection fraction (EF) and Na,K-ATPase concentration was established. A maximum reduction in Na,K-ATPase concentration of 89% was obtained when EF was reduced to 20%. Generally, heart failure associated with heart dilatation, myocardial hypertrophy as well as ischaemic heart disease is associated with reductions in myocardial Na,K-ATPase concentration of around 25%. During digoxin treatment of heart failure patients a further reduction in functional myocardial Na,K-ATPase concentration of 15% has been found. Thus, the total reduction in functional myocardial Na,K-ATPase concentration in digitalised heart failure patients may well be of the magnitude 40%. In conclusion, it has become possible to quantify human myocardial Na,K-ATPase in health and disease. Revealed reductions are in heart failure of importance for contractile function, generation of arrhythmia and for digoxin treatment.     

A new approach for determination of Na,K-ATPase activity: application to intact pancreatic β-cells

It has been postulated that a decrease in Na,K-ATPase-mediated ion gradients may be a contributing mechanism to insulin secretion. However, the precise role of the Na,K-ATPase in pancreatic β-cell membrane depolarization and insulin secretion signalling have been difficult to evaluate, mostly because data reporting changes in enzymatic activity have been obtained in cell homogenates or membrane preparations, lacking intact intracellular signalling pathways. The aim of this work was to develop a method to characterize Na,K-ATPase activity in intact pancreatic β-cells that will allow the investigation of putative Na,K-ATPase activity regulation by glucose and its possible role in insulin secretion signalling. This work demonstrates for the first time that it is possible to determine Na,K-ATPase activity in intact pancreatic β-cells and that this is a suitable method for the study of the mechanisms involved in the Na,K-ATPase regulation and eventually its relevance for insulin secretion signalling.     

Stimulation of Na,K-ATPase activity of frog skeletal muscle by insulin

Na,K-ATPase activity of a plasma membrane fraction obtained from frog skeletal muscles was increased approximately two-fold by exposing muscles to insulin, whereas the addition of insulin to a membrane preparation suspension has no effect on Na,K-ATPase activity. The effect of insulin on Na,K-ATPase activity of whole muscles was specific to insulin and insulin derivatives that had the ability of receptor-binding and was not inhibited by actinomycin D. Insulin also induced a development of Na,K-ATPase activity in muscles whose Na,K-ATPase activity had been blocked by ouabain-pretreating. Such a insulin action was inhibited by monensin. These observations suggest that insulin stimulates the monensin-sensitive intracellular transport of membrane proteins which should be responsible for the increase in Na/K pumping activity.     

Regulation of Na,K-ATPase by synaptosomal factors and neurotransmitters

The identical increase of Na, K-ATPase activity is caused by oxidated and reduced forms of noradrenaline, serotonin and dopamine through the synaptosomal activating factors. The synaptosomal inhibiting factor, orthovanadate and calcium ions independently inhibit Na, K-ATPase activity. The inhibition constant (Ki) for vanadate does not change in the presence of EDTA, whereas in the presence of synaptosomal factors regulating the Na, K-ATPase factors, noradrenaline causes drastic increase of Ki for vanadate. It has been concluded, that the data point to the existence of special regulating system of brain synaptosomal Na, K-ATPase.     

The beta-adrenergic regulation of the Na, K-ATPase activity in the sarcolemma of the heart muscle

Using a sensitive potentiometric method the effect of isoproterenol upon the activity of Na, K-ATPase in cardiomyocytes has been studied. The activity of the enzyme in rat sarcolemma at isoproterenol-induced myocarditis decreases by 42%. A direct action of isoproterenol on the Na, K-ATPase activity in sarcolemma in vitro has been investigated. In the concentration range 10(-9)-10(-3) M (from receptor-binding up to cardiotoxic) a gradual decrease of the activity reaching the complete inhibition at 10(-3) M is revealed. Antagonist of beta-adrenoreceptors propranolol in concentrations required for displacing the agonist (10(-9) M) provides for the recovery of the Na, K-ATPase activity up to 76% of its normal activity. This action transforms into nonspecific inhibition at rising concentration of the antagonist. Possible mechanisms of the beta-adrenergic regulation effect in cardiomyocytes on Na, K-ATPase of the sarcolemma are discussed.     

Distinct role of the N-terminal tail of the Na,K-ATPase catalytic subunit as a signal transducer

Mounting evidence suggests that the ion pump, Na,K-ATPase, can, in the presence of ouabain, act as a signal transducer. A prominent binding motif linking the Na,K-ATPase to intracellular signaling effectors has, however, not yet been identified. Here we report that the N-terminal tail of the Na,K-ATPase catalytic alpha-subunit (alphaNT-t) binds directly to the N terminus of the inositol 1,4,5-trisphosphate receptor. Three amino acid residues, LKK, conserved in most species and most alpha-isoforms, are essential for the binding to occur. In wild-type cells, low concentrations of ouabain trigger low frequency calcium oscillations that activate NF-kappaB and protect from apoptosis. All of these effects are suppressed in cells overexpressing a peptide corresponding to alphaNT-t but not in cells overexpressing a peptide corresponding to alphaNT-t deltaLKK. Thus we have identified a well conserved Na,K-ATPase motif that binds to the inositol 1,4,5-trisphosphate receptor and can trigger an anti-apoptotic calcium signal.     

The sodium pump needs its beta subunit

The sodium pump Na,K-ATPase, located in the plasma membrane of all animal cells, is a member of a family of ion-translocating ATPases that share highly homologous catalytic subunits. In this family, only Na,K-ATPase has been established to be a heterodimer of catalytic (alpha) and glycoprotein (beta) subunits. The beta subunit has not been associated with the pump's transport or enzymatic activity, and its role in Na,K-ATPase function has been, until recently, a puzzle. In this review we describe what is known about the structure of beta and summarize evidence that expression of both alpha and beta subunits is required for Na,K-ATPase activity, that inhibition of glycosylation causes a decrease in accumulation of both alpha and beta subunits, and we provide evidence that pretranslational up-regulation of beta alone can lead to increased abundance of sodium pumps. These findings are all consistent with the hypothesis that the beta subunit regulates, through assembly of alpha beta heterodimers, the number of sodium pumps transported to the plasma membrane.     

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

The heterodimeric Na,K-ATPase has been implicated in vertebrate and invertebrate epithelial cell junctions, morphogenesis and oncogenesis, but the mechanisms involved are unclear. We previously showed that the Drosophila Na,K-ATPase is required for septate junction (SJ) formation and that of the three beta-subunit loci, only Nrv2 isoforms support epithelial SJ barrier function and tracheal tube-size control. Here we show that Nrv1 is endogenously co-expressed with Nrv2 in the epidermis and tracheal system, but Nrv1 has a basolateral localization and appears to be excluded from the Nrv2-containing SJs. When the normally neuronal Nrv3 is expressed in epithelial cells, it does not associate with SJs. Thus, the beta-subunit is a key determinant of Na,K-ATPase subcellular localization as well as function. However, localization of the Na,K-ATPase to SJs is not sufficient for junctional activity because although several Nrv2/Nrv3 chimeric beta-subunits localize to SJs, only those containing the extracellular domain of Nrv2 have junctional activity. Junctional activity is also specific to different alpha-subunit isoforms, with only some isoforms from the major alpha-subunit locus being able to provide full barrier function and produce normal tracheal tubes. Importantly, mutations predicted to inactivate ATPalpha catalytic function do not compromise junctional activity, demonstrating that the Drosophila Na,K-ATPase has an ion-pump-independent role in junction formation and tracheal morphogenesis. These results define new functions for the intensively studied Na,K-ATPase. Strikingly, the rat alpha1 isoform has full junctional activity and can rescue Atpalpha-null mutants to viability, suggesting that the Na,K-ATPase has an evolutionarily conserved role in junction formation and function.