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.     

Characterization of the macrocyclic carbon suboxide factors as potent Na,K-ATPase and SR Ca-ATPase inhibitors

Recently discovered macrocyclic carbon suboxide (MCS) factors with the general formula (C(3)O(2))(n) were found to strongly inhibit rabbit and rat Na,K-ATPase as well as SR Ca-ATPase. Highly active MCS factors were obtained by a base/acid treatment of their lipophilic precursor isolated from plants. In the ESI-MS spectra, the dominant molar mass ion of 431 Da corresponds to a 1:1 complex of the carbon suboxide hexamer (n=6; M(r)=408 Da) with a Na(+) ion. Additional mass ions identified in positive and negative ion mode were assigned as complexes of the MCS hexamer (n=6) and octamer (n=8) with Na(+) or with TFA(-) in various ratios. The dominant mass ion values of these active MCS factors from plants are also found in mass spectra of previously described endogenous digitalis-like factors (EDLF) from animals. This would suggest that ubiquitously distributed MCS factors may function as putative endogenous regulatory substances of Na,K-ATPase and possibly of other ATPases. With the symmetric display of several equivalent carbonyl or hydroxy groups, the structure of MCS factors is particularly suited for interactions with proteins and other bio-molecules. This could explain the high biological activity and the unusual properties of the MCS factors.     

Ouabain-induced blockade of alpha2 isoform of the Na,K-ATPase on electrophysiological and contractile characteristics of the rat diaphragm

In experiments with isolated neuromuscular preparation of the rat diaphragm, selective blockade of alpha2 isoform of the Na,K-ATPase with ouabain (1 mcmol/L) induced steady depolarization of muscle fibers that reached a maximum of 4 mV, a decrease in amplitude of muscle fiber action potential, and prolonged raising and decline phases of the action potential. At the same time, the force, time to peak, and half relaxation time of the isometric muscle twitch were increased, as well as the area under the contraction curve. During continuous fatiguing stimulation (2/s), a more pronounced decline of contraction speed was observed in presence of ouabain; dynamics of the half-relaxation time remaining unchanged. It is suggested that blockade of alpha2 isoform of the Na,K-ATPase impairs excitation-contraction coupling resulting in a delay of Ca2+ release from sarcoplasmic reticulum. The increase in contraction force seems to result from a mechanism similar to that of positive inotropic effect of cardiac glycosides in heart muscle. Physiological significance of the skeletal muscle alpha2 isoform of the Na,K-ATPase in regulation of Ca2+ and Na+ concentrations near triadic junctions and in regulatory processes involving the Na,K-ATPase endogenous modulators or transmitter acetylcholine is discussed.     

The endogenous inhibitor of NCX1 does not resemble the properties of digitalis compound

In our previous study, we ware successful in isolation and purification of an endogenous inhibitor of the Na/Ca exchanger (NCX1) from the calf ventricle extracts. The purified factor has characterized to have strong positive inotropic effect on isometric contractions of isolated ventricle strips of guinea pig. A possibility is that besides the NCX1 the endogenous factor may also interact with other ion-transport systems (e.g., Na,K-ATPase) involved in modulation of muscle contractility-relaxation. Therefore, a primary goal of the present study was to detect a possible effect of newly found NCX1 inhibitor on Na,K-ATPase and Ca-ATPase activities. The preparations of isolated sarcolemma vesicles were used for this goal. Although the crude extracts of calf ventricles can inhibit both the Na/Ca exchange and Na,K-ATPase, these two inhibitory activities can be separated on the Sephadex G-10 column, meaning that different molecular entities might be responsible for inhibition of Na/Ca exchange and Na,K-ATPase. Addition of 100 U of purified endogenous factor to the assay medium results in nearly complete inhibition of forward (Na(i)-dependent Ca-uptake) and reverse (Na(o)-dependent Ca-efflux) modes of Na/Ca exchange. On the other hand, no effect was detected on activities of Na,K-ATPase and Ca-ATPase even in the presence of 500 U of purified factor in the assay medium. In light of the present data, it is concluded that the endogenous inhibitor of NCX1 does not resemble the targeting properties of digitalis like compound. Obviously, more systematic studies are required in the future for resolving a possible interaction of the endogenous inhibitor of NCX1 with other ion-transport systems involved in calcium homeostasis and action potential.     

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.     

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.     

Chronic potassium supplementation of newborn dogs increases cortical Na,K-ATPase but not urinary potassium excretion

The distal nephron of the newborn dog cannot secrete an acute potassium load as efficiently as can that of the adult dog. Distal nephron potassium secretion is dependent upon basolateral Na,K-ATPase activity. Because Na,K-ATPase activity is lower in the immature than the mature distal nephron, it was hypothesized that lower Na,K-ATPase activity may be responsible for the lower potassium secretory capacity of the immature nephron. In the adult, chronic high dietary potassium intake increases renal tubular potassium secretory capacity by increasing Na/K pump abundance in distal nephron segments responsible for potassium secretion. Therefore, in order to test the above hypothesis, renal cortical and outer medullary Na,K-ATPase activity under Vmax conditions (a measure of pump abundance) and urinary potassium excretion during acute potassium loading were determined in 7 age-matched, litter mate pairs (chronically potassium supplemented versus control) newborn dogs. The potassium supplemented member of each pair received 6 mmol.day-1.kg-1 of KCl as a 150 mM solution for 7-21 days after birth and the control member received an equal volume of water for the same period of time. This protocol resulted in a doubling of renal cortical Vmax Na,K-ATPase activity in the potassium supplemented animals (from 369 +/- 186 to 718 +/- 286 nmol Pi liberated.h-1.micrograms DNA-1, P = 0.025). There was no significant change in outer medullary enzyme activity. Contrary to the above hypothesis, this increase in cortical enzyme activity was not associated with increased potassium excretion at baseline or during acute potassium loading.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epithelial Na,K-ATPase expression is down-regulated in canine prostate cancer; a possible consequence of metabolic transformation in the process of prostate malignancy

Background  

An important physiological function of the normal prostate gland is the synthesis and secretion of a citrate rich prostatic fluid. In prostate cancer, citrate production levels are reduced as a result of altered cellular metabolism and bioenergetics. Na, K-ATPase is essential for citrate production since the inward Na⁺ gradients it generates are utilized for the Na⁺ dependent uptake of aspartate, a major substrate for citrate synthesis. The objective of this study was to compare the expression of previously identified Na, K-ATPase isoforms in normal canine prostate, benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma (PCa) using immunohistochemistry in order to determine whether reduced citrate levels in PCa are also accompanied by changes in Na, K-ATPase expression.     

Interaction of hypothalamic Na,K-ATPase inhibitor with isolated human peripheral blood mononuclear cells

A ligand for the digitalis receptor located on the membrane-embedded Na,K-ATPase (NKA; EC 3.6.1.37) has been isolated from bovine hypothalamus (hypothalamic inhibitory factor; HIF) and identified as isomeric ouabain (Tymiaket al, 1993,Proc. Natl. Acad. Sci. 90: 8189–8193). In analogy to cardioactive steroids (CS) derived from plants or from toad, HIF inhibits the Na/K-exchange process and the ATPase activity of isolated Na,K-ATPase although by a different molecular action mechanism. In the present work we show that, as plant-derived ouabain, HIF inhibits⁸⁶Rb-uptake by isolated human lymphocytes with an IC₅₀ of about 20 nM; above this concentration HIF reduces cell viability in contrast to ouabain. The decrease in cell viability by excess HIF is accompanied by discrete morphological alterations (mitochondrial swelling) visible by transmission electron microscopy of ultra-thin sectioned peripheral blood mononuclear cells. Taken together the results show that the hypothalamic NKA inhibitor blocks NKA of isolated human lymphocytes with high potency at nanomolar concentrations without toxicity; concentrations exceeding the ones required to block⁸⁶Rb-uptake reduce cell viability, probably due to leak formation across the NKA molecule. Thus, lymphocytes constitute a potential target for HIF action and by their altered NKA status a possible messenger between the nervous and the immune system.Abbreviations D-PBS Dulbecco's phosphate buffered saline - HBSS Hank's balanced salt Solution - NKA Na,K-ATPase     

Effect of Fe3+ on Na,K-ATPase: Unexpected activation of ATP hydrolysis

Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na⁺ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl₃ increased the intracellular storage of iron, catalase activity, the production of H₂O₂ and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.     

Endogenous digitalis-like factor as a stimulator of endothelin secretion from endothelial cells

Effects of human urine-derived endogenous digitalis-like factor (EDLF) and ouabain on endothelin (ET) secretion were examined in cultured endothelial cells. ET was secreted in a linear fashion over 5 hours from bovine pulmonary artery endothelium into serum-free medium. EDLF stimulated ET secretion in a dose-dependent manner. In contrast, ouabain did not affect ET secretion at the concentration of 10(-9)-10(-5) M. These results indicate that human urine-derived EDLF is distinct from plant-derived ouabain and act as a stimulator of ET secretion by endothelial cells.     

The Na/K-ATPase regulation by neurotransmitters in ontogeny

Activity of the Na/K-ATPase from rat brain synaptic membranes is inhibited by NA (noradrenaline). However, during fractionation of cytozole from nerve endings, two non-homogeneous peaks are found (SF(a), 60-100 kD and SF( i ),;10 kD), which influence the Na/K-ATPase activity, both directly and SF(a) NA-dependently. Joint action of NA and synaptic factors (SF(a) and SF(i)) on the Na/K-ATPase, represents a sum of four different processes: 1) NA, without synaptic factors, inhibits the Na/K-ATPase; 2) At low SF(a) concentrations NA-dependent Na/K-ATPase activatory mechanism is evident; 3) At high SF(a) concentrations NA-independent Na/K-ATPase is activated; 4) The low-molecular SF(i) protein inhibits the Na/K-ATPase. Regulation of the Na/K-ATPase activity by NA, SF(a) and SF( i), obtained in similar conditions from two weeks old and one year old rats, is different. In older rats SF(i) is characterized with strong Na/K-ATPase inhibition; in younger rats SF(i) does not change the Na/K-ATPase activity. The NA- and SF(i) -dependent inhibition and activation ratio is different in young and elder rats. In two week olds NA/SF(i) activatory mechanism is stronger, while in one year olds NA-dependent inhibition of the Na/K-ATPase is prevailing. These experimental data indicate that regulation of the Na/K-ATPase activity has an important role in synaptic transmission and that this process has noteworthy, albeit presently unknown, functional importance in integrative activity of the brain.     

Gill Na,K-ATPase in the spiny lobster Palinurus elephas and other marine osmoconformers. Adaptiveness of enzymes from osmoconformity to hyperregulation

Haemolymph inorganic osmolyte changes and Na,K-ATPase activities in trichobranchiate and epipodite tissues were examined in the spiny lobster Palinurus elephas gradually acclimated from seawater (SW; 38 ppt, salinity; 1291 mOsmol/l) down to dilute seawater (DSW; 20 ppt, salinity; 679 mOsmol/l). During acclimation to DSW haemolymph was only transiently hypoosmotic, becoming isosmotic to the medium over a 24-h period of acclimation. Na,K-ATPase specific activities in homogenates of the trichobranchiate gills from SW- and DSW-acclimated spiny lobsters were in the range of 2-3 μmol Pi/h/mg protein and were not significantly different. It has also been confirmed for the marine stenohaline crustaceans Maja crispata and Dromia personata that gill Na,K-ATPase maintains the same level of specific activity in SW- and DSW-acclimated crabs. The saponin-treated fraction of Na,K-ATPase activity in trichobranchiate gills was 67-89% and epipodites 63-64% over the native homogenates' activity and no differences in enzyme activities upon saponin treatment between SW- and DSW-acclimated spiny lobsters were found. Recovery of 6% and enrichment factor (1.6) of Na,K-ATPase in partially purified plasma membrane fractions of epipodites was relatively low and not different in SW- and DSW-acclimated spiny lobsters. In the hemiepipodite, negative short-circuit current was in the range from -16.7 to -22.7 μA cm(-2) and conductance varied in the range of 205-290 mS cm(-2), values which were not significantly different in spiny lobsters residing in SW or DSW. Very high conductance suggests leakiness of the hemiepipodite epithelium-cuticular complex. In contrast to the group of euryhaline hyperosmoregulating Crustacea in which activation of the specific activity of Na,K-ATPase upon acclimation to dilute seawater occurs, in marine osmoconformers there is no activation of the enzyme in dilute seawater. Based on the literature data and our own results, we have reported a correlation coefficient of 0.65 between specific activity of Na,K-ATPase and the sodium gradient (mmol Na/l; haemolymph-seawater ) between 12 species of osmoconforming and osmoregulating Crustacea. During evolution, hyperosmoregulating Crustacea have achieved internal osmolyte gradients generated by Na,K-ATPase and lowering the gill surface permeability. However these adaptive characteristics are not present in marine osmoconforming Crustacea, restraining them to migrate in the brackish water habitats.     

Region specific regulation of sodium pump isoform and Na,Ca-exchanger expression in the failing human heart--right atrium vs left ventricle.

Na,K-ATPase (NKA, Na-pump), an alphabeta heteromer, is the receptor for cardiac glycosides (CG) which exert a positive inotropic effect by inhibiting enzyme activity, decreasing the driving force for Na,Ca-exchange (NCX) and increasing cellular content and release of Ca2+ during depolarization. Our previous study of regional distribution of NKA in non-failing human hearts demonstrated that Na-pump alpha2-, alpha3- and beta1-isoforms were 30-50% lower in right atrium (RA) compared with left ventricle (LV), resulting in overall lower NKA activity and CG binding site number and increased sensitivity to inotropic stimulation. In failing human heart LV Na-pump alpha1, alpha3 and beta1 proteins were reduced 30-40%, with no change in alpha2 or NCX; NKA activity and CG binding sites decreased 40%, and sensitivity to inotropic stimulation increased, all compared to LV from non-failing hearts. In this study we investigated the influence of region specific factors (e.g. hemodynamics) on the regulation of NKA isoform and NCX expression in heart failure by comparing the pattern of change in right atrial myocardium during heart failure with that previously determined for LV. In RA samples from failing hearts, alpha1-, alpha2- and beta1-isoform protein expression were decreased by 40, 50 and 25%, respectively, with no significant change in alpha3 or NCX levels relative to non-failing hearts (both n= 12). Thus, alphabeta1 decreases in both RA and LV during heart failure, while alpha2beta1 is reduced only in RA and alpha3beta1 only in LV. This indicates that there are not only regional differences in normal cardiac Na-pump isoform expression but also regional differences in the pattern of isoform expression as a function of failure that may have distinct functional consequences in the adaptive process of heart failure. The mechanisms underlying Na,K-ATPase regulation and effect of hemodynamics remain to be investigated.     

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.     

Tissue Localization of Active Na,K-ATPase Isoenzymes by Determination of their Profile of Inhibition with Ouabain,Digoxin, Digitoxigenin and LND 796, A new Aminosteroid Cardiotonic

Abstract

Recently, Na, K-ATPase isoforms with differential affinities for digitalis have been identified that may contribute to different toxicity profiles. Our objectives were to localize them and to define tissue receptor patterns by examining the effect of different glycosides on the Na, K-ATPase activity. The digitalis derivatives used exhibit variation in lipophilicity and rate of enzyme inhibition. Membrane fractions enriched in Na, K-ATPase were prepared from canine heart, brain, aorta and peripheral nerves. The inhibition of enzyme activities indicates a pattern of differential sensitivities with IC₅₀ values starting from 3 nM in heart and 30 nM in brain. Therefore, high and low affinity active forms of the Na, K-ATPase enzyme coexist in these tissues. The data also suggest the existence of two Na, K-ATPase isoforms in aorta and peripheral nerves as identified by the action of digitoxigenin and LND 796 where the predominant expression is that of a high affinity form. The comparison of the patterns of digitalis sensitivities in these different tissues, suggests a more complex molecular interaction than that which can be explained by the presence of only two forms.     

Overexpression of the Polycystin-1 C-Tail Enhances Sensitivity of M-1 Cells to Ouabain

Cells derived from renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) are abnormally sensitive to ouabain, responding to physiological ouabain concentrations with enhanced proliferation and increased forskolin-induced transepithelial fluid secretion. This requires activation of the epidermal growth factor receptor (EGFR), Src kinase and the extracellular signal-regulated kinases MEK and ERK. Here, we have determined if the ADPKD phenotype obtained in mouse cortical collecting duct cells by stable overexpression of the C-terminal domain of polycystin-1 (PC-1 C-tail) also elicits the ADPKD-like response to ouabain in the cells. M-1 C20 cells expressing the PC-1 C-tail and M-1 C17 cells lacking expression of this construct were treated with physiological concentrations of ouabain, and cell proliferation, activation of the EGFR-Src-MEK-ERK pathway, forskolin-induced transepithelial Cl^? secretion and the sensitivity of Na,K-ATPase to ouabain were explored. M-1 C20 cells responded to ouabain with increased cell proliferation and ERK phosphorylation. Ouabain also augmented forskolin-induced and cystic fibrosis transmembrane conductance regulator-mediated apical secretion of Cl^? in M-1 C20 cells. These effects required activation of EGFR, Src and MEK. In contrast, ouabain had no significant effects on M-1 C17 cells. Interestingly, approximately 20 % of the Na,K-ATPase from M-1 C20 cells presented an abnormally increased sensitivity to ouabain. Overexpression of PC-1 C-tail in M-1 C20 cells is associated with an ouabain-sensitive phenotype and an increased ability of the cells to proliferate and secrete anions upon ouabain stimulation. This phenotype mimics the ouabain sensitivity of ADPKD cells and may help promote their cystogenic potential.