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Na + ,K + -ATPase is the active ion transport system that maintains the electrochemical gradients for Na + and K + across the plasma membrane of most animal cells. Na + ,K + -ATPase is constituted by the association of two major subunits, a catalytic α and a glycosylated β subunit, both of which exist as different isoforms (in mammals known as α1, α2, α3, α4, β1, β2 and β3). Na + ,K + -ATPase α and β isoforms assemble in different combinations to produce various isozymes with tissue specific expression and distinct biochemical properties. Na + ,K + -ATPase α4β1 is only found in male germ cells of the testis and is mainly expressed in the sperm flagellum, where it plays a critical role in sperm motility and male fertility. Here, we report the molecular structure of Na + ,K + -ATPase α4β1 at 2.37 Å resolution in the ouabain-bound state and in the presence of beryllium fluoride. Overall, Na + ,K + -ATPase α4 structure exhibits the basic major domains of a P-Type ATPase, resembling Na + ,K + -ATPase α1, but has differences specific to its distinct sequence. Dissimilarities include the site where the inhibitor ouabain binds. Molecular simulations indicate that glycosphingolipids can bind to a putative glycosphingolipid binding site, which could potentially modulate Na + ,K + -ATPase α4 activity. This is the first experimental evidence for the structure of Na + ,K + -ATPase α4β1. These data provide a template that will aid in better understanding the function Na + ,K + -ATPase α4β1 and will be important for the design and development of compounds that can modulate Na + ,K + -ATPase α4 activity for the purpose of improving male fertility or to achieve male contraception.

Na + /K + -ATPase (NKA) is a plasma membrane ion-transporting protein involved in the generation and maintenance of Na + and K + gradients across the cell membrane, which can produce a driving force for the secondary transport of metabolic substrates. NKA also regulates intracellular calcium that is responsible for modulating numerous cellular processes, while it interacts with many other proteins and functions as a signal transducer, with several signaling pathways being involved. Thus, NKA has become an important target for the treatment of human diseases. Cardiac glycosides are well-known NKA inhibitors, of which (+)-digoxin or digoxin has been long used for the treatment of congestive heart failure. Also, digoxin has exhibited potential antitumor activity, by targeting directly HIF-1α, NKA, and NF-κB. Thus, the function of NKA in human cardiovascular diseases and cancer and the therapeutic effects of digoxin on these diseases are summarized in the present review, with the correlations among digoxin, NKA, cardiovascular diseases, and cancer being discussed. Presented herein are also the antitumor potential of monosaccharide cardiac glycoside analogues of digoxin, including (-)-cryptanoside A, (-)-oleandrin, (-)-ouabain, and (+)-strebloside. It is hoped that this contribution will provide some helpful information for the design and discovery of new cardiac glycoside-type therapeutic agents for the treatment of cardiovascular diseases and cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Interaction between ouabain (OUA) and Na + /K + -pump remains in the current focus of hypertension research. This study aimed to find an oligopeptide that would antagonize the inhibitory effect of endogenous OUA on Na + /K + -pump and examine its activity at the cellular and organism levels. To this end, Phage Random 12 Peptide Library was employed to screen for specific polypeptide ligands that interact with M3-M4 extracellular domain of Na + /K + -pump α1 subunit known as OUA-binding site. Synthetic sequence ILEYTWLEAGGGS of extracellular domain M3-M4 of Na + /K + -pump α1 subunit was used as the target. The phage positive clones were screened and identified using the phage library and double sandwich ELISA. DNA was extracted and sequenced to synthesize 3 peptide ligands to Na + /K + -pump: P-A, P-B, and P-C. We also studied the effects of the short peptide with the highest potency for countering OUA on proliferation and apoptosis of EA.hy926 vascular endothelial cells and on systolic BP in spontaneously hypertensive rats (SHR). The effect of peptide P-A on proliferation (stimulation with physiological concentrations of OUA) and on apoptosis (stimulation with OUA in high concentrations) of EA.hy926 vascular endothelial cells was assessed by the MTT test and flow cytometry, respectively. In SHR rats, intravenous injection of P-A decreased systolic BP. Oligopeptide P-A competitively antagonized the inhibitory action of OUA on Na + /K + -pump, OUA-induced proliferation, and OUA-provoked apoptosis of cultured EA.hy926 cells. Our findings open vista for the emergence of novel hypertensive drugs., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Maintenance of Na + and K + gradients across the cell plasma membrane is an essential process for mammalian cell survival. An enzyme responsible for this process, sodium-potassium ATPase (NKA), has been currently extensively studied as a potential anticancer target, especially in lung cancer and glioblastoma. To date, many NKA inhibitors, mainly of natural origin from the family of cardiac steroids (CSs), have been reported and extensively studied. Interestingly, upon CS binding to NKA at nontoxic doses, the role of NKA as a receptor is activated and intracellular signaling is triggered, upon which cancer cell death occurs, which lies in the expression of different NKA isoforms than in healthy cells. Two major CSs, digoxin and digitoxin, originally used for the treatment of cardiac arrhythmias, are also being tested for another indication-cancer. Such drug repositioning has a big advantage in smoother approval processes. Besides this, novel CS derivatives with improved performance are being developed and evaluated in combination therapy. This article deals with the NKA structure, mechanism of action, activity modulation, and its most important inhibitors, some of which could serve not only as a powerful tool to combat cancer, but also help to decipher the so-far poorly understood NKA regulation.

In insects, chilling, anoxia, and dehydration are cues to trigger rapid physiological responses enhancing stress tolerance within minutes. Recent evidence suggests that responses elicited by different cues are mechanistically distinct from each other, though these differences have received little attention. Further, the effects are not well studied in neural tissue. In this study, we examined how brief exposure to desiccation and chilling affect ion homeostatic mechanisms in metathoracic ganglion of the migratory locust, Locusta migratoria. Both desiccation and chilling enhanced resistance to anoxia, though only chilling hastened recovery from anoxic coma. Similarly, only chilling enhanced resistance to pharmacological perturbation of neuronal ion homeostasis. Our results indicate that chilling and desiccation trigger mechanistically distinct responses and, while both may be important for neuronal ion homeostasis, chilling has a larger effect on this tissue. SUMMARY STATEMENT: This is one of few studies to demonstrate the importance of the central nervous system in rapid acclimatory responses in insects., Competing Interests: Declaration of competing interest The authors have no competing interests to report., (Copyright © 2020. Published by Elsevier Inc.)

We demonstrate a new biosensing concept with impact on the development of rapid, point of need cell based sensing with boosted sensitivity and wide relevance for bioanalysis. It involves optogenetic stimulation of cells stably transfected to express light sensitive protein channels for optical control of membrane potential and of ion homeostasis. Time-lapse impedance measurements are used to reveal cell dynamics changes encompassing cellular responses to bioactive stimuli and optically induced homeostasis disturbances. We prove that light driven perturbations of cell membrane potential induce homeostatic reactions and modulate transduction mechanisms that amplify cellular response to bioactive compounds. This allows cell based biosensors to respond more rapidly and sensitively to low concentrations of bioactive/toxic analytes: statistically relevant impedance changes are recorded in less than 30 min, in comparison with >8 h in the best alternative reported tests for the same low concentration (e.g. a concentration of 25 μM CdCl 2 , lower than the threshold concentration in classical cellular sensors). Comparative analysis of model bioactive/toxic compounds (ouabain and CdCl 2 ) demonstrates that cellular reactivity can be boosted by light driven perturbations of cellular homeostasis and that this biosensing concept is able to discriminate analytes with different modes of action (i.e. CdCl 2 toxicity versus ion pump inhibition by ouabain), a significant advance against state of the art cell based sensors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Na + /K + -ATPase α1 was reported to directly interact with and recruit FGF2 (fibroblast growth factor 2), a vital cell signaling protein implicated in angiogenesis, to the inner plasma membrane for subsequent secretion. Cardenolides, a class of cardiac glycosides, were reported to downregulate FGF2 secretion upon binding to Na + /K + -ATPase α1 in a cell system with ectopically expressed FGF2 and Na + /K + -ATPase α1. Herein, we disclose that the cardenolides ouabain and reevesioside A significantly enhance the secretion/release of FGF2 and the phosphorylation of FGFR1 (fibroblast growth factor receptor 1) in a time- and dose-dependent manner, in A549 carcinoma cells. A pharmacological approach was used to elucidate the pertinent upstream effectors. Only the ERK1/2 inhibitor U0126 but not the other inhibitors examined (including those inhibiting the unconventional secretion of FGF2) was able to reduce ouabain-induced FGF2 secretion and FGFR1 activation. ERK1/2 phosphorylation was increased upon ouabain treatment, a process found to be mediated through upstream effectors including ouabain-induced phosphorylated EGFR and a reduced MKP1 protein level. Therefore, at least two independent lines of upstream effectors are able to mediate ouabain-induced ERK1/2 phosphorylation and the subsequent FGF2 secretion and FGFR1 activation. These finding constitute unprecedent insights into the regulation of FGF2 secretion by cardenolides., (Copyright © 2019 Elsevier Inc. All rights reserved.)

There is a renewed interest in the Na + /K + -ATPase (NKA, EC 3.6.3.9) either as a target for new therapeutic uses or for understanding the putative pathophysiological role of its mammalian endogenous ligands. Recent data indicate that bufalin binds to the pig kidney NKA in a way different from ouabain and digoxin, raising the question of a putative class difference between bufadienolides and cardenolides. The purpose of this work was to perform a study of the relationship between structure and both activity and kinetics, focusing mainly on the influence of the lactone ring in C17 (5 vs. 6 membered), the effect of C14-15 cyclization and the carbohydrate moiety in C3. We compared the potency of fourteen related cardiotonic steroids (CTS) for inhibition of the cycling pig kidney NKA in two different concentrations of K + , as well as the affinity for binding to the E2P conformation of the enzyme (Mg-Pi medium) and the potency for inhibiting the E2[2K] conformation of the NKA (K + -pNPPase activity). Cardenolides were clearly sensitive to the antagonistic effect of high K + concentrations whereas bufadienolides were not or less sensitive. The C14-15 cyclization observed in some bufadienolides, such as resibufogenin and marinobufagin, caused a drastic fall in the affinity for binding to the NKA in the E2P conformation and increased the velocity of K + -pNPPase inhibition. The absence of a carbohydrate moiety in C3 increased the velocity of inhibition. Cardenolides were much more dependent on the E2P conformation for binding than bufadienolides since their ratios of E2[2K] IC 50 to E2P K i were higher than for bufadienolides. Therefore, the present data established the remarkable influence of C14-15 cyclization and of the carbohydrate moiety in C3 on both affinity and kinetics of CTS and indicate that, as a class, bufadienolides would harbor qualitative differences from cardenolides with respect to the NKA conformations to which they can bind., (Copyright © 2019. Published by Elsevier Inc.)

Cardiotonic steroids (CTS) are steroidal drugs, processed from the seeds and dried leaves of the genus Digitalis as well as from the skin and parotid gland of amphibians. The most commonly known CTS are ouabain, digoxin, digoxigenin and bufalin. CTS can be used for safer medication of congestive heart failure and other related conditions due to promising pharmacological and medicinal properties. Ouabain isolated from plants is widely utilized in in vitro studies to specifically block the sodium potassium (Na + /K + -ATPase) pump. For checking, whether ouabain derivatives are robust inhibitors of Na + /K + -ATPase pump, molecular docking simulation was performed between ouabain and its derivatives using YASARA software. The docking energy falls within the range of 8.470 kcal/mol to 7.234 kcal/mol, in which digoxigenin was found to be the potential ligand with the best docking energy of 8.470 kcal/mol. Furthermore, pharmacophore modeling was applied to decipher the electronic features of CTS. Molecular dynamics simulation was also employed to determine the conformational properties of Na + /K + -ATPase-ouabain and Na + /K + -ATPase-digoxigenin complexes with the plausible structural integrity through conformational ensembles for 100 ns which promoted digoxigenin as the most promising CTS for treating conditions of congestive heart failure patients.

A Cu(II)-catalyzed diastereoselective Michael/aldol cascade approach is used to accomplish concise total syntheses of cardiotonic steroids with varying degrees of oxygenation including cardenolides ouabagenin, sarmentologenin, 19-hydroxysarmentogenin, and 5- epi-panogenin. These syntheses enabled the subsequent structure activity relationship (SAR) studies on 37 synthetic and natural steroids to elucidate the effect of oxygenation, stereochemistry, C3-glycosylation, and C17-heterocyclic ring. Based on this parallel evaluation of synthetic and natural steroids and their derivatives, glycosylated steroids cannogenol-l-α-rhamnoside (79a), strophanthidol-l-α-rhamnoside (92), and digitoxigenin-l-α-rhamnoside (97) were identified as the most potent steroids demonstrating broad anticancer activity at 10-100 nM concentrations and selectivity (nontoxic at 3 μM against NIH-3T3, MEF, and developing fish embryos). Further analyses indicate that these molecules show a general mode of anticancer activity involving DNA-damage upregulation that subsequently induces apoptosis.

Two isoforms of a ligand-activated nuclear receptor, RORγ and RORγT, have been implicated in various physiological functions, including energy metabolism, circadian rhythm and immune system development. Using a stably transfected reporter cell line, we screened two chemical libraries and identified three cardenolides (natural, plant-derived pesticides) as activators of RORγ-dependent transcription. These compounds increased G6PC and NPAS2 expression in HepG2 cells, accompanied by increased occupancy of RORγ within the promoters of these genes. Further, strophanthidin, digoxigenin and dihydroouabain upregulated IL17A and IL17F expression and enhanced IL17 secretion in Th17 human lymphocytes. Molecular docking analyses of these compounds to the RORγ LBD showed favorable docking scores, suggesting that cardenolides may act as agonists of the receptor. Thus, our results provide new chemical structures for further development of RORγ-selective modulators with virtual therapeutic potential., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Inhibition of the Na,K-ATPase by ouabain potentiates vascular tone and agonist-induced contraction. These effects of ouabain varies between different reports. In this study, we assessed whether the pro-contractile effect of ouabain changes with arterial diameter and the molecular mechanism behind it. Rat mesenteric small arteries of different diameters (150⁻350 µm) were studied for noradrenaline-induced changes of isometric force and intracellular Ca 2+ in smooth muscle cells. These functional changes were correlated to total Src kinase and Src phosphorylation assessed immunohistochemically. High-affinity ouabain-binding sites were semi-quantified with fluorescent ouabain. We found that potentiation of noradrenaline-sensitivity by ouabain correlates positively with an increase in arterial diameter. This was not due to differences in intracellular Ca 2+ responses but due to sensitization of smooth muscle cell contractile machinery to Ca 2+ . This was associated with ouabain-induced Src activation, which increases with increasing arterial diameter. Total Src expression was similar in arteries of different diameters but the density of high-affinity ouabain binding sites increased with increasing arterial diameters. We suggested that ouabain binding induces more Src kinase activity in mesenteric small arteries with larger diameter leading to enhanced sensitization of the contractile machinery to Ca 2+ .

Although the signaling function of Na/K-ATPase has been studied for decades, the chasm between the pumping function and the signaling function of Na/K-ATPase is still an open issue. This article explores the relationship between ion pumping and signaling with attention to the amplification of oxidants through this signaling function. We specifically consider the Na/K-ATPase with respect to its signaling function as a superposition of different states described for its pumping function. We then examine how alterations in the relative amounts of these states could alter signaling through the Src-EGFR-ROS pathway. Using assumptions based on some experimental observations published by our laboratories and others, we develop some predictions regarding cellular oxidant stress.

The information obtained from crystallized complexes of the Na + ,K + -ATPase with cardiotonic steroids (CTS) is not sufficient to explain differences in the inhibitory properties of CTS such as stereoselectivity of CTS binding or effect of glycosylation on the preference to enzyme isoforms. The uncertainty is related to the spatial organization of the hydrophilic cavity at the entrance of the CTS-binding site. Therefore, there is a need to supplement the crystallographic description with data obtained in aqueous solution, where molecules have significant degree of flexibility. This work addresses the applicability of the electron paramagnetic resonance (EPR) method for the purpose. We have designed and synthesized spin-labeled compounds based on the cinobufagin steroid core. The length of the spacer arms between the steroid core and the nitroxide group determines the position of the reporting group (N-O) confined to the binding site. High affinity to Na + ,K + -ATPase is inferred from their ability to inhibit enzymatic activity. The differences between the EPR spectra in the absence and presence of high ouabain concentrations identify the signature peaks originating from the fraction of the spin labels bound within the ouabain site. The degree of perturbations of the EPR spectra depends on the length of the spacer arm. Docking of the compounds into the CTS site suggests which elements of the protein structure might be responsible for interference with the spin label (e.g., steric clashes or immobilization). Thus, the method is suitable for gathering information on the cavity leading to the CTS-binding site in Na + ,K + -ATPase in all conformations with high affinity to CTS., (© 2018 Federation of European Biochemical Societies.)

Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein-protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.

Evidences indicate the relationship between neurotensinergic and dopaminergic systems. Neurotensin inhibits synaptosomal membrane Na + , K + -ATPase activity, an effect blocked by SR 48692, antagonist for high affinity neurotensin receptor (NTS1) type. Assays of high affinity [ 3 H]-ouabain binding (to analyze K + site of Na + , K + -ATPase) show that in vitro addition of neurotensin decreases binding. Herein potential interaction between NTS1 receptor, dopaminergic D2 receptor and Na + , K + -ATPase was studied. To test the involvement of dopaminergic D2 receptors in [ 3 H]-ouabain binding inhibition by neurotensin, Wistar rats were administered i.p.with antipsychotic drugs haloperidol (2mg/kg) and clozapine (3, 10 and 30mg/kg). Animals were sacrificed 18h later, cerebral cortices harvested, membrane fractions prepared and high affinity [ 3 H]-ouabain binding assayed in the absence or presence of neurotensin at a 10 micromolar concentration. No differences versus controls for basal binding or for binding inhibition by neurotensin were recorded, except after 10mg/kg clozapine. Rats were administered with neurotensin (3, 10y 30μg, i.c.v.) and 60min later, animals were sacrificed, cerebral cortices harvested and processed to obtain membrane fractions for high affinity [ 3 H]-ouabain binding assays. Results showed a slight but statistically significant decrease in binding with the 30μg neurotensin dose. To analyze the interaction between dopaminergic D2 and NTS1 receptors, [ 3 H]-neurotensin binding to cortical membranes from rats injected with haloperidol (2mg/kg, i.p.) or clozapine (10mg/kg) was assayed. Saturation curves and Scatchard transformation showed that the only statistically significant change occurred in Bmax after haloperidol administration. Hill number was close to the unit in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modulate the interaction between neurotensin and Na + , K + -ATPase. At the same time, support the notion of an interaction among dopaminergic and neurotensinergic systems and Na + , K + -ATPase at central synapses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The method of purification of Na,K-ATPase from pig kidney is based on a differential centrifugation and SDS-treatment of a microsomal preparation. The yield is 0.4 mg protein per 1 g tissue with the specific (ouabain-sensitive) activity of 25-28 μmol Pi/min per mg protein and nucleotide binding capacity of 3 nmol/mg. The protein/lipid ratio is 1/1 (mg/mg) with a protein purity of ~80 %.

Asthenospermia accounts for about 30% of the causes of male infertility. Currently, most drugs for asthenospermia lack specificity and desirable therapeutic efficiency. An insight into the pathogenesis of asthenospermia is important for the development of specific therapies for this disease. The protein Na+/K(+)- ATPase α4 isoform (NKA4) presents in both mature testis tissue and the sperm tail, the absence or reduced activity of which may significantly decrease sperm motility. Ouabain is a natural inhibitor of NKA4, suppressing its activity by specifically binding the ouabain site in it. The hypothalamus and adrenal cortex excrete an ouabain-like steroid hormone called endogenous ouabain (EO), which may be associated with the pathogenesis of asthenospermia by inhibiting the activity of NKA4, affecting Na+/H+ exchange, Na+/Ca2+ exchange and sperm cell membrane potential, and eventually reducing sperm motility.

Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC50 values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na+/K+-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na+ but not Ca2+, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na+/K+-ATPase, leading to overload of intracellular Na+ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.

Paralytic shellfish poisoning (PSP) toxins are well-known sodium channel-blocking marine toxins, which block the conduction of nerve impulses and lead to a series of neurological disorders symptoms. However, PSP toxins can inhibit the cytotoxicity effect of compounds (e.g., ouabain and veratridine). Under the treatment of ouabain and veratridine, neuroblastoma cell will swell and die gradually, since veratridine causes the persistent inflow of Na(+) and ouabain inhibits the activity of Na(+)/K(+)-ATPases. Therefore, PSP toxins with antagonism effect can raise the chance of cell survival by blocking inflow of Na(+). Based on the antagonism effect of PSP toxins, we designed an improved cell-based assay to detect PSP toxins using a neuroblastoma cell-based impedance biosensor. The results demonstrated that this biosensor showed high sensitivity and good specificity for saxitoxins detection. The detection limit of this biosensor was as low as 0.03 ng/ml, which was lower than previous reported cell-based assays and mouse bioassays. With the improvement of biosensor performance, the neuroblastoma cell-based impedance biosensor has great potential to be a universal PSP screening method., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this work, we discovered that the Na(+)/K(+)-ATPase beta-subunit (ATP1B1) on epidermal cells plays a key role in the peptide-mediated transdermal delivery of macromolecular drugs. First, using a yeast two-hybrid assay, we screened candidate proteins that have specific affinity for the short peptide TD1 (ACSSSPSKHCG) identified in our previous work. Then, we verified the specific binding of TD1 to ATP1B1 in yeast and mammalian cells by a pull-down ELISA and an immunoprecipitation assay. Finally, we confirmed that TD1 mainly interacted with the C-terminus of ATP1B1. Our results showed that the interaction between TD1 and ATP1B1 affected not only the expression and localization of ATP1B1, but also the epidermal structure. In addition, this interaction could be antagonized by the exogenous competitor ATP1B1 or be inhibited by ouabain, which results in the decreased delivery of macromolecular drugs across the skin. The discovery of a critical role of ATP1B1 in the peptide-mediated transdermal drug delivery is of great significance for the future development of new transdermal peptide enhancers.

With the use of the energy of ATP hydrolysis, the Na+/K+-ATPase is able to transport across the cell membrane Na+ and K+ against their electrochemical gradients. The enzyme is strongly inhibited by ouabain and its derivatives, some that are therapeutically used for patients with heart failure (cardiotonic steroids). Using lanthanide resonance energy transfer, we trace here the conformational changes occurring on the external side of functional Na+/K+-ATPases induced by the binding of ouabain. Changes in donor/acceptor pair distances are mainly observed within the α subunit of the enzyme. To derive a structural model matching the experimental lanthanide resonance energy transfer distances measured with bound ouabain, we carried out molecular dynamics simulations with energy restraints applied simultaneously using a novel methodology with multiple non-interacting fragments. The restrained simulation, initiated from the X-ray structure of the E2(2K+) state, became strikingly similar to the X-ray structure of the sodium-bound state. The final model shows that ouabain is trapped within the external ion permeation pathway of the pump., (Published by Elsevier Ltd.)

NMR relaxation enhancement by paramagnetic metals provides powerful restraints on the three-dimensional structures of proteins in solution, and this approach has recently been utilized in several NMR structural investigations of proteins in the solid-state. Here we utilize paramagnetic relaxation enhancement (PRE) by Mn(2+) with cross-polarization magic-angle spinning (CP-MAS) solid-state NMR to investigate the interaction of a membrane-embedded protein the Na,K-ATPase (NKA) with a cardiotonic steroid inhibitor. The inhibitor, a diacetonide derivate of the cardiac glycoside ouabain, with (13)C labelled acetonide groups in the rhamnose sugar and steroid moieties ([(13)C2]ODA), is 1000-fold less potent than the parent compound. It is shown that the (13)C CP-MAS solid-state NMR spectra of the NKA-[(13)C2]ODA complex exhibit distinct signals for the two (13)C labels of the inhibitor when bound to the ouabain site of membrane-embedded NKA. Recent crystal structures of NKA indicate that the catalytic α-subunit binds a single Mn(2+) in a transmembrane site close to the high-affinity ouabain site. Here, complexation of NKA with Mn(2+) broadens the resonance line from the rhamnose group substantially more than the steroid peak, indicating that the rhamnose group is closer to the Mn(2+) site than is the steroid group. These observations agree with computational molecular docking simulations and are consistent with ODA adopting an inverted orientation compared to ouabain in the cardiac glycoside site, with the modified rhamnose group drawn toward the transmembrane centre of the protein. This work demonstrates that PRE can provide unique information on the positions and orientations of ligands within their binding pockets of transmembrane proteins.

The cardiac glycosides ouabain and digitoxin, established Na(+)/K(+) ATPase inhibitors, were found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical genetics screen for cell motility. The Na(+)/K(+) ATPase acts both as an ion-transporter and as a receptor for cardiac glycosides. To delineate which function is related to breast cancer cell migration, structure-activity relationship (SAR) profiles of cardiac glycosides were established at the cellular (cell migration inhibition), molecular (Na(+)/K(+) ATPase inhibition), and atomic (computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a similar profile, a decrease in potency when the parent cardiac glycoside structure was modified, for each activity investigated. Since assays were done at the cellular, molecular, and atomic levels, correlation of SAR profiles across these multiple assays established links between cellular activity and specific protein-small molecule interactions. The observed antimigratory effects in breast cancer cells are directly related to the inhibition of Na(+)/K(+) transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation path formed by transmembrane helices αM1-M6 correlates with the Na(+) pump activity and cell migration. Other Na(+)/K(+) ATPase inhibitors that are structurally distinct from cardiac glycosides also exhibit antimigratory activity, corroborating the conclusion that the antiport function of Na(+)/K(+) ATPase and not the receptor function is important for supporting the motility of MDA-MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific biochemical functions and higher-level cellular processes, particularly for proteins with multiple functions and small molecules with unknown or various modes of action.

The natural product ouabagenin is a complex cardiotonic steroid with a highly oxygenated skeleton. This full account describes the development of a concise synthesis of ouabagenin, including the evolution of synthetic strategy to access hydroxylation at the C19 position of a steroid skeleton. In addition, approaches to install the requisite butenolide moiety at the C17 position are discussed. Lastly, methodology developed in this synthesis has been applied in the generation of novel analogues of corticosteroid drugs bearing a hydroxyl group at the C19 position.

Cisplatin is the most widely used chemotherapeutics for cancer treatment, however, its administration is connected to inevitable adverse effects. Previous studies suggested that cisplatin is able to inhibit Na(+)/K(+)-ATPase (NKA), the enzyme responsible for maintaining electrochemical potential and sodium gradient across the plasma membrane. Here we report a crystallographic analysis of cisplatin bound to NKA in the ouabain bound E2P form. Despite a moderate resolution (7.4 Å and 7.9 Å), the anomalous scattering from platinum and a model representation from a recently published structure enabled localization of seven cisplatin binding sites by anomalous difference Fourier maps. Comparison with NKA structures in the E1P conformation suggested two possible inhibitory mechanisms for cisplatin. Binding to Met151 can block the N-terminal pathway for transported cations, while binding to Met171 can hinder the interaction of cytoplasmic domains during the catalytic cycle., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This paper introduces a strategy to kill selectively multidrug-resistant cells that express the ABCG2 transporter (also called breast cancer resistance protein, or BCRP). The approach is based on specific stimulation of ATP hydrolysis by ABCG2 transporters with subtoxic doses of curcumin combined with stimulation of ATP hydrolysis by Na(+),K(+)-ATPase with subtoxic doses of gramicidin A or ouabain. After 72 h of incubation with the drug combinations, the resulting overconsumption of ATP by both pathways inhibits the efflux activity of ABCG2 transporters, leads to depletion of intracellular ATP levels below the viability threshold, and kills resistant cells selectively over cells that lack ABCG2 transporters. This strategy, which was also tested on a clinically relevant human breast adenocarcinoma cell line (MCF-7/FLV1), exploits the overexpression of ABCG2 transporters and induces caspase-dependent apoptotic cell death selectively in resistant cells. This work thus introduces a novel strategy to exploit collateral sensitivity (CS) with a combination of two clinically used compounds that individually do not exert CS. Collectively, this work expands the current knowledge on ABCG2-mediated CS and provides a potential strategy for discovery of CS drugs against drug-resistant cancer cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Central nervous system (CNS) administration of angiotensin II (Ang II) raises blood pressure (BP). The rise in BP reflects increased sympathetic outflow and a slower neuromodulatory pressor mechanism mediated by CNS mineralocorticoid receptors (MR). We investigated the hypothesis that the sustained phase of hypertension is associated also with elevated circulating levels of endogenous ouabain (EO), and chronic stimulation of arterial calcium transport proteins including the sodium-calcium exchanger (NCX1), the type 6 canonical transient receptor potential protein (TRPC6), and the sarcoplasmic reticulum calcium ATPase (SERCA2). Wistar rats received a chronic intra-cerebroventricular infusion of vehicle (C) or Ang II (A, 2.5 ng/min, for 14 days) alone or combined with the MR blocker, eplerenone (A+E, 5 µg/day), or the aldosterone synthase inhibitor, FAD286 (A+F, 25 µg/day). Conscious mean BP increased (P<0.05) in A (123 ± 4 mm Hg) vs all other groups. Blood, pituitary and adrenal samples were taken for EO radioimmunoassay (RIA), and aortas for NCX1, TRPC6 and SERCA2 immunoblotting. Central infusion of Ang II raised plasma EO (0.58 ± 0.08 vs C 0.34 ± 0.07 nM (P<0.05), but not in A + E and A + F groups as confirmed by off-line liquid chromatography (LC)-RIA and LC-multistage mass spectrometry. Two novel isomers of EO were elevated by Ang II; the second less polar isomer increased >50-fold in the A+F group. Central Ang II increased arterial expression of NCX1, TRPC6 and SERCA2 (2.6, 1.75 and 3.7-fold, respectively; P<0.01)) but not when co-infused with E or F. Adrenal and pituitary EO were unchanged. We conclude that brain Ang II activates a CNS-humoral axis involving plasma EO. The elevated EO reprograms peripheral ion transport pathways known to control arterial Na(+) and Ca(2+) homeostasis; this increases contractility and augments sympathetic effects. The new axis likely contributes to the chronic pressor effect of brain Ang II.

Mutations within ion-transporting proteins may severely affect their ability to traffic ions properly and thus perturb the delicate balance of ion gradients. Somatic gain-of-function mutations of the Na(+),K(+)-ATPase α1-subunit have been found in aldosterone-producing adenomas that are among the causes of hypertension. We used molecular dynamics simulations to investigate the structural consequences of these mutations, namely, Leu97 substitution by Arg (L97R), Val325 substitution by Gly (V325G), deletion of residues 93-97 (Del93-97), and deletion-substitution of residues 953-956 by Ser (EETA956S), which shows inward leak currents under physiological conditions. The first three mutations affect the structural context of the key ion-binding residue Glu327 at binding site II, which leads to the loss of the ability to bind ions correctly and to occlude the pump. The mutated residue in L97R is more hydrated, which ultimately leads to the observed proton leak. V325G mimics the structural behavior of L97R; however, it does not promote the hydration of surrounding residues. In Del93-97, a broader opening is observed because of the rearrangement of the kinked transmembrane helix 1, M1, which may explain the sodium leak measured with the mutant. The last mutant, EETA956S, opens an additional water pathway near the C-terminus, affecting the III sodium-specific binding site. The results are in excellent agreement with recent electrophysiology measurements and suggest how three mutations prevent the occlusion of the Na(+),K(+)-ATPase, with the possibility of transforming the pump into a passive ion channel, whereas the fourth mutation provides insight into the sodium binding in the E1 state.

Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua). Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs) were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days). To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

Aim of this study was to investigate the application of normalized mean of the empirical Higuchi fractal dimension (FD) distributions, as a new approach to analyze the spontaneous bioelectrical activity of garden snail (Helix pomatia) Br neuron. The effect of ouabain on modulation of Br neuron bursting activity before and after the exposure to 10 mT static magnetic field (SMF) was observed by analyzing the following parameters: action potential (AP), interspike interval (ISI) and interbursting interval (IBI) components. Normalized mean of the empirical FD distributions were formed for the following experimental conditions: Control 1, Ouabain 1, Control 2, SMF 2, ASMF 2, Control 3, SMF 3 and Ouabain ASMF 3. Our main results have shown that ouabain without SMF induced increase in participation of AP and a decrease in participation of IBI components compared to the first control condition. However, in the presence of 10 mT SMF, ouabain-induced changes of measured parameters of Br neuron activity were less pronounced compared to the third control condition. We have shown that normalized mean of the empirical FD distributions is a useful method for detecting the changes in AP, ISI, and IBI components of complex bursting activity in altered physiological states.

The Na(+),K(+)-ATPase maintains electrochemical gradients for Na(+) and K(+) that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na(+),K(+)-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na(+),K(+)-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg(2+), and crystallographic studies indicate that Rb(+) and Mn(2+), but not Na(+), bind to this site. Comparison with the low-affinity [K2]E2-MgF(x)-ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742-13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg(2+). K(+) binding at the same site unwinds a turn of αM4, dragging residues Ile318-Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K(+)-Mg(2+) competition and explain the well-known antagonistic effect of K(+) on CTS binding.

Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography., (Copyright © 2013 The Author. Published by Elsevier Inc. All rights reserved.)

Here, we report on a scalable route to the polyhydroxylated steroid ouabagenin with an unusual take on the age-old practice of steroid semisynthesis. The incorporation of both redox and stereochemical relays during the design of this synthesis resulted in efficient access to more than 500 milligrams of a key precursor toward ouabagenin-and ultimately ouabagenin itself-and the discovery of innovative methods for carbon-hydrogen (C-H) and carbon-carbon activation and carbon-oxygen bond homolysis. Given the medicinal relevance of the cardenolides in the treatment of congestive heart failure, a variety of ouabagenin analogs could potentially be generated from the key intermediate as a means of addressing the narrow therapeutic index of these molecules. This synthesis also showcases an approach to bypass the historically challenging problem of selective C-H oxidation of saturated carbon centers in a controlled fashion.

Progesterone and its polar metabolite(s) trigger the meiotic divisions in the amphibian oocyte through a non-genomic signaling system at the plasma membrane. Published site-directed mutagenesis studies of ouabain binding and progesterone-ouabain competition studies indicate that progesterone binds to a 23 amino acid extracellular loop of the plasma membrane α-subunit of Na/K-ATPase. Integral membrane proteins such as caveolins are reported to form Na/K-ATPase-peptide complexes essential for signal transduction. We have characterized the progesterone-induced Na/K-ATPase-caveolin (CAV-1)-steroid 5α-reductase interactions initiating the meiotic divisions. Peptide sequence analysis algorithms indicate that CAV-1 contains two plasma membrane spanning helices, separated by as few as 1-2 amino acid residues at the cell surface. The CAV-1 scaffolding domain, reported to interact with CAV-1 binding (CB) motifs in signaling proteins, overlaps transmembrane (TM) helix 1. The α-subunit of Na/K-ATPase (10 TM helices) contains double CB motifs within TM-1 and TM-10. Steroid 5α-reductase (6 TM helices), an initial step in polar steroid formation, contains CB motifs overlapping TM-1 and TM-6. Computer analysis predicts that interaction between antipathic strands may bring CB motifs and scaffolding domains into close proximity, initiating allostearic changes. Progesterone binding to the α-subunit may thus facilitate CB motif:CAV-1 interaction, which in turn induces helix-helix interaction and generates both a signaling cascade and formation of polar steroids., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na(+), K(+), vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Plant toxins are sequestered by many animals and the toxicity is frequently advertised by aposematic displays to deter potential predators. Such 'unpalatability by appropriation' is common in many invertebrate groups and also found in a few vertebrate groups. However, potentially lethal toxicity by acquisition has so far never been reported for a placental mammal. Here, we describe complex morphological structures and behaviours whereby the African crested rat, Lophiomys imhausi, acquires, dispenses and advertises deterrent toxin. Roots and bark of Acokanthera schimperi (Apocynaceae) trees are gnawed, masticated and slavered onto highly specialized hairs that wick up the compound, to be delivered whenever the animal is bitten or mouthed by a predator. The poison is a cardenolide, closely resembling ouabain, one of the active components in a traditional African arrow poison long celebrated for its power to kill elephants.

The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na(+) and H(2)O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na(+),K(+)-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life.

The Na(+)/K(+) ATPase is an almost ubiquitous integral membrane protein within the animal kingdom. It is also the selective target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to estimate distances between a fluorescent ouabain and a lanthanide binding tag (LBT), which was introduced at five different positions in the Na(+)/K(+) ATPase sequence. These five normally functional LBT-Na(+)/K(+) ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion conditions. The spectroscopic data suggest two mutually exclusive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na(+)/K(+) ATPase constructs, approximate ouabain positions could be determined. Our results suggest that ouabain binds at two sites along the ion permeation pathway of the Na(+)/K(+) ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all functional and structural studies.

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K(+)·P(i) with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na(+), after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na(+) demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway.

The molecular events leading to neuronal dysfunction often associated with mercury toxicity can be complex and is yet to be fully elucidated. Hence, the present study sought to evaluate the interaction of inorganic mercury (Hg(2+)) with the ouabain-sensitive electrogenic pump in partially purified mammalian brain membrane preparations. The results show that Hg(2+) significantly inhibited the transmembrane enzyme in a concentration dependent manner. In addition, Hg(2+) exerts its inhibitory effect on the activity of the enzyme by interacting with groups at the adenosine triphosphate (ATP), Na(+) and K(+) binding sites. However, preincubation of the enzyme with exogenous monothiols, cysteine, prevented the inhibition of Hg(2+) on the pump's activity suggesting that Hg(2+) may be interacting with the thiols at the nucleotide (ATP) and cationic (Na(+) and K(+)) binding sites. In fact, our data show that Hg(2+) oxidizes sulphydryl groups in cysteine in a time dependent fashion in vitro. Finally, we speculate that the small molecular volume of Hg(2+) in comparison with the substrates (ATP, Na(+) and K(+)) of sodium pump, its possibly high reactivity and strong affinity for thiols may account for its high toxicity towards the membrane bound ouabain-sensitive electrogenic pump., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The Na+,K+-ATPase belongs to the P-ATPase family, whose characteristic property is the formation of a phosphorylated intermediate. The enzyme is also a defined target for cardiotonic steroids which inhibit its functional activity and initiate intracellular signaling. Here we describe the 4.6 Å resolution crystal structure of the pig kidney Na+,K+-ATPase in its phosphorylated form stabilized by high affinity binding of the cardiotonic steroid ouabain. The steroid binds to a site formed at transmembrane segments αM1-αM6, plugging the ion pathway from the extracellular side. This structure differs from the previously reported low affinity complex with potassium. Most importantly, the A domain has rotated in response to phosphorylation and αM1-2 move towards the ouabain molecule, providing for high affinity interactions and closing the ion pathway from the extracellular side. The observed re-arrangements of the Na+,K+-ATPase stabilized by cardiotonic steroids may affect protein-protein interactions within the intracellular signal transduction networks., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We show in the present work that the infrared (IR) spectrum of human PC-3 prostate cancer cells exposed to anticancer drugs could offer a unique opportunity to get a fingerprint of all the major biochemical components (DNA, RNA, proteins, lipids, etc.) present in the cells and to identify with high sensitivity the signature of the metabolic changes induced by anticancer drugs. We investigated here the FTIR-related signatures of the effect of 4 structurally-related cardiotonic steroids (CS), i.e. ouabain, 19-hydroxy-2″-oxovoruscharin, hellebrin and 19-hydroxy-hellebrin on PC-3 cancer cells incubated between 0 and 36 h in the absence (control) or the presence of the CS. For each molecule a single spectral signature described the largest part of the time dependent modifications with a possible very minor second component. The spectral signatures characterizing the effects of each of the four CS were unique but very similar when compared to the signature of the effect of an intercalating anticancer drug, i.e. doxorubicin, selected as a positive reference compound in our study, suggesting a fully distinct set of cellular perturbations. The current study thus illustrates that Fourier Transform Infrared (FTIR) analyses can be used to identify, among the perturbations induced on a given cancer cell line, the features common to a group of anticancer compounds as well as features specific to every single drug., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The comparative study of influence of ouabain and calixarene C107, and the structure component of this calixarene--fragment M3, in the conditions of in vitro and chronic action in vivo on Na+, K(+)-ATPase activity was carried out on the fractions of plasmatic membranes (PM) of the rat hepatocytes. A general property in the conditions in vitro is the ability of calixarene C107 and ouabain (both substances were in the concentration of 1 mM) to inhibit PM Na+, K(+)-ATPase of rat hepatocytes. However, in the case of activities of calixarene C107 and ouabain in the conditions in vivo heterogeneous action on Mg2(+)-ATPase and Mg2+, Na+, K(+)-ATPase activities takes place: total activity in the conditions of injection of increased concentrations of ouabain remains without changes, but Mg2(+)-ATPase activity significantly grows; in analogous conditions under the action of calixarene C107 both these activities decrease twice in comparison with control. Both under the in vitro and in vivo conditions, M3 fragment (the structural component of C107) does not change the values of investigated enzymatic activities. The biochemical mechanisms of calixarene C107 action on Na+, K(+)-ATPase activity in PM of rat hepatocytes are discussed.

Aim: To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain., Methods: The inhibitory potency of ouabain and the identified steroid-like compounds on Na(+)/K(+)-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na(+)/K(+)-ATPase., Results: All the examined steroid-like compounds displayed more or less inhibition on Na(+)/K(+)-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na(+)/K(+)-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K(+) binding sites of Na(+)/K(+)-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na(+)/K(+)-ATPase., Conclusion: Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na(+)/K(+)-ATPase.

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. It maintains the transmembrane gradients of Na(+) and K(+) across the cell membrane essential for cell homeostasis. The Na,K-ATPase is specifically inhibited by cardiotonic steroids like ouabain, which bind to the extracellular side of the enzyme and is of significant therapeutic value in the treatment of congestive heart failure. In order to further characterize the binding of cardiotonic steroids to shark Na,K-ATPase, we compared the strength and rate of inhibition at varying pH of two cardiac glycosides with either an unsaturated (ouabain) or saturated (dihydroouabain) lactone ring and three aglycons with either a 5-membered (ouabagenin and digitoxigenin) or a 6-membered (bufalin) lactone. Inhibition by ouabain and dihydroouabain, and especially the aglycon ouabagenin, was found to be strongly dependent on pH with an increase in IC(50) by factors of approximately 6, approximately 20, and approximately 66, respectively, when pH increased from 6.5 to 8.5. The finding that ouabagenin was the most pH-sensitive inhibitor indicates that the steroid hydroxyl side chains are pivotal for this pH effect, whereas the lactone ring saturation was less important. The sugar moiety is important in compensating for the pH effect. In contrast, the IC(50) of the two genins bufalin and digitoxigenin increased by a factor of only approximately 2 when pH increased from 6.5 to 8.5, indicating that the pH effect does not relay on whether the lactone is 5- or 6-membered. The rate of inhibition was retarded much more significantly by increasing pH for the glycosides than for the aglycons. Finally, we demonstrate a change in enzyme subconformations following binding of cardiotonic steroids to Na,K-ATPase phosphoenzymes using fluoride analogues of phosphoenzyme intermediates. The results are discussed with reference to the recent high-resolution crystal structures of shark Na,K-ATPase in the unbound and ouabain-bound conformation.

Aim: To find one kind of peptide that will conjugate with ouabain and inhibit its biological function, and provide a new treatment strategy for primary hypertension., Methods: In this study, ouabain was used as a target to screen ouabain conjugated peptide (OCP) from Ph.D.-7 phage display peptide library. After 3 rounds of bio-panning, the products were identified by ELISA and DNA electrophoresis analysis and sequencing. Peptide was synthesized and analyzed the activity by radioligand binding assay. The inhibitory ratio of cell proliferation was measured by MTT and the cell morphology changing was measured by Hoechst 33342/PI staining. The expression of Na(+)-K(+)-ATPase alpha1-subunit and beta1-subunit were detected by RT-PCR and immunocytochemistry. The levels of the free intracellular Na(+) in EAhy926 cells were measured by laser confocal microscope., Results: The ouabain conjugated peptide was found out, and it was occupied in 0.64 (9/14). The analysis of protein showed that the obtained peptides had no homology with Na(+)-K(+)-ATPase. The amino acid sequence of synthesized peptide was Arg-Cys-Met-Thr-Ser-Arg-Ser. There was binding activity between OCP and (3)H-ouabain. The MTT assay showed that OCP could reverse the inhibition action of ouabain on vascular endothelial EAhy926 cells in a dose and time-dependent manner. The number of apoptotic cells had significantly decreased detected by Hoechst 33342/PI staining. The results of RT-PCR and immunocytochemistry showed that OCP could inhibit the up-regulated expression of Na(+)-K(+)-ATPase alpha1-subunit and down-regulated expression of Na(+)-K(+)-ATPase beta1-subunit induced by ouabain in EAhy926 cells., Conclusion: The OCP could reverse the growth inhibition and death induction of ouabain in EAhy926 cells, which would provide the basis for studying the interaction between ouabain and Na(+)-K(+)-ATPase and explore novel anti-ouabain agents.

The sodium-potassium pump (Na(+),K(+)-ATPase) is responsible for establishing Na(+) and K(+) concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na(+),K(+)-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 A resolution in a state analogous to E2.2K(+).Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K(+), in marked contrast to previous models. Due to antagonism between ouabain and K(+), the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.

There is a growing interest for screening antitumor drugs for their mechanism of action on cancer cells. Yet, screening for "modes of action" presents a technical challenge that is beyond the capability of conventional methods used in cellular or molecular biology. Several studies have highlighted the advantages of using infrared spectroscopy for diagnostic purposes at the clinical level for identifying cell types. In the present work, we suggest that the Fourier Transform Infrared (FTIR) spectrum of cells exposed to anti-cancer drugs could offer a unique opportunity to obtain a fingerprint of all molecules present in the cells and to observe, with a high sensitivity, the metabolic changes induced by potential anti-cancer drugs. Ouabain is one of the most potent cardenolides, which acts by inhibiting sodium pump activity. Cardenolides represent a class of compounds that are intended to soon enter clinical trials in oncology. In order to evaluate the potential of infrared spectroscopy to yield a signature for ouabain action on cancer cells, human prostate cancer PC-3 cells were treated with 36 nM ouabain, a sub-lethal concentration. Using ouabain as a model, we have thus demonstrated the possibility of using IR spectroscopy in the assessment of the global effects of an investigational compound on the cell constituents, thus contributing to setting up a new method for screening for novel anti-cancer agents in general, and potential anti-cancer cardenolides in particular. The most spectacular data obtained strongly suggest a modification in the nature of the cell lipids.

A full account of the total synthesis of ouabagenin and ouabain is described. A highly stereocontrolled anionic cycloaddition for the rapid construction of the basic steroid skeleton is a pivotal conversion for the whole strategy. A careful study was needed to establish the order and the sequence of functional group manipulations. Specific conformational features of the ouabain skeleton allowed us to overcome a few stereochemical problems. Degradation studies on ouabain provided an ultimate proof for a key intermediate, which is used as a relay. Late stage butenolide formation and glycosidation yielded ouabain.