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Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

Spreading depolarization (SD) temporarily shuts down neural processing in mammals and insects. Age is a critical factor for predicting the consequences of SD in humans. We investigated the effect of aging in an insect model of SD and explored the contribution of oxidative stress. Aging slowed the recovery of intact locusts from asphyxia. We monitored SD by recording the DC potential across the blood-brain barrier in response to bath application of the Na+/K+-ATPase inhibitor, ouabain. Ouabain induced changes to the DC potential that could be separated into two distinct components: a slow, permanent negative shift, like the negative ultraslow potential recorded in mammals and human patients, and rapid, reversible negative DC shifts (SD events). Aging had no effect on the slow shift but increased the duration of SD events. This was accompanied by a decrease in the rate of recovery of DC potential at the end of the SD event. An attempt to generate oxidative stress using rotenone was unsuccessful, but pretreatment with the antioxidant, N-acetylcysteine amide, had opposite effects to those of aging, reducing duration, and increasing rate of recovery, suggesting that it prevented oxidative damage occurring during the ouabain treatment. The antioxidant also reduced the rate of the slow negative shift. We propose that the aging locust nervous system is more vulnerable to stress due to a prior accumulation of oxidative damage. Our findings strengthen the notion that insects provide useful models for the investigation of cellular and molecular mechanisms underlying SD in mammals. NEW & NOTEWORTHY: Anoxia and similar energetic crises trigger a shutdown of central neural processing in a process of spreading depolarization (SD) that is generally pathological in mammals and protective in insects. We show that older animals are slower to recover from SD in an insect model. Moreover, preventing oxidative stress with an antioxidant speeds recovery. These findings demonstrate the role of oxidative stress in contributing to the vulnerability of the aging insect central nervous system (CNS) in energetic emergencies. [ABSTRACT FROM AUTHOR]

An unusually large amplitude spontaneous miniature endplate potentials (gMEPPs) occur naturally at low frequency at the vertebrate neuromuscular junction. Unlike the normal miniature endplate potentials (nMEPPs), these gMEPPs have long duration and long time to peak. More strikingly, gMEPPs seem to be independent of extracellular and intracellular Ca+2. and have a greater temperature sensitivity than nMEPPs. They are potentiated by tetrodotoxin but inhibited by acetylcholine (ACh) receptor blockers indicating ACh is the neurotransmitter responsible for gMEPPs. The frequency of gMEPPs is greatly increased in muscles weakened by various drugs, toxins or disease conditions suggesting that gMEPPs may be a part of possible neurotrophic mechanism to preserve effective neuromuscular transmission when the normal function is compromised. [ABSTRACT FROM AUTHOR]

We investigated acute effects of the Na+,K+‐ATPase (NKA) inhibitor, digoxin, on muscle NKA content and isoforms, arterial plasma [K+] ([K+]a) and fatigue with intense exercise. In a randomised, crossover, double‐blind design, 10 healthy adults ingested 0.50 mg digoxin (DIG) or placebo (CON) 60 min before cycling for 1 min at 60% V̇O2peak${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{peak}}}}$ then at 95% V̇O2peak${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{peak}}}}$ until fatigue. Pre‐ and post‐exercise muscle biopsies were analysed for [3H]‐ouabain binding site content without (OB‐Fab) and after incubation in digoxin antibody (OB+Fab) and NKA α1‐2 and β1‐2 isoform proteins. In DIG, pre‐exercise serum [digoxin] reached 3.36 (0.80) nM [mean (SD)] and muscle NKA–digoxin occupancy was 8.2%. Muscle OB‐Fab did not differ between trials, whereas OB+Fab was higher in DIG than CON (8.1%, treatment main effect, P = 0.001), whilst muscle NKA α1‐2 and β1‐2 abundances were unchanged by digoxin. Fatigue occurred earlier in DIG than CON [−7.7%, 2.90 (0.77) vs. 3.14 (0.86) min, respectively; P = 0.037]. [K+]a increased during exercise until 1 min post‐exercise (P = 0.001), and fell below baseline at 3–10 (P = 0.001) and 20 min post‐exercise (P = 0.022, time main effect). In DIG, [K+]a (P = 0.035, treatment effect) and [K+]a rise pre‐fatigue were greater [1.64 (0.73) vs. 1.55 (0.73), P = 0.016], with lesser post‐exercise [K+]a decline than CON [−2.55 (0.71) vs. −2.74 (0.62) mM, respectively, P = 0.003]. Preserved muscle OB‐Fab with digoxin, yet increased OB+Fab with unchanged NKA isoforms, suggests a rapid regulatory assembly of existing NKA α and β subunits exists to preserve muscle NKA capacity. Nonetheless, functional protection against digoxin was incomplete, with earlier fatigue and perturbed [K+]a with exercise. Key points: Intense exercise causes marked potassium (K+) shifts out of contracting muscle cells, which may contribute to muscle fatigue.Muscle and systemic K+ perturbations with exercise are largely regulated by increased activity of Na+,K+‐ATPase in muscle, which can be specifically inhibited by the cardiac glycoside, digoxin.We found that acute oral digoxin in healthy adults reduced time to fatigue during intense exercise, elevated the rise in arterial plasma K+ concentration during exercise and slowed K+ concentration decline post‐exercise.Muscle functional Na+,K+‐ATPase content was not reduced by acute digoxin, despite an 8.2% digoxin occupancy, and was unchanged at fatigue. Muscle Na+,K+‐ATPase isoform protein abundances were unchanged by digoxin or fatigue. These suggest possible rapid assembly of existing subunits into functional pumps.Thus, acute digoxin impaired performance and exacerbated plasma K+ disturbances with intense, fatiguing exercise in healthy participants. These occurred despite the preservation of functional Na+,K+‐ATPase in muscle. [ABSTRACT FROM AUTHOR]

A considerable amount of NaCl reabsorption in proximal tubules (PTs) occurs via the paracellular transport regulated by the tight junction proteins claudins (Cldns). However, the paracellular transport properties in mouse superficial PTs remain unclear. We characterized these properties in superficial PT S1–S3 segments from mice expressing [wild-type (WT, WTS1–WTS3)] or lacking [knockout (KO, KOS1–KOS3)] claudin-2. We isolated and perfused segments with symmetrical solutions in the presence of bath ouabain and measured the diffusion potential upon changing the salt composition of the lumen or bath. Based on the diffusion potential corrected for the liquid junction potential (dVT), we calculated the paracellular Na+ over Cl− permeability (PNa/PCl) ratio. The PNa/PCl values upon reducing luminal NaCl averaged 1.27, 1.04, and 0.85 in WTS1, WTS2, and WTS3 and 0.34, 0.55, and 0.80 in KOS1, KOS2, and KOS3, respectively. The dVT values exhibited a symmetrical response to bidirectional NaCl concentration gradients in WTS1–WTS3 and KOS1–KOS3. WTS1 and WTS3 were monovalent cation-selective, with WTS1 demonstrating stronger cation selectivity. The order of permeabilities relative to Cl− was K+ > Rb+ > Na+ > Li+, whereas both KOS1 and KOS3 exhibited monovalent cation selectivity loss and, consequently, enhanced anion selectivity, especially in KOS1. Protamine addition to the lumen and bath similarly decreased PNa/PCl values upon reduced luminal NaCl in the order of WTS1 > WTS3 > KOS3 > KOS1. Therefore, this study presents evidence of axial heterogeneity in paracellular transport across superficial PTs in mice. NEW & NOTEWORTHY: Research on isolated perfused S2 segments of proximal tubules in mice, both expressing and lacking claudin-2, indicates that claudin-2 forms leaky monovalent cation-selective paracellular channels within the tight junctions of proximal tubules. This study characterized the paracellular transport properties in isolated and perfused superficial proximal tubule S1–S3 segments in both groups of mice. The findings demonstrate, for the first time, functional heterogeneity in the paracellular pathway along the axis of the superficial proximal tubules. [ABSTRACT FROM AUTHOR]

Spreading depolarization (SD) describes the near-complete depolarization of central nervous system (CNS) neural cells as a consequence of chemical, electrical, or metabolic perturbations. It is well established as the central mechanism underlying insect coma and various mammalian neurological dysfunctions. Despite significant progress in our understanding, the question remains: which cation channel, if any, generates SD in the CNS? Previously, we speculated that the sodium-potassium ATPase (NKA) might function as a large-conductance ion channel to initiate SD in insects, potentially mediated by a palytoxin (PLTX)-like endogenous activator. In the current study, we evaluate the effectiveness and properties of PLTX as an SD initiator in Locusta migratoria. Whereas bath-applied PLTX failed to ignite SD, direct injection into the neuropil triggered SD in 57% of the preparations. Notably, PLTX-induced SD onset was significantly more rapid compared with ouabain (OUA) injection and azide controls, though their electrophysiological features remained similar. Furthermore, PLTX-induced SD was recoverable and resulted in a greater frequency of repetitive SD events compared with ouabain. Surprisingly, prior PLTX treatment disrupted the onset and recovery of subsequent SD evoked by other means. PLTX injection could attenuate the amplitude and even completely inhibit the onset of azide-induced SD at higher doses. These results show that PLTX can trigger repetitive and reversible SD-like events in locusts and simultaneously interfere with anoxic SD occurrence. We suggest that the well-documented NKA pump conversion into an open nonselective cationic channel is a plausible mechanism of SD activation in the locust CNS, warranting additional investigations. NEW & NOTEWORTHY: Spreading depolarization (SD) is a critical mechanism underlying central nervous system (CNS) shutdown and injury under stress, yet the initiating ion channel remains unknown. Here, we used the marine poison palytoxin (PLTX), which converts the sodium-potassium ATPase (NKA) into an open channel, to initiate SD in intact locust CNS. We show for the first time that PLTX-induced SD is rapid and recoverable in vivo, providing support that NKA conversion to a channel may be the SD-initiating mechanism. [ABSTRACT FROM AUTHOR]

Diffuse intrinsic pontine glioma (DIPG) is a highly lethal tumor that occurs in the brain stem. At present, there is no effective drug for DIPG. In this work, Na/K‐ATPase inhibitor‐ouabain (OB) is found to efficiently kill H3K27M‐mutated DIPG at low doses, inhibit tumor cell proliferation, reduce tumor stemness for the first time, demonstrating its potential as a chemotherapy drug for DIPG. In order to increase the blood–brain barrier (BBB) permeability and tumor accumulation of OB, acidic‐responsive engineered exosomes loaded with OB (OB@EXO‐LCCP) are constructed, which increase the accumulation of OB within the tumor microenvironment of DIPG and efficiently deliver OB to DIPG cells to achieve targeted killing effect. In in vitro experiments, OB@EXO‐LCCP is able to release OB in an acid‐responsive manner and effectively kill DIPG tumor cells. In in vivo experiments, OB@EXO‐LCCP significantly inhibits DIPG growth and prolongs the overall survival of DIPG‐bearing mice. Overall, OB@EXO‐LCCP can promote the BBB permeability and tumor‐targeting ability of OB, thereby enhancing its DIPG‐killing ability and reducing its toxicity. This study provides technical and theoretical information for the precision therapy of DIPG. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is a common clinical neurodegenerative disorder, primarily characterized by progressive cognitive decline and behavioral abnormalities. The hallmark pathological changes of AD include widespread neuronal degeneration, plaques formed by the deposition of amyloid β-protein (Aβ), and neurofibrillary tangles (NFTs). With the acceleration of global aging, the incidence of AD is rising year by year, making it a major global public health concern. Due to the complex pathology of AD, finding effective interventions has become a key focus of research. Ouabain (OUA), a cardiac glycoside, is well-known for its efficacy in treating heart disease. Recent studies have also indicated its potential in AD therapy, although its exact mechanism of action remains unclear. Methods: This study integrates bioinformatics, multi-omics technologies, and in vivo and in vitro experiments to investigate the effects of OUA on the pathophysiological changes of AD and its underlying molecular mechanisms. Results: This study analyzed the expression of the triggering receptor expressed on myeloid cells 2 (TREM2) across different stages of AD using bioinformatics. Serum samples from patients were used to validate soluble TREM2 (sTREM2) levels. Using an Aβ1-42-induced microglial cell model, we confirmed that OUA enhances the PI3K/AKT signaling pathway activation by upregulating TREM2, which reduces neuroinflammation and promotes the transition of microglia from an M1 proinflammatory state to an M2 anti-inflammatory state. To evaluate the in vivo effects of OUA, we assessed the learning and memory capacity of FAD4T transgenic mice using the Morris water maze and contextual fear conditioning tests. We used real-time quantitative PCR, immunohistochemistry, and Western blotting to measure the expression of inflammation-associated cytokines and to assess microglia polarization. OUA enhances cognitive function in FAD4T mice and has been confirmed to modulate microglial M1/M2 phenotypes both in vitro and in vivo. Furthermore, through bioinformatics analysis, molecular docking, and experimental validation, TREM2 was identified as a potential target for OUA. It regulates PI3K/Akt signaling pathway activation, playing a crucial role in OUA-mediated M2 microglial polarization and its anti-inflammatory effects in models involving Aβ1-42-stimulated BV-2 cells and FAD4T mice. Conclusions: These findings indicate that OUA exerts anti-neuroinflammatory effects by regulating microglial polarization, reducing the production of inflammatory mediators, and activating the PI3K/Akt signaling pathway. Given its natural origin and dual effects on microglial polarization and neuroinflammation, OUA emerges as a promising therapeutic candidate for neuroinflammatory diseases such as AD. [ABSTRACT FROM AUTHOR]

Neurons of the subpostremal nucleus of the solitary tract (NTS) respond to changes in extracellular glucose with alterations in membrane potential with both depolarization and hyperpolarization. From 5 mM glucose, a rapid shift to 0.5 mM glucose produces a membrane depolarization by an unknown mechanism in most neurons. However, the mechanism involved in this response needs to be known. Here, we investigated if the low glucose-induced depolarization could be mimicked by reducing ATP synthesis and possible mediators of this effect. We showed that applying the mitochondrial uncoupler CCCP (1 µM) reproduced the effects of low glucose depolarizing the membrane, generating an inward current, and decreasing membrane resistance. On the other hand, activation of AMPK did not alter these parameters. To test if low glucose and CCCP could depolarize the membrane by affecting the ionic gradient, we inhibited the electrogenic Na/K pump with 10 µM of ouabain. We observed a similar membrane depolarization but not a decrease in membrane resistance. We conclude that perfusion of neurons of the subpostremal NTS with a low glucose solution depolarizes the membrane by probably reducing intracellular ATP, but not by activating AMPK or decreasing the ionic gradient across the membrane. [ABSTRACT FROM AUTHOR]

Background and Aims: Cardiac glycosides (CGs), traditionally used for heart failure, have shown potential as anti‐cancer agents. This study aims to explore their multifaceted mechanisms in cancer cell biology using proteome integral solubility alteration (PISA), focusing on the interaction with key proteins implicated in cellular metabolism and mitochondrial function. Methods: We conducted lysate‐based and intact‐cell PISA assays on cancer cells treated with CGs (Digoxin, Digitoxin, Ouabain) to analyze protein solubility changes. This was followed by mass spectrometric analysis and bioinformatics to identify differentially soluble proteins (DSPs). Molecular docking simulations were performed to predict protein‐CG interactions. Public data including gene expression changes upon CG treatment were re‐analyzed for validation. Results: The PISA assays revealed CGs' broad‐spectrum interactions, particularly affecting proteins like PKM2, ANXA2, SLC16A1, GOT2 and GLUD1. Molecular docking confirmed stable interactions between CGs and these DSPs. Re‐analysis of public data supported the impact of CGs on cancer metabolism and cell signaling pathways. Conclusion: Our findings suggest that CGs could be repurposed for cancer therapy by modulating cellular processes. The PISA data provide insights into the polypharmacological effects of CGs, warranting further exploration of their mechanisms and clinical potential. [ABSTRACT FROM AUTHOR]

m6A modification is a crucial epigenetic regulatory mechanism in diffuse large B-cell lymphoma (DLBCL). Low-dose cardiotonic drugs have been shown to induce apoptosis in DLBCL cells through epigenetic modulation. However, the involvement of the cardiotonic drug ouabain in the malignant progression of DLBCL remains unclear. Our study revealed that ouabain indeed contributes to the malignant progression of DLBCL through m6A modification. Through qPCR analysis, we observed a negative correlation between ouabain concentration and the expression levels of the demethylase ALKBH5 and the m6A-binding protein IGF2BP2 in DLBCL cells. Furthermore, high expression levels of ALKBH5 and IGF2BP2 were identified in both the GEO database and DLBCL patient tissue samples. Notably, elevated ALKBH5 and IGF2BP2 promoted cell proliferation both in vitro and in vivo. Inhibition of their expression rendered DLBCL cells more sensitive to ouabain treatment, resulting in significant suppression of cell proliferation, G1/S phase cell cycle arrest, and increased apoptosis. In summary, our results clarify that the demethylase ALKBH5 and the m6A-binding protein IGF2BP2 are involved in the malignant progression of DLBCL, and that the cardiotonic drug ouabain can inhibit the proliferation of DLBCL cells by inhibiting the expression of ALKBH5 and IGF2BP2, which provides new insights into the targeted treatment of DLBCL. [ABSTRACT FROM AUTHOR]

Hypoxic preconditioning is known to protect against various functional disorders, including skeletal muscle dysfunction. Na,K-ATPase, which plays an important role in adaptation to hypoxia, is critical for maintaining the performance of skeletal muscles, which co-express the α1 and α2 isozymes of the enzyme. Persistent dysfunction of α2 Na,K-ATPase isozyme is characteristic of the motor inactivity of skeletal muscles. In this study, we tested our hypothesis that mild hypoxia is able to improve Na,K-ATPase in disused soleus muscle. Rats were subjected to simulated high-altitude (3000 m above sea level) hypobaric hypoxia (HH) for 3 h using a hypobaric chamber. Then, 18 h after control or HH conditions, rats were subjected to 6 h of hindlimb suspension (HS), a model of skeletal muscle disuse. Isolated soleus muscles were tested. HH itself stably increased the α2 Na,K-ATPase isozyme membrane abundance and its electrogenic activity. These effects were accompanied by a decrease in serum level of circulating endogenous ouabain, a specific ligand for Na,K-ATPase. HS itself caused loss of electrogenic activity of α2 Na,K-ATPase, but pretreatment with HH protected against this impairment. This protective effect was accompanied by an increase in the α2 Na,K-ATPase membrane abundance without a change in total protein content, suggesting an increase in the α2 Na,K-ATPase traffic from the intracellular pool to the sarcolemma; serum ouabain level was significantly increased. We suggest that these findings open a new field for further studies and may have therapeutic implications for disuse-induced skeletal muscle pathology. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is a highly prevalent human gastric pathogen that causes gastritis, ulcer disease, and gastric cancer. It is not yet fully understood how H. pylori injures the gastric epithelium. The Na,K-ATPase, an essential transporter found in virtually all mammalian cells, has been shown to be important for maintaining the barrier function of lung and kidney epithelia. H. pylori decreases levels of Na,K-ATPase in the plasma membrane of gastric epithelial cells, and the aim of this study was to demonstrate that this reduction led to gastric injury by impairing the epithelial barrier. Similar to H. pylori infection, the inhibition of Na,K-ATPase with ouabain decreased transepithelial electrical resistance and increased paracellular permeability in cell monolayers of human gastric cultured cells, 2D human gastric organoids, and gastric epithelium isolated from gerbils. Similar effects were caused by a partial shRNA silencing of Na,K-ATPase in human gastric organoids. Both H. pylori infection and ouabain exposure disrupted organization of adherens junctions in human gastric epithelia as demonstrated by E-cadherin immunofluorescence. Functional and structural impairment of epithelial integrity with a decrease in Na,K-ATPase amount or activity provides evidence that the H. pylori-induced downregulation of Na,K-ATPase plays a role in the complex mechanism of gastric disease induced by the bacteria. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the antihypertensive and antioxidant effects of flaxseed oil in ouabain-induced hypertensive in normal rats Wistar. Experimental hypertension was induced by a daily subcutaneous (sc) injection of 10 μg/kg/day/rat of ouabain diluted in 9 g/L of saline solution (NaCl) for 21 days (n = 16). Animals were then divided into two groups fed an experimental diet containing 20% casein supplemented (Ouab-Oil- Lu) or not (Ouab) with 1% flaxseed oil whereas control rats (n = 8) received only a daily sc injection of NaCl. The results showed that supplementing hypertensive rats with 1% flaxseed oil decreased significantly (P < 0.05) the systolic, diastolic, and mean arterial pressure, ameliorated plasma and liver lipid profile and reduced glucose levels in plasma. Similarly, flaxseed oil decreased TBARS levels in plasma and tissues, as well as carbonyl levels in the heart. Interestingly, this supplementation increased GSH, thiols and nitric oxide levels and improved AchE and antioxidant enzyme activities (SOD and GSH-Px) in the tissues. Our findings suggested that flaxseed oil may provide a rationale for antihypertensive use in traditional medicine. [ABSTRACT FROM AUTHOR]

The sodium pump, or Na+/K+-ATPase (NKA), is an essential enzyme found in the plasma membrane of all animal cells. Its primary role is to transport sodium (Na+) and potassium (K+) ions across the cell membrane, using energy from ATP hydrolysis. This transport creates and maintains an electrochemical gradient, which is crucial for various cellular processes, including cell volume regulation, electrical excitability, and secondary active transport. Although the role of NKA as a pump was discovered and demonstrated several decades ago, it remains the subject of intense research. Current studies aim to delve deeper into several aspects of this molecular entity, such as describing its structure and mode of operation in atomic detail, understanding its molecular and functional diversity, and examining the consequences of its malfunction due to structural alterations. Additionally, researchers are investigating the effects of various substances that amplify or decrease its pumping activity. Beyond its role as a pump, growing evidence indicates that in various cell types, NKA also functions as a receptor for cardiac glycosides like ouabain. This receptor activity triggers the activation of various signaling pathways, producing significant morphological and physiological effects. In this report, we present the results of a comprehensive review of the most outstanding studies of the past five years. We highlight the progress made regarding this new concept of NKA and the various cardiac glycosides that influence it. Furthermore, we emphasize NKA's role in epithelial physiology, particularly its function as a receptor for cardiac glycosides that trigger intracellular signals regulating cell–cell contacts, proliferation, differentiation, and adhesion. We also analyze the role of NKA β-subunits as cell adhesion molecules in glia and epithelial cells. [ABSTRACT FROM AUTHOR]

Abstract Background Glycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na+/K+-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells. Methods Live whole-cell measurements of intracellular sodium [Na+]i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2H NMR to monitor conversion of [6,6-2H2]d-glucose to [2H]-labelled l-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na+]i was titrated using isotonic buffers with varying [Na+] and [K+] and introducing an artificial Na+ plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[18F]fluoroglucose (18F-FDG) positron emission tomography (PET). Results Intracellular [Na+]i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na+]i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na+]i in MDA-MB-231 and 4T1 cells and a Na+-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected. Conclusions Glycolytic flux correlates with Na+-driven NKA activity in breast cancer cells, providing evidence for the ‘centrality of the [Na+]i-NKA nexus’ in the mechanistic basis of the Warburg effect.

Background: Glycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na+/K+-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells. Methods: Live whole-cell measurements of intracellular sodium [Na+]i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2H NMR to monitor conversion of [6,6-2H2]d-glucose to [2H]-labelled l-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na+]i was titrated using isotonic buffers with varying [Na+] and [K+] and introducing an artificial Na+ plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[18F]fluoroglucose (18F-FDG) positron emission tomography (PET). Results: Intracellular [Na+]i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na+]i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na+]i in MDA-MB-231 and 4T1 cells and a Na+-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected. Conclusions: Glycolytic flux correlates with Na+-driven NKA activity in breast cancer cells, providing evidence for the 'centrality of the [Na+]i-NKA nexus' in the mechanistic basis of the Warburg effect. [ABSTRACT FROM AUTHOR]

Cardiotonic steroids (CTSs), such as digoxin, are used for heart failure treatment. However, digoxin permeates the brain–blood barrier (BBB), affecting central nervous system (CNS) functions. Finding a CTS that does not pass through the BBB would increase CTSs' applicability in the clinic and decrease the risk of side effects on the CNS. This study aimed to investigate the tissue distribution of the CTS ouabain following intraperitoneal injection and whether ouabain passes through the BBB. After intraperitoneal injection (1.25 mg/kg), ouabain concentrations were measured at 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h using HPLC–MS in brain, heart, liver, and kidney tissues and blood plasma in C57/black mice. Ouabain was undetectable in the brain tissue. Plasma: Cmax = 882.88 ± 21.82 ng/g; Tmax = 0.08 ± 0.01 h; T1/2 = 0.15 ± 0.02 h; MRT = 0.26 ± 0.01. Cardiac tissue: Cmax = 145.24 ± 44.03 ng/g (undetectable at 60 min); Tmax = 0.08 ± 0.02 h; T1/2 = 0.23 ± 0.09 h; MRT = 0.38 ± 0.14 h. Kidney tissue: Cmax = 1072.3 ± 260.8 ng/g; Tmax = 0.35 ± 0.19 h; T1/2 = 1.32 ± 0.76 h; MRT = 1.41 ± 0.71 h. Liver tissue: Cmax = 2558.0 ± 382.4 ng/g; Tmax = 0.35 ± 0.13 h; T1/2 = 1.24 ± 0.7 h; MRT = 0.98 ± 0.33 h. Unlike digoxin, ouabain does not cross the BBB and is eliminated quicker from all the analyzed tissues, giving it a potential advantage over digoxin in systemic administration. However, the inability of ouabain to pass though the BBB necessitates intracerebral administration when used to investigate its effects on the CNS. [ABSTRACT FROM AUTHOR]

Cardiotonic steroids (CTS), used by certain insects, toads, and rats for protection from predators, became, thanks to Withering's trailblazing 1785 monograph, the mainstay of heart failure (HF) therapy. In the 1950s and 1960s, we learned that the CTS receptor was part of the sodium pump (NKA) and that the Na+/Ca2+ exchanger was critical for the acute cardiotonic effect of digoxin- and ouabain-related CTS. This "settled" view was upended by seven revolutionary observations. First, subnanomolar ouabain sometimes stimulates NKA while higher concentrations are invariably inhibitory. Second, endogenous ouabain (EO) was discovered in the human circulation. Third, in the DIG clinical trial, digoxin only marginally improved outcomes in patients with HF. Fourth, cloning of NKA in 1985 revealed multiple NKA α and β subunit isoforms that, in the rodent, differ in their sensitivities to CTS. Fifth, the NKA is a cation pump and a hormone receptor/signal transducer. EO binding to NKA activates, in a ligand- and cell-specific manner, several protein kinase and Ca2+-dependent signaling cascades that have widespread physiological effects and can contribute to hypertension and HF pathogenesis. Sixth, all CTS are not equivalent, e.g., ouabain induces hypertension in rodents while digoxin is antihypertensinogenic ("biased signaling"). Seventh, most common rodent hypertension models require a highly ouabain-sensitive α2 NKA and the elevated blood pressure is alleviated by EO immunoneutralization. These numerous phenomena are enabled by NKA's intricate structure. We have just begun to understand the endocrine role of the endogenous ligands and the broad impact of the ouabain-binding site on physiology and pathophysiology. [ABSTRACT FROM AUTHOR]

Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 μg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay. [ABSTRACT FROM AUTHOR]

Acanthamoeba, a free-living amoeba, is commonly found in various natural environments, such as rivers and soil, as well as in public baths, swimming pools, and sewers. Acanthamoeba can cause severe illness such as granulomatous amoebic encephalitis and Acanthamoeba keratitis (AK) in humans. AK, the most recognized disease, can cause permanent visual impairment or blindness by affecting the cornea. AK commonly affects contact lens wearers who neglect proper cleaning habits. The symptoms of AK include epithelial and stromal destruction, corneal infiltrate, and intense ocular pain, occasionally necessitating surgical removal of the entire eyeball. Current AK treatment involves the hourly application of eye drops containing polyhexamethylene biocide (PHMB). However, studies have revealed their ineffectiveness against drug-resistant strains. Acanthamoeba can form cysts as a survival mechanism in adverse environments, though the exact mechanism remains unknown. Our experiments revealed that sodium P-type ATPase (ACA1_065450) is closely linked to encystation. In addition, various encystation buffers, such as MgCl2 or NaCl, induced the expression of P-type ATPase. Furthermore, we used ouabain, an ATPase inhibitor, to inhibit the Na+/K+ ion pump, consequently decreasing the encystation rate of Acanthamoeba. Our primary objective is to develop an advanced treatment for AK. We anticipate that the combination of ouabain and PHMB may serve as an effective therapeutic approach against AK in the future.

Introduction. Cardiac glycosides are natural ligands of Na+/K+-ATPase, which regulate its activity and signaling. Intracerebroventricular administration of ouabain has been previously shown to induce hyperlocomotion in C57Bl/6 mice via a decrease in the rate of dopamine reuptake from the synaptic cleft. Materials and methods. This study involved forty C57BL/6 mice. 1.5 μL of 50 μM ouabain was administered daily into the left lateral cerebral ventricle over the course of 4 days. On day 5, open field, beam balance, and ladder rung walking tests were performed to assess the locomotor activity and motor impairments in the mice. We evaluated changes in the activation of signaling cascades, ratios of proapoptotic and antiapoptotic proteins, and the amount of α1 and α3 isoforms of the Na+/K+-ATPase α-subunit in brain tissue using Western blotting. Na+/K+-ATPase activity was evaluated in the crude synaptosomal fractions of the brain tissues. Results. We observed hyperlocomotion and stereotypic behavior during the open field test 24 hours after the last injection of ouabain. On day 5, the completion time and the number of errors made in the beam balance and ladder rung walking tests increased in the mice that received ouabain. Akt kinase activity decreased in the striatum, whereas the ratio of proapoptotic and antiapoptotic proteins and the number of Na+/K+-ATPase α-subunits did not change. Na+/K+-ATPase activity increased in the striatum and decreased in the brainstem. Conclusions. Long-term exposure to ouabain causes motor impairments mediated by changes in the activation of signaling cascades in dopaminergic neurons.

The optimal function of the Na,K-ATPase (NKA) pump is essential for the heart. In ischemic heart disease, NKA activity decreases due to the decreased expression of the pump subunits. Here, we tested whether the hypoxia-inducible transcription factor (HIF-1α), the key signaling molecule regulating the adaptation of cells to hypoxia, is involved in controlling the expression and cellular dynamics of α1- and β1-NKA isoforms and of NKA activity in in-vitro hypoxic H9c2 cardiomyoblasts. HIF-1α was silenced through adenoviral infection, and cells were kept in normoxia (19% O2) or hypoxia (1% O2) for 24 h. We investigated the mRNA and protein expression of α1-, β1-NKA using RT-qPCR and Western blot in whole-cell lysates, cell membranes, and cytoplasmic fractions after labeling the cell surface with NHS-SS-biotin and immunoprecipitation. NKA activity and intracellular ATP levels were also measured. We found that in hypoxia, silencing HIF-1α prevented the decreased mRNA expression of α1-NKA but not of β1-NKA. Hypoxia decreased the plasma membrane expression of α1-NKA and β1- NKA compared to normoxic cells. In hypoxic cells, HIF-1α silencing prevented this effect by inhibiting the internalization of α1-NKA. Total protein expression was not affected. The decreased activity of NKA in hypoxic cells was fully prevented by silencing HIF-1α independent of cellular ATP levels. This study is the first to show that in hypoxic H9c2 cardiomyoblasts, HIF-1α controls the internalization and membrane insertion of α1-NKA subunit and of NKA activity. The mechanism behind this regulation needs further investigation.

Abstract Background Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)—the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored. Methods Digoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation—one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery. Results Primitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation. Conclusions Our data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery. Video Abstract

Ouabain, an organic compound with the ability to strengthen the contraction of the heart muscle, was originally derived from plants. It has been observed that certain mammalian species, including humans, naturally produce ouabain, leading to its classification as a new type of hormone. When ouabain binds to Na+/K+-ATPase, it elicits various physiological effects, although these effects are not well characterized. Previous studies have demonstrated that ouabain, within the concentration range found naturally in the body (10 nmol/L), affects the polarity of epithelial cells and their intercellular contacts, such as tight junctions, adherens junctions, and gap junctional communication. This is achieved by activating signaling pathways involving cSrc and Erk1/2. To further investigate the effects of ouabain within the hormonally relevant concentration range (10 nmol/L), mRNA-seq, a high-throughput sequencing technique, was employed to identify differentially expressed transcripts. The discovery that the transcript encoding MYO9A was among the genes affected prompted an exploration of whether RhoA and its downstream effector ROCK were involved in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells. Supporting this hypothesis, this study reveals the following: (1) Ouabain increases the activation of RhoA. (2) Treatment with inhibitors of RhoA activation (Y27) and ROCK (C3) eliminates the enhancing effect of ouabain on the tight junction seal and intercellular communication via gap junctions. These findings further support the notion that ouabain acts as a hormone to emphasize the epithelial phenotype.

The brilliant red Lilioceris merdigera (Coleoptera, Chrysomelidae) can spend its entire life cycle on the cardenolide-containing plant Convallaria majalis (lily of the valley) and forms stable populations on this host. Yet, in contrast to many other insects on cardenolide-containing plants L. merdigera does not sequester these plant toxins in the body but rather both adult beetles and larvae eliminate ingested cardenolides with the feces. Tracer feeding experiments showed that this holds true for radioactively labeled ouabain and digoxin, a highly polar and a rather apolar cardenolide. Both compounds or their derivatives are incorporated in the fecal shields of the larvae. The apolar digoxin, but not the polar ouabain, showed a deterrent effect on the generalist predatory ant Myrmica rubra, which occurs in the habitat of L. merdigera. The deterrent effect was detected for digoxin both in choice and feeding time assays. In a predator choice assay, a fecal shield derived from a diet of cardenolide-containing C. majalis offered L. merdigera larvae better protection from M. rubra than one derived from non-cardenolide Allium schoenoprasum (chives) or no fecal shield at all. Thus, we here present data suggesting a new way how insects may gain protection by feeding on cardenolide-containing plants. [ABSTRACT FROM AUTHOR]

Ionizing radiation (IR) causes disturbances in the functions of the gastrointestinal tract. Given the therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against IR-induced disturbances in the barrier and transport properties of the jejunum and colon of rats. Male Wistar rats were subjected to 6-day intraperitoneal injections of vehicle or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to total-body X-ray irradiation (10 Gy) or a sham irradiation. Isolated tissues were examined 72 h post-irradiation. Electrophysiological characteristics and paracellular permeability for sodium fluorescein were measured in an Ussing chamber. Histological analysis and Western blotting were also performed. In the jejunum tissue, ouabain exposure did not prevent disturbances in transepithelial resistance, paracellular permeability, histological characteristics, as well as changes in the expression of claudin-1, -3, -4, tricellulin, and caspase-3 induced by IR. However, ouabain prevented overexpression of occludin and the pore-forming claudin-2. In the colon tissue, ouabain prevented electrophysiological disturbances and claudin-2 overexpression. These observations may reveal a mechanism by which circulating ouabain maintains tight junction integrity under IR-induced intestinal dysfunction. [ABSTRACT FROM AUTHOR]

Ouabain, a substance originally obtained from plants, is now classified as a hormone because it is produced endogenously in certain animals, including humans. However, its precise effects on the body remain largely unknown. Previous studies have shown that ouabain can influence the phenotype of epithelial cells by affecting the expression of cell–cell molecular components and voltage-gated potassium channels. In this study, we conducted whole-cell clamp assays to determine whether ouabain affects the activity and/or expression of TRPV4 channels. Our findings indicate that ouabain has a statistically significant effect on the density of TRPV4 currents (dITRPV4), with an EC50 of 1.89 nM. Regarding treatment duration, dITRPV4 reaches its peak at around 1 h, followed by a subsequent decline and then a resurgence after 6 h, suggesting a short-term modulatory effect related to on TRPV4 channel activity and a long-term effect related to the promotion of synthesis of new TRPV4 channel units. The enhancement of dITRPV4 induced by ouabain was significantly lower in cells seeded at low density than in cells in a confluent monolayer, indicating that the action of ouabain depends on intercellular contacts. Furthermore, the fact that U73122 and neomycin suppress the effect caused by ouabain in the short term suggests that the short-term induced enhancement of dITRPV4 is due to the depletion of PIP2 stores. In contrast, the fact that the long-term effect is inhibited by PP2, wortmannin, PD, FR18, and IKK16 suggests that cSrc, PI3K, Erk1/2, and NF-kB are among the components included in the signaling pathways. [ABSTRACT FROM AUTHOR]

Renal cyst progression in autosomal dominant polycystic kidney disease (ADPKD) is highly dependent on agents circulating in blood. We have previously shown, using different in vitro models, that one of these agents is the hormone ouabain. By binding to Na+-K+-ATPase (NKA), ouabain triggers a cascade of signal transduction events that enhance ADPKD cyst progression by stimulating cell proliferation, fluid secretion, and dedifferentiation of the renal tubular epithelial cells. Here, we determined the effects of ouabain in vivo. We show that daily administration of ouabain to Pkd1RC/RC ADPKD mice for 1–5 mo, at physiological levels, augmented kidney cyst area and number compared with saline-injected controls. Also, ouabain favored renal fibrosis; however, renal function was not significantly altered as determined by blood urea nitrogen levels. Ouabain did not have a sex preferential effect, with male and female mice being affected equally. By contrast, ouabain had no significant effect on wild-type mice. In addition, the actions of ouabain on Pkd1RC/RC mice were exacerbated when another mutation that increased the affinity of NKA for ouabain was introduced to the mice (Pkd1RC/RCNKAα1OS/OS mice). Altogether, this work highlights the role of ouabain as a procystogenic factor in the development of ADPKD in vivo, that the ouabain affinity site on NKA is critical for this effect, and that circulating ouabain is an epigenetic factor that worsens the ADPKD phenotype. NEW & NOTEWORTHY This work shows that the hormone ouabain enhances the progression of autosomal dominant polycystic kidney disease (ADPKD) in vivo. Ouabain augments the size and number of renal cysts, the kidney weight to body weight ratio, and kidney fibrosis in an ADPKD mouse model. The Na+-K+-ATPase affinity for ouabain plays a critical role in these effects. In addition, these outcomes are independent of the sex of the mice. [ABSTRACT FROM AUTHOR]

The presence of secondary metabolites in flower nectar can mediate interactions between plants, pollinators, herbivores, and microbes. Milkweeds range in concentrations of cardenolides in flower nectar from ~ 1 to 100 ng μl–1. Using three different behavioral assays with bumblebees Bombus impatiens, we examined the impacts of the commercially available cardenolide ouabain at the range of concentrations at which cardenolides naturally occur in milkweeds. We show that after four days of exposure bees in consumption assays drank more of a nectar solution with a low ouabain concentration of 10 ng μl–1 than the control sucrose nectar, and over the course of the experiment bees consumed less of the 100 ng μl–1 ouabain solution than the control. Bee activity levels in Petri dish arena assays were not impacted by ouabain consumption, even at the highest concentrations; however, in free‐flying choice assays, bees preferentially visited artificial flowers containing 10 ng μl–1 ouabain more than flowers with sucrose control, or flowers with 100 ng μl–1 of ouabain. We therefore conclude that cardenolides may provide plants advantages to pollination at the low end of the naturally occurring range of concentrations for cardenolides, but may be costly to plants at the high end of the range. This research highlights that secondary metabolites in nectar may be under selection to be maintained at low concentrations, and species with high concentrations of secondary metabolites may be under conflicting selection pressures to maintain high circulating levels to deter herbivores at the cost of reduced pollination. [ABSTRACT FROM AUTHOR]

Eugenol is an aromatic compound used in the manufacture of medicines, perfumes, cosmetics and as an anaesthetic due to the ability of the drug to block the neuronal isoform of voltage‐gated Na+ channels (NaV). Some arrhythmias are associated with gain of function in the sodium current (INa) found in cardiomyocytes, and antiarrhythmic sodium channel blockers are commonly used in the clinical practice. This study sought to elucidate the potential mechanisms of eugenol's protection in the arrhythmic model of ouabain‐induced arrhythmias in guinea pig heart. Ex vivo arrhythmias were induced using 50 μM of ouabain. The antiarrhythmic properties of eugenol were evaluated in the ex vivo heart preparation and isolated ventricular cardiomyocytes. The compound's effects on cardiac sodium current and action potential using the patch‐clamp technique were evaluated. In all, eugenol decreased the ex vivo cardiac arrhythmias induced by ouabain. Furthermore, eugenol showed concentration dependent effect upon peak INa, left‐shifted the stationary inactivation curve and delayed the recovery from inactivation of the INa. All these aspects are considered to be antiarrhythmic. Our findings demonstrate that eugenol has antiarrhythmic activity, which may be partially explained by the ability of eugenol to change de biophysical properties of INa of cardiomyocytes. [ABSTRACT FROM AUTHOR]

This study explored the role of the Na/K-ATPase (NKA) in membrane permeabilization induced by nanosecond electric pulses. Using CRISPR/Cas9 and shRNA, we silenced the ATP1A1 gene, which encodes α1 NKA subunit in U937 human monocytes. Silencing reduced the rate and the cumulative uptake of YoPro-1 dye after electroporation by 300-ns, 7–10 kV/cm pulses, while ouabain, a specific NKA inhibitor, enhanced YoPro-1 entry. We conclude that the α1 subunit supports the electropermeabilized membrane state, by forming or stabilizing electropores or by hindering repair mechanisms, and this role is independent of NKA's ion pump function. • ATP1A1 gene encoding for α1 subunit of Na,K-ATPase was silenced by CRISPR and shRNA. • Expression of α1 subunit of Na,K-ATPase enhanced membrane damage by electroporation. • Inhibition of Na,K-ATPase pump function enhanced electropermeabilization. • α1 subunit of Na,K-ATPase contributes to electropore formation and/or longevity. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)—the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored. Methods: Digoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation—one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery. Results: Primitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation. Conclusions: Our data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery. 53GxHmRcFt-KhVdMmSCJrs Video Abstract [ABSTRACT FROM AUTHOR]

Na+/K+-ATPase maintains the neuron's resting potential and the transmembrane gradient of K+ and Na+ cations, thus regulating ion transport and cellular volume. Mutations in Na+/K+-ATPase genes that impair its function can cause significant impairments in the nervous system function, including the development of epilepsy, if not lethal. Different forms of Na+/K+-ATPase are expressed in various classes of neurons and exhibit different characteristics. Thus, the impaired function of Na+/K+-ATPase may differentially affect the functioning of inhibitory and excitatory neurons. This study aims to determine the effects of the Na+/K+-ATPase antagonist ouabain on the electrophysiological characteristics of pyramidal cells and fast-spiking interneurons, as well as its impact on synaptic transmission. The results indicate that exposure to 5 µM ouabain results in depolarization of the resting membrane potential of both types of neurons, as well as decreased amplitude and increased duration of the action potential of pyramidal neurons. Furthermore, ouabain caused a decrease in the amplitude of afterhyperpolarization in fast-spiking interneurons. Moreover, both types of neurons exhibited a decrease in the threshold of action potential generation and the current at which depolarization block occurs. The addition of ouabain did not alter other electrophysiological characteristics of neurons. Furthermore, ouabain rapidly attenuates GABAergic transmission without affecting excitatory synaptic transmission. These new findings on the effects of ouabain on excitatory pyramidal neurons and inhibitory interneurons contribute to the understanding of the mechanism underlying changes in the balance of excitation and inhibition in neural networks under Na+/K+-ATPase function impairment. [ABSTRACT FROM AUTHOR]

Abstract Background Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor prognosis. GMB are highly recurrent mainly because of radio- and chemoresistance. Radiotherapy with Temozolomide (TMZ) is until today the golden standard adjuvant therapy, however, the optimal treatment of recurrent glioblastoma remains controversial. Ouabain belongs to the Cardiotonic Steroids (CTS) the natural ligands of the Na/K-ATPase (NKA). It is established that the NKA represents a signal transducer with either stimulating or inhibiting cell growth, apoptosis, migration and angiogenesis. Over the last decade evidence grew that CTS have anti-tumor properties especially in GBM. Aim Proceeding from recent studies we wanted to further demonstrate a divergent effect of Ouabain on a TMZ-resistant (T98G) as compared to a TMZ-sensitive (LN229) GBM cell line. Methods We analyzed the effect of Ouabain on cell migration and plasma cell membrane potential (PCMP) in the LN229 and T98G GBM cell line as well as underlying mechanisms (Bcl-2 and p-Akt/pan-Akt expression). Moreover, we analyzed the anti-angiogenic effect of Ouabain on human umbilical vein endothelial cells (HUVECs). Results T98G cells showed a significant inhibition of cell migration and a significant depolarization of the PCMP at similar Ouabain concentrations (IC50 = 1.67 × 10–7 M) resp. (IC50 = 2.72 × 10–7 M) with a strong inverse correlation (R2 = 0.95). In contrast, LN229 cells did not respond to Ouabain in these assays at all. Similarly, only T98G but not LN229 cells revealed Bcl-2 down-regulation at nanomolar Ouabain concentrations. This unique response to Ouabain is associated with a down-regulation of pan-Akt in T98G cells 24 h after Ouabain (1.0 × 10–6 M) treatment. For the first time, the anti-angiogenic effect of Ouabain on HUVEC cells (IC50 = 5.49 × 10–8 M) was demonstrated which correlated strongly with the anti-migratory effect (R2 = 0.85). Conclusion The TMZ-resistant T98G cell line as compared to the TMZ-sensitive LN229 cell line shows a high sensitivity towards Ouabain. We consider it as a promising new compound especially in recurrent GBM to overcome the resistance to TMZ and irradiation.

Ouabain (OUA) is a cardiotonic steroid with an immunomodulatory and anti-inflammatory role in different experimental models. Currently, the potential antineoplastic effect of OUA has been studied, however, research is needed to better understand OUA role during tumor development. Therefore, our aim was to investigate the OUA effects on Ehrlich tumor (ET) development in vitro and in vivo. To evaluate the cytotoxic effects of OUA on ET in vitro the cells were incubated with different concentrations of OUA during 24h and 48h and our results showed that only the [1000 ?M] decreased the number and viability of ET cells in the two analyzed times. To study the OUA effects on ET in vivo, Swiss mice were pretreated with 0.56 mg/kg of OUA intraperitoneally (i.p.) for three consecutive days. To develop ET in the solid form, one hour after the last day of pretreatment, ET cells were inoculated subcutaneously into the footpad and the animals were monitored for 13 days. To develop the ascitic form, ET cells were inoculated (i.p.) and the animals were monitored for 3 days. OUA was able to reduce the thickness and weight of the tumor paw, in addition to reduce the weight of the popliteal lymph node. In the ascitic tumor, OUA reduced the number of neutrophils and macrophages and increased the lymphocytes in the peritoneum. Thus, we demonstrated that OUA affects ET development both in vitro and in vivo. Our results suggest a new perspective in the anti-inflammatory, immunomodulatory and a possible anti-cancer role of ouabain and brings new concepts about the pathophysiological role of this substance.

Preeclampsia (PE), the most severe presentation of hypertensive disorders of pregnancy, is the major cause of morbidity and mortality linked to pregnancy, affecting both mother and fetus. Despite advances in prophylaxis and managing PE, delivery of the fetus remains the only causative treatment available. Focus on complex pathophysiology brought the potential for new treatment options, and more conservative options allowing reduction of feto-maternal complications and sequelae are being investigated. Endogenous digitalis-like factors, which have been linked to the pathogenesis of preeclampsia since the mid-1980s, have been shown to play a role in the pathogenesis of various cardiovascular diseases, including congestive heart failure and chronic renal disease. Elevated levels of EDLF have been described in pregnancy complicated by hypertensive disorders and are currently being investigated as a therapeutic target in the context of a possible breakthrough in managing preeclampsia. This review summarizes mechanisms implicating EDLFs in the pathogenesis of preeclampsia and evidence for their potential role in treating this doubly life-threatening disease. [ABSTRACT FROM AUTHOR]

Na/K-ATPase maintains transmembrane ionic gradients and acts as a signal transducer when bound to endogenous cardiotonic steroids. At subnanomolar concentrations, ouabain induces neuroprotection against calcium overload and apoptosis of neurons during excitotoxic stress. Here, the role of lipid rafts in interactions between Na/K-ATPase, sodium–calcium exchanger (NCX), and N-methy-D-aspartate receptors (NMDARs) was investigated. We analyzed 0.5–1-nanometer ouabain's effects on calcium responses and miniature post-synaptic current (mEPSCs) frequencies of cortical neurons during the action of NMDA in rat primary culture and brain slices. In both objects, ouabain attenuated NMDA-evoked calcium responses and prevented an increase in mEPSC frequency, while the cholesterol extraction by methyl-β-cyclodextrin prevented the effects. The data support the conclusions that (i) ouabain-induced inhibition of NMDA-elicited calcium response involves both pre- and post-synapse, (ii) the presence of astrocytes in the tripartite synapse is not critical for the ouabain effects, which are found to be similar in cell cultures and brain slices, and (iii) ouabain action requires the integrity of cholesterol-rich membrane microdomains in which the colocalization and functional interaction of NMDAR-transferred calcium influx, calcium extrusion by NCX, and Na/K-ATPase modulation of the exchanger occur. This regulation of the molecules by cardiotonic steroids may influence synaptic transmission, prevent excitotoxic neuronal death, and interfere with the pharmacological actions of neurological medicines. [ABSTRACT FROM AUTHOR]

Bipolar disorder (BD) is a complex and severe mental disorder that affects 1–3 % of the world population. Studies have suggested the involvement of oxidative stress in the physiopathology of this psychiatry disorder. Folic acid (FA), a vitamin from the B complex, is a nutraceutical that has recently been researched as a possible treatment for BD since folate is reduced in patients with the disorder. The present study aimed to evaluate the effects of lithium (Li) and FA on behavioral changes and oxidative stress parameters in an animal model of mania induced by ouabain (OUA). Wistar rats received a single intracerebroventricular (ICV) injection of OUA or artificial cerebrospinal fluid (aCSF). From the day following ICV injection, the rats were treated for seven days with gavage injections of Li (47.5 mg/kg/mL), FA (50 mg/kg/mL), or water (1 mL/kg). On the 7th day after OUA injection, locomotor activity was measured using the open-field test. In addition, the oxidative stress parameters were evaluated in rats' frontal cortex, striatum, and hippocampus. OUA induced mania-like behavior and oxidative stress in rats' brains, but Li could reverse these alterations. FA did not affect behavior parameters; however, it presents an antioxidant effect on the brain structures evaluated. The study was only evaluated male rats and ICV injection is an invasive procedure. These results indicate that even though FA has an effect against the oxidative stress induced by OUA, this effect was not strong enough to interfere with behavior parameters. • Ouabain induces hyperactivity and oxidative stress in Wistar rats. • Lithium reverted hyperactivity and oxidative stress induced by ouabain. • Folic acid did not revert hyperactivity induced by ouabain. • Folic acid protected the brain against oxidative damage induced by ouabain. [ABSTRACT FROM AUTHOR]

Na+/K+ ATPase is a protein involved in the active transport of ions across the cellular membrane. Ouabain is a cardiotonic glycoside that, by inhibiting the Na+/K+ pump, interferes with cell processes mediated directly by the pump, but also indirectly influences other cellular processes such as cell cycle and proliferation, growth, cell differentiation, angiogenesis, migration, adhesion, and invasion. We used the SK-BR-3 breast cancer cell line, mesenchymal stem cells (MSCs), and tumor-associated fibroblasts (TAFs) in vitro to determine the effects of ouabain exposure on these cellular types. The results showed a multi-level effect of ouabain mainly on tumor cells, in a dose-dependent manner, while the TAFs and their normal counterparts were not significantly influenced. Following exposure to ouabain, the SK-BR-3 cells changed their morphologic appearance, decreased the expression of immunophenotypic markers (CD29, Her2, VEGF), the proliferation rate was significantly decreased (Ki67 index), the cells were blocked in the G0 phase of the cell cycle and suffered necrosis. These data were correlated with the variable expression of α and β Na+/K+ pump subunits in tumor cells, resulting in decreased ability to adhere to the VCAM-1 substrate in functional flow chamber studies. Being indicative of the pro-apoptotic and inhibitory effect of ouabain on tumor invasion and metastasis, the results support the addition of ouabain to the oncological therapeutic arsenal, trailing the "repurposing drugs" approach. [ABSTRACT FROM AUTHOR]

Simple Summary: Medicinal treatment of chronic pain prompts finding novel approaches to the creation of safe and effective analgesics. Ouabain, a cardiotonic steroid detected in the human organism at extremely low concentrations, has been previously shown by us to switch off the pain signal produced by peripheral neurons. The current manuscript elucidates the mechanism of ouabain binding to its molecular target, the Na,K-ATPase enzyme. Application of very sensitive physiological techniques demonstrated a complete loss of the ouabain effect upon removal of the rhamnosyl residue, a structural element of the ouabain molecule. Theoretical modeling made it possible to determine the contribution of the rhamnosyl residue to the process of ouabain binding with Na,K-ATPase. It was concluded that intermolecular bonds between the rhamnosyl residue of ouabain and Na,K-ATPase amino acid residues identified through modeling are required for the analgesic effect of ouabain to manifest itself. To facilitate creation of fundamentally new safe and effective analgesics, the mechanisms of their binding to the corresponding molecular receptors should be clarified at the atomic level. The signaling or non-pumping Na,K-ATPase function was first observed by us in the nociceptive neuron; Na,K-ATPase transduced the signals from the opioid-like receptors to NaV1.8 channels. This study elucidates the role of the rhamnosyl residue of ouabain in the activation of the Na,K-ATPase signaling function. The effects resulting from activation of Na,K-ATPase signaling by the Ca2+ chelate complex of ouabain (EO) are not manifested upon removal of the rhamnosyl residue, as demonstrated in viable cells by the highly sensitive patch-clamp and organotypic cell culture methods. Docking calculations show that the rhamnosyl residue is involved in five intermolecular hydrogen bonds with the Na,K-ATPase α1-subunit, which are fundamentally important for activation of the Na,K-ATPase signaling function upon EO binding. The main contribution to the energy of EO binding is provided by its steroid core, which forms a number of hydrogen bonds and hydrophobic interactions with Na,K-ATPase that stabilize the ligand–receptor complex. Another critically important role in EO binding is expected to be played by the chelated Ca2+ cation, which should switch on strong intermolecular ionic interactions between the EO molecule and two α1-Na,K-ATPase amino acid residues, Glu116 and Glu117. [ABSTRACT FROM AUTHOR]

In recent years, enough evidence has accumulated to assert that cardiotonic steroids, Na+,K+-ATPase ligands, play an integral role in the physiological and pathophysiological processes in the body. However, little is known about the function of these compounds in the central nervous system. Endogenous cardiotonic steroids are involved in the pathogenesis of affective disorders, including depression and bipolar disorder, which are linked to dopaminergic system dysfunction. Animal models have shown that the cardiotonic steroid ouabain induces mania-like behavior through dopamine-dependent intracellular signaling pathways. In addition, mutations in the alpha subunit of Na+,K+-ATPase lead to the development of neurological pathologies. Evidence from animal models confirms the neurological consequences of mutations in the Na+,K+-ATPase alpha subunit. This review is dedicated to discussing the role of cardiotonic steroids and Na+,K+-ATPase in dopaminergic system pathologies—both the evidence supporting their involvement and potential pathways along which they may exert their effects are evaluated. Since there is an association between affective disorders accompanied by functional alterations in the dopaminergic system and neurological disorders such as Parkinson's disease, we extend our discussion to the role of Na+,K+-ATPase and cardiotonic steroids in neurodegenerative diseases as well. [ABSTRACT FROM AUTHOR]

Biliary tract cancer is a deadly disease with limited therapeutic options. Ouabain is a well-known inhibitor of the pumping function of Na+/K+-ATPase, though there is evidence that low concentrations of ouabain lead to a reduction of cell viability of cancer cells independent of its inhibition of the pumping function of the Na+/K+-ATPase. Regarding the impact of ouabain on biliary tract cancer, no data is currently available. Therefore, we aimed for a first-time investigation of ouabain as a potential anti-neoplastic biliary tract cancer agent using comprehensive human biliary tract cancer in vitro models. We found that ouabain has a strong cell line-dependent cytotoxic effect with IC50 levels in the (low) nanomolar-range and that this effect was not associated with the mRNA expression levels of the Na+/K+-ATPase α, β and fxyd-subunits. Regarding the mode of cytotoxicity, we observed induction of apoptosis in biliary tract cancer cells upon treatment with ouabain. Interestingly, cytotoxic effects of ouabain at sub-saturating (< μM) levels were independent of cellular membrane depolarization and changes in intracellular sodium levels. Furthermore, using a 3D cell culture model, we found that ouabain disturbs spheroid growth and reduces the viability of biliary tract cancer cells within the tumor spheroids. In summary, our data suggest that ouabain possesses anti-biliary tract cancer potential at low μM-concentration in 2D and 3D in vitro biliary tract cancer models and encourage further detailed investigation. [ABSTRACT FROM AUTHOR]

Highly oxygenated cardiotonic steroids, such as ouabain, possess a wide spectrum of biological functions and remain significant synthetic challenges. Herein, we have applied an unsaturation‐functionalization strategy and developed a synthetic method in addressing the C19‐hydroxylation issue for efficient synthesis of polyhydroxylated steroids. An effective asymmetric dearomative cyclization allowed the construction of the C19‐hydroxy unsaturated steroidal skeleton in only four steps from the Hajos‐Parrish ketone ketal 7. The synthetic sequence featured C3−OH‐directed hydrogenation/epoxidation, m‐CPBA‐triggered epoxidation/SN2′ nucleophilic substitution, Birch reduction of an enone, and regioselective LiAlH4 reduction to furnish the polyhydroxy functionalities on the steroid skeleton with high stereochemical control and efficiency. This approach ultimately enabled the total synthesis of 19‐hydroxysarmentogenin and ouabagenin in 18 and 19 steps, respectively, overall. The synthesis of these polyhydroxylated steroids offers synthetic versatility and practicality in the search for new therapeutic agents. [ABSTRACT FROM AUTHOR]

The nature of odoroside A, a cardiac glycoside (CG) extracted from Nerium oleander, as well as its chemical structure is quite similar to a well-known CG, ouabain possessing a steroid skeleton, a five-membered unsaturated lactone ring, and a sugar moiety as a common structure. Like ouabain, odoroside A inhibits the activity of Na+/K+-ATPase (NKA) and shows significant anticancer activity, however its inhibitory mechanism remains unknown. CGs show various physiological activities, including cardiotonic and anticancer activities, through the inhibition of NKA by direct interaction. Additionally, X-ray crystallographic analysis revealed the inhibitory mechanism of ouabain and digoxin in relation to NKA. By using different molecular modeling techniques, docking simulation of odoroside A and NKA was conducted based on the results of these X-ray crystallographic analyses. Furthermore, a comparison of the results with the binding characteristics of three known CGs (ouabain, digoxin, and digitoxin) was also conducted. Odoroside A fitted into the CG binding pocket on the α-subunit of NKA revealed by X-ray crystallography. It had key interactions with Thr797 and Phe783. Also, three known CGs showed similar interactions with Thr797 and Phe783. Interaction modes of odoroside A were quite similar to those of ouabain, digoxin, and digitoxin. Docking simulations indicated that the sugar moiety enhanced the interaction between NKA and CGs, but did not show enhanced NKA inhibitory activity because the sugar moiety was placed outside the entrance of active site. Thus, these results suggest that the inhibitory mechanism of odoroside A to NKA is the same as the known CGs. [ABSTRACT FROM AUTHOR]

Recently, we have developed software that allows, using a minimum of required experimental data, to find the characteristics of ion homeostasis and a list of all unidirectional fluxes of monovalent ions through the main pathways in the cell membrane both in a balanced state and during the transient processes. Our approach has been successfully validated in human proliferating lymphoid U937 cells during transient processes after stopping the Na/K pump by ouabain and for staurosporine-induced apoptosis. In present study, we used this approach to find the characteristics of ion homeostasis and the monovalent ion fluxes through the cell membrane of human erythrocytes in a resting state and during the transient processes after stopping the Na/K pump with ouabain and in response to osmotic challenge. Due to their physiological significance, erythrocytes remain the object of numerous studies, both experimental and computational methods. Calculations showed that, under physiological conditions, the K+ fluxes through electrodiffusion channels in the entire erythrocyte ion balance is small compared to the fluxes through the Na/K pump and cation–chloride cotransporters. The proposed computer program well predicts the dynamics of the erythrocyte ion balance disorders after stopping the Na/K pump with ouabain. In full accordance with predictions, transient processes in human erythrocytes are much slower than in proliferating cells such as lymphoid U937 cells. Comparison of real changes in the distribution of monovalent ions under osmotic challenge with the calculated ones indicates a change in the parameters of the ion transport pathways through the plasma membrane of erythrocytes in this case. The proposed approach may be useful in studying the mechanisms of various erythrocyte dysfunctions. [ABSTRACT FROM AUTHOR]