Arrhythmia onsets triggered by acute myocardial ischemia are not mediated by lysophosphoglycerides accumulation in ventricular myocardium.

Lysophosphoglycerides (LPLs) have been reported to accumulate in myocardium and serve as a cause of arrhythmias in acute myocardial ischemia. However, in this study we found that LPLs level in the ventricular myocardium was decreased by the onset of acute myocardial ischemia in vivo in rats. Decreasing of LPLs level in left ventricular myocardium, but not right, was observed within 26 min of left myocardial ischemia, regardless of whether arrhythmias were triggered. Lower LPLs level in the ventricular myocardium was also observed in aconitine-simulated ventricular fibrillation (P < 0.0001) and ouabain-simulated III° atrioventricular block (P < 0.0001). Shot-lasting electric shock, e.g., ≤ 40 s, decreased LPLs level, while long-lasting, e.g., 5 min, increased it (fold change = 2.27, P = 0.0008). LPLs accumulation was observed in long-lasting myocardial ischemia, e.g., 4 h (fold change = 1.20, P = 0.0012), when caspase3 activity was elevated (P = 0.0012), indicating increased cell death, but not coincided with higher frequent arrhythmias. In postmortem human ventricular myocardium, differences of LPLs level in left ventricular myocardium was not observed among coronary artery disease- and other heart diseases-caused sudden death and non-heart disease caused death. LPLs level manifested a remarkable increasing from postmortem 12 h on in rats, thus abolishing the potential for serving as biomarkers of sudden cardiac death. Token together, in this study we found that LPLs in ventricular myocardium were initially decreased by the onset of ischemia, LPLs accumulation do not confer arrhythmogenesis during acute myocardial ischemia. It is necessary to reassess the roles of LPLs in myocardial infarction.

Involvement of the Na+, K+-ATPase α1 Isoform and Endogenous Cardiac Steroids in Depression- and Manic-like Behaviors.

Bipolar disorder (BD) is a severe and common chronic mental illness characterized by recurrent mood swings between depression and mania. The biological basis of the disease is poorly understood, and its treatment is unsatisfactory. Na+, K+-ATPase is a major plasma membrane transporter and signal transducer. The catalytic α subunit of this enzyme is the binding site for cardiac steroids. Three α isoforms of the Na+, K+-ATPase are present in the brain. Previous studies have supported the involvement of the Na+, K+-ATPase and endogenous cardiac steroids (ECS) in the etiology of BD. Decreased brain ECS has been found to elicit anti-manic and anti-depressive-like behaviors in mice and rats. However, the identity of the specific α isoform involved in these behavioral effects is unknown. Here, we demonstrated that decreasing ECS through intracerebroventricular (i.c.v.) administration of anti-ouabain antibodies (anti-Ou-Ab) decreased the activity of α1+/- mice in forced swimming tests but did not change the activity in wild type (wt) mice. This treatment also affected exploratory and anxiety behaviors in α1+/- but not wt mice, as measured in open field tests. The i.c.v. administration of anti-Ou-Ab decreased brain ECS and increased brain Na+, K+-ATPase activity in wt and α1+/- mice. The serum ECS was lower in α1+/- than wt mice. In addition, a study in human participants demonstrated that serum ECS significantly decreased after treatment. These results suggest that the Na+, K+-ATPase α1 isoform is involved in depressive- and manic-like behaviors and support that the Na+, K+-ATPase/ECS system participates in the etiology of BD.

Sensational site: the sodium pump ouabain-binding site and its ligands.

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 ß 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.

A century of exercise physiology: effects of muscle contraction and exercise on skeletal muscle Na+,K+-ATPase, Na+ and K+ ions, and on plasma K+ concentration-historical developments.

This historical review traces key discoveries regarding K+ and Na+ ions in skeletal muscle at rest and with exercise, including contents and concentrations, Na+,K+-ATPase (NKA) and exercise effects on plasma [K+] in humans. Following initial measures in 1896 of muscle contents in various species, including humans, electrical stimulation of animal muscle showed K+ loss and gains in Na+, Cl- and H20, then subsequently bidirectional muscle K+ and Na+ fluxes. After NKA discovery in 1957, methods were developed to quantify muscle NKA activity via rates of ATP hydrolysis, Na+/K+ radioisotope fluxes, [3H]-ouabain binding and phosphatase activity. Since then, it became clear that NKA plays a central role in Na+/K+ homeostasis and that NKA content and activity are regulated by muscle contractions and numerous hormones. During intense exercise in humans, muscle intracellular [K+] falls by 21 mM (range - 13 to - 39 mM), interstitial [K+] increases to 12-13 mM, and plasma [K+] rises to 6-8 mM, whilst post-exercise plasma [K+] falls rapidly, reflecting increased muscle NKA activity. Contractions were shown to increase NKA activity in proportion to activation frequency in animal intact muscle preparations. In human muscle, [3H]-ouabain-binding content fully quantifies NKA content, whilst the method mainly detects α2 isoforms in rats. Acute or chronic exercise affects human muscle K+, NKA content, activity, isoforms and phospholemman (FXYD1). Numerous hormones, pharmacological and dietary interventions, altered acid-base or redox states, exercise training and physical inactivity modulate plasma [K+] during exercise. Finally, historical research approaches largely excluded female participants and typically used very small sample sizes.

Empagliflozin prevents oxidative stress in human coronary artery endothelial cells via the NHE/PKC/NOX axis.

BACKGROUND: Empagliflozin (EMPA) ameliorates reactive oxygen species (ROS) generation in human endothelial cells (ECs) exposed to 10 % stretch, but the underlying mechanisms are still unclear. Pathological stretch is supposed to stimulate protein kinase C (PKC) by increasing intracellular calcium (Ca2+), therefore activating nicotinamide adenine dinucleotide phosphate oxidase (NOX) and promoting ROS production in human ECs. We hypothesized that EMPA inhibits stretch-induced NOX activation and ROS generation through preventing PKC activation. METHODS: Human coronary artery endothelial cells (HCAECs) were pre-incubated for 2 h before exposure to cyclic stretch (5 % or 10 %) with either vehicle, EMPA or the PKC inhibitor LY-333531 or PKC siRNA. PKC activity, NOX activity and ROS production were detected after 24 h. Furthermore, the Ca2+ chelator BAPTA-AM, NCX inhibitor ORM-10962 or NCX siRNA, sodium/potassium pump inhibitor ouabain and sodium hydrogen exchanger (NHE) inhibitor cariporide were applied to explore the involvement of the NHE/Na+/NCX/Ca2+ in the ROS inhibitory capacity of EMPA. RESULTS: Compared to 5 % stretch, 10 % significantly increased PKC activity, which was reduced by EMPA and PKC inhibitor LY-333531. EMPA and LY-333531 showed a similar inhibitory capacity on NOX activity and ROS generation induced by 10 % stretch, which was not augmented by combined treatment with both drugs. PKC-ß knockdown inhibits the NOX activation induced by Ca2+ and 10 % stretch. BAPTA, pharmacologic or genetic NCX inhibition and cariporide reduced Ca2+ in static HCAECs and prevented the activation of PKC and NOX in 10%-stretched cells. Ouabain increased ROS generation in cells exposed to 5 % stretch. CONCLUSION: EMPA reduced NOX activity via attenuation of the NHE/Na+/NCX/Ca2+/PKC axis, leading to less ROS generation in HCAECs exposed to 10 % stretch.

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status.

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.

Development of Reproducible and Scalable Culture Conditions for In Vitro Maintenance of Pig Embryonic Stem Cells Using the Sandoz Inbred Swiss Mouse Thioguanine-Resistant Ouabain-Resistant Cell Line as a Feeder Layer.

Feeder cells play a crucial role in maintaining the pluripotency of embryonic stem cells (ESCs) by secreting various extrinsic regulators, such as extracellular matrix (ECM) proteins and growth factors. Although primary mouse embryonic fibroblasts (MEFs) are the most widely used feeder cell type for the culture of ESCs, they have inevitable disadvantages such as batch-to-batch variation and labor-intensive isolation processes. Here, we revealed that the Sandoz inbred Swiss Mouse (SIM) thioguanine-resistant ouabain-resistant (STO) cell line, an immortalized cell line established from mouse SIM embryonic fibroblasts, can be used as a feeder layer for in vitro culture of authentic pig ESCs instead of primary MEFs. First, the expression of genes encoding ECM proteins and growth factors was analyzed to compare their secretory functions as feeder cells. Quantitative real-time polymerase chain reaction (qPCR) showed that the gene expression of these pluripotency-associated factors was downregulated in STO cells compared to primary MEFs of similar density. Therefore, subsequent optimization of the culture conditions was attempted using higher STO cell densities. Notably, pig ESCs cultured on STO cell density of 3 × (187,500 cells/cm2) exhibited the most similar pluripotent state to pig ESCs cultured on primary MEF density of 1 × (62,500 cells/cm2), as determined by alkaline phosphatase staining, qPCR, and immunocytochemistry. In addition, pig ESCs cultured on STO cell density of 3 × formed complex teratoma containing multiple types of tissues derived from all three germ layers. Our culture conditions using optimal STO cell density can be applied to fields requiring reproducible and scalable production of pig ESCs, such as preclinical research and cellular agriculture.

Unidirectional fluxes of monovalent ions in human erythrocytes compared with lymphoid U937 cells: Transient processes after stopping the sodium pump and in response to osmotic challenge.

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.

Metabolomics analysis reveals cytotoxic effects of ouabain towards psoriatic keratinocytes via impairment of glutathione metabolism.

Ouabain is a cardiac glycoside long studied for treating heart diseases, but the attempts to evaluate its anti-psoriatic activity have not been reported. We aimed to explore the effects of ouabain on proliferation and metabolism towards psoriatic keratinocytes. In human HaCaT keratinocytes, ouabain potently decreased viability, promoted apoptosis and caused G2/M cycle arrest. Metabolomics analysis indicated that ouabain markedly impaired glutathione metabolism. The solute carrier family 7 member 11 (SLC7A11) is an amino acid transporter highly specific to cysteine, which is critical for glutathione synthesis. Ouabain downregulated SLC7A11, reduced cysteine uptake and subsequently inhibited glutathione synthesis, probably through inhibiting Akt/mTOR/beclin axis that regulate protein activity of SLC7A11. The impaired glutathione synthesis and oxidative stress caused by ouabain may contribute to its cytotoxicity towards psoriatic keratinocytes. Our results provide experimental evidence supporting further study of ouabain as a potential anti-psoriatic agent.

Compound-Specific Behavioral and Enzymatic Resistance to Toxic Milkweed Cardenolides in a Generalist Bumblebee Pollinator.

Plant secondary metabolites that defend leaves from herbivores also occur in floral nectar. While specialist herbivores often have adaptations providing resistance to these compounds in leaves, many social insect pollinators are generalists, and therefore are not expected to be as resistant to such compounds. The milkweeds, Asclepias spp., contain toxic cardenolides in all tissues including floral nectar. We compared the concentrations and identities of cardenolides between tissues of the North American common milkweed Asclepias syriaca, and then studied the effect of the predominant cardenolide in nectar, glycosylated aspecioside, on an abundant pollinator. We show that a generalist bumblebee, Bombus impatiens, a common pollinator in eastern North America, consumes less nectar with experimental addition of ouabain (a standard cardenolide derived from Apocynacid plants native to east Africa) but not with addition of glycosylated aspecioside from milkweeds. At a concentration matching that of the maximum in the natural range, both cardenolides reduced activity levels of bees after four days of consumption, demonstrating toxicity despite variation in behavioral deterrence (i.e., consumption). In vitro enzymatic assays of Na+/K+-ATPase, the target site of cardenolides, showed lower toxicity of the milkweed cardenolide than ouabain for B. impatiens, indicating that the lower deterrence may be due to greater tolerance to glycosylated aspecioside. In contrast, there was no difference between the two cardenolides in toxicity to the Na+/K+-ATPase from a control insect, the fruit fly Drosophila melanogaster. Accordingly, this work reveals that even generalist pollinators such as B. impatiens may have adaptations to reduce the toxicity of specific plant secondary metabolites that occur in nectar, despite visiting flowers from a wide variety of plants over the colony's lifespan.

Sophoridine manifests as a leading compound for anti-arrhythmia with multiple ion-channel blocking effects.

BACKGROUND: Sophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively inadequate, which limits the development of SR as an antiarrhythmic candidate. PURPOSE: To facilitate the development process of SR as an antiarrhythmic candidate, we performed integrated studies on the electrophysiological properties of SR in vitro and ex vivo to gain more comprehensive insights into the multi-ion channel blocking effects of SR, which provided the foundation for the further drugability studies in antiarrhythmic and safety studies. Firstly, SR's electrophysiological properties and antiarrhythmic potentials were recorded and assessed at the cell and tissue levels by comprehensively integrating the patch clamp with the Electrical and Optical Mapping systems. Subsequently, the antiarrhythmic effects of SR were validated by aconitine and ouabain-induced arrhythmia in vivo. Finally, the safety of SR as an antiarrhythmic candidate compound was evaluated based on the guidelines of the Comprehensive in Vitro Proarrhythmia Assay (CiPA). STUDY DESIGN: The antiarrhythmic effect of SR was evaluated at the in vitro, ex vivo, and in vivo levels. METHODS: Isolated primary cardiomyocytes and stable cell lines were prepared to explore the electrophysiologic properties of being a multiple ion-channel blocker in vitro by whole-cell patch clamp. Using electrical and optical mapping, the negative chronotropic effect of SR was determined in langendorff-perfused rat or guinea-pig hearts.The antiarrhythmic activity of SR was assessed by the ex vivo tachyarrhythmia models induced by left coronary artery ligation (LCAL) and isoproterenol (ISO). Canonical models of aconitine and ouabain-induced arrhythmia were used to verify the antiarrhythmic effects in vivo. Finally, the pro-arrhythmic risk of SR was detected in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hSCCMs) using a Microelectrode array (MEA). RESULTS: Single-cell patch assay validated the multiple ion-channel blockers of SR in transient outward current potassium currents (Ito), l-type calcium currents (ICa-l), and rapid activation delayed rectifier potassium currents (IKr). SR ex vivo depressed heart rates (HR) and ventricular conduction velocity (CV) and prolonged Q-T intervals in a concentration-dependent manner. Consistent with the changes in HRs, SR extended the active time of hearts and increased the action potential duration measured at 90% repolarization (APD90). SR could also significantly lengthen the onset time and curtail the duration of spontaneous ventricular tachycardia (VT) in the ex vivo arrhythmic model induced by LCAL. Meanwhile, SR could also significantly upregulate the programmed electrical stimulation (PES) frequency after the ISO challenge in forming electrical alternans and re-entrant excitation. Furthermore, SR exerted antiarrhythmic effects in the tachyarrhythmia models induced by aconitine and ouabain in vivo. Notably, the pro-arrhythmic risk of SR was shallow for a moderate inhibition of the human ether-à-go-go-related gene (hERG) channel. Moreover, SR prolonged field potential duration (FPDc) of hSCCMs in a concentration-dependent manner without early after depolarization (EAD) and arrhythmia occurrence. CONCLUSION: Our results indicated that SR manifested as a multiple ion-channel blocker in the electrophysiological properties and exerts antiarrhythmic effects ex vivo and in vivo. Meanwhile, due to the low pro-arrhythmic risk in the hERG inhibition assay and the induction of EAD, SR has great potential as a leading candidate in the treatment of ventricular tachyarrhythmia.

Effects of renal denervation on endogenous ouabain in spontaneously hypertensive rats.

PURPOSE: To investigate the role of renal denervation (RDN) on endogenous ouabain (EO) secretion in spontaneously hypertensive rats (SHR). METHODS: Sixteen 12-week-old male SHR were randomly separated into the renal denervation group (RDNX group) and sham operation group (sham group), and eight age-matched Wistar Kyoto rats (WKY) were served as control group. EO concentrations, the Na+- K+-ATPaseactivity, and the expression of Na+-K+-ATPase were assessed. RESULTS: EO levels in serum, kidneys and hypothalamus of sham group were higher than in RDNX group (p < 0.05). Renal Na+-K+-ATPase activity subjected to denervation surgery showed significantly reduction when compared with the sham groups (p < 0.05). A positive correlation existed between norepinephrine (NE) content and Na+-K+-ATPase activity in the kidney (r2 = 0.579). Renal Na+-K+-ATPase α1 subunit mRNA expression was down-regulated in the RDNX group compared with the sham group (P < 0.05), while renal Na+-K+-ATPase α1 subunit mRNA expression was no statistical significance between the groups (P = 0.63). Immunohistochemical analysis showed that there were significant differences in the renal expression of Na+-K+-ATPasebetween the three groups (P < 0.05). CONCLUSIONS: These experiments demonstrate that RDN exerted an anti-hypertensive effect with reduction of EO levels and Na+-K+-ATPase activity and Na+-K+-ATPase α1 subunit expression of kidney in SHR.

Short-Term Mild Hypoxia Modulates Na,K-ATPase to Maintain Membrane Electrogenesis in Rat Skeletal Muscle.

The Na,K-ATPase plays an important role in adaptation to hypoxia. Prolonged hypoxia results in loss of skeletal muscle mass, structure, and performance. However, hypoxic preconditioning is known to protect against a variety of functional impairments. In this study, we tested the possibility of mild hypoxia to modulate the Na,K-ATPase and to improve skeletal muscle electrogenesis. The rats were subjected to simulated high-altitude (3000 m above sea level) hypobaric hypoxia (HH) for 3 h using a hypobaric chamber. Isolated diaphragm and soleus muscles were tested. In the diaphragm muscle, HH increased the α2 Na,K-ATPase isozyme electrogenic activity and stably hyperpolarized the extrajunctional membrane for 24 h. These changes were accompanied by a steady increase in the production of thiobarbituric acid reactive substances as well as a decrease in the serum level of endogenous ouabain, a specific ligand of the Na,K-ATPase. HH also increased the α2 Na,K-ATPase membrane abundance without changing its total protein content; the plasma membrane lipid-ordered phase did not change. In the soleus muscle, HH protected against disuse (hindlimb suspension) induced sarcolemmal depolarization. Considering that the Na,K-ATPase is critical for maintaining skeletal muscle electrogenesis and performance, these findings may have implications for countermeasures in disuse-induced pathology and hypoxic therapy.

Chronic Ouabain Prevents Radiation-Induced Reduction in the α2 Na,K-ATPase Function in the Rat Diaphragm Muscle.

The damaging effect of ionizing radiation (IR) on skeletal muscle Na,K-ATPase is an open field of research. Considering a therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against the IR-induced disturbances of Na,K-ATPase function in rat diaphragm muscle that co-expresses the α1 and α2 isozymes of this protein. Male Wistar rats (n = 26) were subjected to 6-day injections of vehicle (0.9% NaCl) or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to one-time total-body X-ray irradiation (10 Gy), or a sham irradiation. The isolated muscles were studied 72 h post-irradiation. IR decreased the electrogenic contribution of the α2 Na,K-ATPase without affecting its protein content, thereby causing sarcolemma depolarization. IR increased serum concentrations of ouabain, IL-6, and corticosterone, decreased lipid peroxidation, and changed cellular redox status. Chronic ouabain administration prevented IR-induced depolarization and loss of the α2 Na,K-ATPase electrogenic contribution without changing its protein content. This was accompanied with an elevation of ouabain concentration in circulation and with the lack of IR-induced suppression of lipid peroxidation. Given the crucial role of Na,K-ATPase in skeletal muscle performance, these findings may have therapeutic implications as countermeasures for IR-induced muscle pathology.

The extent of involvement of ouabain, hippocampal expression of Na+/K+-ATPase, and corticosterone/melatonin receptors ratio in modifying stress-induced behavior differs according to the stressor in context.

The aim of this study was to assess the effect of two types of stressors, regarding the extent of involvement of ouabain (OUA), hippocampal sodium/potassium ATPase (NKA) expression, and the hippocampal corticosterone receptors (CR)/melatonin receptors (MR) expression ratio, on the behavioral and cardiovascular responses and on the hippocampal cornu ammonis zone 3 (CA3) and dentate gyrus (DG). Thirty adult male Wistar albino rats aged 7-8 months were exposed to either chronic immobilization or a disturbed dark/light cycle and treated with either ouabain or vehicle. In the immobilized group, in the absence of hippocampal corticosterone (CORT) changes, rats were non-responsive to stress, despite experiencing increased pulse rate, downregulated hippocampal sodium/potassium pump, and enhanced hippocampal CR/MR expression ratio. Prolonged darkness precipitated a reduced upright attack posture, with elevated CORT against hippocampal MR downregulation. Both immobilization and, to a lesser extent, prolonged darkness stress resulted in histopathological and ultrastructural neurodegenerative changes in the hippocampus. OUA administration did not change the behavioral resilience in restrained rats, despite persistence of the underlying biochemical derangements, added to decreased CORT. On the contrary, with exposure to short photoperiods, OUA reverted the behavior towards a combative reduction of inactivity, with unvaried CR/MR and CORT, while ameliorating hippocampal neuro-regeneration, with co-existing NKA and MR repressions. Therefore, the extent of OUA, hippocampal NKA expression, and CR/MR expression, and subsequent behavioral and cardiac responses and hippocampal histopathology, differ according to the type of stressor, whether immobilization or prolonged darkness.

Monovalent ions and stress-induced senescence in human mesenchymal endometrial stem/stromal cells.

Monovalent ions are involved in growth, proliferation, differentiation of cells as well as in their death. This work concerns the ion homeostasis during senescence induction in human mesenchymal endometrium stem/stromal cells (hMESCs): hMESCs subjected to oxidative stress (sublethal pulse of H2O2) enter the premature senescence accompanied by persistent DNA damage, irreversible cell cycle arrest, increased expression of the cell cycle inhibitors (p53, p21) cell hypertrophy, enhanced ß-galactosidase activity. Using flame photometry to estimate K+, Na+ content and Rb+ (K+) fluxes we found that during the senescence development in stress-induced hMESCs, Na+/K+pump-mediated K+ fluxes are enhanced due to the increased Na+ content in senescent cells, while ouabain-resistant K+ fluxes remain unchanged. Senescence progression is accompanied by a peculiar decrease in the K+ content in cells from 800-900 to 500-600 µmol/g. Since cardiac glycosides are offered as selective agents for eliminating senescent cells, we investigated the effect of ouabain on ion homeostasis and viability of hMESCs and found that in both proliferating and senescent hMESCs, ouabain (1 nM-1 µM) inhibited pump-mediated K+ transport (ID50 5 × 10-8 M), decreased cell K+/Na+ ratio to 0.1-0.2, however did not induce apoptosis. Comparison of the effect of ouabain on hMESCs with the literature data on the selective cytotoxic effect of cardiac glycosides on senescent or cancer cells suggests the ion pump blockade and intracellular K+ depletion should be synergized with target apoptotic signal to induce the cell death.

Pharmacological inhibition of Na+/K+-ATPase induces neurovascular degeneration and glial cell alteration in the rat retina.

Na+/K+-ATPase (NKA) plays an important role in ion homeostasis and neurotransmitter uptake. In the retina, multidirectional communications among neurons, glia, and blood vessels (that is, neuro-glio-vascular interaction) are crucial for maintaining tissue homeostasis. We investigated the role of NKA in the elements of neuro-glio-vascular unit in neonatal and adult rat retinas. Male Sprague-Dawley rats (1- and 8-week-old) were injected intravitreally with ouabain (20 nmol/eye), an inhibitor of NKA. Morphological changes in retinal neurons, glia, and blood vessels were examined. The intravitreal injection of ouabain decreased the number of cells in the ganglion cell layer, as well as the thicknesses of the inner plexiform and inner nuclear layers in neonatal and adult rats compared to age-matched controls. The ouabain-induced neuronal cell damage was partially prevented by D-(-)-2-amino-5-phosphonopentanoic acid, an antagonist of N-methyl-D-aspartic acid receptors. In the deep retinal vascular plexus of the ouabain-injected eyes, angiogenesis was delayed in neonatal rats, whereas capillary degeneration occurred in adult rats. The immunoreactivity of glutamine synthetase and vascular endothelial growth factor (VEGF) decreased in the retinas of neonatal and adult rats injected intravitreally with ouabain. The immunoreactivity of glial fibrillary acidic protein was enhanced in the retinas of ouabain-injected adult eyes. After the ouabain injection, CD45-positive leukocytes and Iba1-positive microglia increased in the inner retinal layer of neonatal rats, whereas they increased in the middle retinal layer of adult rats. These results suggest that the inhibition of NKA induces the degeneration of neuronal and vascular cells and alteration of glial cells in both neonatal and adult retinas. In addition to the direct effects of NKA inhibition, the disturbance of retinal glutamate metabolism and decreased VEGF expression may contribute to neurovascular degeneration. The activity of NKA is crucial for maintaining elements of neuro-glio-vascular unit in the retina.

A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase.

The highly conserved, cardiotonic steroid binding site (also termed ouabain binding site) on the primary α subunit of Na,K-ATPase plays a receptor signaling role in a range of vital cell processes and is a therapeutic target for human disease. Mouse lines with altered affinity for cardiotonic steroids on the α1 or α2 subunit isoform of Na,K-ATPase, without any change in pump activity, were developed by the late Jerry B Lingrel and are a valuable tool for studying its physiological roles and drug actions. In one model, the normally ouabain resistant α1 isoform was rendered sensitive to ouabain binding. In a second model, the normally sensitive α2 isoform was rendered resistant to ouabain binding. Additional useful models are obtained by mating these mice. To further advance their use, we developed a rapid, real-time PCR method that detects mutant alleles using specific primers and fluorescent probes. PCR is performed in fast mode with up to 15 samples processed in 40 min. The method was validated by Sanger sequencing using mice of known genotype, and by comparing results with a previous two-step method that used PCR amplification followed by gel electrophoresis. In addition, we clarified inconsistencies in published sequences, updated numbering to current reference sequences, and confirmed the continued presence of the mutations in the colony. It is expected that a wider availability of these models and a more efficient genotyping protocol will advance studies of the Na,K-ATPase and its cardiotonic steroid receptor.

Inhibition of the Na+/K+-ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation.

Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumor microenvironment on immune checkpoint expression and function. Here we describe a mechanistic link between Na+/K+-ATPase (NKA) inhibition and activity of the immune checkpoint protein indoleamine-pyrrole 2',3'-dioxygenase 1 (IDO1). We found that IDO1 was necessary and sufficient for production of kynurenine, a downstream tryptophan metabolite, in cancer cells. We developed a spectrophotometric assay to screen a library of 31 model ion transport-targeting compounds for potential effects on IDO1 function in A549 lung and MDA-MB-231 breast cancer cells. This revealed that the cardiac glycosides ouabain and digoxin inhibited kynurenine production at concentrations that did not affect cell survival. NKA inhibition by ouabain and digoxin resulted in increased intracellular Na+ levels and downregulation of IDO1 mRNA and protein levels, which was consistent with the reduction in kynurenine levels. Knockdown of ATP1A1, the ɑ1 subunit of the NKA and target of cardiac glycosides, increased Na+ levels to a lesser extent than cardiac glycoside treatment and did not affect IDO1 expression. However, ATP1A1 knockdown significantly enhanced the effect of cardiac glycosides on IDO1 expression and kynurenine production. Mechanistically, we show that cardiac glycoside treatment resulted in curtailing the length of phosphorylation-mediated stabilization of STAT1, a transcriptional regulator of IDO1 expression, an effect enhanced by ATP1A1 knockdown. Our findings reveal cross talk between ionic modulation via cardiac glycosides and immune checkpoint protein expression in cancer cells with broad mechanistic and clinical implications.

The Janus face of ouabain in Na+ /K+ -ATPase and calcium signalling in neurons.

Na+ /K+ -ATPase, a transmembrane protein essential for maintaining the electrochemical gradient across the plasma membrane, acts as a receptor for cardiotonic steroids such as ouabain. Cardiotonic steroids binding to Na+ /K+ -ATPase triggers signalling pathways or inhibits Na+ /K+ -ATPas activity in a concentration-dependent manner, resulting in a modulation of Ca2+ levels, which are essential for homeostasis in neurons. However, most of the pharmacological strategies for avoiding neuronal death do not target Na+ /K+ -ATPase activity due to its complexity and the poor understanding of the mechanisms involved in Na+ /K+ -ATPase modulation. The present review aims to discuss two points regarding the interplay between Na+ /K+ -ATPase and Ca2+ signalling in the brain. One, Na+ /K+ -ATPase impairment causing illness and neuronal death due to Ca2+ signalling and two, benefits to the brain by modulating Na+ /K+ -ATPase activity. These interactions play an essential role in neuronal cell fate determination and are relevant to find new targets for the treatment of neurodegenerative diseases. LINKED ARTICLES: This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc.