Concentrations Of Ouabain Research Articles

Ouabain prevents pathological cardiac hypertrophy and heart failure through activation of phosphoinositide 3-kinase α in mouse.

Background Use of low doses of digitalis to prevent the development of heart failure was advocated decades ago, but conflicting results of early animal studies dissuaded further research on this issue. Recent discoveries of digitalis effects on cell signal pathways prompted us to reexamine the possibility of this prophylactic action of digitalis. The specific aim of the present study was to determine if subinotropic doses of ouabain would prevent pressure overload-induced cardiac remodeling in the mouse by activating phosphoinositide 3-kinase α (PI3Kα).ResultsStudies were done on an existing transgenic mouse deficient in cardiac PI3Kα (p85-KO) but with normal cardiac contractility, a control mouse (Con), and on cultured adult cardiomyocytes. In Con myocytes, but not in p85-KO myocytes, ouabain activated PI3Kα and Akt, and caused cell growth. This occurred at low ouabain concentrations that did not activate the EGFR-Src/Ras/Raf/ERK cascade. Con and p85-KO mice were subjected to transverse aortic constriction (TAC) for 8 weeks. A subinotropic dose of ouabain (50 µg/kg/day) was constantly administrated by osmotic mini-pumps for the first 4 weeks. All mice were monitored by echocardiography throughout. Ouabain early treatment attenuated TAC-induced cardiac hypertrophy and fibrosis, and improved cardiac function in TAC-operated Con mice but not in TAC-operated p85-KO mice. TAC downregulated α2-isoform of Na+/K+-ATPase but not its α1-isoform in Con hearts, and ouabain treatment prevented the downregulation of α2-isoform. TAC-induced reduction of α2-isoform did not occur in p85-KO hearts.ConclusionsOur results show that (a) safe doses of ouabain prevent or delay cardiac remodeling of pressure overloaded mouse heart; and (b) these prophylactic effects are due to ouabain binding to α2-isoform resulting in the selective activation of PI3Kα. Our findings also suggest that potential prophylactic use of digitalis for prevention of heart failure in man deserves serious consideration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-015-0053-7) contains supplementary material, which is available to authorized users.

Application of atomic force microscopy for studying intracellular signalization in neurons

The first attempt is made at applying the method of atomic force microscopy (AFM) for determining the molecular mechanisms of intracellular signalization with the participation of Na+, K+-ATPhase playing an important role of a signal transductor (amplifier). The AFM method combined with the organotypic cultivation makes it possible to obtain quantitative information on the Young moduli of living neurons and cells subjected to the action of very low concentrations of ouabain. This substance is known to trigger in this case the intracellular signalization processes by transferring a molecular signal to the genome of a cell. The cell response is manifested in a sharp intensification of protein synthesis accompanied by a rearrangement of the cytoskeleton and activation of enzyme signal pathways in a cytosol. AFM measurements of the images of the cell surface relief are performed using the PeakForce quantitative nanomechanical properties mapping PeakForce QNM mode. The Young moduli of control neurons and of sensory neurons under the action of ouabain are measured simultaneously. It is found that the activation of the signal-transducing function of Na+, K+-ATPhase triggers intracellular signal cascades, which increase the cell stiffness. The application of the AFM method in further studies of the mechanisms of intracellular molecular processes appears as promising. Its combination with inhibitory analysis will clarify the role of individual molecules (e.g., a number of ferments) in regulation of growth and development of living organisms.

Abstract 205: Ouabain Activates the NF-κB Pathway in Macrophages via Na/K-ATPase/TLR4 Complex Leading to Pro-Inflammatory Cytokine Production

Cardiotonic steroids such as ouabain, digoxin, and marinobufagenin are known ligands for the plasma membrane receptor Na/K-ATPase (NKA). These ligands are able to stimulate interaction of the NKA with other membrane and cytosolic proteins leading to cellular events such as activation of kinase cascades and gene transcription. Endogenous cardiotonic steroids have been detected in human circulation and interestingly their levels were elevated in human patients with hypertension, congestive heart failure and diabetes, all of which were associated with chronic systemic inflammation. However, the role of cardiotonic steroids in systemic inflammation and immunity has not been well studied. We previously discovered that ouabain stimulated macrophages to produce pro-inflammatory cytokines, many of which are known targets of the transcription factor, NF-κB. Therefore, we hypothesized that ouabain activates NF-κB pathway leading to pro-inflammatory cytokine production in macrophages. Using Western blot and densitometry analysis, we showed that physiological concentrations of ouabain promoted IκBα degradation (as low as 5 nM ouabain decreased IκBα level by 66.8%±7.4%, n=4). This was accompanied by NF-κB translocation from cytoplasm to the nuclei as shown by immunocytochemistry (% of nuclei NF-κB signals increased from 30.5%±2.3% in control to 62.2%±2.6% in ouabain-treated macrophages, n>25). Moreover, via quantitative RT-PCR (n=3), we found that ouabain increased mRNA levels of pro-inflammatory cytokines such as MCP-1 (3.2±1.1 fold), TNF-α (59.7±35.6 fold), and CXCL-10 (2.8±1.6 fold), all of which are known NF-κB targets. Consistent with the increase in mRNA level, we found that MCP-1 protein levels were elevated in both cell lysates (1.8±0.3 fold) and culture media (1.4±0.1 fold; n=4). Addition of an NF-κB inhibitor blocked MCP-1 production induced by ouabain (n=4). Mechanistically, ouabain stimulated interaction between NKA and TLR4 as shown by Co-Immunoprecipitation (n=3). Blockade of TLR4 signaling using a specific peptide inhibitor, CLI-095, abolished the ouabain effect on NF-κB activation (n=3). We conclude that ouabain activates NF-κB through NKA/TLR4 complex leading to pro-inflammatory cytokine production by macrophages.

Ouabain signaling requires angiotensin II type 1 receptor activation.

Low dose ouabain stimulates sodium transport in proximal tubules. Patients with congestive heart failure and CKD show significantly elevated concentrations (pM range) of endogenous ouabain (EO), and exogenous ouabain increases blood pressure in rats. IP of ouabain bound proteins from proximal tubule cells showed association with the angiotensin II type 1 receptor (AT1R), suggesting a direct pathway linking EO and RAS in mediating salt sensitive hypertension. We hypothesize that low dose ouabain signaling is dependent on AT1R activation. We treated human (HKC11), mouse (mRPT) and porcine (LLC-PK1) kidney cells with pathophysiological concentrations of ouabain and performed confocal, Total Internal Reflection Fluorescence (TIRF), and Forster's Resonance Electron Transfer (FRET) microscopy to examine associations between AT1R, ouabain, and Na-K ATPase a1 subunit (NKA). TIRF and FRET microscopy showed that ouabain binds directly to NKA but not to AT1R. NKA and AT1R associated directly with each other and treatment with ouabain increased association between NKA and AT1R. Ouabain increased phosphorylation of signaling proteins (Src, EGFR, and ERK), and increased activity (86Rb uptake), tyrosine phosphorylation (IP), and surface expression (biotinylation) of NKA in cells expressing AT1R but not in cells that lack AT1R expression. Treatment with candesartan, an AT1R antagonist, prevented the ouabain-stimulated effects on signaling and NKA. These results suggest that pathophysiological concentrations of ouabain may stimulate renal salt reabsorption through an AT1R-dependent pathway.

Apoptosis caused by excessive mitochondrial albumin uptake in renal cells is initiated by increased mitochondrial calcium concentration

Recent advances in the development of organelle-targeted low affinity Ca2+ sensitive dyes have lead to new understanding of the role of Ca2+ in subcellular compartments. Here we report that the onset of the mitochondrial apoptotic pathway is preceded by an increase in mitochondrial Ca2+. Excessive Ca2+ uptake in renal cells causes apoptosis and is a major contributor to the progression of chronic kidney disease. Primary rat proximal tubule cells (RPTC) were incubated with 10mg/ml lipid-free albumin and monitored with regard to mitochondrial Ca2+ concentration ([Ca2+]mt), mitochondrial membrane potential (Δψm) and mitochondrial accumulation of the apoptotic factor Bax. To monitor changes in [Ca2+]mt we expressed RCaMP1h, a mitochondria targeted genetic Ca2+ indicators in RPTC. [Ca2+]mt started to increase significantly after 5 min of albumin incubation and continued to increase during the observation period. At 30 min it was 20 % higher than during control conditions. The increase in [Ca2+]mt was followed by a significant Δψm depolarization after 45 min, and mitochondrial accumulation of the apoptotic factor Bax, which became significant after 30 min. Low concentration of ouabain (10 nM) protected from depolarization and Bax accumulation by activating a Na+, K+-ATPase/IP3R signalling pathway. Thapsigargin, an inhibitor of the SERCA pump causes apoptosis in all cells but its mechanism of action has been elusive. Here we found that exposure of RPTC to cyclopiazonic acid, an analogue of thapsigargin, caused an increase in mitochondrial Ca2+ that paralleled the increase in cytosolic Ca2+. Conclusion Selective sustained increase of mitochondrial Ca2+ concentration may be a major factor responsible for the onset of the intrinsic apoptotic pathway.

ATP1A1-mediated Src signaling inhibits coronavirus entry into host cells.

In addition to transporting ions, the multisubunit Na(+),K(+)-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na(+),K(+)-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na(+),K(+)-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections. Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na(+),K(+)-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na(+),K(+)-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.

Ouabain induces nitric oxide release by a PI3K/Akt-dependent pathway in isolated aortic rings from rats with heart failure.

Ouabain occurs in nanomolar concentrations in myocardial infarction and heart failure (HF). However, the effects of ouabain in vascular function in HF conditions were not investigated yet. Therefore, we analyzed the effects of acute administration of 3 nM ouabain in isolated aortic rings from rats with HF 4 weeks after myocardial infarction. Rats were submitted to sham operation or coronary artery occlusion. In HF rats, left ventricular positive and negative derivatives of intraventricular pressure reduced and left ventricular end diastolic pressure increased. Phenylephrine responses increased in HF rings when compared with controls. Ouabain incubation for 45 minutes reduced phenylephrine-induced contraction in both groups. Endothelial removal increased more phenylephrine response in ouabain-treated rings of sham rats. Ouabain potentiated the effect of L-NAME in both groups but more in sham rats. Wortmannin increased the phenylephrine response only in HF rings. The effect of tetraethylammonium was potentiated by ouabain only in HF rings. Ouabain increased phenylephrine-stimulated nitric oxide production in rings from both groups but increased the activation of Akt only in vessels from HF rats. Results demonstrate that low ouabain concentration can decrease vascular reactivity of aortic rings from HF rats. Ouabain was able to increase nitric oxide production in HF rats by triggering a signal transduction PI3K/Akt-dependent pathway and increasing an endothelium-hyperpolarizing factor release.

Functional Interaction Between Na/K-ATPase and NMDA Receptor in Cerebellar Neurons.

NMDA receptors play a crucial role in regulating synaptic plasticity and memory. Activation of NMDA receptors changes intracellular concentrations of Na(+) and K(+), which are subsequently restored by Na/K-ATPase. We used immunochemical and biochemical methods to elucidate the potential mechanisms of interaction between these two proteins. We observed that NMDA receptor and Na/K-ATPase interact with each other and this interaction was shown for both isoforms of α subunit (α1 and α3) of Na/K-ATPase expressed in neurons. Using Western blotting, we showed that long-term exposure of the primary culture of cerebellar neurons to nanomolar concentrations of ouabain (a cardiotonic steroid, a specific ligand of Na/K-ATPase) leads to a decrease in the levels of NMDA receptors which is likely mediated by the α3 subunit of Na/K-ATPase. We also observed a decrease in enzymatic activity of the α1 subunit of Na/K-ATPase caused by NMDA receptor activation. This effect is mediated by an increase in intracellular Ca(2+). Thus, Na/K-ATPase and NMDA receptor can interact functionally by forming a macromolecular complex which can be important for restoring ionic balance after neuronal excitation. Furthermore, this interaction suggests that NMDA receptor function can be regulated by endogenous cardiotonic steroids which recently have been found in cerebrospinal fluid or by pharmacological drugs affecting Na/K-ATPase function.

Why isn't endogenous ouabain more widely accepted?

A new idea is the most quickly acting antigen known to science . —Wilfred Trotter During the past few years, several distinguished senior colleagues who visited our department and inquired about the status of endogenous ouabain (EO) have asked, “How come I didn't know about these [published]

Contribution of auditory nerve fibers to compound action potential of the auditory nerve.

Sound-evoked compound action potential (CAP), which captures the synchronous activation of the auditory nerve fibers (ANFs), is commonly used to probe deafness in experimental and clinical settings. All ANFs are believed to contribute to CAP threshold and amplitude: low sound pressure levels activate the high-spontaneous rate (SR) fibers, and increasing levels gradually recruit medium- and then low-SR fibers. In this study, we quantitatively analyze the contribution of the ANFs to CAP 6 days after 30-min infusion of ouabain into the round window niche. Anatomic examination showed a progressive ablation of ANFs following increasing concentration of ouabain. CAP amplitude and threshold plotted against loss of ANFs revealed three ANF pools: 1) a highly ouabain-sensitive pool, which does not participate in either CAP threshold or amplitude, 2) a less sensitive pool, which only encoded CAP amplitude, and 3) a ouabain-resistant pool, required for CAP threshold and amplitude. Remarkably, distribution of the three pools was similar to the SR-based ANF distribution (low-, medium-, and high-SR fibers), suggesting that the low-SR fiber loss leaves the CAP unaffected. Single-unit recordings from the auditory nerve confirmed this hypothesis and further showed that it is due to the delayed and broad first spike latency distribution of low-SR fibers. In addition to unraveling the neural mechanisms that encode CAP, our computational simulation of an assembly of guinea pig ANFs generalizes and extends our experimental findings to different species of mammals. Altogether, our data demonstrate that substantial ANF loss can coexist with normal hearing threshold and even unchanged CAP amplitude.

Combination of ouabain and a specific ROCK inhibitor Y27632 in HeLa cells: implication of therapeutic benefits (797.2)

In our previous work we have observed that while the concentrations of ouabain above 100 nM had cytotoxic effects and 30 nM ouabain induced senescence in HeLa cells, ouabain had no noticeable morphological effects on cells at 10 nM and lower concentrations. Therefore in the current work we evaluated the effect of 10nM ouabain on the migration of HeLa cells and the possible involvement of ROCK in this process. The xCELLigence RTCA DP system with CIM‐plates was performed to detect the effect of drugs on cell migration. HeLa cells were exposed to 10 µM Y27632 for 30 min and 0.5 µg/ml CT04 (Rho A inhibitor) for 2h. After preincubation cells were treated with 10 nM ouabain. Cell index was monitored every 15 min for up to 48 h. Our results showed that while 10 nM ouabain had no inhibitory effect on cell migration, combination of ouabain with Y27632 significantly reduced the migrated cell number of HeLa cells. Also Y27632 incubation by itself inhibited the migration of cells. Moreover similar results obtained from CT04 treatment. In contrast with data reported that ouabain has anti migratory properties at nanomolar concentrations, we demonstrated that low nanomolar concentration of the cardiac glycoside did not inhibit the HeLa cell migration. It has been known that ROCK inhibitors have some anticancer properties. In addition, cardiac glycosides have been developed and tested in clinical trial for the treatment of cancer. Therefore our findings imply that combination therapy of ouabain and Y27632 may be beneficial for cancer patients.Grant Funding Source: This study is supported by TUBITAK in 110S426 project.

No endogenous ouabain is detectable in human plasma by ultra-sensitive UPLC-MS/MS

BackgroundThe presence of a binding site for cardiac glycosides, such as digitoxin and digoxin, in the sodium–potassium–ATPase, stimulated attempts to isolate endogenous cardiotonic steroids. Using immunoassays, clinical studies found the cardenolide ouabain to be secreted endogenously in response to exercise and untreated hypertension and to be correlated with severity of clinical conditions such as kidney failure and dilated cardiomyopathy. The assays used were not standardized and the mean concentrations of endogenous ouabain reported for healthy controls ranged from 60 to 530pmol/l. None of these immunoassays is available any more. Therefore, the aim of this study was to develop a highly specific and reliable method for measurement of ouabain in human plasma based on isotope dilution liquid chromatography tandem-mass spectrometry (ID-LC-MS/MS). MethodAn ultra-sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed which applied solid phase extraction of plasma for sample preparation. ResultsThe method was comprehensively validated and had a lower limit of quantification of 1.7pmol/l. However, despite this very low detection limit ouabain was not observed in plasma samples from patients with and without heart failure. ConclusionOur results suggest that immunoassays previously used to quantify assumed endogenous ouabain detected compounds which are not structurally identical with ouabain. Cross reactivity of structurally related compounds of endogenous origin may cause these discrepancies between immunological and mass spectrometric analyses. Conclusive characterization of assumed endogenous counterparts of digoxin in a biomarker discovery approach seems to require distinct analytical techniques.

Relationship between ouabain and asthenozoospermia

A growing number of researches have shown that ouabain can regulate mammalian sperm function and male reproduction by modulating the sperm motility, capacitation and acrosome reaction in vitro. This study further examined the relationship between ouabain and asthenozoospermia. In this study, the rat was intraperitoneally injected with ouabain at different concentrations (low-dose ouabain group: 12.5 μg/kg body weight per day, and high-dose ouabain group: 25 μg/kg body weight per day) for 30 days to establish the asthenozoospermia model. The sperms from 60 males with normal fertility were incubated with ouabain of gradient concentrations (10(-7)-10(-2) mol/L) for 4 h. The sperm motility was evaluated under a microscope. Moreover, the endogenous ouabain (EO) level was determined in seminal plasma of mild or severe asthenozoospermia patients and males with normal fertility by competitive inhibition ELISA. The results showed that the sperm motility was significantly diminished in the rats treated with different concentrations of ouabain. The number of motile sperms (grades a and b) was decreased greatly in a time- and dose-dependent manner in 10(-5)-10(-2) mol/L ouabain groups (P<0.01), while no obvious change in sperm motility was observed in 10(-7)-10(-6)mol/L groups even for 4-h incubation (P>0.05). Furthermore, the EO level was significantly increased in asthenozoospermia patients as compared with that in males with normal fertility (25.27±1.71 μg/L in mild asthenozoospermia patients, 26.52±1.82 μg/L in severe asthenozoospermia patients, 19.31±1.45 μg/L in normal fertility men) (P<0.01). In conclusion, rat asthenozoospermia was successfully established by intraperitoneal injection of ouabain, and 10(-5) mol/L ouabain was sufficient enough to inhibit sperm motility in vitro. Moreover, EO, a normal constituent of seminal plasma, was highly expressed in asthenozoospermia males as compared with normal fertility ones.

Pathophysiologically-Relevant Levels of Endogenous Cardiotonic Steroids Inhibit the Cardiac Na/K ATPase and Activate ERK1/2 Hypertrophic Signaling In Vivo and In Vitro

Type-4 cardiorenal syndrome patients develop heart failure as result of kidney failure. While the mechanism of cardiac dysfunction is unknown, elevated levels of circulating endogenous Na/K ATPase inhibitors (NKAi) are detected in patients and animals with kidney failure. We investigated the pathophysiological consequences of NKAi elevation in rat myocytes and hearts of animals with chronic kidney disease (CKD).Methods: Intracellular Na concentration ([Nai]) was measured using fluorescent ratio-imaging microscopy and ERK1/2 phosphorylation by western blotting, in adult rat ventricular myocytes. Cardiac Na/K ATPase (NKA) activity was measured using biochemical NKA assays. CKD was induced in rats via 5/6 partial-nephrectomy (PN) or in mice via folic-acid administration (FA). Creatinine serum levels were increased in both models, with kidney dysfunction (and cardiac hypertrophy) being more severe in PN model.Results: Treatment of field-stimulated rat cardiac myocytes with bufalin (10nM) or ouabain (100nM) significantly increased [Nai] by 1.2±0.3 and 1.9±0.2mM, respectively, whereas, concentrations of marinobuganenin below 1μM did not. Furthermore, NKAi activated hypertrophic signaling cascades (assessed by ERK1/2 phosphorylation) with a potency order of bufalin (EC50=70nM)>ouabain (EC50=93nM)> marinobuganenin (EC50=6.4μM). Despite small but significant increases in NKA alpha-1/2 expression, NKA activity in hearts of animals with kidney dysfunction was reduced by 45±19% in PN (6 weeks post-PN) and 34±9% in FA animals (12 weeks post FA-injection). NKA inhibition was reversed by incubation with a polyclonal anti-Digoxin antibody (binds most structurally-related NKAi), confirming involvement of NKAi.Conclusions: Pathophysiologically-relevant concentrations of bufalin and ouabain, but not marinobufagenin, raise [Nai] and activate hypertrophic signaling pathways in the heart, thus contributing to Na and diastolic Ca overload and hypertrophy, during CKD.

Validation of Na,K-ATPase Pump Function of Corneal Endothelial Cells for Corneal Regenerative Medicine

Tissue-engineering approaches to cultivate corneal endothelial cells (CECs) or induce CECs from stem cells are under investigation for the treatment of endothelial dysfunction. Before clinical application, a validation method to determine the quality of these cells is required. In this study, we quantified the endothelial pump function required for maintaining the corneal thickness using rabbit CECs (RCECs) and a human CEC line (B4G12). The potential difference of RCECs cultured on a permeable polyester membrane (Snapwell), B4G12 cells on Snapwell, or B4G12 cells on a collagen membrane (CM6) was measured by an Ussing chamber system, and the effect of different concentrations of ouabain (Na,K-ATPase specific inhibitor) was obtained. A mathematical equation derived from the concentration curve revealed that 2 mM ouabain decreases pump function of RCECs to 1.0 mV, and 0.6 mM ouabain decreases pump function of B4G12 on CM6 to 1.0 mV. Ouabain injection into the anterior chamber of rabbit eyes at a concentration of <2 mM maintained the corneal thickness, while those over 3 mM significantly increased the corneal thickness. B4G12 cell sheets transplanted into rabbit eyes treated with 0.6 mM ouabain maintained the corneal thickness, while 3.5 mM ouabain significantly increased the corneal thickness. Taken together, pump function >1.0 mV is required to maintain the corneal thickness. These results can be used for standardization of CEC pump function and validation of tissue-engineered CEC sheets for clinical use.

Nanomolar ouabain elicits apoptosis through a direct action on HeLa cell mitochondria

The steroid Na+/K+ ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30–50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca2+ concentration ([Ca2+]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca2+ store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria.

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

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

Ouabain activates NFκB through an NMDA signaling pathway in cultured cerebellar cells

Na,K-ATPase, an ion pump, has been shown to interact with other proteins in signaling complexes in cardiac myocytes, renal and glial cells, and several other cell types. Our previous in vivo studies indicated that intrahippocampal administration of ouabain (OUA), an inhibitor of Na,K-ATPase, induces NFκB activation, leading to an increase in mRNA levels of target genes of this transcription factor in the rat hippocampus. The present work investigated whether OUA can regulate NF-κB in primary cultured rat cerebellar cells. Cells were treated with different concentrations of OUA (1, 10 or 100 μM) for different periods of time (1, 2 and 4 h). OUA induced a time- and concentration-dependent activation of NFκB (peak of activation: 10 μM, 2 h), involving both p50/p65 and p50/p50 NFκB dimers. OUA (10 μM, 2 h) induced upregulation of tumor necrosis factor α (Tnf-α), interleukin-1β (Il-1β), and brain derived neurotrophic factor (Bdnf) mRNA levels. Both NFκB activation and gene expression activation induced by OUA (10 μM) were abolished when cells were pre-treated for 20 min with MK-801 (N-Methyl-D-Aspartate (NMDA) receptor antagonist), manumycin A (farnesyltransferase inhibitor), PP-1(Src-family tyrosine kinase inhibitor) and PD98059 (mitogen-activated protein kinase (MAPK) inhibitor). OUA (10 μM) alone or in the presence of MK-801, PP-1, PD98059 did not cause cell death or DNA fragmentation. These findings suggest that OUA activates NFκB by NMDA-Src-Ras-like protein through MAPK pathways in cultured cerebellar cells. This pathway may mediate an adaptive response in the central nervous system.

Na+/K+ ATPase 1 and 3 Isoforms Are Differentially Expressed in - and -Motoneurons

The Na(+)/K(+) ATPase (NKA) is an essential membrane protein underlying the membrane potential in excitable cells. Transmembrane ion transport is performed by the catalytic α subunits (α1-4). The predominant subunits in neurons are α1 and α3, which have different affinities for Na(+) and K(+), impacting on transport kinetics. The exchange rate of Na(+)/K(+) markedly influences the activity of the neurons expressing them. We have investigated the distribution and function of the main isoforms of the α subunit expressed in the mouse spinal cord. NKAα1 immunoreactivity (IR) displayed restricted labeling, mainly confined to large ventral horn neurons and ependymal cells. NKAα3 IR was more widespread in the spinal cord, again being observed in large ventral horn neurons, but also in smaller interneurons throughout the dorsal and ventral horns. Within the ventral horn, the α1 and α3 isoforms were mutually exclusive, with the α3 isoform in smaller neurons displaying markers of γ-motoneurons and α1 in α-motoneurons. The α3 isoform was also observed within muscle spindle afferent neurons in dorsal root ganglia with a higher proportion at cervical versus lumbar regions. We confirmed the differential expression of α subunits in motoneurons electrophysiologically in neonatal slices of mouse spinal cord. γ-Motoneurons were excited by bath application of low concentrations of ouabain that selectively inhibit NKAα3 while α-motoneurons were insensitive to these low concentrations. The selective expression of NKAα3 in γ-motoneurons and muscle spindle afferents, which may affect excitability of these neurons, has implications in motor control and disease states associated with NKAα3 dysfunction.

A specific and essential role for Na,K‐ATPase α3 in neurons co‐expressing α1 and α3

Most neurons co‐express two catalytic isoforms of Na,K‐ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here we used electrophysiology and sodium imaging to study the role of α3 in neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside ouabain resulted in a modest increase in the intracellular sodium concentration ([Na+]i), accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na+]i, similar to what could be expected following suprathreshold neuronal activity, inhibition of α3 almost completely abolished the capacity to restore [Na+]i in dendrites. Recordings of Na,K‐ATPase‐specific current supported the notion that α3 is the predominant isoform, responsible for rapid extrusion of Na+. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A (PKA) consensus site, which is structurally associated with a Na+ binding site. Activation of PKA significantly attenuated both the α3 dependent current and restoration of dendritic [Na+]i, indicating that α3 is a target for phosphorylation and may participate in short‐term regulation of neuronal function.