Concentrations Of Ouabain Research Articles

Cardiotonic steroids trigger non-classical testosterone signaling in Sertoli cells via the α4 isoform of the sodium pump

The α4 isoform of the Na+,K+-ATPase (sodium pump) is known to be expressed in spermatozoa and to be critical for their motility. In the investigation presented here, we find that the rat-derived Sertoli cell line 93RS2 also expresses considerable amounts of the α4 isoform in addition to the α1 isoform. Since Sertoli cells are not motile, one can assume that the function of the α4 isoform in these cells must differ from that in spermatozoa. Thus, we assessed a potential involvement of this isoform in signaling pathways that are activated by the cardiotonic steroid (CTS) ouabain, a highly specific sodium pump ligand. Treatment of 93RS2 cells with ouabain leads to activation of the c-Src/c-Raf/Erk1/2 signaling cascade. Furthermore, we show for the first time that the activation of this cascade by ouabain results in phosphorylation and activation of the transcription factor CREB. This signaling cascade is induced at low nanomolar concentrations of ouabain, consistent with the involvement of the α4 isoform. This is further supported by experiments involving siRNA: silencing of α4 expression entirely blocks ouabain-induced activation of Erk1/2 whereas silencing of α1 has no effect. The findings of this study unveil new aspects in CTS/sodium pump interactions by demonstrating for the first time ouabain-induced signaling through the α4 isoform. The c-Src/c-Raf/Erk1/2/CREB cascade activated by ouabain is identical to the so-called non-classical signaling cascade that is normally triggered in Sertoli cells by testosterone. Taking into consideration that CTS are produced endogenously, our results may help to gain new insights into the physiological mechanisms associated with male fertility and reproduction.

Tolperisone potently inhibits ouabaine-induced increase of cortical excitability in mouse brain slices

Event Abstract Back to Event Tolperisone potently inhibits ouabaine-induced increase of cortical excitability in mouse brain slices I. Tarnawa1*, B. Farkas1 and P. Kocsis1 1 Gedeon Richter Plc.,, Pharmacology and Drug Safety Research, Hungary Tolperisone is a centrally acting muscle relaxant drug, which has long been used for the treatment of muscle spasticity and pain associated with rheumatic diseases. It is thought to act via inhibition of sodium and calcium channels. Cortical spreading depression (CSD) is a pathological phenomenon characterized by abnormally increased neuronal activity and excitability. It is implicated in the pathomechanism of some central nervous system diseases such as migraine or ischemic brain damage due to injury or stroke. In a mouse brain slice model, we investigated the effect of tolperisone on the threshold concentration of ouabain to induce CSD. Tissue DC potential changes associated with CSD were recorded with glass electrodes. Slices were perfused with artificial cerebrospinal fluid. Thirty min later perfusion was switched to a solution containing ouabain so that the concentration of the excitatory substance in the slice chamber was elevated continuously in a nearly linear manner. When ouabain exceeded a threshold concentration a CSD was elicited. In control solution the latency of CSD initiation was 12.3±0.2 min, which corresponded to a ouabain concentration of about 100 µM. In the presence of tolperisone (12.5-200 µM) the latency increased dose-dependently, with a minimum effective concentration of 12.5 µM, where the latency increased to 14.5±0.4. Our model seems to be suitable for testing drug effect on cortical excitability in vitro. Keywords: Neurophysiology, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Neurophysiology Citation: Tarnawa I, Farkas B and Kocsis P (2011). Tolperisone potently inhibits ouabaine-induced increase of cortical excitability in mouse brain slices. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00054 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. I. Tarnawa, Gedeon Richter Plc.,, Pharmacology and Drug Safety Research, Budapest, Hungary, tarnawa@richter.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers I. Tarnawa B. Farkas P. Kocsis Google I. Tarnawa B. Farkas P. Kocsis Google Scholar I. Tarnawa B. Farkas P. Kocsis PubMed I. Tarnawa B. Farkas P. Kocsis Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Ouabain-induced modifications of prostate cancer cell lipidome investigated with mass spectrometry and FTIR spectroscopy

Fourier transform infrared (FTIR) spectroscopy was used to investigate modifications of prostate cancer PC-3 cell lipidome after exposure to sub-lethal concentrations of ouabain. FTIR spectroscopy offered an overview of the lipid classes present in the whole sample. The method is simple, label free and some features can be detected on entire cells. We compared the achievements of FTIR spectroscopy with data obtained by mass spectrometry (MS) on the same samples. It appears that FTIR spectroscopy could identify content variations in some lipid classes, e.g., these containing choline head groups such as phosphatidylcholine and sphingomyelin. MS analysis could confirm this result as indicated by principal component analysis and 2D heterocorrelation maps. FTIR spectra were also able to report changes in ester/choline/phosphate ratios characterizing lipid changes induced by ouabain. Furthermore, quantization of major lipid classes (PC, PE, PG, SM) could be obtained by curve fitting of the FTIR spectra. Yet, FTIR failed to resolve lipid classes for which the polar heads do not display specific IR features such as phosphatidylglycerol and cardiolipin.

Signaling through the Na/K-ATPase: implications for cardiac fibrosis

the cardiotonic effects of digitalis have been exploited in clinical practice for many years. However, while the existence of endogenous digitalis-like substances (also called cardiotonic steroids) has been accepted for some time, their exact chemical nature has been only recently demonstrated ([1][

The Ouabain-Binding Site of the 2 Isoform of Na,K-ATPase Plays a Role in Blood Pressure Regulation During Pregnancy

The cardiotonic steroid/ouabain-binding site of the α subunit of Na,K-ATPase is thought to play an important role in cardiovascular homeostasis. Previously, we demonstrated the cardiotonic steroid-binding site of the α2 Na,K-ATPase is involved in adrenocorticotropic hormone (ACTH)-induced hypertension by using gene-modified α2(R/R) mice in which the cardiotonic steroid-binding site is relatively resistant to ouabain compared to the ouabain-sensitive wild-type α2(S/S) mice. To further explore the importance of this site in the cardiovascular system, we investigated blood pressure regulation during pregnancy in mice with the α2(R/R) isoform. The systolic blood pressure (SBP) of the α2(S/S) and α2(R/R) mice was measured before and during pregnancy by tail-cuff. The expression of the α isoforms of Na, K-ATPase in various tissues and plasma endogenous ouabain contents were assessed prior to pregnancy as well as days 7 and 17 of gestation. The α2(S/S) mice showed a gradual decrease in the SBP during the first two trimesters, followed by an increase above the preconceptional level in the third trimester. However, the α2(R/R) mice exhibited a lower blood pressure in the third trimester. The cardiac expression of the α2 Na,K-ATPase in the α2(S/S) mice was significantly less than that of the α2(R/R) mice throughout the pregnancy. The plasma endogenous ouabain concentration significantly increased by twofold at day 17 of pregnancy in the α2(R/R) mice but not in the α2(S/S) mice. The cardiotonic steroid-binding site of the α2 Na,K-ATPase plays a role in maintaining normal SBP during pregnancy.

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2+ level in SH-SY5Y cells

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na(+)/K(+)-ATPase, leads to the elevation of intracellular Ca(2+) level as observed in cells treated with cardiac glycosides. Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca(2+) levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na(+)/Ca(2+) exchanger inhibitor) and 2-APB (IP(3) receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na(+)/K(+)-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope. severe toxicity was observed in cells treated with ouabain of concentration higher than 1 micromol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca(2+) levels were substantially elevated by MLB (1 micromol/L) and ouabain (1 micromol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 micromol/L) or 2-APB (100 micromol/L). Equivalent interaction with the binding cavity of Na(+)/K(+)-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 micromol/L), but not MLB (1 mumol/L), induced dendritic shrink of SH-SY5Y cells. Comparable to ouabain, MLB leads to the elevation of intracellular Ca(2+) level presumably via the same mechanism by inhibiting Na(+)/K(+)-ATPase. The elevated Ca(2+) levels seem to be supplied by Ca(2+) influx through the reversed mode of the Na(+)/Ca(2+) exchanger and intracellular release from endoplasmic reticulum.

A low concentration of ouabain (0.18 µg/kg) enhances hypertension in spontaneously hypertensive rats by inhibiting the Na+ pump and activating the renin-angiotensin system

We investigated the effects of low ouabain concentrations on systolic (SAP) and diastolic (DAP) arterial pressures and on pressor reactivity in 3-month-old male spontaneously hypertensive rats (SHR). Arterial blood pressure (BP) and pressor reactivity to phenylephrine (PHE) were investigated before and after 0.18 microg/kg ouabain administration (N = 6). The influence of hexamethonium (N = 6), canrenone (N = 6), enalapril (N = 6), and losartan (N = 6) on ouabain actions was evaluated. Ouabain increased BP (SAP: 137 +/- 5.1 to 150 +/- 4.7; DAP: 93.7 +/- 7.7 to 116 +/- 3.5 mmHg; P<0.05) but did not change PHE pressor reactivity. Hexamethonium reduced basal BP in control but not in ouabain-treated rats. However, hexamethonium + ouabain increased DAP sensitivity to PHE. Canrenone did not affect basal BP but blocked ouabain effects on SAP. However, after canrenone + ouabain administration, DAP pressor reactivity to PHE still increased. Enalapril and losartan reduced BP and abolished SAP and DAP responses to ouabain. Enalapril + ouabain reduced DAP reactivity to PHE, while losartan + ouabain reduced SAP and DAP reactivity to PHE. In conclusion, a small dose of ouabain administered to SHR increased BP without altering PHE pressor reactivity. Although the renin-angiotensin system (RAS), Na+ pump and autonomic reflexes are involved in the effects of ouabain on PHE reactivity, central mechanisms might blunt the actions of ouabain on PHE pressor reactivity. The effect of ouabain on SAP seems to depend on the inhibition of both Na+ pump and RAS, whereas the effect on DAP seems to depend only on RAS.

Ouabain modulates epithelial cell tight junction

Epithelial cells treated with high concentrations of ouabain (e.g., 1 microM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell-cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K(+) pumping nor disturb the K(+) balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (J(DEX)). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, J(DEX), and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell-cell contacts.

Alcohol Excites Cerebellar Golgi Cells by Inhibiting the Na+/K+ ATPase

Alcohol-induced alterations of cerebellar function cause motor coordination impairments that are responsible for millions of injuries and deaths worldwide. Cognitive deficits associated with alcoholism are also a consequence of cerebellar dysfunction. The mechanisms responsible for these effects of ethanol are poorly understood. Recent studies have identified neurons in the input layer of the cerebellar cortex as important ethanol targets. In this layer, granule cells (GrCs) receive the majority of sensory inputs to the cerebellum through the mossy fibers. Information flow at these neurons is gated by a specialized pacemaker interneuron known as the Golgi cell, which provides divergent GABAergic input to thousands of GrCs. In vivo electrophysiological experiments have previously shown that acute ethanol exposure abolishes GrC responsiveness to sensory inputs carried by mossy fibers. Slice electrophysiological studies suggest that ethanol causes this effect by potentiating GABAergic transmission at Golgi cell-to-GrC synapses through an increase in Golgi cell excitability. Using patch-clamp electrophysiological techniques in cerebellar slices and computer modeling, we show here that ethanol excites Golgi cells by inhibiting the Na(+)/K(+) ATPase. Voltage-clamp recordings of Na(+)/K(+) ATPase currents indicated that ethanol partially inhibits this pump and this effect could be mimicked by low concentrations of ouabain. Partial inhibition of Na(+)/K(+) ATPase function in a computer model of the Golgi cell reproduced these experimental findings. These results establish a novel mechanism of action of ethanol on neuronal excitability, which likely has a role in ethanol-induced cerebellar dysfunction and may also contribute to neuronal functional alterations in other brain regions.

Selectivity of Digitalis Glycosides for Isoforms of Human Na,K-ATPase

There are four isoforms of the alpha subunit (alpha1-4) and three isoforms of the beta subunit (beta1-3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. alpha2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An alpha2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of alpha2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing alpha1beta1, alpha2beta1, and alpha3beta1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, beta-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for alpha2/alpha3 over alpha1 (K(D) alpha1 > alpha2 = alpha3). By contrast, ouabain shows moderate selectivity ( approximately 2.5-fold) for alpha1 over alpha2 (K(D) alpha1 <or= alpha3 < alpha2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested are indistinguishable (K(D) alpha1 = alpha3 = alpha2), showing that the sugar determines isoform selectivity. Selectivity patterns for inhibition of Na,K-ATPase activity of the purified isoform proteins are consistent with binding selectivities, modified somewhat by different affinities of K(+) ions for antagonizing cardiac glycoside binding on the three isoforms. The mechanistic insight on the role of the sugars is strongly supported by a recent structure of Na,K-ATPase with bound ouabain, which implies that aglycones of cardiac glycosides cannot discriminate between isoforms. In conclusion, several digitalis glycosides, but not ouabain, are moderately alpha2-selective. This supports a major role of alpha2 in cardiac contraction and cardiotonic effects of digitalis glycosides.

Regulation of the sperm EGF receptor by ouabain leads to initiation of the acrosome reaction

The sperm acrosome reaction occurs after the binding of the capacitated sperm to the egg zona pellucida. This study describes a novel mode of regulation of the sperm epidermal growth factor receptor (EGFR) under physiological conditions and its relevance to the acrosome reaction. Ouabain, a known Na/K ATPase blocker is present in the blood and in the female reproductive tract. We show here that physiological concentrations (nM) of ouabain enhance phosphorylation of EGFR on tyr-845, stimulate Ca 2+ influx and induce the acrosome reaction in sperm. These effects could be seen only in the presence of very low concentrations of EGF (0.1 ng/ml or 0.016 nM) added together with nano-molar ouabain. Phosphorylation, Ca 2+ influx, and the acrosome reaction are inhibited by an EGFR blocker, suggesting that trans-activation of the EGFR is involved. Moreover, our data revealed that protein kinase A and the family of tyrosine kinase, SRC, shown before to be involved in EGFR activation in sperm, mediate the acrosome reaction induced by ouabain. Ouabain alone (without EGF) at relatively high concentration (10 µM) could enhance EGFR phosphorylation, Ca 2+ influx and acrosome reaction, and these processes were inhibited by EGFR blockers. Moreover, we show here that PKA and SRC family are involved in the activation of EGFR by 10 µM ouabain, further demonstrating that ouabain induces the acrosome reaction by a mechanism mediated by the trans-activation of EGFR. In conclusion, this study describes an interesting regulatory path of EGFR by physiological concentrations of ouabain and EGF found in the female reproductive tract. Neither of these compounds can activate the EGFR alone at such low physiological levels; however, when both are present, the interaction of ouabain with the Na/K ATPase leads to the priming of the EGFR, which undergoes its full activation by EGF.

Increased Na, K-ATPase α2 isoform gene expression by ammonia in astrocytes and in brain in vivo

In mouse astrocyte cultures identical to those used in the present study ammonia increases the production of ouabain-like compounds and Na, K-ATPase activity (Kala et al., 2000). Increased activity of Na, K-ATPase could be the result of enhanced production of ouabain-like compounds, since cultured rat astrocytes react to prolonged exposure to a high concentration of ouabain with an upregulation of the Na, K-ATPase α1 isoform (Hosoi et al., 1997). However, unlike astrocytes in brain in vivo and mouse primary cultures, cultured rat astrocytes do not express the astrocyte-specific α2 isoform, which shows a higher affinity for ouabain (EC50 ∼0.1μM) than the α1 isoform (EC50∼10μM). In the present study we have investigated (i) effects of ammonia on mRNA and protein expression of α1 and α2 isoforms in primary cultures of mouse astrocytes; (ii) effects of hyperammonia obtained by urease injection on mRNA and protein expression of α1 and α2 isoforms in the brain in vivo; and (iii) effect on observed upregulation of gene expression of AG1478, an inhibitor of the EGF receptor-tyrosine kinase, PP1, an inhibitor of Src, and GM6001, an inhibitor of Zn2+-dependent metalloproteinases in the cultured cells. It was established that α2 mRNA and protein expression, but not α1 expression, was upregulated in cultured astrocytes by 1–4 days of exposure to 3 or 5mM ammonia and that similar upregulation, contrasted by a downregulation of the neuronal α3 subunit occurred in the hyperammonemic brain. Based on the effects of the inhibitors and literature data it is concluded that ammonia activates formation of an endogenous ouabain-like compound, which binds to the Na, K-ATPase, activating Src, which in turn stimulates the receptor-tyrosine kinase of the EGF receptor, leading to activation of the Ras, Raf, MEK pathway and phosphorylation of ERK1/2, which eventually causes upregulation of α2 gene expression.

Sympathetic nerves and the endothelium influence the vasoconstrictor effect of low concentrations of ouabain in pressurized small arteries

We hypothesized that in salt-dependent forms of hypertension, endogenous ouabain acts on arterial smooth muscle to cause enhanced vasoconstriction. Here, we tested for the involvement of the arterial endothelium and perivascular sympathetic nerve terminals in ouabain-induced vasoconstriction. Segments of rat mesenteric or renal interlobar arteries were pressurized to 70 mmHg at 37 degrees C and exposed to ouabain (10(-11)-10(-7) M). Removal of the endothelium enhanced ouabain-induced vasoconstriction by as much as twofold (at an ouabain concentration of 10(-9) M). A component of the ouabain-induced vasoconstriction is due to the enhanced spontaneous release of norepinephrine (NE) from nerve terminals in the arterial wall. The alpha(1)-adrenoceptor blocker prazosin (10(-6) M) decreased ouabain-induced vasoconstrictions by as much as 50%. However, neither the contraction induced by sympathetic nerve activity (SNA) nor the NE release evoked by SNA (measured directly by carbon fiber amperometry) was increased by ouabain (<10(-7) M). Nevertheless, the converse case was true: after brief bursts of SNA, vasoconstrictor responses to ouabain were transiently increased (1.75-fold). This effect may be mediated by neuropeptide Y and Y(1) receptors on smooth muscle. In arteries lacking the endothelium and exposed to prazosin, ouabain (10(-11) M and greater) caused vasoconstriction, indicating a direct effect of very "low" concentrations of ouabain on arterial smooth muscle. In conclusion, in intact arteries, the endothelium opposes ouabain (10(-11)-10(-7)M)-induced vasoconstriction, which is caused by both enhanced spontaneous NE release and direct effects on smooth muscle. Ouabain (<10(-7)M) does not enhance SNA-mediated contractions, but SNA enhances ouabain-induced contractions. The effects of endogenous ouabain may be accentuated in forms of hypertension that involve sympathetic nerve hyperactivity and/or endothelial dysfunction.

Sympathetic Nerves and Endothelium Influence the Vasoconstrictor Effect of Low Concentrations of Ouabain in Pressurized Small Arteries

Here, we tested the involvement of arterial endothelium and peri‐vascular sympathetic nerve terminals (NT) in ouabain‐induced vasoconstriction. Segments of rat arteries were pressurized and exposed to ouabain (10−11M–10−7M). Endothelium removal enhanced ouabain‐induced (10−9M) vasoconstriction as much as 2‐fold. A component of the ouabain‐induced vasoconstriction is due to enhanced spontaneous release of norepinephrine (NE) from NT's in the arterial wall. The α1‐adrenoceptor blocker, prazosin (10−6M) decreased ouabain induced vasoconstrictions by as much as 50%. The electrically evoked release of NE from sympathetic NT was not affected by ouabain (&lt; 10−7M). However, neither the ouabain induced contraction nor the NE release (measured directly by carbon fiber amperometry) was increased by sympathetic nerve activity (SNA). However, following brief bursts of SNA, vasoconstrictor responses to ouabain were transiently increased (1.75‐fold). In arteries lacking endothelium and exposed to prazosin, ouabain (&gt;10−11M) caused vasoconstriction, indicating a direct effect on arterial smooth muscle. Conclusions: Endothelium opposes ouabain induced vasoconstriction, which is caused by both enhanced spontaneous NE release and direct effects on smooth muscle. Ouabain does not enhance SNA mediated contractions, but SNA enhances ouabain‐induced contraction.NIH grants R01‐HL073094 &amp; P01‐HL078870

Sodium pump α1 and α3 subunit isoforms mediate distinct responses to ouabain and are both essential for survival of human neuroblastoma

Using SK-N-AS human neuroblastoma cells, which co-express the alpha1 and alpha3 isoforms of the sodium pump alpha subunit, we selectively silenced either the alpha1 or alpha3 subunit by means of transfection with small interfering RNA, and investigated cell survival and the cellular response to ouabain. We found that both of the alpha subunits are essential for cell survival, indicating that substitution of one subunit for the other is not sufficient. In the presence of both alpha subunits, ouabain causes sustained activation of extracellular signal-regulated kinases 1 and 2 (Erk1/2). This activation is not affected when the alpha1 subunit is silenced. However, when alpha3 expression is silenced, ouabain-induced activation of Erk1/2 does not occur, even at a high concentration of ouabain (1 microM). Thus, ouabain-induced Erk1/2 activation is mediated in SK-N-AS cells by alpha3 only, and alpha1 does not participate in this event. This is a clear demonstration of selective involvement of a specific sodium pump alpha subunit isoform in ouabain-induced signaling.

Different neuronal Na+/K+‐ATPase isoforms are involved in diverse signaling pathways

Inhibition of rat neuronal Na(+)/K(+)-ATPase alpha3 isoform at low (100 nM) ouabain concentration led to activation of MAP kinase cascade via PKC and PIP(3) kinase. In contrast to ouabain-sensitive alpha3 isoform of Na(+)/K(+)-ATPase, an ouabain-resistant alpha1 isoform (inhibition with 1 mM of ouabain) of Na(+)/K(+)-ATPase regulates MAP kinase via Src kinase dependent reactions. Using of Annexin V-FITC apoptotic test to determine the cells with early apoptotic features allows to conclude that alpha3 isoform stimulates and alpha1 suppresses apoptotic process in cerebellum neurons. These data are the first demonstration showing participation of ouabain-resistant (alpha1) and ouabain-sensitive (alpha3) Na(+)/K(+)-ATPase isoforms in diverse signaling pathways in neuronal cells.

Dual effects of ouabain, digoxin and proscillaridin A on the regulation of apoptosis in human fibroblasts

In this study, we looked at the effect of ouabain, digoxin and proscillaridin A on human fibroblasts. These data show that low concentrations of ouabain, digoxin and proscillaridin A can activate proliferation of human fibroblasts, suggesting that the Na+, K+-adenosine triphosphatase complex may act as a transducing receptor. It was shown that 30 nM ouabain, digoxin and proscillaridin A stimulated an antiapoptotic action by the increase in the level of phosphorylated extracellular signal-regulated kinases (P-ERK 1/2). Ouabain, digoxin and proscillaridin A only at the relatively high concentration of 300 nM increased intracellular Ca2+ concentration, activated caspase-3 and induced apoptosis in human fibroblasts. In terms of reduction in cell viability, antiproliferative and apoptotic activity, these cardiac glycosides rank in the order proscillaridin A >digoxin >ouabain.

Sodium Pump A1 And A3 Subunit Isoforms Mediate Distinct Responses To Ouabain And Are Both Essential For Human Neuroblastoma

The sodium pump (Na+,K+-ATPase) maintains the sodium gradient across plasma membranes of animal cells. By hydrolyzing ATP the enzyme transports 3 Na+ ions out of the cell in exchange for 2 K+ ions that are taken into the cytosol. This activity can be interrupted by cardiotonic steroids (CTS). Recent publications have, however, established that CTS not only inhibit the sodium pump but that they also induce signalling cascades that influence the physiology of cells in various ways.Sodium pumps are composed of α and β subunits (and additionally in some tissues of γ subunits) that appear is several isoforms. In some cells different α subunit isoforms are coexpressed, giving rise to the question about the need for their co-existence.Using human neuroblastoma cells SK-N-AS that co-express α1 and α3 isoforms of the sodium pump α subunit, we selectively silenced either the α1 or α3 subunit by means of small interfering RNA and investigated cell survival and the cellular response to ouabain, a widely used CTS. We find that both of the two α subunit isoforms are essential for cell survival, indicating that substitution of one subunit for the other is not sufficient. In the presence of both α subunits ouabain causes a sustained Erk1/2 activation. This activation is not affected when the α1 subunit is silenced. When α3 expression is silenced, ouabain-induced activation of Erk1/2 does not occur, even at a high concentration of ouabain (1 μM). Thus, ouabain-induced Erk1/2 activation is mediated in SK-N-AS cells by α3 only, and α1 does not participate in this event. This is the first demonstration of selective involvement of a specific sodium pump α subunit isoform in ouabain-induced signaling.

Na/K-ATpase α2-Subunit Preferentially Modulates Ca Transients and SR Ca Release in Cardiac Myocytes

Na/K-ATPase (NKA) is essential in regulating [Na]i, and thus cardiac myocyte Ca and contractility via Na/Ca exchange. Different NKAα-subunit isoforms are present in heart and may differ functionally, depending on differential membrane localization. In smooth muscle and astrocytes, NKA-α2 is located at the junctions with endo(sarco)plasmic reticulum, where they could regulate local Na, and indirectly junctional cleft [Ca]. In contrast, NKA-α1 is ubiquitously distributed and may regulate bulk [Na]i. It is controversial whether this model holds for cardiac myocytes. We measured the effect of selective NKA-α2/NKA-α1 inhibition with low concentrations of ouabain on [Na]i, Ca transients and fractional SR Ca release in cardiac myocytes from wild-type (WT) mice and transgenic mice in which NKA-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant (SWAP mice), respectively. Different [ouabain] (5 and 0.1 μM, respectively) were used to attain a similar level of total NKA inhibition in WT and SWAP mice. Ouabain increased Ca transients in WT (F/F0=1.84±0.13 vs. 1.54±0.12 under control conditions) but not in SWAP mice (1.49±0.15 vs. 1.46±0.12), despite a similar and modest increase in [Na]i (≤2 mM). Ca transient increase in WT mice was mediated specifically by NKA-α2 inhibition, because 5 μM ouabain had no effect in transgenic mice where both NKA-α1 and NKA-α2 are ouabain-resistant. Ouabain also significantly increased the fractional SR Ca release in WT mice (from 23±2% to 28±2%) but not SWAP mice (23±3% with and without ouabain). Dual-color immunofluorescence measurements coupled with spatial cross-correlation analysis revealed that NKA-α2 is co-localized to a significant extent with both Na/Ca exchanger (43% co-localization) and ryanodine receptors (43% co-localization) in cardiac myocytes. In conclusion, our data indicate that NKA-α2 has a more prominent role (vs. NKA-α1) in modulating Ca transients and SR Ca release in cardiac myocytes.

Effects of the confluence rate on the FTIR spectrum of PC-3 prostate cancer cells in culture

Recently, the possibility of using IR spectroscopy to fingerprint the mode of action of potent antitumor drugs on cancer cells at sub-lethal concentrations has been demonstrated by comparing spectra recorded from untreated or drug-treated cells. The present study investigates the potential interference of the cell culture confluence rate on cell FTIR signature. Significant spectral differences were observed on cells harvested at different confluence rates. Yet, these differences were weak when compared to those induced by sub-lethal ouabain concentrations, used as a model of cardenolide drug. Furthermore, principal component analysis reveals that the impact of the confluence rate, above 50% coverage, on the FTIR spectra is essentially unique and orthogonal to the one induced by the drug model.