Ouabain resistance of a human trophoblast cell line is not related to its reactivity to ouabain

Ouabain is a specific inhibitor of sodium, potassium-dependent adenosine triphosphatase (Na,K-ATPase), a P-type ion-transporting ATPase which is essential for the maintenance of adequate concentrations of intracellular Na+ and K+ ions. The present study describes the establishment of a ouabain-resistant mutant, TLouaR, from a human trophoblast cell line TL. Morphologically TL and TLouaR are indistinguishable, but, TLouaR is about 1000 times more resistant to the cytotoxic effect of ouabain and > 2000 times to that of bufalin and yet ouabain can retard the growth of the TLouaR cells and in parallel reduce its cloning efficiency in a time- and dose-dependent manner. Furthermore, Na,K-ATPase activity from TLouaR cells is inhibitable by ouabain albeit with lower efficiency. [3H]ouabain binding studies reveal that TLouaR cells have less (P < 0.05) ouabain binding sites (1.7 +/- 0.15 x 10(4)/cell vs. 2.3 +/- 0.115 x 10(4)/cell in the control). However, affinities (dissociation constants Kd) to ouabain for TL and TLouaR cells are not significantly different. Lastly, Na,K-ATPase activity (1.375 +/- 0.25 micromole ATP/min mg protein) of TLouaR cells is significantly higher (P < 0.05) than that of the TL cells (0.895 +/- 0.12 micromole ATP/min x mg protein). These studies show that the interactions between ouabain and Na,K-ATPase can be mediated through different pathways resulting in diverse phenotypic characteristics. In addition, ouabain resistance does not necessarily reflect the lack of response to the digitalis drug. The exact mechanisms of ouabain resistance observed in the present study remain to be determined but the TLouaR cells may be the best tool to uncover the many functional characteristics of Na,K-ATPase.     

Na+ effects on mitochondrial respiration and oxidative phosphorylation in diabetic hearts

Intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control. We hypothesized that the increase in Na+i activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to loss of intramitochondrial Ca2+, with a subsequent alteration (generally depression) in bioenergetic function. To further evaluate this hypothesis, mitochondria were isolated from hearts of control and streptozotocin-induced (4 weeks) diabetic rats. Respiratory function and ATP synthesis were studied using routine polarography and 31P-NMR methods, respectively. While addition of Na+ (1-10 mM) decreased State 3 respiration and rate of oxidative phosphorylation in both diabetic and control mitochondria, the decreases were significantly greater for diabetic than for control. The Na+ effect was reversed by providing different levels of extramitochondrial Ca2+ (larger Ca2+ levels were needed to reverse the Na+ depressant effect in diabetes mellitus than in control) and by inhibiting the Na+/Ca2+ exchanger function with diltiazem (a specific blocker of Na+/Ca2+ exchange that prevents Ca2+ from leaving the mitochondrial matrix). On the other hand, the Na+ depressant effect was enhanced by Ruthenium Red (RR, a blocker of mitochondrial Ca2+ uptake, which decreases intramitochondrial Ca2+). The RR effect on Na+ depression of mitochondrial bioenergetic function was larger in diabetic than control. These findings suggest that intramitochondrial Ca2+ levels could be lower in diabetic than control and that the Na+ depressant effect has some relation to lowered intramitochondrial Ca2+. Conjoint experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels in diabetic rats, but produced smaller changes in control. These data support our hypothesis that in diabetic cardiomyocytes, increased Na+ leads to abnormalities of oxidative processes and subsequent decrease in ATP levels, and that these changes are related to Na+ induced depletion of intramitochondrial Ca2+.     

Na pump and plasma membrane structure in L-cell fibroblasts expressing rat liver fatty acid binding protein.

Although the intracellular fatty acid binding proteins have been investigated for nearly two decades and purified proteins are now available, little is known regarding the function of these proteins in intact cells. Therefore, L-cell fibroblasts transfected with cDNA encoding for rat liver fatty acid binding protein (L-FABP) were examined as to whether L-FABP expression in intact cells modifies plasma membrane enzyme activities, fluidity, and lipids. Plasma membrane Na/K-ATPase activity was 65.9 +/- 18.7 and 38.6 +/- 22.8 (P less than 0.001) nmol/mg protein x min for control and high-expression transfected cells, respectively. Consistent with this observation, [3H] ouabain binding to whole cells was significantly decreased from 3.7 +/- 0.3 to 2.0 +/- 0.8 pmol ouabain bound/mg cell protein in control and high-expression cells, respectively, whereas the cell's affinity for ouabain was not significantly altered. Unexpectedly, Western blot analysis indicated that transfected cells had higher levels of Na+, K(+)-ATPase protein; in contrast, the activities of 5'-nucleotidase and Mg-ATPase were unaltered. The effects of L-FABP expression on plasma membrane Na/K-ATPase function appeared to be mediated through alterations in plasma membrane lipids and/or structure. The plasma membrane cholesterol/phospholipid ratio decreased and the bulk plasma membrane fluidity increased in the high-expression cells. In conclusion, plasma membrane Na/K-ATPase activity in L cells may be regulated in part through expression of cytosolic L-FABP.     

Protective effect of a low K+ cardioplegic solution on myocardial Na,K-ATPase activity.

Long duration ischemia in hypothermic conditions followed by reperfusion alters membrane transport function and in particular Na,K-ATPase. We compared the protective effect of two well-described cardioplegic solutions on cardiac Na,K-ATPase activity during reperfusion after hypothermic ischemia. Isolated perfused rat hearts (n = 10) were arrested with CRMBM or UW cardioplegic solutions and submitted to 12 hr of ischemia at 4 degrees C in the same solution followed by 60 min of reperfusion. Functional recovery and Na,K-ATPase activity were measured at the end of reperfusion and compared with control hearts and hearts submitted to severe ischemia (30 min at 37 degrees C) followed by reflow. Na,K-ATPase activity was not altered after 12 hr of ischemia and 1 hr reflow when the CRMBM solution was used for preservation (55 +/- 2 micromolPi/mg prot/hr) compared to control (53 +/- 2 micromol Pi/mg prot/hr) while it was significantly altered with UW solution (44 +/- 2 micromol Pi/mg prot/hr, p < 0.05 vs control and CRMBM). Better preservation of Na,K-ATPase activity with the CRMBM solution was associated with higher functional recovery compared to UW as represented by the recovery of RPP, 52 +/- 12% vs 8 +/- 5%, p < 0.05 and coronary flow (70 +/- 2% vs 50 +/- 8%, p < 0.05). The enhanced protection provided by CRMBM compared to UW may be related to its lower K+ content.     

Effects of detergents on Na+ + K+-dependent ATPase activity in plasma-membrane fractions prepared from frog muscles. Studies of insulin action on Na+ and K+ transport.

The increase in Na+/K+ transport activity in skeletal muscles exposed to insulin was analysed. Plasma-membrane fractions were prepared from frog (Rana catesbeiana) skeletal muscles, and examination of the Na,K-ATPase (Na+ + K+-dependent ATPase) activity showed that it was insensitive to ouabain. In contrast, plasma-membrane fractions prepared from ouabain-pretreated muscles, by the same procedures, showed extremely low Na,K-ATPase activity. On adding saponin to the membrane suspension, the Na,K-ATPase activity increased, according to the detergent concentration. The maximum activity was about twice the control value, at 0.33 mg of saponin/mg of protein. Thus saponin makes vesicle membranes leaky, allowing ouabain in assay solutions to reach receptors on the inner surface of vesicles. Addition of insulin to saponin-treated membrane suspensions had no effect on the Na,K-ATPase activity, whereas the maximum activity of Na,K-ATPase in whole muscles was stimulated by exposure to insulin. The results show that the stimulation of Na+/K+ transport by insulin is not directly due to insulin binding to receptors on the cell surface, but rather support the view that the increase in the Na,K-ATPase induced by insulin requires an alteration of intracellular events.     

High-Mr glycoprotein profiles in human milk serum and fat-globule membrane.

In the standard [3H]ouabain-binding assay for quantification of the Na,K-ATPase (Na+ + K+-dependent ATPase) concentration in rat skeletal muscles, samples are incubated for 2 X 60 min in 1 microM-[3H]ouabain at 37 degrees C followed by a wash-out for 4 X 30 min at 0 degree C. To obtain accurate determinations, values determined by this standard assay should be corrected for non-specific uptake and retention of [3H]ouabain (11% overestimation), loss of specifically bound [3H]ouabain during wash-out (21% underestimation), evaporation from muscle samples during weighing (4% overestimation), impurity of [3H]ouabain (5% underestimation) and incomplete saturation of [3H]ouabain binding sites (6% underestimation). Thus corrected the standard [3H]ouabain-binding assay determines the total Na,K-ATPase concentration. Hence, in the soleus muscle of 12-week-old rats the total [3H]ouabain-binding-site concentration is 278 +/- 20 pmol/g wet wt. This is at variance with the evaluation of the Na,K-ATPase concentration from Na,K-ATPase activity measurements in muscle membrane fractions, where the recovery of Na,K-ATPase is only 2-18%. Quantification of the total Na,K-ATPase concentration is of particular importance since it is a prerequisite for the discussion of quantitative aspects of the Na,K-ATPase.     

Roles of the Na,K-ATPase alpha4 isoform and the Na+/H+ exchanger in sperm motility

The Na,K-ATPase generates electrochemical gradients that are used to drive the coupled transport of many ions and nutrients across the plasma membrane. The functional enzyme is comprised of an alpha and beta subunit and families of isoforms for both subunits exist. Recent studies in this laboratory have identified a biological role for the Na,K-ATPase alpha4 isoform in sperm motility. Here we further investigate the role of the Na,K-ATPase carrying the alpha4 isoform, showing again that ouabain eliminates sperm motility, and in addition, that nigericin, a H+/K+ ionophore, and monensin, a H+/Na+ ionophore, reinitiate motility. These data, along with the observation that the K+ ionophore valinomycin has no effect on the motility of ouabain-inhibited sperm, suggest that ouabain may change intracellular H+ levels in a manner that is incompatible with sperm motility. We have also localized NHE1 and NHE5, known regulators of intracellular H+ content, to the same region of the sperm as the Na,K-ATPase alpha4 isoform. These data highlight the important role of the Na,K-ATPase alpha4 isoform in regulating intracellular H(+) levels, and provide evidence suggesting the involvement of the Na+/H+ exchanger, which is critical for maintaining normal sperm motility.     

Ankyrin B modulates the function of Na,K-ATPase/inositol 1,4,5-trisphosphate receptor signaling microdomain

Na,K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappaB activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na,K-ATPase/IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na,K-ATPase and IP3R. We identify the N terminus tail of the Na,K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na,K-ATPase and IP3R, reduced the number of cells responding to pm doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappaB. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na,K-ATPase and no effect on the distribution and apparent mobility of Na,K-ATPase in the plasma membrane.     

Murine lung injury caused by Leptospira interrogans glycolipoprotein,a specific Na/K-ATPase inhibitor

Background

Leptospiral glycolipoprotein (GLP) is a potent and specific Na/K-ATPase inhibitor. Severe pulmonary form of leptospirosis is characterized by edema, inflammation and intra-alveolar hemorrhage having a dismal prognosis. Resolution of edema and inflammation determines the outcome of lung injury. Na/K-ATPase activity is responsible for edema clearance. This enzyme works as a cell receptor that triggers activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathway. Therefore, injection of GLP into lungs induces injury by triggering inflammation.

Methods

We injected GLP and ouabain, into mice lungs and compared their effects. Bronchoalveolar lavage fluid (BALF) was collected for cell and lipid body counting and measurement of protein and lipid mediators (PGE₂ and LTB₄). The levels of the IL-6, TNFα, IL-1B and MIP-1α were also quantified. Lung images illustrate the injury and whole-body plethysmography was performed to assay lung function. We used Toll-like receptor 4 (TLR4) knockout mice to evaluate leptospiral GLP-induced lung injury. Na/K-ATPase activity was determined in lung cells by nonradioactive rubidium incorporation. We analyzed MAPK p38 activation in lung and in epithelial and endothelial cells.

Results

Leptospiral GLP and ouabain induced lung edema, cell migration and activation, production of lipid mediators and cytokines and hemorrhage. They induced lung function alterations and inhibited rubidium incorporation. Using TLR4 knockout mice, we showed that the GLP action was not dependent on TLR4 activation. GLP activated of p38 and enhanced cytokine production in cell cultures which was reversed by a selective p38 inhibitor.

Conclusions

GLP and ouabain induced lung injury, as evidenced by increased lung inflammation and hemorrhage. To our knowledge, this is the first report showing GLP induces lung injury. GLP and ouabain are Na/K-ATPase targets, triggering intracellular signaling pathways. We showed p38 activation by GLP-induced lung injury, which was may be linked to Na/K-ATPase inhibition. Lung inflammation induced by GLP was not dependent on TLR4 activation.     

Comparison of the substrate dependence properties of the rat Na,K-ATPase alpha 1, alpha 2, and alpha 3 isoforms expressed in HeLa cells.

The role of multiple isoforms for the alpha subunit of Na,K-ATPase is essentially unknown. To examine the functional properties of the three alpha subunit isoforms, we developed a system for the heterologous expression of Na,K-ATPase in which the enzymatic activity of each isoform can be independently analyzed. Ouabain-resistant forms of the rat alpha 2 and alpha 3 subunits were constructed by site-directed mutagenesis of amino acid residues at the extracellular borders of the first and second transmembrane domains (L111R and N122D for alpha 2 and Q108R and N119D for alpha 3). cDNAs encoding the rat alpha 1 subunit, which is naturally ouabain-resistant, and rat alpha 2 and alpha 3, which were mutated to ouabain resistance (designated rat alpha 2* and rat alpha 3*, respectively) were cloned into an expression vector and transfected into HeLa cells. Resistant clones were isolated and analyzed for ouabain-inhibitable ATPase activity in the presence of 1 microM ouabain, which inhibits the endogenous Na,K-ATPase present in HeLa cells (I50 approximately equal to 10 nM). The remaining activity corresponds to Na,K-ATPase molecules containing the transfected rat alpha 1, rat alpha 2*, or rat alpha 3* isoforms. Utilizing this system, we examined Na+, K+, and ATP dependence of enzyme activity. Na,K-ATPase molecules containing rat alpha 1 and rat alpha 2* exhibited a 2-3-fold higher apparent affinity for Na+ than those containing rat alpha 3* (apparent KNa+ (millimolar): rat alpha 1 = 1.15 +/- 0.13; rat alpha 2* = 1.05 +/- 0.11; rat alpha 3* = 3.08 +/- 0.06). Additionally, rat alpha 3* had a slightly higher apparent affinity for ATP (in the millimolar concentration range) compared with rat alpha 1 or rat alpha 2* (apparent K0.5 (millimolar): rat alpha 1 = 0.43 +/- 0.12; rat alpha 2* = 0.54 +/- 0.15; rat alpha 3* = 0.21 +/- 0.04) and all three isoforms has similar apparent affinities for K+ (apparent KK+: rat alpha 1 = 0.45 +/- 0.01; rat alpha 2* = 0.43 +/- 0.004; rat alpha 3* = 0.27 +/- 0.01). This study represents the first comparison of the functional properties of the three Na,K-ATPase alpha isoforms expressed in the same cell type.     

Evidence of time-dependent changes in renal medullary Na,K-ATPase activity and expression in diabetic rats.

The medullary thick ascending limb (MTAL) of the kidney displays structural changes during long term diabetes. After twelve weeks of diabetes, there is controversy over the changes in Na,K-ATPase activity. To observe the long-term changes, we studied MTAL Na,K-ATPase activity and protein expression in diabetic animals 6 (6W) and 12 weeks (12W) after induction of diabetes with streptozotocin. Three groups were studied, one control group, one group 6W after, and one group 12W after induction of diabetes. Membrane fractions from the inner strip of the outer medulla representing MTAL were isolated. Na,K-ATPase activity and western blottings of alpha1- and beta1-subunits were carried out. 6W diabetes resulted in an increase, and 12W in a decrease in the MTAL Na,K-ATPase activity versus the control group (respectively 63.3 +/- 21.2; 7.5 +/- 2.4 and 31.6 +/- 11.4; micromol Pi/mg prot/hr +/- SEM). The Na,K-ATPase subunit expression was increased at 6W, and decreased after 12W, resulting in amounts below control values for both alpha1- and beta1-subunits. Our results confirm a diabetes-induced biphasic time-dependent alteration MTAL Na,K-ATPase activity, supported by similar changes in alpha1 and beta1 Na,K-ATPase subunits-expression.     

Cell signaling microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations

Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP3R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP3R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na,K-ATPase catalytic alpha1-subunit abolished Ca2+ oscillations and downstream activation of NF-kappaB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.     

Expression of functional Na,K-ATPase isozymes in normal human cardiac biopsies.

In human heart failure, disturbances in Ca2+ homeostasis are well known but the fate of the Na,K-ATPase isoforms (alpha1beta1, alpha2beta1 and alpha3beta1), the receptors for cardiac glycosides, still remains under study. Microsomes have been purified from non-failing human hearts. As judged by the sensitivities of Na,K-ATPase activity to ouabain (IC50 values: 7.0 +/- 2.5 and 81 +/- 11 nM), 3H-ouabain-binding measurements at equilibrium with and without 10 mM K+ and by a biphasic ouabain dissociation process, at least two finctionally active Na,K-ATPase isozymes coexist in normal human hearts. These are demonstrated as a very high- and a high affinity ouabain-binding site. The KD values are 3.6 +/- 1.6 nM and 17 +/- 6 nM, respectively. The two dissociation rate constants are 42 x 10(4) min(-1) and 360 x 10(-4) min(-1). Addition of 10 mM K+ ions shifted the respective KD values for ouabain from 3.6 +/- 1.6 to 20 +/- 5 nM and from 17 +/- 6 nM to 125 +/- 25 nM, respectively. The isozymes involved are identified by comparing these three pharmacological parameters to those of each alpha/beta-isozyme separately expressed in Xenopus oocytes (9). In human heart, the very high affinity site for ouabain is the alpha1beta1 dimer and the high affinity site is alpha2beta1.     

Na,K-ATPase Activity Is Required for Formation of Tight Junctions, Desmosomes, and Induction of Polarity in Epithelial Cells

Na,K-ATPase is a key enzyme that regulates a variety of transport functions in epithelial cells. In this study, we demonstrate a role for Na,K-ATPase in the formation of tight junctions, desmosomes, and epithelial polarity with the use of the calcium switch model in Madin-Darby canine kidney cells. Inhibition of Na,K-ATPase either by ouabain or potassium depletion prevented the formation of tight junctions and desmosomes and the cells remained nonpolarized. The formation of bundled stress fibers that appeared transiently in control cells was largely inhibited in ouabain-treated or potassium-depleted cells. Failure to form stress fibers correlated with a large reduction of RhoA GTPase activity in Na,K-ATPase-inhibited cells. In cells overexpressing wild-type RhoA GTPase, Na,K-ATPase inhibition did not affect the formation of stress fibers, tight junctions, or desmosomes, and epithelial polarity developed normally, suggesting that RhoA GTPase is an essential component downstream of Na,K-ATPase-mediated regulation of these junctions. The effects of Na,K-ATPase inhibition were mimicked by treatment with the sodium ionophore gramicidin and were correlated with the increased intracellular sodium levels. Furthermore, ouabain treatment under sodium-free condition did not affect the formation of junctions and epithelial polarity, suggesting that the intracellular Na(+) homeostasis plays a crucial role in generation of the polarized phenotype of epithelial cells. These results thus demonstrate that the Na,K-ATPase activity plays an important role in regulating both the structure and function of polarized epithelial cells.     

The alpha2 isoform of Na,K-ATPase mediates ouabain-induced cardiac inotropy in mice

Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be the major mechanism by which this class of drugs increases heart contractility. However, direct evidence demonstrating this is lacking. Furthermore it is unknown which specific alpha isoform of Na,K-ATPase is responsible for the effect of cardiac glycosides. Several studies also suggest that cardiac glycosides, such as ouabain, function by mechanisms other than inhibition of the Na,K-ATPase. To determine whether Na,K-ATPase, specifically the alpha2 Na,K-ATPase isozyme, mediates ouabain-induced cardiac inotropy, we developed animals expressing a ouabain-insensitive alpha2 isoform of the Na,K-ATPase using Cre-Lox technology and analyzed cardiac contractility after administration of ouabain. The homozygous knock-in animals were born in normal Mendelian ratio and developed normally to adulthood. Analysis of their cardiovascular function demonstrated normal heart function. Cardiac contractility analysis in isolated hearts and in intact animals demonstrated that ouabain-induced cardiac inotropy occurred in hearts from wild type but not from the targeted animals. These results clearly demonstrate that the Na,K-ATPase and specifically the alpha2 Na,K-ATPase isozyme mediates ouabain-induced cardiac contractility in mice.     

Porcine kidney extract contains a specific inhibitor of the ouabain-sensitive alpha2-isoform of Na, K-ATPase present in rat diaphragm fibres

In experiments on isolated rat diaphragm muscle, acetylcholine (100 nmol/l) hyperpolarized muscle fibres due to activation of the alpha 2 isoform of Na,K-ATPase. This hyperpolarization was blocked in a dose-dependent manner by ouabain (K0.5 = 8 +/- 4 nmol/l) as well as by a solution of porcine kidney extract (10 kDa cut-off filtration), with the K0.5 approximately equal to a 1:20,000-fold dilution. The inhibitory activity of the developed slowly over a period of 3 hours and, in contrast to ouabain, was still present after 1 hour of washing. Ouabain, but not the extract, inhibits Rb+ uptake in human erythrocytes that only express the alpha = 1 isoform of Na, K-ATPase. Our data suggest that in rat skeletal muscle the alpha 1 isoform of Na,K-ATPase is primarily responsible for ionic homeostasis, while the alpha 2 isoform provides a "regulatable" function and may be controlled by cholinergic stimulation and/or endogenous digitalis-like factors (EDLFs). Porcine kidney extract contains a factor (M. W. < 10 kDa) that selectively inhibits the rat alpha 2 isoform and differs from ouabain. Our experimental protocol can be used as a highly sensitive physiological assay for factors that selectively inhibit the alpha 2 isoform of Na,K-ATPase.     

Investigation of mechanism of p38 MAPK activation in renal epithelial cell from distal tubules triggered by cardiotonic steroids

Ouabain and other cardiotonic steroids (CTS) kill renal epithelial cells from distal tubules (C7-MDCK) via interaction with Na,K-ATPase but independently of inhibition of Na,K-ATPase-mediated ion fluxes. Recently, we demonstrated that modest intracellular acidification and inhibition of p38 MAPK suppress death of C7-MDCK cells triggered by ouabain. In the present study we investigate the mechanism of p38 MAPK activation in renal epithelial cell from distal tubules evoked by cardiotonic steroids. Using Na⁺/K⁺ ionophores (monensin, nigericin) and media with different content of monovalent cations, we revealed that p38 MAPK phosphorylation in ouabain-treated renal epithelial cells is not caused by Na,K-ATPase inhibition and inversion of the [Na⁺]_i/[K⁺]_i ratio. We also demonstrated that attenuation of pH from 7.45 to 6.75 did not alter the level of p38 MAPK phosphorylation observed in ouabain-treated cells. Inhibitors of PKA, PKC, and PKG as well as protein phosphatases were unable to abolish p38 MAPK activation triggered by ouabain. Using phosphotyrosine antibodies we did not detect any effect of ouabain on activation of tyrosine kinases. Thus, our results show that activation of p38 MAPK and cytotoxic action of CTS are independent of intracellular Na⁺, K⁺, and H⁺ concentrations. The molecular origin of intermediates of death signaling induced by CTS via conformation changes of Na,K-ATPase with following activation of p38 MAPK should be examined further.     

Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).     

The role of conformational changes in the functioning of brain Na,K-ATPase]

In the experiments with enzyme preparations of Na,K-ATPase from normal brain tissue (NBT) and tumorous brain tissue (TBT) the following data were established: 1) the cooperativity of Na,K-ATPase with Na+ from NBT is temperature-dependent, the Hill coefficient (nH) at 37, 27.0-30.5 and 20-22 degrees C being 1.80 +/- 0.07, 1.30 +/- 0.09 and 1.10 +/- 0.08, respectively; the cooperativity of Na+ with Na,K-ATPase from TBT was absent; 2) the cooperativity for ouabain (nH-1.30 +/- 0.05) was revealed only in the case of Na-pump from TBT; 3) the protective effect of ATP against the inhibitory action of pCMB is temperature-dependent and differs significantly in enzyme preparations from NBT and TBT; 4) the parameters of the temperature inactivation of enzyme preparations at 45-52 degrees C, especially the change of entropy (delta S*) were different in the case of NBT and TBT; 5) a peptide fraction isolated from sheep brain differently inhibited the Na,K-ATPase from NBT and TBT. In conclusion, these data demonstrate that there are significant differences in functioning of Na,K-ATPase from NBT and TBT, and that besides lipid-protein interactions the local domenic conformational changes in the enzyme molecule may play a definite role in these differences.     

Kinetic analysis of Na,K-activated adenosine triphosphatase induced by low external K+ in a rat liver cell line

Exposure of ARL 15 cells to medium containing reduced concentrations of K+ (0.65 mM) elicited a 50-100% increase in Na,K-ATPase activity. The inhibition by ouabain of both the basal and the induced enzyme conformed to a single-site model (KI = 1 x 10(-4) M). The low K+-induced increment in Na,K-ATPase activity was accompanied by an equivalent increase in the abundance of Na,K-pump sites estimated by ouabain-stabilized ("back-door") phosphorylation, such that the calculated catalytic turnover number of approximately 8000/min was minimally changed. Comparison of the dependence of ouabain-inhibitable K+ uptake on intracellular Na+ and on extracellular K+ concentrations in control and low K+-treated cells revealed no change in the respective half-maximal stimulatory concentrations for these cations, whereas the maximal rate of active K+ uptake in cells exposed to low external K+ increased by nearly 100%. The derived Hill coefficients for active K+ transport rate were also unchanged by the low K+ treatment (i.e. approximately 1.4 for extracellular K+ and 2.6 for intracellular Na+). Na,K-ATPase activity of basal and low K+-induced cells calculated from the measured maximal Na,K transport rate closely approximated the Na,K-ATPase activity measured enzymatically in unfractionated cell lysates under Vmax conditions, suggesting that all or most of the Na,K-ATPase enzymatic units present in both basal and stimulated states are functionally active. Northern blot analysis of RNA isolated from control cells indicated the presence of the Na,K-ATPase alpha-I isoform of the enzyme which increased by nearly 200% following incubation of the cells in low-K+ medium. By contrast, the alpha-II and alpha-III mRNAs were undetectable in either the basal or low K+-stimulated state. These results indicate that the Na,K-ATPase induced by incubation of ARL 15 cells in low-K+ medium is kinetically and functionally indistinguishable from the basal enzyme, and that only the alpha-I isoform is expressed under control and low-K+ conditions.