Temperature-activity relationships of cation activation and ouabain inhibition of (Na+ + K+)-ATPase.

The nonlinear temperature-activity relationship of membrane preparations of (Na⁺ + K⁺)-ATPase gives rise to discontinuities in Arrhenius plots of this enzyme. The different apparent energies of activation of (Na⁺ + K⁺) — ATPase which are observed above and below the critical temperature of the system have been considered to result from different conformational forms of the enzyme protein. Because both activation of (Na⁺ + K⁺)-ATPase by cations, and its specific inhibition by cardiac glycosides may be influenced by the conformational form of the enzyme protein, we have reexamined the effect of temperature upon the activation energy of the system under the different experimental conditions of cation activation and ouabain inhibition.Our results indicate that the activation of (Na⁺ + K⁺)-ATPase by cations, is less influenced by change in temperature than is inhibition of the enzyme by ouabain. In addition, mild lipolysis by phospholipase-A had a marked effect upon the ouabain-dependent response of the enzyme to temperature, but not upon the cation-dependent response. The effect of phospholipase-A can be overcome by reincubation of the treated preparation with phosphatidyl serine.We conclude that the ouabain-dependent temperature effects of (Na⁺ + K⁺)-ATPase are more dependent upon the integrity and nature of the membrane lipids than are the cation-dependent responses. It is possible that phosphatidyl serine plays a unique role in this regard.     

Effects of nucleotides and Na + on ouabain activation of the phosphatase associated with Na + , K + -ATPase

Ouabain activation of the phosphatase associated with Na⁺,K⁺-ATPase is a time-dependent process which is stimulated by ATP and other nucleotides. Further stimulation by Na⁺ is observed under certain conditions. The stimulatory effect of ATP was found to be due to an increase in the affinity of the enzyme for ouabain. The time required for maximal ouabain activation to be achieved was decreased by ATP and further decreased by ATP + Na⁺.These conditions for maximal activation by ouabain are similar to those required for maximal ouabain binding and suggest that the same ouabain site is responsible for activation of Mg²⁺-dependent phosphatase and for inhibition of Na⁺,K⁺-ATPase and K⁺-phosphatase.     

Ouabain-induced Internalization and Lysosomal Degradation of the Na+/K+-ATPase

Internalization of the Na⁺/K⁺-ATPase (the Na⁺ pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na⁺/K⁺-ATPase molecule or more generally by the disruption of cation homeostasis (Na⁺, K⁺, Ca²⁺) due to the partial inhibition of active Na⁺ and K⁺ transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K⁺-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na⁺/K⁺-ATPase complex.     

Sodium-dependent amino acid transport in preimplantation mouse embryos. III. Na+-k+-atpase-linked mechanism in blastocysts.

Mouse blastocysts collapse in cytochalasin B (CB), reexpand (accumulate fluid) in control medium, but cannot reexpand in ouabain, an inhibitor of Na⁺K⁺-ATPases. These ATPases, then, seem to be necessary for fluid accumulation in blastocysts. Since intact blastocysts are relatively insensitive to ouabain, CB seems to make it possible for ouabain to reach the Na⁺K⁺-ATPases localized on the blastocoelic surface. CB-Collapsed blastocysts were found to transport alanine and lysine at the same rate as intact blastocysts, indicating that, in 1 hr, amino acids are transported into the cells of the intact blastocyst, and not into the fluid-filled blastocoel. Transport rates in CB-collapsed blastocysts do not exceed those in intact blastocysts, suggesting that hypothetical amino acid carriers are located only on the external blastocyst surface. Most important, ouabain strongly inhibits sodium-dependent alanine transport in CB-collapsed blastocysts, but not in intact blastocysts, providing strong evidence that Na⁺K⁺-ATPases, localized on the blastocoelic surface, are necessary for this transport. Ouabain does not inhibit sodium-independent lysine transport in CB-collapsed blastocysts. Thus, the dependency of both sodium-dependent amino acid transport and fluid accumulation upon Na⁺K⁺-ATPases, and the separate localization of amino acid carriers and these ATPases, provides functional evidence for an epithelial tissue type of mechanism for sodium-dependent amino acid transport in mouse blastocysts.     

Differential Properties Between an Endogenous Brain Na+, K+-ATPase Inhibitor and Ouabain

By means of a Sephadex G-50 column and anionic exchange HPLC a cerebral cortex soluble fraction (II-E) which highly inhibits neuronal Na⁺-K⁺-ATPase activity has been previously obtained. Herein, II-E properties are compared with those of the cardenolide ouabain, the selective and specific Na⁺, K⁺-ATPase inhibitor. It was observed that alkali treatment destroyed II-E but not ouabain inhibitory activity. II-E presented a maximal absorbance at 265 nm both at pH 7 and pH 2 which diminished at pH 10. Ouabain showed a maximum at 220 nm which was not altered by alkalinization. II-E was not retained in a C-18 column, indicating its hydrophilic nature, whereas ouabain presented a 26-min retention time in reverse phase HPLC. Therefore, it is concluded that the inhibitory factor present in II-E is structurally different to ouabain.     

The effect of sodium on inorganic phosphate- and p-nitrophenyl phosphate-facilitated ouabain binding to (Na+ + K+)-activated ATPase

The effect of the hydrolysis product P_i and the artificial substrate p-nitrophenyl phosphate (p-nitrophenyl-P) on ouabain binding to (Na⁺ + K⁺)-activated ATPase was investigated.The hypothesis that (Mg²⁺ + p-nitrophenyl-P)-supported ouabain binding might be due to P_i release and thus (Mg²⁺ + P_i)-supported could not be confirmed.The enzyme · ouabain complexes obtained with different substrates were characterized according to their dissociation rates after removal of the ligands facilitating binding. The character of the enzyme · ouabain complex is determined primarily by the monovalent ion present during ouabain binding, but, qualitatively at least, it is immaterial whether binding was obtained with p-nitrophenyl phosphate or P_i.The presence or absence of Na⁺ during binding has a special influence upon the character of the enzyme · ouabain complex. Without Na⁺ and in the presence of Tris ions the complex obtained with (Mg²⁺ + P_i) and that obtained with (Mg²⁺ + p-nitrophenyl-P) behaved in a nearly identical manner, both exhibiting a slow decay. High Na⁺ concentration diminished the level of P_i-supported ouabain binding, having almost no effect on p-nitrophenyl phosphate-supported binding. Both enzyme · ouabain complexes, however, now resembled the form obtained with (Na⁺ + ATP), as judged from their dissociation rates and the K⁺ sensitivity of their decay. The complexes obtained at a high Na⁺ concentration underwent a very fast decay which could be slowed considerably after adding a low concentration of K⁺ to the resuspension medium. The most stable enzyme · ouabain complex was obtained in the presence of Tris ions only, irrespective of whether p-nitrophenyl phosphate or P_i facilitated complex formation. The presence of K⁺ gave rise to a complex whose dissociation rate was intermediate between those of the complexes obtained in the presence of Tris and a high Na⁺ concentration.It is proposed that the different ouabain dissociation rates reflect different reactive state of the enzyme. The resemblance between the observations obtained in phosphorylation and ouabain binding experiments is pointed out.     

Ouabain switches Na+ transport to Na+/Na+ equivalent exchange in normal and apoptotic lymphoid cells

A theory of change of the ionic fluxes in the lymphoid cells in their transition from normal to apoptosis we have developed previously is applied to the analysis of Na⁺/Na⁺ exchange fluxes in human lymphoid cells U937 exposed to ouabain. We solve a system of equations describing changes in the intracellular concentrations of Na⁺, K⁺ and Cl^?, membrane potential and cell volume. It is shown that the Na⁺ influx (I _Na/Na) and output flux through the Na⁺/Na⁺ tract increased 4 times in 8 h after disconnecting Na⁺/K⁺-ATPase for normal cell U937. These fluxes increased 2.6 times for apoptotic cells. The value of I _Na/Na after 8 h off pump by ouabain is 97% of the total Na⁺ input for both cell types. It is concluded that ouabain not only inhibits the Na⁺/K⁺-ATPase, but also increases Na⁺ exchange fluxes through the Na⁺/Na⁺ tract, thereby switching sodium transport across the membrane of lymphoid cells to Na⁺/Na⁺ equivalent exchange.     

Estimation of ouabian specifically bound to dog renal (Na+ + K+)-ATPase in vivo

It is not known whether ouabain injected into the kidney in vivo is bound exclusively to the (Na⁺ + K⁺)-ATPase and whether the reduction of sodium pumping capacity is large enough to account for the reduction in sodium reabsorption. In the present study on dogs the total amount of parenchymal ouabain was therefore estimated and the specific renal binding compared to the reduction in (Na⁺ + K⁺)-ATPase activity. Ouabain, 120 nmol/kg body weight, was injected into the renal artery in vivo reducing the (Na⁺ + K⁺)-ATPase activity by 3lmost 80%. After nephrectomy, tissue ouabain could be quantified by radioimmunoassay after heating the homogenate to 70°C for 30 min; negligible amounts were detectable without heating. No correlation between ouabain binding and tissue volume, protein content, DNA content or Mg²⁺-ATPase content could be found when comparing the following four fractions of the kidney: outer cortex, inner cortex, outer medulla and papilla. For the whole kidney, mean parenchymal tissue concentration of ouabain equalled 0.58 ± 0.03 μmol/100 g wet tissue. Only 21.3 ± 1.2% of the ouabain was confined to the outer medulla corresponding to 54 ± 4 nmol giving a tissue concentration of 1.08 ± 0.05 μmol/100 g wet tissue. The renal ouabain concentrations were highly correlated to the reduction in (Na⁺ + K⁺)-ATPase activity, giving a ratio between the reduction in hydrolysis rate and bound ouabain (turnover number) of 6105 min^?1 which is close to the value of 7180 min^?1 found by in vitro Scatchard analysis. No ouabain seems to be bound to other tissue components than the (Na⁺ + K⁺)-ATPase and the present method is therefore a simple way of measuring the number of inhibited (Na⁺ + K⁺)-ATPase molecules after in vivo injection of ouabain.     

Ouabain receptor interactions in (Na + K)-ATPase preparations. III. On the stability of the ouabain receptor against physical treatment, hydrolases and SH reagents

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant (K_D) of 13 nM was found in the presence of (Mg²⁺ + P_i) and (Na⁺ + Mg²⁺ + ATP). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of (Mg²⁺ + P_i), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of (Na⁺ + K⁺)-ATPase. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).     

In vitro binding of inorganic mercury to the plasma membrane of rat platelet affects Na+-K+-ATPase activity and platelet aggregation

Hg²⁺ binding to ouabain-sensitive Na⁺-K⁺-ATPase of rat platelet membrane was specific with a K_a of 1.3×10⁹ moles and B_max of 3.8 nmoles/mg protein. The binding of mercury to Na⁺-K⁺-ATPase also inhibits the enzyme significantly (P<0.001), which is greater than its ouabain sensitivity. Further in the cytosol of washed platelets conjugation of reduced glutathione (GSH) to Hg²⁺ is correlated dose dependently (25, 50 and 100 pmoles) to enhanced GSH-S-transferase (GST) activity. It may be concluded from the present in vitro experiments that mercury binds specifically to thiol groups present in the platelet membrane Na⁺-K⁺-ATPase, inhibits the enzyme and induces changes in platelet function, namely, platelet aggregation by interfering with the sodium pump.     

Characterization of the stimulation of neuronal Na+, K+-ATPase activity by low concentrations of ouabain

Low concentrations (< 10^?7 M) of ouabain stimulate the activity of Na⁺, K⁺-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentrations of ouabain which induces maximal stimulation is also highly variable and ranges between 10^?9 to 10^?7 M. The ouabain stimulation disappears following 1:50 dilution and 2 h preincubation or freezing and thawing of the membranes or their treatment with deoxycholate. “Aging” of a preparation of ATPase also results in loss of its ability to be stimulated by ouabain but ouabain inhibition is preserved. No stimulation of enzyme activity by ouabain is observed in rat brain microsomal fraction. The β-adrenergic blocker propranolol does not inhibit the ouabain induced stimulation of ATPase activity. It is suggested that the stimulation of Na⁺, K⁺-ATPase activity by low concentrations of cardiac glycosides if a result of either the displacement of an endogenous ouabain-like compound from the enzyme or an indirect effect by changing membrane surrounding environment of the Na⁺, K⁺-ATPase.     

Na+/K+ATPase regulates sperm capacitation through a mechanism involving kinases and redistribution of its testis‐specific isoform

Incubation of bovine sperm with ouabain, an endogenous cardiac glycoside that inhibits both the ubiquitous (ATP1A1) and testis‐specific α4 (ATP1A4) isoforms of Na⁺/K⁺ATPase, induces tyrosine phosphorylation and capacitation. The objectives of this study were to investigate: (1) fertilizing ability of bovine sperm capacitated by incubating with ouabain; (2) involvement of ATP1A4 in this process; and (3) signaling mechanisms involved in the regulation of sperm capacitation induced by inhibition of Na⁺/K⁺ATPase activity. Fresh sperm capacitated by incubating with ouabain (inhibits both ATP1A1 and ATP1A4) or with anti‐ATP1A4 immunoserum fertilized bovine oocytes in vitro. Capacitation was associated with relocalization of ATP1A4 from the entire sperm head to the post‐acrosomal region. To investigate signaling mechanisms involved in oubain‐induced regulation of sperm capacitation, sperm preparations were pre‐incubated with inhibitors of specific signaling molecules, followed by incubation with ouabain. The phosphotyrosine content of sperm preparations was determined by immunoblotting, and capacitation status of these sperm preparations were evaluated through an acrosome reaction assay. We inferred that Na⁺/K⁺ATPase was involved in the regulation of tyrosine phosphorylation in sperm proteins through receptor tyrosine kinase, nonreceptor type protein kinase, and protein kinases A and C. In conclusion, inhibition of Na⁺/K⁺ATPase induced tyrosine phosphorylation and capacitation through multiple signal transduction pathways, imparting fertilizing ability in bovine sperm. To our knowledge, this is the first report documenting both the involvement of ATP1A4 in the regulation of bovine sperm capacitation and that fresh bovine sperm capacitated by the inhibition of Na⁺/K⁺ATPase can fertilize oocytes in vitro. Mol. Reprod. Dev. 77: 136–148, 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of Na+, K+-ATPase in Cultured and Separated Neuronal and Glial Cells from Rat Cerebellum

Abstract: The activities of certain properties of sodium, potassium-activated adenosine triphosphatase (Na ⁺, K⁺- ATPase; EC 3.6.1.3) were examined in cultures and peri- karya fractions enriched in rat cerebellar nerve cells or astrocytes, in comparison with preparations from whole immature and adult rat cerebellum and derived synapto- somal fractions, as well as nonneural tissue such as the kidney. The specific activity of Na ⁺, K⁺-ATPase was markedly higher in the freshly isolated astrocytes than in the nerve cells (3–15-fold greater depending on neuronal cell type). In contrast, the specific activity of the enzyme was about twice as high in the primary neuronal as in the a'strocytic cultures after 14 days in vitro. In membrane preparations from the whole cerebellum, synaptosomal fractions, and total perikarya suspensions the inhibition of enzyme activity by ouabain indicated complex kinetics, which were consistent with the presence of two forms of the Na ⁺, K⁺-ATPase (apparent A_j values of about 10–⁷M and 10–⁴-10–⁵M, respectively), the high- affinity form accounting for 60–75% of the total activity. The interaction of the enzyme with ouabain was apparently similar in perikarya preparations of granule neurones, Purkinje cells, and astrocytes. Differences were, however, observed in the properties of the Na ⁺,K ⁺ - ATPase of cultured neurones and astrocytes. The latter contained predominantly, but not exclusively, an Na⁺,K⁺-ATPase with low affinity for ouabain (73% of the total) that is similar to the single enzyme form in the kidney. This form constituted a significantly smaller proportion of the Na ⁺, K⁺-ATPase in the cultured neuronal preparations (55%). It would appear, therefore, that in membrane fractions from preparations enriched in different separated and cultured neural cell types both the high- and the low-affinity forms of the enzyme, in terms of interaction with ouabain, are expressed. Depending on the class of cells these enzyme forms constituted a different proportion of the total activity, but both forms seemed to be present in every type of cell examined, even after taking into acc.ount the contribution in the enriched preparations of the contaminating cell types. In contrast with the results on the Na⁺, K⁺-ATPase activity determined under optimal conditions in preparations derived from disrupted cells, differences could not be detected between the cultured cell types when the effect of ouabain on the uptake of ⁸⁶Rb into “live cells” was estimated as a measure of in situ ion pump activity. Besides the interaction with ouabain, the K⁺ dependence of the Na⁺, K⁺-ATPase activity was also investigated in crude particulate preparations from cultured cerebellar neurones and astrocytes. Differences were observed as nearly maximal enzyme activity was obtained in the as- trocyte preparations at 1 mM KCl, when only about one- third of the maximal activity was displayed by the cultured nerve cells.     

Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain

Effects of long-term, subtotal inhibition of Na⁺-K⁺ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K⁺ medium, are described for HeLa cells. After prolonged growth in 2 × 10^?8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing amodest induction of Na⁺, K⁺-ATPase. In contrast, after long-term growth in low K⁺ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na⁺] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na⁺]_i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, V_max for transport, and specific phosphorylation. Parallel exposure of cryptic Na⁺, K⁺-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na⁺, K⁺-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.     

Cardiotonic steroids as potential Na+/K+-ATPase inhibitors – a computational study

Abstract

Cardiotonic steroids (CTS) are steroidal drugs, processed from the seeds and dried leaves of the genus Digitalis as well as from the skin and parotid gland of amphibians. The most commonly known CTS are ouabain, digoxin, digoxigenin and bufalin. CTS can be used for safer medication of congestive heart failure and other related conditions due to promising pharmacological and medicinal properties. Ouabain isolated from plants is widely utilized in in vitro studies to specifically block the sodium potassium (Na⁺/K⁺-ATPase) pump. For checking, whether ouabain derivatives are robust inhibitors of Na⁺/K⁺-ATPase pump, molecular docking simulation was performed between ouabain and its derivatives using YASARA software. The docking energy falls within the range of 8.470?kcal/mol to 7.234?kcal/mol, in which digoxigenin was found to be the potential ligand with the best docking energy of 8.470?kcal/mol. Furthermore, pharmacophore modeling was applied to decipher the electronic features of CTS. Molecular dynamics simulation was also employed to determine the conformational properties of Na⁺/K⁺-ATPase-ouabain and Na⁺/K⁺-ATPase-digoxigenin complexes with the plausible structural integrity through conformational ensembles for 100?ns which promoted digoxigenin as the most promising CTS for treating conditions of congestive heart failure patients.     

Molecular species of a soluble nucleoside diphosphokinase related to the Na + ,K + -ATPase

Soluble nucleosidediphosphokinase (NDK) activity is prepared from purified kidney membranes rich in Na⁺, K⁺-ATPase. The soluble NDK differs from the bound activity by the lack of sensitivity to Na⁺, K⁺ and ouabain. Sucrose gradient centrifugation of the soluble NDK shows the presence of three major species with molecular weights of 21,000, 92,000 and 138,000. Increasing the urea concentration in the sucrose gradient centrifugation decreases the proportion of the enzyme in the heavier form. This effect of urea is reversible. These data suggest that the higher molecular weight forms are tetramers and hexamers of the 21,000 dalton form of the soluble NDK.     

Ouabain receptor interactions in (Na+ + K+)-ATPase preparations. II. Effect of cations and nucleotides on rate constants and dissociation constants

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant ($\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of 13 nM was found in the presence of ($\text{Mg}{}^{\mathrm{2+}}\text{+ P}{}_{\mathrm{i}}$) and ($\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}\text{+ ATP}$). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of ($\text{Mg}{}^{\mathrm{2+}}\text{+ P}{}_{\mathrm{i}}$), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).     

Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated Na i + /K i + -independent signaling

Recent studies demonstrate that cytotoxic actions of ouabain and other cardiotonic steroids (CTS) on renal epithelial cells (REC) are triggered by their interaction with the Na⁺,K⁺-ATPase α-subunit but not the result of inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio. This study examined the role of mitogen-activated protein kinases (MAPK) in the death of ouabain-treated REC. Exposure of C7-MDCK cells that resembled principal cells from canine kidney to 3 μM ouabain led to phosphorylation of p38 without significant impact on phosphorylation of ERK and JNK MAPK. Maximal increment of p38 phosphorylation was observed at 4 h followed by cell death at 12 h of ouabain addition. In contrast to ouabain, neither cell death nor p38 MAPK phosphorylation were affected by elevation of the [Na⁺]_i/[K⁺]_i ratio triggered by Na⁺,K⁺-ATPase inhibition in K⁺-free medium. p38 phosphorylation was noted in all other cell types exhibiting death in the presence of ouabain, such as intercalated cells from canine kidney and human colon rectal carcinoma cells. We did not observe any action of ouabain on p38 phosphorylation in ouabain-resistant smooth muscle cells from rat aorta and endothelial cells from human umbilical vein. Both p38 phosphorylation and death of ouabain-treated C7-MDCK cells were suppressed by p38 inhibitor SB 202190 but were resistant to its inactive analogue SB 202474. Our results demonstrate that death of CTS-treated REC is triggered by Na_i⁺,K_i⁺—independent activation of p38 MAPK.     

The effect of Na+-K+ ATPase inhibition by ouabain on histamine release from human cutaneous mast cells

Background: There are controversial reports on the effect of sodium-potassium adenosine triphosphatase (Na⁺-K⁺ ATPase) inhibition on mast cell mediator release. Some of them have indicated that ouabain (strophanthin G), a specific Na⁺-K⁺ ATPase inhibitor, inhibited the release, whereas the others have shown that ouabain had no effect or even had a stimulatory effect on the mediator secretion. Most of these studies have utilized animal-derived mast cells. The aim of this study was to determine the effect of Na⁺-K⁺ ATPase inhibition on human skin mast cells. Methods: Unpurified and purified mast cells were obtained from newborn foreskins and stimulated by calcium ionophore A23187 (1 μM) for 30 min following a 1 hr incubation with various concentrations (10⁻⁴ to 10⁻⁸ M) of ouabain. Histamine release was assayed by enzyme-linked immunosorbent assay (ELISA). Results: The results indicated that ouabain had no significant effect on the non-immunologic histamine release from human skin mast cells, in vitro. Conclusions: Na⁺-K⁺ ATPase inhibition by ouabain had no significant effect on the non-immunologic histamine release from human cutaneous mast cells and suggested differences between human and animal mast cells.     

Purification and characterization of an (Na++K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain

Highly purified lamb kidney (Na⁺+K⁺)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na⁺+K⁺)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (γ1 and γ2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that γ1 is an aggregated form of γ2. The total yield was 0.8–1.5 mol of γ component per mol of catalytic subunit recovered. This indicates that the γ component is present in stoichiometric amounts in the (Na⁺+K⁺)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.