Differential effects of ouabain on human cell-mediated cytotoxicity. I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets.

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed ⁵¹Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10^?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.     

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNA and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [³H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K⁺ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K⁺, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K⁺ transport, controlling mitosis at distinct phases of the cell cycle.     

Ultrastructural study of biochemically modulated ADCC in HSV-1 infected and uninfected Chang liver cells

The effect of cytochalasin B, ouabain and 25-OH cholesterol on specific lysis due to antibody dependent cellular cytotoxicity (ADCC) in allogeneic and xenogeneic systems was studied using Herpes simplex I infected Chang liver cells. Cytochalasin B reduced both cytotoxicity and lymphocyte/target (LT) binding in the allogeneic system whereas cytotoxicity but not LT binding was reduced in the xenogeneic system. Ouabain inhibited ADCC in both systems as well as LT binding in the allogeneic system; however, binding in the xenogeneic system was not significantly reduced. The 25-OH cholesterol produced a marked decrease in ADCC in both systems but had no significant effect on LT binding in either system. The biochemical and ultrastructural data suggest that the modulators act at different stages in the ADCC response and that there may be more than one mechanism of ADCC to handle different types of target antigens.     

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells

Cardiotonic glycosides, like ouabain, inhibit Na⁺-K⁺-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser⁴⁷³ phosphorylation, as evidenced by an increase in phospho-Akt Ser⁴⁷³ band density. Ouabain-stimulated Akt Ser⁴⁷³ phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser⁴⁷³ phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser⁴⁷³ phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in ⁸⁶Rb uptake but did not significantly alter Na⁺-K⁺-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na⁺-K⁺-ATPase-mediated ion transport. Na⁺-K⁺-ATPase; opossum kidney cells     

Differential effects of ouabain on human cell-mediated cytotoxicity. II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets.

We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10^?5M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10^?9M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.     

Ion transport by ouabain resistant and sensitive ehrlich ascites carcinoma cells

Cell division, net Na⁺-K⁺ and amino-acid transport of cultured Ehrlich ascites is reversibly inhibited by Ouabain at a final concentration of 1 × 10^–3M. A line of Ehrlich ascites cells resistant to the growth inhibiting effects of Ouabain has been developed. These cells behave similarly to Ouabain-sensitive cells in the following respects doubling time, S phase time, chromosome number, cell surface charge density, rate of incorporation of C¹⁴ Uridine and ³H-Thymidine, sensitivity to Digoxin and Digitoxin, steady state Na⁺, K⁺ levels and rate of loss of K⁺ and gain of Na⁺ in cold. Ouabain resistant cells differ from sensitive cells only with respect to the effect of ouabain on active Na⁺, K⁺ transport. Although Ouabain inhibits active Na⁺, K⁺ transport in sensitive cells it has no significant effect in resistant cells.     

Polyadnylic acid sequences in RNA able to transfer specific delayed type hypersensitivity in vitro.

Antibody-depedent cell-mediated cytotoxicity (ADCC) could be initiated without protein synthesis [human peripheral blood lymphocytes as effector cells incubated with 10^?3M cycloheximide, (Cy)], although the reaction did not achieve its full lytic ability. This partial inhibition of ADCC was dependent on the dose of Cy. Both ADCC and protein synthesis returned to normal values after removal of the inhibitor. The kinetics of the reaction carried out by Cy-treated effector cells for short periods was similar to that of controls. After this time, the percentage of lysed target cells increased continuously in controls, while the cytotoxiciy of Cy-treated effector cells reached a plateau. When effector cells carried out ADCC in the presence of Cy, their lytic mechanism was “wasted,” and it could be recovered only by removal of the inhibitor. Our results indicate that effector cells have a preformed lytic mechanism operative in ADCC. This lytic mechanism is consumed during the reaction and its recovery requires protein synthesis.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNa and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [3H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K+ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K+, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K+ transport, controlling mitosis at distinct phases of the cell cycle.     

Inhibition of lymphocyte activation by ouabain Interference with the early activation of membrane phospholipid metabolism

Activation of lymphocytes by antigens and mitogens can effectively be prevented by ouabain, a known inhibitor of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$. Recently it was shown that lowering of intracellular levels of monovalent cations is not involved in the inhibitory effect of ouabain. $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$ was found to be closely associated with acylCoA: lysophosphatidylcholine acyltransferase in the plasma membrane of lymphocytes. Both enzymes are activated as an immediate consequence of mitogen binding. Human peripheral lymphocytes were stimulated with concanavalin A. Ouabain suppressed the induction of RNA and DNA synthesis in a concentration-dependent way. Increase of RNA synthesis was suppressed only if the glycoside were added within the first hours of activation. If ouabain was added later, incorporation of uridine remained at the rate that was reached at the time of glycoside administration, pointing to an early event where ouabain may be operative. Ouabain, in a dose-dependent manner similar to that affecting RNA and DNA synthesis, inhibited the increase in the incorporation of oleate into phospholipids in stimulated lymphocytes, whereas the turnover of phospholipid fatty acids in resting lymphocytes was unaffected. Increasing extracellular K⁺ concentrations reversed the binding of ouabain to lymphocytes. Simultaneously, the inhibition of stimulated RNA synthesis was decreased and the inhibition of oleate incorporation was reversed. These results suggest that the suppression of lymphocyte activation by ouabain is due to the inhibition of membrane phospholipid metabolism mediated by the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$.     

Localization of sodium pump sites in frog urinary bladder

[³H]Ouabain binding in frog and toad urinary bladder was investigated by short-circuit current (SCC), scintillation counting and authoradiographic techniques. SCC data and analysis of tissue digests following serosal exposure to ouabain showed that ouabain binding and inhibition of Na⁺ transport was completely reversible in toad bladder whereas, in frog bladder, [³H] ouabain was tightly bound and Na⁺ transport remained suppressed even after a 60-min washout. Mucosal exposure of frog bladder to [³H]ouabain or serosal exposure after preincubation with unlabeled ouabain led to a marked reduction in binding. Specificity of binding was assessed further by adjusting the concentration of cecrtain (Na⁺?K⁺)-ATPase ligands (K⁺, ATP) to levels known to reduce ouabain binding. High K⁺ concentrations and depletion of endogenous ATP by incubation under anoxic conditions resulted in a significant drop in [³H]ouabain binding. Autoradiographic analysis showed that grains are localized primarily to the basolateral plasma membranes of the granular cells, providing direct morphological evidence for the location of Na⁺ pumps at these sites. Although autoradiographs did not provide sufficient resolution to rule out unequivocally ouabain binding to the mitochondria-rich cell, morphological evidence suggests that grain densities are significatly higher between adjacent granular cells than between granular cell-mitochondria-rich cell interfaces.     

低浓度哇巴因对负鼠肾小管上皮细胞增殖的影响

目的:用低血清培养液来模拟肾脏供血不足的营养不良状态,研究低浓度哇巴因对低血清培养下OK细胞(负鼠肾小管上皮细胞)增殖的影响。方法:用低浓度哇巴因(1-30n M)处理0.2%血清培养下OK细胞,MTT实验和Brdu掺入法检测哇巴因对OK细胞增殖的影响;Western blot检测Akt和ERK1/2的磷酸化水平;用LY294002和PD98059分别抑制PI3K/Akt和ERK1/2蛋白激酶活性,观察抑制PI3K/Akt和ERK1/2对哇巴因促进OK细胞增殖的影响。结果:低浓度哇巴因(1-30n M)促进OK细胞的增值,上调OK细胞中Akt和ERK1/2磷酸化水平。用LY294002和PD98059特异抑制Akt和ERK1/2的活化能够抑制哇巴因的促增殖作用。结论:低浓度哇巴因(1-10n M)能够促进OK细胞的增值,PI3K/Akt和ERK1/2信号通路参与哇巴因对OK细胞促增殖作用的调节。     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

Characterization of temperature-sensitive mutants of animal cells

An early increase in lymphocyte plasma membrane K⁺ transport is essential for PHA stimulated lymphocytes to divide. Little is known about the specific source and amount of energy required to support the increased transport by activated lymphocytes. Since ouabain, a cardiac glycoside, specifically inhibits the transport ATPase, we have measured the decrement in glycolysis and tricarboxylic acid cycle activity when untreated and PHA treated lymphocytes were exposed to ouabain. This metabolic decrement represents the portion of metabolism associated with monovalent cation transport and closely related processes. Since TCA cycle activity accounted for only 0.2% of glucose consumption, aerobic glycolysis was the major source of energy, i.e., ATP, for increased transport. Approximately one-third of the total lactate production in both control and PHA stimulated lymphocytes was ouabain-sensitive. Ouabain sensitive lactate production in control, 105 μmol/10¹⁰ cells/hour, increased 1.8-fold to 193 μmol/10¹⁰ cells/hour after PHA treatment. Active K⁺ influx in similar cell populations increased from 40 μmol/10¹⁰ cells/hour to 74 μmol/10¹⁰ cells/hour (1.9-fold) after PHA treatment. The increment in ouabain-sensitive energy production and K⁺ transport were closely correlated and, therefore, 0.38 moles of K⁺ are transported for each mole of ATP generated in both control and PHA treated cells. The increased requirement for transport related energy is provided by increasing the ouabain-sensitive ATP production rather than altering the efficiency of ATP transduction.     

Virus-mediated induction in human lymphocytes of antibody-independent cytotoxicity (VDCC) and enhancement of antibody-dependent cytotoxicity (ADCC) against natural killer-resistant tumor target cells

The effect of Parotis virus on the in vitro cytotoxicity of human lymphocytes against NK-resistant mouse mastocytoma cells was studied. In the ⁵¹Cr-release assay, treatment of lymphocytes with virus induced a rapid cytotoxicity in the absence of anti-P8 15 antibody (virus-dependent cellular Cytotoxicity, VDCC) and strongly enhanced antibody-dependent cytotoxicity (ADCC). At the effector cell level, virus treatment was found to increase the frequency of target-binding cells (TBC) as well as the proportion thereof mediating VDCC and/ or ADCC, indicating recruitment of active effector cells. The recruited cells were heterogeneous but contained a major fraction bearing the T-cell-associated antigen T3. Virus was found to decrease rather than to increase the recycling capacity of the cytotoxic lymphocytes, suggesting that VDCC induction and ADCC enhancement were due to a virus-mediated improvement of effector cell-target cell interactions. VDCC and ADCC enhancement may be of protective importance in early phases of virus infection as well as for the production of nonspecific tissue injuries associated with viral disease.     

Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway

Endogenous cardiotonic glycosides bind to the inhibitory binding site of the plasma membrane sodium pump (Na⁺/K⁺-ATPase). Plasma levels of endogenous cardiotonic glycosides increase in several disease states, such as essential hypertension and uremia. Low concentrations of ouabain, which do not inhibit Na⁺/K⁺-ATPase, induce cell proliferation. The mechanisms of ouabain-mediated response remain unclear. Recently, we demonstrated that in opossum kidney (OK) proximal tubular cells, low concentrations of ouabain induce cell proliferation through phosphorylation of protein kinase B (Akt) in a calcium-dependent manner. In the present study, we identified ERK as an upstream kinase regulating Akt activation in ouabain-stimulated cells. Furthermore, we provide evidence that low concentrations of ouabain stimulate Na⁺/K⁺-ATPase-mediated ⁸⁶Rb uptake in an Akt-, ERK-, and Src kinase-dependent manner. Ouabain-mediated ERK phosphorylation was inhibited by blockade of intracellular calcium release, calcium entry, tyrosine kinases, and phospholipase C. Pharmacological inhibition of phosphoinositide-3 kinase and Akt failed to inhibit ouabain-stimulated ERK phosphorylation. Ouabain-mediated Akt phosphorylation was inhibited by U0126, a MEK/ERK inhibitor, suggesting that ouabain-mediated Akt phosphorylation is dependent on ERK. In an in vitro kinase assay, active recombinant ERK phosphorylated recombinant Akt on Ser⁴⁷³. Moreover, transient transfection with constitutively active MEK1, an upstream regulator of ERK, increased Akt phosphorylation and activation, whereas overexpression of constitutively active Akt failed to stimulate ERK phosphorylation. Ouabain at low concentrations also promoted cell proliferation in an ERK-dependent manner. These findings suggest that ouabain-stimulated ERK phosphorylation is required for Akt phosphorylation on Ser⁴⁷³, cell proliferation, and stimulation of Na⁺/K⁺-ATPase-mediated ⁸⁶Rb uptake in OK cells. opossum kidney cells; sodium/potassium adenosine triphosphatase; extracellular signal-regulated kinase; cell proliferation     

Ouabain-induced apoptosis and Rho kinase: a novel caspase-2 cleavage site and fragment of Rock-2

Ouabain, a specific Na⁺/K⁺-ATPase inhibitor, has recently been identified as a mammalian hormone. Its elevated concentrations in human plasma have also been associated with pathogenesis of several diseases. Recent studies have shown that ouabain induces aponecrotic cell death in a cell-type- and dose-dependent manner. However, the exact mechanism of ouabain-induced cell death is not fully understood. The Rho GTPase effectors Rho kinases-1 and -2 (Rock-1 and Rock-2) which play central roles in the organization of the actin cytoskeleton, involve in several models of apoptosis. In this study, we investigated the possible involvement of Rocks in ouabain-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Ouabain treatment resulted in loss of cell–cell and cell–substratum adhesion and apoptotic blebbing. Pretreatment of cells with Y-27632, a specific Rock inhibitor, resulted in the inhibition of cell-to-cell detachment and formation of membrane blebs. However, Y-27632 did not prevent ouabain-induced cell–substratum detachment. Instead, treatment with Y-27632 actually accelerated this process. Ouabain treatment induced cleavage of Rock-1 and Rock-2, which was prevented by caspase-3 and caspase-2 specific inhibitors z-DEVD-fmk and z-VDVAD-fmk, respectively. Ouabain-induced Rock-2 cleavage generated a fragment of approximately 140 kDa corresponding to the consensus sequence of caspase-2 on the carboxy terminus of Rock-2. Although it has been previously shown that Rock-2 was cleaved by caspase-2, we have identified here a novel cleavage site and fragment of Rock-2. Our data indicate that ouabain induces both Rock-1 and Rock-2 cleavage via caspase-dependent mechanisms and provide evidence that Rocks are involved in ouabain-induced cell-to-cell detachment and apoptosis.     

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 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.