Inhibitory Effect Of Ouabain Research Articles

In Vitro Study of the Multimodal Effect of Na+/K+ ATPase Blocker Ouabain on the Tumor Microenvironment and Malignant Cells.

Na+/K+ ATPase is a protein involved in the active transport of ions across the cellular membrane. Ouabain is a cardiotonic glycoside that, by inhibiting the Na+/K+ pump, interferes with cell processes mediated directly by the pump, but also indirectly influences other cellular processes such as cell cycle and proliferation, growth, cell differentiation, angiogenesis, migration, adhesion, and invasion. We used the SK-BR-3 breast cancer cell line, mesenchymal stem cells (MSCs), and tumor-associated fibroblasts (TAFs) in vitro to determine the effects of ouabain exposure on these cellular types. The results showed a multi-level effect of ouabain mainly on tumor cells, in a dose-dependent manner, while the TAFs and their normal counterparts were not significantly influenced. Following exposure to ouabain, the SK-BR-3 cells changed their morphologic appearance, decreased the expression of immunophenotypic markers (CD29, Her2, VEGF), the proliferation rate was significantly decreased (Ki67 index), the cells were blocked in the G0 phase of the cell cycle and suffered necrosis. These data were correlated with the variable expression of α and β Na+/K+ pump subunits in tumor cells, resulting in decreased ability to adhere to the VCAM-1 substrate in functional flow chamber studies. Being indicative of the pro-apoptotic and inhibitory effect of ouabain on tumor invasion and metastasis, the results support the addition of ouabain to the oncological therapeutic arsenal, trailing the "repurposing drugs" approach.

Inhibitory Effect of Ouabain on Resistance to Imatinib in Chronic Myeloid Leukemia K562/G01 Cells

Inhibitory Effect of Ouabain on Resistance to Imatinib in Chronic Myeloid Leukemia K562/G01 Cells

Ouabain reduces the expression of the adhesion molecule CD18 in neutrophils

Ouabain, a hormone that inhibits Na+/K+-ATPase, modulates many aspects of the inflammatory response. It has been previously demonstrated that ouabain inhibits neutrophil migration in several inflammation models in vivo, but little is known about the mechanisms underlying this effect. Thus, this work aimed to evaluate the effect of ouabain on molecules related to neutrophil migration. For this purpose, neutrophils obtained from mouse bone marrow were treated with ouabain (1, 10, and 100nM) in vitro. Neutrophil viability was assessed by annexin V/propidium iodide staining. Ouabain treatment did not affect neutrophil viability at different times (2, 4, and 24h). However, basal neutrophil viability was decreased after 4h. Thus, we assessed the effect of ouabain on the adhesion molecule CD18, an integrin β2 chain protein, and on the chemokine receptor CXCR2 after 2h of treatment. CD18 expression was reduced (by 30%) by 1nM ouabain. However, the expression of CXCR2 on the neutrophil membrane was not affected by ouabain treatment (1, 10, and 100nM). Moreover, ouabain (1, 10, and 100nM) did not modulate the zymosan-induced secretion of CXCL1 (a chemokine receptor CXCR2 ligand) in macrophage cultures. These data suggest that the inhibitory effect of ouabain on neutrophil migration is related to reduced CD18 expression, indicating a novel mechanism of action.

Ouabain signaling in oral squamous cell carcinoma cells.

Accurate evaluation of the anti-cancer effects of ouabain, a cardiac glycoside, requires an understanding of its signaling pathway. This study examined the effects of ouabain stimulation on spontaneous interleukin (IL)-8 and IL-1α secretion in the HSC3 oral squamous cell carcinoma cell line. IL-8 secretion was reduced and IL-1α secretion was increased in the cells. Real-time polymerase chain reaction confirmed that these changes were regulated at the transcriptional level. Further analysis revealed that ouabain stimulation induced phosphorylation of activator protein (AP)-1 components (c-Jun and c-Fos) but not nuclear factor kappa B (NF-κB) components (p65 and p50). A luciferase assay demonstrated that the NF-κB-binding site located at 1 kb upstream of the TATA box in the IL-8 gene contributed to the reduction in IL-8 secretion. Pre-incubation of the cells with BAPTA-AM and L-glutathione increased IL-8 secretion, which indicates that Ca2+ ions and reactive oxygen species are associated with the ouabain-mediated reduction in IL-8 levels. The inhibitory effect of ouabain was attributed to reduced nuclear translocation of the NF-κB p65 subunit. Taken together, these findings indicate that ouabain exerts opposing effects on transcription factors NF-κB and AP-1.

Effects of exogenous ouabain on endothelium‐dependent vascular function

Endogenous ouabain is an inhibitor of sodium‐potassium adenosine triphosphatase (Na+/K+‐ATPase, also known as sodium‐pump). It is released by high sodium chloride concentrations from the hypothalamus and the adrenal cortex. Upon its release in the circulation, endogenous ouabain increases arterial constriction and peripheral resistance hence arterial blood pressure. In the clinical setting, digoxin, targeting the sodium pump as well, is an orally active agent exerting inotropic effects to treat heart failure or atrial arrhythmias. But toxicity often occurs due to overdose. The present study aimed to investigate the local effects when the sodium pump is inhibited by exogenous ouabain in acute settings. Aortae isolated from male Sprague Dawley rats (12 weeks old) were cut into rings and incubated in the organ chambers with Krebs‐Ringer solution for isometric tension recording, in the absence or presence of ouabain. They were contracted with phenylephrine and relaxed with increasing concentrations of endothelium‐dependent and ‐independent vasodilators, acetylcholine (ACh) and DETA NONOate, or exogenous arachidonic acid (AA). Ouabain inhibited endothelium‐dependent relaxations to ACh and AA in a concentration‐dependent manner. It reduced the maximal relaxation to ACh in aortae with endothelium where the nitric oxide signaling pathway plays a dominant role in vasodilatation, and increased the half maximal effective concentration (EC50) of DETA NONOate. The inhibitory effect of ouabain on AA‐induced relaxations was not observed in the presence of indomethacin (cyclooxygenase inhibitor). Ouabain potentiated the relaxations to AA when endothelial potassium channels involved in endothelium‐dependent hyperpolarization (EDH) signalings [namely intermediate‐ and small‐conductance calcium‐activated potassium channels (IKCa and SKCa, respectively)] were blocked by TRAM‐34 and UCL1684, respectively; the relaxation was not affected by the inhibition, with barium, of inwardly‐rectifying potassium channels (KIR, which also play a role in EDH pathway). Taken in conjunction, ouabain likely impairs the nitric oxide and/or cyclooxygenase pathways to reduce endothelium‐dependent relaxations. When the major mechanisms for the generation of endothelium‐derived relaxing signals (namely, nitric oxide, prostanoids and EDH) are inhibited, ouabain unmasks the relaxation to AA that is independent of KIR activation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inactivation of Src-to-ezrin pathway: a possible mechanism in the ouabain-mediated inhibition of A549 cell migration.

Ouabain, a cardiac glycoside found in plants, is primarily used in the treatment of congestive heart failure and arrhythmia because of its ability to inhibit Na+/K+-ATPase pump. Recently ouabain has been shown to exert anticancer effects but the underlying mechanism is not clear. Here, we explored the molecular mechanism by which ouabain exerts anticancer effects in human lung adenocarcinoma. Employing proteomic techniques, we found 7 proteins downregulated by ouabain in A549 including p-ezrin, a protein associated with pulmonary cancer metastasis in a dose-dependent manner. In addition, when the relative phosphorylation levels of 39 intracellular proteins were compared between control and ouabain-treated A549 cells, p-Src (Y416) was also found to be downregulated by ouabain. Furthermore, western blot revealed the ouabain-mediated downregulation of p-FAK (Y925), p-paxillin (Y118), p130CAS, and Na+/K+-ATPase subunits that have been shown to be involved in the migration of cancer cells. The inhibitory effect of ouabain and Src inhibitor PP2 on the migration of A549 cells was confirmed by Boyden chamber assay. Anticancer effects of ouabain in A549 cells appear to be related to its ability to regulate and inactivate Src-to-ezrin signaling, and proteins involved in focal adhesion such as Src, FAK, and p130CAS axis are proposed here.

Cap-dependent translation initiation factor, eIF4E, is the target for Ouabain-mediated inhibition of HIF-1α

Hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that mediates the adaptation of tumor cells and tissues to the hypoxic microenvironment, has attracted considerable interest as a potential therapeutic target. Recently, HIF-1α has been recognized as the critical target of cardiac glycosides for cancer therapy, but the molecular mechanism of cardiac glycosides’ inhibition of HIF-1α is still poorly understood. In the present study, we observed that neither HIF-1α mRNA levels nor HIF-1α protein degradation are affected by Ouabain. However, Ouabain was found to be associated with the regulation of HIF-1α translation. Basing on in silico, in vitro and ex vivo models of translation processing, further studies revealed that eIF4E plays a critical role in the inhibitory effect of Ouabain on HIF-1α protein synthesis, rather than mTORC1, eIF2α signaling or Na+/K+-ATPase inhibition. Mechanistically, Ouabain directly binds eIF4E, disrupts eIF4E/eIF4G association (200μM, Inhibit rate =61±3%) but not the eIF4E/mRNA complex formation (200μM, Inhibit rate =18±5%) both in vitro and in cells, thereby inhibiting the intracellular cap-dependent translation. The association between Ouabain and eIF4E not only raises the hope of using cardiac glycosides for cancer therapeutics more rational, but also offers a pharmacologic means for developing novel anti-cancer HIF-1α antagonists.

The inhibitory effect of ouabain on the response of slowly adapting pulmonary stretch receptors to hyperinflation in the rabbit

The combined effects of ouabain (Na +–K + ATPase inhibitor) and hyperinflation (inflation volume = three tidal volumes) on slowly adapting pulmonary stretch receptors (SARs) were studied before and after administration of nifedipine (an L-type Ca 2+ channel blocker) and KB-R7943 (a reverse-mode Na +–Ca 2+ exchanger blocker) in anesthetized, artificially ventilated rabbits after bilateral vagotomy. Before ouabain administration, hyperinflation stimulated SAR activity. After 20 min of ouabain administration (30 μg/kg) the SARs increased discharge rates in normal inflation. Under these conditions, hyperinflation initially stimulated SAR activity but subsequently inhibited the activity at peak inflation. Additional administration of 60 μg/kg ouabain (total dose = 90 μg/kg) caused a further stimulation of SAR activity, but 20 min later both normal inflation and hyperinflation resulted in a greater inhibition of the receptor activity. The hyperinflation-induced SAR inhibition in the presence of ouabain (30 μg/kg) was not significantly altered by administration of either nifedipine (2 and 4 mg/kg) or KB-R7943 (1 and 3 mg/kg). In another series of experiments, we further examined the combined effects of ouabain and hyperinflation in veratridine (a Na + channel opener, 40 μg/kg)-treated animals. After recovery from the veratridine effect on SAR activity, which vigorously stimulated the receptor activity, ouabain treatment (30 μg/kg) that silenced the receptor activity at peak inflation greatly inhibited hyperinflation-induced SAR stimulation. These results suggest that hyperinflation-induced SAR inhibition in the presence of ouabain may be related to a Na + overload, but not to a Ca 2+ influx via activation of L-type Ca 2+ channels, in the SAR endings.

Ouabain exacerbates activation-induced cell death in human peripheral blood lymphocytes

Lymphocytes activated by mitogenic lectins display changes in transmembrane potential, an elevation in the cytoplasmic Ca2+ concentrations, proliferation and/or activation induced cell death. Low concentrations of ouabain (an inhibitor of Na+,K+-ATPase) suppress mitogen-induced proliferation and increases cell death. To understand the mechanisms involved, a number of parameters were analyzed using fluorescent probes and flow cytometry. The addition of 100 nM ouabain to cultures of peripheral blood lymphocytes activated with 5 microg/ml phytohemagglutinin (PHA) did not modify the increased expression of the Fas receptor or its ligand FasL induced by the mitogen. However, treatment with ouabain potentiated apoptosis induced by an anti-Fas agonist antibody. A synergy between ouabain and PHA was also observed with regard to plasma membrane depolarization. PHA per se did not induce dissipation of mitochondrial membrane potential but when cells were also exposed to ouabain a marked depolarization could be observed, and this was a late event. It is possible that the inhibitory effect of ouabain on activated peripheral blood lymphocytes involves the potentiation of some of the steps of the apoptotic process and reflects an exacerbation of the mechanism of activation-induced cell death.

ROLE OF SODIUM‐POTASSIUM ATPASE IN PHOSPHATE TRANSPORT BY THE EVERTED SACS OF MOUSE INTESTINE

1. This study investigated the uptake and release of phosphate from everted intestinal sacs of mice. 2. When the activity of sodium-potassium ATPase was altered (by changing the cationic concentrations in the medium by the addition of ouabain and the manipulation of metabolism by the use of selective inhibitors or stimulants), the release of phosphate appeared to be much more affected by the changes than the uptake of phosphate. 3. A small sodium gradient allowed uptake to be maintained. The addition of ouabain in the presence of an attenuated gradient significantly reduced the uptake and release of phosphate. The inhibitory effect of ouabain on phosphate release was partly reversed by an increase in the potassium concentration in serosal fluid. 4. These results indicate a role for sodium-potassium ATPase in phosphate release from the intestine.

Ouabain, na +-free and k +-free solutions and relaxations to nitric oxide and nitrovasodilators

1. 1. Experiments were designed to determine the effect of inhibition of Na +, K + ATPase by ouabain, Na +-free and K +-free solutions on the relaxations induced by nitric oxide, nitroglycerin and sodium nitroprusside in coronary smooth muscle. 2. 2. Rings without endothelium of canine left anterior descending coronary artery were suspended for isometric tension recording in modified Krebs-Ringer bicarbonate solution, in the presence of indomethacin, propranolol, and phenoxybenzamine. The rings were incubated with ouabain, Na +-free or K +-free solutions prior to contractions with prostaglandin F 2α. 3. 3. Nitric oxide induced transient relaxations which were significantly depressed by ouabain but were not affected by Na +- or K +-free solutions. Thus, the inhibitory effect of ouabain on relaxations to nitric oxide is not due to inhibition of the Na +, K + pump. 4. 4. The relaxation induced by sodium nitroprusside was impaired both in the presence of ouabain and after incubation in Na +- or K +-free solution, suggesting that it may involve in part activation of Na +, K + pumping. 5. 5. Only high concentration of ouabain and Na +-free solution decreased the relaxation evoked by nitroglycerin, indicating that the mechanism of action of nitroglycerin on vascular smooth muscle does not involve the Na +-K + pump.

Bafilomycin A 1 is a potent inhibitor of urine formation by Malpighian tubules of Drosophila hydei: Is a vacuolar-type ATPase involved in ion and fluid secretion?

The effects of bafilomycin A 1, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), N-ethylmaleimide (NEM), vanadate and ouabain on urine formation by isolated larval Malpighian tubules of Drosophila hydei were studied. Of these reagents, bafilomycin was the most effective inhibitor of the secretory activity of the tubules. When added to the bathing saline, the secretory rates of fluid, K + and Na + were rapidly decreased in a dose-dependent manner, at 1 μM bafilomycin to about 40–50%. NBD-Cl and NEM at concentrations of 10 and 50 times higher than bafilomycin, respectively, showed inhibitory effects comparable to bafilomycin, but their time course of inhibition was characterized by a progressive decline of secretion rates. When vanadate was added, the fluid secretion persisted for about 15 min and then decreased dose-dependently, at 1 mM vanadate to about 34%. In contrast to bafilomycin, which enhanced the Na + concentration of the secreted fluid slightly to a relatively stable value (at 1 μM to about 120%), both vanadate (1 mM) and ouabain (0.1 mM) increased the Na + concentration steadily to about 200 and 250%, respectively. Contrary to vanadate, the inhibitory effect of ouabain on fluid secretion within the experimental time (45 min) was relatively small. Considering the different effects of bafilomycin and vanadate on fluid and ion secretion a new working hypothesis for a transport model is proposed.

Cytochrome P450-Dependent Arachidonic Acid Metabolites, 19− and 20-Hydroxyeicosatetraenoic Acids, Enhance Sodium-Potassium ATPase Activity in Vascular Smooth Muscle

Several cytochrome P450-dependent arachidonic acid metabolites have been shown to affect Na+,K(+)-ATPase activity. In the present study, we tested the effect of omega- and omega - 1-hydroxylated products, i.e., 19- and 20-hydroxyeicosatetraenoic acids (19- and 20-HETE), on the K-induced relaxation in rat aortic rings. 19-HETE and 20-HETE increased the magnitude of the potassium-induced relaxation in a dose-dependent fashion (10(-7)-10(-5) M). The inhibitory effect of ouabain on the potassium-induced relaxation was reversed by both 19- and 20-HETE. In addition, indomethacin fully inhibited the stimulatory effect of 19- and 20-HETE on relaxation induced by potassium. Vascular ouabain-sensitive 86Rb uptake was also increased by 19- and 20-HETE. These observations suggest that 19- and 20-HETE stimulate vascular Na+,K(+)-ATPase via their conversion by cyclooxygenase to prostaglandin-like material.

Concentrative uptake of digoxin by slices of chicken renal cortex

The purpose of this investigation was to define, under controlled in vitro conditions, the processes contributing to the uptake and accumulation of [ 3H]digoxin by incubated slices of chicken renal cortex. Progressive uptake was evident in time-course experiments with the slice-to-medium concentration ratio (S/M) reaching 5.25 after 120 min. No metabolism was evident. Increasing the ratio of unlabeled to labeled digoxin resulted in a concentration-dependent decrease in relative uptake of the label, suggesting saturability. Incubation under conditions of metabolic inhibition reduced digoxin S/M by about 50%, indicating that both energy-requiring and passive mechanisms contribute to the overall accumulation process. The structural nature of the uptake process was explored by incubating digoxin in the presence of potential inhibitors of transport. The organic cation quinine and the non-glycosidic steroids digoxigenin and spironolactone were without effect even at greater than 1000-fold excess compared to digoxin. Similarly, the sugar digitoxose had no inhibitory activity on digoxin accumulation by the slices. On the other hand, the glycosides digitoxin, digoxigenin-bis-digitoxoside and digoxigenin-mono-digitoxoside inhibited dogoxin uptake in a concentration-dependent manner. These results indicate a structural preference for an intact glycoside rather than for either the steroidal or sugar portion of the molecule alone. An inhibitory effect of ouabain and a stimulatory effect of reduced medium potassium concentration suggest a possible role for Na +, K +-ATPase in the uptake of digoxin by the renal cortex.

The protective action of R56865 against ouabain-induced intoxication in rat heart isolated atria and ventricles

The action of R56865 has been examined on the contractile effects produced by ouabain concentrations interacting with high (3 μM) and low (300 μM) affinity digitalis receptors on electrically stimulated ventricular strips isolated from rat. R56865 1 μM reduced the increase in resting tension produced by ouabain 300 μM and left unalterated the inotropic effect evoked by ouabain 3 and 300 μM was reduced by higher concentrations (3 and 6 μM) of R56865. The action of R56865 was also studied on ouabain-induced intoxication in electrically stimulated and spontaneously beating atria of rat. On electrically stimulated (3 Hz) whole atria, R56865 0.3 μM reduced the maximal increase in resting tension produced by ouabain 300 μM and delayed the time to onset of the ouabain-induced arrhythmias but did not affect ouabain's inotropic effect. Higher concentrations of R56865 were required to reduce the inotropic effect of ouabain. The protective action of R56865 against ouabain-induced intoxication was most pronounced on spontaneously beating atria where it reduced spontaneous rate of beats. Experiments in electrically driven left atria indicated that only a part of the protective effect of R56865 could be related to its bradycardic action. The effect of R56865 was also examined on ouabain-induced inhibition of sodium pump in human red blood cells. R56865 μM did not modify the inhibition produced by ouabain (from 0.3 to 10 nM), this indicates that the protective action of R56865 against ouabain-induced intoxication is not due to an interaction with the inhibitory effect of ouabain on sodium pump.

Effect of Ouabain on the Responses to Vasoconstrictor Agents in Isolated Perfused Rat Tail Arteries

In isolated rat tail arteries perfused with Krebs-Henseleit solution, ouabain, in concentrations of less than 10(-5) M, caused a concentration-dependent, noncompetitive inhibition of the vasoconstrictor response to phenylephrine. Concentrations of 10(-8) and 10(-6) M caused 30 and 61% inhibition, respectively. The inhibition was gradual in onset, with a maximal effect at 45 min. In concentrations greater than 10(-5) M, ouabain caused a parallel shift to the left of the phenylephrine dose-response curve, indicating potentiation. The potentiation was rapid in onset (within 1 min), and was approximately 1.4-fold. The cutoff point between inhibition and potentiation of vasoconstrictors occurred at a ouabain concentration of 10(-5) M. Ouabain-induced potentiation was nonspecific in regard to the vasoconstrictor used. Phenylephrine, clonidine, and serotonin were each potentiated. Potentiation probably resulted from a reduction in the smooth muscle membrane potential as a consequence of inhibition of Na-K ATPase. The mechanism of the inhibitory effect of ouabain on vasoconstrictors was not resolved but may operate via stimulation of Na-K ATPase. The results are consistent with a "partial agonist" action of ouabain on Na-K ATPase.

Effects of Na +, K +-pump inhibitors and membrane depolarizing agents on acetylcholine-induced endothelium-dependent relaxation and cyclic GMP accumulation in rat aorta

The purpose of this study was to investigate the effects of inhibitors of the Na +,K +-pump and membrane depolarizing agents on endothelium-dependent relaxation and elevated cyclic GMP levels induced by acetylcholine in rat thoracic aorta. Ouabain or exposure to K +-free or Mg 2+-free Krebs-Ringer bicarbonate solution, agents and procedures known to inhibit the Na +,K +-pump, inhibited acetylcholine-induced relaxation and the associated increased levels of cyclic GMP. However, the inhibitory effect of ouabain on cyclic GMP levels was abolished in the absence of norepinephrine or in the presence of norepinephrine and the α-adrenergic receptor antagonist phentolamine. The membrane depolarizing agents KCl and tetraethylammonium also inhibited the acetylcholine-induced relaxation and the elevated cyclic GMP levels. Exposure to norepinephrine reduced the increased levels of cyclic GMP due to acetylcholine as compared to rested controls. This effect was inhibited by prior exposure to phentolamine, but not by the β-adrenergic receptor antagonist, propranolol. These results suggest that (1) increased activity of the Na +,K +-pump may mediate, in part, endothelium-dependent relaxation; (2) inhibition of relaxation may be due to membrane depolarization; (3) the endothelium-dependent increased levels of cyclic GMP may increase Na +,K +-pump activity; (4) a complex interaction exists between membrane polarization, the Na +,K +-pump and α-adrenergic stimulation in regulation of cyclic GMP accumulation and relaxation.

Species Differences in the Inhibitory Effect of Ouabain on High K-Induced Contraction in the Ileal Longitudinal Muscle

Effects of ouabain on a high K-induced contraction and intracellular Na and K contents of the ileal longitudinal muscle preparations isolated from various animal species was compared. In muscles of various animals (monkey, dog, cat, rabbit, guinea-pig, rat and mouse), the high K-induced contraction was inhibited by verapamil (5 X 10(-8) M) or ouabain (3 X 10(-6) M), and both the inhibitions were antagonized by an increase in external Ca in a competitive manner. Species differences were shown in the ouabain effect but not in the verapamil one. Regarding the sensitivity to ouabain, the muscles were divided into two groups, that is, the muscle from monkey, rabbit, guinea-pig or dog belongs to the high sensitivity group, and that from cat, rat or mouse belongs to the low one. The order of sensitivities of the muscles to ouabain in the relaxation was consistent with that in Na,K-ATPase inhibition, as reported by Repke et al. (1965). Intracellular Na contents of muscles were increased by an addition of ouabain to the high K solution, and the rate and amount of the accumulation of intracellular Na varied in these muscles. Except for the cat muscle, a high correlation was noted between the ouabain-induced relaxation and the amount of intracellular Na accumulation. However, the regression coefficients were variable: 4.1 in the monkey muscle, 2.2 in the rat one, and about 1.0 in the others. That is, the monkey muscle showing the high regression coefficient value probably has a high sensitivity to the inhibition of intracellular Na in the high K-induced contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence against an involvement of Na+, K+-ATPase in antiarrhythmic mechanism of phenytoin.

Phenytoin (Diphenylhydantoin, DPH) did not activate Na+,K+-ATPase activity prepared from both canine cardiac and renal tissues at any ratio of NA+ to K+ in standard assay medium and this drug failed to relieve the inhibitory effect of ouabain on Na+,K+-ATPase. With the Na+,K+-ATPase partially purified from the cardiac tissue the maximum number of ouabain binding sites was 50 pmol ouabain per mg enzyme and the dissociation constant (Kd) was 4 X 10(-8) mol/l. Scatchard analysis of ouabain binding to Na+,K+-ATPase indicates that DPH did not significantly alter these parameters. The release of ouabain from Na+,K+-ATPase and ouabain complex was also not significantly influenced by DPH which indicates that the antiarrhythmic action of DPH against digitalis-induced arrhythmia is not due to a simple displacement of ouabain from Na+,K+-ATPase molecules.

The role of the cellular sodium pump in human granulopoiesis in vitro

The specific sodium pump antagonist ouabain was used to study the effect of sodium pump inhibition on granulopoietic colony formation by normal human bone marrow cells cultured in soft agar for 7 d. Suppression of colony formation was dose-dependent and occurred at a low and reproducible concentration. The use of an extended range of concentrations (1 X 10(-19) M to 1 X 10(-4) M) revealed no additional effect. Inhibition occurred when colony formation was stimulated by either normal human white blood cells in a soft agar underlayer, or the inclusion of pre-formed colony-stimulating activity. Sensitivity to inhibition was similar under either set of conditions, the 50% inhibitory concentrations being 2.70 +/- 0.43 (SE) X 10(-8) M and 2.83 +/- 0.21 (SE) X 10(-8) M respectively. This is interpreted as showing an effect primarily on the colony-forming cells rather than on the cellular production of colony-stimulating activity. Inhibition of colony formation by ouabain was opposed by the addition of extra potassium to the culture medium, confirming that the inhibition was mediated via a perturbation of monovalent cation exchange. Inhibition by 2 X 10(-7) M ouabain appeared to be reversible by simple washing after exposure for up to 24 h, suggesting that the inhibitory effect of ouabain is not primarily cytotoxic.