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Journal of Nuclear Medicine : Official... Sep 1989Effects of hypoxia and ouabain on transcapillary exchange of [99mTc]hexakis (2-methoxyisobutylisonitrile) [SESTAMIBI, also known as MIBI or HEXAMIBI] and 201TI were... (Comparative Study)
Comparative Study
Effects of hypoxia and ouabain on transcapillary exchange of [99mTc]hexakis (2-methoxyisobutylisonitrile) [SESTAMIBI, also known as MIBI or HEXAMIBI] and 201TI were investigated with indicator-dilution studies using isolated rabbit hearts. Peak myocardial extraction (Emax), permeability-surface area products (PScap), and net myocardial extraction (Enet) were compared among serial injections during constant coronary flows. Overall, measures of transcapillary transport (Emax and PScap) for SESTAMIBI were significantly lower (p less than 0.001) than those simultaneously determined for thallium, but estimates of tissue retention (Enet) for SESTAMIBI and thallium were not statistically distinguishable. Hypoxia had no significant effect on mean (+/- s.d.) Emax for SESTAMIBI (0.31 +/- 0.13) or thallium (0.59 +/- 0.11), nor on mean PScap or Enet values. Ouabain (1.5 X 10(-7) M and 1.5 X 10(-6) M) had no effect on SESTAMIBI or thallium Emax (respectively, 0.29 +/- 0.08 and 0.60 +/- 0.05) or on PScap for SESTAMIBI. Thallium PScap was depressed with higher ouabain dose (control, 1.22 +/- 0.40; high ouabain, 1.06 +/- 0.41 ml/min/g; p less than 0.01). Ouabain also caused a significant and progressive increase in average SESTAMIBI Enet (control, 0.23 +/- 0.10 to high ouabain, 0.33 +/- 0.12; p less than 0.05), but depressed thallium Enet (control, 0.38 +/- 0.14 to high ouabain, 0.32 +/- 0.18; p less than 0.01). These results suggest myocardial metabolic and/or functional status have minor influence on transcapillary transport of SESTAMIBI and thallium, but significantly affects cellular retention.
Topics: Animals; Heart; Hypoxia; In Vitro Techniques; Myocardium; Organometallic Compounds; Ouabain; Rabbits; Radionuclide Imaging; Technetium; Technetium Tc 99m Sestamibi; Thallium Radioisotopes
PubMed: 2527973
DOI: No ID Found -
FEBS Letters Sep 2015Ion pump, Na,K-ATPase specifically binds cardiotonic steroids (CTS), which leads to inhibition of the enzyme activity and activation of signaling network in the cell. We...
Ion pump, Na,K-ATPase specifically binds cardiotonic steroids (CTS), which leads to inhibition of the enzyme activity and activation of signaling network in the cell. We have studied interaction of Na,K-ATPase with CTS of two different types - marinobufagenin and ouabain. We have shown that both CTS inhibit activity of Na,K-ATPase with the same Ki values, but binding of ouabain is sensitive to the conformation of Na,K-ATPase while binding of marinobufagenin is not. Furthermore, binding of ouabain and marinobufagenin results in different structural changes in Na,K-ATPase. Our data allow to explain the diversity of effects on the receptor function of Na,K-ATPase caused by different types of CTS.
Topics: Bufanolides; Enzyme Inhibitors; Hydrolysis; Models, Molecular; Ouabain; Protein Binding; Protein Structure, Tertiary; Sodium-Potassium-Exchanging ATPase; Thermodynamics
PubMed: 26297827
DOI: 10.1016/j.febslet.2015.08.011 -
Proceedings of the National Academy of... Jul 1991The plasma membrane sodium-potassium pumps that regulate intracellular sodium in most animal cells have specific, high-affinity receptors for the digitalis glycosides...
The plasma membrane sodium-potassium pumps that regulate intracellular sodium in most animal cells have specific, high-affinity receptors for the digitalis glycosides and their aglycones. This has fostered speculation that there is an endogenous ligand. We have purified and structurally identified by mass spectroscopy an endogenous substance from human plasma that binds with high affinity to this receptor and that is indistinguishable from the cardenolide ouabain. This human ouabain-like compound (OLC) displaces [3H]ouabain from its receptor, inhibits Na,K-ATPase and ouabain-sensitive 86Rb+ uptake, and has cardiotonic actions quantitatively similar to commercial ouabain. Immunoreactive OLC was detected in the plasma of many mammals, and high concentrations were found in the adrenals. The circulating OLC may modulate intracellular Na+ and affect numerous Na+ gradient-dependent processes including intracellular Ca2+ and pH homeostasis in many tissues. Furthermore, altered circulating levels of OLC may be associated with the pathogenesis of certain forms of hypertension.
Topics: Adrenal Glands; Animals; Cattle; Cells, Cultured; Dogs; Erythrocytes; Humans; Kidney; Mass Spectrometry; Ouabain; Rats; Rubidium; Sodium-Potassium-Exchanging ATPase
PubMed: 1648735
DOI: 10.1073/pnas.88.14.6259 -
Journal of Medicinal Chemistry Mar 2018Na,K-ATPase α4 is a testis-specific plasma membrane Na and K transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete...
Na,K-ATPase α4 is a testis-specific plasma membrane Na and K transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase α4 is characterized by a high affinity for the cardiac glycoside ouabain. With the goal of discovering selective inhibitors of the Na,K-ATPase α4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue 25, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding α4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase α4 and sperm function.
Topics: Animals; Contraception; Drug Design; Enzyme Inhibitors; Humans; Isoenzymes; Male; Mice; Ouabain; Sodium-Potassium-Exchanging ATPase; Sperm Motility; Spermatozoa; Structure-Activity Relationship; Testis
PubMed: 29291372
DOI: 10.1021/acs.jmedchem.7b00925 -
PloS One 2023Biliary tract cancer is a deadly disease with limited therapeutic options. Ouabain is a well-known inhibitor of the pumping function of Na+/K+-ATPase, though there is...
Biliary tract cancer is a deadly disease with limited therapeutic options. Ouabain is a well-known inhibitor of the pumping function of Na+/K+-ATPase, though there is evidence that low concentrations of ouabain lead to a reduction of cell viability of cancer cells independent of its inhibition of the pumping function of the Na+/K+-ATPase. Regarding the impact of ouabain on biliary tract cancer, no data is currently available. Therefore, we aimed for a first-time investigation of ouabain as a potential anti-neoplastic biliary tract cancer agent using comprehensive human biliary tract cancer in vitro models. We found that ouabain has a strong cell line-dependent cytotoxic effect with IC50 levels in the (low) nanomolar-range and that this effect was not associated with the mRNA expression levels of the Na+/K+-ATPase α, β and fxyd-subunits. Regarding the mode of cytotoxicity, we observed induction of apoptosis in biliary tract cancer cells upon treatment with ouabain. Interestingly, cytotoxic effects of ouabain at sub-saturating (< μM) levels were independent of cellular membrane depolarization and changes in intracellular sodium levels. Furthermore, using a 3D cell culture model, we found that ouabain disturbs spheroid growth and reduces the viability of biliary tract cancer cells within the tumor spheroids. In summary, our data suggest that ouabain possesses anti-biliary tract cancer potential at low μM-concentration in 2D and 3D in vitro biliary tract cancer models and encourage further detailed investigation.
Topics: Humans; Ouabain; Biliary Tract Neoplasms; Antineoplastic Agents; Apoptosis; Sodium-Potassium-Exchanging ATPase
PubMed: 37390071
DOI: 10.1371/journal.pone.0287769 -
Hypertension Research : Official... Nov 2001Ouabain has recently been identified as an endogenous Na+-K+ pump inhibitor having a close association with hypertension. However, some patients with hypertention do not...
Ouabain has recently been identified as an endogenous Na+-K+ pump inhibitor having a close association with hypertension. However, some patients with hypertention do not show high levels of endogenous ouabain (EO), and patients with high EO levels do not necessarily suffer from hypertention. It is believed that the Na+-K+-ATPase activity in essential hypertension does not undergo homogenous change. The present study was designed, therefore, to investigate the expression and the significance of the Na+-K+-ATPase alpha-subunit isoforms in kidney tissue in ouabain-hypertensive rats. Ouabain was administered chronically to establish a model of ouabain-hypertensive rats. Biochemical analysis, cytobiology and sABC immunohistochemistry were they used to assay for expression of Na+-K+-ATPase alpha-subunit isoforms in kidney tissue. After the first week of receiving ouabain, 65% (n=13) of rats had hypertension. After the second week, the blood pressure of these 13 hypertensive rats was increased significantly compared to the baseline and control levels (p<0.05). The plasma renin activity was normal, and angiotensin II and aldosterone levels were increased significantly in these rats (p<0.05). But in the other 35% (n=7) of rats of the experimental group, there was no apparent increase in blood pressure after receiving ouabain. The plasma ouabain level in the non-hypertensive subgroup was significantly higher than that in the hypertensive subgroup, but the 86Rb intake and the number of 3H-ouabain binding sites did not decrease. The Na+-K+-ATPase activity showed non-homogeneous changes. In hypertensive rats, the expression levels of ouabain paralleled the degree of hypertension (r=0.88, p<0.05). The positive granules were mainly scattered in the cytoblastoma of the reticular zone of adrenal cortex. There were thus different levels of expression of Na+-K+-ATPase alpha-subunit isoforms in this model. In the hypertension subgroup the alpha1 was most strongly expressed, followed by the alpha2 and alpha3 isforms. But in the non-hypertensive subgroup the order was alpha3 > alpha2 > alpha1. The positive granular was mainly scattered in the convoluted tubules of the kidney. These results suggest that the high level of ouabain and the change of the Na+-K+-ATPase alpha-subunit isoforms may play a critical role in hypertension.
Topics: Adrenal Glands; Animals; Binding Sites; Blood Pressure; Electrolytes; Enzyme Inhibitors; Erythrocytes; Hypertension; Male; Ouabain; Rats; Rats, Sprague-Dawley; Reference Values; Sodium-Potassium-Exchanging ATPase
PubMed: 11768735
DOI: 10.1291/hypres.24.729 -
Cells Aug 2023Na/K-ATPase maintains transmembrane ionic gradients and acts as a signal transducer when bound to endogenous cardiotonic steroids. At subnanomolar concentrations,...
Na/K-ATPase maintains transmembrane ionic gradients and acts as a signal transducer when bound to endogenous cardiotonic steroids. At subnanomolar concentrations, ouabain induces neuroprotection against calcium overload and apoptosis of neurons during excitotoxic stress. Here, the role of lipid rafts in interactions between Na/K-ATPase, sodium-calcium exchanger (NCX), and N-methy-D-aspartate receptors (NMDARs) was investigated. We analyzed 0.5-1-nanometer ouabain's effects on calcium responses and miniature post-synaptic current (mEPSCs) frequencies of cortical neurons during the action of NMDA in rat primary culture and brain slices. In both objects, ouabain attenuated NMDA-evoked calcium responses and prevented an increase in mEPSC frequency, while the cholesterol extraction by methyl-β-cyclodextrin prevented the effects. The data support the conclusions that (i) ouabain-induced inhibition of NMDA-elicited calcium response involves both pre- and post-synapse, (ii) the presence of astrocytes in the tripartite synapse is not critical for the ouabain effects, which are found to be similar in cell cultures and brain slices, and (iii) ouabain action requires the integrity of cholesterol-rich membrane microdomains in which the colocalization and functional interaction of NMDAR-transferred calcium influx, calcium extrusion by NCX, and Na/K-ATPase modulation of the exchanger occur. This regulation of the molecules by cardiotonic steroids may influence synaptic transmission, prevent excitotoxic neuronal death, and interfere with the pharmacological actions of neurological medicines.
Topics: Rats; Animals; Ouabain; Calcium; N-Methylaspartate; Neurons; Cholesterol; Adenosine Triphosphatases
PubMed: 37566090
DOI: 10.3390/cells12152011 -
The Journal of Physiology Oct 19891. We have examined whether the apparently ouabain-resistant fraction of cellular volume regulation in liver slices under isosmotic conditions is due to a failure of...
1. We have examined whether the apparently ouabain-resistant fraction of cellular volume regulation in liver slices under isosmotic conditions is due to a failure of ouabain to cause complete inhibition of the coupled transport of Na+ and K+. The ion and water contents of rat and rabbit liver slices were altered by pre-incubation at 1 degree C and then allowed to recover at 38 degrees C, with or without ouabain or other inhibitors. The net movements of ions and water were determined during the recovery. The influx of 86Rb under steady-state conditions was taken as a measure of unidirectional influx of K+. 2. Concentrations of ouabain for half-maximal inhibition of 86Rb influx were 0.15 mM for rat and 0.15 microM for rabbit liver slices, with maximal inhibition at 2 mM and 10 microM respectively. Inhibition of net K+ reaccumulation closely followed inhibition of 86Rb influx. 3. The 86Rb influx persisting in the presence of maximally inhibiting concentrations of ouabain was not reduced by inhibitors of cellular respiration or glycolysis. 4. In rat liver slices, about 50% of net water extrusion was resistant to 2 mM-ouabain; rabbit liver showed a much smaller, but statistically significant, extrusion of water in the presence of 10 microM-ouabain. 5. In rat liver slices, a small, net uptake of K+ continued in the presence of amytal alone, when water extrusion was completely inhibited; by contrast, ouabain gave complete inhibition of K+ uptake while permitting 50% of the control water extrusion. 6. Isolated rat hepatocytes in primary culture were pre-incubated at 4 degrees C for 20 h. They recovered their original K+ content within 60 min of restoration to 37 degrees C. Ouabain, 1-2 mM, completely prevented this recovery. 7. The results imply that ouabain completely inhibits the coupled transport of Na+ and K+ in both rat and rabbit liver slices. Thus, the fraction of total water extrusion continuing in the presence of maximally inhibiting concentrations of ouabain is the consequence of a truly ouabain-resistant mechanism.
Topics: Animals; In Vitro Techniques; Liver; Male; Ouabain; Potassium; Rabbits; Rats; Rats, Inbred Strains
PubMed: 2621601
DOI: 10.1113/jphysiol.1989.sp017808 -
FASEB Journal : Official Publication of... Oct 2014Up-regulation of placental soluble fms-like tyrosine kinase 1 (sFlt1) contributes to the pathogenesis of preeclampsia. To evaluate novel upstream pathways that regulate...
Up-regulation of placental soluble fms-like tyrosine kinase 1 (sFlt1) contributes to the pathogenesis of preeclampsia. To evaluate novel upstream pathways that regulate placental sFlt1 production, we screened a library of natural compounds (n=502) in human placental cell lines. Here, we report 3 compounds in the cardiac glycoside family, ouabain, gitoxigenin, and digitoxin, that inhibit placental sFlt1 production at nanomolar concentrations in vitro. We further characterized ouabain and demonstrated that it inhibits sFlt1 mRNA and protein expression in human placental cytotrophoblasts and explant cultures in a dose- and time-dependent manner. Ouabain down-regulated sFlt1 production by inhibiting hypoxia-inducible factor 1 (HIF-1α) protein expression in the placenta. Furthermore, we found that phosphorylation of heat-shock protein 27 (HSP27) was necessary for ouabain to inhibit HIF-1α translation. In a rat model of pregnancy-induced hypertension, ouabain reduced mean arterial pressure and enhanced placental HSP27 phosphorylation without any adverse effects on pups. Further studies are needed to explore the usefulness of targeting HIF-1α/HSP27 pathway in preeclampsia.
Topics: Animals; Blood Pressure; Cardenolides; Cardiotonic Agents; Cells, Cultured; Digitoxin; Dose-Response Relationship, Drug; Female; HSP27 Heat-Shock Proteins; Humans; Hypertension, Pregnancy-Induced; Hypoxia-Inducible Factor 1, alpha Subunit; Ouabain; Phosphorylation; Placenta; Pregnancy; RNA, Messenger; Rats; Rats, Sprague-Dawley; Vascular Endothelial Growth Factor Receptor-1
PubMed: 24970393
DOI: 10.1096/fj.14-252684 -
Molecular Genetics and Genomics : MGG May 2023Ouabain is a cardiac glycoside long studied for treating heart diseases, but the attempts to evaluate its anti-psoriatic activity have not been reported. We aimed to...
Ouabain is a cardiac glycoside long studied for treating heart diseases, but the attempts to evaluate its anti-psoriatic activity have not been reported. We aimed to explore the effects of ouabain on proliferation and metabolism towards psoriatic keratinocytes. In human HaCaT keratinocytes, ouabain potently decreased viability, promoted apoptosis and caused G2/M cycle arrest. Metabolomics analysis indicated that ouabain markedly impaired glutathione metabolism. The solute carrier family 7 member 11 (SLC7A11) is an amino acid transporter highly specific to cysteine, which is critical for glutathione synthesis. Ouabain downregulated SLC7A11, reduced cysteine uptake and subsequently inhibited glutathione synthesis, probably through inhibiting Akt/mTOR/beclin axis that regulate protein activity of SLC7A11. The impaired glutathione synthesis and oxidative stress caused by ouabain may contribute to its cytotoxicity towards psoriatic keratinocytes. Our results provide experimental evidence supporting further study of ouabain as a potential anti-psoriatic agent.
Topics: Humans; Ouabain; Cysteine; Keratinocytes; Antineoplastic Agents; Apoptosis; Glutathione; Psoriasis; Cell Proliferation
PubMed: 36856826
DOI: 10.1007/s00438-023-02001-9