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1
Fu, Yong, Dalian Ding, Lei Wei, Haiyan Jiang, and Richard Salvi. "Ouabain-Induced Apoptosis in Cochlear Hair Cells and Spiral Ganglion NeuronsIn Vitro." BioMed Research International 2013 (2013): 1–16. http://dx.doi.org/10.1155/2013/628064.
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Ouabain is a common tool to explore the pathophysiological changes in adult mammalian cochleain vivo. In prior studies, locally administering ouabain via round window membrane demonstrated that the ototoxic effects of ouabainin vivovaried among mammalian species. Little is known about the ototoxic effectsin vitro. Thus, we prepared cochlear organotypic cultures from postnatal day-3 rats and treated these cultures with ouabain at 50, 500, and 1000 μM for different time to elucidate the ototoxic effects of ouabainin vitroand to provide insights that could explain the comparative ototoxic effects of ouabainin vivo. Degeneration of cochlear hair cells and spiral ganglion neurons was evaluated by hair-cell staining and neurofilament labeling, respectively. Annexin V staining was used to detect apoptotic cells. A quantitative RT-PCR apoptosis-focused gene array determined changes in apoptosis-related genes. The results showed that ouabain-induced damagein vitrowas dose and time dependent. 500 μM ouabain and 1000 μM ouabain were destructively traumatic to both spiral ganglion neurons and cochlear hair cells in an apoptotic signal-dependent pathway. The major apoptotic pathways in ouabain-induced spiral ganglion neuron apoptosis culminated in the stimulation of the p53 pathway and triggering of apoptosis by a network of proapoptotic signaling pathways.
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Curras, M. C., and J. A. Boulant. "Effects of ouabain on neuronal thermosensitivity in hypothalamic tissue slices." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 257, no. 1 (July 1, 1989): R21—R28. http://dx.doi.org/10.1152/ajpregu.1989.257.1.r21.
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To determine the role of the electrogenic Na+-K+ pump in neuronal thermosensitivity, single-unit activity was recorded in rat hypothalamic tissue slices before, during, and after perfusions containing 10(-5) or 10(-6) M ouabain, a specific pump inhibitor. Most neurons were recorded in the preoptic-anterior hypothalamus. Some neurons were also tested with high magnesium-low calcium perfusions to determine ouabain's effects on neuronal activity during synaptic blockade. When the neurons were characterized according to thermosensitivity, 24% were warm sensitive, 8% were cold sensitive, and 68% were temperature insensitive. Ouabain increased the firing rate of 60% of all neurons. Ouabain did not reduce the thermosensitivity of cold-sensitive and warm-sensitive neurons; however, temperature-insensitive neurons became more warm sensitive during ouabain perfusion. This increase in warm sensitivity did not occur with ouabain plus high Mg2+-low Ca2+ perfusion, suggesting that Ca2+ is important in this response. These results indicate that the Na-K pump is not responsible for the thermosensitivity of hypothalamic cold-sensitive or warm-sensitive neurons; however, this pump may be actively employed by many neurons that remain insensitive to temperature changes.
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Blanco, Gustavo, and Darren P. Wallace. "Novel role of ouabain as a cystogenic factor in autosomal dominant polycystic kidney disease." American Journal of Physiology-Renal Physiology 305, no. 6 (September 15, 2013): F797—F812. http://dx.doi.org/10.1152/ajprenal.00248.2013.
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The classic role of the Na-K-ATPase is that of a primary active transporter that utilizes cell energy to establish and maintain transmembrane Na+ and K+ gradients to preserve cell osmotic stability, support cell excitability, and drive secondary active transport. Recent studies have revealed that Na-K-ATPase located within cholesterol-containing lipid rafts serves as a receptor for cardiotonic steroids, including ouabain. Traditionally, ouabain was viewed as a toxin produced only in plants, and it was used in relatively high concentrations to experimentally block the pumping action of the Na-K-ATPase. However, the new and unexpected role of the Na-K-ATPase as a signal transducer revealed a novel facet for ouabain in the regulation of a myriad of cell functions, including cell proliferation, hypertrophy, apoptosis, mobility, and metabolism. The seminal discovery that ouabain is endogenously produced in mammals and circulates in plasma has fueled the interest in this endogenous molecule as a potentially important hormone in normal physiology and disease. In this article, we review the role of the Na-K-ATPase as an ion transporter in the kidney, the experimental evidence for ouabain as a circulating hormone, the function of the Na-K-ATPase as a signal transducer that mediates ouabain's effects, and novel results for ouabain-induced Na-K-ATPase signaling in cystogenesis of autosomal dominant polycystic kidney disease.
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McLaughlin, Charles W., Sylvia Zellhuber-McMillan, Anthony D. C. Macknight, and Mortimer M. Civan. "Electron microprobe analysis of rabbit ciliary epithelium indicates enhanced secretion posteriorly and enhanced absorption anteriorly." American Journal of Physiology-Cell Physiology 293, no. 5 (November 2007): C1455—C1466. http://dx.doi.org/10.1152/ajpcell.00205.2007.
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The rate of aqueous humor formation sequentially across the pigmented (PE) and nonpigmented (NPE) ciliary epithelial cell layers may not be uniform over the epithelial surface. Because of the tissue's small size and complex geometry, this possibility cannot be readily tested by conventional techniques. Rabbit iris-ciliary bodies were divided, incubated, quick-frozen, cryosectioned, and freeze-dried for electron probe X-ray microanalysis of the elemental contents of the PE and NPE cells. We confirmed that preincubation with ouabain to block Na+,K+-ATPase increases Na+ and decreases K+ contents far more anteriorly than posteriorly. The anterior and posterior regions were the iridial portion of the primary ciliary processes and the pars plicata, respectively. Following interruption of gap junctions with heptanol, ouabain produced smaller changes in anterior PE cells, possibly reflecting higher Na+ or K+ permeability of anterior NPE cells. Inhibiting Na+ entry selectively with amiloride, benzamil, or dimethylamiloride reduced anterior effects of ouabain by ∼50%. Regional dependence of net secretion was also assessed with hypotonic stress, which stimulates ciliary epithelial cell regulatory volume decrease (RVD) and net Cl− secretion. In contrast to ouabain's actions, the RVD was far more marked posteriorly than anteriorly. These results suggest that 1) enhanced Na+ reabsorption anteriorly, likely through Na+ channels and Na+/H+ exchange, mediates the regional dependence of ouabain's actions; and 2) secretion may proceed primarily posteriorly, with secondary processing and reabsorption anteriorly. Stimulation of anterior reabsorption might provide a novel strategy for reducing net secretion.
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Lewis, Lynley K., Timothy G. Yandle, Philip J. Hilton, Berit P. Jensen, Evan J. Begg, and M. Gary Nicholls. "Endogenous Ouabain Is Not Ouabain." Hypertension 64, no. 4 (October 2014): 680–83. http://dx.doi.org/10.1161/hypertensionaha.114.03919.
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Donaldson, S. K. "Peeled mammalian skeletal muscle fibers. Possible stimulation of Ca2+ release via a transverse tubule-sarcoplasmic reticulum mechanism." Journal of General Physiology 86, no. 4 (October 1, 1985): 501–25. http://dx.doi.org/10.1085/jgp.86.4.501.
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Single muscle fibers from rabbit soleus and adductor magnus and from semitendinosus muscles were peeled to remove the sarcolemma and then stimulated to release Ca2+ by (a) caffeine application or (b) ionic depolarization accomplished via substitution of choline chloride for potassium propionate at constant [K+] X [Cl-] in the bathing solution. Each stimulus, ionic or caffeine, elicited an isometric tension transient that appeared to be due to Ca2+ released from the sarcoplasmic reticulum (SR). The peak magnitude of the ionic (Cl- -induced) tension transient increased with increasing Cl- concentration. The application of ouabain to fibers after peeling had no effect on either type of tension transient. However, soaking the fibers in a ouabain solution before peeling blocked the Cl- -induced but not the caffeine-induced tension transient, which suggests that ouabain's site of action is extracellular, perhaps inside transverse tubules (TTs). Treating the peeled fibers with saponin, which should disrupt TTs to a greater extent than SR membrane, greatly reduced or eliminated the Cl- -induced tension transient without significantly altering the caffeine-induced tension transient. These results suggest that the Cl- -induced tension transient is elicited via stimulation of sealed, polarized TTs rather than via ionic depolarization of the SR.
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Gomez-Sanchez, Elise P., Mark F. Foecking, Deborah Sellers, Mary S. Blankenship, and Celso E. Gomez-Sanchez. "Is the Circulating Ouabain-like Compound Ouabain?" American Journal of Hypertension 7, no. 7_Pt_1 (July 1994): 637–46. http://dx.doi.org/10.1093/ajh/7.7.637.
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Gomez-Sanchez, Elise P., Mark F. Foecking, Sellers Deborah, Mary S. Blankenship, and Celso E. Gomez-Sanchez. "Is the Circulating Ouabain-like Compound Ouabain?" American Journal of Hypertension 7, no. 7_Pt_1 (July 1994): 647–50. http://dx.doi.org/10.1093/ajh/7.7.647.
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Murrell, Julie R., Jeffrey D. Randall, James Rosoff, Ji-liang Zhao, Roderick V. Jensen, Steven R. Gullans, and Garner T. Haupert. "Endogenous Ouabain." Circulation 112, no. 9 (August 30, 2005): 1301–8. http://dx.doi.org/10.1161/circulationaha.105.554071.
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Hamlyn, John M., and Mordecai P. Blaustein. "Endogenous Ouabain." Hypertension 68, no. 3 (September 2016): 526–32. http://dx.doi.org/10.1161/hypertensionaha.116.06599.
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Blaustein, Mordecai P., and John M. Hamlyn. "Ouabain, endogenous ouabain and ouabain-like factors: The Na+ pump/ouabain receptor, its linkage to NCX, and its myriad functions." Cell Calcium 86 (March 2020): 102159. http://dx.doi.org/10.1016/j.ceca.2020.102159.
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Kjeldsen, K. "Homogeneity of [3H]ouabain-binding sites in rat soleus muscle." Biochemical Journal 249, no. 2 (January 15, 1988): 481–85. http://dx.doi.org/10.1042/bj2490481.
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Homogeneity or heterogeneity of rat soleus-muscle Na,K-ATPase (Na+ + K+-dependent ATPase) with respect to affinity for [3H]ouabain was evaluated. Since the standard method for measuring specific [3H]ouabain binding to rat skeletal-muscle samples includes subtraction of a value for non-specific [3H]ouabain uptake and retention, and a wash-out in the cold to remove [3H]ouabain from the extracellular phase, it was possible that these procedures could hide a class of [3H]ouabain-binding sites either with low affinity or with a rapid dissociation of [3H]ouabain. However, measurements of [3H]ouabain uptake and retention over the range 0.1-5 mM, as well as the omission of wash-out, gave no evidence for heterogeneity of [3H]ouabain-binding sites in rat soleus muscle. Furthermore, the observation of agreement between the uptake and retention of non-specific [3H]ouabain and of [14C]sucrose gave no evidence for the existence of a major pool of [3H]ouabain-binding sites with low affinity for [3H]ouabain. Assuming homogeneity, the total concentration of [3H]ouabain binding sites in soleus-muscle samples from 12-week-old rats is 278-359 pmol/g wet wt.
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Dostanic, Iva, Richard J. Paul, John N. Lorenz, Steven Theriault, James W. Van Huysse, and Jerry B. Lingrel. "The α2-isoform of Na-K-ATPase mediates ouabain-induced hypertension in mice and increased vascular contractility in vitro." American Journal of Physiology-Heart and Circulatory Physiology 288, no. 2 (February 2005): H477—H485. http://dx.doi.org/10.1152/ajpheart.00083.2004.
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Although ouabain is known to induce hypertension, the mechanism of how this cardiac glycoside affects blood pressure is uncertain. The present study demonstrates that the α2-isoform of the Na-K-ATPase mediates the pressor effects of ouabain in mice. To accomplish this, we analyzed the effect of ouabain on blood pressure in wild-type mice, where the α2-isoform is sensitive to ouabain, and genetically engineered mice expressing a ouabain-insensitive α2-isoform of the Na-K-ATPase. Thus differences in the response to ouabain between these two genotypes can only be attributed to the α2-isoform of Na-K-ATPase. As the α1-isoform is naturally resistant to ouabain in rodents, it will not be inhibited by ouabain in either genotype. Whereas prolonged administration of ouabain increased levels of ouabain in serum from both wild-type and targeted animals, hypertension developed only in wild-type mice. In addition, bolus intravenous infusion of ouabain increased the systolic, mean arterial, and left ventricular blood pressure in only wild-type anesthetized mice. In vitro, ouabain increased vascular tone and thereby phenylephrine-induced contraction of the aorta in intact and endothelium-denuded wild-type mice but in α2-resistant mice. Ouabain also increased the magnitude of the spontaneous contractions of portal vein and the basal tone of the intact aorta from only wild-type mice. The increase in aortic basal tone was dependent on the presence of endothelium. Our studies also demonstrate that the α2-isoform of Na-K-ATPase mediates the ouabain-induced increase in vascular contractility. This could play a role in the development and maintenance of ouabain-induced hypertension.
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Anwer, M. S. "Effects of ion substitution on transport and choleretic effect of ouabain." American Journal of Physiology-Gastrointestinal and Liver Physiology 252, no. 3 (March 1, 1987): G357—G364. http://dx.doi.org/10.1152/ajpgi.1987.252.3.g357.
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The role of inorganic ions in hepatic transport and choleretic effect of ouabain was studied in isolated perfused rat liver to verify whether Na+-coupled ouabain uptake into hepatocytes is responsible for the choleretic effect. Hepatic uptake and clearance of ouabain were not significantly affected when perfusate Na+ was replaced by Li+ or choline+, chloride by nitrate or isethionate, or bicarbonate by tricine. However, these ion substitutions, with the exception of Li+, significantly reduced ouabain-induced choleresis and biliary electrolyte excretion. When ouabain was infused at different rates followed by perfusion without ouabain, changes in bile flow paralleled biliary excretion of ouabain rather than hepatic uptake. These results indicate that hepatic uptake of ouabain is not Na+ dependent and that the osmotic effect of biliary excreted ouabain is responsible for its choleretic effect. A part of the choleretic effect (30%) must also involve other mechanisms, since a permeable anion-like nitrate failed to substitute for perfusate chloride. Results of infusion studies also showed that ouabain was concentrated in liver (liver/perfusate = 30) and in bile (bile/liver = 15), indicating that ouabain is transported against its concentration gradient across both sinusoidal and canalicular membranes.
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Khundmiri, Syed J., Melissa A. Metzler, Mohamed Ameen, Vishal Amin, Madhavi J. Rane, and Nicholas A. Delamere. "Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells." American Journal of Physiology-Cell Physiology 291, no. 6 (December 2006): C1247—C1257. http://dx.doi.org/10.1152/ajpcell.00593.2005.
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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.
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Raina, Hema, Qingli Zhang, Albert Y. Rhee, Thomas L. Pallone, and W. Gil Wier. "Sympathetic nerves and the endothelium influence the vasoconstrictor effect of low concentrations of ouabain in pressurized small arteries." American Journal of Physiology-Heart and Circulatory Physiology 298, no. 6 (June 2010): H2093—H2101. http://dx.doi.org/10.1152/ajpheart.01045.2009.
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We hypothesized that in salt-dependent forms of hypertension, endogenous ouabain acts on arterial smooth muscle to cause enhanced vasoconstriction. Here, we tested for the involvement of the arterial endothelium and perivascular sympathetic nerve terminals in ouabain-induced vasoconstriction. Segments of rat mesenteric or renal interlobar arteries were pressurized to 70 mmHg at 37°C and exposed to ouabain (10−11–10−7 M). Removal of the endothelium enhanced ouabain-induced vasoconstriction by as much as twofold (at an ouabain concentration of 10−9 M). A component of the ouabain-induced vasoconstriction is due to the enhanced spontaneous release of norepinephrine (NE) from nerve terminals in the arterial wall. The α1-adrenoceptor blocker prazosin (10−6 M) decreased ouabain-induced vasoconstrictions by as much as 50%. However, neither the contraction induced by sympathetic nerve activity (SNA) nor the NE release evoked by SNA (measured directly by carbon fiber amperometry) was increased by ouabain (<10−7 M). Nevertheless, the converse case was true: after brief bursts of SNA, vasoconstrictor responses to ouabain were transiently increased (1.75-fold). This effect may be mediated by neuropeptide Y and Y1 receptors on smooth muscle. In arteries lacking the endothelium and exposed to prazosin, ouabain (10−11 M and greater) caused vasoconstriction, indicating a direct effect of very “low” concentrations of ouabain on arterial smooth muscle. In conclusion, in intact arteries, the endothelium opposes ouabain (10−11–10−7M)-induced vasoconstriction, which is caused by both enhanced spontaneous NE release and direct effects on smooth muscle. Ouabain (<10−7M) does not enhance SNA-mediated contractions, but SNA enhances ouabain-induced contractions. The effects of endogenous ouabain may be accentuated in forms of hypertension that involve sympathetic nerve hyperactivity and/or endothelial dysfunction.
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Emanuel, J. R., S. Graw, D. Housman, and R. Levenson. "Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity." Molecular and Cellular Biology 9, no. 9 (September 1989): 3744–49. http://dx.doi.org/10.1128/mcb.9.9.3744.
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To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.
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Emanuel, J. R., S. Graw, D. Housman, and R. Levenson. "Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity." Molecular and Cellular Biology 9, no. 9 (September 1989): 3744–49. http://dx.doi.org/10.1128/mcb.9.9.3744-3749.1989.
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To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.
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Bauer, N., G. Scheiner-Bobis, and W. Schoner. "„Endogenes Digitalis” – der lange Weg vom herzwirksamen pflanzlichen Toxin zum Hormon der Säuger." Tierärztliche Praxis Ausgabe K: Kleintiere / Heimtiere 34, no. 06 (2006): 389–97. http://dx.doi.org/10.1055/s-0037-1622553.
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ZusammenfassungEndogene Herzglykoside wurden kürzlich aus Blut, Urin, Nebennieren und Hypothalamus von Säugetieren isoliert und in ihrer Struktur aufgeklärt. Zu den endogenen Herzglykosiden zählen so gut bekannte Hemmstoffe der Natriumpumpe wie Ouabain (g-Strophanthin), Digoxin und Marinobufagenin. Endogenes Ouabain und Digoxin werden in der Nebennierenrinde der Säuger aus Progesteron und Pregnenolon synthetisiert. Ouabain wird bei Kreislaufbelastung rasch freigesetzt, seine Konzentration fällt bei Ruhe innerhalb weniger Minuten wieder ab. ACEInhibitoren und β-Blocker verhindern bei Hunden diesen Anstieg. Ouabain wird durch ACTH, Adrenalin und Angiotensin II aus Nebennierenrindenzellen in Kultur freigesetzt. Nanomolare Ouabain-Konzentrationen stimulieren die Proliferation von glatten Muskelzellen. An schwerer dilatativer Kardiomyopathie erkrankte Hunde haben im Vergleich zu gesunden Hunden signifikant erniedrigte Ouabain-Blutwerte. Beim Menschen und Ratten führt eine lang dauernde zu hohe NaCl-Zufuhr über die Nahrung zum Konzentrationsanstieg von endogenem Ouabain im Blut und zum Bluthochdruck. Eine über lange Zeit durchgeführte Infusion von Ouabain, aber nicht von Digoxin, erzeugt bei Ratten Bluthochdruck. Digoxin senkt den Ouabain-induzierten Bluthochdruck. Da bei ca. 50% der Hochdruckpatienten erhöhte Ouabain-Werte vorliegen, ist es von großer medizinischer Bedeutung, dass mit dem Ouabain-Antagonisten Rostafuroxin ein neues Prinzip und eine neue Gruppe von Blutdrucksenkern gefunden wurde. Marinobufagenin, dessen Konzentration bei Herzinfarkt akut ansteigt, hat auf die Niere eine natriuretische Wirkung. Im Gehirn wird Ouabain im Hypothalamus synthetisiert und bei einer erhöhten intrazellulären Natriumkonzentration freigesetzt.
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Vakkuri, Olli, Sighvatur S. Arnason, Päivi Joensuu, Jorma Jalonen, Olli Vuolteenaho, and Juhani Leppäluoto. "Radioiodinated Tyrosyl-Ouabain and Measurement of a Circulating Ouabain-like Compound." Clinical Chemistry 47, no. 1 (January 1, 2001): 95–101. http://dx.doi.org/10.1093/clinchem/47.1.95.
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Abstract Background: Assays for endogenous ouabain, a cardiac glycoside believed to be involved in blood pressure and volume regulation, are characterized by laboratory-specific plasma values that are measured by different assays. Because of this variability, our study focused on the development of a new 125I-labeled ouabain derivative for RIA of high sensitivity. Methods: We generated rabbit antisera against a ouabain-thyroglobulin conjugate. A tyrosylated ouabain derivative for radioiodination was synthesized using periodate and sodium cyanoborohydride reagents. Results: Mass spectrometric analyses showed that the main product of the tyrosylating reaction was tyrosyl-ouabain (molecular mass, 702 Da). This was radioiodinated with Chloramine-T and used as a tracer in a RIA, which gave an assay detection limit of 5 pmol/L (4 ng/L), 2–100 times lower than that in the corresponding 3H-RIAs and 2–20 times lower than ouabain ELISAs, making it possible to measure low plasma concentrations of immunoreactive ouabain. Different amounts of SepPak C18-extracted plasma samples displaced the 125I-labeled tyrosyl-ouabain tracer at the same rate at which authentic ouabain was displaced. Plasma immunoreactive ouabain coeluted with authentic ouabain in two different HPLC conditions. Using the new RIA, we found plasma ouabain concentrations, assayed as immunoreactive equivalents, of 10.0 ± 1.3 pmol/L in healthy women and 12.0 ± 0.9 pmol/L in healthy men (mean ± SE; n = 10), as well as 41.2 ± 9.6 pmol/L in rats. The concentrations were 2–90 times lower than those previously reported using different assay methods. Conclusions: Our ouabain 125I-RIA enables reliable measurements of low endogenous concentrations of a ouabain-like compound for both physiological and clinical purposes.
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Cao, Chunhua, Kristie Payne, Whaseon Lee-Kwon, Zhong Zhang, Sun Woo Lim, John Hamlyn, Mordecai P. Blaustein, H. Moo Kwon, and Thomas L. Pallone. "Chronic ouabain treatment induces vasa recta endothelial dysfunction in the rat." American Journal of Physiology-Renal Physiology 296, no. 1 (January 2009): F98—F106. http://dx.doi.org/10.1152/ajprenal.90429.2008.
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Descending vasa recta (DVR) are 15-μm vessels that perfuse the renal medulla. Ouabain has been shown to augment DVR endothelial cytoplasmic Ca2+ ([Ca2+]CYT) signaling. In this study, we examined the expression of the ouabain-sensitive Na-K-ATPase α2 subunit in the rat renal vasculature and tested effects of acute ouabain exposure and chronic ouabain treatment on DVR. Immunostaining with antibodies directed against the α2 subunit verified its expression in both DVR pericytes and endothelium. Acute application of ouabain (100 or 500 nM) augmented the DVR nitric oxide generation stimulated by acetylcholine (ACh; 10 μM). At a concentration of 1 mM, ouabain constricted microperfused DVR, whereas at 100 nM, it was without effect. Acute ouabain (100 nM) did not augment constriction by angiotensin II (0.5 or 10 nM), whereas l-nitroarginine methyl ester-induced contraction of DVR was slightly enhanced. Ouabain-hypertensive (OH) rats were generated by chronic ouabain treatment (30 μg·kg−1·day−1, 5 wk). The acute endothelial [Ca2+]CYT elevation by ouabain (100 nM) was absent in DVR endothelia of OH rats. The [Ca2+]CYT response to 10 nM ACh was also eliminated, whereas the response to 10 μM ACh was not. The endothelial [Ca2+]CYT response to bradykinin (100 nM) was significantly attenuated. We conclude that endothelial responses may offset the ability of acute ouabain exposure to enhance DVR vasoconstriction. Chronic exposure to ouabain, in vivo, leads to hypertension and DVR endothelial dysfunction, manifested as reduced [Ca2+]CYT responses to both ouabain- and endothelium-dependent vasodilators.
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Weiss, D. N., D. J. Podberesky, J. Heidrich, and M. P. Blaustein. "Nanomolar ouabain augments caffeine-evoked contractions in rat arteries." American Journal of Physiology-Cell Physiology 265, no. 5 (November 1, 1993): C1443—C1448. http://dx.doi.org/10.1152/ajpcell.1993.265.5.c1443.
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Chronic parenteral administration of ouabain to normal rats raises plasma ouabain concentrations to low nanomolar levels and induces hypertension [C. M. Yuan, P. Manunta, J. M. Hamlyn, S. W. Chen, E. Bohen, J. Yeun, F. J. Haddy, and M. B. Pamnani. Hypertension 22: 178-187, 1993 and see also M. P. Blaustein. Am. J. Physiol. 264 (Cell Physiol. 33): C1367-C1387, 1993]. To determine whether rat arteries are sensitive to these low ouabain levels, we tested the effects of various ouabain concentrations on caffeine-evoked contractions (CEC) in rat aortic and small mesenteric artery rings. CEC amplitude was used as a measure of the sarcoplasmic reticulum (SR) Ca2+ content. Ouabain increased CEC in aortic as well as mesenteric artery rings, but the effects in the aorta were difficult to quantitate because the CEC were often oscillatory. Mesenteric artery, under control conditions and after sensitization with 10-30 nM phenylephrine (PE), exhibited biphasic ouabain dose-CEC response curves. Low concentrations of ouabain (0.1-10 nM) caused small significant increases in CEC, but a further effect was observed only with > or = 10 microM ouabain. PE shifted the ouabain dose-response curve toward lower ouabain concentrations; conversely, ouabain shifted the PE dose-response curve toward lower PE concentrations. It appears that nanomolar concentrations of ouabain can influence vascular responsiveness to vasoconstrictors. We conclude that rat vascular smooth muscle contains both high- and low-affinity ouabain receptors, possibly corresponding to Na+ pumps with alpha 3- and alpha 1-subunit isoforms, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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Stimers, J. R., L. A. Lobaugh, S. Liu, N. Shigeto, and M. Lieberman. "Intracellular sodium affects ouabain interaction with the Na/K pump in cultured chick cardiac myocytes." Journal of General Physiology 95, no. 1 (January 1, 1990): 77–95. http://dx.doi.org/10.1085/jgp.95.1.77.
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Whether a given dose of ouabain will produce inotropic or toxic effects depends on factors that affect the apparent affinity (K0.5) of the Na/K pump for ouabain. To accurately resolve these factors, especially the effect of intracellular Na concentration (Nai), we have applied three complementary techniques for measuring the K0.5 for ouabain in cultured embryonic chick cardiac myocytes. Under control conditions with 5.4 mM Ko, the value of the K0.5 for ouabain was 20.6 +/- 1.2, 12.3 +/- 1.7, and 6.6 +/- 0.4 microM, measured by voltage-clamp, Na-selective microelectrode, and equilibrium [3H]ouabain-binding techniques, respectively. A significant difference in the three techniques was the time of exposure to ouabain (30 s-30 min). Since increased duration of exposure to ouabain would increase Nai, monensin was used to raise Nai to investigate what effect Nai might have on the apparent affinity of block by ouabain. Monensin enhanced the rise in Na content induced by 1 microM ouabain. In the presence of 1 microM [3H]ouabain, total binding was found to be a saturating function of Na content. Using the voltage-clamp method, we found that the value of the K0.5 for ouabain was lowered by nearly an order of magnitude in the presence of 3 microM monensin to 2.4 +/- 0.2 microM and the magnitude of the Na/K pump current was increased about threefold. Modeling the Na/K pump as a cyclic sequence of states with a single state having high affinity for ouabain shows that changes in Nai alone are sufficient to cause a 10-fold change in K0.5. These results suggest that Nai reduces the value of the apparent affinity of the Na/K pump for ouabain in 5.4 mM Ko by increasing its turnover rate, thus increasing the availability of the conformation of the Na/K pump that binds ouabain with high affinity.
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Boulanger, B. R., M. P. Lilly, J. M. Hamlyn, J. Laredo, D. Shurtleff, and D. S. Gann. "Ouabain is secreted by the adrenal gland in awake dogs." American Journal of Physiology-Endocrinology and Metabolism 264, no. 3 (March 1, 1993): E413—E419. http://dx.doi.org/10.1152/ajpendo.1993.264.3.e413.
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Ouabain has been identified in the plasma and adrenal glands of several mammals, including humans. To investigate possible adrenal secretion of ouabain in vivo, at rest, and in response to acute blood volume changes, we prepared trained adult dogs (n = 10) with splenectomy and unilateral adrenal venous (AV) cannulation. Two days later, after an overnight fast, dogs had either 1) 20% hemorrhage (hem) or 2) 20% blood volume expansion (exp; 6% Dextran 70, 0.9% NaCl) in random order. In AV and arterial plasma (ART), ouabain was measured by a ouabain-specific immunoassay, and cortisol and aldosterone were measured by radioimmunoassay. ART and AV ouabain concentration did not change after hem or exp [P = not significant (NS)]. In 94 of 97 paired samples, the concentration of ouabain in AV was greater than that in ART (Wilcoxon, P < 0.001), and the mean ouabain concentration was greater in AV (756.4 +/- 85.7 pmol/l) than ART (235.4 +/- 18.5 pmol/l; P < 0.001). The mean AV-to-ART ouabain concentration ratio was 5.7 +/- 1.29. Adrenal secretion of ouabain was not influenced by hem or exp (analysis of variance, P = NS). Adrenal secretion of cortisol and aldosterone increased after hem (P < 0.05) and was unaltered by exp (P = NS). This study demonstrates that ouabain is secreted by the adrenal gland in the awake dog. However, adrenal ouabain secretion and arterial blood ouabain are not altered by acute hem or exp.
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Tamura, M., D. W. Piston, M. Tani, M. Naruse, E. J. Landon, and T. Inagami. "Ouabain increases aldosterone release from bovine adrenal glomerulosa cells: role of renin-angiotensin system." American Journal of Physiology-Endocrinology and Metabolism 270, no. 1 (January 1, 1996): E27—E35. http://dx.doi.org/10.1152/ajpendo.1996.270.1.e27.
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To evaluate the potential physiological significance of ouabain or a ouabainlike substance, we investigated the effect of nanomolar concentrations of ouabain on aldosterone release by cultured bovine adrenal glomerulosa cells. Ouabain (10 nM) increased aldosterone release from 0.35 to 0.89 ng.mg-1.4 h-1 in the serum-containing medium. Losartan prevented this increase. When angiotensinogen was added to the nonserum medium, 10 nM ouabain enhanced the aldosterone release. Losartan again blocked the increase. These findings together with a stimulation of renin release by ouabain indicate that angiotensin II generated by the adrenal cell renin-angiotensin system in the presence of exogenous serum or exogenous angiotensinogen is necessary for the ouabain-induced stimulation of aldosterone release. Ouabain (10 nM) enhanced the intracellular calcium concentration increase elicited by 0.1 nM angiotensin II severalfold. Addition of 1 nM ouabain enhanced the aldosterone secretion resulting from the addition of 1 nM angiotensin II. Nanomolar levels of ouabain, therefore, interact with both locally formed and exogenous angiotensin II to stimulate aldosterone production. A suggested mechanism is that ouabain increases calcium stores in the endoplasmic reticulum, thereby increasing the agonist response.
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Brownlee, A. A., Paul Johnson, and Ivor H. Mills. "Actions of bufalin and cinobufotalin, two bufadienolides respectively more active and less active than ouabain, on ouabain binding and 86Rb uptake by human erythrocytes." Clinical Science 78, no. 2 (February 1, 1990): 169–74. http://dx.doi.org/10.1042/cs0780169.
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1. We have reported that the bufadienolide, bufalin (purified from toad skin), was more potent than ouabain in inhibiting the sodium/potassium-dependent adenosine triphosphatase from canine kidney (Sigma) [Brownlee, A. A., Lee, G. & Mills, I. H. J. Physiol. (London) 1987; 390, 94P]. 2. The activities of bufalin and cinobufotalin were compared with ouabain in the [3H]ouabain binding assay and on 86Rb uptake in human erythrocytes. 3. When the percentage binding of ouabain-sensitive [3H]ouabain was plotted against the log of the concentration of drug in mol/l, it was shown that the bufalin curve was shifted to the left of that of ouabain and that of cinobufotalin was to the right 4. Linear regression lines were fitted to the data transformed as the log of (p/1–p) plotted against the log of the drug concentration, where p is the proportion of maximal ouabain-sensitive activity at the drug concentration being considered. The IC50 (the concentration of drug producing a 50% change in the maximal ouabain-sensitive response) was 1.4 × 10−9 mol/l for bufalin, 9.7 × 10−9 mol/l for ouabain and 1.70 × 10−7 mol/l for cinobufotalin. 5. The introduction of bufalin 1 h before ouabain reduced the binding of [3H]ouabain to 23.4 ± 1.5% (P < 0.001). Bufalin added in the second hour reduced the ouabain-sensitive binding from 100 ± 1.9% to 87.4 ± 2.9% (P < 0.01). 6. The oubain-sensitive 86Rb uptake curves showed that that of bufalin was to the left of ouabain and that of cinobufotalin was to the right. 7. Regression lines were fitted to the 86Rb uptake data as described for the ouabain-binding data. The IC50 was 1.05 × 10−8 mol/l for bufalin, 4.4 × 10−8 mol/l for ouabain and 4.3 × 10−7 mol/l for cinobufotalin. 8. The differences in potencies are not all explained by what is known of the effects of structure on the cardiac toxicity of cardenolides and bufadienolides.
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Zeng, Weian, Shuji Dohi, Hiroyuki Shimonaka, and Toshio Asano. "Spinal Antinociceptive Action of Na+-K+Pump Inhibitor Ouabain and Its Interaction with Morphine and Lidocaine in Rats." Anesthesiology 90, no. 2 (February 1, 1999): 500–508. http://dx.doi.org/10.1097/00000542-199902000-00026.
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Background The Na+,K+-adenosine triphosphatase is a ubiquitous enzyme system that maintains the ion gradient across the plasma membrane of a variety of cell types, including cells in the central nervous system. We investigated the antinociceptive effect of intrathecally administered ouabain and examined its potential interaction with spinal morphine and lidocaine. Methods Using rats chronically implanted with lumbar intrathecal catheters, the ability of intrathecally administered ouabain, morphine, and lidocaine and of mixtures of ouabain-morphine and ouabain-lidocaine to alter tail-flick latency was examined. To characterize any interactions, isobolographic analysis was performed. The effects of pretreatment with intrathecally administered atropine or naloxone also were tested. Results Intrathecally administered ouabain (0.1-5.0 microg), morphine (0.2-10.0 microg), and lidocaine (25-300 microg) given alone produced significant dose- and time-dependent antinociception, but systemic administration of ouabain did not produce such an effect. The median effective dose (ED50) values for intrathecally administered ouabain, morphine, and lidocaine were 2.3, 5.0, and 227.0 microg, respectively. Isobolographic analysis exhibited a synergistic interaction after the coadministration of ouabain and morphine. With ouabain and lidocaine, there was no such evidence of synergism. Intrathecally administered atropine, but not naloxone, completely blocked the antinociceptive effect of ouabain and attenuated its interaction with spinally administered morphine. Conclusions Intrathecally administered ouabain produces antinociception, at least in part, via an enhancement of cholinergic transmission in the spinal nociceptive processing system. The results of the interaction of ouabain with morphine and lidocaine suggest that modulation of Na+-,K+-electrochemical gradients and thus subsequent release of neurotransmitters in the spinal cord are likely to play important roles in the spinal antinociceptive effect of intrathecally administered ouabain.
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Mandal, Amritlal, Nicholas A. Delamere, and Mohammad Shahidullah. "Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes." American Journal of Physiology-Cell Physiology 295, no. 1 (July 2008): C100—C110. http://dx.doi.org/10.1152/ajpcell.90636.2007.
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Sodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cytoplasmic pH fell abruptly, then gradually recovered toward baseline. Ouabain (1 μM) did not change cell sodium content, but the rate of pH recovery increased by 68%. Ouabain speeded pH recovery both in the presence and absence of bicarbonate. In bicarbonate-free medium, dimethylamiloride, an NHE inhibitor, eliminated the effect of 1 μM ouabain on pH recovery. Western blot analysis showed an NHE1 immunoreactive band but not NHE2, NHE3, or NHE4. Immunoprecipitation studies showed phosphorylation of NHE1 in cells treated with 1 μM ouabain. Ouabain evoked an increase of cAMP, and the effect of 1 μM ouabain on pH recovery was abolished by H-89, a protein kinase A inhibitor. 8-Bromoadenosine-cAMP increased the pH recovery rate, and this recovery was not further increased by ouabain. Although 1 μM ouabain did not alter cytoplasmic calcium concentration, it stimulated calcium entry after store depletion, a response abolished by 2-APB. Ouabain-induced stimulation of pH recovery was suppressed by inhibitors of capacitative calcium entry, SKF-96365, and 2-APB, as well as the cytoplasmic calcium chelator BAPTA. The cAMP increase in ouabain-treated cells was abolished by BAPTA and 2-APB. Taken together, the results are consistent with increased capacitative calcium entry and subsequent cAMP-PKA-dependent stimulation of NHE1 in ouabain-treated cells.
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Nesher, Maoz, Yan Bai, Daxiang Li, Haim Rosen, David Lichtstein, and Lijun Liu. "Interaction of atrial natriuretic peptide and ouabain in the myocardium." Canadian Journal of Physiology and Pharmacology 90, no. 10 (October 2012): 1386–93. http://dx.doi.org/10.1139/y2012-112.
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Natriuretic peptides and digitalis-like compounds serve as regulators of homeostasis, including control of volume expansion and blood pressure. The aim of the present study was to explore possible interactions between atrial natriuretic peptide (ANP) and ouabain in the heart. ANP (1 nmol/L) had no effect in papillary muscle preparations from guinea pigs. Ouabain (1 µmol/L) induced positive inotropic effect. The addition of ANP prior to ouabain resulted in a significant decrease in the ouabain-induced positive inotropic effect, manifested as an attenuated increase in twitch maximal upward force slope and resting muscular tension. In addition, ANP caused an increase in Na+–K+-ATPase activity in heart microsomal preparations. The effect of ouabain on Na+–K+-ATPase activity was shown in a biphasic manner. Ouabain (0.01–1 nmol/L) had a small but significant increase on pump activity, but higher doses of ouabain inhibited activity. ANP attenuated ouabain-induced Na+–K+-ATPase activity. Furthermore, ouabain (50 nmol/L) or ANP (10 nmol/L) alone induced Akt activation in cardiomyocytes. However, ANP blocked ouabain-induced Akt activation. These results point to the existence of interactions between ANP and ouabain on Na+–K+-ATPase signaling and function in the heart, which may be mediated by regulation of Na+–K+-ATPase activity and (or) signal transduction mechanisms.
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Nascimento, C. R., R. C. Valente, J. Echevarria-Lima, C. F. L. Fontes, L. de Araujo-Martins, E. G. Araujo, and V. M. Rumjanek. "The Influence of Ouabain on Human Dendritic Cells Maturation." Mediators of Inflammation 2014 (2014): 1–15. http://dx.doi.org/10.1155/2014/494956.
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Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua). Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs) were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days). To investigate Ouabain role on DC activation, DCs were stimulated with TNF-αfor 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.
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Shao, Chao, Shengwei Lin, Sudan Liu, Peipei Jin, Wenbin Lu, Na Li, Yan Zhang, Lulong Bo, and Jinjun Bian. "HIF1α-Induced Glycolysis in Macrophage Is Essential for the Protective Effect of Ouabain during Endotoxemia." Oxidative Medicine and Cellular Longevity 2019 (April 28, 2019): 1–10. http://dx.doi.org/10.1155/2019/7136585.
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Ouabain, a steroid binding to the Na+/K+-ATPase, has several pharmacological effects. In addition to the recognized effects of blood pressure, there is more convincing evidence suggesting that ouabain is involved in immunologic functions and inflammation. Hypoxia-inducible factor 1α (HIF-1α) is a metabolic regulator which plays a considerable role in immune responses. Previous studies had shown that HIF-1α-induced glycolysis results in functional reshaping in macrophages. In this study, we investigated the role of glycolytic pathway activation in the anti-inflammatory effect of ouabain. We found that ouabain is involved in anti-inflammatory effects both in vivo and in vitro. Additionally, ouabain can inhibit LPS-induced upregulation of GLUT1 and HK2 at the transcriptional level. GM-CSF pretreatment almost completely reversed the inhibitory effect of ouabain on LPS-induced release of proinflammatory cytokines. Alterations in glycolytic pathway activation were required for the anti-inflammatory effect of ouabain. Ouabain can significantly inhibit the upregulation of HIF-1α at the protein level. Our results also revealed that the overexpression of HIF-1α can reverse the anti-inflammatory effect of ouabain. Thus, we conclude that the HIF-1α-dependent glycolytic pathway is essential for the anti-inflammatory effect of ouabain.
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de Vasconcelos, Danielle Ingrid Bezerra, Jacqueline Alves Leite, Luciana Teles Carneiro, Márcia Regina Piuvezam, Maria Raquel Vitorino de Lima, Liana Clébia Lima de Morais, Vivian Mary Rumjanek, and Sandra Rodrigues-Mascarenhas. "Anti-inflammatory and Antinociceptive Activity of Ouabain in Mice." Mediators of Inflammation 2011 (2011): 1–11. http://dx.doi.org/10.1155/2011/912925.
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Ouabain, an inhibitor of the Na+/K+-ATPase pump, was identified as an endogenous substance of human plasma. Ouabain has been studied for its ability to interfere with various regulatory mechanisms. Despite the studies portraying the ability of ouabain to modulate the immune response, little is known about the effect of this substance on the inflammatory process. The aim of this work was to study the effects triggered by ouabain on inflammation and nociceptive models. Ouabain produced a reduction in the mouse paw edema induced by carrageenan, compound 48/80 and zymosan. This anti-inflammatory potential might be related to the inhibition of prostaglandin E2, bradykinin, and mast-cell degranulation but not to histamine. Ouabain also modulated the inflammation induced by concanavalin A by inhibiting cell migration. Besides that, ouabain presented antinociceptive activity. Taken these data together, this work demonstrated, for the first time, that ouabain presentedin vivoanalgesic and anti-inflammatory effects.
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Nguyen, Anh-Nguyet T., Kyle Jansson, Gladis Sánchez, Madhulika Sharma, Gail A. Reif, Darren P. Wallace, and Gustavo Blanco. "Ouabain activates the Na-K-ATPase signalosome to induce autosomal dominant polycystic kidney disease cell proliferation." American Journal of Physiology-Renal Physiology 301, no. 4 (October 2011): F897—F906. http://dx.doi.org/10.1152/ajprenal.00095.2011.
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The Na-K-ATPase is part of a cell signaling complex, the Na-K-ATPase signalosome, which upon activation by the hormone ouabain regulates the function of different cell types. We previously showed that ouabain induces proliferation of epithelial cells derived from renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD cells). Here, we investigated the signaling pathways responsible for mediating the effects of ouabain in these cells. Incubation of ADPKD cells with ouabain, in concentrations similar to those found in blood, stimulated phosphorylation of the epidermal growth factor receptor (EGFR) and promoted its association to the Na-K-ATPase. In addition, ouabain activated the kinase Src, but not the related kinase Fyn. Tyrphostin AG1478 and PP2, inhibitors of EGFR and Src, respectively, blocked ouabain-dependent ADPKD cell proliferation. Treatment of ADPKD cells with ouabain also caused phosphorylation of the caveolar protein caveolin-1, and disruption of cell caveolae with methyl-β-cyclodextrin prevented Na-K-ATPase-EGFR interaction and ouabain-induced proliferation of the cells. Downstream effects of ouabain in ADPKD cells included activation of B-Raf and MEK and phosphorylation of the extracellular regulated kinase ERK, which translocated into the ADPKD cell nuclei. Finally, ouabain reduced expression of the cyclin-dependent kinase inhibitors p21 and p27, which are suppressors of cell proliferation. Different from ADPKD cells, ouabain showed no significant effect on B-Raf, p21, and p27 in normal human kidney epithelial cells. Altogether, these results identify intracellular pathways of ouabain-dependent Na-K-ATPase-mediated signaling in ADPKD cells, including EGFR-Src-B-Raf-MEK/ERK, and establish novel mechanisms involved in ADPKD cell proliferation.
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Rajendran, Vazhaikkurichi M., Satish K. Singh, John Geibel, and Henry J. Binder. "Differential localization of colonic H+-K+-ATPase isoforms in surface and crypt cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 274, no. 2 (February 1, 1998): G424—G429. http://dx.doi.org/10.1152/ajpgi.1998.274.2.g424.
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Two distinct colonic H+-K+-adenosinetriphosphatase (H+-K+-ATPase) isoforms can be identified in part on the basis of their sensitivity to ouabain. The colonic H+-K+-ATPase α-subunit (HKcα) was recently cloned, and its message and protein are present in surface (and the upper 20% of crypt) cells in the rat distal colon. These studies were performed to establish the spatial distribution of the ouabain-sensitive and ouabain-insensitive components of both H+-K+-ATPase activity in apical membranes prepared from surface and crypt cells and K+-dependent intracellular pH (pHi) recovery from an acid load both in isolated perfused colonic crypts and in surface epithelial cells. Whereas H+-K+-ATPase activity in apical membranes from surface cells was 46% ouabain sensitive, its activity in crypt apical membranes was 96% ouabain sensitive. Similarly, K+-dependent pHi recovery in isolated crypts was completely ouabain sensitive, whereas in surface cells K+-dependent pHi recovery was insensitive to ouabain. These studies provide compelling evidence that HKcα encodes the colonic ouabain-insensitive H+-K+-ATPase and that a colonic ouabain-sensitive H+-K+-ATPase isoform is present in colonic crypts and remains to be cloned and identified.
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Teixeira, Mariana Pires, and Vivian Mary Rumjanek. "Ouabain Affects the Expression of Activation Markers, Cytokine Production, and Endocytosis of Human Monocytes." Mediators of Inflammation 2014 (2014): 1–10. http://dx.doi.org/10.1155/2014/760368.
Full textAbstract:
Ouabain is a steroid capable of binding to and inhibiting Na+,-K+-ATPase. Studies have demonstrated some actions of ouabain on immune cells, which indicated both pro- and anti-inflammatory properties of this molecule. Nevertheless, its effects on human monocytes are still poorly understood. Thus, the present work investigated effects of ouabain in the activation and function of human adherent monocytes. Our results show that there is an increase in intracellular calcium levels already 5 minutes following monocyte treatment with 10−7 M of ouabain. Furthermore, monocytes expressed increased amounts of surface activation markers such as CD69, HLA-DR, CD86, and CD80 and also presented an augmented endocytic activity of dextran-FITC particles after 24 hours of culture in the presence of ouabain. However, monocytes treated with ouabain did not have an increased stimulatory capacity in allogeneic mixed leukocyte reaction. Ouabain-treated monocytes produced higher levels of IL-1βand TNF-αas reported before. A novel observation was the fact that ouabain induced IL-10 and VEGF as well. Collectively, these results suggest that ouabain impacts monocyte activation and modulates monocyte functions, implying that this steroid could act as an immunomodulator of these cells.
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de Wardener, H. E. "Ouabain and hypertension." Nephrology Dialysis Transplantation 12, no. 3 (March 1, 1997): 384–85. http://dx.doi.org/10.1093/ndt/12.3.384.
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Goto, Atsuo, and Kaoru Yamada. "Ouabain-like factor." Current Opinion in Nephrology and Hypertension 7, no. 2 (March 1998): 189–96. http://dx.doi.org/10.1097/00041552-199803000-00008.
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Cai, Haiping, Liang Wu, Weikai Qu, Deepak Malhotra, Zijian Xie, Joseph I. Shapiro, and Jiang Liu. "Regulation of apical NHE3 trafficking by ouabain-induced activation of the basolateral Na+-K+-ATPase receptor complex." American Journal of Physiology-Cell Physiology 294, no. 2 (February 2008): C555—C563. http://dx.doi.org/10.1152/ajpcell.00475.2007.
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The long-term effects of ouabain on transepithelial Na+ transport involve transcriptional downregulation of apical Na+/H+ exchanger isoform 3 (NHE3). The aim of this study was to determine whether ouabain could acutely regulate NHE3 via a posttranscriptional mechanism in LLC-PK1 cells. We observed that the basolateral, but not apical, application of ouabain for 1 h significantly reduced transepithelial Na+ transport. This effect was not due to changes in the integrity of tight junctions or increases in the intracellular Na+ concentration. Ouabain regulated the trafficking of NHE3 and subsequently inhibited its activity, a process independent of intracellular Na+ concentration. Ouabain-induced NHE3 trafficking was abolished by either cholesterol depletion or Src inhibition. Moreover, ouabain increased the intracellular Ca2+ concentration. Pretreatment of cells with the intracellular Ca2+ chelator BAPTA-AM blocked ouabain-induced trafficking of NHE3. Also, blockade of Na+-K+-ATPase endocytosis by a phosphatidylinositol 3-kinase inhibitor was equally effective in attenuating ouabain-induced NHE3 trafficking. These data indicate that ouabain acutely stimulates NHE3 trafficking by activating the basolateral Na+-K+-ATPase signaling complex. Taken together with our previous observations, we propose that ouabain can simultaneously regulate basolateral Na+-K+-ATPase and apical NHE3, leading to inhibition of transepithelial Na+ transport. This mechanism may be relevant to proximal tubular Na+ handling during conditions associated with increases in circulating endogenous cardiotonic steroids.
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Oh, V. M. S., E. A. Taylor, J. L. Ding, N. A. Boon, J. K. Aronson, and D. G. Grahame-Smith. "Enhancement of specific [3H]ouabain binding and ouabain sensitive 86rubidium influx in intact human lymphocytes by a dialysable factor in human and fetal calf serum." Clinical Science 72, no. 1 (January 1, 1987): 71–79. http://dx.doi.org/10.1042/cs0720071.
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1. We have measured specific [3H]ouabain binding and ouabain sensitive 86rubidium influx in intact human lymphocytes incubated for up to 7 days in media containing different concentrations of fetal calf serum and human serum. 2. Incubation for periods of up to 7 days with fetal calf serum and human serum produced increases in both specific [3H]ouabain binding and ouabain sensitive 86rubidium influx that were dependent on concentration and time. 3. Neither specific [3H]ouabain binding nor ouabain sensitive 86rubidium influx was altered when dialysed serum was used, suggesting that both fetal calf serum and human serum contain a dialysable factor or factors which stimulate specific [3H]ouabain binding and ouabain sensitive 86rubidium influx in intact human lymphocytes. 4. To further elucidate the mechanisms underlying these changes we also measured the activity of two other enzymes of the lymphocyte plasma membrane, 5′-nucleotidase and γ-glutamyltransferase, the uptake of [3H]thymidine by the intact cells, and the effects of cycloheximide, puromycin, and anisomycin, inhibitors of protein synthesis. 5. The activity of 5′-nucleotidase was increased after incubation of the lymphocytes in fetal calf serum for 72 h, but the activity of γ-glutamyltransferase was not changed, suggesting some selectivity of the stimulatory effect. 6. Measurements of [3H]thymidine uptake by the lymphocytes showed that the major part of the observed changes in specific [3H]ouabain binding and ouabain sensitive 86rubidium influx was not attributable to transformation of the lymphocytes to lymphoblasts. 7. All three inhibitors of protein synthesis prevented the increase in specific [3H]ouabain binding due to fetal calf serum.
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Vakkuri, O., SS Arnason, A. Pouta, O. Vuolteenaho, and J. Leppaluoto. "Radioimmunoassay of plasma ouabain in healthy and pregnant individuals." Journal of Endocrinology 165, no. 3 (June 1, 2000): 669–77. http://dx.doi.org/10.1677/joe.0.1650669.
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Ouabain was recently isolated from human plasma, bovine hypothalamus and bovine adrenal in attempts to identify endogenous substances inhibiting the cell membrane sodium pump. A number of radioimmunoassays have been developed in order to study the clinical significance of ouabain. The results have been controversial with regard to the presence and chemical nature of plasma ouabain-like immunoreactivity. We have now measured ouabain in healthy and pregnant individuals using solid-phase extraction of plasma samples followed by a new radioimmunoassay with the extraordinary sensitivity of at least 2 fmol/tube (5 pmol/l). Plasma extracts, a previously isolated human plasma ouabain-like compound and bovine hypothalamic inhibitory factor displaced the tracer in parallel and eluted identically with ouabain in high-performance liquid chromatography. Plasma ouabain immunoreactivity was found to be much lower than reported previously: 12.6+/-1.3 pmol/l in healthy men (mean+/-s.e., n=20) and 9.4+/-0.7 pmol/l in women (n=14). In pregnant women (n=28) plasma ouabain concentration was 16.3+/-4.0 pmol/l during the first trimester, 18.8+/-4.3 pmol/l during the second trimester and 24.3+/-4.0 pmol/l during the third trimester (all P<0.01 compared with non-pregnant women). Plasma ouabain 3-5 days after the delivery was 13.6+/-1.1 pmol/l (n=10, P<0.05-0.01 compared with second and third trimesters). The pregnancy-related changes in the plasma concentrations of ouabain resembled those of cortisol. Therefore cortisol was measured from the same plasma samples and a significant positive correlation was found (r=0.512, P=0.006). The similar profiles of plasma ouabain and cortisol during pregnancy and their rapid decreases postpartum are consistent with the adrenal cortical origin of ouabain and also show that the secretions of these hormones are possibly under the control of same factors.
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Pasdois, Philippe, Casey L. Quinlan, Abraham Rissa, Liliane Tariosse, Béatrice Vinassa, Alexandre D. T. Costa, Sandrine V. Pierre, Pierre Dos Santos, and Keith D. Garlid. "Ouabain protects rat hearts against ischemia-reperfusion injury via pathway involving src kinase, mitoKATP, and ROS." American Journal of Physiology-Heart and Circulatory Physiology 292, no. 3 (March 2007): H1470—H1478. http://dx.doi.org/10.1152/ajpheart.00877.2006.
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We showed recently that mitochondrial ATP-dependent K+ channel (mitoKATP) opening is required for the inotropic response to ouabain. Because mitoKATP opening is also required for most forms of cardioprotection, we investigated whether exposure to ouabain was cardioprotective. We also began to map the signaling pathways linking ouabain binding to Na+-K+-ATPase with the opening of mitoKATP. In Langendorff-perfused rat hearts, 10–80 μM ouabain given before the onset of ischemia resulted in cardioprotection against ischemia-reperfusion injury, as documented by an improved recovery of contractile function and a reduction of infarct size. In skinned cardiac fibers, a ouabain-induced protection of mitochondrial outer membrane integrity, adenine nucleotide compartmentation, and energy transfer efficiency was evidenced by a decreased release of cytochrome c and preserved half-saturation constant of respiration for ADP and adenine nucleotide translocase-mitochondrial creatine kinase coupling, respectively. Ouabain-induced positive inotropy was dose dependent over the range studied, whereas ouabain-induced cardioprotection was maximal at the lowest dose tested. Compared with bradykinin (BK)-induced preconditioning, ouabain was equally efficient. However, the two ligands clearly diverge in the intracellular steps leading to mitoKATP opening from their respective receptors. Thus BK-induced cardioprotection was blocked by inhibitors of cGMP-dependent protein kinase (PKG) or guanylyl cyclase (GC), whereas ouabain-induced protection was not blocked by either agent. Interestingly, however, ouabain-induced inotropy appears to require PKG and GC. Thus 5-hydroxydecanoate (a selective mitoKATP inhibitor), N-(2-mercaptopropionyl)glycine (MPG; a reactive oxygen species scavenger), ODQ (a GC inhibitor), PP2 (a src kinase inhibitor), and KT-5823 (a PKG inhibitor) abolished preconditioning by BK and blocked the inotropic response to ouabain. However, only PP2, 5-HD, and MPG blocked ouabain-induced cardioprotection.
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Zhang, Lin, Christian Aalkjaer, and Vladimir Matchkov. "The Na,K-ATPase-Dependent Src Kinase Signaling Changes with Mesenteric Artery Diameter." International Journal of Molecular Sciences 19, no. 9 (August 23, 2018): 2489. http://dx.doi.org/10.3390/ijms19092489.
Full textAbstract:
Inhibition of the Na,K-ATPase by ouabain potentiates vascular tone and agonist-induced contraction. These effects of ouabain varies between different reports. In this study, we assessed whether the pro-contractile effect of ouabain changes with arterial diameter and the molecular mechanism behind it. Rat mesenteric small arteries of different diameters (150–350 µm) were studied for noradrenaline-induced changes of isometric force and intracellular Ca2+ in smooth muscle cells. These functional changes were correlated to total Src kinase and Src phosphorylation assessed immunohistochemically. High-affinity ouabain-binding sites were semi-quantified with fluorescent ouabain. We found that potentiation of noradrenaline-sensitivity by ouabain correlates positively with an increase in arterial diameter. This was not due to differences in intracellular Ca2+ responses but due to sensitization of smooth muscle cell contractile machinery to Ca2+. This was associated with ouabain-induced Src activation, which increases with increasing arterial diameter. Total Src expression was similar in arteries of different diameters but the density of high-affinity ouabain binding sites increased with increasing arterial diameters. We suggested that ouabain binding induces more Src kinase activity in mesenteric small arteries with larger diameter leading to enhanced sensitization of the contractile machinery to Ca2+.
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Shin, Hye Kyoung, Byung Jun Ryu, Sik-Won Choi, Seong Hwan Kim, and Kyunglim Lee. "Inactivation of Src-to-Ezrin Pathway: A Possible Mechanism in the Ouabain-Mediated Inhibition of A549 Cell Migration." BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/537136.
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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.
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Pierre, Sandrine V., Yoann Sottejeau, Jean-Michel Gourbeau, Gladis Sánchez, Amjad Shidyak, and Gustavo Blanco. "Isoform specificity of Na-K-ATPase-mediated ouabain signaling." American Journal of Physiology-Renal Physiology 294, no. 4 (April 2008): F859—F866. http://dx.doi.org/10.1152/ajprenal.00089.2007.
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The ion transporter Na-K-ATPase functions as a cell signal transducer that mediates ouabain-induced activation of protein kinases, such as ERK. While Na-K-ATPase composed of the α1-polypeptide is involved in cell signaling, the role of other α-isoforms (α2, α3, and α4) in transmitting ouabain effects is unknown. We have explored this using baculovirus-directed expression of Na-K-ATPase polypeptides in insect cells and ERK phosphorylation as an indicator of ouabain-induced signaling. Ouabain addition to Sf-9 cells coexpressing Na-K-ATPase α1- and β1-isoforms stimulated ERK phosphorylation. In contrast, expression of the α1- and β1-polypeptides alone resulted in no effect, indicating that the αβ-complex is necessary for Na-K-ATPase signaling. Moreover, the ouabain effect was sensitive to genistein, suggesting that Na-K-ATPase-mediated tyrosine kinase activation is a critical event in the intracellular cascade leading to ERK phosphorylation. In addition, the Na-K-ATPases α3β1- and α4β1-isozymes, but not α2β1, responded to ouabain treatment. In agreement with the differences in ouabain affinity of the α-polypeptides, α1β1 required 100- to 1,000-fold more ouabain to signal than did α4β1 and α3β1, respectively. These results confirm the role of the Na-K-ATPase in ouabain signal transduction, show that there are important isoform-specific differences in Na-K-ATPase signaling, and demonstrate the suitability of the baculovirus expression system for studying Na-K-ATPase-mediated ouabain effects.
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Cunningham, M. J., C. S. Apstein, E. O. Weinberg, and B. H. Lorell. "Deleterious effect of ouabain on myocardial function during hypoxia." American Journal of Physiology-Heart and Circulatory Physiology 256, no. 3 (March 1, 1989): H681—H687. http://dx.doi.org/10.1152/ajpheart.1989.256.3.h681.
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The effect of cardiac glycosides on myocardial function during hypoxia is controversial. Accordingly, we studied left ventricular performance during hypoxia and reoxygenation in the presence of a mildly inotropic, nontoxic dose of ouabain using isolated, isovolumic, buffer-perfused rabbit hearts. After 15 min of hypoxia, left ventricular developed pressure was less in the ouabain-treated group than in controls (35 +/- 4 vs. 55 +/- 3 mmHg, P less than 0.025). Left ventricular end-diastolic pressure (LVEDP) increased more during hypoxia in the presence of ouabain (9 +/- 1 to 32 +/- 7 with ouabain vs. 9 +/- 1 to 14 +/- 3 mmHg without ouabain, P less than 0.005) despite comparable degrees of coronary vasodilatation and myocardial lactate production in the two groups. When coronary flow was abruptly reduced to zero to eliminate the coronary turgor contribution to diastolic pressure, LVEDP after 15 min of hypoxia in the presence of ouabain was greater than that in control hearts that did not receive ouabain (13 +/- 4 vs. 4 +/- 1 mmHg, P less than 0.05), implicating greater diastolic myocardial fiber tension in the ouabain group during hypoxia. With reoxygenation, recovery of developed pressure was less and end-diastolic pressure remained elevated in the ouabain-treated group when compared with controls. We conclude that a modestly inotropic dose of ouabain exacerbates the decrease in diastolic ventricular distensibility induced by hypoxia, worsens the decline in developed pressure during hypoxia, and impairs recovery during reoxygenation.
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Khundmiri, Syed J., Vishal Amin, Jeff Henson, John Lewis, Mohamed Ameen, Madhavi J. Rane, and Nicholas A. Delamere. "Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway." American Journal of Physiology-Cell Physiology 293, no. 3 (September 2007): C1171—C1180. http://dx.doi.org/10.1152/ajpcell.00535.2006.
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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.
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Bai, Yan, Jian Wu, Daxiang Li, Eric E. Morgan, Jiang Liu, Xiaochen Zhao, Aaron Walsh, et al. "Differential roles of caveolin-1 in ouabain-induced Na+/K+-ATPase cardiac signaling and contractility." Physiological Genomics 48, no. 10 (October 1, 2016): 739–48. http://dx.doi.org/10.1152/physiolgenomics.00042.2016.
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Binding of ouabain to cardiac Na+/K+-ATPase initiates cell signaling and causes contractility in cardiomyocytes. It is widely accepted that caveolins, structural proteins of caveolae, have been implicated in signal transduction. It is known that caveolae play a role in Na+/K+-ATPase functions. Regulation of caveolin-1 in ouabain-mediated cardiac signaling and contractility has never been reported. The aim of this study is to compare ouabain-induced cardiac signaling and contractility in wild-type (WT) and caveolin-1 knockout (cav-1 KO) mice. In contrast with WT cardiomyocytes, ouabain-induced signaling e.g., activation of phosphoinositide 3-kinase-α/Akt and extracellular signal-regulated kinases (ERK)1/2, and hypertrophic growth were significantly reduced in cav-1 KO cardiomyocytes. Interactions of the Na+/K+-ATPase α1-subunit with caveolin-3 and the Na+/K+-ATPase α1-subunit with PI3K-α were also decreased in cav-1 KO cardiomyocytes. The results from cav-1 KO mouse embryonic fibroblasts also proved that cav-1 significantly attenuated ouabain-induced ERK1/2 activation without alteration in protein and cholesterol distribution in caveolae/lipid rafts. Intriguingly, the effect of ouabain induced positive inotropy in vivo (via transient infusion of ouabain, 0.48 nmol/g body wt) was not attenuated in cav-1 KO mice. Furthermore, ouabain (1–100 μM) induced dose-dependent contractility in isolated working hearts from WT and cav-1 KO mice. The effects of ouabain on contractility between WT and cav-1 KO mice were not significantly different. These results demonstrated differential roles of cav-1 in the regulation of ouabain signaling and contractility. Signaling by ouabain, in contrast to contractility, may be a redundant property of Na+/K+-ATPase.
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Akimova, Olga A., James Van Huysse, Johanne Tremblay, and Sergei N. Orlov. "Low efficiency of functional translation of ouabain-resistant α2-Na+,K+-ATPase mRNA in C7-MDCK epithelial cells." Canadian Journal of Physiology and Pharmacology 90, no. 1 (January 2012): 83–88. http://dx.doi.org/10.1139/y11-113.
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Na+,K+-ATPase is a heterodimer consisting of catalytic α1–α4 and regulatory β1–β3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na+,K+-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K+ (86Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na+,K+-ATPase. α2R mRNA in transfected cells was ∼8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10 µmol/L ouabain led to complete inhibition of 86Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of 86Rb influx in α1R-transfectd cells was observed at 1000 µmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3 µmol/L ouabain , α1R-cells survived after 24 h incubation with 1000 µmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na+,K+-ATPase subunit mRNA and immunoreactive protein does not contribute to Na+/K+ pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na+,K+-ATPase on Na+/K+ pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.
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Pidgeon, G. B., A. M. Richards, M. G. Nicholls, C. J. Charles, M. T. Rademaker, K. L. Lynn, R. R. Bailey, L. K. Lewis, and T. G. Yandle. "Chronic ouabain infusion does not cause hypertension in sheep." American Journal of Physiology-Endocrinology and Metabolism 270, no. 3 (March 1, 1996): E386—E392. http://dx.doi.org/10.1152/ajpendo.1996.270.3.e386.
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Ouabain is claimed to be a hormone of adrenal origin, capable of raising arterial pressure in rats. We infused ouabain in conscious sheep under carefully controlled circumstances to determine its effects on blood pressure, urine electrolytes, and vasoactive hormones. Eight healthy ewes were studied while taking a constant intake of dietary sodium and potassium. Ouabain infusion at 0.25 mg daily over 22 days reduced heart rate and arterial pressure and had no effect on pressor responsiveness to incremental intravenous infusions of angiotensin II. Ouabain induced minor, but statistically significant, decrements in urine volume, urinary sodium excretion, plasma renin and angiotensin II concentrations, and a rise in plasma aldosterone and cortisol. Plasma ouabain levels averaged 1.37 +/- 0.28 nmol/l during ouabain infusion. In conclusion, high-dose chronic ouabain infusion in sheep did not elevate arterial pressure or alter pressor responsiveness to angiotensin II, was antidiuretic and antinatriuretic, and induced minor perturbations in circulating renin, angiotensin II, aldosterone, and cortisol.
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Pidgeon, G. B., A. M. Richards, M. G. Nicholls, L. K. Lewis, and T. G. Yandle. "Acute Effects of Intravenous Ouabain in Healthy Volunteers." Clinical Science 86, no. 4 (April 1, 1994): 391–97. http://dx.doi.org/10.1042/cs0860391.
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1. The influence of single-dose intravenous ouabain on haemodynamics, renal function and vasoactive hormones was investigated in healthy subjects. 2. After ouabain injection there was a significant reduction in minimum and average hourly heart rates. Diastolic blood pressure fell immediately after ouabain, without a significant change in systolic blood pressure. 3. There was no change in renal blood flow, glomerular filtration rate, hourly urine volume or sodium and potassium excretion. 4. Ouabain injection was associated with a fall in plasma angiotensin II levels and a rise in plasma adrenaline levels, but plasma noradrenaline levels were unchanged. Both plasma atrial and brain natriuretic peptide levels were increased after ouabain administration. 5. The present study demonstrates that ouabain has significant although minor, acute effects on plasma levels of vasoactive hormones. It fails, however, to confirm that ouabain is an acute pressor or natriuretic substance. If it has an important influence on electrolyte and haemodynamic homoeostasis, its actions are likely to be slowly developed.