This list of abstracts of recent publications, which give an overview of the current state of research on endogenous ouabain will be updated as new interesting articles are published.

 

No endogenous ouabain is detectable in human plasma by ultra-sensitive UPLC-MS/MS.

Baecher S, Kroiss M, Fassnacht M, Vogeser M. Clin Chim Acta. 2014 Feb 7;431C:87-92. doi: 10.1016/j.cca.2014.01.038

 

BACKGROUND:

The presence of a binding site for cardiac glycosides, such as digitoxin and digoxin, in the sodium-potassium-ATPase, stimulated attempts to isolate endogenous cardiotonic steroids. Using immunoassays, clinical studies found the cardenolideouabain to be secreted endogenously in response to exercise and untreated hypertension and to be correlated with severity of clinical conditions such as kidney failure and dilated cardiomyopathy. The assays used were not standardized and the mean concentrations of endogenous ouabain reported for healthy controls ranged from 60 to 530pmol/l. None of these immunoassays is available any more. Therefore, the aim of this study was to develop a highly specific and reliable method for measurement of ouabainin human plasma based on isotope dilution liquid chromatography tandem-mass spectrometry (ID-LC-MS/MS).

METHOD:

An ultra-sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed which applied solid phase extraction of plasma for sample preparation.

RESULTS:

The method was comprehensively validated and had a lower limit of quantification of 1.7pmol/l. However, despite this very low detection limit ouabain was not observed in plasma samples from patients with and without heart failure.

CONCLUSION:

Our results suggest that immunoassays previously used to quantify assumed endogenous ouabain detected compounds which are not structurally identical with ouabain. Cross reactivity of structurally related compounds of endogenous origin may cause these discrepancies between immunological and mass spectrometric analyses. Conclusive characterization of assumedendogenous counterparts of digoxin in a biomarker discovery approach seems to require distinct analytical techniques.

 

Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning. Belliard A, Sottejeau Y, Duan Q, Karabin JL, Pierre SV. Am J Physiol Heart Circ Physiol. 2013 Jan 1;304(1):H94-103.

 

Abstract

Na(+),K(+)-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na(+),K(+)-ATPase α(1)- and α(3)-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α(1)-cell surface abundance was significantly decreased. Na(+),K(+)-ATPase-activity in crude homogenates and (86)Rb(+) transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 μM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na(+),K(+)-ATPase activity and surface expression. This model also revealed that Na(+),K(+)-ATPase-mediated (86)Rb(+) uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na(+),K(+)-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na(+),K(+)-ATPase α(1) known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na(+),K(+)-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na(+),K(+)-ATPase may be independent of ion transport.

 

Pongrakhananon V, Chunhacha P, Chanvorachote P. Ouabain suppresses the migratory behavior of lung cancer cells. PLoS One. 2013 Jul 10;8(7):e68623. doi: 10.1371/journal.pone.0068623. Print 2013.

 

Abstract

The migratory capability of cancer cells is one of the most important hallmarks reflecting metastatic potential. Ouabain, an endogenous cardiac glycoside produced by the adrenal gland, has been previously reported to have anti-tumor activities; however, its role in the regulation of cancer cell migration remains unknown. The present study has revealed that treatment with ouabain at physiological concentrations is able to inhibit the migratory activities of human lung cancer H292 cells. The negative effects of ouabain were found to be mediated through the suppression of migration regulatory proteins, such as focal adhesion kinase (FAK), ATP-dependent tyrosine kinase (Akt), and cell division cycle 42 (Cdc42). We found that the observed actions of ouabain were mediated via a reactive oxygen species (ROS)-dependent mechanism because the addition of ROS scavengers (N-acetylcysteine and glutathione) could reverse the effect of ouabain on cell migration. Furthermore, ouabain was shown to inhibit the spheroidal tumor growth and decrease the cancer cell adhesion to endothelial cells. However, the compound had no significant effect on anoikis of the cells. Together, these findings shed light on the understanding of cancer cell biology by exploring the novel function of this endogenous human substance.

 

Blanco G, Wallace DP, Novel role of ouabain as a cystogenic factor in autosomal dominant polycystic kidney disease. Am J Physiol Renal Physiol. 2013 Jun 12. [Epub ahead of print]

 

Abstract

The classical 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 (ADPKD).

 

Silva E, Soares-da-Silva P, New insights into the regulation of Na+,K+-ATPase by ouabain. Int Rev Cell Mol Biol. 2012;294:99-132. doi: 10.1016/B978-0-12-394305-7.00002-1.

 

Abstract Cardiac glycosides have been widely used in the treatment of congestive heart failure and cardiac arrhythmias. The mechanism of their positive ionotropic effect is well characterized and is based on the inhibition of Na(+),K(+)-ATPase. However, recently it has been discovered that circulating endogenous cardiotonic steroids bind to Na(+),K(+)-ATPase, activate cell signaling pathways, and regulate diverse cellular functions. Thus, they play an important role in both physiological and pathological processes. Circulating endogenous ouabain has been reported to activate several signaling pathways upon binding to Na(+),K(+)-ATPase, and altered plasma levels of this endogenous cardiotonic steroid have been found in several pathologies. This review focuses on ouabain-mediated activation of cell signaling pathways and secondary messengers that conversely may modulate Na(+),K(+)-ATPase function.

 

Li J, Khodus GR, Kruusmägi M, Kamali-Zare P, Liu XL, Eklöf AC, Zelenin S, Brismar H, Aperia A. Ouabain protects against adverse developmental programming of the kidney. Nat Commun. 2010 Jul 27;1:42. doi: 10.1038/ncomms1043.

 

Abstract

The kidney is extraordinarily sensitive to adverse fetal programming. Malnutrition, the most common form of developmental challenge, retards the formation of functional units, the nephrons. The resulting low nephron endowment increases susceptibility to renal injury and disease. Using explanted rat embryonic kidneys, we found that ouabain, the Na,K-ATPase ligand, triggers a calcium-nuclear factor-κB signal, which protects kidney development from adverse effects of malnutrition. To mimic malnutrition, kidneys were serum deprived for 24 h. This resulted in severe retardation of nephron formation and a robust increase in apoptosis. In ouabain-exposed kidneys, no adverse effects of serum deprivation were observed. Proof of principle that ouabain rescues development of embryonic kidneys exposed to malnutrition was obtained from studies on pregnant rats given a low-protein diet and treated with ouabain or vehicle throughout pregnancy. Thus, we have identified a survival signal and a feasible therapeutic tool to prevent adverse programming of kidney development.

 

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