Ouabain

Ouabain is a selective Na+/K+, -ATPase inhibitor, binds to α2 /α3 subunit with K_i of 41 nM/15 nM.

Pivotal role of alpha2 Na(+) pumps and their high affinity ouabain binding site in cardiovascular health and disease.[Pubmed:27350568]

J Physiol. 2016 Nov 1;594(21):6079-6103.

Reduced smooth muscle (SM)-specific alpha2 Na(+) pump expression elevates basal blood pressure (BP) and increases BP sensitivity to angiotensin II (Ang II) and dietary NaCl, whilst SM-alpha2 overexpression lowers basal BP and decreases Ang II/salt sensitivity. Prolonged Ouabain infusion induces hypertension in rodents, and Ouabain-resistant mutation of the alpha2 Ouabain binding site (alpha2(R/R) mice) confers resistance to several forms of hypertension. Pressure overload-induced heart hypertrophy and failure are attenuated in cardio-specific alpha2 knockout, cardio-specific alpha2 overexpression and alpha2(R/R) mice. We propose a unifying hypothesis that reconciles these apparently disparate findings: brain mechanisms, activated by Ang II and high NaCl, regulate sympathetic drive and a novel neurohumoral pathway mediated by both brain and circulating endogenous Ouabain (EO). Circulating EO modulates Ouabain-sensitive alpha2 Na(+) pump activity and Ca(2+) transporter expression and, via Na(+) /Ca(2+) exchange, Ca(2+) homeostasis. This regulates sensitivity to sympathetic activity, Ca(2+) signalling and arterial and cardiac contraction.

Ouabain-induced alterations in ABCB1 of mesenteric lymph nodes and thymocytes of rats and mice.[Pubmed:28105236]

Oncol Lett. 2016 Dec;12(6):5275-5280.

Ouabain is a glycoside with immunomodulating properties, and recent studies have suggested its use in adjuvant therapy for cancer treatment. Ouabain is known to modulate the immune system in vitro, and previous studies have revealed that Ouabain can modulate the expression and activity of ABCB1, a protein associated with multidrug resistance present in immune system. Therefore, the present study investigated alterations in the expression and activity of ABCB1 in the thymi, peripheral blood monocytes and lymph nodes of Wistar rats and Swiss mice treated acutely or chronically with Ouabain. A decrease of almost 45% in the monocyte count and an increase of 55% in the basophil count were observed. A significant decrease (75% reduction) in the amount of cells with ABCB1 activity was found in the thymocytes of Ouabain-treated rats and mice. The possible implications of these results for cancer treatment are discussed.

Ouabain induces apoptosis and autophagy in Burkitt's lymphoma Raji cells.[Pubmed:27894666]

Biomed Pharmacother. 2016 Dec;84:1841-1848.

The steroid Na+/K+-ATPase blocker Ouabain has been shown to exhibit cytotoxic effects in various tumor cell systems. This study aimed to determine the effects of Ouabain on Burkitt's lymphoma Raji cells. Ouabain treatment of Raji cells significantly inhibited cell proliferation in a dose-dependent manner and increased the morphological changes associated with apoptosis. Additionally, increased numbers of both early and late apoptotic cells were observed by annexin V-FITC/PI flow cytometry assay. Increased levels of caspase-3 and cleaved-caspase-3, higher Bax activity and decreased expression of the anti-apoptotic protein Bcl-2 were detected in Ouabain-treated Raji cells. Vacuole accumulation was also observed in transmission electron microscope (TEM) images of Ouabain-treated Raji cells, indicating that these cells were undergoing autophagy. Expression of the autophagy-related proteins LC3-II and Beclin-1 was upregulated in Ouabain-treated Raji cells. These results suggest that Ouabain may promote cell death in Raji cells by inducing pathways associated with apoptosis and autophagy. Our study also provides novel evidence that Ouabain may be an effective agent for treating Burkitt's lymphoma.

Intense interval training in healthy older adults increases skeletal muscle [(3)H]ouabain-binding site content and elevates Na(+),K(+)-ATPase alpha2 isoform abundance in Type II fibers.[Pubmed:28373411]

Physiol Rep. 2017 Apr;5(7). pii: 5/7/e13219.

Young adults typically adapt to intense exercise training with an increased skeletal muscle Na(+),K(+)-ATPase (NKA) content, concomitant with reduced extracellular potassium concentration [K(+)] during exercise and enhanced exercise performance. Whether these changes with longitudinal training occur in older adults is unknown and was investigated here. Fifteen older adults (69.4 +/- 3.5 years, mean +/- SD) were randomized to either 12 weeks of intense interval training (4 x 4 min at 90-95% peak heart rate), 3 days/week (IIT, n = 8); or no exercise controls (n = 7). Before and after training, participants completed an incremental cycle ergometer exercise test until a rating of perceived exertion of 17 (very hard) on a 20-point scale was attained, with measures of antecubital venous [K(+)]v Participants underwent a resting muscle biopsy prior to and at 48-72 h following the final training session. After IIT, the peak exercise work rate (25%), oxygen uptake (16%) and heart rate (6%) were increased (P < 0.05). After IIT, the peak exercise plasma [K(+)]v tended to rise (P = 0.07), while the rise in plasma [K(+)]v relative to work performed (nmol.L(-1)J(-1)) was unchanged. Muscle NKA content increased by 11% after IIT (P < 0.05). Single fiber measurements, increased in NKA alpha2 isoform in Type II fibers after IIT (30%, P < 0.05), with no changes to the other isoforms in single fibers or homogenate. Thus, intense exercise training in older adults induced an upregulation of muscle NKA, with a fiber-specific increase in NKA alpha2 abundance in Type II fibers, coincident with increased muscle NKA content and enhanced exercise performance.

Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding.[Pubmed:9346307]

Eur J Biochem. 1997 Sep 1;248(2):488-95.

Random mutagenesis with Ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the alpha1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter Ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent Ouabain selection, 24 Ouabain-resistant clones have been identified. All previously described amino acids that confer Ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest Ouabain resistance, such as Gln111-->Arg, Asp121-->Gly, Asp121-->Glu, Asn122-->Asp, and Thr797-->Ala were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that affect Ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330-->Gln, Ala331-->Gly, Thr338-->Ala, and Thr338-->Asn in the H4 transmembrane domain and Phe982-->Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1-7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase alpha1 subunit to identify amino acids residues that are important in Ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs.

Isoform-specific up-regulation by ouabain of Na+,K+-ATPase in cultured rat astrocytes.[Pubmed:9349566]

J Neurochem. 1997 Nov;69(5):2189-96.

There are two alpha-subunit isoforms (alpha1 and alpha2) and two beta-subunit isoforms (beta1 and beta2) of Na+,K+-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5-1.0 mM) increased the levels of alpha1 and beta1 mRNAs, whereas it decreased those of alpha2 and beta2 mRNAs in cultured rat astrocytes. The increases in alpha1 and beta1 mRNAs were observed at 6-48 h after addition of the inhibitor. Immunochemical analyses showed that Ouabain increased alpha1 and beta1, but not alpha2 and beta2, proteins, and that the isoforms in control and Ouabain-treated cultures were of glial origin. Low extracellular K+ and monensin (20 microM) mimicked the effect of Ouabain on alpha1 mRNA. The Ouabain-induced increase in alpha1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 microM), the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (30 microM), and the calcineurin inhibitor FK506 (1 nM). These findings indicate that chronic inhibition of Na+,K+-ATPase up-regulates the alpha1 and beta1, but not alpha2 and beta2, isoforms in astrocytes, suggesting a functional coupling of alpha1beta1 complex. They also suggest that intracellular Na+, Ca2+, and calcineurin may be involved in Ouabain-induced up-regulation of the enzyme in astrocytes.

Ouabain improves cardiac function in vivo in rats with heart failure after chronic but not acute treatment.[Pubmed:9272726]

Naunyn Schmiedebergs Arch Pharmacol. 1997 Aug;356(2):203-9.

Rats are generally believed to be insensitive for cardiac glycosides. However, like in humans, the hemodynamic effects may be related to the pathophysiological condition. Since the hemodynamic effects of cardiac glycosides have never been investigated in rats with heart failure, the aim of the present experiments was to investigate the role of the pathophysiological condition in the rat. Therefore, hemodynamic and cardiac effects of Ouabain were investigated both in normal rats and rats with heart failure due to myocardial infarction (MI). Since the effects of Ouabain may also depend on the treatment scheme, rats were treated either for a short-term period or a long-term period. Three weeks after sham surgery or ligation of the left coronary artery (MI), Wistar rats were treated for two weeks with Ouabain (14.4 mg/kg.d s.c.), either continuously (osmotic minipumps) or intermittently (once daily). A separate group of rats was treated for 45-60 min (1-100 microg/kg.min Ouabain; i.v. infusion 5 weeks after MI). Hemodynamic measurements were performed at rest and after volume loading in conscious rats, chronically instrumented with an electromagnetic flow probe and catheters. Induction of MI resulted in a significant increase in total peripheral resistance (TPR), and a significant decrease in basal and maximal cardiac output following volume loading (basal CO: sham, 92 +/- 5; MI, 74 +/- 5 ml/min; maximal CO: sham, 152 +/- 4; MI, 105 +/- 7 ml/min; n = 7-11). Chronic intermittent Ouabain treatment further increased TPR in MI rats. In contrast, continuous Ouabain treatment normalized TPR in rats. Only in continuously treated MI rats, basal and maximal CO improved significantly (basal: 83 +/- 4; maximal: 134 +/- 7 ml/min; n = 7). Acute treatment dose-dependently worsened the hemodynamic conditions of MI rats, since TPR and MAP increased and CO and stroke volume decreased significantly. These experiments demonstrate that Ouabain can improve cardiac function in rats, although only in MI rats and strongly depending on the delivery regimen. Thus, in contrast to the general belief, the presently used rat model is suitable for investigation of cardiac glycosides in heart failure. The preferential improvement of cardiac function in MI rats continuously treated with Ouabain may depend upon changes in Na+,K+-ATPase or altered neurohumoral conditions due to MI and chronic treatment.