Ouabain Articles 2

© 2011

Endogenous sodium pump inhibitors and age-associated increases in salt sensitivity of blood pressure in normotensives

            (Anderson, Fedorova et al. 2008) Download

Factors that mediate increases in salt sensitivity of blood pressure with age remain to be clarified. The present study investigated 1) the effects of high-NaCl intake on two Na pump inhibitors, endogenous ouabain (EO) and marinobufagenin (MBG), in middle-aged and older normotensive Caucasian women; and 2) whether individual differences in EO and MBG are linked to variations in sodium excretion or salt sensitivity. A change from 6 days of a lower (0.7 mmol.kg(-1).day(-1))- to 6 days of a higher (4 mmol.kg(-1).day(-1))-NaCl diet elicited a sustained increase in MBG excretion that directly correlated with an increase in the fractional Na excretion and was inversely related to age and to an age-dependent increase in salt sensitivity. In contrast, EO excretion increased only transiently in response to NaCl loading and did not vary with age or correlate with fractional Na excretion or salt sensitivity. A positive correlation of both plasma and urine levels of EO and MBG during salt loading may indicate a casual link between two Na pump inhibitors in response to NaCl loading, as observed in animal models. A linear mixed-effects model demonstrated that age, dietary NaCl, renal MBG excretion, and body mass index were each independently associated with systolic blood pressure. Thus, a sustained increase in MBG in response to acutely elevated dietary NaCl is inversely linked to salt sensitivity in normotensive middle-aged and older women, and a relative failure of MBG elaboration by these older persons may be involved in the increased salt sensitivity with advancing age.

Folic acid administration prevents ouabain-induced hyperlocomotion and alterations in oxidative stress markers in the rat brain

            (Brocardo, Budni et al. 2010) Download

OBJECTIVE: Bipolar disorder (BD) is a chronic, prevalent, and highly debilitating psychiatric illness. Folic acid has been shown to have antidepressant-like effects in preclinical and clinical studies and has also been suggested to play a role in BD. The present work investigates the therapeutic value of folic acid supplementation in a preclinical animal model of mania induced by ouabain. METHODS: Male Wistar rats were treated twice daily for seven days with folic acid (10, 50, and 100 mg/kg, p.o.) or the mood stabilizer lithium chloride (LiCl) (45 mg/kg, p.o.). One day after the last dose was given, the animals received an i.c.v. injection of ouabain (10 microM), a Na(+),K(+)-ATPase-inhibiting compound. Locomotor activity was assessed in the open-field test. Thiobarbituric acid-reactive substance (TBARS) levels, glutathione peroxidase (GPx), and glutathione reductase (GR) activities were measured in the cerebral cortex and hippocampus. RESULTS: Ouabain (10 microM, i.c.v.) significantly increased motor activity in the open-field test, and seven days of pretreatment with folic acid (50 mg/kg, p.o.) or LiCl (45 mg/kg, p.o.) completely prevented this effect. Ouabain treatment elicited lipid peroxidation (increased TBARS levels) and reduced GPx activity in the hippocampus. GR activity was decreased in the cerebral cortex and hippocampus. These effects were prevented by pretreatment with folic acid and LiCl. CONCLUSIONS: Our results show that folic acid, similarly to LiCl, produces a clear antimanic action and prevents the neurochemical alterations indicative of oxidative stress in an animal model of mania.

On the structure of endogenous ouabain

            (Kawamura, Guo et al. 1999) Download

The ouabain-like sodium pump inhibitor in mammals (so-called "endogenous ouabain") has been considered a subtle structural isomer of ouabain. Its structural investigation, however, has long been hindered by the paucity of sample material. Our recent purification of endogenous ouabain (3 micrograms) from bovine hypothalamus allowed the measurement of its 1H-NMR. The obtained spectrum as well as reexamination of past microscale structural studies on endogenous ouabain led us to identify the purified material as ouabain in an unusual manner. It turned out that the structural analysis had been complicated by a facile ouabain-borate complexation in borosilicate glassware. In retrospect, it is not surprising that the polyhydroxylated ouabain molecule serves as a polydentate ligand to inorganic species. In its physiological environment, ouabain may exist as some unknown complex. The chemical species giving rise to the reported biological activities of hypothalamic inhibitory factor preparations remain to be clarified.

Liquid chromatography mass spectrometric analysis of ouabainlike factor in biological fluid

            (Komiyama, Nishimura et al. 2000) Download

Ouabainlike factor (OLF), assayed as ouabainlike immunoreactivity (OLI), is a probable endogenous digitalislike factor (EDLF). Liquid chromatography/mass spectrometry (LC/MS) is one of the most highly sensitive tools for obtaining structural information regarding low-molecular weight materials in a target compound, and to measure the concentrations of these materials. We have previously reported that OLI can be isolated from the culture supernatant of the rat pheochromocytoma cell line, PC12, by several reverse-phase chromatography and LC/MS techniques. The present study was performed to characterize OLF from biological fluids such as plasma and culture supernatant of PC12 cells by LC/MS. The previous applications of LC/MS to OLI in plasma have been limited to structural identification at the final stages of isolation, in which the starting volume of plasma has been over 10 I. In the present study, we tried to minimize the volume of plasma, and to develop a new preclearing step to gain adequate LC/MS characterization using MS/MS analysis. The plasma was acidified, and OLI was purified by ODS column chromatography. OLI in chromatographic fractions from plasma was assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. After Sep-Pak treatment and two rounds of ODS column chromatography, OLI was identified from 80 ml of plasma. The structure of the purified OLI was identical to authentic ouabain and digoxin, as assessed by LC/MS. In conclusion, we identified the chemically or structurally clarified ouabain and digoxin as the circulating form in plasma by LC/MS.

Endogenous ouabain and the renin-angiotensin-aldosterone system: distinct effects on Na handling and blood pressure in human hypertension

            (Manunta, Hamlyn et al. 2011) Download

OBJECTIVE: To evaluate whether the renin-angiotensin-aldosterone system (RAAS) and endogenous ouabain system differently affect renal Na handling and blood pressure. METHODS: Three hundred and one patients in whom we compared blood pressure, and renal Na tubular reabsorption in the basal condition and 2 h (T120) after saline infusion. RESULTS: Following multivariate-adjusted linear and quartiles analysis, baseline mean blood pressure (MBP) was significantly higher (113.7 +/- 1.33 mmHg) in the fourth versus the first endogenous ouabain quartile (103.8 +/- 1.04 mmHg) and the trend across the quartiles was highly significant (beta = 0.23, P = 3.53e-04). In contrast, an inverse relationship was present in the renin activity (PRA) quartiles with MBP highest in the first (112.5 +/- 1.26) and lowest in the fourth PRA quartile (107.6 +/- 1.48, P = 0.039). Following an acute saline load, changes in MBP and the slope of the pressure-natriuresis relationship were inversely related across the PRA quartiles. The fractional excretion of sodium (FENa) showed a negative linear trend going from the first to the third endogenous ouabain quartiles (2.35 +/- 0.17 and 1.90 +/- 0.14%, P = 0.05). Patients in the fourth endogenous ouabain quartile (>323 pmol/l) showed increased FENa T120 (2.78 +/- 0.18%, P < 0.01) and increased Na tubular rejection fraction (P = 0.007) after Na load. After the saline load, there was a biphasic relationship between plasma endogenous ouabain and FENa favoring Na retention at low endogenous ouabain and Na excretion at high endogenous ouabain levels. CONCLUSION: The RAAS and endogenous ouabain system are two independent and complementary systems having an inverse (RAAS) or a direct (endogenous ouabain system) relationship with hemodynamic parameters.

Endogenous ouabain: upregulation of steroidogenic genes in hypertensive hypothalamus but not adrenal

            (Murrell, Randall et al. 2005) Download

BACKGROUND: Mammalian tissues contain a presumed endogenous Na+, K(+)-ATPase inhibitor that binds reversibly to the Na+ pump with high affinity and specificity. The inhibitor has been linked to the pathogenesis of experimental volume-expanded and human essential hypertension. This compound has been isolated from mammalian hypothalamus and appears to be an isomer of the plant-derived cardiac glycoside ouabain, if not ouabain itself. The objective of this study was to test the hypothesis that a biosynthetic pathway exists in mammalian tissues to produce a steroid derivative closely related to plant cardiac glycosides. METHODS AND RESULTS: Using bioinformatics and genomic techniques, Milan hypertensive rat tissues were studied because this strain has a 10-fold increase in hypothalamic ouabain-like compound that is linked to the pathogenesis of the hypertension. A putative steroid biosynthetic pathway was constructed and candidate genes encoding enzymes in this pathway were identified from sequence databases. Differential expression of selected genes in the pathway was studied by microarray analysis and quantitative polymerase chain reaction, with functional validation by gene silencing using small interfering RNAs. Marked upregulation of genes coding for P450 side chain cleavage and Delta5-3beta-hydroxysteroid dehydrogenase/Delta5-Delta4- isomerase enzymes in hypertensive hypothalamus but not adrenal was found, compared with normotensive Milan rats. Knockdown of the latter gene decreased production of ouabain-like factor from neural tissue. CONCLUSIONS: Our findings support the possibility that a unique steroid biosynthetic circuit exists in Milan rat brain, functioning independently from adrenal, which could account for the overproduction of the hypothalamic ouabain-like compound in this species.

Independent and incremental prognostic value of endogenous ouabain in idiopathic dilated cardiomyopathy

            (Pitzalis, Hamlyn et al. 2006) Download

Increased circulating levels of endogenous ouabain (EO) have been observed in some heart failure patients, but their long term clinical significance is unknown. This study investigated the prognostic value of EO for worsening heart failure among 140 optimally treated patients (age 50+/-14 years; 104 male; NYHA class 1.9+/-0.7) with idiopathic dilated cardiomyopathy. Plasma EO was determined by RIA and by liquid chromatography mass spectrometry, values were linearly correlated (r = 0.89) in regression analysis. During follow-up (13+/-5 months), heart failure progression was defined as worsening clinical condition leading to one or more of the following: sustained increase in conventional therapies, hospitalization, cardiac transplant, or death. NYHA functional class, age, LVEF, peak VO2 and plasma levels of EO were predictive for heart failure progression. Heart failure worsened 1.5 fold (HR: 1.005; 95% CI: 1.001-1.007; p<0.01) for each 100 pmol/L increase in plasma EO. Moreover, those patients with higher plasma EO values had an odds ratio of 5.417 (95% CI: 2.044-14.355; p<0.001) for heart failure progression. Following multivariate analysis, LVEF, NYHA class and plasma EO remained significantly linked with clinical events. This study provides the first evidence that circulating EO is a novel, independent and incremental marker that predicts the progression of heart failure.

Modulation of the immune system by ouabain

            (Rodrigues-Mascarenhas, Da Silva de Oliveira et al. 2009) Download

Ouabain, a known inhibitor of the Na,K-ATPase, has been shown to regulate a number of lymphocyte functions in vitro and in vivo. Lymphocyte proliferation, apoptosis, cytokine production, and monocyte function are all affected by ouabain. The ouabain-binding site occurs at the alpha subunit of the enzyme. The alpha subunit plays a critical role in the transport process, and four different alpha-subunit isoforms have been described with different sensitivities to ouabain. Analysis by RT-PCR indicates that alpha1, alpha2, and alpha3 isoforms are all present in murine lymphoid cells obtained from thymus, lymph nodes, and spleen. In these cells ouabain exerts an effect at concentrations that do not induce plasma membrane depolarization, suggesting a mechanism independent of the classical inhibition of the pump. In other systems, the Na,K-ATPase acts as a signal transducer in addition to being an ion pump, and ouabain is capable of inducing the activation of various signal transduction cascades. Neither resting nor concanavalin A (Con A)-activated thymocytes had their levels of phosphorylated-extracellular signal-regulated kinase (P-ERK) modified by ouabain. However, ouabain decreased p38 phosphorylation induced by Con A in these cells. The pathway induced by ouabain in lymphoid cells is still unclear but might vary with the type and state of activation of the cell.

The Use of Circulatory Stimulants in the Care of the Sick

            (Rudolf 1922) Download

Endogenous ouabain in Meniere's disease

            (Teggi, Zagato et al. 2010) Download

OBJECTIVE: Endogenous Ouabain (EO) has been demonstrated to modulate the activity of Na+, K+ -ATPase. Our purpose was to measure plasma levels of EO in Meniere's Disease (MD) subjects as a possible predisposing factor to developing and maintaining hydrops. STUDY DESIGN: Case-control study. SETTINGS: University hospital. PATIENTS: Thirty-nine MD subjects and 29 controls with a lifetime negative history for vertigo and dizziness. MAIN OUTCOME MEASURES: Plasma levels of EO. RESULTS: Plasma EO in MD subjects was in the range between 33 and 504 pmol/L (median, 135.5 pmol/L), whereas in the control group, plasma EO varied between 70 and 724 pmol/L (median, 205 pmol/L). The Mann-Whitney U test detected a statistically significant difference (p = 0.0001). CONCLUSION: Low plasma levels of EO have been proposed to augment Na-K pump activity, whereas high EO levels show an inhibitory effect on the pump activity. A proper pump activity may be necessary to keep the right ionic amount and osmolarity in endolymph. Although other possibilities may be considered, we suggest that altered control mechanisms of pump activity may be related to the pathogenesis and maintenance of MD.

Progesterone derivatives that bind to the digitalis receptor: synthesis of 14 beta-hydroxyprogesterone. A novel steroid with positive inotropic activity

            (Templeton, Kumar et al. 1987) Download

The synthesis of 14-hydroxy-14 beta-pregn-4-ene-3,20-dione (14 beta-hydroxyprogesterone) is described. This novel steroid is about 10 times more potent than progesterone and one-tenth as potent as ouabagenin in an [3H]ouabain radioligand binding assay and is the first in a series of progesterone congeners that interact at the cardiac glycoside receptor both to possess the C/D cis ring junction and to enhance contractility of isolated cardiac tissue.

Radioimmunoassay of plasma ouabain in healthy and pregnant individuals

            (Vakkuri, Arnason et al. 2000) Download

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.

Long-Term Ouabain Treatment Impairs Vascular Function in Resistance Arteries

            (Wenceslau, Davel et al. 2011) Download

Background/Aims: The purpose of this study was to examine the cardiovascular effects of long-term ouabain treatment at different time points. Methods: Systolic blood pressure (SBP) was measured by tail-cuff method in male Wistar rats treated with ouabain (approx. 8.0 mug.day(-1)) or vehicle for 5, 10 and 20 weeks. Afterwards, vascular function was assessed in mesenteric resistance arteries (MRA) using a wire myograph. ROS production and COX-1 and COX-2, TNF-alpha, and IL-6 protein expression were investigated. Results: SBP was increased by ouabain treatment up to the 6th week and remained stable until the 20th week. However, noradrenaline-induced contraction increased only in MRA in rats treated with ouabain for 20 weeks. NOS inhibition and endothelium removal increased the noradrenaline response, but to a smaller magnitude in MRA in the ouabain group. Moreover, inhibition of COX-2 or incubation with superoxide dismutase restores noradrenaline-induced contraction in the 20-week ouabain group to control levels. ROS production as well as COX-2, IL-6 and TNF-alpha protein expression increased in MRA in this group. Conclusion: Although ouabain treatment induced hypertension in all groups, a larger noradrenaline induced contraction was observed over 20 weeks of treatment. This vascular dysfunction was related to COX-2-derived prostanoids and oxidative stress, increased pro- inflammatory cytokines and reduced NO bioavailability.

Characterization of ouabain-like immunoreactivity in human urine

         (Worgall, Hanze et al. 1996) Download

OBJECTIVE: To characterize ouabain-like immunoreactivity in human urine. METHODS: Sensitive radioimmunoassay for ouabain characterized by high-performance liquid chromatography. RESULTS: Serial dilution of urinary immunoreactive ouabain paralleled the standard curve, but not so plasma immunoreactive ouabain. Intravenous administration of 86 nmol (62.5 micrograms) ouabain caused a rapid rise in ouabain immunoreactivity in plasma of healthy volunteers with a maximum of 1.7 nmol/l 8 min after injection and returned to basal levels after 6 h. Ouabain immunoreactivity rose to 36 nmol/l in urine, suggesting that exogenously administered ouabain can be measured reliably in plasma and urine. Analytical reversed-phase high-performance liquid chromatography (isopropanol-propanol biphasic gradient; linear acetonitrile gradient) of sample extracts before assay demonstrated measurable amounts of ouabain-related material only in native urine, but not in plasma. When plasma and urine were spiked with ouabain standard or normal volunteers were injected with ouabain, the assay reliably measured ouabain. CONCLUSION: A substance closely related to ouabain can be detected in urine, but circulates, if at all, in small amounts in human plasma.



Anderson, D. E., O. V. Fedorova, et al. (2008). "Endogenous sodium pump inhibitors and age-associated increases in salt sensitivity of blood pressure in normotensives." Am J Physiol Regul Integr Comp Physiol 294(4): R1248-54.

Brocardo, P. S., J. Budni, et al. (2010). "Folic acid administration prevents ouabain-induced hyperlocomotion and alterations in oxidative stress markers in the rat brain." Bipolar Disord 12(4): 414-24.

Kawamura, A., J. Guo, et al. (1999). "On the structure of endogenous ouabain." Proc Natl Acad Sci U S A 96(12): 6654-9.

Komiyama, Y., N. Nishimura, et al. (2000). "Liquid chromatography mass spectrometric analysis of ouabainlike factor in biological fluid." Hypertens Res 23 Suppl: S21-7.

Manunta, P., J. M. Hamlyn, et al. (2011). "Endogenous ouabain and the renin-angiotensin-aldosterone system: distinct effects on Na handling and blood pressure in human hypertension." J Hypertens 29(2): 349-56.

Murrell, J. R., J. D. Randall, et al. (2005). "Endogenous ouabain: upregulation of steroidogenic genes in hypertensive hypothalamus but not adrenal." Circulation 112(9): 1301-8.

Pitzalis, M. V., J. M. Hamlyn, et al. (2006). "Independent and incremental prognostic value of endogenous ouabain in idiopathic dilated cardiomyopathy." Eur J Heart Fail 8(2): 179-86.

Rodrigues-Mascarenhas, S., A. Da Silva de Oliveira, et al. (2009). "Modulation of the immune system by ouabain." Ann N Y Acad Sci 1153: 153-63.

Rudolf, R. D. (1922). "The Use of Circulatory Stimulants in the Care of the Sick." Can Med Assoc J 12(10): 697-701.

Teggi, R., L. Zagato, et al. (2010). "Endogenous ouabain in Meniere's disease." Otol Neurotol 31(1): 153-6.

Templeton, J. F., V. P. Kumar, et al. (1987). "Progesterone derivatives that bind to the digitalis receptor: synthesis of 14 beta-hydroxyprogesterone. A novel steroid with positive inotropic activity." J Med Chem 30(8): 1502-5.

Vakkuri, O., S. S. Arnason, et al. (2000). "Radioimmunoassay of plasma ouabain in healthy and pregnant individuals." J Endocrinol 165(3): 669-77.

Wenceslau, C. F., A. P. Davel, et al. (2011). "Long-Term Ouabain Treatment Impairs Vascular Function in Resistance Arteries." J Vasc Res 48(4): 316-326.

Worgall, S., J. Hanze, et al. (1996). "Characterization of ouabain-like immunoreactivity in human urine." J Hypertens 14(5): 623-8.