Ouabain-like Factor Research Articles

Ouabain, endogenous ouabain and ouabain-like factors: The Na+ pump/ouabain receptor, its linkage to NCX, and its myriad functions

In this brief review we discuss some aspects of the Na+ pump and its roles in mediating the effects of ouabain and endogenous ouabain (EO): i) in regulating the cytosolic Ca2+ concentration ([Ca2+]CYT) via Na/Ca exchange (NCX), and ii) in activating a number of protein kinase (PK) signaling cascades that control a myriad of cell functions. Importantly, [Ca2+]CYT and the other signaling pathways intersect at numerous points because of the influence of Ca2+ and calmodulin in modulating some steps in those other pathways. While both mechanisms operate in virtually all cells and tissues, this article focuses primarily on their functions in the cardiovascular system, the central nervous system (CNS) and the kidneys.

Aldosterone and Mineralocorticoid Receptors-Physiology and Pathophysiology.

Aldosterone is a uniquely terrestrial hormone, first appearing in lungfish, which have both gills and lungs. Mineralocorticoid receptors (MRs), on the other hand, evolved much earlier, and are found in cartilaginous and bony fish, presumptive ligand cortisol. MRs have equivalent high affinity for aldosterone, progesterone, and cortisol; in epithelia, despite much higher cortisol circulating levels, aldosterone selectively activates MRs by co-expression of the enzyme 11β-hydroxysteroid dehydrogenase, Type 11. In tissues in which the enzyme is not expressed, MRs are overwhelmingly occupied but not activated by cortisol, which normally thus acts as an MR antagonist; in tissue damage, however, cortisol mimics aldosterone and acts as an MR agonist. The risk profile for primary aldosteronism (PA) is much higher than that in age-, sex-, and blood pressure-matched essential hypertensives. High levels of aldosterone per se are not the problem: in chronic sodium deficiency, as seen in the monsoon season in the highlands of New Guinea, plasma aldosterone levels are extraordinarily high, but cause neither hypertension nor cardiovascular damage. Such damage occurs when aldosterone levels are out of the normal feedback control, and are inappropriately elevated for the salt status of the individual (or experimental animal). The question thus remains of how excess salt can synergize with elevated aldosterone levels to produce deleterious cardiovascular effects. One possible mechanism is through the agency of the elusive ouabain-like factors (OLFs). Such factors are secreted from the adrenal in response to ACTH (adrenalocortical tropic hormone), to angiotensin via AT2R, and—the polar opposite of aldosterone—to sodium loading. They act on blood vessels to cause vasoconstriction and thus elevate blood pressure to dump excess sodium through pressure natriuresis. Their levels are chronically elevated in PA in response to the continually elevated sodium status, and they thus act to constrict coronary and systemic arteries. In the context of the elevated blood volume and total body sodium in a PA patient, this raises blood pressure and acts as the proximate cause of cardiovascular damage. If this is the case, it would appear to offer new insights into therapy for PA. One would be the use of digibindin, or its more recent successors as antagonists of OLFs acting on Na/K ATPase at the vessel wall. A second would be to routinely combine a low dose MR antagonist, an ENaC inhibitor, and sodium restriction as first-line therapy for bilateral aldosterone overproduction. Finally, for unilateral cases post-surgery, there is good reason to include low-dose MRs in drug therapy if required, given the ability of cortisol in damaged blood vessels to mimic aldosterone vasoconstrictor action.

Identification of Putative Natriuretic Hormones Isolated from Human Urine.

This brief review describes some representative methodological approaches to the isolation of putative endogenous inhibitors of epithelial sodium transport – i.e., as ouabain-like factors (OLF) that inhibit the sodium transport enzyme Na-K-ATPase or inhibit the epithelial sodium channel (ENaC). Gel chromatography and reverse-phase (RP)-high performance liquid chromatography (HPLC) of lyophilized and reconstituted 24 h-urine from salt-loaded healthy humans led to two active fractions, a hydrophilic OLF-1 and a lipophilic OLF-2, whose mass (Ms)-spectroscopic data indicate a Mr of 391 (1, 2). Further identification was attempted by Ms-, infrared (IR)-, ultraviolet (UV)-, and 1H-NMR-spectroscopy. OLF-1 and OLF-2 may be closely related if not identical to (di)ascorbic acid or its salts such as vanadium (V)-Vv-diascorbate with Mr 403 (3) and VIV-diascorbate. OLF-1 and Vv-diascorbate are about 10-fold stronger inhibitors of Na-K-ATPase than OLF-2 and VIV-diascorbate, respectively. In conscious rats, i.v. infusion of OLF-1 and OLF-2 resulted in a strong natriuresis. In a similar study, Cain et al. (4) isolated a sodium transport inhibitor from the urine of uremic patients by gel chromatography and RP-HPLC. In uremic rats, a natriuretic response to the injection of the active material was found. Xanthurenic acid 8-O-β-d-glucoside (Mr 368) and xanthurenic acid 8-O-sulfate (Mr 284) were identified as endogenous inhibitors of sodium transport acting, e.g., by ENaC blockade. No definite relation to blood pressure, body fluid volume, or sodium balance has been reported for any of these above factors, and further studies to identify the natriuretic and/or ouabain-like compound(s) or hormone(s) will be needed.

Why isn't endogenous ouabain more widely accepted?

A new idea is the most quickly acting antigen known to science . —Wilfred Trotter During the past few years, several distinguished senior colleagues who visited our department and inquired about the status of endogenous ouabain (EO) have asked, “How come I didn't know about these [published]

An ouabain-like factor is secreted from immortalized hypothalamic cells in an aldosterone-dependent manner

Ouabain-like factor (OLF) modulates blood pressure via sodium pump inhibition in the central nervous system and in the peripheral circulation. Ouabain-like factor (OLF) is thought to be produced in the adrenal gland and hypothalamus, and it may relate locally to the renin–angiotensin–aldosterone system. However, the evidence for the latter was obtained from in vivo experiments using animals. In the present study, we investigated ouabain production in the immortalized hypothalamic cell line N1. First, cell culture supernatant was collected from the immortalized hypothalamic cell line N1 at 0.5, 4, 8, and 24h. A newly developed enzyme-linked immunosorbent assay (ELISA) that used anti-ouabain antibody showed that immunoreactivity in the supernatant was increased significantly at 24 vs. 0.5h (0.01±0.004 vs. 0.16±0.033pmol/mg protein, p<0.01). A combination of HPLC and ELISA was used to characterize N1 cell-derived OLI, showing that the highest peak of OLI had the same retention time as authentic ouabain. Thereafter, N1 cells were cultured with (1–10μM) aldosterone, and supernatant was collected after 24h of culture. In addition, N1 cells were cultured with 5μM eplerenone, a mineralocorticoid receptor blocker, plus aldosterone. OLI was significantly increased in the supernatant of the cells cultured with 10μM aldosterone (0.40±0.078pmol/mg protein), and this increase was abolished by the addition of the aldosterone antagonist eplerenone (0.12±0.030pmol/mg protein). These data suggest that the immortalized hypothalamic N1 cells secrete OLF and that aldosterone stimulates its secretion via mineralocorticoid receptors.

Isolation of marinobufotoxin from the supernatant of cultured PC12 cells

1. Digitalis-like factors (DLFs) are believed to be involved in sodium metabolism via inhibition of Na(+) /K(+) -ATPase and may cause hypertension. Yet, the source and regulation of secretion of DLFs remain unknown. Recently, marinobufagenin (MBG) was isolated in mammals and implicated in renal sodium and water metabolism. More recently, we isolated marinobufotoxin (MBT), a suberoyl arginine ester of MBG, in Y-1 cells. We have developed an ELISA to measure MBG-like immunoreactivity (MBG-IR) and have characterized MBG-IR using chromatography. We have also identified a ouabain-like factor in cultured PC12 cells from a phaeochromocytoma cell line. In the present study, we examined whether MBT was produced in the adrenal medulla. 2. PC12 cells were cultured in serum-free medium and culture supernatants were collected over a period of 24 h. The supernatants were analysed by ELISA and HPLC to determine MBG-IR content. The HPLC fraction containing the main peak of MBG-IR was characterized by LC/MS. 3. Compared with samples collected at 0.5 h, the concentration of MBG-IR in culture supernatants increased significantly after 2 h and continued to increase until 24 h. The fraction with the highest ELISA peak for MBG-IR had the same HPLC elution time as authentic MBT. Furthermore, tandem mass spectrometry indicated that each fraction of MBT and MBG had the correct specific daughter ions. 4. The results indicate that MBT and MBG are produced and/or secreted by adrenomedullary cells.

Aberrant regulation of endogenous ouabain-like factor in bipolar subjects

Ill phases of bipolar illness are associated with abnormalities in ion regulation and intracellular ion concentrations. Previously, it has been reported that mania is characterised by lower circulating levels of ion regulating endogenous cardenolides, and that bipolar subjects lack the normal seasonal variation of these factors. Since endogenous cardenolides are elaborated in settings of extensive physical activity, euthymic bipolar and psychiatrically normal control subjects were asked to exercise to exhaustion. Plasma concentrations of endogenous cardenolides were measured at baseline, 60 min, peak exercise and post-recovery. Ouabain-like immunoreactive factor (OLF) was lower at baseline (0.005 ± S.D. 0.01 ng/mL in bipolar vs. 0.072 ± 0.06 ng/mL in normal control subjects, P = 0.019), lower at 60 min (0.007 ± S.D. 0.02 ng/mL in bipolar vs. 0.075 ± 0.06 ng/mL in normal control subjects, P = 0.029), and tended to be lower at peak exercise (0.009 ± S.D. 0.02 ng/mL in bipolar vs. 0.131 ± 0.21 ng/mL in normal control subjects, P = 0.15) in bipolar subjects compared to non-psychiatric controls. Other endogenous cardenolides did not vary significantly. The endogenous cardenolide, OLF, may be aberrantly controlled in bipolar illness.

Ouabain stimulates Na-K-ATPase through a sodium/hydrogen exchanger-1 (NHE-1)-dependent mechanism in human kidney proximal tubule cells

Recent investigations demonstrate increased Na/H exchanger-1 (NHE-1) activity and plasma levels of ouabain-like factor in spontaneously hypertensive rats. At nanomolar concentrations, ouabain increases Na-K-ATPase activity, induces cell proliferation, and activates complex signaling cascades. We hypothesize that the activity of NHE-1 and Na-K-ATPase are interdependent. To test whether treatment with picomolar ouabain regulates Na-K-ATPase through an NHE-1-dependent mechanism, we examined the role of NHE-1 in ouabain-mediated stimulation of Na-K-ATPase in kidney proximal tubule cell lines [opossum kidney (OK), HK-2, HKC-5, and HKC-11] and rat kidney basolateral membranes. Ouabain stimulated Na-K-ATPase activity and tyrosine phosphorylation in cells that express NHE-1 (OK, HKC-5, and HKC-11) but not in HK-2 cells that express very low levels of NHE-1. Inhibition of NHE-1 with 5 microM EIPA, a NHE-1-specific inhibitor, prevented ouabain-mediated stimulation of (86)Rb uptake and Na-K-ATPase phosphorylation in OK, HKC-5, and HKC-11 cells. Expression of wild-type NHE-1 in HK2 cells restored regulation of Na-K-ATPase by picomolar ouabain. Treatment with picomolar ouabain increased membrane expression of Na-K-ATPase and enhanced NHE-1-Na-K-ATPase alpha1-subunit association. Treatment with ouabain (1 microg x kg body wt(-1) x day(-1)) increased Na-K-ATPase activity, expression, phosphorylation, and association with NHE-1 increased in rat kidney cortical basolateral membranes. Eight days' treatment with ouabain (1 microg x kg body wt(-1) x day(-1)) resulted in increased blood pressure in these rats. These results suggest that the association of NHE-1 with Na-K-ATPase is critical for ouabain-mediated regulation of Na-K-ATPase and that these effects may play a role in cardioglycoside-stimulated hypertension.

Mammalian Cardenolides in Cancer Prevention and Therapeutics

Digoxin-like immunoreactive factor (DLIF) and ouabain-like factor (OLF) are the mammalian counterparts to the plant-derived cardiotonic steroids digoxin and ouabain. Compelling evidence indicates that the cardiotonic steroids may have anticancer properties. Recent evidence indicates that low (nanomolar) concentrations of DLIF selectively induce cell death in transformed cells, while sparing normal cells, and is even more potent than the plant-derived compounds. The discovery that these endogenous molecules may play a role in the regulation of cancer cell proliferation provides a potentially new paradigm for the physiologic role of DLIF and OLF. In addition, the possible use of digoxin itself as a therapeutic agent in cancer has been explored, and evidence suggests that its conversion to dihydrodigoxin may be involved in regulating anticancer activity. The mechanism(s) for the pro-apoptotic property of these compounds is not known. In this brief review, we will discuss the proposed mechanism of action of digoxin, ouabain, DLIF, and OLF as anticancer compounds and discuss the effects that metabolic conversion to their dihydro-derivatives may have on this activity. From the perspective of therapeutic drug monitoring, these findings suggest some potential new challenges in the need to measure concentrations of digoxin and dihydrodigoxin as well as their endogenous counterparts DLIF and OLF in serum.

Pregnancy Blocks the Hypertensinogenic Effect of Ouabain in Sprague‐Dawley Rats

Ouabain‐like inhibitors are elevated in ∼50% of patients with essential hypertension and patients with pre‐eclampsia (PE). Prolonged administration of ouabain induces hypertension in Sprague‐Dawley rats. Accordingly, we hypothesized that ouabain might induce PE in otherwise normal pregnant rats. We implanted ouabain pellets subcutaneously in pregnant (E10) and non‐pregnant rats and measured their blood pressures (BP) weekly by tail cuff. After delivery, trunk blood was collected to determine ouabain‐like activity by radioreceptor assay. Ouabain pellets raised BP significantly in non‐pregnant rats (+18.3 mmHg, p&lt;0.05) compared to non‐pregnant controls (+5.4 mmHg). Surprisingly, there was no significant difference in BP between pregnant rats with ouabain and their pregnant controls. Moreover, there was a significant increase in ouabain‐like activity in all pregnant (57.0 ± 3.1 nmoles plasma equiv/L) verses non‐pregnant (12.4 ± 1.1 nmoles plasma equiv/L) rats, irrespective of BP. These data show 1) that normal pregnant rats become resistant to the hypertensive effect of ouabain; 2) the pregnant state is associated with elevated levels of polar ouabain‐like materials that compete with 3H‐ouabain for binding to Na/K‐ATPase. These ouabain‐like factors may act to prevent increases in BP, or oppose the hypotensive effects of pregnancy. Funded by NHLBI 078870/75584, NIGMS Initiative for Minority Student Development Grant (R25‐GM55036), and Procter and Gamble

Ouabain modulation of cellular calcium stores and signaling

Ouabain-like factors modulate intracellular Ca2+ concentrations and Ca2+ stores. Recently, a role for Na+-K+-ATPase Na+ transport inhibition as a pivotal event in ouabain signaling was questioned (Kaunitz JD. Am J Physiol Renal Physiol 290: F995-F996, 2006). In the present study, we used a mathematical model of Ca2+ trafficking in cytoplasm and subplasmalemmal microdomains to simulate the pathways through which ouabain can affect Ca2+ signaling: inhibition of active transport by Na+-K+-ATPase alpha1- and alpha2-isoforms, activation of inositol trisphosphate (IP3) production, and increased IP3 receptor (IP3R) conductance. A fundamental prediction is that Na+-K+-ATPase inhibition favors sarcoplasmic reticulum Ca2+ store loading, whereas Src-mediated increases in IP3 production and IP3R sensitization favor store depletion. The model predicts that alpha2-isoform inhibition generates a peak-and-plateau pattern of cytosolic Ca2+ concentration ([Ca2+](cyt)) elevation, whereas alpha1-isoform inhibition yields a monophasic rise. The effects of ouabain-mediated increases in IP3 production or IP3R conductance on [Ca2+](cyt) depend on their relative distributions between cellular microdomains and the bulk cytoplasm. Simulations suggest that the intracellular localization of IP3 production is a pivotal determinant of the changes in compartmental Ca2+ concentrations that can be induced by ouabain. As a consequence of sequestration of the ouabain-sensitive alpha2-isoform into microdomains, inhibition of the alpha2-isoform in rodents is not predicted to significantly affect cytosolic Na+ concentration. Model simulations support the hypothesis that ouabain can enhance agonist-evoked [Ca2+](cyt) transients when its predominant effect is to inhibit alpha2-isoform Na+ transport and, thereby, increase Ca2+ loading into sarcoplasmic reticulum stores.

Erythrocyte sodium pump stimulation by ouabain and an endogenous ouabain‐like factor

Cardiac glycosides inhibit the sodium pump. However, some studies suggest that nanomolar ouabain concentrations can stimulate the activity of the sodium pump. In this study, using the Na(+)/K(+)-ATPase of human erythrocytes, we compared the effect of digoxin, ouabain and an ouabain like-factor (OLF), on (86)Rb uptake. Ouabain concentrations below 10(-9) M significantly stimulate Rb(+) uptake, and the maximal increase above base-line values is 18 +/- 5% at 10(-10) M ouabain. No stimulation is observed in the same conditions by digoxin. OLF behaved like ouabain, producing an activation of Rb(+) flux at concentrations lower than 10(-9) M ouabain equivalents (14 +/- 3% at 10(-10) M). Western blot analysis revealed the presence of both alpha(1) and alpha(3) pump isoforms in human erythrocytes. Our data confirm the analogies between OLF and ouabain and suggest that Na(+)/K(+)-ATPase activation may be related to the alpha(3) isoform. In addition, we investigated whether ouabain at different concentrations was effective in altering the intracellular calcium concentration of erythrocytes. We found that ouabain at concentration lower than 10(-9) M did not affect this homeostasis.

Endogenous Ouabain

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. 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. 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.

Ouabain-like factor and acute salt loading in low-renin hypertension

OR-39 Key Words: Ouabain-Like-Factor, Salt, Renin

Elevated Blood Levels of Endogenous Ouabain-Like Factor in Preterm versus Mature Newborns at Birth

Compared to healthy adults, elevated levels of endogenous ouabain-like factor (OLF) have already been shown in the cord blood, and a role of OLF in the maintenance of high Na⁺ excretion by reducing tubular sodium reabsorption during intrauterine life was suggested. In this study, we aimed to measure OLF cord blood levels of premature and mature newborns to provide further data on the possible physiological significance of this compound in neonates. OLF was assessed in the cord blood of newborns (28–41 weeks) and in the blood of healthy adults using ouabain radioimmunoassay. HPLC was employed to isolate endogenous OLF. Newborns had about twelve times higher OLF levels than healthy adults (41.96 ± 4.64 vs. 3.1 ± 1.1 pg/ml, p < 0.001). Further, there was a highly significant correlation (p < 0.004) between maturity and OLF concentration; OLF level increased with gestational age, however there was a rapid drop in its concentration at week 39 (43.26 ± 7 vs. 35.47 ± 1.84 pg/ml, p = NS). Further studies are needed to evaluate the physiological relevance of higher OLF in preterm versus mature newborns.

Endogenous ouabain-like factor (OLF) secretion is modulated by nicotinic mechanisms in rat adrenocortical cells

This study tested the hypothesis that rat adrenocortical secretion of endogenous ouabain-like factor (OLF) is regulated by nicotinic mechanisms. OLF secreted by dispersed cell suspensions of zona glomerulosa (ZG) and fasciculata/reticularis (ZFR) cells was found to co-elute with authentic ouabain by reverse phase HPLC; OLF concentrations in cell supernatants were measured by radioimmunoassay. Nicotine (10 −6 − 10 −3 M) stimulated significant OLF secretion in rat adrenocortical cells. Acetylcholine (10 −7 − 10 −4 M) and eserine (10 −7 − 10 −3 M) stimulated OLF secretion in ZG cells at lower concentrations and stimulated at higher concentrations. Acetylcholine had no effect on ZFR secretion of OLF, but eserine stimulated OLF secretion. ACTH (10 −8 M) strongly potentiated the OLF stimulatory effect of nicotine in ZG cells; however significant interactions between nicotine and ACTH or angiotensin II on OLF secretion in ZFR cells were not apparent. The ganglionic blockers hexamethonium and mecamylamine further potentiated the effect of nicotine, implicating nicotinic acetylcholine receptors (nAChRs) in regulation of OLF secretion. The α7-receptor antagonist methyllycaconitine (MLA) dose-dependently inhibited the effect of nicotine in the ZG cells, and in ZFR cells MLA potentiated nicotine-induced OLF secretion. These data suggest that nicotinic regulation may underlie OLF secretion by rat adrenocortical cells, and strongly suggest presence of functional nicotinic acetylcholine receptors on these cells.

Quantification of alpha-subunit isoforms of Na,K-ATPase in rat resistance vessels.

Rat mesenteric resistance vessels (RV) were characterized with respect to concentration of individual alpha-subunit isoforms of Na,K-ATPase. Total vessel homogenates were used to avoid any loss or subfractionation of membranes. They were applied to sodium dodecyl sulphate gels and, for calibration, in parallel lanes were run purified rat Na,K-ATPase preparations with known isoform distribution and content. The capacity per mg protein for Na+-dependent 32P-phosphorylation of Na,K-ATPase isolated from rat kidney was used for alpha1 calibration and that for high-affinity (3H)ouabain binding of Na,K-ATPase isolated from rat brain was used for (alpha2 + alpha3) calibration. Western blots containing homogenate proteins and reference enzyme were incubated with isoform-specific antibodies and radiolabelled secondary antibodies. The signals from adjacent alpha spots were used for qualitative and quantitative characterization of rat vessels. A concentration of 100.7 +/- 14.4 pmol (n = 11) per g wet weight of the alpha1-isoform containing Na,K-ATPase was found in RV from 12-14-week rats. A much lower and more unreliable content of alpha2- and alpha3-isoforms was found. These ouabain-sensitive isoforms seem to represent a maximum of 5-10% each compared with the ouabain-insensitive rat alpha1-isoform. The isoform pattern in RV, in which the isoform with high/intermediate Na+-affinity is the absolutely dominating one representing nearly all sodium pumps in this tissue, is very different from that seen in rat skeletal muscles. Due to the high content of the ouabain-insensitive alpha1-isoform in rat RV this species would seem a less relevant model in studies addressing a role of cardiac glycosides and putative endogenous ouabain-like factors in hypertension.

Effect of canrenone on the digitalis site of Na+/K(+)-ATPase in human placental membranes and in erythrocytes.

It has been reported that canrenone, which is used in hypertensive therapy as an antialdosteronic drug, may also act as a blocker of ouabain effects. Several studies suggest that human plasma contains an endogenous ouabain-like factor similar to ouabain, which may be increased in hypertension, in pregnancy, and in the neonatal state. This study evaluated (1) the effect of canrenone on Na+/K(+)-ATPase in relation to ouabain in human placental membranes and erythrocytes by 3H-ouabain binding assay; (2) the capacity of canrenone (10 microM) to reverse the inhibition of Na+/K(+)-ATPase by ouabain and by ouabain-like factor (from umbilical cord plasma) in human erythrocytes employing a 86Rb uptake assay. Increasing concentrations of canrenone (0-350 microM) partially competed with 3H-ouabain binding in placental membrane (40%) and erythrocytes (60%). Scatchard plot from radioreceptor assay in placental membrane showed that ouabain and canrenone compete for the same binding site. In erythrocytes, canrenone completely reversed the inhibition caused by ouabain (5 x 10(-9) M) and ouabain-like factor (2 x 10(-9) M ouabain equivalents). A reduction of inhibition of about 50% was observed with ouabain and ouabain-like factor respectively at a concentration of 5 x 10(-8) M and 2 x 10(-8) M (ouabain equivalents). Our results thus provide evidence that canrenone, at therapeutical concentrations, is a partial competitive agonist of ouabain and of ouabain-like factor in human placental membranes and erythrocytes.

Human Adrenal Cells in Culture Produce Both Ouabain-like and Dihydroouabain-like Factors

Ouabain-like factor (OLF) and its newly discovered reduced species, dihydroouabain-like factor (Dh-OLF), are mammalian cardenolides whose structural and functional characteristics are similar to the plant-derived compounds ouabain and dihydroouabain. These endogenous compounds are believed to be produced by the adrenals and to constitute part of an hormonal axis that may regulate the catalytic activity of the alpha-subunit of Na(+),K(+)-ATPase. We developed antibodies sufficiently specific to distinguish between OLF and Dh-OLF, and in this study demonstrate the selective secretion of OLF and Dh-OLF from human H295R-1 adrenocortical cells in culture. We used reversed-phase HPLC, inhibition of Na(+),K(+)-ATPase catalytic activity, and two enzyme immunoassays developed with antibodies specific to ouabain and dihydroouabain to purify and characterize the secretion of these two compounds by human adrenal cells in culture. Purified antisera had high titers (1 x 10(6) for ouabain and 8 x10(5) for dihydroouabain) and were specific to their corresponding antigens. Human H295R-1 cells grown in serum-free medium secreted 0.18 +/- 0.03 pmol of OLF and 0.39 +/- 0.04 pmol of Dh-OLF per 10(6) cells in 24 h. Both OLF and Dh-OLF inhibited the ouabain-sensitive catalytic activity of the sodium pump (0.03 micro mol/L OLF inhibited 29% of the catalytic activity; 0.07 micro mol/L Dh-OLF inhibited 17%). Stimulation of the cell culture by dibutryl cAMP increased the secretion of Dh-OLF 50% over control (unstimulated), whereas the secretion of OLF did not increase significantly. OLF and Dh-OLF are secreted by human adrenal cells, and antibodies specific to these two compounds can be developed, using the plant-derived counterparts as antigens. The secretion of Dh-OLF is responsive to a cAMP-dependent stimulation mechanism, whereas OLF is not. Our data suggest that either the secretory or biosynthetic pathways for production of these two compounds by human adrenal cells may have different control mechanisms or that they may be linked via a precursor-product relationship.

Endogenous ouabain-like factor predicts progression toward heart failure in patients with idiopathic dilated cardiomyopathy

Endogenous ouabain-like factor predicts progression toward heart failure in patients with idiopathic dilated cardiomyopathy