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PubMed1991

Binding properties and biological effects of oxidized-ouabain on cultured neonatal-rat cardiac myocytes. Implications on the mechanism of action of the digitalis-glycosides.

Mild oxidation of ouabain with NaIO4, causes the cleavage of the bond between C2' and C3' of the rhamnose ring, leaving the steroid moiety intact. The oxidized ouabain (ox-ouabain) was examined on spontaneously contracting cultured rat-cardiac myocytes. Two classes of binding sites, with high and low affinities, were detected for both ox-ouabain and unmodified ouabain. The dissociation constants (KD) were found to be similar for both compounds, but the rate constants of association (ka) and dissociation (kd) of the low affinity sites were higher for ox-ouabain as compared with ouabain. Displacement experiments showed that ox-ouabain and ouabain bind to the same sites. The effects of ox-ouabain and ouabain on the activity of Na+, K(+)-ATPase were determined in microsomal preparations. Similar dose-response curves for the inhibition of the enzyme activity were determined for both drugs. Inhibition was observed only at concentrations above 10(-6) M. The biological effects of the drugs were examined by their capacity to induce positive inotropic or toxic effects. Concentrations of ox-ouabain which induced positive inotropic effects (increase in amplitude of systolic cell motion), ranged from 5 x 10(-8) M to 5 x 10(-6) M, as compared with 10(-7) M to 5 x 10(-7) M with ouabain. "Toxic" effects (decrease in the amplitude of systolic motion, increased beating frequencies and elevation in the position of maximal relaxation) was observed only with 10(-5) M ox-ouabain as compared with 10(-6) M ouabain. The mechanism of the inotropic action of ox-ouabain at the lower concentration range was investigated by measuring the effect of the drugs on 86Rb+ (analogue of K+) influx. Dose-response curves of effects of ouabain and ox-ouabain on 86Rb+ influx were bi-phasic. At low concentrations stimulation was observed, whereas at high concentrations 86Rb+ influx was inhibited. Ox-ouabain stimulated 86Rb+ influx by lower concentrations and to a greater extent than ouabain. A part of 86Rb+ influx into cardiac myocytes is mediated by the K+/Na+/Cl- cotransporter, which can be inhibited by loop diuretic drugs such as bumetanide. We have previously shown that ouabain, at low concentrations, stimulates the activity of the cotransporter. It is shown in the present work that ox-ouabain stimulates the activity of the cotransporter by lower concentrations and to a greater extent than ouabain.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1969

The ouabain-sensitive fluxes of sodium and potassium in squid giant axons.

1. Fifty to ninety per cent of the Na efflux from axons of Loligo forbesi is inhibited by ouabain. The properties of the ouabain-sensitive component of the Na efflux are different from those of the ouabain-insensitive component.2. In unpoisoned axons with an average Na content of 75 m-mole/kg axoplasm the bulk of the ouabain-sensitive Na efflux is dependent on external K.3. In the presence of 460 mM Na in the external medium, raising the external K concentration from 0 to 100 mM increases the ouabain-sensitive Na efflux along a sigmoid curve which shows signs of saturating at high K concentrations.4. The curve relating ouabain-sensitive K influx to external K concentration is similar in shape to that for the ouabain-sensitive Na efflux. At all K concentrations examined the ouabain-sensitive K influx was less than the ouabain-sensitive Na efflux.5. Potassium-free sea water acts rapidly in reducing the Na efflux. There is no appreciable difference between the rates of action of K-free sea water on the Na pump and Na-free sea water on the action potential.6. Caesium and Rb can replace external K in activating the ouabain-sensitive Na efflux. Both the affinity and maximum rate of the Na efflux mechanism are lower when Cs replaces K as the activating cation.7. Isosmotic replacement of external Na by either choline or dextrose, but not Li, increases the affinity of the ouabain-sensitive Na efflux mechanism for external K without appreciably affecting the maximum rate of pumping. External Li behaves like external Na and exerts an inhibitory action on the Na efflux.8. There is a large ouabain-sensitive Na efflux into K-free choline or dextrose sea waters. Addition of either Na or Li to the external medium reduces this efflux along a section of a rectangular hyperbola. The properties of this efflux suggest that there is a residual K concentration of up to 2 mM immediately external to the pumping sites in the axolemma.9. Over the range of internal Na concentrations studied (16-140 m-mole/kg axoplasm) the ouabain-sensitive Na efflux increased linearly with Na concentration.10. Tetrodotoxin (10(-6) g/ml.) reduces the Na influx by about half, but does not affect the ouabain-sensitive Na efflux.11. Isobutanol (1% v/v) reversibly decreases both the ouabain-sensitive and ouabain-insensitive components of the Na efflux.12. Application of 2 mM cyanide to axons immersed in K-free sea water produces a transient rise in the Na efflux. This rise is not seen if ouabain is included in the sea water. The rise in efflux occurs at a time when the axons are partially poisoned and contain adenosine triphosphate (ATP) but no arginine phosphate (ArgP). A similar, but maintained rise can be obtained after application of dinitrophenol (DNP) at pH 8.0. The increased Na efflux in these partially poisoned axons is also inhibited by ouabain.13. Under conditions of partial-poisoning by alkaline DNP, there is a ouabain-sensitive Na influx from K-free sea water. The ouabain-sensitive Na influx is of similar size to the ouabain-sensitive Na efflux. These results show that in partially-poisoned axons immersed in K-free sea water intracellular Na exchanges with extracellular Na in a one-for-one manner by a ouabain-sensitive route. External Li cannot replace external Na in maintaining this process.14. Axons partially poisoned with alkaline DNP are not insensitive to external K. In the absence of external Na their response to external K is essentially the same as that seen in unpoisoned axons.15. Possible mechanisms are discussed for the appearance of Na-Na exchange in partially poisoned axons.

Animals↗
PubMed1996

Ouabain production by cultured adrenal cells.

The adrenal cortex releases a sodium pump inhibitor. The present studies tested whether this material was endogenous and identical to ouabain by 1) studying the production of ouabain in long term cultures of adrenocortical cells, 2) seeking evidence that ouabain might be taken up from exogenous sources by adrenocortical cells, 3) examining the release of adrenocortical cells loaded with exogenous ouabain, 4) attempting to stimulate ouabain steroidogenesis in cultured adrenocortical cells, and 5) performing further chemical analysis on ouabain immunoreactivity released by cultured adrenocortical cells. Our results indicate that ouabain immunoreactivity is present in conditioned medium from both murine Y-1 adrenocortical cultures and primary bovine adrenocortical cell (BAC) cultures. We also found that BACs bind and internalize [3H]ouabain. Bound [3H]ouabain is released from BACs by both receptor dissociation and cytoplasmic release of internalized [3H]ouabain. Only one isoform of membrane sodium, potassium-adenosine triphosphatase, alpha 1, was expressed in the adrenal. Authentic ouabain was not metabolized during membrane binding or while present intracellularly. Stimulation of steroidogenesis in Y-1 and BAC with 22R-hydroxycholesterol and 25-hydroxycholesterol was performed and confirmed increased steroidogenesis; however, there was no effect on ouabain immunoreactivity content or release. Comparison of the ouabain binding density in cultured BAC, hepatoma cells, and 3T3 fibroblasts indicated that adrenocortical cells have a high ouabain-binding capacity. HPLC studies of the ouabain immunoreactivity released by bovine adrenocortical cells indicated that essentially no authentic ouabain was secreted. The present studies confirm that both BAC and Y-1 cultures release a ouabain-like material that differs in structure from authentic plant ouabain and is not a product of cholesterol side-chain cleavage.

Adrenal Cortex↗
PubMedDate not supplied

Influence of pretreatment with phenytoin, lidocaine and quinidine on the cardiodynamic and electrophysiological effects of ouabain in the cat heart-lung preparation.

The effect of pretreatment with phenytoin (diphenylhydantoin), lidocaine, and, for comparison, quinidine, on the doses of ouabain which produce a maximal inotropic effect, onset of arrhythmias and cardiac arrest, was explored in the cat heart-lung preparation. Ouabain was administered as an infusion (0.5 micrograms/min) either alone or after treatment with phenytoin (0.095 +/- 0.012 mM), lidocaine (0.090 +/- 0.004 mM) or quinidine (0.028 +/- 0.006 mM) and the cardiodynamic and electrophysiological changes monitored. Phenytoin, lidocaine and quinidine were administered in doses which were maximally tolerated by the preparations to ensure full effect, as evidenced by early cardiac depression. Ouabain alone produced a maximal increase in contractility prior to the development of arrhythmias at a blood concentration of 0.212 +/- 0.014 microM, onset of arrythmias at 0.227 +/- 0.015 microM, stable ventricular tachycardia at 0.269 +/- 0.010 microM and cardiac arrest at 0.342 +/- 0.014 microM. Pretreatment with phenytoin or lidocaine did neither modify these values nor change the pattern of the arrhythmias or the terminal cardiac event. Pretreatment with quinidine prevented the development of ventricular extrasystoles and aberrant ventricular conduction, which were the earliest arrhythmias in all other series. It also made the preparations develop stable ventricular tachycardia at an ouabain blood concentration of 0.246 +/- 0.007 microM, which was not significantly different from the concentration at which early arrhythmias were noted in the other series. In addition, quinidine decreased the dose of ouabain producing cardiac arrest by 13% but did not modify the terminal event. Pretreatment with phenytoin, lidocaine and quinidine did not affect the electrocardiographic pattern, but at the maximal increase in contractility with ouabain prior to the development of arrhythmias, the PR interval increased to comparable limits with ouabain alone and ouabain after quinidine and lidocaine. However, with ouabain after phenytoin, this increase was 61% less than that with ouabain alone and 31% less than that with ouabain after quinidine. Ouabain given alone or after phenytoin, lidocaine or quinidine produced comparable maximal effects on dp/dt, -dp/dt and left atrial pressure. It may be concluded that pretreatment with phenytoin and lidocaine does not modify the maximal inotropic dose of ouabain prior to the development of arrhythmias, the arrhythmogenic dose or the dose producing cardiac arrest, and that phenytoin partly counters the ouabain-induced depression of AV conduction. Quinidine has an additive effect on the ouabain-induced depression of AV conduction, prevents the ouabain-induced increase in idioventricular rhythm responsible for extrasystoles but not that responsible for ventricular tachycardia, and reduces the dose of ouabain producing cardiac arrest.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

In vivo assessment of the inotropic and toxic effects of oxidized ouabain.

Oxidized ouabain, a product of the oxidative cleavage of the rhamnose ring in ouabain has been found to have a higher inotropic toxic ratio in cultured cardiac myocytes. The purpose of our study was to evaluate the efficacy and toxicity of oxidized ouabain in comparison with ouabain in intact animals. Drugs were infused to healthy cats; the positive inotropic effect, and the time-course of development of arrhythmia were followed and recorded until death. Oxidized ouabain was associated with a higher increase in arterial blood pressure, a mean increase of 41 +/- 19% as compared with 21 +/- 8% in the ouabain group (p < 0.10). There were no significant differences in maximal increases of dP/dt or dP/dt/P (65 +/- 29%, 28 +/- 10% for oxidized ouabain and 49 +/- 16%, 27 +/- 11% for ouabain, respectively). The mean doses causing persistent arrhythmia (toxic dose) were 93 +/- 23 micrograms/kg of oxidized ouabain vs 39 +/- 14 micrograms/kg of ouabain. Lethal arrhythmias were produced by 215 +/- 46 micrograms/kg of oxidized ouabain and 62 +/- 16 micrograms/kg of ouabain. The ratio of toxic to lethal doses was 0.62 +/- 0.11 for ouabain vs 0.45 +/- 0.09 for oxidized ouabain (p < 0.05), but the inotropic to toxic dose ratios were not different. We conclude that oxidized ouabain acts similarly to the known cardiac glycosides in doses which produce inotropic effects in cats, has a lower potency as compared to ouabain, and appears to have a more benign course of intoxication.

Animals↗
PubMed1990

Role of Na-H exchange in the inotropic action of Bay K 8644 and of ouabain in guinea-pig isolated atria.

1. The inotropic effects of two concentrations of ouabain and of Bay K 8644 have been studied in isolated left atria of the guinea-pig in physiological solutions at pH lowered from 7.4 to 6.0 and in the presence of ethylisopropylamiloride (EIPA) an inhibitor of Na+/H+ exchange. The low concentration of ouabain (300 nM) was chosen to saturate the high affinity binding sites (it occupied about 7% of the low affinity sites). The high concentration of ouabain saturated both high and low binding sites. Bay K 8644 evoked a positive inotropic effect of a magnitude similar to ouabain (300 nM). 2. When comparing the positive inotropic effects of equi-effective concentrations of ouabain (300 nM) and of Bay K 8644 (100 nM), it was observed that extracellular acidification specifically depressed the inotropic effect of ouabain 300 nM; the positive inotropic effect of the high concentration of ouabain (3 microM) was barely affected by extracellular acidification. 3. EIPA 10 microM depressed the positive inotropic effect of ouabain 300 nM, but did not affect the peak response to Bay K 8644. The depressant action of EIPA on the positive inotropic effect of ouabain was concentration-dependent and was much more obvious on the effect of ouabain 300 nm than on ouabain 3 microM. 4. An increase in diastolic tension was evoked by 3 microM but not by 300 nM ouabain. This increase in tone was reduced dose-dependently by EIPA (10-30 microM). It was also significantly reduced when the extracellular pH was equal to 6.4 or 6.0. 5. Ouabain (300 nM) evoked a gain in tissue Na and an equivalent loss in tissue K. Acidification of the extracellular pH down to pH 6.0 evoked a pH-dependent reduction of Na gain but left K loss unaltered. EIPA 10, 20 and 30 microM evoked a significant reduction of Na gain without significantly affecting K loss. 6. Ouabain (3 microM) evoked a large gain in tissue Na and an equivalent loss of K. Tissue Ca content was also increased. Acidification of the extracellular pH from pH 7.4 to pH 6.9 evoked a significant reduction of Na and Ca gain; changes in tissue K were not significant. Acidification down to pH 6.0 increased reduction of Na and Ca gain, but not that of K loss which nevertheless became statistically significant. 7. These results show that tissue Na gain observed after inhibition of the sodium pump by ouabain may be related to activation of Na-H exchange. They also indicate that blockade of Na-H exchange selectively reduced the inotropic effect of ouabain in guinea-pig atria resulting from interaction of the glycoside with high affinity binding sites. This confirms previous observations in rat heart.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Endogenous ouabain, sodium balance and blood pressure: a review and a hypothesis.

OBJECTIVE: To assess possible relationships between endogenous ouabain, sodium balance and blood pressure. CONTENT: This review concerns the structure of endogenous ouabain, circulating levels of this steroid in various disorders of fluid and electrolyte balance, recent evidence for the association of endogenous ouabain with human hypertension, the influence of sodium and volume factors on ouabain-induced hypertension, and possible mechanisms for the hypertensinogenic activity of ouabain. CONCLUSIONS: The human circulation contains a closely related isomer of ouabain of putative adrenocortical origin. Elevated circulating levels of this 'endogenous ouabain' are common but not universal in physiologic and pathologic states associated with positive sodium balance or high blood pressure, or both. In the absence of adrenal hyperfunction, elevating circulating levels of endogenous ouabain appear to be secondary to impaired renal clearance. Prolonged elevation of circulating ouabain in the rat induces sustained hypertension. This model exhibits normal plasma renin activity, increased levels of ouabain in the hypothalamus, pituitary, and kidney, and responds to angiotensin converting enzyme inhibitor. In rats with normal kidney function, ouabain-induced hypertension is primarily sodium-insensitive although maneuvers that hinder renal sodium excretion augment the pressor effect of this steroid. Prolonged administration of ouabain into the brain ventricles augments sympathetic nervous system activity and induces sustained hypertension. These observations lead us to propose the following hypothesis. Among Caucasian patients with essential hypertension, a large fraction have elevated circulating levels of endogenous ouabain, possibly caused by an inherited or acquired renal defect in clearance of this steroid. In these patients, and in rats with ouabain-induced hypertension, increased local generation of, or increased target organ sensitivity to, angiotensin II, or both, may contribute critically to heightened vasoconstriction and a sustained increase in blood pressure. Investigations of the efferent pressor mechanisms and the renal handling of endogenous ouabain are novel approaches to the etiology and therapy of several common cardiovascular disorders.

Adrenal Gland Neoplasms↗
PubMed2006

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells.

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 Ser(473) phosphorylation, as evidenced by an increase in phospho-Akt Ser(473) band density. Ouabain-stimulated Akt Ser(473) 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 Ser(473) 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 Ser(473) 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 (86)Rb 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.

Animals↗
PubMed2004

The alpha2-isoform of Na-K-ATPase mediates ouabain-induced hypertension in mice and increased vascular contractility in vitro.

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 alpha2-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 alpha2-isoform is sensitive to ouabain, and genetically engineered mice expressing a ouabain-insensitive alpha2-isoform of the Na-K-ATPase. Thus differences in the response to ouabain between these two genotypes can only be attributed to the alpha2-isoform of Na-K-ATPase. As the alpha1-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 alpha2-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 alpha2-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.

Anesthesia↗
PubMed1994

Is ouabain an authentic endogenous mammalian substance derived from the adrenal?

Ouabain has recently been reported to be an endogenous mammalian substance released by the adrenal cortex and present in normal plasma. We have attempted to confirm and extend this observation. Using a ouabain radioimmunoassay developed in this laboratory, we fractionated by high-performance liquid chromatography (HPLC) normal human plasma from healthy volunteers to determine the presence of ouabain immunoreactivity and compare this immunoreactivity with authentic ouabain. In most subjects no ouabain immunoreactivity that coeluted with authentic ouabain was observed. Some subjects had ouabain-immunoreactive material present at low levels, but it was largely attributable to cross-reactivity with diverse substances found not to be ouabain. Similar results were obtained after analysis of plasma collected from 10 patients entering a medical intensive care unit. Studies of serum-free medium conditioned by bovine adrenocortical cells showed some ouabain immunoreactivity. To determine whether this material might be a steroid product of cholesterol side-chain cleavage, we performed chemical blockade of steroidogenesis, which effectively suppressed progesterone production by these cells but had no consistent effect on ouabain immunoreactivity in this medium. Stimulation of steroidogenesis with 22-R-OH-cholesterol in bovine adrenocortical cells did not produce any increase in the ouabain immunoreactivity present in conditioned medium. Subsequent HPLC studies of ouabain immunoreactivity in bovine adrenocortical cell-conditioned medium indicated that authentic ouabain did not account for most of the ouabain immunoreactivity in serum-free medium. Studies with bovine adrenocortical cells incubated in a minimal salt and glucose medium indicated a small peak of immunoreactivity that may correspond to authentic ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1999

Digoxin prevents ouabain and high salt intake-induced hypertension in rats with sinoaortic denervation.

Digoxin prevents ouabain-induced hypertension in rats. In the present study, we tested whether this effect of digoxin depends on its sensitizing effect on baroreflex function or is due to an antagonistic action on exogenous ouabain or endogenous ouabain-like activity ("ouabain") in the brain. In Wistar rats, resting mean arterial pressure (MAP) was significantly increased by long-term subcutaneous (SC) ouabain (75 microg/d) plus high salt (8%) intake for 12 days (but not after only 5 days). In rats with chronic sinoaortic denervation (SAD), MAP was increased within 5 days of ouabain treatment to the same extent as MAP after 12 days of treatment in intact rats. The effect of ouabain and high salt was prevented when digoxin was given SC concomitantly via osmotic minipump (200 microg x kg(-1) x d(-1)). Resting MAP was not changed in rats treated with digoxin alone. In a second set of rats with chronic SAD or sham surgery, high salt intake was given for 14 days, with or without SC digoxin (200 microg x kg(-1) x d(-1)) or intracerebroventricular (ICV) antibody Fab fragments (200 microg/d), which bind "ouabain" with high affinity. On day 14, MAP, central venous pressure, heart rate, and renal sympathetic nerve activity were recorded in conscious rats at rest and in response to air-jet stress, IV phenylephrine and nitroprusside, and acute volume expansion with 5% dextrose IV. In rats with SAD versus sham surgery, high salt significantly increased resting MAP as well as excitatory responses of MAP, heart rate, and renal sympathetic nerve activity to air stress. These effects of high salt in rats with SAD were prevented by digoxin or Fab fragments. Arterial baroreflex function was blunted but cardiopulmonary baroreflex function was not affected in rats with SAD. Digoxin and Fab fragments had no effects on either function. In an in vitro assay for the inhibitory effects on Na+, K(+)-ATPase activity, 20 ng of ouabain caused 29% inhibition, but 20 ng of ouabain plus 13 or 53 ng of digoxin caused only 16% or 4% inhibition, respectively. These data indicate that the arterial baroreflex opposes sympathoexcitatory responses to ouabain and "ouabain" in the brain, thereby delaying ouabain- and preventing high salt-induced hypertension in Wistar rats. In addition to possible effects on the arterial baroreflex, digoxin appears to act centrally to prevent the sympathoexcitatory and pressor effects of increased brain "ouabain" or ouabain.

Animals↗