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PubMed1989

The protective action of R56865 against ouabain-induced intoxication in rat heart isolated atria and ventricles.

The action of R56865 has been examined on the contractile effects produced by ouabain concentrations interacting with high (3 microM) and low (300 microM) affinity digitalis receptors on electrically stimulated ventricular strips isolated from rat. R56865 1 microM reduced the increase in resting tension produced by ouabain 300 microM and left unalterated the inotropic effect evoked by ouabain 3 and 300 microM that was reduced by higher concentrations (3 and 6 microM) of R56865. The action of R56865 was also studied on ouabain-induced intoxication in electrically stimulated and spontaneously beating atria of rat. On electrically stimulated (3 Hz) whole atria, R56865 0.3 microM reduced the maximal increase in resting tension produced by ouabain 300 microM and delayed the time to onset of the ouabain-induced arrhythmias but did not affect ouabain's inotropic effect. Higher concentrations of R56865 were required to reduce the inotropic effect of ouabain. The protective action of R56865 against ouabain-induced intoxication was most pronounced on spontaneously beating atria where it reduced spontaneous rate of beats. Experiments in electrically driven left atria indicated that only a part of the protective effect of R56865 could be related to its bradycardic action. The effect of R56865 was also examined on ouabain-induced inhibition of sodium pump in human red blood cells. R56865 6 microM did not modify the inhibition produced by ouabain (from 0.3 to 10 nM), this indicates that the protective action of R56865 against ouabain-induced intoxication is not due to an interaction with the inhibitory effect of ouabain on sodium pump.

Animals↗

PubMed1998

Persistent hyperactivity following a single intracerebroventricular dose of ouabain.

Intracerebroventricular (i.c.v.) administration of ouabain has been shown to alter motor activity in the rat. It has been purported that this may model the behavioral abnormalities of human manic-depressive (bipolar) illness. Since manic-depression is a recurrent condition, we elected to investigate the effects of the multiple administration of i.c.v. ouabain. Male Sprague-Dawley rats were allowed to acclimate to the animal facility for 7-10 days after which time i.c.v. cannulae were placed. Animals received two i.c.v. injections of either ouabain (10[-3] M) or artificial cerebrospinal fluid (aCSF) 9 days apart, so that 6 rats received aCSF-aCSF, 6 received ouabain-aCSF, and 6 received ouabain-ouabain. Behavioral activity was evaluated in an open field (86 x 86 cm subdivided into sixteen 21.5 x 21.5-cm squares) for 20 min at baseline and immediately following each i.c.v. injection. After the last behavioral test, the animals were killed, and the brains were rapidly harvested and dissected over ice. Specific ouabain binding and sodium pump activity were determined. A single dose of ouabain produced a marked increase (297.0%, p = 0.002) in open field activity compared to both baseline behavior and to aCSF injected animals. The effects of ouabain appeared to last for 9 days. A second i.c.v. injection of either ouabain (136.5 +/- 60.4 SEM) or aCSF (108.0%, p < 0.01) had no effect on the activity level which was intermediate between the initial ouabain hyperactivity and the baseline level. Nine days after ouabain administration, hippocampal ouabain binding was increased relative to the control group (5477 +/- 485.7 vs. 3579 +/- 518.6, p < 0.05) and sodium pump activity was relatively lower (2293.8 +/- 265.5 vs. 3174.2 +/- 410.5, p < 0.05).

Animals↗

PubMed1999

Spinal antinociceptive action of Na+-K+ pump inhibitor ouabain and its interaction with morphine and lidocaine in rats.

BACKGROUND: The Na+,K+-adenosine triphosphatase is a ubiquitous enzyme system that maintains the ion gradient across the plasma membrane of a variety of cell types, including cells in the central nervous system. We investigated the antinociceptive effect of intrathecally administered ouabain and examined its potential interaction with spinal morphine and lidocaine. METHODS: Using rats chronically implanted with lumbar intrathecal catheters, the ability of intrathecally administered ouabain, morphine, and lidocaine and of mixtures of ouabain-morphine and ouabain-lidocaine to alter tail-flick latency was examined. To characterize any interactions, isobolographic analysis was performed. The effects of pretreatment with intrathecally administered atropine or naloxone also were tested. RESULTS: Intrathecally administered ouabain (0.1-5.0 microg), morphine (0.2-10.0 microg), and lidocaine (25-300 microg) given alone produced significant dose- and time-dependent antinociception, but systemic administration of ouabain did not produce such an effect. The median effective dose (ED50) values for intrathecally administered ouabain, morphine, and lidocaine were 2.3, 5.0, and 227.0 microg, respectively. Isobolographic analysis exhibited a synergistic interaction after the coadministration of ouabain and morphine. With ouabain and lidocaine, there was no such evidence of synergism. Intrathecally administered atropine, but not naloxone, completely blocked the antinociceptive effect of ouabain and attenuated its interaction with spinally administered morphine. CONCLUSIONS: Intrathecally administered ouabain produces antinociception, at least in part, via an enhancement of cholinergic transmission in the spinal nociceptive processing system. The results of the interaction of ouabain with morphine and lidocaine suggest that modulation of Na+-,K+-electrochemical gradients and thus subsequent release of neurotransmitters in the spinal cord are likely to play important roles in the spinal antinociceptive effect of intrathecally administered ouabain.

Analgesics↗

PubMed1996

Characterization of ouabain-like immunoreactivity in human urine.

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.

Adult↗

PubMed1980

On the release of catecholamines and dopamine-beta-hydroxylase evoked by ouabain in the perfused cat adrenal gland.

1 Secretion of catecholamines (CA) and dopamine-beta-hydroxylase (DBH) activity from the retrogradely perfused cat adrenal gland was studied following ouabain infusion. Perfusion with ouabain (10(-4) M) for 10 min caused a gradual release of CA in the effluent which reached its peak 30 min after the ouabain pulse, and was maintained constant for at least 1 h. The effect of ouabain seemed to be irreversible. 2 Mecamylamine, while blocking the CA secretory effects of acetylcholine (ACh) perfusion, did not affect the secretion of CA evoked by ouabain. In denervated adrenal glands, ouabain-induced CA secretion was similar to that in the contralateral, innervated gland. However, physostigmine perfusion potentiated the CA secretory effects of ouabain. 3 The release of CA evoked by ouabain was accompanied by a proportional release of DBH activity. The time course of appearance of DBH activity followed the pattern of CA release. 4 The CA and DBH outputs in response to a pulse of ouabain were suppressed in the absence of calcium. Calcium reintroduction to a calcium-free perfused, ouabain-treated gland not only restored but greatly potentiated the release of CA and DBH. The amplitude of the secretory response to calcium reintroduction in ouabain-treated glands was proportional to the extracellular calcium concentration, and was antagonized by an external sodium-deficient medium. 5 These data demonstrate that ouabain releases CA from the perfused cat adrenal gland by a calcium-dependent exocytotic mechanism. The secretory effect of ouabain is not secondary to the release of ACh from cholinergic nerve terminals present in the adrenal gland, but due to a direct action on the chromaffin cell itself. In addition, the results suggest that this action is exerted through redistribution of monovalent cations secondary to the inhibition by the glycoside of the sodium pump. Such monovalent cation redistribution may cause a rise of intracellular ionized calcium levels through the activation of an internal sodium-dependent calcium influx system probably located in the chromaffin cell membrane.

Adrenal Glands↗

PubMed1984

Action of ouabain on rat heart: comparison with its effect on guinea-pig heart.

The inotropic dose-response curve of ouabain in rat cardiac ventricular strips exceeded a concentration range of two decades (1 X 10(-7) M to 3 X 10(-5) M) displaying an intermediate plateau phase. In guinea-pig ventricular strips the inotropic ouabain concentrations spanned only one decade (1 X 10(-7) M-1 X 10(-6) M). Ouabain-intoxication in guinea-pig ventricular strips occurring at 3 X 10(-6) M consisted of arrhythmia and contracture, while in rat ventricular strips at the toxic concentration of 1 X 10(-4) M only a progressive increase in diastolic tension was observed. By means of atomic absorption spectroscopy the ouabain-induced loss of cellular potassium and gain of sodium in rat ventricular strips was detected only at concentrations of ouabain higher than 10(-4) M. Ouabain reduced the activity of Na/K-ATPase prepared from rat and guinea-pig cardiac ventricles to half of its maximum at 6.5 X 10(-5) M in rat and 1.0 X 10(-6) M in guinea-pig, rat heart Na/K-ATPase thus being about 60 fold less sensitive towards ouabain. Specific [3H]-ouabain binding to membrane suspensions prepared from rat and guinea-pig ventricles was characterized by a similar affinity in rat (KD = 4 X 10(-8) M) and guinea-pig (KD = 13 X 10(-8) M). The number of ouabain binding sites in rat membranes was only about 10% of the number found in guinea-pig membranes. In rat the presence of additional ouabain-binding with low affinity and high capacity seemed possible, but could not be verified for methodological reasons. In the light of the biochemical results and binding data, the wider range of ouabain concentration exerting a positive inotropic effect in the rat may be attributed to the existence in the latter of two populations of receptors with different affinities for ouabain and different capacities. In contrast, in the guinea-pig, there is a single population. Nevertheless it is probable that all the receptors in both species are part of the Na/K-ATPase complex and mediate a positive inotropic effect after ouabain-binding in an identical manner.

Animals↗

PubMed1972

Effect of prolonged ouabain treatment of Na, K, Cl and Ca concentration and fluxes in cultured human cells.

1. Girardi and Hela cells (derived from human heart and cervix respectively) were grown as monolayer cultures in B.M.E. (Eagles basal medium) containing concentrations of ouabain up to 5 x 10(-8)M for periods ranging up to 5 days. The cell sizes, numbers, Na, K, Cl, and Ca concentrations and fluxes were then measured.2. Twenty-four hours incubation in ouabain concentrations equal to or less than 5 x 10(-8)M caused a rise in [Na](i) and an almost equal fall in [K](i) to new steady levels. The concentrations so reached were linearly related to the ouabain concentrations, such that in 5 x 10(-8)M ouabain [Na](i) rose to 124 m-mole/l. intracellular water and [K](i) fell to 55 m-mole/l. i.c. water in Girardi cells. In Hela cells the changes were smaller at any particular ouabain concentration. These levels were maintained constant for at least 5 days.3. In cells in the logarithmic phase of growth, raising [Na](i) and lowering [K](i) by ouabain caused a slowing of growth rate proportional to the ouabain concentration used. In cells in the stationary phase there was no change in the cell numbers over 24 hr. The volume of the cells was not directly affected by the treatment.4. Reducing [K](o) from the normal value of 5.4 to 2.5 mM increased the effect of any ouabain concentration, whereas increasing [K](o) to 7.5 decreased the effect of ouabain.5. Reduction of [K](o) to 2.5 mM had no effect on the [K](i) or [Na](i) but halved the cell numbers, probably by a reduction in the growth rate. The mechanism of this effect is obscure.6. In Girardi cells raising [Na](i) and lowering [K](i) by prolonged treatment increases the total Na fluxes and decreases the total K fluxes but keeps the total Na + K flux constant. High-Na, low-K cells had a reduced Na:K exchange compared to fresh cells and also had a Na:K pumped ratio nearer 4:1 than the 3:2 normally found.7. These cells also show ouabain-sensitive and ouabain-insensitive Na:Na exchanges. In high-Na, low-K cells the ouabain sensitive Na:Na exchange is the same as in fresh cells. The effect of treatment on the ouabain insensitive Na:Na exchanges has not been elucidated.8. The Cl content and fluxes are not altered by prolonged ouabain treatment. From this it is inferred that the membrane potential in high-Na, low-K cells is the same as in normal cells.9. High-Na, low-K cells have the same calcium content and fluxes as fresh cells. From this it is concluded that there is no Na:Ca coupling in these cells.

Biological Transport↗

PubMed1993

Nanomolar ouabain augments caffeine-evoked contractions in rat arteries.

Chronic parenteral administration of ouabain to normal rats raises plasma ouabain concentrations to low nanomolar levels and induces hypertension [C. M. Yuan, P. Manunta, J. M. Hamlyn, S. W. Chen, E. Bohen, J. Yeun, F. J. Haddy, and M. B. Pamnani. Hypertension 22: 178-187, 1993 and see also M. P. Blaustein. Am. J. Physiol. 264 (Cell Physiol. 33): C1367-C1387, 1993]. To determine whether rat arteries are sensitive to these low ouabain levels, we tested the effects of various ouabain concentrations on caffeine-evoked contractions (CEC) in rat aortic and small mesenteric artery rings. CEC amplitude was used as a measure of the sarcoplasmic reticulum (SR) Ca2+ content. Ouabain increased CEC in aortic as well as mesenteric artery rings, but the effects in the aorta were difficult to quantitate because the CEC were often oscillatory. Mesenteric artery, under control conditions and after sensitization with 10-30 nM phenylephrine (PE), exhibited biphasic ouabain dose-CEC response curves. Low concentrations of ouabain (0.1-10 nM) caused small significant increases in CEC, but a further effect was observed only with > or = 10 microM ouabain. PE shifted the ouabain dose-response curve toward lower ouabain concentrations; conversely, ouabain shifted the PE dose-response curve toward lower PE concentrations. It appears that nanomolar concentrations of ouabain can influence vascular responsiveness to vasoconstrictors. We conclude that rat vascular smooth muscle contains both high- and low-affinity ouabain receptors, possibly corresponding to Na+ pumps with alpha 3- and alpha 1-subunit isoforms, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Elevated concentrations of endogenous ouabain in patients with congestive heart failure.

BACKGROUND: An endogenous digitalis-like compound in mammals has long been postulated, but only recently has a substance indistinguishable from ouabain been identified in human plasma. Because of the potential significance of such a substance in patients with congestive heart failure, we sought to evaluate the pathophysiology of endogenous ouabain in these individuals. METHODS AND RESULTS: Using an immunoassay, we determined plasma ouabain concentrations in 51 patients with heart failure and in 19 control subjects. Plasma ouabain concentrations in control subjects ranged from 0.16 to 0.77 nM (mean, 0.44 +/- 0.20 nM). In 19 matched heart failure patients receiving digoxin, the mean ouabain was significantly elevated at 1.59 +/- 2.2 nM (range, 0.17-8.76 nM, p less than 0.05 versus control subjects). The ouabain concentration correlated inversely with both cardiac index (r = -0.62, p less than 0.005) and mean arterial pressure (r = -0.51, p less than 0.05). However, there was no correlation between ouabain and left ventricular filling (r = 0.19, NS) or right atrial pressures (r = 0.20, NS). In 16 heart failure patients not receiving digoxin, the mean ouabain was 1.52 +/- 2.58 nM. No relation between renal function and ouabain was detected. CONCLUSIONS: The unanticipated lack of correlation of ouabain with atrial pressures indicates that volume is not the chief determinant of ouabain concentration in patients with congestive heart failure. However, the significant relations of plasma ouabain concentration with cardiac index and mean arterial pressure imply that endogenous ouabain may be an important homeostatic factor in humans.

Digoxin↗

PubMed1993

Long-term ouabain administration produces hypertension in rats.

Ouabain has recently been identified as an endogenous Na(+)-K+ pump inhibitor. We administered ouabain chronically to normotensive rats with varying degrees of reduced renal mass (RRM) and to normal two-kidney rats to see whether hypertension could be produced. Normal male Wistar rats and rats with 25%, 60%, and 70% RRM received ouabain (13.9 micrograms/kg per day IP) in normal saline for 4 weeks followed by ouabain (27.8 micrograms/kg per day IP) for 3 to 4 more weeks. Respective control animals received vehicle only. Blood pressure was recorded weekly by tail plethysmography. Animals received tap water and standard rat chow, except for 70% RRM rats, which received distilled water and sodium-free chow. After 6 to 8 weeks of treatment, with rats under thiobutabarbital anesthesia, direct blood pressure was determined. Plasma, tissue, and urinary ouabain levels were measured with a specific radioimmunoassay. Animals receiving ouabain developed significant increases in mean blood pressure compared with control animals (70% RRM, 147 +/- 4 vs 116 +/- 4 mm Hg; 60% RRM, 140 +/- 4 vs 107 +/- 3 mm Hg; 25% RRM, 131 +/- 5 vs 100 +/- 2 mm Hg; no RRM, 116 +/- 4 vs 98 +/- 5 mm Hg). Plasma ouabain levels measured 24 hours after the last ouabain dose were not different in animals receiving ouabain vs those receiving vehicle. However, kidney tissue ouabain levels were significantly greater (6.39 +/- 1.17 vs 2.36 +/- 0.52 micrograms/kg, P < .05) in animals receiving ouabain. In conclusion, ouabain, given chronically, is associated with the development of hypertension in RRM rats as well as in normal rats. Blood pressure was greater in animals with greater degrees of RRM for a given ouabain dose.

Animals↗

PubMed2000

Differential regulation of the sodium pump alpha-subunit isoform gene by ouabain and digoxin in tissues of rats.

The effects of ouabain and digoxin on both the systolic blood pressure (SBP) and sodium pump alpha-subunit expression in some tissues of rats were compared. Normal rats were injected with ouabain, digoxin, and normal saline (NS), respectively, everyday, and indirect SBP was recorded once a week. Six weeks later, all the rats were killed, and sodium pump alpha1-, alpha2-, and alpha3-subunit mRNA levels were detected in the myocardium, kidney, adrenal gland, aortic smooth muscle, and hypothalamus by the RT-PCR method. The results showed that the SBP of rats infused with ouabain increased significantly at the end of week 6, while no difference in SBP was found between the digoxin and NS groups. The effects of ouabain and digoxin on sodium pump alpha-subunit isoform expression were also different. Myocardium: both ouabain and digoxin stimulated expression of the alpha3-isoform whereas alpha2 was unchanged. Levels of the alpha1 isoform decreased significantly in the ouabain group and decreased slightly in the digoxin group, respectively. Kidney: digoxin had the same effects as ouabain. alpha1 levels increased, but those of alpha2 and alpha3 remained unchanged. Adrenal gland: alpha2 and alpha3 levels increased, but those of alpha1 decreased in the ouabain group. alpha1 and alpha3 levels increased and those of alpha2 remained unchanged in the digoxin group. Aortic smooth muscle: both ouabain and digoxin increased alpha1 and alpha3 expression. alpha2 levels decreased in the digoxin group but remained unchanged in the ouabain group. Hypothalamus: both ouabain and digoxin stimulated alpha1 expression, while alpha2 and alpha3 levels remained unchanged. The results of this study have shown that ouabain and digoxin have the different effects on both the systolic blood pressure and expression of sodium pump alpha-subunit isoforms in some tissues in rats. Further studies on the expression of sodium pump alpha-subunit isoforms might be helpful for the understanding of the physiological role of endogenous ouabain and the molecular mechanisms involved in the pathogenesis of hypertension.

Animals↗

PubMed1988

Reduction of doxorubicin cytotoxicity by ouabain: correlation with topoisomerase-induced DNA strand breakage in human and hamster cells.

The cardiac glycoside ouabain, which is a specific inhibitor of the Na+,K+-pump, confers dramatic protection from the cytotoxic effects of doxorubicin (Adriamycin). This effect was documented in cultured A549 cells (human lung adenocarcinoma). CCL210 cells (human fibroblasts), and V79 cells (hamster fibroblasts). Maximum protection from doxorubicin cytotoxicity was achieved using 1 microM ouabain for A549 and CCL210 cells and 300 microM ouabain for V79 cells. These concentrations correlated well with the concentrations of ouabain required to induce Na+,K+-pump blockade, which was assessed using the K+ analogue 86Rb+. This suggests that protection is mediated by pump blockade. Addition of ouabain at the same time as doxorubicin was just as protective as preincubation with ouabain for an hour, demonstrating that the ouabain acts rapidly. Ouabain treatment affected neither influx nor efflux of doxorubicin. Ouabain also had no effect on verapamil-induced inhibition of doxorubicin efflux. However, ouabain partially blocked the verapamil-induced potentiation of the cytotoxic effects of doxorubicin. Therefore, ouabain does not protect by affecting intracellular doxorubicin levels. Fluorescence microscopy showed that the ability of doxorubicin to reach the nucleus was not influenced by ouabain. Alkaline elution studies demonstrated that ouabain greatly decreased doxorubicin-induced DNA strand breakage. Protection from cytotoxicity correlated closely with this decrease in strand breakage. These studies suggest that the stabilization of DNA-topoisomerase II complexes is closely linked to the mechanism of doxorubicin cytotoxicity and that this stabilization is influenced by the intracellular ionic milieu.

Animals↗

PubMed1984

Two ouabain binding sites in guinea pig heart Na+-K+-ATPase. Differentiation by sodium and erythrosin B.

3H-ouabain binding to guinea pig heart NaK-ATPase resulted in two different cardiac glycoside binding sites: A high affinity, low capacity binding was obtained at a KD = 2.6 X 10(-7) mol/l (about 40% of the binding sites) whereas a low affinity, high capacity binding was established at a KD = 1.3 X 10(-6) mol/l (about 60% of the binding sites). Similar results were obtained when studying the effect of increasing Na+-concentrations on ouabain binding: again two distinct processes involved in the Na+/ouabain interaction could be demonstrated. Whereas one ouabain binding process was activated at rather low Na+-concentrations (K0.5 = 4.5 mmol/l), the other ouabain binding process was predominant at high Na+-concentrations only (K0.5 = 69 mmol/l). Comparing these data with the ouabain action on contractile force and with NaK-ATPase activity in guinea pig heart, the high affinity binding of ouabain seems to correlate with the inotropic action, whereas the low affinity ouabain binding is more related to NaK-ATPase inhibition. To further discriminate the two cardiac glycoside binding sites operative in guinea pig heart muscle, erythrosin B, shown to be an inhibitor of the high affinity ouabain binding in this preparation, was applied to isolated guinea pig left atria in the presence of increasing ouabain concentrations. There was no change in the ouabain induced inotropy or toxicity by erythrosin B. These results could indicate that the low affinity ouabain binding is responsible for both inotropic and toxic actions in guinea pig heart. However, the functional significance of the high affinity ouabain binding would then remain obscure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1975

Influence of ouabain and dihydroouabain on the circular dichroism of cardiac plasmalemmal microsomes.

1. The influence of ouabain on the tertiary structure of cardiac plasmalemmal proteins was investigated by means of circular dichroism measurement. Purified plasmalemmal microsomes were obtained by sucrose gradient centrifugation. The CD-spectra of the membranal proteins were shifted to the red and the amplitudes were smaller than those of the same proteins after solubilization. 2. Ouabain induced an increase of the ellipticity bands at 210 and 222 nm of about 50% above the level yielded with microsomes after sonication. At 222 nm ouabain exhibited the half maximum effect at a concentration of 5 X 10(-9) M. The effect could, however, only be exerted if the inside of the microsomes was exposed to ouabain by sonication, thus reflecting the inside-out nature of the plasmalemmal microsomes. 3. The high specificity of the ouabain effect was underlined by the following experiments: a) Dihydroouabain, a much less cardioactive derivative of ouabain proved to be ineffective in corresponding concentrations, b) ouabain had no influence upon the CD spectrum of microsomes derived from cardiac sarcoplasmic reticulum, c) a detergent-like action of ouabain underlying the observed effect can be excluded since highly active tensides, i.e. desoxycholate and dodecylsulfate, only influence the CD spectra at concentrations exceeding 10(-3) M, d) electronmicrographs of microsomes exposed to ouabain demonstrated no alteration of either the appearance or size of the microsomes. 4. The magnitude of the observed ouabain effect indicates that a large portion of the membrane-bound proteins is involved. The number of binding sites and their isolated structural alteration induced by ouabain are not sufficient to account quantitatively for the enhanced amplitudes of the CD spctra. This suggests that ouabain evokes structural changes of membrane proteins different from actual binding sites. It seems, therefore highly improbable that changes of the Na-K-ATPase present in the plasmalemmal microsomes are responsible for the observed effect.

Animals↗

PubMed1986

Frequent stimulation of the guinea-pig myocardium raises the inotropic efficacy of tissue-bound ouabain.

3H-Ouabain binding to frequently (1 Hz) stimulated papillary muscles from reserpine-pretreated guinea pigs was evaluated at ouabain concentrations of 18.5 and 200 nmol/l. Myocardial activity increased the amount of 3H-ouabain bound to the tissue in comparison with quiescent preparations. Since the shape of the time course of ouabain binding changed with frequent stimulation, a greater number of ouabain-accessible binding sites of the Na pump as induced by the rise in intracellular Na with frequent stimulation cannot be the sole mechanism of the frequency dependence. In view of their stimulatory properties on the Na pump the effects of intracellular Na and extracellular K could be equivalent. By contrast, both interventions were differently effective. The K antagonism on 3H-ouabain binding was independent from stimulation frequency. Furthermore, the shape of the time course of binding was not altered by [K]o. As evidenced by the dependence of half-times to steady-state effect on muscle diameter, the apparent rate of diffusion of ouabain was accelerated with the frequency of contractions. This acceleration could have interfered with the time course of binding at frequent stimulation. After correlating the time courses of positive inotropic effect and ouabain binding (concentration of ouabain in the medium 200 nmol/l), frequent stimulation was found to raise the inotropic efficacy of tissue-bound ouabain. The relation of excitation-dependent Na influx to the saturable, ouabain-inhibited, Na pump explained the frequency dependence of the intropic efficacy of ouabain; that is, the observed change of efficacy was consistent with Na-pump saturation in dependence on intracellular Na.

Animals↗

PubMed1985

Transport functions of the liver. Lack of correlation between hepatocellular ouabain uptake and binding to (Na+ + K+)-ATPase.

Ouabain uptake was studied on isolated rat hepatocytes. Hepatocellular uptake of the glycoside is saturable (Km = 348 mumol/l, Vmax = 1.4 nmol/mg cell protein per min), energy dependent and accumulative. Concentrative ouabain uptake is not present on permeable hepatocytes, Ehrlich ascites tumor cells and AS-30D ascites hepatoma cells. There is no correlation between ouabain binding to rat liver (Na+ + K+)ATPase and ouabain uptake into isolated rat hepatocytes. While ouabain uptake is competitively inhibited by cevadine, binding to (Na+ + K+)-ATPase is not affected by the alkaloid. Although the affinities of digitoxin and ouabain to (Na+ + K+)-ATPase are similar, digitoxin is 10000-times more potent in inhibiting [3H]ouabain uptake as compared to ouabain. That binding to (Na+ + K+)-ATPase appears to be no precondition for ouabain uptake was also found in experiments with plasmamembranes derived from Ehrlich ascites tumor cells and AS-30D hepatoma cells. While tumor cell (Na+ + K+)-ATPase is ouabain sensitive, the intact cells are transport deficient. Hepatic ouabain uptake might be related to bile acid transport. Several inhibitors of the bile acid uptake system also inhibit ouabain uptake.

Animals↗

PubMed1976

Ouabain-binding and phosphorylation of (Na+ + K+) ATPase treated with N-ethylmaleimide or oligomycin.

Ouabain-binding and phosphorylation of (Na+ mk+)-ATPase (EC 3.6.1.3) of the plasma membranes from kidney were investigated after treatment with N-ethylmaleimide or oligomycin. Either of these inhibitors brought about the following changes: the phosphoenzyme, formed in the presence of Na+, Mg2+ and ATP became essentially insensitive to splitting by K+ but was split by ADP. One mole of this ADP-sensitive phosphoenzyme bound one mole of ouabain but the enzyme-ouabain complex was less stable than in the native enzyme primarily because the rate of its dissociation increased. Ouabain was bound to the ADP-sensitive phosphoenzyme in the presence of Mg2+ alone and addition of inorganic phosphate enhanced both the rate of formation and the steady-state level of the enzyme-ouabain complex. The inhibitors did not affect the properties of this second type of complex. Both in the native enzyme and in the enzyme treated with the two inhibitors inorganic phosphate enhanced ouabain binding by phosphorylating the active center of the enzyme as shown (a) by mapping the labeled peptides from the enzyme after peptic digestion, (b) by inhibition of this phosphorylation with Na+ and (c) by the 1:1 stoichiometric relation between this phosphorylation and the amount of bound ouabain. Unlike the phosphoenzyme, the binding of ouabain remained sensitive to K+ in the enzyme treated with the inhibitors. K+ slowed ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding than to stimulate dephosphorylation. This finding is interpreted as being an indication of separate sites for K+ on the enzyme: a site(s) with high K+-affinity which stimulates dephosphorylation, another site(s) with moderate K+-affinity which inhibits ouabain-binding. Inhibitors may enhance formation of the ADP-sensitive phosphoenzyme by blocking interaction between K+ and the site(s) with high affinity.

Adenosine Triphosphatases↗

PubMed1986

Kinetic evaluation of carrier-mediated transport of ouabain and taurocholic acid in isolated rat hepatocytes. Evidence for independent transport systems.

Ouabain and taurocholic acid (Tch) share in common the cyclopentanophenanthrene (steroid) nucleus, and both are concentrated in the liver by hepatic sinusoidal carrier-mediated transport processes. Multiple transport systems for Tch uptake have been implicated, and Tch is an effective inhibitor of ouabain uptake. To determine if the ouabain transport system is related to transport of Tch, kinetic studies were conducted to examine the nature of cross-inhibition of ouabain and Tch. Tch was found to inhibit ouabain uptake in a competitive manner, with a Ki approximately ten times less than the Km for ouabain. However, ouabain failed to inhibit total Tch uptake in a competitive manner when added to the system at the same time as the Tch substrate. Preincubation of cells with ouabain resulted in noncompetitive inhibition of sodium-dependent Tch uptake but had no effect on sodium-independent Tch uptake. Ouabain had a weak competitive inhibitory effect on sodium-independent transport of Tch, but the Ki was approximately ten times greater than the Km for ouabain. These results demonstrate that the ouabain transport system is distinct from both the sodium-dependent and sodium-independent transport systems for Tch. Tch apparently binds competitively to the ouabain transport system but is not effectively transported across the cell membrane by this system.

Animals↗