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PubMed1977

Enhancement (by ATP, insulin, and lack of divalent cations) of ouabain inhibition of cation transport and ouabain binding in frog skeletal muscle; effect of insulin and ouabain on sarcolemmal (Na + K)MgATPase.

Using small, intact frog muscles, the basic properties of Na+ and K+ transport were shown to resemble those of the (Na+ + K+)Mg2+ATPase (EC 3.6.1.3) isolated from skeletal muscle. (a) External K+ is essential for Na+ exit and K+ entry after the muscles are Na+-loaded and K+-depleted; (b) the ouabain concentration causing maximum inhibition of recovery is the same for transport as for the inhibition of the isolated enzyme. Ouabain causes a decrease in the sorbitol space and causes muscle fibre swelling. Absence of Ca2+ and Mg2+ inhibits recovery of normal Na+ and K+ concentrations and increases the sorbitol space. Insulin stimulates K+ uptake and Na+ loss in intact muscles but has no effect on the isolated sarcolemmal (Na+ + K+)Mg2+ATPase. Absence of divalent cations, addition of external ATP and of insulin enhance the ouabain inhibition of recovery. Bound ouabain was measured using [3H]ouabain and [14C]sorbitol (to measure the extracellular space). The process of binding was slowly reversible and was saturable within a range of ouabain concentrations from 1.48 X 10(-7) to 5.96 X 10(-7) M. From the nonexchangeable ouabain bound, the density of glycoside receptors was estimated to be 650 molecules per square micrometre of membrane surface. The absence of divalent cations, addition of external ATP and of insulin significantly enhanced the amount of ouabain bound. Substitution of Na+ and K+ by choline greatly reduced the bound ouabain.

Adenosine Triphosphatases↗

PubMed1993

Role of ouabain-like factors in hypertension: effects of ouabain and certain endogenous ouabain-like factors in hypertension.

Several reports suggest the presence of sodium-potassium pump inhibitor in plasma and various tissues, particularly during volume-expanded state and low-renin hypertension. It has been hypothesized that by inhibiting the cardiovascular muscle-cell Na(+)-K+ pump, this inhibitor can constrict blood vessels, enhance vasoconstriction, and increase cardiac contractility, thereby raising blood pressure. Only two such endogenous inhibitors have been chemically characterized: the bufodienolide derivative, resibufogenin, obtained from toad skin and plasma; and a factor with the same structure (based on mass spectral analysis) as ouabain, from human plasma. However, unlike bufalin (aglycone), which is almost structurally identical to resibufogenin, neither ouabain nor ouabagenin (aglycone of ouabain) caused a sustained increase in blood pressure when infused in equimolar doses during a 30-min period in rats. Because the rat is 10(4)-fold less sensitive to ouabain than the human is, we wondered whether the absence of a response to ouabain was due to the short infusion time. Therefore, in new experiments ouabain was administered chronically during a 6- to 7-week period to two-kidney normal rats and rats with 70, 60, and 25% reduced renal mass. Reduced renal mass rats were used because these rats have decreased sodium excretion capacity, and thus we hoped the action of exogenous ouabain would be potentiated in these volume-expanded rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1972

Uptake of ( 3 H)ouabain and Na pump turnover rates in cells cultured in ouabain.

1. The binding of [(3)H]ouabain to fresh Girardi and Hela cells and to those cultured in low concentrations of ouabain for 24 hr has been measured.2. Fresh cells bind 1.6-2.2 x 10(6) molecules of ouabain(*) per cell from K-free Krebs, but less than 0.3 x 10(6) molecules from 15 mM-[K](o) Krebs. The ouabain(*) binds with a t((1/2)) of about 8 min from K-free 2 x 10(-7)M ouabain(*) and is released with a t((1/2)) of about 20 hr. Cells in a poor condition probably exchange ouabain more quickly.3. Cells incubated in ouabain(*) for 24 hr bind ouabain in amounts dependent on the [ouabain] and the external [K]. At the highest [ouabain] used the total amount bound exceeds that bound by fresh cells. Lowering [K](o) in the medium increases the maximum ouabain which is bound.4. Cells incubated in ouabain(*) for 24 hr bind an additional amount of ouabain when exposed to 2 x 10(-7)M ouabain(*) in K-free Krebs.5. There is a close relationship between the% of the total ouabain bound in 24 hr and the% inhibition of the Na efflux suggesting that this ouabain is bound to the Na pumps.6. Radiochromatography of the counts recovered from the cells showed that it migrated to the same place as the applied [(3)H]ouabain. The wash-off rate of ouabain bound to cells during incubation is similar to that from fresh cells, both tests suggesting that the ouabain exists in the same state in the cells.7. The number of Na ions extruded per pump is constant at about 60 sec(-1) in fresh cells and those pre-incubated in ouabain.8. The total ouabain bound by the cells is closely related to the [Na](i) in cells pre-incubated in ouabain but is unaffected by it in fresh cells where [Na](i) is raised acutely.9. These results are compatible with the hypothesis that partial blocking of Na pumps leads to the production of more pumping sites by the cell.

Binding Sites↗

PubMed1985

Effect of ouabain on tone, membrane potential and sodium efflux compared with [3H]ouabain binding in rat resistance vessels.

To investigate the mechanism by which ouabain causes acute increase in peripheral resistance, the effect of ouabain on vessel tone, 22Na efflux and membrane potential has been compared with the binding of [3H]ouabain in rat isolated mesenteric resistance vessels (lumen diameter approximately 200 microns). Ouabain at concentrations between 1 X 10(-8) and 1 X 10(-3) M had no effect on the tone of resting vessels but caused potentiation of the tone of vessels which were submaximally contracted with noradrenaline. In the entire concentration range, these acute potentiating effects of ouabain on vessel tone correlated well with an acute inhibitory effect on 22Na efflux and with acute [3H]ouabain binding, consistent with the vasoconstrictor effect of ouabain being associated with inhibition of the Na-K pump. At all concentrations, in both resting and activated vessels, ouabain exposure caused an acute concentration-dependent depolarization of the membrane. At low ouabain concentrations (1 X 10(-6) and 1 X 10(-5) M) the potentiating effect on vessel tone and the inhibitory effect on 22Na efflux were transient, so that after 10 min no effect of ouabain was seen. This was consistent with these ouabain concentrations failing to cause a measurable increase in the intracellular Na concentration ([Na]i) after longer term (up to 2 h) exposure. The recovery of 22Na efflux was due to a recovery of the ouabain-sensitive part of the 22Na efflux, suggesting that the transitory nature of these effects is due to stimulation of uninhibited Na-K pumps. The transient effects of low ouabain concentrations contrasted with binding experiments where, with 1 X 10(-6) M-ouabain, the binding first peaked after 30 min with only a small decline thereafter. With higher concentrations of ouabain (1 X 10(-4) and 1 X 10(-3) M), the potentiating effect was also transitory, but 22Na efflux was permanently inhibited and the [Na]i rose. The possibility of there being two populations of ouabain binding sites having different affinities, was supported by the observation of shoulders in the ouabain concentration-effect characteristics obtained with respect to the mechanical potentiation of noradrenaline responses, the 22Na efflux and the acute ouabain binding. In all cases a shoulder was seen between 1 X 10(-6) and 1 X 10(-5) M-ouabain. The results are consistent with the vasoconstrictor effect of ouabain being due to depolarization of the membrane as a consequence of inhibition of the Na-K pump.

Animals↗

PubMed1987

Ouabain-like and non-ouabain-like factors in plasma of patients with essential hypertension.

Circulating inhibitor of Na+,K+-ATPase and ouabain-like immunoreactivity were studied in patients with essential hypertension. In the plasma of patients, two types of Na+,K+-ATPase inhibitors (ouabain-like and non-ouabain-like inhibitors) and ouabain-like immunoreactivity were detected. Ouabain-like inhibitor was clearly detected at a low KCl concentration (0.1 mM) in the assay buffer, and non-ouabain-like inhibitor was detected at a high KCl concentration (10 mM). The plasma level of ouabain-like inhibitor correlated significantly with that of ouabain-like immunoreactivity (p less than 0.001) and with a mean blood pressure (p less than 0.01). The plasma level of non-ouabain-like inhibitor was not correlated with the levels of either ouabain-like immunoreactivity or mean blood pressure. The level of plasma ouabain-like inhibitor did not correlate with that of plasma non-ouabain like inhibitor. Both ouabain-like inhibitor and ouabain-like immunoreactivity in the plasma of patients with essential hypertension were significantly higher than those in normotensive subjects, but the plasma level of non-ouabain-like inhibitor in patients with essential hypertension was not higher than that in normotensive subjects. These results suggest that the plasma from patients with essential hypertension contains ouabain-like factor(s) which is important to maintain the high blood pressure.

Adult↗

PubMed2001

Radioiodinated tyrosyl-ouabain and measurement of a circulating ouabain-like compound.

BACKGROUND: Assays for endogenous ouabain, a cardiac glycoside believed to be involved in blood pressure and volume regulation, are characterized by laboratory-specific plasma values that are measured by different assays. Because of this variability, our study focused on the development of a new (125)I-labeled ouabain derivative for RIA of high sensitivity. METHODS: We generated rabbit antisera against a ouabain-thyroglobulin conjugate. A tyrosylated ouabain derivative for radioiodination was synthesized using periodate and sodium cyanoborohydride reagents. RESULTS: Mass spectrometric analyses showed that the main product of the tyrosylating reaction was tyrosyl-ouabain (molecular mass, 702 Da). This was radioiodinated with Chloramine-T and used as a tracer in a RIA, which gave an assay detection limit of 5 pmol/L (4 ng/L), 2-100 times lower than that in the corresponding (3)H-RIAs and 2-20 times lower than ouabain ELISAs, making it possible to measure low plasma concentrations of immunoreactive ouabain. Different amounts of SepPak C(18)-extracted plasma samples displaced the (125)I-labeled tyrosyl-ouabain tracer at the same rate at which authentic ouabain was displaced. Plasma immunoreactive ouabain coeluted with authentic ouabain in two different HPLC conditions. Using the new RIA, we found plasma ouabain concentrations, assayed as immunoreactive equivalents, of 10.0 +/- 1.3 pmol/L in healthy women and 12.0 +/- 0. 9 pmol/L in healthy men (mean +/- SE; n = 10), as well as 41.2 +/- 9. 6 pmol/L in rats. The concentrations were 2-90 times lower than those previously reported using different assay methods. CONCLUSIONS: Our ouabain (125)I-RIA enables reliable measurements of low endogenous concentrations of a ouabain-like compound for both physiological and clinical purposes.

Adult↗

PubMed1992

Ouabain-resistant transfectants of the murine ouabain resistance gene contain mutations in the alpha-subunit of the Na,K-ATPase.

A 6.5-kilobase murine genomic DNA fragment isolated by Levenson et al. (Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1489-1493) (called the ouabain resistance gene) has been shown to produce ouabain resistance in primate cells. Preliminary sequence information has revealed no homology with the coding sequence of the Na,K-ATPase. We have introduced this murine sequence into monkey and murine cells in an attempt to characterize its mechanism of action. In our experiments, transfection of this DNA fragment is associated with the low frequency (1 in 8 x 10(5) cells) appearance of ouabain-resistant clones of CV1, COS, and NIH 3T3 cells, an event not seen in control transfections. Characterization of a new clone of ouabain-resistant CV1 cells (called OR8 cells) revealed a 5-fold increase in the IC50 for ouabain inhibition of rubidium uptake and a 10-fold increase in cell survival on ouabain. Although the murine sequence was detectable in Southern blots of ouabain-resistant cells soon after transfection, this exogenous DNA was rapidly lost despite continued exposure to ouabain. Furthermore, we were unable to detect message expression by this genomic sequence in any of the three cell types tested. Instead, we found that all three ouabain-resistant cell lines exhibited point mutations in a domain of the alpha-subunit that has been implicated in ouabain sensitivity (H1-H2). One of these mutations (Asp121-Asn121 in OR8 cells) has been previously reported to cause ouabain resistance (Price, E.M., Rice, D.A., and Lingrel, J.B. (1989) J. Biol. Chem. 264, 21902-21906). Other novel mutations in the H2 transmembrane domain were also detected. We postulate that the "ouabain resistance gene" is important in the early selection process on ouabain but that the permanent ouabain-resistant phenotype is due to a stable mutation in one allele of the alpha-subunit of the Na,K-ATPase.

3T3 Cells↗

PubMed1986

Erythroid differentiation and the Na+,K+-pump in ouabain-sensitive and ouabain-resistant Friend erythroleukemia cell lines.

The selection and biochemical characterization of ouabain-resistant erythroleukemia cell lines are described. Treatment of ouabain-resistant Friend erythroleukemia cell (FLC) lines with 1 mM ouabain demonstrated a reduced ouabain-sensitive 86Rb+-uptake after Na+-preloading in comparison with ouabain-sensitive cells. The ouabain- and diuretic (piretanide)-insensitive component of the 86Rb+-uptake (residual influx) was significantly enhanced in the ouabain-resistant FLC clones. Measurements of the Na+,K+-ATPase activity (E.C. 3.6.1.3) in plasma membrane preparations of the ouabain-resistant FLC clone B6/2 indicated that a ouabain-resistant Na+,K+-ATPase activity of about 20% of the total enzyme activity existed in the presence of 1 mM ouabain. Further experiments showed that the Na+,K+-ion-gradient in ouabain-resistant B6/2 cells was unaffected by ouabain exposure whereas the gradient collapsed in wild type 12 N cells. Another property of the ouabain-resistant cell lines was a decrease of the 86Rb+-uptake due to the Na+,K+, 2Cl(-)-cotransport system measured as piretanide-sensitive 86Rb+-uptake. The data on ion transport mechanisms in QuaR and QuaS FLC are discussed with respect to mutagen-induced and spontaneous cellular ouabain resistance. In addition, the role of altered ion transport mechanisms is considered for induced erythroid differentiation.

Animals↗

PubMedDate not supplied

Ouabain-resistant (Na+,K+)-ATPase enzyme activity in chemically induced ouabain-resistant C3H/10T1/2 cells.

To further understand the molecular nature of changes leading to chemically induced ouabain resistance in C3H/10T1/2 Cl 8 (10T1/2) cells, we isolated plasma membranes from wild-type and ouabain-resistant (Ouar) 10T1/2 cells and characterized (Na,K)-ATPase activity in the plasma membrane fraction. (Na+,K+)-ATPase enzyme activity in membrane fractions extracted from wild-type 10T1/2 cells was inhibited in a concentration-dependent manner by ouabain and was completely inhibited by 2.4 mM ouabain. Lineweaver-Burke and Eisenthal/Cornish-Bowden analysis indicated that the inhibition was uncompetitive. Ten to 45% of (Na+,K+)-ATPase enzyme activity extracted from three Ouar 10T1/2 cell lines cultured in 1 mM ouabain was resistant to 2.4 mM ouabain, depending on the cell line. Resistance of (Na+,K+)-ATPase activity in the plasma membrane fraction of Ouar cells to inhibition by ouabain and resistance of cultured Ouar cells to the cytotoxicity of ouabain occurred over similar concentrations of ouabain (0.1-3mM). Two ouabain-resistant cell lines, Ouar MNNG Cl 2 and Ouar MCA Cl 16-7, demonstrated the same total (Na+,K+)-ATPase specific activity as 10T1/2 cells, but the fraction of Ouar enzyme activity increased (from 18 to 40% in MNNG Cl 2 cells and from 10 to 25% in Sp Ouar Cl 16 cells) when the cells were cultured in ouabain. Thermal denaturation profiles and pH dependence profiles of (Na+,K+)-ATPase activity in plasma membranes from wild-type and Ouar 10T1/2 cells were identical. A 3.9-kb (Na,K)-ATPase alpha subunit mRNA transcript was found in 10T1/2 cells, and in the Ouar MNNG Cl 2 cell line cultured in the presence or absence of ouabain. There was no amplification of the gene coding for the alpha subunit of (Na+,K+)-ATPase in the chemically induced Ouar MNNG Cl 2 cell line, whether this cell line was cultured in the presence or absence of ouabain. These studies provide further evidence that the Ouar phenotype of chemically induced and spontaneous Ouar 10T1/2 cell lines derives from Ouar (Na+,K+)-ATPase activity, and that this Ouar (Na+,K+)-ATPase activity increases further in some cell lines cultured in the presence of ouabain.

Animals↗

PubMed1988

CV-1 cell recipients of the mouse ouabain resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids.

The cation-transporting activity and Na,K-ATPase activity of CV-1 cell recipients of the mouse ouabain resistance gene (ouaR6, or OR6 cells; see Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493) have been further characterized. OR6 cells grown in strophanthidin (a cardiac aglycon which may be removed rapidly from the Na,K-ATPase) possess both ouabain-sensitive and -insensitive 86Rb+ uptake activities. The ouabain-sensitive 86Rb+ uptake activity of these cells (OR6-S cells) exhibits the same Ki for ouabain as that of the CV-1 parent cells (Ki(app) = 3 x 10(-7) M ouabain), but accounts for only approximately 30% of total 86Rb+ uptake into Na+-loaded OR6-S cells, compared to 80% for CV-1 cells. Most of the ouabain-resistant 86Rb+ uptake in OR6-S cells is dependent on internal Na+ and is insensitive to furosemide, suggesting that it is due to an ouabain-resistant Na,K pump. In OR6-S cell lysates, 50% of Na+-dependent ATPase activity is insensitive to 1 mM ouabain, compared to less than 5% in CV-1 cell lysates. In addition, purified plasma membranes from OR6-S cells contain a 100-kDa protein which is transiently phosphorylated by ATP in an Na+-dependent, K+-sensitive manner, like the alpha subunit of the CV-1 Na,K-ATPase and the canine renal Na,K-ATPase, but which is unaffected by preincubation in 1 mM ouabain. All of these data suggest that OR6-S cells possess a ouabain-insensitive Na,K pump with characteristics similar to the ouabain-sensitive pump of CV-1 parent cells. Since the mouse ouabain resistance gene does not encode either subunit of the Na,K-ATPase, these results suggest that the ouabain resistance gene product may modify the ouabain sensitivity of the endogenous CV-1 Na,K pump.

Animals↗

PubMed1994

Ouabain-induced hypertension in the rat: relationships among plasma and tissue ouabain and blood pressure.

OBJECTIVES: To determine the steady-state dose-dependence of blood pressure, plasma and tissue ouabain during continuous infusion of ouabain in the rat, and to evaluate the adrenal dependence and effect of a high salt intake on this form of hypertension. DESIGN AND METHODS: Ouabain was administered, via subcutaneous osmotic pumps, to normal and adrenalectomized male Sprague-Dawley rats for 5 weeks. Blood pressure, plasma renin and aldosterone, and circulating and tissue levels of ouabain were determined. RESULTS: Following a latent period, blood pressures and circulating ouabain were significantly elevated dose-dependently in glycoside-infused rats at 5 weeks. Upon withdrawal of the ouabain infusion, blood pressure and plasma ouabain levels normalized within 1 week. In rats that received 30 micrograms ouabain/kg per day, the circulating, kidney, hypothalamic and anterior pituitary levels of ouabain were increased significantly (by 7-, 15-, 2.8- and 2.1-fold, respectively), whereas the content of other tissues tested was unchanged. Blood pressure and plasma levels of ouabain correlated with hypothalamic and kidney glycoside content in the infused rats. High-performance liquid chromatography of the adrenal, renal, hypothalamic and pituitary extracts showed one major peak of ouabain immunoreactivity, with a retention time equivalent to that of commercial ouabain. Plasma renin activity was normal, whereas aldosterone levels were increased significantly to 2.9- and sevenfold in rats that received 10 and 30 micrograms ouabain/kg per day, respectively. Dietary salt loading suppressed aldosterone and did not exacerbate hypertension. In bilaterally adrenalectomized rats the ambient circulating and kidney levels of ouabain were low and ouabain infusion raised glycoside levels and blood pressure significantly. CONCLUSIONS: Prolonged infusion of ouabain in the normal rat raises the circulating, kidney, hypothalamic and anterior pituitary levels and induces a reversible hypertension with normal plasma renin activity. Although characterized by raised aldosterone levels, the hypertension does not require the adrenal glands and is not salt-sensitive. This model may be useful for exploring novel mechanisms of long-term regulation of blood pressure.

Adrenalectomy↗

PubMed1990

Actions of bufalin and cinobufotalin, two bufadienolides respectively more active and less active than ouabain, on ouabain binding and 86Rb uptake by human erythrocytes.

1. We have reported that the bufadienolide, bufalin (purified from toad skin), was more potent than ouabain in inhibiting the sodium/potassium-dependent adenosine triphosphatase from canine kidney (Sigma) [Brownlee, A.A., Lee, G. & Mills, I.H.J. Physiol. (London) 1987; 390, 94P]. 2. The activities of bufalin and cinobufotalin were compared with ouabain in the [3H]ouabain binding assay and on 86Rb uptake in human erythrocytes. 3. When the percentage binding of ouabain-sensitive [3H]ouabain was plotted against the log of the concentration of drug in mol/l, it was shown that the bufalin curve was shifted to the left of that of ouabain and that of cinobufotalin was to the right. 4. Linear regression lines were fitted to the data transformed as the log of (p/1--p) plotted against the log of the drug concentration, where p is the proportion of maximal ouabain-sensitive activity at the drug concentration being considered. The IC50 (the concentration of drug producing a 50% change in the maximal ouabain-sensitive response) was 1.4 x 10(-9) mol/l for bufalin, 9.7 x 10(-9) mol/l for ouabain and 1.70 x 10(-7) mol/l for cinobufotalin. 5. The introduction of bufalin 1 h before ouabain reduced the binding of [3H]ouabain to 23.4 +/- 1.5% (P less than 0.001). Bufalin added in the second hour reduced the ouabain-sensitive binding from 100 +/- 1.9% to 87.4 +/- 2.9% (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1983

Effects of ouabain on [3H]ouabain binding, 86Rb uptake, cellular sodium and potassium, and parathyroid hormone secretion in dispersed bovine parathyroid cells.

We have compared the effects of the cardiac glycoside ouabain on [3H]ouabain binding, 86Rb uptake, cellular sodium and potassium, and PTH secretion in dispersed bovine parathyroid cells. [3H]ouabain binds reversibly to a single class of binding sites with an affinity of 6.1 X 10(-8) M and a binding capacity of 5.8 X 10(5) sites/cell. Ouabain also inhibits the uptake of 86Rb, an analog of K, by 90% with half-maximal inhibition at 7.2 X 10(-8) M. There is a concomitant, ouabain-induced increase in cellular sodium and a reduction in cellular potassium. The half-maximal effect on cellular monovalent cations takes place at 1.4 X 10(-7 M ouabain. Finally, ouabain causes a dose- and time-dependent inhibition of low calcium-stimulated PTH secretion. This inhibition does not require extracellular calcium; half-maximal inhibition occurs at 1.1 X 10(-7) M ouabain. These results show that dispersed bovine parathyroid cells contain abundant binding sites for ouabain, a known inhibitor of Na+-K+-ATPase. Moreover, the ouabain-induced reduction in 86Rb uptake and alterations in cellular sodium and potassium support an inhibition of this enzyme in parathyroid cells by the cardiac glycoside. Finally, the close correspondence between ouabain binding and effects on Rb uptake, cellular monovalent cations, and PTH release suggest a role for Na+-K+-ATPase per se or for monovalent cations in PTH secretion. A change in sodium-calcium exchange due to the elevation in cellular sodium is a potential mechanism by which ouabain might inhibit PTH secretion.

Animals↗

PubMed1988

Voltage dependence of the Na-K ATPase: measurements of ouabain-dependent membrane current and ouabain binding in oocytes of Xenopus laevis.

Ouabain- or dihydroouabain(DHO)-sensitive membrane currents and binding of 3H-ouabain were investigated under voltage-clamp conditions in full-grown prophase-arrested oocytes of Xenopus laevis. (1) The ouabain-sensitive current is outwardly directed and usually exhibits a maximum at about +20 mV. The occurrence of the maximum is not affected by application of blockers for passive K+ currents. In the presence of Ba2+ as a K+ channel blocker, the KI value for inhibition of the Na-K ATPase by ouabain is reduced by an order of magnitude, the number of binding sites is not affected. In K+-free solution (which inhibits the normal reaction cycle of the Na-K ATPase), addition of DHO has no significant effect on the remaining currents. (2) The voltage-dependence of the ouabain-sensitive current can be modulated. Reduction of extracellular Na+ increases the pump current at the resting potential and reduces the positive slope of the I-V curve. Simultaneously, the number of binding sites for ouabain is reduced by about 25%. Seasonal variations of an unknown factor affect the negative slope. (3) Modulation of the voltage-clamp conditions has no effect on the number of binding sites for ouabain, on current inhibition by ouabain, or on ouabain binding at different concentrations of the inhibitor. It is concluded that the ouabain-sensitive current is not significantly affected by passive permeabilities and that its current-voltage dependence reflects the voltage dependence of current generated by the Na-K pump. Since ouabain binding, also at non-maximal binding concentrations, is not affected by membrane potential, steps that affect the probability of the E2P state should be voltage-insensitive.

Animals↗

PubMed2001

Biological implication of conformational flexibility in ouabain: observations with two ouabain phosphate isomers.

Ouabain is a highly polar and unusually potent sodium pump inhibitor that possesses uncommon conformational flexibility in its steroid A-ring moiety. The biological significance of ring flection in the cardiotonic steroids has not been described. Accordingly, we prepared ouabain 1,5,19- and 1,11,19-phosphates. The former stabilizes the steroid A-ring chair conformation and the latter locks the A-ring in the half-boat conformation and decreases flection of the ABC-ring moiety. Using a dog kidney cell line (MDCK) in a pH microphysiometer (Cytosensor), ouabain and its 1,5,19-phosphate at 10(-5) M reduced the rate of extracellular acidification by 15-20%. During inhibitor washout, the rate of recovery from the 1,5,19-phosphate analogue was approximately 3 times faster than ouabain. The 1,11,19-phosphate at 10(-4) M elicited a weak ( approximately 7%) response, and the effects reversed approximately 44-fold faster than ouabain. Studies with purified Na(+),K(+)-ATPase showed that ouabain and its 1,5,19-phosphate analogue were of similar efficacy (EC(50) = 1.1 and 5.2 x 10(-7) M, respectively) and >100-fold more potent than the 1,11,19-phosphate analogue. Studies of the binding kinetics showed that the 1,5,19-phosphate analogue bound 3-fold and dissociated 16-fold faster from the purified Na(+),K(+)-ATPase than ouabain. Both analogues were competitive inhibitors of 3H-ouabain binding. Taken together, these results suggest that the marked conformational flexibility of the A-ring in ouabain ordinarily slows the initial binding of this steroid to the sodium pump. However, once ouabain is bound, flection of the steroidal A- and BC-rings is critical for the maintenance of high-affinity binding. Our results indicate that the ouabain-binding site is comprised of structurally mobile elements and highlight the roles that synchronization between receptor and ligand dynamics play as determinants of biological activity in this system.

Animals↗

PubMed1987

Enhancement of specific [3H]ouabain binding and ouabain sensitive 86rubidium influx in intact human lymphocytes by a dialysable factor in human and fetal calf serum.

We have measured specific [3H]ouabain binding and ouabain sensitive 86rubidium influx in intact human lymphocytes incubated for up to 7 days in media containing different concentrations of fetal calf serum and human serum. Incubation for periods of up to 7 days with fetal calf serum and human serum produced increases in both specific [3H]ouabain binding and ouabain sensitive 86rubidium influx that were dependent on concentration and time. Neither specific [3H]ouabain binding nor ouabain sensitive 86rubidium influx was altered when dialysed serum was used, suggesting that both fetal calf serum and human serum contain a dialysable factor or factors which stimulate specific [3H]ouabain binding and ouabain sensitive 86rubidium influx in intact human lymphocytes. To further elucidate the mechanisms underlying these changes we also measured the activity of two other enzymes of the lymphocyte plasma membrane, 5'-nucleotidase and gamma-glutamyltransferase, the uptake of [3H]thymidine by the intact cells, and the effects of cycloheximide, puromycin, and anisomycin, inhibitors of protein synthesis. The activity of 5'-nucleotidase was increased after incubation of the lymphocytes in fetal calf serum for 72 h, but the activity of gamma-glutamyltransferase was not changed, suggesting some selectivity of the stimulatory effect. Measurements of [3H]thymidine uptake by the lymphocytes showed that the major part of the observed changes in specific [3H]ouabain binding and ouabain sensitive 86rubidium influx was not attributable to transformation of the lymphocytes to lymphoblasts. All three inhibitors of protein synthesis prevented the increase in specific [3H]ouabain binding due to fetal calf serum.

5'-Nucleotidase↗

PubMed2000

Ouabain resistance of a human trophoblast cell line is not related to its reactivity to ouabain.

Ouabain is a specific inhibitor of sodium, potassium-dependent adenosine triphosphatase (Na,K-ATPase), a P-type ion-transporting ATPase which is essential for the maintenance of adequate concentrations of intracellular Na+ and K+ ions. The present study describes the establishment of a ouabain-resistant mutant, TLouaR, from a human trophoblast cell line TL. Morphologically TL and TLouaR are indistinguishable, but, TLouaR is about 1000 times more resistant to the cytotoxic effect of ouabain and > 2000 times to that of bufalin and yet ouabain can retard the growth of the TLouaR cells and in parallel reduce its cloning efficiency in a time- and dose-dependent manner. Furthermore, Na,K-ATPase activity from TLouaR cells is inhibitable by ouabain albeit with lower efficiency. [3H]ouabain binding studies reveal that TLouaR cells have less (P < 0.05) ouabain binding sites (1.7 +/- 0.15 x 10(4)/cell vs. 2.3 +/- 0.115 x 10(4)/cell in the control). However, affinities (dissociation constants Kd) to ouabain for TL and TLouaR cells are not significantly different. Lastly, Na,K-ATPase activity (1.375 +/- 0.25 micromole ATP/min mg protein) of TLouaR cells is significantly higher (P < 0.05) than that of the TL cells (0.895 +/- 0.12 micromole ATP/min x mg protein). These studies show that the interactions between ouabain and Na,K-ATPase can be mediated through different pathways resulting in diverse phenotypic characteristics. In addition, ouabain resistance does not necessarily reflect the lack of response to the digitalis drug. The exact mechanisms of ouabain resistance observed in the present study remain to be determined but the TLouaR cells may be the best tool to uncover the many functional characteristics of Na,K-ATPase.

Adenosine Triphosphatases↗

PubMed1994

Is the circulating ouabain-like compound ouabain?

The evidence is very strong for a circulating inhibitor of the sodium, potassium ATPase in volume-expanded hypertension. Recently, this inhibitor was isolated from human plasma and identified as ouabain. We are reporting our results using a very specific and sensitive immunoassay for ouabain with which we were unable to detect or able to detect only very low levels of circulating immunoreactive ouabain. Immunoassay of 5 mL of human and rat plasma, incubation fluid from bovine and human adrenal cell cultures extracted using a C-18 solid phase column, and HPLC separation did not detect a peak corresponding to ouabain. This procedure could easily detect authentic ouabain added to these extracts at a concentration slightly below that reported to be present by others. The extract from the adrenal cultures had clearly detectable sodium, potassium ATPase using an assay based on inhibition of tritiated ouabain binding to human red cells. Extraction of bovine adrenals detected a very small amount of immunoassayable ouabain which did not elute at a time corresponding to that of ouabain. This study indicates that the postulated sodium, potassium ATPase inhibitor that circulates in plasma is not ouabain, but it is likely to be structurally similar to ouabain, as it appears to cross-react with some antibodies against ouabain.

Adrenal Glands↗