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PubMed1987

Autoradiographic visualization and characterization of [3H]ouabain binding to the Na+,K+-ATPase of rat brain and pineal.

Ouabain binds to the catalytic subunit of Na+,K+-ATPase and specific [3H]ouabain binding can be used as a measure of the number of active enzyme molecules present in a given tissue. Specific [3H]ouabain binding can be demonstrated in frozen, cryostat sections from rat brain and pineal and these sites have the characteristics of Na+,K+-ATPase. Incubations carried out in the absence of ATP or the presence of excess unlabeled ouabain reduces specific binding by greater than or equal to 98%. The addition of K+ or omission of Mg2+ also result in a decrease in specific binding. Strophanthidin, digoxin and digoxigenin displace [3H]ouabain binding with IC50 values of 0.73, 0.48 and 1.4 microM, respectively. Scatchard analyses of specific [3H]ouabain binding in brain sections shows a single class of non-interacting binding sites with an apparent affinity (Kd) of 339 nM and a maximal binding capacity (Bmax) of 34.9 pmol/mg protein. [3H]Ouabain binding is unevenly distributed throughout the brain with the olfactory nuclei, superior colliculus, dentate gyrus, pontine nuclei and pineal gland having a relatively high density of binding sites. The outer layers (1-3) of the cerebral cortex show more labeling than the inner layers (4-6) and most other brain areas have intermediate levels of [3H]ouabain binding sites, whereas white matter has virtually no specific binding. Computer-assisted densitometry was used to measure changes in specific [3H]ouabain binding after kainic acid injection into the caudate nucleus. An initial increase in [3H]ouabain binding was observed at 1 and 24 h after lesioning and a decrease in [3H]ouabain binding was evident by 9 days after lesioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Nanomolar concentrations of ouabain block ethanol-inducible Na+,K(+)-ATPase activity in brain.

The effect of low concentrations of ethanol on Na+,K(+)-ATPase activity, defined as ouabain-inhibitable 86Rb+ (K+) uptake, was investigated in a crude synaptosome preparation which was subject to minimal subcellular fractionation procedures. Moderate (20-30%) but potent (EC50 = 3.8 mM) stimulation of total ouabain (1 mM)-inhibitable K+ uptake by ethanol was observed following incubation periods of up to 20 min. The activity of the ethanol-induced component of K+ uptake was antagonized by nanomolar concentrations of ouabain. Thus, the moderate stimulation of total ouabain-inhibitable K+ uptake by ethanol was attributable to the activation of a component of K+ uptake which was very sensitive (VS; IC50 = 2.8 x 10(-10) M) to inhibition by ouabain. Slightly higher concentrations of ouabain (10(-9) - 10(-6.6) M) stimulated K+ uptake above control (no ethanol or ouabain) in both the absence and presence of ethanol. The selectivity of the VS-ethanol interaction was demonstrated by the lack of any ethanol effect on two other components of ouabain-inhibitable K+ uptake which accounted for inhibition of K+ uptake by concentrations of ouabain above 10(-6.6) M and were defined as sensitive (S; IC50 = 10(-6) M) and insensitive (I; IC50 = 10(-4) M) to ouabain. These results define the ethanol-inducible component of ouabain-inhibitable Na+,K(+)-ATPase activity and promote the view that changes in Na+,K(+)-ATPase-dependent ion translocation may contribute to ethanol intoxication in vivo.

Animals↗

PubMed1996

Reduction of cerebral cortical [3H]ouabain binding site (Na+,K(+)-ATPase) density in dementia as evaluated in fresh human cerebral cortical biopsies.

Na+,K(+)-ATPase density in human cerebral cortex was for the first time studied by vanadate facilitated [3H]ouabain binding to intact samples. Fresh human cerebral cortical biopsies were obtained as a result of diagnostic frontal lobe biopsy from patients with normal pressure hydrocephalus (NPH) syndrome and associated dementia. For control measurements post-mortem samples were obtained from patients without clinically observed dementia. [3H]ouabain binding kinetics were evaluated: when incubating samples in 1 microM [3H]ouabain binding equilibrium was obtained after 6 h of incubation, non-specific uptake and retention amounted to only 2.3% of total uptake and retention of [3H]ouabain and release of specifically bound [3H]ouabain during washout in the cold occurred only slowly (T1/2 = 37 h). Evaluation of receptor affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. [3H]Ouabain binding was significantly reduced after frozen storage of samples before measurements. Post-mortem degradation of cerebral [3H]ouabain binding sites occurred only slowly (T1/2 = 75 h). No significant variation in [3H]ouabain binding site density was observed between the cerebral lobes with occipital, parietal and temporal values (means +/- S.E.M., n = 5) amounting to 10281 +/- 649, 11267 +/- 1011 and 9263 +/- 615 pmol/g wet wt., respectively. [3H]Ouabain binding measured in frontal cortical samples gave values of (means +/- S.E.M., n = 5) 4274 +/- 1020 and 11397 +/- 976 pmol/g wet wt. delta % = 62; P < 0.05) in patients with dementia and controls, respectively. Human cerebral cortical capacity for active K+ uptake was around 37- and 16-fold greater than in skeletal muscular and myocardial tissue, respectively.

Aged↗

PubMed2003

Phe783, Thr797, and Asp804 in transmembrane hairpin M5-M6 of Na+,K+-ATPase play a key role in ouabain binding.

Ouabain is a glycoside that binds to and inhibits the action of Na+,K+-ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H+,K+-ATPase and Na+,K+-ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na+,K+-ATPase in a backbone of H+,K+-ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H+,K+-ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na+,K+-ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H+,K+-ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na+,K+-ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na+,K+-ATPase suggests that Asp804, in contrast to Phe783 and Thr797, does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.

Amino Acid Sequence↗

PubMed1983

[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts.

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high-affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta-gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.

Adenosine Diphosphate↗

PubMed2003

Mechanisms of ouabain toxicity.

The suggested involvement of ouabain in hypertension raised the need for a better understanding of its cellular action, but the mechanisms of ouabain toxicity are only now being uncovered. In the present study, we show that reduced glutathione (GSH) protected ouabain-sensitive (OS) cells from ouabain-induced toxicity and that the inhibition of GSH synthesis by D, L-buthionine-(S,R)-sulfoximine (BSO) sensitized ouabain-resistant (OR) cells. We could not observe formation of *OH or H2O2, but there was an increase in O2*-only in OS cells. Unexpectedly, an increased number of OR cells depolarized after treatment with ouabain, and BSO blocked this depolarization. Moreover, GSH increased ouabain-induced depolarization in OS cells. A sustained increase in tyrosine phosphorylation (P-Tyr) and Ras expression was observed after treatment of OS cells, and GSH prevented it. Conversely, BSO induced P-Tyr and Ras expression in ouabain-treated OR cells. The results obtained have three major implications: There is no direct correlation between membrane depolarization and ouabain-induced cell death; ouabain toxicity is not directly related to its classical action as a Na+, K+-ATPase inhibitor but seems to be associated to signal transduction, and GSH plays a major role in preventing ouabain-induced cell death.

Animals↗

PubMed1995

Immunoreactive endogenous ouabain in primary aldosteronism and essential hypertension: relationship with plasma renin, aldosterone and blood pressure levels.

OBJECTIVE: To investigate the role of ouabain in human hypertension and to establish whether immunoreactive endogenous ouabain is secreted by the adrenal gland under the influence of dopaminergic regulation. METHODS: We measured plasma levels of endogenous ouabain by immunoassay, together with other variables, including plasma renin activity and aldosterone levels, in 91 clinically selected hypertensives and 19 healthy volunteers. We also measured endogenous ouabain in adrenal venous blood and the effect of DA2 dopaminergic receptor blockade and stimulation. After a thorough clinical evaluation, 64 patients were diagnosed with essential hypertension and 24 with primary aldosteronism. RESULTS: Plasma levels of endogenous ouabain were higher in essential hypertensives than in controls. Multiple regression analysis showed a significant relationship of mean blood pressure with plasma endogenous ouabain, age and body mass index, but not with other measured parameters. The plasma levels of endogenous ouabain were more than two standard deviations above the mean value for normotensives in 45% of patients with essential hypertension in whom plasma renin activity was normal. Higher plasma levels of endogenous ouabain were found in patients with aldosterone excess, specifically affecting 56% of 17 patients with surgically confirmed adrenal cortical adenoma and one (14%) of seven patients with idiopathic causes. Removal of adenomas lowered blood pressure in half of the patients in whom plasma levels of endogenous ouabain normalized after surgery. Plasma endogenous ouabain levels were similar in venous blood from the adrenal and inferior vena cava, and plasma levels were not influenced by DA2 dopaminergic blockade and stimulation. CONCLUSION: Approximately half of Caucasian patients with essential hypertension and with hyperaldosteronism exhibit elevated circulating levels of endogenous ouabain. The latter do not appear to be secondary to hypertension, are unrelated to plasma renin activity, and may not involve adrenal type-2 dopaminergic receptors.

Aldosterone↗

PubMed2003

Ouabain changes arterial blood pressure and vascular reactivity to phenylephrine in L-NAME-induced hypertension.

Ouabain is an endogenous compound that has been associated with the genesis and maintenance of hypertension. This compound inhibits the Na+ pump activity, which leads to an accumulation of intracellular Na and ultimately might increase vascular tone. In nanomolar concentrations, it enhances vasopressor responses to phenylephrine in some vascular beds from normotensive and hypertensive rats. However, it is not known whether this action of ouabain is a common mechanism for all models of hypertension. The aim of this work was to determine whether ouabain can alter pressor responses to phenylephrine in rats with Nomega-nitro-l-arginine methyl ester (L-NAME)-induced hypertension. In anesthetized rats, ouabain (0.18 microg/kg, i.v.) increased arterial blood pressure in L-NAME-treated rats but not in controls. Ganglionic blockade by hexamethonium (5 mg/kg, i.v.) prevented the increase in arterial blood pressure produced by ouabain in L-NAME-treated rats. Additional studies using isolated perfused tail artery preparations were performed to investigate which factors are involved in the action of ouabain in L-NAME-treated rats. The effects of 10 nM ouabain on the vasoconstrictor actions of phenylephrine were determined on preparations with intact or damaged endothelium or in the presence of tetraethylammonium (a K+-channel blocker). Ouabain reduced pressor actions of phenylephrine in preparations with an intact endothelium. However, after endothelial damage or infusing tetraethylammonium, the response to phenylephrine was increased after ouabain. In tails from L-NAME-treated rats, the functional activity of the Na, K+-ATPase was reduced, and 10 nM ouabain did not produce any further reduction. In conclusion, in this model of hypertension, a low dose of ouabain (0.18 microg/kg) increased arterial blood pressure in vivo probably as a result of increased sympathetic tone. However, this effect was not accompanied by an enhanced action of phenylephrine on the tail vascular bed with an intact endothelium. The results suggest that this was due to the release of an endothelium-derived K+-channel opener.

Animals↗

PubMed1981

Correlation between catecholamine secretion from bovine isolated chromaffin cells and [3H]-ouabain binding to plasma membranes.

1 Secretion of catecholamines (CA) evoked by ouabain, chlormadinone acetate (CMA), phenoxybenzamine (Pbz) and vanadate, four agents known to inhibit Na(+), K(+)-dependent Mg(2+)-activated adenosine triphosphatase (ATPase) activity has been studied in suspensions of bovine isolated adrenal medullary cells.2 Acetylcholine (ACh) evoked a 5 fold increase of the basal CA secretion from isolated cells suspended in oxygenated Krebs-bicarbonate solution kept at 27 degrees C. Secretion was antagonized by Ca(2+)-deprivation or hexamethonium, indicating good functional viability of the cells.3 Ouabain (10(-7) to 10(-4) M) evoked a progressive, dose-dependent release of CA from cell suspensions. Study of the time course of the secretory response for 2 h allowed the separation of two components in the secretory response at all doses studied: a slow initial component (0.011 pg/min CA) and a second faster component (0.032 pg/min CA).4 CMA evoked a clear-cut CA secretory response. The ED(50) for CMA was 10(-4) M, as compared to 3 x 10(-6) M for ouabain. Pbz and vanadate did not induce CA release.5 [(3)H]-ouabain was taken up and bound to intact isolated cells by a non-saturable binding process. However, in semi-purified plasma membranes from bovine adrenal medulla a saturable specific [(3)H]-ouabain binding process was observed with a K(D) of 8.1 nM. Binding to the membranes was ATP-dependent and antagonized by K(+).6 [(3)H]-ouabain specific binding to membranes was antagonized by ouabain and CMA, but not by Pbz or vanadate; the ID(50) for ouabain and CMA were 10(-6) and 10(-5) M respectively.7 Ouabain partially inhibited, in a dose-dependent manner, Na(+), K(+)-Mg(2+) ATPase activity of the semi-purified plasma membranes.8 The results demonstrate a good correlation between the ability of different drugs, known to inhibit ATPase activity, to displace [(3)H]-ouabain binding to adreno-medullary plasma membranes and their capacity to evoke a CA secretory response from isolated chromaffin cells. The data also suggest that the CA secretory effects of ouabain may not be due simply to inhibition of the Na(+) pump and the subsequent ionic redistribution across the plasma membrane; a second mechanism may also be involved.

Acetylcholine↗

PubMed1981

Differential effects of ouabain and 2,4-dinitrophenol on contractile tension of and on sodium and calcium efflux from frog heart ventricular strips.

1 The efflux, from heart ventricular strips of Rana pipiens, of sodium ((22)Na) and calcium ((45)Ca) was measured simultaneously.2(22)Na efflux could be resolved into two first order kinetic components: k(I) = 0.105 min(-1), thought to represent efflux from the extracellular space, and k(II) = 0.0182 min(-1) representing efflux from the cells.3(45)Ca efflux was also resolved into an extracellular component, k(I) = 0.1216 min(-1); and an intracellular one, k(II) = 0.0102 min(-1). (45)Ca k(II) was greatly increased by 2,4-dinitrophenol (DNP), but unchanged by caffeine. This suggests that it represents a mitochondrial calcium compartment.4(22)Na k(II) was not changed by DNP. This indicates that, at the time of DNP addition, (22)Na was passively bound to undefined intracellular components.5 Ouabain (10(-6) M) decreased (45)Ca efflux (k(II)) initially but at later periods slightly increased it. The former effect is thought to be due to an action at the plasma membrane level, while the latter probably represents an increased exchangeability of mitochondrial calcium. The same effects were always found when ouabain was applied at different times of strip superfusion.6 Ouabain (0.25 to 4 muM) did not decrease the k(II) of (22)Na efflux. Kinetic reasons are presented which indicate that, in this preparation, the activity of the sodium pump may be too fast to be measured by means of (22)Na efflux, therefore these findings do not necessarily mean that ouabain does not inhibit active sodium transport.7 The time course of the inotropic effect of ouabain was also studied in ventricular strips of Rana pipiens heart that were stimulated at 0.2 Hz with biphasic, 2 ms pulses of supramaximal intensity, and incubated in Ringer solution containing 1.1 mM calcium, or in ;calcium-free' Ringer (residual calcium: 5.2 muM), or in ;calcium-free' Ringer with 0.1 mM of the calcium chelator ethyleneglycol bis (beta-aminoethylether) N,N'-tetraacetic acid (EGTA).8 In Ringer, the inotropic effect of ouabain was already observed at 5-10 s after steroid addition, even with the lowest concentration tested (0.25 muM), while signs of toxicity appeared only after 15 min in 4 muM ouabain, the highest concentration used.9 When the strips were incubated in ;calcium-free' Ringer solution, force of contraction decayed to 1-2% of that in 1.1 mM calcium. Addition of 4 muM ouabain to these hypodynamic strips led to a progressive increase in contractile force of up to 300%, that started after a 50 s latency period. No signs of toxicity were observed.10 Incubation of the strips in EGTA-Ringer also reduced contractile force to about 2% of that in Ringer, and 4 muM ouabain also increased force of contraction by approximately the same amount as seen in ;calcium-free' Ringer, but the effect began after a 10 min latency period. The concentration of calcium ion (Ca(2+)) in the extracellular space of strips incubated in EGTA-Ringer, was approximately 800 fold lower than in Ringer, and 60 fold lower than in ;calcium-free' Ringer solution.11 Caffeine (20 mM) induced, in strips previously incubated for 1 h in 4.4 mM calcium Ringer solution plus 10(-6) M ouabain, a marked initial contracture, that relaxed spontaneously, and was followed by slow waves of contracture. This was not observed if the strips were incubated, prior to caffeine, in 4.4 mM calcium Ringer without ouabain, or in 1.1 mM calcium Ringer solution that contained 10(-6) M ouabain.12 Based on these findings, a hypothesis that can explain the inotropic effect of cardioactive steroids is presented.

2,4-Dinitrophenol↗

PubMed1991

Modulation of ouabain binding in beating ventricular myocardium from guinea-pigs: effects of lidocaine and monensin.

To further elucidate the interdependence between digitalis sensitivity and the cellular Na+ load, the influence of two Na+ load modifying drugs, monensin and lidocaine, on the concentration-dependence of ouabain binding and ouabain effects was studied in beating ventricular strips from guinea-pig heart. Monensin (3 x 10(-6)M), a Na+ ionophore known to elevate Na+ influx, enhanced 3H-ouabain binding (by approximately 40%) as well as the ouabain effect at non-toxic ouabain concentrations, and it shifted the threshold for toxicity towards threefold lower ouabain concentrations. Lidocaine (2 x 10(-4)M), a Na+ channel blocker, lowered ouabain binding by about one third, and it extended the ouabain concentration range tolerated without toxicity by a factor of three. In the concentrations used, neither compound exerted any direct effect on ouabain binding studied in isolated cardiac membranes. The binding data obtained in the muscle strips were well fitted by a mathematical model which quantifies the dependence of ouabain binding on the underlying (Na+ +K+)-ATPase activity. These findings provide evidence for an indirect drug-induced modulation of ouabain binding via the interference with the cellular Na+ load.

Animals↗

PubMed1978

The effect of ouabain on noradrenaline output from peripheral adrenergic neurones of isolated guinea-pig vas deferens.

1. The effect of ouabain on the noradrenaline output from peripheral adrenergic neurones has been studied using isolated guinea-pig vasa deferentia.2. Exposure to ouabain (10(-4)M) causes a gradual increase in the noradrenaline output. The effect occurs after a delay of 20 min and reaches a maximum during the period from 40-60 min.3. In the absence of external Ca, exposure to ouabain fails to produce an increase in the noradrenaline output. However, the reintroduction of Ca (2.5 mM) after a 1 hr exposure to ouabain in Ca-free media causes a rapid rise in noradrenaline output which reaches a maximum within the first 20 min.4. After a 1 hr exposure to a low concentration of ouabain (10(-5)M) the reintroduction of Ca is almost ineffective in increasing the noradrenaline output. When the concentration of ouabain is increased, the reintroduction of Ca becomes effective and causes a maximum effect with 10(-4)M ouabain. In the presence of a constant amount of ouabain (10(-4)M) the noradrenaline output induced by the reintroduction of Ca increases over the range 0.2-2.5 mM.5. In the presence of ouabain (10(-4)M) the Ca-induced noradrenaline output increases in a linear fashion with increasing Na concentrations from 25 to 143 mM, as long as NaCl is replaced with equimolar choline chloride or isotonic sucrose.6. In the presence of the lowest effective concentration of sodium (25 mM) the noradrenaline output induced by the reintroduction of Ca after a 1 hr exposure to ouabain is potentiated by LiCl. However, in the complete absence of Na(+) ions, there is no Li-dependent increase in the Ca-induced noradrenaline output.7. It is suggested that ouabain may cause an increase in noradrenaline output by an effect on the Na-dependent Ca influx system.

Adrenergic Fibers↗

PubMed1979

The interaction of ouabain and salicylate on sheep cardiac muscle.

1. The action potential duration in sheep ventricular fibres is rapidly diminished on exposure to 10(-6) M-ouabain. However, if 10--20 mM-sodium salicylate is added to the ouabain solution, glycoside-induced shortening is prevented, and a substantial increase in duration then occurs. Sodium salicylate also reverses the shortening effect of ouabain if applied after the glycoside has been allowed to act alone. 2. Sodium salicylate alone produces a much smaller prolongation than in the presence of ouabain. Alone and in the presence of ouabain it eventually increases the threshold and produces inexcitability. 3. Three other surface charge agents have been compared with salicylate: aminonaphthalene sulphonate, sodium dodecylsulphate and salicylamide, were unable to counter the actions of ouabain at the concentrations used. Since they also produced no changes in excitability it is likely that they did not bind significantly to the cell membrane. 4. In Purkinje fibres the reversal potential for the pacemaker current, iK2, is initially shifted in a negative direction in the presence of 10(-6) M-ouabain and 10 mM-salicylate instead of the positive shift expected with ouabain alone at this concentration. 5. In guinea-pig ventricle, salicylate alone reduces the duration of the action potential. This effect is rapidly reversible. Toxic levels of ouabain also reduce the action potential duration but this effect takes several hours to reverse. By contrast, the effects of salicylate and ouabain applied together are readily reversible. 6. It is suggested that the mechanism of these effects may depend on the ability of a surface negative charge agent like salicylate to increase the surface K+ concentration at the membrane and so protect the sodium-potassium pump from inhibition by large doses of ouabain.

Action Potentials↗

PubMed1980

Separate and combined effects of ouabain and extracellular potassium on renin secretion from rat renal cortical slices.

1. Renin secretion of rat renal cortical slices was measured as a function of extracellular K and ouabain concentrations in the incubation medium.2. A sigmoid relationship was found between renin secretion and log K concentration over the range 1.0-4.0 mM. Secretion was maximal at about 2.25 mM-K and half-maximal at about 1.43 mM-K.3. In media containing 4.0 mM-K, ouabain at 10(-8), 10(-7), and 10(-6)M did not affect renin secretion. Higher concentrations of ouabain inhibited secretion. A sigmoid relationship was found between% inhibition of secretion and log ouabain concentration (10(-6)-10(-3)M). Inhibition was half-maximal at 2.3 x 10(-5)M and complete at 10(-3)M-ouabain.4. Lowering extracellular K concentration from 4.0 to 2.25 mM shifted the dose-effect curve of ouabain to the left. At 2.25 mM-K, inhibition of renin secretion was half-maximal at 10(-5)M-ouabain.5. The inhibitory effect of 2 x 10(-5)M-ouabain (twice the dose for 50% inhibition) in media containing 2.25 mM-K was nearly identical to the combined effect of lowering K to 1.43 mM (the concentration required for 50% inhibition) and adding 10(-5)M-ouabain. This observation suggests that ouabain and low extracellular K act at a common site, presumably on Na, K-ATPase activity, to inhibit renin secretion.6. Neither 10(-3)M-ouabain nor K-free medium inhibited renin secretion when the concentration of free Ca in the medium was lowered to < 10(-8)M. Therefore it is proposed that as a result of Na, K-ATPase inhibition, (a) intracellular Na increases, (b) intracellular Ca increases via Na-Ca exchange, provided that extracellular Ca exceeds 10(-8)M, and that (c) Ca accumulation, in some unknown manner, inhibits renin secretion from rat renal cortical slices.

Animals↗

PubMed1983

Effects of ouabain on carotid body chemoreceptor activity in the cat.

The effects of infusions of ouabain on chemoreceptor activity recorded from the peripheral end of a sectioned carotid sinus nerve were studied in cats anaesthetized with pentobarbitone. Ouabain caused a marked increase in chemoreceptor discharge followed by a decline in discharge to frequencies near or below the pre-ouabain level; during the latter period further administration of ouabain had no effect. Infusion of ouabain during hypoxia further increased the chemoreceptor discharge, but this effect was short-lasting. On intracarotid administration ouabain was less effective in cats with the ganglioglomerular (sympathetic) nerves cut, whereas on intravenous administration no significant difference was observed. Following intravenous administration of ouabain the chemoreceptor peak discharge occurred with dose levels similar to those needed to cause cardiac arrhythmias, but following intracarotid administration the chemoreceptor discharge peaked at doses about 40% of those causing arrhythmias. During ouabain-induced excitation the stimulatory action of NaCN, CO2-equilibrated Locke solution and acetylcholine was potentiated, as was the chemo-inhibition induced by dopamine. During the post-excitatory period the responses evoked by these substances were reduced or abolished. Neither mecamylamine, a nicotinic antagonist, nor physostigmine, an anti-cholinesterase, affected the response of the carotid chemoreceptors to ouabain. The major finding of this study was that ouabain initially 'sensitizes' the carotid body chemoreceptors and then 'desensitizes' them. The most likely mechanism responsible for these effects is the well established Na+--K+-ATPase-inhibiting property of ouabain.

Action Potentials↗

PubMed1987

Effects of ion substitution on transport and choleretic effect of ouabain.

The role of inorganic ions in hepatic transport and choleretic effect of ouabain was studied in isolated perfused rat liver to verify whether Na+-coupled ouabain uptake into hepatocytes is responsible for the choleretic effect. Hepatic uptake and clearance of ouabain were not significantly affected when perfusate Na+ was replaced by Li+ or choline+, chloride by nitrate or isethionate, or bicarbonate by tricine. However, these ion substitutions, with the exception of Li+, significantly reduced ouabain-induced choleresis and biliary electrolyte excretion. When ouabain was infused at different rates followed by perfusion without ouabain, changes in bile flow paralleled biliary excretion of ouabain rather than hepatic uptake. These results indicate that hepatic uptake of ouabain is not Na+ dependent and that the osmotic effect of biliary excreted ouabain is responsible for its choleretic effect. A part of the choleretic effect (30%) must also involve other mechanisms, since a permeable anion-like nitrate failed to substitute for perfusate chloride. Results of infusion studies also showed that ouabain was concentrated in liver (liver/perfusate = 30) and in bile (bile/liver = 15), indicating that ouabain is transported against its concentration gradient across both sinusoidal and canalicular membranes.

Animals↗

PubMed2002

Ouabain-binding protein(s) from human plasma.

Conservation of the binding site on mammalian Na+,K+-ATPase for cardiac glycosides and the importance of the Na+ pump in mammalian cellular physiology has stimulated the search for a mammalian analog of these plant compounds. One candidate, isolated from brain and blood, appears to be ouabain itself or a closely related isomer, the ouabain-like compound. Little is known about the circulating form. Because human steroid hormones circulate with carrier proteins, we produced a ouabain-specific monoclonal antibody (mAb 1-10) and used it to probe normal human plasma for ouabain-protein carrier complex. Ouabain-like biological activity was isolated in association with protein bands of 80, 50, and 25 kDa. These proteins appear to be human immunoglobulins or immunoglobulin-like because they are recognized by anti-human immunoglobulin antibodies, but not by anti-mouse immunoglobulin antibodies. The protein-containing fractions inhibit the binding of mAb 1-10 to immobilized ouabain, and with further purification on protein A, the immunoglobulin-like protein binds radioactive ouabain with an IC50 of 200 to 600 nmol/L, but binds digoxin with 100-fold less affinity, suggesting specificity for ouabain or its isomer. Active protein fractions after purification on C18 inhibit Na+ pump activity in human erythrocytes (IC50 approximately 4 nmol/L, ouabain equivalents), and this chromatography appears to dissociate the ouabain-like compound from the immunoglobulin protein(s). These immunoglobulin-like molecules may represent a subset of immunoglobulins (< or =0.5% of total protein A immunoglobulin) that function as a reservoir and delivery system for ouabain-like compounds in the modulation of human Na+, K+-ATPase in vivo.

Animals↗

PubMed1994

Chronic central versus peripheral ouabain, blood pressure, and sympathetic activity in rats.

To assess whether chronic ouabain administration causes hypertension by increasing sympathetic activity, we recorded arterial blood pressure and heart rate at rest and after ganglionic blockade in conscious Wistar rats following 10 to 14 days of central or peripheral administration of ouabain. Intracerebroventricular or intravenous infusion of ouabain (10 micrograms/d for both) as well as subcutaneous ouabain pellets (releasing 25 micrograms ouabain/d per pellet) increased mean arterial pressure by 20 to 30 mm Hg and heart rate by 40 to 60 beats per minute. Ouabain pellets increased blood pressure and heart rate in a dose-related manner. After 2 weeks of all ouabain treatments, ouabainlike activity in plasma was not changed but increased significantly in hypothalamus and adrenals. Ouabainlike activity in the adrenals was increased more by intravenous than subcutaneous or intracerebroventricular ouabain treatment, but the different treatment modes caused similar increases in the hypothalamus. Concomitant central infusion of antibody Fab fragments against ouabain prevented the ouabain pellet-induced increases in blood pressure and heart rate. Ganglionic blockade by intravenous hexamethonium normalized blood pressure and heart rate in ouabain-treated rats. These data suggest that in normotensive rats exogenous ouabain, regardless of the mode of administration, may act centrally to cause sympathoexcitation and thus hypertension.

Animals↗