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PubMed1982

Interaction between ouabain and the phosphorylated intermediate of Na,K-ATPase.

After phosphorylation of electric eel Na,K-ATPase by Na+, Mg2+, and ATP was terminated by removing the unbound Mg2+, the phosphoenzyme was able to bind ouabain upon the addition of 2 mM ouabain under certain conditions. This binding was demonstrated by a 50% inhibition of ATPase after the removal of unbound ouabain by a Sephadex G-50 column (ouabain trapping method). At 4 degrees, this ouabain binding was observed on the K+- sensitive phosphoprotein (E2P) formed in the presence of 10 mM Na+ but was not observed on the ADP-sensitive phosphoprotein (E1P) formed in the presence of 1000 mM Na+. The increase in the dephosphorylation rate of E2P with various concentrations of K+ paralleled the decrease in inhibition by the addition of 2 mM ouabain after the termination of phosphorylation. In 50-200 mM Na+, the eel enzyme used here formed the E1P-rich phosphoprotein, but this phosphoprotein could bind with ouabain, even though the presence of ADP or oligomycin, which prevents the conversion of E1P to E2P, partially interfered with this ouabain binding. At 25 degrees, ouabain binding with E1P-rich phosphoprotein was observed in higher yield (up to 71%), but in each of these cases ADP or oligomycin strongly inhibited ouabain binding. Moreover, ouabain binding with E2P-rich phosphoprotein did not significantly change with temperature, but ouabain binding with E1P-rich phosphoprotein increased more than 6 times at temperatures from 4 degrees to 25 degrees. From these results, it can be concluded that E2P can bind with ouabain in the absence of free Mg2+ whereas E1P cannot, and that the interconversion between E1P and E2P can be stimulated with ouabain binding and accelerated with elevation of temperature. ADP- and K+ -insensitive phosphoprotein probably is only a minor intermediate for ouabain binding.

Animals↗

PubMed1995

Age-dependency and dietary influence on the hypothalamic ouabain-like factor in Milan hypertensive rats.

AIM: Previous studies have demonstrated that the hypothalamus of the adult Milan hypertensive rat strain (MHS) contains a higher proportion of ouabain-like factor than Milan normotensive (MNS) controls. The present study was designed to demonstrate that the rat standard diet contains a ouabain-like factor similar to that extracted from rat tissue and to investigate the influence of low or high dietary ouabain-like factor content on tissue ouabain-like factor levels at different ages in MHS and MNS rats. MATERIALS AND METHODS: MHS and MNS rats were reared on two controlled batches of standard rat diet containing low (batch A 0.09 mu g/kg) and high (batch B 0.7 mu g/kg) concentrations of ouabain-like factor. The mothers of these rats had also been fed with the diet throughout pregnancy and lactation. The hypothalamic content of ouabain-like factor was measured in both strains at 21, 30 and 90 days of age by high performance liquid chromatography fractionation. RESULTS: (1) The dietary ouabain-like factor content did not influence either the hypothalamic ouabain-like factor yield or systolic blood pressure, either in MHS or MNS rats. (2) As a function of age, the hypothalamic ouabain-like factor content was constant between 21 and 30 days of age in MHS rats, and then decreased by 60% at 90 days. In MNS rats, ouabain-like factor was decreased by 80 and 90%, respectively, at 30 and 90 days, compared to the age of 21 days. (3) At the age of 21 days, MHS rats had 30% lower levels of ouabain-like factor than MNS rats, but 60% higher levels at 30 and 90 days. CONCLUSIONS: Hypothalamic ouabain-like factor and systolic blood pressure are not influenced by dietary ouabain-like factor, thus excluding a process of passive tissue accumulation. Different mechanisms regulate the age-dependent endogenous ouabain-like factor production and accumulation in MHS and MNS rats, suggesting that the maintenance of higher ouabain-like factor levels in MHS than in MNS at the age of 30 and 90 days contributes to the development and maintenance of hypertension in this strain.

Age Factors↗

PubMed1982

Studies on ouabain-complexed (Na+ +K+)-ATPase carried out with vanadate.

Vanadate is able to promote the binding of ouabain to (Na+ +K+)-ATPase and it is shown that vanadate is trapped in the enzyme-ouabain complex. Also ouabain-bound enzyme, the formation of which was facilitated by (Mg2+ +Na+ +ATP) or (Mg2+ +Pi), is accessible to vanadate when washed free of competing ligands used for the promotion of ouabain binding. For vanadate binding to (Na+ +K+)-ATPase and to enzyme-ouabain complexes a divalent cation (Mg2+ or Mn2+) is indispensable, indicating that the cation does not remain attached to the ouabain-bound enzyme. K+ further increases vanadate binding in the absence of ouabain, but seems to have no additional role in case of vanadate binding to enzyme-ouabain complexes. Mn2+ is more efficient than Mg2+ in promoting binding of vanadate and ouabain to (Na+ +K+)-ATPase. That K+ in combination with Mn2+, in analogy with the effect in combination with Mg2+, increases the equilibrium binding level of vanadate and decreases that of ouabain does not seem to favour the hypothesis of selection of a special E2-subconformation by Mn2+. The vanadate-trapped enzyme-ouabain complex was examined for simultaneous nucleotide binding which could demonstrate a two-substrate mechanism per functional unit of the enzyme. The acceleration by (Na+ +ATP) of ouabain release from the (Mg2+ +Pi)-facilitated enzyme-ouabain complex does not, as anticipated, support such a mechanism. On the other hand, the deceleration of vanadate release as well as of ouabain release from a (Mg2+ +vanadate)-promoted complex could be consistent with a two-substrate mechanism working out-of-phase.

Animals↗

PubMed1976

Induction by ouabain of hemoglobin synthesis in cultured Friend erythroleukemic cells.

Induction of erythroid differentiation in ouabain-resistant murine erythroleukemia cells by ouabain is reported. Ouabain induction results in the appearance of hemoglobin-containing cells 12-24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide. The concentration of ouabain required to induce hemoglobin synthesis depends upon the K+ ion levels in the culture medium. Lowering the extracellular K+ ion concentration 2-4 fold reduced by 10-40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis. In low K+ medium (1.8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0.02 mM. This K+ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K+ ions in the culture medium. These results suggest that the binding of ouabain to the plasma membrane enzyme, Na/K ATPase, is required for the induction of erythroid differentiation by ouabain. A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells. The observation that the binding of ouabain to the Na/K ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K+ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells.

Acetamides↗

PubMed1981

Ouabain binding to chick embryo neuroretina during development in ovo and in monolayer cultures.

Eight-day-old embryo neuroretinas (NR) were dissociated and cultured as monolayer. [3H]Ouabain binding was investigated during the culture period. [3H]Ouabain binding to NR increases continuously until around the tenth day of culture, but after that it gradually decreased. On the other hand, [3H]ouabain binding to NR during in ovo development continued to increase until the embryo was 20 days old. Na-K-ATPase activity was also compared with [3H]ouabain binding during development in ovo and in monolayer culture. It was found that changes in Na-K-ATPase activity of NR in vitro and in ovo were different from those of [3H]ouabain binding activity. Exposure of NR at the second day of culture to 0.1 mM ouabain for 8 h resulted in the degeneration of some differentiated neurons (rosette-formed cells), but new rosettes were soon re-formed after the withdrawal of ouabain, and [3H]ouabain binding to the ouabain-treated NR was decreased by 30%. Ouabain-treatment of NR in 10-day-old cultures completely destroyed the aggregated neurons, but did not apparently injure the glial cell layer. [3H]Ouabain binding decreased more than 90% in the ouabain-treated NR. These results suggest that [3H]ouabain binding activity of NR and the sensitivity to ouabain toxicity are useful indicators for differentiation and development of neurons in the cultured retinal cells.

Animals↗

PubMed1978

Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na, K-ATPase.

We have synthesized 2-nitro-5-azidobenzoyl (NAB) derivatives of ouabain as photoaffinity labels of the cardiac glyocoside binding site of Na, K-ATPase. [3HzNAB-ouabain was found to bind to the same number of sites on Na, K-ATPase (purified from pig kidney outer medulla) as ouabain (1.9 nmol/mg), with approximately the same affinity (Kk(ouabain)/Kd(NAB-ouabain) congruent to 1.6), and ouabain was fully competitive uith NAB-ouabain at these sites. NAB-ouabain binding and inhibition were reversible in the dark, but on exposure to ultraviolet light (310-370 nm) 30-40% of the binding and ihibition became irreversible; this binding was shown to be covalent by stability to trichloroacetic acid, organic solvents, and heat denaturation. Covalent labeling was prevented by photolysis of NAB-ouabain prior to the experiment, or by prior incubation of the enzyme with ouabain. On sodium dodecyl suffate-polyacrylamide gels of labeled Na,K-ATPase, about half of the covalently bound [3H]NAB-ouabain migrated with the large polypeptide (molecular weight congruent to 95 000), and half migrated with a small polypeptide (molecular weight congruent to 12 000); noncovalently bound NAB-ouabain (60-70% of total label) ran with the tracking dye. A similar labeling pattern was obtained utilizing NaI microsomes prepared from pig kidney outer medulla. The small polypeptide was characterized as an acidic proteolipid by extractability into acid chloroform/methanol; labeling of this component by NAB-ouabain is the first demonstration that it is directly associated with the Na,K-ATPase. The results of our characterization of NAB-ouabain show that it has the required specificity, covalency, and efficiency of labeling for application in structural studies of Na,K-ATPase subunit interactions.

Affinity Labels↗

PubMed2004

Contribution of the endothelin and renin-angiotensin systems to the vascular changes in rats chronically treated with ouabain.

Renin-angiotensin and endothelin systems are involved in the cardiovascular effects produced by treatment with ouabain. We recently demonstrated that the contractile response to phenylephrine is decreased in ouabain-treated rats. The present study investigated whether endothelin-1 (ET-1) and angiotensin II (Ang II) contributes to the vascular changes observed in rats chronically treated with ouabain. Wistar rats were treated with ouabain (8.0 microg day(-1), s.c. pellets for 5 weeks) alone or in combination with an endothelin type A receptor (ET(A)) antagonist, BMS182874 (40 mg kg(-1) day(-1), per gavage) or an angiotensin type 1 (AT(1)) receptor antagonist, losartan (15 mg kg(-1) day(-1), p.o.). Treatment with ouabain increased systolic blood pressure and treatment with either losartan or BMS182874 prevented the development of ouabain-induced hypertension. The sensitivity and maximal response for phenylephrine were reduced in aortic rings from ouabain-treated rats. Removal of the endothelium or in vitro exposure to an inhibitor of nitric oxide synthase (NOS), N-nitro-L-arginine methyl ester (L-NAME, 100 microM) increased the responses to phenylephrine, an effect that was more pronounced in aortas from ouabain-treated rats. Endothelial NOS protein (eNOS) expression was increased after ouabain treatment. Treatment with BMS182874, but not with losartan, prevented the effects of ouabain on the reactivity of phenylephrine and in eNOS protein expression. Gene expression of pre-pro-ET-1 and ET(A) receptors was increased in aortic rings from ouabain-treated rats. ET(B) receptor gene expression was not altered by ouabain treatment. In conclusion, our results suggest that endothelin and angiotensin systems play an important role in the development of ouabain-induced hypertension. However, ET-1, by activation of ET(A) receptors, but not Ang II, contributes to changes in vascular reactivity to phenylephrine induced by chronic treatment with ouabain.

Animals↗

PubMed1980

Isolation of diploid human lymphoblast mutants presumably homozygous for ouabain resistance.

From a mass culture of diploid human lymphoblasts we have isolated a subline resistant to 1 muM ouabain, and from this, a subline resistant to 10 muM ouabain. These sublines occurred spontaneously, but similar mutants were induced with 10-fold increased frequency by treatment with mutagens, in which case they could be selected as clones in soft agarose. Scatchard plot analyses of ouabain binding indicated that the subline resistant to 1 muM ouabain retained an average of 39% of the high-affinity ouabain receptors of the parental lymphoblast line, and the subline resistant to 10 muM ouabain retained an average of 8.4%. The ouabain binding site is known to be located in the Na(+),K(+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) molecule, an essential cell membrane enzyme that mediates ion transport. Studies on Na(+),K(+)-ATPase activity, using (86)Rb in the absence and presence of ouabain, indicated that our parental lymphoblasts contained one population of Na(+),K(+)-ATPase molecules highly sensitive to ouabain inhibition, the subline resistant to 10 muM ouabain contained one population relatively insensitive to ouabain, and the subline resistant to 1 muM ouabain contained both populations. Thus, the moderately resistant subline appears to be heterozygous for ouabain resistance, probably containing a structural mutation in the ouabain receptor region of the Na(+),K(+)-ATPase molecule in one of the two homologous loci for this enzyme, whereas the highly resistant subline derived from it appears to be homozygous, containing an additional mutation in the other Na(+),K(+)-ATPase locus.

Binding Sites↗

PubMed1990

Intracellular sodium affects ouabain interaction with the Na/K pump in cultured chick cardiac myocytes.

Whether a given dose of ouabain will produce inotropic or toxic effects depends on factors that affect the apparent affinity (K0.5) of the Na/K pump for ouabain. To accurately resolve these factors, especially the effect of intracellular Na concentration (Nai), we have applied three complementary techniques for measuring the K0.5 for ouabain in cultured embryonic chick cardiac myocytes. Under control conditions with 5.4 mM Ko, the value of the K0.5 for ouabain was 20.6 +/- 1.2, 12.3 +/- 1.7, and 6.6 +/- 0.4 microM, measured by voltage-clamp, Na-selective microelectrode, and equilibrium [3H]ouabain-binding techniques, respectively. A significant difference in the three techniques was the time of exposure to ouabain (30 s-30 min). Since increased duration of exposure to ouabain would increase Nai, monensin was used to raise Nai to investigate what effect Nai might have on the apparent affinity of block by ouabain. Monensin enhanced the rise in Na content induced by 1 microM ouabain. In the presence of 1 microM [3H]ouabain, total binding was found to be a saturating function of Na content. Using the voltage-clamp method, we found that the value of the K0.5 for ouabain was lowered by nearly an order of magnitude in the presence of 3 microM monensin to 2.4 +/- 0.2 microM and the magnitude of the Na/K pump current was increased about threefold. Modeling the Na/K pump as a cyclic sequence of states with a single state having high affinity for ouabain shows that changes in Nai alone are sufficient to cause a 10-fold change in K0.5. These results suggest that Nai reduces the value of the apparent affinity of the Na/K pump for ouabain in 5.4 mM Ko by increasing its turnover rate, thus increasing the availability of the conformation of the Na/K pump that binds ouabain with high affinity.

Animals↗

PubMed1995

Long-term ouabain administration does not alter blood pressure in conscious Sprague-Dawley rats.

1. We tested the ability of ouabain to cause chronic hypertension by continuously infusing ouabain for 28 days (miniosmotic pump implantation; i.p.). The blood pressure and metabolic effects of sham (150 mmol/L NaCl; n = 12) or ouabain infusion (10 micrograms/kg per day; n = 14; 100 micrograms/kg per day; n = 14) were examined in conscious Sprague-Dawley rats. 2. Plasma ouabain concentrations measured after 28 days of ouabain infusion were as follows: sham, not detectable (n = 11); ouabain 10 micrograms/kg per day, 0.60 +/- 0.07 nmol/L (n = 14); and ouabain 100 micrograms/kg per day, 7.17 +/- 0.57 nmol/L (n = 14; P < 0.001). 3. Sham or ouabain infusion did not alter food intake, bodyweight, water intake or urine output in conscious rats. 4. Blood pressure was not altered by sham treatment. Ouabain at 10 micrograms/kg per day or 100 micrograms/kg per day did not produce consistent rises in blood pressure. Ouabain at 10 micrograms/kg per day increased blood pressure on treatment day 12 only (+6 mmHg; P < 0.05), while at 100 micrograms/kg per day blood pressure increased on treatment days 16 (+9 mmHg; P < 0.05) and day 18 (+8 mmHg; P < 0.05) only. There was no significant difference in blood pressure between sham and ouabain groups. 5. Renal blood flow was decreased in rats infused with ouabain at 10 micrograms/kg per day (2.0 +/- 0.3 mL/min per 100 g bodyweight; n = 5; P < 0.01) and 100 micrograms/kg per day (2.2 +/- 0.4 mL/min per 100 g bodyweight; n = 7; P < 0.05) compared with sham treatment (3.5 +/- 0.2 mL/min per 100 g bodyweight; n = 6). Renal vascular resistance was increased in rats treated with ouabain at 10 micrograms/kg per day (65.5 +/- 12.6 mmHg/mL per min per 100 g bodyweight; n = 5; P < 0.01) and 100 micrograms/kg per day (66.0 +/- 15.6 mmHg/mL per min per 100 g bodyweight; n = 7; P < 0.05) compared with sham treatment (32.6 +/- 2.5 mmHg/mL per min per 100 g bodyweight; n = 6). 6. High plasma concentrations of ouabain do not cause consistent increases in blood pressure in conscious Sprague-Dawley rats.

Animals↗

PubMed2000

Different effects of in vivo ouabain and digoxin on renal artery function and blood pressure in the rat.

To investigate vascular mechanisms in hypertension, we isolated renal arterial rings from rats with ouabain-dependent hypertension and studied their function. In rats infused with ouabain for 5 weeks, systolic and mean blood pressures (BP) were increased relative to controls. Contractions evoked by high KCl solutions were greater in rings from ouabain-infused rats whereas the threshold concentrations and EC50s for KCl and the peak caffeine contractures were not different. KCl contractures were not affected by 5 microM prazosin. Phenylephrine contractures were increased marginally in ouabain-infused rats, while acetylcholine-induced relaxation was normal. In vitro superfusion of rings with 10 nM ouabain or digoxin did not affect the measured parameters. Plasma ouabain, BP, and all evoked responses were normal one week following interruption of the ouabain infusion. In a second study, BP increased in ouabain (15 microg/kg/day, n= 23), but not digoxin (30 microg/kg/day, n=12), or vehicle-infused (n=16) rats. KCl contractures were greater in rings from ouabain-but decreased in rings from digoxin-infused rats, respectively and correlated with systolic and mean BP (r=0.69, n=30, p<0.005). Peak caffeine (25 mM) responses were similar but the area under the contraction was reduced in the vessels from ouabain-infused rats and correlated inversely with MBP (r=-0.47, n=33, p<0.02). We conclude that a voltage-dependent component of tone in the rat renal artery is reversibly and specifically augmented by in vivo administration of ouabain whereas it is diminished by in vivo digoxin. Vascular production of and response to nitric oxide does not appear to be impaired in the ouabain model. Alterations of intracellular Ca2+ storage and Ca2+ influx in response to in vivo ouabain may underlie the increase in renal vascular resistance and hypertension in this model. The opposite effects of ouabain and digoxin on the hemodynamic and vascular parameters in this study indicate that these agents have novel mechanisms of action in vivo that may not be mediated exclusively by sodium-potassium pumps.

Acetylcholine↗

PubMed1999

Monoclonal antibodies that distinguish between two related digitalis glycosides, ouabain and digoxin.

The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine gamma-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12beta (digoxin) or 16beta (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo.

Animals↗

PubMed1980

Affinity labeling of the digitalis receptor with p-nitrophenyltriazene-ouabain, a highly specific alkylating agent.

Three derivatives of ouabain have been synthesized which alkylate the digitalis receptor. These derivatives were formed through reductive amination of p-nitrophenyltriazene (NPT) ethylenediamine to the periodate-oxidized rhamnose moiety of ouabain. The non-covalent binding of the ouabain derivatives (NPT-ouabain, designated I, II, and III) was followed (i) by their ability to inhibit the activity of sodium- and potassium-activated ATPase ((Na+,K+)-ATPase) purified from the electric organ of Electrophorus electricus, (ii) by the binding of [3H]NPT-ouabain I to the enzyme, and (iii) by the inhibition of [3H]ouabain binding with unlabeled NPT-ouabain I. Covalent modification of the digitalis site of (Na+,K+)-ATPase occurs after long periods of time. At pH 7.5 (25 degrees C) the best alkylating derivative, NPT-ouabain I, gives maximum covalent labeling after 6 h. Only the large polypeptide chain (Mr = 93,000) of the purified enzyme is specifically labeled with [3H]NPT-ouabain I while the glycoprotein chain (Mr = 47,000) is not significantly labeled. Labeling of a microsomal fraction of the electric organ with [3H]NPT-ouabain I gave the same type of gel pattern as that observed with the purified enzyme. [3H]NPT-ouabain I was also used to label the digitalis receptor in highly purified axonal membranes and in cardiac membranes prepared from embryonic chick heart. Although the (Na+,K+)-ATPase in both types of membranes has a low affinity for ouabain, [3H]NPT-ouabain I proved to be a very efficient affinity label for the digitalis receptor. In the complex mixture of polypeptides found in these membrane preparations, only a single polypeptide chain having a Mr = 93,000 is specifically labeled by [3H]NPT-ouabain I.

Affinity Labels↗

PubMed1979

Phenotypic characterization of ouabain-resistant Aedes albopictus cells.

The phenotype of a ouabain-resistant Aedes albopictus cell line has been partially characterized. Treatment of ouabain-sensitive cells with 0.005-1.0 mM ouabain resulted in an 80% reduction in the uptake of 86rubidium (86Rb+), an ion with an affinity for the K+ pump binding site; ouabain-resistant cells showed only a 40% reduction with 1.0 mM ouabain. When ouabain-sensitive cells were incubated in the presence of ouabain (0.1 mM) for one and one-half to three hours, the molar ratio of intracellular Na+/K+ rose from 0.2 to 4.2. In ouabain-resistant cells, a similar treatment had very little effect. Based on [3H] ouabain-binding studies, ouabain-resistant cells were estimated to have 60% fewer binding sites per cell than ouabain-sensitive cells. The spontaneous mutation rate from ouabain sensitivity to ouabain resistance was calculated to be 1-6 x 10(-8) mutations/cell/generation, a value similar to that reported for mammalian cells at the analogous locus.

Aedes↗

PubMed1990

Characterization of ouabain-induced phosphoinositide hydrolysis in brain slices of the neonatal rat.

The effect of the Na/K-ATPase inhibitor ouabain on phosphoinositide (Ptdlns) hydrolysis was studied in rat brain cortical slices. Ouabain induced a dose-dependent accumulation of inositol phosphates (InsPs) which was much higher in neonatal rats (1570 +/- 40% of basal) than in adult animals (287 +/- 18% of basal). For this reason, all experiments were conducted with 7 day-old rats. Strophantidin caused a similar stimulation of Ptdlns hydrolysis, although it was less potent than ouabain. The order of potency for ouabain-stimulated InsPs accumulation in brain areas was hippocampus greater than cortex greater than brainstem greater than cerebellum. The effect of ouabain was not blocked by antagonists for the muscarinic, alpha1 -adrenergic and glutamate receptors. Also ineffective were the K+ channel blockers 4-aminopyridine and tetraethylammonium, the sodium channel blocker tetrodotoxin, and the calcium channel blocker verapamil, whereas the Na/Ca exchanger blocker amiloride partially antagonized the effect of ouabain. The accumulation of InsPs induced by ouabain was additive to that of carbachol and norepinephrine, as well as to that induced by high K+ and veratrine, but not to that of glutamate. Removal of Na+ ions from the incubation buffer completely prevented the accumulation of InsPs induced by ouabain. The effect of ouabain was also dependent upon extracellular calcium and was under negative feedback control of protein kinase C. Despite the higher effect of ouabain on Ptdlns hydrolysis of immature rats, the density of [3H]ouabain binding sites, as well as the activity of Na/K-ATPase were higher in adult animals. Furthermore, a poor correlation was found between ouabain-stimulated Ptdlns hydrolysis and [3H]ouabain binding in brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1984

Requirement of Cl- and Na+ for the ouabain-resistant control of cell volume in slices of rat liver.

The ability of liver cells to control their volume in the presence of ouabain has been studied in tissue slices that were recovering at 38 degrees C from a period of swelling at 1 degrees C. Morphological observations were made in conjunction with measurements of the net movement of water and ions. Extrusion of water in the presence of ouabain (2 mM) was accompanied by a net loss of Na+ and Cl- and by the formation of characteristic, rounded vesicles in the peri-canalicular regions of the hepatocytes; bile canaliculi were patent. When incubation was carried out in a medium in which either NO-3 or SO2-4 replaced Cl-, ouabain-resistant water extrusion was prevented and the cytoplasmic vesicles normally found with ouabain were almost totally absent. When these slices were subsequently transferred to Cl- medium with ouabain, extrusion of intracellular water was initiated and cytoplasmic vesicles reappeared. Replacement of medium Na+ by Li+ mimicked the effects of ouabain on water and ion movements and ultrastructure. In addition, the ouabain-resistant extrusion of water and Cl- was reduced and there was some diminution in the number of vesicles induced by ouabain. Furosemide (2 mM) had little effect on water movement or ultrastructure in the absence of ouabain, but it slowed the net water loss and substantially reduced the formation of cytoplasmic vesicles in the presence of ouabain. The results show a close relationship between ouabain-resistant water extrusion and the formation of the cytoplasmic vesicles that are characteristic of treatment with ouabain. They further suggest that a cotransport of Na+ and Cl- forms an important part of the mechanism underlying ouabain-resistant water extrusion and, specifically, that this cotransport may take place across the membranes of the cytoplasmic vesicles.

Animals↗

PubMed2003

Effects of ouabain on human bronchial muscle in vitro.

The effects of ouabain, an inhibitor of the plasmalemmal Na(+)/K(+)-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with -logEC(50)=7.16+/-0.11 and maximal effect of 67+/-4% of the response to acetylcholine (1 mM). Ouabain (10 microM)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H(1)-receptors and alpha-adrenoceptors, or neuronal Na(+) channel blockade. The inhibition of ouabain contraction in tissues bathed in K(+)-free medium, and the inhibition by ouabain of the K(+)-induced relaxation confirm that the contractile action of ouabain is mediated by inhibition of Na(+)/K(+)-ATPase. Furthermore, depolarization (16.4+/-0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 microM)-induced contractions were abolished by a Ca(2+)-free solution but not by blockers of L-type Ca(2+) channels. In human cultured bronchial smooth muscle cells, ouabain (10 microM) produced a sustained increase in [Ca(2+)](i) (116+/-26 nM) abolished in Ca(2+)-free medium. Incubation with a Na(+)-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of ouabain thus suggesting the role of Na(+)/Ca(2+) exchange in ouabain contraction while selective inhibitors of Na(+)/H(+)-antiport, Na(+)/K(+)/Cl(-)-antiport, or protein kinase C had no effect. Ouabain (10 microM) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 microM) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca(2+) entry unrelated to L-type channels and involving the Na(+)/Ca(2+)-antiporter.

Acetylcholine↗

PubMed1982

Interactions between the autonomic nervous system and the cardiovascular effects of ouabain in guinea-pigs.

Anaesthetized guinea-pigs were intoxicated with an intravenous infusion of ouabain. This infusion induced a marked pressor response which was reduced in bilaterally adrenalectomized or pithed animals. Ouabain produced initial bradyarrhythmias in 60% of guinea-pigs. Bilateral vagotomy or pretreatment with atropine abolished the bradyarrhythmias and sensitized the animals to the arrhythmic effects of ouabain. Pithing or beta-adrenoceptor blockade reduced the potency of ouabain for producing arrhythmias, but bilateral adrenalectomy did not give protection. Preferential alpha 2-adrenoceptor stimulation with clonidine (10-300 micrograms . kg-1 i.v.) also reduced the arrhythmogenic effects of ouabain, whereas no protection was found with St 91, a clonidine related compound which does not cross the blood-brain barrier. The effect of clonidine was antagonized by piperoxan. Preferential alpha 2-adrenoceptor blockade with piperoxan (6 mg . kg-1 i.v.) did not change the pressor response to ouabain, but sensitized the animals to the arrhythmogenic effects of ouabain. In contrast, the preferential alpha 1-antagonistic agent AR-C 239 (0.3 mg . kg-1 i.v.) abolished the pressor response to ouabain and in addition increased the dose of ouabain required to produced ventricular premature beats and ventricular fibrillation. These experiments indicate: (i) that ouabain produces in guinea-pigs a pressor response which seems to be due to catecholamine release from the adrenal medulla probably by an action on the central nervous system; (ii) that the ventricular arrhythmias induced by ouabain are due in part to stimulation of the central nervous system leading to an increase in beta-adrenoceptor activity; (iii) that central alpha-adrenoceptors appear to be involved in the arrhythmogenic effects of ouabain, as clonidine reduced these effects. On the other hand piperoxan, a preferential alpha 2-adrenoceptor antagonist did not change the pressor response to ouabain and increased the arrhythmogenic effects whereas AR-C 239 had opposite effects.

Adrenalectomy↗