Inhibition of sarcolemmal Na,K, Mg ATPase from the guinea pig heart is not compatible with a homogeneous population of non-interacting ouabain receptors

Summary The inhibition of sarcolemmal Na, K, Mg ATPase from the guinea pig heart by ouabain was evaluated with a coupled enzyme assay. Models of negative cooperativity and of two independent receptors fitted the inhibition data equally well. The analysis was not compatible with a homogeneous population of non-interacting ouabain receptors.Acknowledgments. The technical assistance of P. Mayr and G. Ruhland is gratefully acknowledged.     

Comparative effects of ouabain, natriuretic factor and ammonium chloride in the toad urinary bladder

Electrical changes and direct effects on Na-K ATPase activity induced by an endogenous digitalis-like natriuretic factor (NF), NH4Cl and ouabain were studied in toad bladders. NF inhibited the SCC and the Na-K ATPase activity in a similar manner to ouabain, but induced a greater increase in calculated direct current resistance (R) (p less than 0.05). NH4Cl was a weak inhibitor of Na-K ATPase activity, although it produced steeper SCC inhibition slopes than those observed with ouabain or NF (p less than 0.01). The data suggested the same mechanism of action of NF and ouabain on the sodium pump, with an additional effect of the former on apical sodium permeability of the cells and/or closure of paracellular routes leading to an increased tissue resistance. In contrast, the effects of NH4Cl were mostly compatible with intracellular inhibition of apical sodium entry into the cell.     

The role of distinct membrane components of the enzyme in the ouabain sensitivity of Na+/K+ ATPase]

Purified plasma membranes from a cell line derived from the murine plasmocytoma MOPC 173 exhibited a 50% inhibition of the Na+K+ ATPase by 120 muM ouabain. EDTA treatment of such membranes induced a drop in the ouabain concentration needed for 50% inhibition to 0.4 muM. Supernatants obtained after EDTA treatment were able to complement with Ca(++) + Mg++ the washed EDTA treated membranes which recover their original resistance.     

Inhibitory effect of taurine on decrease in the inotropic action of ouabain at high concentrations in isolated atria

Summary In vitro, taurine was shown to inhibit the decrease in the inotropic effect of ouabain at large doses in the normal and also low K⁺ medium in which this decrease in the inotropism of ouabain was facilitated. This inhibitory effect of taurine was, at least in part, due to the inhibition of the efflux of intracellular K⁺ in the isolated heart.     

Effects of inhibition and stimulation of Na+-K+ active transport on the resting membrane input conductance of the guinea-pig ventricle

The effects of inhibition by ouabain and stimulation by high frequency drive of the sarcolemmal Na+-K+ active transport system on the resting input conductance (gi) of guinea-pig ventricular muscles were determined. Although both pump inhibition and stimulation were associated with changes in electrophysiological properties of the muscles, neither had a significant effect on gi.     

Effect of prolactin on human red cell sodium transport

Incubation of red cells with higher concentrations of prolactin in vitro enhanced the cellular sodium level and produced a significant reduction in erythrocyte membrane adenosine triphosphatase activity. This effect was dose and time-dependent. It is the result of an inhibition of the active sodium pump similar to that produced by ouabain, suggesting altered red cell function and electrolyte balance in hyperprolactinemic states.     

Synaptosomal adenosine triphosphatase (ATPase) inhibition by organophosphates.

Chicken spinal cord adenosine triphosphatases (both Na+, K+ stimulated and ouabain insensitive) were inhibited by tri-o-tolyl phosphate (TOTP, a neurotoxic organophosphate which is not a cholinesterase inhibitor) and mevinphos (a non-neurotoxic compound but inhibitor of cholinesterases). The inhibition was concentration and time dependent, with an initial rapid drop in activity followed by a gradual exponential decline.     

Effect of prolactin on human red cell sodium transport

Summary Incubation of red cells with higher concentrations of prolactin in vitro enhanced the cellular sodium level and produced a significant reduction in erythrocyte membrane adenosine triphosphatase activity. This effect was dose and time-dependent. It is the result of an inhibition of the active sodium pump similar to that produced by ouabain, suggesting altered red cell function and electrolyte balance in hyperprolactinemic states.Acknowledgments. Ovine prolactin was generously supplied by Ferring (Malmö, Sweden) and NPA, NIH (Maryland, USA). Thanks are also due to Prof. S. Dutta, Wayne State University, Detroit, for the gift, of ouabain octahydrate and Dr M. Ramachandran and Prof. S. Ramakrishnan, Department of Biochemistry, JIPMER, Pondicherry, for their encouragement and keen interest in this study.     

Inhibition of microsomal Na+ K+ ATPase of the brain by extracts of Mansonia altissima]

Water extracts of the bark of Mansonia altissima var altissima inhibit the activity of the Na+, K+-ATPase of Rabbit brain microsomes. The kinetics of hydrolysis of ATP show non-competitive inhibition analogous to that produced by ouabain.     

Effects of inhibition and stimulation of Na+−K+ active transport on the resting membrane input conductance of the guinea-pig ventricle

Summary The effects of inhibition by ouabain and stimulation by high frequency drive of the sarcolemmal Na⁺–K⁺ active transport system on the resting input conductance (g_i) of guinea-pig ventricular muscles were determined. Although both pump inhibition and stimulation were associated with changes in electrophysiological properties of the muscles, neither had a significant effect on g_i.Supported by a grant from the North Carolina Heart Association.     

Correlation between inhibition of stimulatory membrane repolarization and positive inotropic response caused by ouabain in rat heart]

During a stimulus train, the diastolic membrane potential of rat atria exhibits a depolarization phase followed by a slower repolarization phase which has been attributed to the activation of an electrogenic sodium pump (ATPase Na+, K+). This pump seems to be all the more active as stimulation frequency is higher. The parallel evolution of the sodium pump inhibition and a positive inotropic effect in response to ouabain perfusion, suggests that the enzymatic inhibition is directly involved in the development of the cardiotonic effect of digitalis.     

Synaptosomal adenosine triphosphatase (ATPase) inhibition by organophosphates

Summary Chicken spinal cord adenosine triphosphatases (both Na⁺, K⁺ stimulated and ouabain insensitive) were inhibited by tri-o-tolyl phosphate (TOTP, a neurotoxic organophosphate which is not a cholinesterase inhibitor) and mevinphos (a non-neurotoxic compound but inhibitor of cholinesterases). The inhibition was concentration and time dependent, with an initial rapid drop in activity followed by a gradual exponential decline.     

A molecular recognition hypothesis for nonpeptides: Na+ K+ ATPase and endogenous digitalis-like peptides

The molecular recognition hypothesis for peptides is that binding sites of ligands and their receptors are encoded by short, complementary segments of DNA. A corollary hypothesis for nonpeptide ligands posited here is that peptide replicas may be encoded by the DNA segment complementary to the receptor binding sites for nonpeptides. This corollary was tested for digitalis, a family of cardiotonic and natriuretic steroids including ouabain. A hexapeptide (ouabain-like peptide, OLP) complementary to a ouabain binding site on sodium/potassium dependent adenosine triphosphatase (Na⁺ K⁺ ATPase) exhibited activity in a digitalis bioassay. Antisera to the complementary peptide (OLP) stained the neurohypophysis in an immunocytochemical procedure. The complementary peptide was found to share an identical 4-amino acid region with the 39-amino acid glycopeptide moiety of the vasopressin-neurophysin precursor. This glycopeptide was isolated from pituitary extracts; it exhibited digitalis-like activity in the submicromolar range and cross-reacted with complementary peptide antibodies. Another digitalis-like substance with high activity also was detected in the extracts. These results demonstrate that the vasopressin-neurophysin glycopeptide has digitalis-like activity. Moreover, the findings are consistent with the hypothesis that peptide mimetics of nonpeptides are encoded in the genome. Received 23 November 1998; received after revision 18 January 1999; accepted 19 February 1999     

Selection of cell lines resistant to ouabain from murine plasmacytoma MOPC 173. Cross resistance to cyclic AMP, theophylline, and concanavalin A]

Using ouabain as a selective agent, we obtained from a contact inhibited and a non contact inhibited cell layer derived from the same plasmocytoma, different resistant cell lines. All of them were ouabain resistant but were also able to grow in presence of normallly toxic levels of cAMP, theophylline and concanavalin A.     

Influence of medium amino acids on the ouabain sensitive and insensitive 86Rb+-fluxes in HeLa cells

Components of the 86Rb+-influx in HeLa cells were investigated in Joklik minimal essential medium, or in Earle's balanced salt solution with and without medium amino acids. The presence of amino acids led to the stimulation of the ouabain sensitive 86Rb+-uptake and inhibition of the diuretic-sensitive and residual 86Rb+-fluxes. These results show that the presence of amino acids is an important regulator of the K+/Rb+-fluxes under normal conditions in growth medium.     

Inhibitory effect of ouabain on in vitro and in vivo gastric acid secretion in the frog and the rat

The in vitro and in vivo effects of ouabain on gastric acid secretion in the frog and the rat, the 2 species known to have different sensitivity to ouabain, were studied. It was found that ouabain was a potent inhibitor of histamine-stimulated acid secretion in the isolated frog gastric mucosa. Ouabain administered i.v. at dose levels far below the lethal range also produced a marked and significant reduction of histamine-stimulated gastric acid secretion in the anesthetized frogs and rats. It is considered that the inhibitory effect of ouabain on acid secretion could be partly related to its specific antagonizing action on the Na+ -K+ -ATPase in the gastric mucosa.     

Vascular reactivity in hypertension: Altered effect of ouabain

Summary Ouabain inhibits the relaxing effect of Ca²⁺ (but not of Mn²⁺) on contractile responses in tail artery strips isolated from spontaneously hypertensive and normotensive rats. The magnitude of ouabain inhibition was greater in vascular strips from hypertensive rats suggesting a significant difference in basic membrane function in hypertensive vascular smooth muscle.These studies were supported by National Heart, Lung, and Blood Institute grants HL-03765 and HL-18575-02-03. R.C. Webb is a Postdoctoral Research Fellow of the Michigan Heart Association.     

Inhibitory effect of ouabain on in vitro and in vivo gastric acid secretion in the frog and the rat

Summary The in vitro and in vivo effects of ouabain on gastric acid secretion in the frog and the rat, the 2 species known to have different sensitivity to ouabain, were studied. It was found that ouabain was a potent inhibitor of histamine-stimulated acid secretion in the isolated frog gastric mucosa. Ouabain administered i.v. at dose levels far below the lethal range also produced a marked and significant reduction of histamine-stimulated gastric acid secretion in the anesthetized frogs and rats. It is considered that the inhibitory effect of ouabain on acid secretion could be partly related to ist specific antagonizing action on the Na⁺-K⁺-ATPase in the gastric mucosa.     

Ouabain action on sodium,chloride and fluid transport in rabbit jejunum and ileum

Riassunto É stato studiato l'effetto della ouabaina sul trasporto di Na, Cl e fluido in digiuno e ileo isolato di coniglio. Mentre in entrambi i tratti intestinali il flusso mucosa-serosa di Na è inibito dal glicoside, il flusso nello stesso senso di Cl, di cui è stata dimostrata una componente attiva, non viene modificato. Poichè inoltre il trasporto di fluido viene inibito dalla ouabaina in misura diversa in digiuno e in ileo, si suggerisce la presenza di sistemi ATPasici non ouabaino-sensibili accanto alla Na–K ATPasi sensibile alla ouabaina che si ammette essere responsabile del trasporto di Na.

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Experiments not reported in this paper show that ouabain 5×10^–4 M does not enhance the ileal Na influx inhibition and also does not modify the Cl influx. Moreover ouabain 10^–4 M affects jejunal Na influx to a less significant extent than 5×10^–4 M.     

Inhibition of potassium (86Rb) influx in Ehrlich ascites cells by bilirubin and ouabain

Summary Bilirubin inhibited influx of potassium into Ehrlich ascites cells without altering efflux. The data showed that compared with ouabain, net potassium influx components were impaired in a higher degree by bilirubin. The reversal of this effect was shown, in our experimental conditions, only for ouabain.Acknowledgments. This work was supported by a grant from the Consejo Nacional de Investigaciones Cientificas y Técnicas, República Argentina. The authors wish to thank Miss Marta S. Göthje for techical assistance.