Inhibitory Action Of Ouabain Research Articles

Inhibition by ouabain and veratridine of acetylcholine-evoked phasic contraction in the guinea-pig taenia coli

Inhibitory action of ouabain and veratridine on acetylcholine (ACh)-evoked phasic contraction was examined in the guinea-pig taenia coli. ACh (5 x 10(-4)M) produced a biphasic (phasic and tonic) contraction. As the phasic contraction, but not the tonic contraction, was resistant to gallopamil (D600, 2 x 10(-7)M), a blocker of voltage-dependent Ca2+ channels, all experiments were carried out in the presence of gallopamil at this concentration. Addition of high Ca2+ (30 mM) to the solution containing ACh induced a sustained contraction, which increased in the presence of ouabain (10(-5)M), an inhibitor of the Na(+)-K+ exchange system. On the other hand, the ACh-evoked phasic contraction was suppressed by ouabain (10(-7)-10(-5) M) in a time- and dose-dependent manner. The suppression by ouabain (10(-6)M) of the phasic contraction was transiently potentiated by the addition of veratridine (10(-6)M), an activator of Na+ channel. In contrast, the greater suppression by ouabain (10(-5)M) of the contraction was antagonized by amiloride (10(-4)M), a blocker of Na+ channel. This antagonism by amiloride was transiently inhibited in the presence of veratridine. In the absence of ouabain, the amplitude of the phasic contraction was transiently reduced by adding veratridine but was increased by amiloride. In addition, the phasic contraction by ACh increased 80 min after exposure to Na(+)-free isotonic high-K+ solution (K+, 143 mM), which elicited greater depolarization of the cell membrane. In a fluorescence study with Fura-2, an intracellular free-Ca2+ indicator, ACh increased the fluorescence intensity from the tissue by excitative light at 340 nm, which coupled with the phasic contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement by cytochalasin B of ouabain-stimulated catecholamine secretion from cultured bovine adrenal chromaffin cells: possible relation to alteration in Na+/K(+)-pump activity.

1. Catecholamine secretion evoked by ouabain from cultured bovine adrenal chromaffin cells has previously been shown to be markedly enhanced by pretreatment of the cells with cytochalasin B (Morita et al., 1988). To elucidate a possible mechanism of this enhancement, the stimulatory action of ouabain on Ca2+ influx as well as catecholamine secretion was then examined in the cells pretreated with or without cytochalasin B. The effect of cytochalasin B pretreatment on the inhibitory action of ouabain on the Na+/K+ pump was also examined by measuring 86Rb+ uptake into the cells. 2. Pretreatment of the cells with cytochalasin B caused enhancement of ouabain-induced catecholamine secretion, and this enhancement was accompanied by the elevation of ouabain-stimulated 45Ca2+ uptake into the cells. The inhibitory action of ouabain on 86Rb+ uptake was significantly enhanced by pretreatment of the cells with cytochalasin B under the same conditions. 3. These results indicate that the enhancement of ouabain-induced catecholamine secretion caused by cytochalasin B pretreatment may be due to the increase in ouabain-stimulated Ca2+ influx into the cells and, furthermore, suggest the possibility that this increase in Ca2+ influx may be attributed to the potentiation of the inhibitory action of ouabain on the Na+/K+ pump in the adrenal chromaffin cell. Thus, the present study provides an evidence for a possible role of microfilaments as one of the intrinsic factors modulating the plasma membrane functions.

Directional immobilization of sodium- and potassium-activated ATPase to expose its cytoplasmic part to the liquid phase on microtiter plates by wheat germ agglutinin

A convenient method for highly efficient and directional immobilization of intact sodium- and potassium-activated ATPase (Na,K-ATPase) using wheat germ agglutinin linked on microtiter plates was developed. Wheat germ agglutinin, which bound tightly to the β-subunit of Na,K-ATPase and had no effect on the Na,K-ATPase activity, the potassium-activated p-nitrophenylphosphatase activity, or the inhibitory action of ouabain, was covalently linked to microtiter plates and used as an immobilizer of the enzyme. The amount of Na,K-ATPase coupled to microtiter plates in this immobilizing system was more than 10-fold greater than that used in the direct immobilizing system (O. Urayama, M. Nakao, H. Nagamune, and H. Sugiyama, (1984) Anal. Biochem. 141, 194–198). Also in this system, the cytoplasmic domain of Na,K-ATPase was exposed to the liquid phase. This technique was useful for investigating the reactivities of monoclonal antibody specific for the cytoplasmic domain of the enzyme. Moreover, because this technique was used successfully in the immobilization of periodic acid-Schiff positive staining glycoprotein 1 prepared from human erythrocytes and human α2-macroglobulin, the technique should also be useful for other membrane or secreted proteins that possess N-linked sugar chains containing bisecting N-acetylglucosamine or a high amount of sialic acid.

Effet de l'ouabaïne sur l'action lactogene de la prolactine et sur le niveau des récepteurs prolactiniques mammaires

Ouabain added to the culture medium of rabbit mammary gland inhibits prolactin action on the initiation of lactose and casein synthesis. The degree of inhibition is a function of the ouabain concentration in the medium. Likewise, ouabain blocks the accumulation of casein mRNA supported by prolactin. In addition, ouabain provokes a rapid disappearance of prolactin receptors. Conversely prolactin keeps its capacity to enhance the concentration of casein mRNA and the parallel casein synthesis when K+ ions are totally absent from the culture medium. These results suggest that although prolactin induces a modification of the K+/Na+ ratio in the mammary cell and ouabain prevents this effect of prolactin, the inhibitory action of ouabain on lactogenesis can be explained essentially by its effect on the hormone receptors.

Effect of ouabain on the Ca 2+-dependent increase in K + permeability in depleted guinea-pig red cells

1. 1. The hypothesis that the inhibitory action of ouabain on the Ca 2+-dependent increase in K + permeability observed in depleted human red cells is mediated by changes in the intracellular level of ATP was tested by measuring simultaneously the ouabain sensitive K + loss and the concentration of ATP in depleted guinea-pig red cells in the presence and absence of external Ca 2+. 2. 2. The results showed that ouabain has opposite effects on the K + loss. Thus, in the presence of external K +, when the forward running of the Na + pump contributes to the breakdown of ATP, ouabain reduces K + loss, as observed in human red cells; in a K + free, high Na + medium when the pump running backwards induces a net synthesis of ATP (which can be detected in guinea-pig red cells but not in human red cells), ouabain increases K + loss. 3. 3. The results suggest that the action of ouabain is mediated through the changes in the intracellular level of ATP induced by the forward or reverse operation of the Na + pump.

Effects of Na+ and K+ on the uptake of metaraminol by rabbit ventricular slices.

1. The ionic dependence of [(3)H]-metaraminol transport by rabbit ventricular slices was studied.2. Transport was Na(+) dependent. Choline, Li(+), K(+), Rb(+) or Cs(+) could not be substituted for Na(+).3. Transport was K(+) dependent. Rb(+) and Cs(+), but not Li, could be substituted for K(+), their relative potencies being K(+) = or >Rb(+)>Cs(+)>>Li(+). Higher concentrations of K(+), Rb(+), Cs(+) and Li inhibited [(3)H]-metaraminol transport.4. The inhibitory effects on transport of either a marked reduction in the concentration of Na(+) or of omission of K(+) were rapidly reversible on exposure of slices to Krebs solution.5. The inhibitory effect of ouabain on [(3)H]-metaraminol transport was markedly time dependent, being significantly increased by preincubation with ouabain. The inhibitory action of ouabain was not affected, however, by the Na(+) concentration present during preincubation.6. An inwardly directed Na(+) gradient did not increase [(3)H]-metaraminol transport in slices in which Na(+) pumping had been prevented by 10(-5)M ouabain, 4 degrees C, omission of K(+) or by metabolic inhibition.7. These findings provide additional evidence that the Na(+) and K(+) activated adenosine triphosphatase may participate in the transport of metaraminol and related amines. In the absence of Na(+) pumping, induced Na(+) gradients were unable to produce transport of amine as would be predicted by the co-transport model proposed by Bogdanski & Brodie (1969).

Inhibition of Aldosterone Secretion by Ouabain in Dog Adrenal Cortical Tissue

Steroid secretion in vitro by adrenal cortical slices from hypophysectomized and nephrectomized dogs was studied. The addition of 5.5×10–5M ouabain to the medium of some flasks reduced the final content of aldosterone without alteration of 17-OH corticosteroids. Prior salt depletion of the donor animals produced enhanced aldosterone without change in 17-OH corticosteroid compared to the controls. Addition to the medium of ouabain produced sticking reductions in aldosterone without change in corticosterone or 17-OH corticosteroids in the media. Addition of 4 mEq/1 of KC1 to otherslices from the same salt-depleted, hypophysectomized, nephrectomized dogs produced increases in aldosterone. When ouabain was added to the high K medium, similar reductions in aldosterone without change in corticosterone or 17-OH corticosteroids occurred. In studies with 42K, ouabain produced decreased 42K uptakes, decreased tissue K and reduced “net K exchanged” /90 min. It is suggested that the inhibitory action of ouabain on al...

Diphenylhydantoin and human myocardial microsomal (Na +, K +)-ATPase

Diphenylhydantoin has no effect on the activity of human myocardial microsomal (Na +, K +)-ATPase or on the inhibitory action of ouabain.

Potassium and sodium content of guinea-pig ureteral muscle

1. 1. Guinea-pig ureteral smooth muscle showed the following figures (per kg wet tissue): water content, 0·835 ± 0·007. ( n = 10); inulin space, 0·461 ± 0·271. ( n = 10); K + content, 59·3 ± 2·0 m-mole ( n = 10); and Na + content, 81·3 ± 2·2 m-mole ( n = 20). Estimated intracellular concentrations were K +, 154 m-mole/1 and Na +, 33 m-mole/1. 2. 2. Resting potentials determined under normal and modified conditions were less than the values which would be expected from a pure potassium electrode. 3. 3. Radioisotope studies confirmed the ionic distribution and inhibitory action of ouabain on K + uptake.

Insulin-like action of ouabain II. Primary antilipolytic effect through inhibition of adenyl cyclase

1. 1. In the absence of added glucose, ouabain had a strong inhibitory action upon hormone-stimulated lipolysis. By contrast, the presence of ouabain resulted in little or no inhibition of lipolysis induced by dibutyryl-3',5'-AMP or caffein, indicating that the inhibitory action was exerted at the level of the formation of cyclic AMP. 2. 2. The inhibitory action of ouabain on hormone-stimulated lipolysis in the absence of glucose can be reproduced by removing K+ from the incubation medium. The inhibitory effect of ouabain and that of K+ lack could be overcome by increasing the concentrations of the lipolytic hormone. 3. 3. Using an assay system for adenyl cyclase described here, it could be shown that the adenyl cyclase activity of adipose tissue pretreated with ouabain was reduced by 60–70% when compared to that of the untreated tissue. Similarly, the removal of K+ from the incubation medium resulted in a marked decrease of the adenyl cyclase activity. By contrast, ouabain did not influence the activity of phosphodiesterase. 4. 4. These results are consistent with a decreased formation of cyclic AMP induced by both ouabain and/or lack of K+. Althrough the mechanism of this effect is unknown, it is possible that the affinity between lipolytic hormones and adenyl cyclase is regulated by the concentrations of cations and that the inhibitory effect of ouabain on hormone-stimulated lipolysis may be the result of decreasing intracellular K+ levels. Furthermore, it would seem that the effects of insulin and of ouabain have some features in common, since the presence of both agents may lead to decreased levels of cyclic AMP and decreased intracellular concentrations of free fatty acids.

Influence of calcium and ouabain upon the potassium influx in human erythrocytes

The influence of calcium and ouabain upon the potassium influx was studied in human erythrocytes. Changing the Ca concentration of the suspension medium from 1.1 to 8.2 μEq/ml did not influence the K influx or the inhibitory action of ouabain (1.36 × 10 −5 M) on K influx. Potassium influx was inhibited, however, by increasing the Ca concentration within the erythrocytes from 0.7 to 8.4 μEq/ml cells. The inactivation of the K transport by ouabain was diminished by an increase of the Ca concentration in the erythrocytes. It seems justified to conclude that the site of action of Ca and of ouabain upon the K/Na transporting system is on the inside of the erythrocyte membrane.