Inhibitory Effects Of Ouabain Research Articles

Ouabain Exhibited Strong Anticancer Effects in Melanoma Cells via Induction of Apoptosis, G2/M Phase Arrest, and Migration Inhibition.

BackgroundMalignant melanoma was characterized by insensitive chemotherapy, drug resistance, and high metastatic ability, which resulted in the main reason for the mortality among skin-related cancers. The current agents were not sufficient to improve the treatment status of melanoma patients, and it was still needed to develop new chemotherapeutic drugs for melanoma. Our study aimed to study the anticancer effects and potential mechanisms of ouabain on melanoma cells.MethodsThe inhibitory effects of ouabain were determined by CCK8 and colony formation assays, and the morphological changes of melanoma cells were observed by inverted microscope. The apoptosis induction and cell cycle distribution were detected by annexin V/PI double staining and PI staining, respectively. The expression of the biomarker proteins in apoptosis and G2/M phase were determined by Western blotting analysis. The effects of ouabain on the migration of melanoma cells were measured by transwell migration assay and wound closure analysis. The potential mechanisms of ouabain in melanoma cells were analyzed by transcriptome sequencing.ResultsOur present study demonstrated that ouabain exhibited strong inhibitory effects on cell proliferation and triggered dramatical morphological changes of melanoma cells. Moreover, ouabain induced significant apoptosis in A375 rather than SK-Mel-28 cells via upregulation of bax expression and downregulation of bcl-2 expression. Consistently, ouabain treatment induced cell cycle arrest at G2/M phase in both A375 and SK-Mel-28 cells via upregulation of cyclin B1 and downregulation of cdc2 and cdc25c. Importantly, ouabain suppressed the migration of A375 and SK-Mel-28 cells. Furthermore, the transcriptome sequencing demonstrated that p53 and MAPK signaling pathway might play important roles in the inhibitory effects of ouabain.ConclusionOur study revealed that ouabain exhibited dramatical anticancer effects, which provided a novel application for cardiac glycoside drugs in the clinical treatment of melanoma.

Inhibitory effects of ouabain on early heart development and cardiomyogenesis in the chick embryo

Pericardial cavity formation and epithelialization of the cardiac precursor cell population constitute a critical developmental period that precedes stable cardiac cell commitment and differentiation. These events delineate the myocardial and endocardial precursor population in the embryo. Restriction of Na/K-ATPase (the sodium pump) expression to the pre-cardiomyocyte lateral membranes coincides with these events. Na/K-ATPase has been implicated developmentally in cavitation and in maintaining membrane potential. Experiments were undertaken to determine if the effects of perturbing sodium pump activity will affect pericardial cavity formation and, in turn, whether heart formation and/or cardiac cell commitment will be affected. We incubated whole chick embryos in vitro between stages 5 to 8 in the presence of the highly specific Na/K-ATPase inhibitor ouabain. Exposure of whole embryos to 10 microM ouabain (10(-5) M) demonstrated that heart development and precardiomyocyte differentiation are inhibited principally between stage 5 through stage 7. In each stage the degree of inhibition follows a rostrocaudal gradient as development proceeds along the anterior to posterior axis. After stage 8 ouabain no longer affects heart development or cardiomyogenesis. The inhibition is concentration- and developmental stage-dependent. The inhibition is reversible by elevating the outside potassium ion concentration [Ko] in the culture medium or by transferring the embryos into normal medium minus ouabain even after 20 hr of ouabain exposure. The results also suggest that the regulation of the formation of the three-dimensional organ is independent from regulation of myogenesis.

Plasma potassium may protect sodium pumps of toad hearts from an endogenous inhibitor.

Resibufogenin (3-hydroxy-14,15-epoxy-20,22-dienolide glycoside) is a potent sodium pump inhibitor present in toad toxin. It is present in the skin of the cane toad (Bufo marinus) at a concentration equivalent to ouabain of approximately 1 mM. Because toads, like other amphibians, have permeable skin, resibufogenin is also found in high concentrations in the blood. In the cane toad the blood concentration is estimated to be 1 microM (D. Lichtstein, S. Kachalsky, and J. Deutsch. Life Sci. 38: 1261-1270, 1986; D. Lichtstein, S. Samuelov, J. Deutsch, H. Xu, R. A. Lutz, S. S. Chernick and S. S. Chernick. Klin. Wochenschr. 65, Suppl. 8: 40-48, 1987), a concentration thousands of times that required to produce toxicity in humans (J. S. Flier, E. Matatos-Flier, J. A. Pallotta, and D. McIsaac. Nature Lond. 279: 341-343, 1979). In examining how the cane toad avoids inhibiting its own sodium pumps, work on the heart showed that 1) cane toads possess a similar number of cardiac sodium pumps as other vertebrates, and 2) normal plasma K+ levels completely prevent ouabain, and presumably resibufogenin, from binding to cardiac sodium pumps of the cane toad. Other species, i.e., rat (Rattus norvegicus) and salamander (Ambystoma mexicanum), did not show K+ protection of their cardiac ouabain binding sites up to normal plasma K+ levels. These species do not possess the high level of endogenous ouabain-like substance found in the toad. K+ demonstrated a capacity to protect the enzymatic activity of the toad heart sodium pumps from the inhibitory effects of ouabain.

Inhibition by ouabain of palytoxin-induced catecholamine secretion and calcium influx into cultured bovine adrenal chromaffin cells

The effect of ouabain on palytoxin (PTX)-induced catecholamine secretion from cultured bovine adrenal chromaffin cells was examined in relation to its effect on calcium (Ca 2+) influx into the cells. Ouabain showed concentration-dependent inhibition of catecholamine secretion induced by PTX. Ouabain also inhibited [ 45Ca] 2+ influx induced by PTX, this inhibition being parallel with that of catecholamine secretion. The inhibitory effects of ouabain on PTX-induced catecholamine secretion and [ 45Ca] 2+ influx were both overcome by increasing the concentrations of PTX, indicating that ouabain inhibited the actions of PTX in a competitive manner. These results suggest that the ouabain-sensitive (or -binding) site on the cell membrane might be the target site of action of PTX, which causes an increase in Ca 2+ permeability and initiation of catecholamine secretion.

Comparative aspects of Na +-K + and Ca 2+-Mg 2+ ATPase in erythrocyte membranes of various mammals

This work is a comparative study of Na +-K + and Ca 2+-Mg 2+ ATPase associated with the erythrocyte plasma membranes in different mammals. The method used to test the activity of these enzymes is based on quantitative measurements of ADP released during the reaction with HPLC: the Chromatographie type is an Ion-Pair Reversed Phase. We have found that the levels of Ca 2+ stimulated ATPase are higher than those of Na +-K + ATPase in red blood cells of all the different mammalian species, with the only exception being lamb erythrocytes where the values of both the ATPase activités are almost equal. The results obtained have shown different levels of the Na +-K + ATPase as well as of the Ca 2+ stimulated ATPase activity. Furthermore, we have made a comparative study of the ATPase activities in different red blood cells with specific reference to the optimum pH, thermostability, kinetic characteristics and the inhibitory effects of ouabain and vanadate.

Anion-dependent transport of thallous ions through human erythrocyte membrane.

Undirectional fluxes of 204Tl+ through the human red blood cell membrane were measured. The inward rate coefficient measured in a K(+)-free saline was 15.6 +/- 0.6 hr-1. The influx of Tl+ could be partially inhibited with 0.1 mM ouabain (by 28%), 0.1 mM DIDS (by 50%) or 1 mM furosemide (by 51%). The inhibitory effects of ouabain and DIDS or furosemide were additive. Half-maximal responses were seen at 0.72 microM and 0.22 mM concentrations of DIDS and furosemide, respectively. A similar action of these blockers on Tl+ influx was observed in the erythrocytes incubated in MgCl2-sucrose media. The outward rate coefficient of 204Tl was also inhibited by DIDS and furosemide (by 65 and 52%, respectively). Rate coefficients of 204Tl influx and efflux decreased significantly in the red cells exposed to Cl(-)-free media (NaNO3 or Mg(NO3)2-sucrose). Under these conditions addition of DIDS and furosemide led to only a small inhibition of Tl+ fluxes. There was a linear increase in Tl+ influx with rising of external Cl- concentration within 80-155 mM or HCO3- concentration from 20 to 40 mM when the sum of anions was kept constant (155 mM) with NO3-. The HCO3(-)-stimulated Tl+ influx was completely blocked by 0.05 mM DIDS but only 67% by 1 mM furosemide. The present study provides direct evidence for the occurrence of Cl- (HCO3-)-dependent, DIDS-sensitive movement of Tl+ across the human erythrocyte membrane in both directions. Under physiological conditions, about half of net Tl+ fluxes occurs due to an anion exchange mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of sodium-potassium pump inhibitors and membrane-depolarizing agents on sodium nitroprusside-induced relaxation and cyclic guanosine monophosphate accumulation in rat aorta.

The purpose of this study was to investigate the relationship between sodium nitroprusside-induced relaxation, inhibition of the sodium-potassium pump, and cyclic guanosine monophosphate. Exposure of rat thoracic aorta to ouabain, or potassium- or magnesium-free Krebs-Ringer bicarbonate solution, procedures which presumably inhibit the sodium-potassium pump, or to potassium chloride or tetraethylammonium, membrane-depolarizing agents, inhibited relaxation to nitroprusside. These conditions had little or no effect on the elevated cyclic guanosine monophosphate levels at a concentration of nitroprusside (0.1 microM) that relaxed norepinephrine contracted tissues by 80%. However, at a maximum relaxant concentration of nitroprusside (1.0 microM), these conditions decreased the elevation of cyclic guanosine monophosphate. The inhibition of elevated cyclic guanosine monophosphate levels was independent of the endothelium, extracellular calcium, and the cyclic guanosine monophosphate phosphodiesterase inhibitor, M&B 22,948. The inhibitory effects of ouabain and of potassium- and magnesium-free solution on the increased levels of cyclic guanosine monophosphate caused by 1.0 microM nitroprusside were abolished when tissues were incubated without norepinephrine, or with norepinephrine in the presence of the alpha-adrenergic blocker, phentolamine. In contrast, a beta-adrenergic blocker, propranolol, had no effect on the ouabain-induced inhibition of elevated cyclic guanosine monophosphate levels, with norepinephrine present. These results are consistent with the hypothesis that membrane events regulate cyclic guanosine monophosphate synthesis. At nitroprusside concentrations greater than 0.1 microM, the formation of cyclic guanosine monophosphate appears to be coupled to the status of the smooth muscle cell membrane and integrity of the sodium-potassium pump.(ABSTRACT TRUNCATED AT 250 WORDS)

A quantitative relationship between cellular Na accumulation and relaxation produced by ouabain in the depolarized smooth muscle of guinea-pig taenia coli.

1. An investigation was made of the effect of oubain on the membrane potential, tension development,45Ca uptake and intracellular Na and K contents of smooth muscle of the guinea-pig taenia coli depolarized by high-K solution. The results were compared with the effects of 2,4-dinitrophenol (DNP) and verapamil. 2. All drugs produced concentration-dependent relaxations of high-K induced contractures without affecting the depolarization of the membrane. After removal of Na from the medium, the contracture was inhibited by DNP or verapamil but not by ouabain. The inhibitory effects of ouabain and verapamil were antagonized by raising the concentration of external Ca ([Ca]0), but the relaxation produced by DNP was independent of [Ca]0. 3. Estimates of cellular Na were made by bathing the tissues in cold Li-solution. After an initial rapid loss of Na due to an exchange of extracellular Na with Li, the tissue Na reached a steady state. The residual after 1 h was regarded as cellular Na. 4. During relaxation induced by ouabain, the depolarized muscle gained cellular Na. The increase in cellular Na was dependent upon the concentration of ouabain. A smaller increase was noted with DNP, while verapamil had no effect on cellular Na. 5. The relaxation produced by ouabain was related to the intracellular Na content of the tissues. Also, the magnitude of the contraction produced by adding 1 mM Ca to Ca-free K-depolarized tissues was inversely correlated with the logarithm of the intracellular Na content, and an increase in the Na from 7.3–26.6 mmole/kg wet wt. halved the size of the Ca contracture. 6. 45Ca uptake, which was measured by a modified “La-method”, was increased in high-K solution. This increase was inhibited by pretreatments with ouabain, DNP and verapamil. 7. It is suggested that the action of ouabain is closely related to an accumulation of cellular Na, which may cause the relaxation mainly by inhibiting Ca influx in response to depolarization with high-K. The data also suggest that the mechanism of relaxation induced by DNP or verapamil is different from that induced by ouabain.

The effect of ouabain on amino acid and orthophosphate influxes in squid giant axons.

1. Some effects of ouabain on the uptake of 14C-labelled amino acids and 32P-labelled orthophosphate into squid axons have been investigated. 2. Ouabain in artificial sea water reduced the uptake of glycine, L-alanine, L-arginine, L-aspartate and L-glutamate. After a period of 3 hr in ouabain the inhibition of glycine and L-glutamate uptake was appreciable. 3. After 3 hr in ouabain the axoplasmic Na concentration had increased by only about 14% and the K concentration had fallen by only about 9%. It therefore seems unlikely that changes in the Na and K gradients across the membrane due to ouabain are the reason for the inhibition of amino acid uptake. This was confirmed when the axons were made to conduct impulses at 200/sec for 40 min. There was no subsequent inhibition of glycine and L-glutamate uptake, even though the axoplasmic Na increased by about 42% and the K decreased by about 37%. 4. A detailed investigation of the time course of the inhibition of amino acid uptake by ouabain in which intracellular scintillator techniques were used, showed that there was no inhibition for 30--100 min, and then it occurred fairly quickly. This course of events was not affected by the protein synthesis inhibitor cycloheximide. It is unlikely that the inhibition of orthophosphate uptake is subject to a similar delay since this had reached a maximum after 40 min. 5. It is concluded that the inhibitory effects of ouabain on amino acid uptake in squid axons are not due to effects on the Na and K gradients brought about by inhibition of the Na-K pump. The delay in the inhibiton may reflect the operation of an intracellular regulatory mechanis, which is initiated when ouabain inhibits the pump but which takes time to operate. If the formation of an inhibitory second messenger molecule is involved this is unlikely to be a protein.

A Comparative Study on Inhibitory Effects of Ouabain and Organic Mercuriais on Microsomal ATPase of Pt enal Cortex in Rabbits

A Comparative Study on Inhibitory Effects of Ouabain and Organic Mercuriais on Microsomal ATPase of Pt enal Cortex in Rabbits

Ouabain inhibition of ion transport and respiration in renal cortical slices of ground squirrels and hamsters

1. 1. The inhibitory effects of ouabain on oxygen consumption, K + uptake and Na + extrusion by renal cortical slices of ground squirrels and hamsters were determined. 2. 2. The maximum inhibition of K + uptake at 38° in slices of kidney cortex of ground squirrel which had been leached of K + occurred in 25 μM ouabain. Inhibition was actually less in higher concentrations of ouabain. 3. 3. The reduction of respiration at 38° due to ouabain was parallel to the inhibition of K + uptake. The uninhibited respiration was 18.0 μl O 2/mg dry wt./h and the minimal O 2 uptake was 11.9 in 25 μM ouabain. Extrusion of Na + was inhibited by ouabain in a manner parallel to that of K + uptake, but in 25 μM ouabain about half the normal extrusion of Na + occurred. 4. 4. At 5° the sensitivity of ground squirrel kidneys to ouabain was less, so that 25 μM ouabain caused only slight inhibition and 3125 μM was required for maximal inhibition of K + uptake, of respiration and of Na + extrusion. There was greater total inhibition of Na + extrusion by ouabain at the lower temperature than at 38°. 5. 5. The reduced sensitivity at low temperature was apparently due to reduced attachment or access of inhibitor to its active site. When slices were pretreated in low concentration of ouabain (25 μM) at 38° they were completely inhibited at 5° with or without additional inhibitor. 6. 6. The inhibition due to ouabain at 38° cannot be readily reversed by washing. 7. 7. Hamster kidney cortex was relatively insensitive to ouabain. In 25 μM ouabain at 38° there was negligible inhibition of K + uptake and a concentration of 3125 μM was required for a maximal effect.