SEARCH · PubMed Health

Results for “OUABAIN”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources

PubMed2001

The change and significance of the Na+-K+-ATPase alpha-subunit in ouabain-hypertensive rats.

Ouabain has recently been identified as an endogenous Na+-K+ pump inhibitor having a close association with hypertension. However, some patients with hypertention do not show high levels of endogenous ouabain (EO), and patients with high EO levels do not necessarily suffer from hypertention. It is believed that the Na+-K+-ATPase activity in essential hypertension does not undergo homogenous change. The present study was designed, therefore, to investigate the expression and the significance of the Na+-K+-ATPase alpha-subunit isoforms in kidney tissue in ouabain-hypertensive rats. Ouabain was administered chronically to establish a model of ouabain-hypertensive rats. Biochemical analysis, cytobiology and sABC immunohistochemistry were they used to assay for expression of Na+-K+-ATPase alpha-subunit isoforms in kidney tissue. After the first week of receiving ouabain, 65% (n=13) of rats had hypertension. After the second week, the blood pressure of these 13 hypertensive rats was increased significantly compared to the baseline and control levels (p<0.05). The plasma renin activity was normal, and angiotensin II and aldosterone levels were increased significantly in these rats (p<0.05). But in the other 35% (n=7) of rats of the experimental group, there was no apparent increase in blood pressure after receiving ouabain. The plasma ouabain level in the non-hypertensive subgroup was significantly higher than that in the hypertensive subgroup, but the 86Rb intake and the number of 3H-ouabain binding sites did not decrease. The Na+-K+-ATPase activity showed non-homogeneous changes. In hypertensive rats, the expression levels of ouabain paralleled the degree of hypertension (r=0.88, p<0.05). The positive granules were mainly scattered in the cytoblastoma of the reticular zone of adrenal cortex. There were thus different levels of expression of Na+-K+-ATPase alpha-subunit isoforms in this model. In the hypertension subgroup the alpha1 was most strongly expressed, followed by the alpha2 and alpha3 isforms. But in the non-hypertensive subgroup the order was alpha3 > alpha2 > alpha1. The positive granular was mainly scattered in the convoluted tubules of the kidney. These results suggest that the high level of ouabain and the change of the Na+-K+-ATPase alpha-subunit isoforms may play a critical role in hypertension.

Adrenal Glands↗

PubMed2006

Ouabain binds with high affinity to the Na,K-ATPase in human polycystic kidney cells and induces extracellular signal-regulated kinase activation and cell proliferation.

In autosomal dominant polycystic kidney disease (ADPKD), cyst formation and enlargement require proliferation of mural renal epithelial cells and the transepithelial secretion of fluid into the cyst cavity. Na,K-ATPase is essential for solute and water transport in ADPKD cells, and ouabain blocks fluid secretion in these cells. By binding to the Na,K-ATPase, ouabain also induces proliferation in some cell types. Surprisingly, it was found that nanomolar concentrations of ouabain, similar to those circulating in blood, induced ADPKD cell proliferation but had no statistically significant effect on normal human kidney (NHK) cells. Ouabain, acting from the basolateral side of the cells, also caused an increase in the level of phosphorylated extracellular signal-regulated kinases (ERK). Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocked ouabain-induced ERK activation and cell proliferation, suggesting that ouabain effect is mediated through the MEK-ERK pathway. In contrast to NHK cells, the dose-response curve for ouabain inhibition of Na,K-ATPase activity indicated that approximately 20% of the enzyme in ADPKD cells exhibits a higher affinity for ouabain. The increased ouabain affinity of ADPKD cells was not due to differences in Na,K-ATPase isoform expression because these cells, like NHK cells, possess only the alpha1 and beta1 subunits. The gamma variants of the Na,K-ATPase also are expressed in the cells but are elevated in ADPKD cells. Currently, the basis for the differences in ouabain sensitivity of NHK and ADPKD cells is unknown. It is concluded that ouabain stimulates proliferation in ADPKD cells by binding to the Na,K-ATPase with high affinity and via activation of the MEK-ERK pathway.

Base Sequence↗

PubMed2001

Dual effects of ouabain on the regulation of proliferation and apoptosis in human prostatic smooth muscle cells.

PURPOSE: To elucidate the role of ouabain in the pathophysiology of benign prostatic hyperplasia we examined the effects of ouabain on the proliferation and apoptosis of human prostatic smooth muscle cells. MATERIALS AND METHODS: Primary cultures of human prostatic smooth muscle cells were obtained from 7 patients with bladder outlet obstruction caused by benign prostatic enlargement. A cell proliferation study was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay method to examine the effects of different concentrations of ouabain and various inhibitors. Western blot analysis was done to determine mitogen activated protein kinase (MAPK) activation. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction method and caspase-3 activity assay were also performed to examine the apoptotic mechanism. RESULTS: Ouabain exhibited a modest but significant proliferative effect in nanomolar concentrations; whereas it induced cell apoptosis at higher concentrations. Ouabain caused rapid activation of p42/44 MAPKs. The proliferative effect of ouabain was completely flattened by W-7 and MAPK kinase (MEK) inhibitor, suggesting the requirement of Ca(2+) mobilization and the involvement of the MEK-p42/44 MAPK cascade. The cytotoxic effect by ouabain was defined as apoptosis and necrosis using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction technique and lactate dehydrogenase release assay, respectively. In addition, ouabain induced profound caspase-3 activity in the cytotoxic concentrations and DEVD-CHO reversed the cytotoxic action to ouabain, demonstrating the involvement of caspase-3 activation in the cytotoxic action. CONCLUSIONS: Ouabain at different concentrations caused dual effects on proliferation and apoptosis in human prostatic smooth muscle cells. At low concentrations ouabain promoted cell proliferation via a Ca(2+) dependent mechanism and activation of the MEK-p42/44 MAPK pathway; whereas it induced cell apoptosis via the activation of caspase-3 activity at higher concentrations.

Analysis of Variance↗

PubMed1983

Inhibition of mitogen-induced lymphocyte proliferation by ouabain: interference with interleukin 2 production and interleukin 2 action.

Since the discovery of IL 2 it has been possible to dissect the mitogenic activation of T lymphocytes into two steps: first, the production of IL 2; and second, the response of IL 2-dependent T cell blasts to IL 2. We investigated the influence of ouabain, a known inhibitor of T cell activation, on the production of and the response to IL 2 in mouse T lymphocytes. Ouabain dose-dependently inhibited DNA synthesis in Con A activated C57BL/6 spleen cells; inhibition below background levels occurred at a concentration of 5 X 10(-4) mol/liter. The action of IL 2 was assayed on a mouse T cell line, named M 2, derived from C57BL/6 spleen cells and maintained in continuous IL 2-dependent culture for more than 21 mo. Ouabain dose-dependently inhibited the response of these cells to mouse IL 2 with optimal inhibition at 10(-3) mol/liter ouabain. The production of IL 2 by Con A-stimulated C57BL/6 spleen cells was also totally inhibited by 10(-3) mol/liter ouabain; 10(-4) mol/liter ouabain inhibited IL 2 production partially, and 10(-5) mol/liter and 10(-6) mol/liter ouabain had no inhibitory effect. The ouabain-mediated inhibition of IL 2 production and of IL 2 action was not due to a nonspecific toxicity, because the preincubation of spleen cells with 10(-3) mol/liter ouabain for 24 hr did not impair their ability to produce IL 2, nor was the response of M 2 cells to exogenously added IL 2 abrogated by the preincubation of the cells with 10(-3) mol/liter ouabain. The data suggest that ouabain acts on two steps during the induction of T cell activation.

Animals↗

PubMed1997

Comparative study of the effects of ouabain and digoxin on blood pressure of rats.

OBJECTIVE: To compare the effect of ouabain on the blood pressure of rats with that of digoxin to find the evidences of the relationship between endogenous ouabain (EO) and development of hypertension. METHODS: Sprague-Dawley rats, which were divided into 3 groups, were infused with ouabain (23 x 75 micrograms.kg-1/day, i.p.), digoxin (36 x 84 micrograms.kg-1/day, i.p.) and normal saline (NS) once a day respectively. Systolic blood pressure and body weight were recorded weekly. Five weeks later, rats of ouabain group were randomly assigned to three infusion subgroups: Oc group, continued with ouabain infusion; Od group, added digoxin (73 x 68 micrograms.kg-1/day, i.p.) and Os group, stopped administration of ouabain. Another week later, direct blood pressure was recorded in aorta. Systolic and diastolic cardiac function, plasma renin activity and aldosterone levels of all the rats were measured. RESULTS: After a latent period of one week, blood pressure of Ouabain group increased significantly [95.4 +/- 11.8 mmHg (1 mmHg = 0.133 kPa) at the beginning of the experiment vs 122.5 +/- 16.9 mmHg at the end of week 6, P < 0.05] with normal plasma renin activity and higher aldosterone (1.28 +/- 0.45 ng/ml vs 0.69 +/- 0.27 ng/ml, P < 0.05). The blood pressure decreased after either withdrawal of ouabain or addition of digoxin (116.3 +/- 14.4 mmHg vs 100 +/- 10.7 mmHg, P < 0.05; 123.9 +/- 13.9 vs 103.3 +/- 10.5 mmHg, P < 0.05, respectively). No difference of blood pressure was found between the digoxin and NS group. CONCLUSIONS: Our results suggested that EO might be one of the causes of the development of hypertension. Aldosterone might play some role in the mechanism of ouabain-induced hypertension. Digoxin can not induce hypertension. There is a great difference between the effect of ouabain and digoxin on the blood pressure. Moreover, digoxin can reverse the hypertension induced by ouabain.

Animals↗

PubMed1971

Ouabain-insensitive sodium movements in the human red blood cell.

Red blood cells exposed to ouabain are capable of net Na outflux against an electrochemical gradient; the net outflux is inhibited by the diuretic, furosemide. In ouabain-treated cells, both the unidirectional Na outflux and the unidirectional Na influx are inhibited by furosemide. Furosemide also inhibits the ouabain-sensitive Na-Na exchange accomplished by the Na-K pump in K-free solutions. From the interaction of extracellular K, furosemide, and ouabain with the transport system, it seems possible that the ouabain-insensitive Na outflux is accomplished by the same mechanism that is responsible for the ouabain-sensitive Na-K exchange. The ouabain-insensitive Na outflux is increased by extracellular Na, and the influx increases as the intracellular Na increases. In fresh cells, high extracellular K concentrations decrease the ouabain-insensitive Na outflux and increase the ouabain-insensitive Na influx. When the rate constant for sodium outflux and the rate constant for sodium influx in ouabain-treated cells are plotted against the extracellular K concentration, the curves obtained are mirror images of each other. In starved cells, extracellular K increases the ouabain-insensitive Na outflux as does extracellular Na, and it has little effect on the Na influx.

Biological Transport, Active↗

PubMed2001

Plasma ouabain-like factor during acute and chronic changes in sodium balance in essential hypertension.

An ouabain-like factor has been implicated repeatedly in salt-sensitive hypertension as a natriuretic agent. However, the response of plasma ouabain-like factor to acute and chronic variation of body sodium is unclear. We studied 138 patients with essential hypertension who underwent an acute volume expansion/contraction maneuver (2 days) and 20 patients who entered a blind randomized crossover design involving chronically controlled sodium intake and depletion (170 to 70 mmol/d; 2 weeks each period). In both studies, plasma levels of ouabain-like factor were higher during sodium depletion (acute: 338.8+/-17.4 and 402.7+/-22.8 pmol/L for baseline and low sodium, respectively, P<0.01; chronic: 320.4+/-32.0 versus 481.0+/-48.1 pmol/L, P=0.01). No significant change in plasma ouabain-like factor was observed after a 2-hour saline infusion (333.4+/-23.9 pmol/L) or controlled sodium (402.1+/-34.9 pmol/L). When patients were divided into salt-sensitive or salt-resistant groups, no differences in plasma ouabain-like factor were observed in the 2 groups at baseline or in response to the 2 protocols: salt resistant (n=69, 340.1+/-25.9 pmol/L) versus salt sensitive (n=69, 337.4+/-23.6 pmol/L) and chronic salt resistant (n=11, 336.0+/-53.2) versus salt sensitive (n=9, 301.1+/-331.4 pmol/L). However, circulating ouabain-like factor was increased by sodium depletion in both groups. These results demonstrate that circulating ouabain-like factor is raised specifically by maneuvers that promote the loss of body sodium. Acute expansion of body fluids with isotonic saline is not a stimulus to plasma ouabain-like factor. Moreover, basal levels of plasma ouabain-like factor do not differ among patients with salt-sensitive or salt-resistant hypertension. Taken together, these new results suggest that ouabain-like factor is involved in the adaptation of humans to sodium depletion and argue against the hypothesis that ouabain-like factor is a natriuretic hormone.

Adult↗

PubMed2005

Inhibitory effects of digoxin and ouabain on aldosterone synthesis in human adrenocortical NCI-H295 cells.

The present study was to investigate the effects and action mechanisms of digoxin and ouabain on steroidogenesis in human adrenocortical NCI-H295 cells. Administration of digoxin or ouabain for 24 h decreased the basal and angiotensin II (Ang II)-stimulated release of aldosterone by NCI-H295 cells. The conversions of corticosterone (substrate of cytochrome P450 aldosterone synthase, P450c11AS) to aldosterone or deoxycortisol (substrate of cytochrome P450 11beta-hydroxylase, P450c11beta) to cortisol were reduced by digoxin or ouabain. The basal and 22-hydroxy-cholesterol (a membrane-permeable cholesterol, substrate of cytochrome P450 side-chain cleavage enzyme, P450scc)-stimulated pregnenolone release in mitochondria was inhibited by digoxin or ouabain. Digoxin or ouabain suppressed the basal and Ang II-stimulated protein expression of steroidogenic acute regulatory (StAR) protein and P450scc. Incubation of digoxin or ouabain for 24 h reduced P450c11AS mRNA expression in NCI-H295 cells. Digoxin or ouabain (10(-6) M, 24 h)-treated cells showed a lower resting intracellular Ca2+ concentration ([Ca2+]i) and an attenuated response of [Ca2+]i to Ang II. Since no significant cytotoxicity was observed at 10(-6) M digoxin or ouabain, the digoxin- or ouabain-induced decrease of aldosterone or cortisol release was not associated with cytotoxicity. These results demonstrate that digoxin or ouabain inhibits the aldosterone or cortisol release via reduction of P450c11AS or P450c11beta and P450scc activities, inhibition of StAR and P450scc protein expression, suppression of P450c11AS mRNA expression, and attenuation of Ca2+ mobilization in NCI-H295 cells.

Adrenal Cortex Neoplasms↗

PubMed1990

Characteristics of ouabain binding to isolated trout hepatocytes.

Na+, K+ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10(-4) M produced maximal inhibition (95%) of K+ uptake and enhanced intracellular Na+ accumulation, showing that active fluxes account for a very large proportion of Na+ and K+ exchanges. Inhibition of the Na-K pump by ouabain was significant at low concentrations (10(-8) M). When external K+ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC50) of K+ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [3H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K+ concentration in the medium, the other linear and independent of external K+. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5 x 10(5) per cell) and independent of the external K+ concentration (7, 0.5 or 0 mM), while the dissociation constant (KD) decreased from 4.2 microM to 7.3 nM when K+ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min-1), as estimated from ouabain-sensitive K+ flux, in comparison to those described in other cell types of higher vertebrates. At each external K+ concentration studied, the inhibition of K+ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.

Animals↗

PubMed1992

The effects of cAMP, Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine responses in Helix neurons.

1. Using internal perfusion and concentration-clamp procedures applied to Helix neurons, the effects of cAMP, Ca2+, and phorbol esters on ouabain-induced depression of acetylcholine Cl-dependent responses were determined. 2. Intracellular cAMP (10(-4) M) depressed those acetylcholine responses which were blocked by ouabain but had no effect on ouabain-insensitive acetylcholine responses. In the presence of elevated intracellular cAMP, ouabain had no further depressant effect on these acetylcholine responses. Both elevated cAMP and ouabain reduced the acetylcholine response without altering the current-voltage curves. 3. An increase in intracellular Ca2+ concentration depressed the amplitude of current induced by application of acetylcholine in neurons with ouabain-sensitive responses and shifted the dose-response relationship to the right. However, elevated Ca2+ did not reduce the maximal response induced by acetylcholine, nor did it prevent the reduction of that response by ouabain. 4. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of protein kinase C activity, caused depression of both the ouabain-sensitive and the ouabain-insensitive acetylcholine responses. The inhibitory effect of TPA was markedly enhanced after addition of ATP to the intracellular medium and was greatly reduced by cooling to 5 degrees C. The blocking effect of ouabain, however, reexamined in the presence of TPA. 5. These observations are consistent with the hypothesis that the depression of acetylcholine induced Cl--responses in Helix neurons is a result of an increase in intracellular cAMP concentration but is unrelated to activation of protein kinase C or increases in intracellular Ca2+.

Acetylcholine↗

PubMed1981

Ouabain-resistant mechanism of volume control and the ultrastructural organization of liver slices recovering from swelling in vitro.

We have studied the net extrusion of water by liver slices recovering from swelling at 1 degree C and have attempted to relate this to ultrastructural alterations. Special attention was paid to the ouabain-resistant extrusion of water. The restoration of many details of intracellular architecture was dissociated from the net loss of water, since an osmotic stimulus (produced by 5% inulin) caused a passive withdrawal of water with little recovery of structure. Also, a similar recovery of structure was produced during active extrusion of water in the absence and presence of ouabain, even though ouabain reduced the water extrusion by 50%. The time-course of water extrusion in the presence of ouabain was correlated with the formation of cytoplasmic vesicles. Incubation without K+ in the medium had similar effects to those caused by ouabain. Colchicine had little effect on water extrusion in presence or absence of ouabain except at concentrations which reduced tissue ATP levels and caused much necrosis. Cytochalasin B alone had little effect on water extrusion, but led to the accumulation of many vesicles in the cytoplasm and appeared to abolish the access of such vesicles to the canaliculi. In the presence of ouabain, cytochalasin B had a similar effect on ultrastructure, and totally prevented the ouabain-resistant water extrusion. Ni2+ had rather similar effects to cytochalasin B both in the presence and absence of ouabain, although to a smaller degree. The results support our previous suggestion that the ouabain-resistant water extrusion proceeds by secretion of water into cytoplasmic vesicles, followed by the exocytotic expulsion of the vesicular contents into the bile canaliculi. Microfilaments appear to play an important role in the process.

Animals↗

PubMed2000

Influence of apoptosis on the enhancement of radiotoxicity by ouabain.

BACKGROUND: The Na+, K(+)-ATPase inhibitor ouabain enhances the toxicity of irradiation and we have previously demonstrated that the drug suppresses repair capacity. The influence of ouabain on apoptosis is not known and is examined in this study. MATERIALS AND METHODS: Seven human cell lines of defined TP53 status were irradiated with 60Co-gamma irradiation in the presence and absence of 10(-7) M ouabain. Cell survival was determined by the clonogenic assay, apoptosis by acridine orange staining and cell cycle delays by flow cytometry. RESULTS: The ouabain-induced enhancement of radiotoxicity, expressed as the ratio of SF2's, is independent of TP53 status and ranges from 1.1 to 2.8 depending upon cell line. Ouabain prolongs the irradiation-induced G2 delay in TP53 mutant tumor cell lines by a factor greater than 2, but not in the normal lung fibroblast L132, where the cell recovery is not altered in the presence of ouabain. Twenty hours post irradiation, ouabain enhances apoptosis induced by irradiation by factors of 1.3 to 1.7 depending on the cell line. CONCLUSION: Ouabain preferentially enhances the radiotoxicity in tumor cells irrespective of TP53 status. In the pattern of DNA damage responses which are influenced by ouabain we show that the G2 cell cycle delay is prolonged and that early apoptosis events are upregulated in TP53 wild type and TP53 mutant cells. It is concluded that apoptosis plays a significant role in the enhancement of radiotoxicity by ouabain.

Animals↗

PubMed2006

Repeated electroconvulsive shock induces changes in high-affinity [3H]-ouabain binding to rat striatal membranes.

Repeated electroconvulsive shock is an effective treatment for affective disorders. Striatum, hippocampus and brainstem are involved in affective disorders. Sodium-potassium/ATPase is of paramount importance for the proper functioning of the brain and its involvement in the affective disorders has been claimed for a long time. Sodium-potassium/ATPase has an extracellular regulatory binding site to which cardiotonic glycosides, such as ouabain, bind to, thus regulating the activity of the enzyme. Endogenous "ouabain-like" substances exist in the brain and their actions on the sodium-potassium/ATPase resemble ouabain biological properties. The aim of this work was to determine if electroconvulsive shock (ECS) would induce changes in the high-affinity binding of ouabain to the sodium-potassium/ATPase from rat brain regions. Adult, male Wistar rats received one (ECSx1 group) or seven electroshocks (ECSx7 group) delivered daily through ear-clips electrodes. Control rats received the same manipulations; however, no current was delivered through the electrodes (SHAMx1 and SHAMx7 groups). All groups were sacrificed 24 h after the last ECS session. The B (max) and K (D) of high-affinity [(3)H]-ouabain binding were determined in crude membrane preparations from the striatum, hippocampus and brainstem. The results obtained showed a statistically significant increase in the affinity of [(3)H]-ouabain (lower K (D)) to striatal membranes in those rats receiving seven ECS. In the striatum there was no change in the K (D) after one ECS; as well as there was no change in the B (max) after a single or seven ECS. High-affinity [(3)H]-ouabain binding to hippocampus and brainstem did not reveal any significant differences either in K (D) or B (max) after one or seven ECS. The increased affinity of ouabain to the striatal sodium-potassium/ATPase induced by repeated ECS suggests an increased interaction in vivo of the endogenous "ouabain-like" substances with the enzyme and the involvement of the extracellular regulatory allosteric ouabain binding site in the striatal sodium-potassium/ATPase in the effects of electroconvulsive shock.

Animals↗

PubMed1978

The effect of sodium on inorganic phosphate- and p-nitrophenyl phosphate-facilitated ouabain binding to (Na+ + K+)-activated ATPase.

The effect of the hydrolysis product Pi and the artificial substrate p-nitrophenyl phosphate (p-nitrophenyl-P) on ouabain binding to (Na+ + K+)-activated ATPase was investigated. The hypothesis that (Mg2+ + p-nitrophenyl-P)-supported ouabain binding might be due to Pi release and thus (Mg2+ + Pi)-supported could not be confirmed. The enzyme . ouabain complexes obtained with different substrates were characterized according to their dissociation rates after removal of the ligands facilitating binding. The character of the enzyme . ouabain complex is determined primarily by the monovalent ion present during ouabain binding, but, qualitatively at least, it is immaterial whether binding was obtained with p-nitrophenyl phosphate or Pi. The presence or absence of Na+ during binding has a special influence upon the character of the enzyme . ouabian complex. Without Na+ and in the presence of Tris ions the complex obtained with (Mg2+ + Pi) and that obtained with (Mg2+ + p-nitrophenyl-P) behaved in a nearly identical manner, both exhibiting a slow decay. High Na+ concentration diminished the level of Pi-supported ouabain binding, having almost no effect on p-nitrophenyl phosphate-supported binding. Both enzyme . ouabain complexes, however, now resembled the form obtained with (Na+ + ATP), as judged from their dissociation rates and the K+ sensitivity of their decay. The complexes obtained at a high Na+ concentration underwent a very fast decay which could be slowed considerably after adding a low concentration of K+ to the resuspension medium. The most stable enzyme . ouabain complex was obtained in the presence of Tris ions only, irrespective of whether p-nitrophenyl phosphate of Pi facilitated complex formation. The presence of K+ gave rise to a complex whose dissociation rate was intermediate between those of the complexes obtained in the presence of Tris and a high Na+ concentration. It is proposed that the different ouabain dissociation rates reflect different reactive states of the enzyme. The resemblance between the observations obtained in phosphorylation and ouabain binding experiments is pointed out.

Adenosine Triphosphatases↗

PubMed1979

Sodium influx rate and ouabain-sensitive rubidium uptake in isolated guinea pig atria.

1. Ouabain-sensitive 86Rb+ uptake by tissue preparations has been used as an estimate of Na+ pump activity. This uptake, however, may be a measure of the Na+ influx rate, rather than capacity of the Na+ pump, since intracellular Na+ concentration is a determinant of the active Na+/Rb+ exchange reaction under certain conditions. This aspect was examined by studying the effect of altered Na+ influx rate on ouabain-sensitive 86Rb+ uptake in atrial preparations of guinea pig hearts. 2. Electrical stimulation markedly enhanced ouabain-sensitive 86Rb+ uptake without affecting nonspecific, ouabain-insensitive uptake. Paired-pulse stimulation studies indicate that the stimulation-induced enhancement of 86Rb+ uptake is due to membrane depolarizations, and hence related to the rate of Na+ influx. 3. Alterations in the extracellular Ca2+ concentration failed to affect the 86Rb+ uptake indicating that the force of contraction does not influence 86Rb+ uptake. 4. Reduced Na+ influx by low extracellular Na+ concentration decreased 86Rb+ uptake, and an increased Na+ influx by a Na+-specific ionophore, monensin, enhanced 86Rb+ uptake in quiescent atria. 5. Grayanotoxins, agents that increase transmembrane Na+ influx, and high concentrations of monensin appear to have inhibitory effects on ouabain-sensitive 86Rb+ uptake in electrically stimulated and in quiescent atria. 6. Electrical stimulation or monensin enhanced ouabain binding to (Na+ + K+)-ATPase and also increased the potency of ouabain to inhibit 86Rb+ uptake indicating that the intracellular Na+ available to the Na+ pump is increased under these conditions. 7. The ouabain-sensitive 86Rb+ uptake in electrically stimulated atria was less sensitive to alterations in the extracellular Na+ concentration, temperature and monensin than that in quiescent atria. 8. These results indicate that the rate of Na+ influx is the primary determinant of ouabain-sensitive 86Rb+ uptake in isolated atria. Electrical stimulation most effectively increases the Na+ available to the Na+ pump system. The ouabain-sensitive 86Rb+ uptake by atrial preparations under electrical stimulation at a relatively high frequency seems to represent the maximal capacity of the Na+ pump in this tissue.

Animals↗

PubMed1981

The insect brain (Na+ + K+)-ATPase. Binding of ouabain in the hawk moth, Manduca sexta.

(1) A quantitative study has been made of the binding of ouabain to the (Na+ + K+)-ATPase in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain (Na+ + K+)-ATPase will bind ouabain either in the presence of Mg2+ and Pi, ('Mg2+, Pi' conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide ('nucleotide' conditions) as is the case for the bovine brain (Na+ + K+)-ATPase. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the KD) of ouabain to the M. sexta brain (Na+ + K+)-ATPase under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta (Na+ + K+)-ATPase has a higher KD for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta (Na+ + K+)-ATPase can bind ouabain.

Animals↗

PubMed1976

Cardiac NaK ATPase activity during positive inotropic and toxic actions of ouabain.

In order to define pharmacological actions of ouabain in the dog heart, ouabain uptake and subcellular distribution and its effect on NaK ATPase (MG2+ dependent, Na+-K+-activated adenosinetriphosphate phosphohydrolase, E.C. 3.6.1.3), have been investigated in 21 open-chest dogs. A continuous infusion of ouabain (0.036 mug/kg/min) after a loading dose (20 mug/kg) produced a relatively constant plasma concentration of approximately 10(-8) M (6 ng/ml) ouabain, which induced a sustained positive inotropic response for the 300 min experimental period. In these hearts much greater binding of ouabain was noted in the NaK ATPase and microsomal fractions than in other myocardial fractions. No statistically significant inhibition of NaK ATPase activity was noted. Doubling the loading and infusion doses of ouabain raised the plasma level of ouabain to approximately 3 X 10(-8) M and produced various types of arrhythmia within an hour, which persisted for the rest of the 5 h experimental period. Under this experimental protocol there was a significant inhibition of NaK ATPase activity and increased binding of ouabain to this enzyme. This study does not support the hypothesis that there is a causal relationship between inotropic response to ouabain and NaK ATPase inhibition. It was concluded that NaK ATPase inhibition might be causally related to the development of ouabain toxicity.

Adenosine Triphosphatases↗

PubMed1978

Effects of ouabain and potassium-free solution on the contraction of isolated blood vessels.

Ouabain and K-free solution affected the tension development of isolated vessels as follows. In rabbit aorta, ouabain induced a transient contraction followed by a delayed sustained contraction whereas K-free solution was almost ineffective; both ouabain and K-free solution produced a sustained contraction in guinea pig aorta; rat aorta rarely responded to ouabain but developed a contraction in K-free solution. Phentolamine inhibited the transient contraction produced by ouabain in rabbit aorta. In rabbit aorta, ouabain produced a concentration-dependent potentiation of norepinephrine contraction. In denervated rabbit aorta, however, the potentiation only occurred in the presence of 5 X 10(-7) to 5 X 10(-6) M ouabain. K-free solution inhibited the norepinephrine contraction in denervated rabbit aorta. Ouabain potentiated the norepinephrine contraction in guinea pig aorta but not in rat aorta. It is concluded that ouabain and K-free solution affect contraction both indirectly via the release of endogenous catecholamines and directly by acting on vascular smooth muscle, and that there are species differences in the sensitivity to ouabain.

Animals↗