Specific 3H-ouabain Binding Research Articles

Novel receptors for ouabain: studies in adrenocortical cells and membranes.

Sodium-potassium pumps (Na pumps) are the only known plasma membrane receptors for cardiac glycosides. However, adrenocortical cells secrete an endogenous ouabain via an unknown mechanism that is subject to feedback inhibition via the cell surface. In addition, recent studies suggest that the induction of sustained hypertension by ouabain analogs in rats may be independent of Na pump inhibition. Accordingly, we used bovine adrenocortical cells and membranes to search for novel binding sites for ouabain. In high extracellular potassium solutions, the binding of ouabain to the Na pumps of cultured cells was suppressed, yet residual specific binding of (3)H-ouabain was observed. In high extracellular potassium, Scatchard analyses revealed a novel class of ouabain binding sites with high affinity (<50 nmol/L, < 2.5 x 10(5) sites/cell) that was distinct from the low-affinity Na pump sites (>1 micromol/L, 4.5 x 10(6) sites/cell). Analysis of the kinetics for the dissociation of (3)H-ouabain from intact cells revealed components whose t(0.5) values were 6.5 minutes, 3.3 hours, and 33 hours and associated with novel sites, Na pumps, and lysosomal recycling, respectively. Studies with isolated membranes under ligand conditions where the participation of Na pumps was minimized revealed specific ouabain binding to novel sites that was saturable, time-dependent, of high affinity (K(d) approximately 15 nmol/L), and of low density (apparent B(max)=0.23 pmol/mg, c.f., Na pumps=10.2 pmol/mg). Ouabain binding to the novel sites was stimulated by high concentrations of KCl but was not affected by aldosterone or cortisol up to 30 micromol/L. Novel sites were not detected in skeletal muscle or liver membranes. Photoaffinity studies followed by SDS-PAGE showed ouabain-protectable labeling of membrane polypeptides with apparent molecular weights of 143, 113, and 65 kDa. We conclude that adrenocortical cells express ouabain receptors that are distinct from Na pumps. These novel receptors may be involved in the regulation and/or secretion of endogenous ouabain.

Brefeldin A influences the cell surface abundance and intracellular pools of low and high ouabain affinity Na+, K(+)-ATPase alpha subunit isoforms in articular chondrocytes.

The catalytic alpha isoforms of the Na+, K(+)-ATPase and stimuli controlling the plasma membrane abundance and intracellular distribution of the enzyme were studied in isolated bovine articular chondrocytes which have previously been shown to express low and high ouabain affinity alpha isoforms (alpha 1 and alpha 3 respectively; alpha 1 >> alpha 3). The Na+, K(+)-ATPase density of isolated chondrocyte preparations was quantified by specific 3H-ouabain binding. Long-term elevation of extracellular medium [Na+] resulted in a significant (31%; p < 0.05) upregulation of Na+, K(+)-ATPase density and treatment with various pharmacological inhibitors (Brefeldin A, monensin and cycloheximide) significantly (p < 0.001) blocked the upregulation. The subcellular distribution of the Na+, K(+)-ATPase alpha isoforms was examined by immunofluorescence confocal laser scanning microscopy which revealed predominantly plasma membrane immunostaining of alpha subunits in control chondrocytes. In Brefeldin A treated chondrocytes exposed to high [Na+], Na+, K(+)-ATPase alpha isoforms accumulated in juxta-nuclear pools and plasma membrane Na+, K(+)-ATPase density monitored by 3H-ouabain binding was significantly down-regulated due to Brefeldin A mediated disruption of vesicular transport. There was a marked increase in intracellular alpha 1 and alpha 3 staining suggesting that these isoforms are preferentially upregulated following long-term exposure to high extracellular [Na+]. The results demonstrate that Na+, K(+)-ATPase density in chondrocytes is elevated in response to increased extracellular [Na+] through de novo protein synthesis of new pumps containing alpha 1 and alpha 3 isoforms, delivery via the endoplasmic reticulum-Golgi complex constitutive secretory pathway and insertion into the plasma membrane.

CHARACTERIZATION OF THE Na+K+-ATPase IN ISOLATED BOVINE ARTICULAR CHONDROCYTES; MOLECULAR EVIDENCE FOR MULTIPLE α AND β ISOFORMS

We have used isoform-specific antibodies against the Na+K+-ATPase αα1, α2 and α3) and ββ1 and β2) subunit isoforms in order to establish their specific localization in isolated bovine articular chondrocytes. Immunoblotting confirmed the presence of the α1 and α3 isoforms, although α1 expression was significantly greater than α3 as assessed by immunofluorescence confocal laser scanning microscopy and PCR. A similar approach revealed the presence of the β1 and β2 isoforms in chondrocytes, although β2 immunostaining on the plasma membrane was more punctate than β1 which in contrast predominated in a subcellular compartment. The plasma membrane abundance of the Na+K+-ATPase was found to be sensitive to the extracellular ionic concentration and long-term elevation of extracellular Na+concentration significantly upregulated Na+K+-ATPase density as measured by specific3H-ouabain binding. Our observations suggest that the expression of α3 and β2 is not restricted to excitable tissues as previously reported. The physiological relevance of α3 expression in chondrocytes may be related to its low affinity for intracellular Na+in an extracellular environment where Na+concentration is unusually high (260–350mm) compared to other cell types (140mm). Glycoproteins and their branched carbohydrates have been implicated in cell recognition events, thus the β2 subunit glycoprotein may allow the chondrocyte to detect changes in its extracellular environment by physically interacting with components of the cellular cytoskeleton and matrix macromolecules.

Role of protein phosphatase in the regulation of Na+-K+-ATPase by vasopressin in the cortical collecting duct.

In the cortical collecting duct (CCD), arginin vasopressin (AVP) has been shown to increase the number and activity of basolateral Na+-K+-ATPase by recruiting or activating a latent pool of pumps. However, the precise mechanism of this phenomenon is still unknown. The aim of this study was to investigate whether this AVP-induced increase in basolateral Na+-K+-ATPase could depend on a dephosphorylation process. To this purpose, the effect of protein serine/threonine phosphatase (PP) inhibitors was examined on both the specific 3H-ouabain binding (to evaluate the number of pumps in the basolateral membrane) and the ouabain-dependent 86Rb uptake (to evaluate pump functionality) in the presence or absence of AVP. In addition, the activity of two PP, PP1 and PP2A, was measured and the influence of AVP was examined on both enzymes. Experiments have been performed on mouse CCD isolated by microdissection. Results show that inhibition of PP2A prevents the AVP-induced increase in the number and activity of Na+-K+-ATPases, independent of an effect on the apical cell sodium entry. In addition, AVP rapidly increased the activity of PP2A without effect on PP1. These data suggest that PP2A is implied in the regulation of Na+-K+-ATPase activity by AVP in the CCD and that the AVP-dependent increase in the number of Na+-K+-ATPases is mediated by a PP2A-dependent dephosphorylation process.

Synergistic action of vasopressin and aldosterone on basolateral Na(+)-K(+)-ATPase in the cortical collecting duct.

The respective effects of aldosterone and arginine vasopressin (AVP) were examined on the number of active Na(+)-K(+)-ATPase and their pumping activity in nonperfused microdissected mouse cortical collecting tubules (CCD) by measuring specific 3H-ouabain binding and ouabain-sensitive 86Rb uptake. In adrenalectomized (ADX) animals, incubation of CCD with AVP (10(-8) M for 5 min) had no effect on the number of pumps. In contrast, in ADX animals replete with aldosterone, AVP induced a approximately equal to 40% increase in the number of pumps. This was accompanied by a approximately equal to 60-65% increase in ouabain-sensitive Rb uptake. AVP effect was dose-dependent (10(-10)-10(-8) M) and was reproduced by dDAVP, forskolin and 8-Br cAMP, indicating a V2 pathway. It was inhibited by amiloride 10(-5) M, and did not occur in CCD incubated in hyperosmotic solution, suggesting that the signal was transmitted via apical sodium entry and cell swelling. Finally, the AVP-dependent increase in the number of pumps was rapid (within 5 min) and transient (< 25 min). These results demonstrate that, in the CCD, aldosterone and AVP act synergistically to increase not only the apical sodium entry but also the basolateral Na(+)-K(+)-ATPase transport capacity: AVP allows a rapid recruitment and/or activation of an aldosterone-dependent pool of latent Na(+)-K(+)-ATPase.

Doppler blood flow measurements of umbilical artery of the in vitro perfused human placenta

Lymphoblastoid cell lines established from patients suffering from bipolar manic depression have been used to study the possible involvement of cation transport in the aetiology of this illness. No significant difference was found in the K+ fluxes mediated by the ouabain-sensitive sodium pump, the diuretic-sensitive cotransport system and the passive leak pathway of cell lines established from either control or bipolar subjects. The mean value for the specific binding of 3H-ouabain (sodium pump site number) was significantly higher in the bipolar group (approximately 30%) than in the control group.

Digitalis-like activity in human plasma: Relation to blood pressure and sodium balance

purpose: On the assumption that renal tubular cells are more important as the target cells for a natriuretic factor than blood cells, we used a well-characterized cultured renal tubular cell line, Madin-Darby canine kidney (MDCK) cells, to monitor the circulating digitalis-like factor in human plasma and examine its role in the regulation of blood pressure and sodium balance. subjects and methods: We investigated the effects of plasma on binding of radioactive ouabain to monolayered MDCK cells in order to determine the level of a circulating digitalis-like factor. First, we measured specific 3H-ouabain binding to MDCK cells in the presence of plasma from 71 outpatients (34 normotensive subjects and 37 hypertensive patients) after incubation for 4 hours. Second, we measured specific 3H-ouabain binding after incubation of cells with plasma from 16 hospitalized subjects (eight normotensive subjects and eight hypertensive patients) receiving low and high sodium diets. results: In Study 1, ouabain binding was lower by 30% with plasma from hypertensive patients than with plasma from normotensive subjects (p <0.01). There was a significant negative correlation between individual subject's systolic or mean blood pressure and ouabain binding (r = −0.34, p <0.01 or r = −0.29, p <0.01). In Study 2, ouabain binding was also significantly reduced by 25% in the presence of plasma from hypertensive subjects as compared with plasma from normotensive subjects irrespective of sodium intake (p <0.01). A significant negative correlation was also found for all subjects between either systolic, diastolic, or mean blood pressure and ouabain binding (r = −0.58, p <0.01, r = −0.51, p <0.01, or r = −0.55, p <0.01, respectively). With the changes from low to high sodium intake, there was a corresponding decrease in ouabain binding (p <0.01) and an increase in sodium excretion (p <0.01). A significant negative correlation was observed between these two parameters (r = −0.47, p <0.05). conclusions: These findings suggest that a circulating digitalis-like factor, which may act on renal tubular cells as the ouabain-displacing compound, is increased in patients with essential hypertension and also demonstrate that plasma levels may be influenced by changes in dietary sodium intake.

The specific 3H-ouabain binding to Na +/K +-ATPase of the human placental tissue is inhibited by newborn plasma extracts

The specific 3H-ouabain binding to Na +/K +-ATPase of the human placental tissue is inhibited by newborn plasma extracts

The effect of ethanol and acetaldehyde on Na pump function in cultured rat heart cells

To further define the sarcolemmal effects of ethanol and acetaldehyde, their effects on Na pump function were studied in synchronously contracting monolayers of neonatal rat myocardial cells. The effects of ethanol (10 mg/dl to 1000 mg/dl: 2 X 10(-3) M-0.2 M) and acetaldehyde (10(-6) M to 10(-4) M) on total 42K influx, ouabain-sensitive 42K influx, Na pump density (from specific 3H-ouabain binding) and pump turnover rates were measured. Applied acutely ethanol had no effect on 42K influx but after 30 min of treatment 42K influx was decreased by 13%, 23% and 48% in 100 mg/dl, 300 mg/dl and 1000 mg/dl ethanol respectively. This primarily reflected a decrease in mean ouabain-sensitive K+ influx from a control of 12.54 to 9.90, 8.95 and 6.68 (p-mol/cm2/s) in 100, 300 and 1000 mg/dl (2 X 10(-2) M, 6 X 10(-2) M) ethanol. Acetaldehyde in the concentrations tested had no effect on K+ influx. Ethanol treatment produced a decrease in Na pump density, maximum within 30 min and dose-dependent, at concentrations of 100 mg/dl (22%), 300 mg/dl (37%) and 1000 mg/dl (55%). Acetaldehyde had no effect on Na pump density. In the presence of ethanol (300 mg/dl and 1000 mg/dl) intracellular Na+ increased significantly and the Na+ efflux declined in parallel with the K+ influx. From the ouabain-sensitive K+ and Na+ fluxes and the Na pump density individual pump turnover rates were calculated at 62.5/s in control cells and 66/s and 84/s in cells treated with 300 mg/dl (6 X 10(-2) M) and 1000 mg/dl (0.2 M) respectively. We conclude that ethanol, but not acetaldehyde has a depressant effect on sarcolemmal Na pump function. The results suggest this is due primarily to a decrease in the number of sarcolemmal Na pump sites.

Kinetics of triiodothyronine action on Na-K-ATPase in single segments of rabbit nephron.

This study was initiated to define the dose- and time-dependence of triiodothyronine (T3) action on Na-K-ATPase in single microdissected nephron segments. For this purpose, the activity and the number of catalytic sites of Na-K-ATPase, as determined by the specific binding of 3H-ouabain, were measured following a single injection of T3 to rabbits thyroidectomized since 8-12 days. Triiodothyronine restored both the activity and the number of catalytic sites of Na-K-ATPase in a dose-dependent manner in all nephron segments where the enzyme was decreased following thyroidectomy, i.e., the proximal and the collecting tubule. At a dose of 50 micrograms/kg bw, T3 restored Na-K-ATPase activity and 3H-ouabain binding with the same kinetics. However, the kinetics depended on the nephron segments: in the proximal tubule, Na-K-ATPase stimulation occurred after a 12 h period of latency and was completed within 24 h whereas in the collecting tubule, the stimulation was biphasic with a first increase within the first 3 h and a second increase concomitantly to that observed in the proximal tubule. These results indicate that thyroid hormones regulate Na-K-ATPase activity by altering the number of catalytic sites of the enzyme. This control depends on two different mechanisms which differ by their time-dependence.

Sites of thyroid hormone action on Na?K-ATPase along the rabbit nephron

Although Ismail-Beigi and Edelman demonstrated in 1971 that thyroid hormones control the activity of Na-K-ATPase in the mammalian kidney, the actual site of this regulation inside the organ was not located. We therefore decided to study the relationship between thyroid hormones and Na-K-ATPase activity in individual nephron segments obtained by microdissection of collagenase-treated rabbit kidneys. For this purpose, the changes in the activity and number of catalytic sites of Na-K-ATPase in response to thyroidectomy or triiodothyronine administration were examined. Eight to 12 days after thyroidectomy, Na-K-ATPase activity had dropped by 40 to 80% in the convoluted and straight portions of the proximal tubules, and in the cortical and outer medullary collecting tubules, but not in the thick ascending limbs of Henle's loops or distal convoluted tubules. The apparent number of catalytic sites for Na-K-ATPase, as measured by specific binding of 3H-ouabain, decreased in parallel with Na-K-ATPase activity, and therefore this enzyme's specific activity was not altered. Fourty eight hours after injection of thyroidectomized animals with a single dose of either 100 or 500 micrograms/kg triiodothyronine, Na-K-ATPase activity in target segments was restored to the level measured in control animals. These effects of thyroid hormone were specific for Na-K-ATPase, since the activity of adenylate cyclase, another marker of the basolateral membrane, was not altered by thyroidectomy. The results obtained indicate that triiodothyronine controls Na-K-ATPase activity in specific nephron segments, by altering the number of this enzyme's catalytic sites.

Specific activity of Na-K-ATPase after adrenalectomy and hormone replacement along the rabbit nephron.

Both aldosterone and dexamethasone are known to stimulate renal Na-K-ATPase activity although their action is restricted to specific nephron segments: the collecting tubule, the target site for mineralocorticoids, and the thick ascending limb and distal convoluted tubule, the target sites for glucocorticoids. As this stimulation by corticosteroids is very fast, we attempted to establish whether it occurs through de novo synthesis of new Na-K-ATPase units or by increasing the specific activity of the Na-K-ATPase units already present. For this purpose we studied the effects of aldosterone and dexamethasone on Na-K-ATPase specific activity in microdissected nephron segments from adrenalectomized rabbits. This specific activity was determined by the ratio of ATPase activity over the apparent number of catalytic units, as measured by specific 3H ouabain binding. In the proximal tubule, neither adrenalectomy nor steroid replacement altered Na-K-ATPase activity or the apparent number of catalytic sites. In other nephron segments, adrenalectomy reduced Na-K-ATPase activity and specific 3H ouabain binding concomitantly, and therefore left this enzyme's specific activity unaltered. In the cortical and outer medullary collecting tubules, 10 micrograms/kg aldosterone simultaneously restored both the activity and apparent number of catalytic units of Na-K-ATPase to their control levels, and therefore did not modify the specific activity of the pump. Conversely, 100 micrograms/kg dexamethasone increased Na-K-ATPase activity in the thick ascending limb and distal convoluted tubule without changing the apparent number of catalytic units.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced number of digitalis receptor sites in the hypertrophied pig myocardium

Left ventricular hypertrophy was established in young pigs (35-40 kg) after 4 weeks of supravalvular aortic stenosis. Isolated sarcolemmal membrane fraction was characterized for specific 3H-ouabain binding. Analysis revealed a single class of receptor sites of 5.7 p moles/mg protein and a dissociation constant (Kd) of the order of 6.25 X 10(-8) M. Measurement of specific 3H-ouabain binding with sarcolemmal fraction isolated from hypertrophied heart revealed a 26% reduction of digitalis receptor after 4 weeks of aortic stenosis, without any significant change in the affinity of receptor sites. This change in the number of digitalis receptors was maintained between 4 and 8 weeks of aortic stenosis and may be an important adaptive mechanism.

Adaptation of the cardiac muscle sodium pump to chronic potassium deficiency.

During chronic dietary K+ depletion in rabbits there is a significant reduction of intracellular K+ concentration [( K+]i) in skeletal muscle, but not in the heart. After 1 week of subsequent K+ repletion both plasma [K+] and skeletal muscle [K+]i had returned to control levels. Cardiac muscle on the other hand showed a significant increase in [K+]i above control. It has been suggested that the heart is protected during K+ depletion by some modification to the sarcolemmal Na+ pump. The Na+ pump density was estimated in membrane preparations from normal and K+ depleted rabbits by the specific binding of 3H-ouabain. 3H-ouabain binding in cardiac muscle preparations was increased significantly after 2 weeks of K+ depletion, and remained so for a further 2 weeks of depletion. There was no significant change in 3H-ouabain binding to skeletal muscle preparations after 4 weeks of K+ depletion. After 1 week of subsequent K+ repletion 3H-ouabain binding was still increased in cardiac muscle preparations, corresponding to the overshoot in cardiac muscle [K+]i. These results support the hypothesis that cardiac muscle is protected against K+ loss during chronic K+ depletion by an adaptive increase in Na+ pump density.

Two receptors for cardiac glycosides in the heart.

Specific binding of 3H-ouabain to rat cardiac cell membranes revealed a high affinity and a low affinity site. In order to test the pharmacological significance of these different types of binding sites, specific 3H-ouabain binding, force of contraction and 86Rb+-uptake were measured simultaneously in contracting cat papillary muscles and in guinea pig atria. The results in the digitalis sensitive cat shows one type of cardiac glycoside receptors with high affinity (KD approximately 10(-7) M) for ouabain. The occupation of this receptor runs parallel with an increase in force of contraction and an inhibition of 86Rb+-uptake. In the rather digitalis insensitive guinea pig, 3H-ouabain binding also runs parallel with increased force of contraction, 86Rb+-uptake, however, is only inhibited at toxic glycoside concentrations. Thus, in rat and guinea pig heart there exist at least two different digitalis receptors--the high affinity receptor seems to be coupled to inotropic effects, the low affinity receptor is linked to inhibition of (Na+ + K+)-ATPase.

Binding of dihydrodigitoxin to beef and human cardiac (Na + + K +)-Atpase: Evidence for two binding sites in cell membranes

The specific binding of three cardiac glycosides, 3H-ouabain, 3H-digitoxin and 3H-dihydrodigitoxin, to beef cardiac (Na + + K +)-ATPase was compared. Non-specific binding was defined as that in the presence of 0.1 mM unlabelled compound, or in the absence of ligands. The dissociation constants ( K D -values) calculated from the inhibition of 3H-ouabain binding were: ouabain, 2.9 × 10 −9M; digitoxin, 1.1 × 10 −9M; and dihydrodigitoxin 2.7 × 10 −8M. The concentrations which inhibited beef cardiac (Na + + K +)-ATPase by 50% were: ouabain, 5.9 × 10 −9M; digitoxin, 1.6 × 10 −9M; and dihydrodigitoxin, 2.5 × 10 −8M. Ouabain and digitoxin showed straight Scatchard plots for one site of high affinity (ouabain, K D = 2.6 × 10 −9 M; digitoxin, K D = 1.7 × 10 −9 M). However, dihydrodigitoxin gave a curved Scatchard plot. Analysis of this binding by the methods of M.J. Weidemann, H. Erdelt and M. Klingenberger ( Eur. J. Biochem. 16, 313 (1970)) for two binding sites gave the following results: for Mg 2+,P i-supported binding, the K D of the high affinity site was 1.6 × 10 −8 M with a capacity similar to that for ouabain of about 30 pmole/mg protein. For binding supported by Na +,ATP,Mg 2+, the K D -value of the high affinity site was 5.3 × 10 −8 M of similar capacity. The low affinity binding site ( K D = 4.0 × 10 −6 M for Mg 2+,P i; K D = 5.5 × 10 −6 M for Na +,ATP,Mg 2+) bound about 350 pmole/mg protein. The low affinity site but not the high affinity site was also present in heat-denatured enzyme. Bincling supported by Mg 2+,P i showed one low affinity site only for ouabain and dihydrodigitoxin in the presence of 200 mM Na +. The high affinity sites for these three cardiac glycosides were further characterized by measurement of the association and dissociation rate constants. The specific binding of 3H-ouabain and 3H-dihydrodigitoxin to human cardiac (Na + + K +)-ATPase was measured. 3H-Ouabain showed a straight Scatchard plot for one high affinity site only ( K D = 4.5 × 10 −9 M, capacity about 15 pmole/mg protein). 3H-Dihydrodigitoxin gave two binding sites: a high affinity site ( K D = 1.8 × 10 −8 M) of similar capacity to ouabain, and a low affinity site ( K D = 2.0 × 10 −6 M) of about 10-fold greater capacity. We conclude that there are two binding sites for some cardiac glycosides in cardiac (Na + + K +)-ATPase from the digitalis-sensitive beef and human: a dihydrodigitoxin high affinity site also occupied by ouabain and digitoxin, and a low affinity binding site, for dihydrodigitoxin only, whose biological significance has to be investigated further.