3H-ouabain Binding Research Articles

Inward currents through somatic Na/K‐ATPase α1‐subunit mutants are not required for hyperaldosteronism

Hyperaldosteronism (HA), the most common cause of secondary hypertension, is often initiated by an aldosterone‐producing adenoma within the zona glomerulosa of the adrenal cortex. Somatic mutations to several genes were identified in these adenomas, including to the Na/K‐ATPase (NKA) α1‐subunit. The membrane potential (Vm) of cells harboring these NKA‐α1 mutations is depolarized, which is believed to underlie the pathophysiological aspects of HA.At physiological Na+ and K+ concentrations, wild type NKA activity generates outward current due to the export of 3 Na+ and import of 2 K+ per cycle against their gradients. Under similar conditions, all HA‐associated NKA mutants evaluated thus far have dramatically reduced activity and an inward current, mediated by Na+ or H+, proposed to be a ‘gain‐of‐function’ required for Vm depolarization. The outward current of wild type NKA typically contributes minimally to Vm. Thus, for the inward current through HA‐associated NKA mutants to significantly depolarize Vm, its amplitude is expected to be significantly larger than the amplitude of wild type NKA outward current. Assuming similar expression levels, the transport rate of inward current through NKA mutant pumps should be larger than the pumping turnover rate of a single wild type NKA.To calculate the turnover rate of inward current through human NKA mutants, we utilized two‐electrode voltage clamp (TEVC) of Xenopus oocytes heterologously expressing HA‐associated mutants to measure the ouabain‐sensitive inward current amplitude in the presence of Na+, and 3H‐ouabain binding assays on the same oocyte. As ouabain binds to NKA at a 1:1 ratio, its quantity is equivalent to the number of pumps in the oocyte's plasma membrane. The ratio of current at −50 mV (a physiologically‐relevant voltage) to the moles of ouabain bound to the oocyte yielded inward current turnover rates of 22.0 ± 1.8 s−1 for L104R, 417.3 ± 29.4 s−1 for delF100‐L104, 60.7 ± 5.8 s−1 for V332G, and 19.1 ± 1.4 s−1 for EETA963S, while turnover rate of wild type K+‐induced pump current was 32.0 ± 1.5 s−1. This indicates that the inward current of several NKA mutants may not suffice to significantly depolarize Vm, and that the pathophysiological effects may simply reflect lack of function. Furthermore, oocytes expressing G99R, a mutant recently associated with HA, lacked inward current under physiological conditions, instead presenting K+‐induced outward pump current. TEVC measurements of extracellular K+‐induced current in G99R revealed a ~3‐fold reduction of K+ apparent affinity compared to wild type, while measurements of intracellular Na+‐dependent, ATP‐activated currents in giant inside‐out patches showed a ~3‐fold reduction of Na+ apparent affinity, demonstrating impaired function under physiological conditions. These results suggest that lack of function is the main mechanism leading to HA in all mutants.Support or Funding InformationSupported by NSF MCB‐1515434

Canonical Bcl-2 Motifs of the Na+/K+ Pump Revealed by the BH3 Mimetic Chelerythrine: Early Signal Transducers of Apoptosis?

Background/Aims: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na⁺/K⁺ pump/ATPase [NKP, NKA], Na⁺-K⁺-2Cl⁺ [NKCC] and K⁺-Cl^- [KCC] cotransporters, and channel-supported K⁺ loss) in human lens epithelial cells [LECs]. Methods: K⁺ loss and K⁺ uptake, using Rb⁺ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl^- replacement by NO₃^ˉ to determine KCC. ³H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. Results: CET inhibited NKP and NKCC by >90% (IC₅₀ values ∼35 and ∼15 µM, respectively) without significant KCC activity change, and stimulated K⁺ loss by ∼35% at 10-30 µM. Neither ATP levels nor phosphorylation of the NKA α1 subunit changed. ³H-ouabain was displaced from pig renal NKA only at 100 fold higher CET concentrations than the ligand. Sequence alignments of NKA with BH1- and BH3-like motifs containing pro-survival Bcl-2 and BclXl proteins showed more than one BH1-like motif within NKA for interaction with CET or with BH3 motifs. One NKA BH1-like motif (ARAAEILARDGPN) was also found in all P-type ATPases. Also, NKA possessed a second motif similar to that near the BH3 region of Bcl-2. Conclusion: Findings support the hypothesis that CET inhibits NKP by binding to BH1-like motifs and disrupting the α₁ subunit catalytic activity through conformational changes. By interacting with Bcl-2 proteins through their complementary BH1- or BH3-like-motifs, NKP proteins may be sensors of normal and pathological cell functions, becoming important yet unrecognized signal transducers in the initial phases of apoptosis. CET action on NKCC1 and K⁺ channels may involve PKC-regulated mechanisms; however, limited sequence homologies to BH1-like motifs cannot exclude direct effects.

Age-Dependent Brain Tissue Hydration, Ca Exchange and their Dose- Dependent Ouabain Sensitivity

Tissue hydration, dose-dependent 3H-ouabain binding, 45Ca2+ exchange in rati?½s brain cortex, subcortex and cerebellum were studied in three age groups. Age-dependent tissue dehydration in all three zones of brain was due to inhibition of Na+/K+ pump. The age-dependence of cell hydration in cortex was more expressed. The curve of dose-dependent ouabain binding consists of three components corresponding to Na+/K+ pump isoforms (α1, α2, α3). Age-dependency of these isoforms was more expressed in cortex than in subcortex and cerebellum. High affinity receptors were depressed in old ratsi?½ brain tissues. Initial 45Ca2+ uptake in three brain zones of old rats was depressed as compared to that of young animals. Ouabain at 10-9 M has activation effect on 45Ca2+ uptake, which was also age dependent. Initial 45Ca2+ efflux in cortex and subcortex tissue in old rats was significantly depressed as compared to young ones while in cerebellum the opposite age-dependence was observed. The curves of dose-dependent ouabain effect on 45Ca2+ efflux and cell hydration consist of 6 components. However, close correlation between kinetics of 45Ca2+ efflux and cell hydration was not observed. It is suggested that brain tissue dehydration in aged animals is a consequence of Na+/K+ pump dysfunction induced intracellular calcium elevation. It is suggested that α3 receptors are functionally connected with intracellular Ca2+ buffering systems through intracellular signaling systems and their dysfunction in aged brain is a consequence of [Ca2+]i increase. Obtained data allow us to conclude that endogen nanomolar ouabain-like species circulating in mammalsi?½ blood removing Ca2+ from cells could have a beneficial effect on brain of old animals.

Atrial Na,K-ATPase Increase and Potassium Dysregulation Accentuate the Risk of Postoperative Atrial Fibrillation

Background: Postoperative atrial fibrillation is a common complication to cardiac surgery. Na,K-ATPase is of major importance for the resting membrane potential and action potential. The purpose of the present study was to evaluate the importance of Na,K-ATPase concentrations in human atrial biopsies and plasma potassium concentrations for the development of atrial fibrillation. Methods: Atrial myocardial biopsies were obtained from 67 patients undergoing open chest cardiac surgery. Na,K-ATPase was quantified using vanadate-facilitated ³H-ouabain binding. Plasma potassium concentration was measured with ion-selective electrode. Results: In patients with preoperative sinus rhythm, ³H-ouabain-binding site concentration was 16% higher in patients developing postoperative atrial fibrillation compared to patients maintaining sinus rhythm [302 ± 15 pmol/g wet weight (n = 20) vs. 261 ± 11 mmol/g wet weight (n = 33), p = 0.03]. Also with multivariable analysis, ³H-ouabain-binding site concentration was significantly associated with the development of atrial fibrillation. High increase in plasma potassium concentration during the perioperative period and surgery was associated with postoperative atrial fibrillation. Conclusions: The present study supports the increasing evidence of dysregulation of the potassium homeostasis as an important factor in the development of cardiac arrhythmias. High atrial Na,K-ATPase and sudden plasma potassium concentration increase may contribute to precipitate atrial fibrillation.

Subcellular redistribution of trimeric G-proteins – potential mechanism of desensitization of hormone response; internalisation, solubilization, down-regulation

Agonist-induced subcellular redistribution of G-protein coupled receptors (GPCR) and of trimeric guanine-nucleotide binding regulatory proteins (G-proteins) represent mechanisms of desensitization of hormone response, which have been studied in our laboratory since 1989. This review brings a short summary of these results and also presents information about related literature data covering at least small part of research carried out in this area. We have also mentioned sodium plus potassium dependent adenosine triphosphatase (Na, K-ATPase) and 3H-ouabain binding as useful reference standard of plasma membrane purity in the brain.

Presence of a functional TSH receptor on human erythrocytes

The presence of thyrotropin receptors (TSHR) has been reported in some extrathyroidal tissues but its physio-pathological role still remains unclear. TSH (seems to) affects the human erythrocytes Na +/K +-ATPase in vitro, however receptors on erythrocytes have not yet been described. In this work the effect of recombinant human TSH (rhTSH) on sites number and activity of erythrocyte Na +/K +-ATPase was investigated in a group of thyroidectomized patient enrolled for rhTSH test. As detected by 3H-ouabain binding, rhTSH administration induced a significant increase in the number of sites ( p = 0.005) and in the Kd ( p = 0.006) of Na +/K +-ATPase. rhTSH did not induce significant difference in Na +/K +-ATPase activity measured by 86Rb uptake. 125I-TSH binding studies and Western blotting data showed the existence of TSHR in the erythrocytes of healthy donors. In conclusion The TSH action on Na +/K +-ATPase of human erythrocytes can be explained by the presence of TSHR

Involvement of Na +, K +-ATPase and Endogenous Digitalis-Like Compounds in Depressive Disorders

Sodium and potassium-activated adenosine triphosphatase (Na(+), K(+)-ATPase) and endogenous digitalis-like compounds (DLC) in the brain have been implicated in the pathogenesis of mood disorders. This hypothesis was examined by the determination of Na(+), K(+)-ATPase/DLC system in parietal cortex of patients with different mood disorders and two animal models of depression. Na(+), K(+)-ATPase concentrations in human brain synaptosomal fractions, from patients with mood disorders, schizophrenia, and normal individuals, were determined by (3)H-ouabain binding assay. Alpha isoforms were quantified by Western blotting. Brain DLC were measured using sensitive enzyme linked immunosorbant assay (ELISA). The effects of ouabain and ouabain-antibodies on behavior were determined in two animal models of depression. (3)H-ouabain binding in bipolar patients was significantly lower than in major depressed and schizophrenic patients. Na(+), K(+)-ATPase alpha isoforms in synaptosomal fractions were not different among the groups. DLC levels in the parietal cortex of bipolar patients were significantly higher than in normal individuals and depressed patients. Injection of lipopolysaccharide (intraperitoneally) to rats elicited depression-like symptoms, which were significantly attenuated by pre-injection of ouabain-antibodies. Injection of ouabain and ouabain-antibodies (intracerebroventricular) reduced depression-like symptoms in the forced swimming test in rats. The results support the possibility that Na(+), K(+)-ATPase and endogenous DLC participate in the pathogenesis of depressive disorders.

Functional human corneal endothelial cell sheets harvested from temperature‐responsive culture surfaces

This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen-coated dishes and, after several passages, expanded cells were then seeded onto novel temperature-responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+-ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A 3H-ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2 and 3070 cells/mm2. We also confirmed Na+, K+-ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.

Protein kinases A and C stimulate the Na+ active transport in frog skeletal muscle without an appreciable change in the number of sarcolemmal Na+ pumps

The activation of both protein kinases A (PKA) and protein kinases C (PKC) in some cell types increases and in others reduces active Na+ efflux. These effects have been ascribed to either a change in the rate of ionic translocation by a fixed number of Na+ pumps or, a change in the number of plasma membrane pumps. The purpose of the present experiments was to study the effect of activating PKA and PKC on the Na+ extrusion by the Na+ pump in frog skeletal muscle. Na+ (22Na+) fluxes and ouabain (3H-ouabain) binding were measured in frog sartorius muscles. Both activation of PKA and PKC increased the active Na+ extrusion by a factor of two; these effects were not additive. Ouabain binding experiments indicated that the pump stimulation by activation of these kinases is not associated with any significant increase in the number of plasma membrane pumps. Stimulation of the active Na+ efflux by protein kinase activation (no change in the number of sarcolemmal pumps) and by hypotonicity (increase in the number of pumps) could be elicited in the same preparation and they were additive. It is concluded that in frog skeletal muscle fibres, (1) activation of both PKA and PKC stimulate the Na+ pump by increasing its rate of ionic translocation; and (2) two modes of Na+ active transport (with and without an increase in the number of pumps) are operative, and can be at work simultaneously, a phenomenon to be reckoned with.

Amiodarone inhibits the 3,5,3'-triiodothyronine-dependent increase of sodium/potassium adenosine triphosphatase activity and concentration in human atrial myocardial tissue.

In animal models the function of the sodium pump (sodium/potassium-adenosine triphosphatase [Na+/K(+)-ATPase]) is enhanced by 3,5,3'-triiodothyronine (T3) and inhibited by the antiarrhythmic agent amio. However, it is still unclear whether the effect of the drug on Na+/K(+)-ATPase depends on the interference with thyroid hormone action. We evaluated the interaction of T3 with amiodarone on Na+/K(+)-ATPase activity and site number in human myocardium. Right atrial slices were cultured with (T3+) and without (T3-) 3 nM T3 in presence and absence of amiodarone at therapeutical dose (1.5 microM). When compared to T3+, T3- preparations showed decreased 3H-ouabain binding (p < 0.004) and lower 20-minute and 45-minute 86Rb-uptake (p < or = 0.004). Amiodarone caused an average 49% reduction of the T3-dependent 3H-ouabain binding and decreased the Western blot signal for the Na+/K(+)-ATPase alpha1 subunit. The drug also inhibited T3-dependent increase in 86Rb-influx at 20 and 45 minutes by 66% and 42%, respectively, without affecting the affinity of the pump for K+. No differences were found in the 3H-ouabain binding and 86Rb-uptake of T3-, T3- amio and T3(+)-amio. In conclusion, T3 stimulates the Na+/K(+)-ATPase in human atrial myocardium by increasing the number of ouabain-binding sites, whereas amiodarone decreases the sodium pump function secondarily to the antagonism with thyroid hormone.

Quantification of alpha-subunit isoforms of Na,K-ATPase in rat resistance vessels.

Rat mesenteric resistance vessels (RV) were characterized with respect to concentration of individual alpha-subunit isoforms of Na,K-ATPase. Total vessel homogenates were used to avoid any loss or subfractionation of membranes. They were applied to sodium dodecyl sulphate gels and, for calibration, in parallel lanes were run purified rat Na,K-ATPase preparations with known isoform distribution and content. The capacity per mg protein for Na+-dependent 32P-phosphorylation of Na,K-ATPase isolated from rat kidney was used for alpha1 calibration and that for high-affinity (3H)ouabain binding of Na,K-ATPase isolated from rat brain was used for (alpha2 + alpha3) calibration. Western blots containing homogenate proteins and reference enzyme were incubated with isoform-specific antibodies and radiolabelled secondary antibodies. The signals from adjacent alpha spots were used for qualitative and quantitative characterization of rat vessels. A concentration of 100.7 +/- 14.4 pmol (n = 11) per g wet weight of the alpha1-isoform containing Na,K-ATPase was found in RV from 12-14-week rats. A much lower and more unreliable content of alpha2- and alpha3-isoforms was found. These ouabain-sensitive isoforms seem to represent a maximum of 5-10% each compared with the ouabain-insensitive rat alpha1-isoform. The isoform pattern in RV, in which the isoform with high/intermediate Na+-affinity is the absolutely dominating one representing nearly all sodium pumps in this tissue, is very different from that seen in rat skeletal muscles. Due to the high content of the ouabain-insensitive alpha1-isoform in rat RV this species would seem a less relevant model in studies addressing a role of cardiac glycosides and putative endogenous ouabain-like factors in hypertension.

Effect of canrenone on the digitalis site of Na+/K(+)-ATPase in human placental membranes and in erythrocytes.

It has been reported that canrenone, which is used in hypertensive therapy as an antialdosteronic drug, may also act as a blocker of ouabain effects. Several studies suggest that human plasma contains an endogenous ouabain-like factor similar to ouabain, which may be increased in hypertension, in pregnancy, and in the neonatal state. This study evaluated (1) the effect of canrenone on Na+/K(+)-ATPase in relation to ouabain in human placental membranes and erythrocytes by 3H-ouabain binding assay; (2) the capacity of canrenone (10 microM) to reverse the inhibition of Na+/K(+)-ATPase by ouabain and by ouabain-like factor (from umbilical cord plasma) in human erythrocytes employing a 86Rb uptake assay. Increasing concentrations of canrenone (0-350 microM) partially competed with 3H-ouabain binding in placental membrane (40%) and erythrocytes (60%). Scatchard plot from radioreceptor assay in placental membrane showed that ouabain and canrenone compete for the same binding site. In erythrocytes, canrenone completely reversed the inhibition caused by ouabain (5 x 10(-9) M) and ouabain-like factor (2 x 10(-9) M ouabain equivalents). A reduction of inhibition of about 50% was observed with ouabain and ouabain-like factor respectively at a concentration of 5 x 10(-8) M and 2 x 10(-8) M (ouabain equivalents). Our results thus provide evidence that canrenone, at therapeutical concentrations, is a partial competitive agonist of ouabain and of ouabain-like factor in human placental membranes and erythrocytes.

Ambient Salinity Modulates the Expression of Sodium Pumps in Branchial Mitochondria-Rich Cells of Mozambique Tilapia, Oreochromis mossambicus

Na,K-ATPase (sodium pumps) provide the primitive driving force for ion transport in branchial epithelial cells. Immunoblots of epithelial homogenates of both seawater (SW)- and freshwater (FW)-adapted tilapia gills as well as rat brain homogenate, a positive control, revealed one major band with a molecular weight of about 100 kDa. SW-adapted tilapia gills possessed larger (about 2-fold) amounts of sodium pumps compared with FW-adapted tilapia gills. (3)H-ouabain binding representing functional binding sites of Na,K-ATPase was also higher (about 3.5-fold) in gills of SW-adapted tilapia compared to that of FW-adapted fish. Moreover, specific activities of SW fish were higher (about 2-fold) than those of FW fish. Double labeling of Na,K-ATPase and Con-A, a fluorescent marker of MR cells, in tilapia gills followed by analysis with confocal microscopy showed that sodium pumps were localized mainly in MR cells, including the SW type and different FW types. Although more-active expression of Na,K-ATPase was demonstrated in gills of SW-adapted tilapia, no significant differences in densities of apical openings of MR cells were found between SW- and FW-adapted fish. These results indicate that, during salinity challenge, tilapia develop more "functional" Na,K-ATPase in SW-type MR cells to meet physiological demands.

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.

The alpha1 isoform of Na+,K+-ATPase in rat soleus and extensor digitorum longus.

A novel immunochemical method was used for determination of the concentration of Na,K-adenosine triphosphatase (ATPase) containing the ouabain-insensitive alpha1 peptide in rat m. soleus and extensor digitorum longus (EDL). Homogenates of soleus and EDL from 4-week or 10-11-week rats were run on sodium dodecyl sulphate (SDS) gels and in parallel lanes was run a well-characterized preparation of Na,K-ATPase isolated from rat kidney that is known to contain only the alpha1 isoform. After electroblotting to PVDF membranes blots were incubated with the alpha1 specific monoclonal antibody 3B, then with an 125I-coupled secondary antibody, and finally the specific labelling of adjacent alpha spots was analysed by means of an electronic autoradiography system (Packard InstantImager). As the alpha1 content of reference Na,K-ATPase was known from the specific Na+-dependent 32P-phosphorylation capacity, the alpha1 content of adjacent alpha spots in homogenates from soleus and EDL could be calculated. In soleus and EDL from 4-week rats an alpha1 concentration of 135-220 pmol (g tissue)(-1) was found, dependent on the conditions of the experiments but without significant differences between the two types of muscle. In 10-11-week rats a significantly lower concentration of 70-80 and 40-60 pmol (g tissue)(-1) in soleus and EDL, respectively, was found. Ouabain-insensitive Na,K-ATPase containing the alpha1 peptide may thus represent 15-25% of the total number of pumps in skeletal muscle if another 20-30% has to be added to the pool known from (3H)ouabain binding.

Canrenone competes for the same site of ouabain on Na+/K+-ATPase

Recently several studies suggested the existence in human plasma of an endogenous digitalis-like factors (EDLF) similar to ouabain which could have a role in the pathogenesis of some forms of hypertension. Canrenone is a metabolic product of the antialdosteronic drug spironolactone, used in hypertensive therapy and recently described as a blocker of ouabain effects. The aim of this study was to evaluate the effect of canrenone on Na+/K+-ATPase in relation to ouabain investigating its interaction with the ouabain receptor and with the sodium-potassium pump activity. For this purpose we employed a3H ouabain receptor assay in human placental membranes and a 86Rb uptake assay in human erythrocytes. Increasing concentration of canrenone (0-180 μM) partially inhibited the ouabain sensitive 86Rb uptake in red blood cells until 40%. Moreover canrenone partially competed for 30% with 3H ouabain binding in placental membrane receptors in absence and in presence of 5 mM K+, respectively at a concentration of 350 μM and 70 μM. When the displacement of ouabain (10-7M) by canrenone (180 μM) was valuated, measuring ouabain sensitive 86Rb uptake, a recovery of 86Rb uptake was obtained only in presence of 5 mM K+. Finally Scatchard plot from radioreceptor assay showed that ouabain in absence and presence of canrenone intersected at a common point on the abscissal axis. These results provide evidence that canrenone is a partial competitive agonist of ouabain and interacts with the same receptor site suggesting that it could be used in hypertensive states with high levels of endogenous ouabain.

Biological implication of conformational flexibility in ouabain: observations with two ouabain phosphate isomers.

Ouabain is a highly polar and unusually potent sodium pump inhibitor that possesses uncommon conformational flexibility in its steroid A-ring moiety. The biological significance of ring flection in the cardiotonic steroids has not been described. Accordingly, we prepared ouabain 1,5,19- and 1,11,19-phosphates. The former stabilizes the steroid A-ring chair conformation and the latter locks the A-ring in the half-boat conformation and decreases flection of the ABC-ring moiety. Using a dog kidney cell line (MDCK) in a pH microphysiometer (Cytosensor), ouabain and its 1,5,19-phosphate at 10(-5) M reduced the rate of extracellular acidification by 15-20%. During inhibitor washout, the rate of recovery from the 1,5,19-phosphate analogue was approximately 3 times faster than ouabain. The 1,11,19-phosphate at 10(-4) M elicited a weak ( approximately 7%) response, and the effects reversed approximately 44-fold faster than ouabain. Studies with purified Na(+),K(+)-ATPase showed that ouabain and its 1,5,19-phosphate analogue were of similar efficacy (EC(50) = 1.1 and 5.2 x 10(-7) M, respectively) and >100-fold more potent than the 1,11,19-phosphate analogue. Studies of the binding kinetics showed that the 1,5,19-phosphate analogue bound 3-fold and dissociated 16-fold faster from the purified Na(+),K(+)-ATPase than ouabain. Both analogues were competitive inhibitors of 3H-ouabain binding. Taken together, these results suggest that the marked conformational flexibility of the A-ring in ouabain ordinarily slows the initial binding of this steroid to the sodium pump. However, once ouabain is bound, flection of the steroidal A- and BC-rings is critical for the maintenance of high-affinity binding. Our results indicate that the ouabain-binding site is comprised of structurally mobile elements and highlight the roles that synchronization between receptor and ligand dynamics play as determinants of biological activity in this system.

Effect of strenuous strength training on the Na-K pump concentration in skeletal muscle of well-trained men.

This study examined how strenuous strength training affected the Na-K pump concentration in the knee extensor muscle of well-trained men and whether leg muscle strength and endurance was related to the pump concentration. First, the pump concentration, taken as 3H-ouabain binding, was measured in top alpine skiers since strength training is important to them. Second, well-trained subjects carried out strenuous eccentric resistance training either 1, 2, or 3 times.week-1 for 3 months. The Na-K pump concentration, the maximal muscle strength in a full squat lift (one repetition maximum, 1 RM), and the muscle endurance, taken as the number of full squat lifts of a mass of 70% of the 1 RM load, were measured before and after the training period. The mean pump concentration of the alpine skiers was 425 (SEM 11) nmol.kg-1 wet muscle mass. The subjects in part two increased their maximal strength in a dose-dependent manner. The muscle endurance increased for all subjects but independently of the training programme. From a mean starting value of 356 (SEM 6) nmol.kg-1 the mean Na-K pump concentration increased by 54 (SEM 15) nmol.kg-1 (+15%, P < 0.001) when the results for all subjects were pooled. The effect was larger for those who had trained twice a week than for those who had trained only once a week (P = 0.025), suggesting that the effect of strength training depended on the amount of training carried out. The muscle strength and endurance were not related to the pump concentration, suggesting that the pumping power of this enzyme did not limit the performance during heavy lifting. However, the individual improvements in the endurance test during the training period correlated with the individual changes in the pump concentration (rSpearman = 0.5; P = 0.01) which could mean that a common factor both increases the pump concentration and makes the muscles more adapted to repeated heavy lifting.

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.

Receptor occupancy with digoxin vs receptor occupancy with a putative endogenous digitalislike factor.

The possibility that an endogenous ligand for the digitalis receptor might exist has been a source of speculation resulting in efforts over the past decades to identify such a hormone. In the current context it is of interest that prolonged wash of myocardial and skeletal muscular samples from subjects who were not in digoxin treatment generally resulted in small tendencies to increase 3H-ouabain binding ranging from 2 to 9% and from -2 to 7%, respectively. It may be appreciated that neither wet weight nor water content of left ventricular or skeletal muscular samples have been found to change as a result of the prolonged wash. Although these tendencies most likely are a mere play of chance, it may be argued that the studies did not entirely rule out the possibility of the existence of a quantitatively small amount of endogenous digitalislike factor. However, based on the evaluations of receptor occupancy with digoxin during treatment of 24-34% in the left ventricle and 9-13% in skeletal muscle, it would seem reasonable to expect that a comparatively larger fraction of digitalis receptors should be occupied by a putative endogenous digitalislike factor, if such a factor were to be of any physiological significance.