A protein whose binding to Na,K-ATPase is regulated by ouabain

Immunoprecipitation of Na,K-ATPase from kidney homogenate by antibodies against alpha1-subunit results in the precipitation of several proteins together with the Na,K-ATPase. A protein with molecular mass of about 67 kD interacting with antibodies against melittin (melittin-like protein, MLP) was found in the precipitate when immunoprecipitation was done in the presence of ouabain. If immunoprecipitation was done using antibodies against melittin, MLP and Na,K-ATPase alpha1-subunit were detected in the precipitate, and the amount of alpha1-subunit in the precipitate was increased after the addition of ouabain to the immunoprecipitation medium. MLP was purified from mouse kidney homogenate using immunoaffinity chromatography with antibodies against melittin. The addition of MLP to purified FITC-labeled Na,K-ATPase decreases fluorescence in medium with K+ and increases it in medium with Na+. The enhancement of fluorescence depends upon the MLP concentration. The N-terminal sequence of MLP determined by the Edman method is the following: HPPKRVRSRLNG. No proteins with such N-terminal sequence were found in the protein sequence databases. However, we revealed five amino acid sequences that contain this peptide in the middle part of the chain at distance 553 amino acids from the C-terminus (that corresponds to protein with molecular mass of about 67 kD). Analysis of amino acid sequence located between C-terminus and HPPKRVRSRLNG in all found sequences has shown that they were highly conservative and include WD40 repeats. It is suggested that the 67-kD MLP either belongs to the found protein family or was a product of proteolysis of one of them.     

Role of cell membrane Na,K-ATPase for survival of human lymphocytes in vitro

Lymphocytes are primordial immune cells with variable life times. Besides genetic programming, extracellular factors interacting with cell surface receptors might alter cell survival. We investigated whether the activity of the membrane-embedded Na,K-ATPase (EC 3.6.1.37) or sodium pump (NKA) plays a role for cell survival since this ubiquitous system establishes the vital transmembrane Na and K gradients as well as the resulting high intracellular K/Na ratio required for macromolecule synthesis; furthermore, the system exposes an extracellular inhibitory receptors for cardioactive steroids and palytoxin. Isolated human lymphocytes were incubated in vitro and their viability assessed by exclusion of trypan blue. Various incubation conditions were compared; in RPMI-1640 medium cell viability was preserved for 30 h at 37 °C. Externally added ouabain, a hydrophilic cardioactive steroid, blocked the [⁸⁶Rb]potassium uptake at nanomolar concentrations. Despite pump inhibition ouabain did not alter lymphocyte survival, even at 10 mM for 30 h. By contrast, the hydrophilic toxin palytoxin, the most potent animal poison described so far, killed all cells within 2 h at 10 nM; this toxin is known to act via the sodium pump and to provoke deadly cation-leaks by unmasking a channel component. Intracellular Na increased and K decreased as measured by atomic absorption spectrometry in presence of palytoxin; cell swelling was seen by electron microscopy. Ouabain protected the cells from the toxic effect of palytoxin. The results reveal a pivotal role of NKA integrity for lymphocyte survival.Abbreviations BCA bicinchonic acid - D-PBS Dulbecco's Phosphate Buffered Saline - HBSS Hanks' Balanced Salt Solution - PYX palytoxin (used in figures only) - NKA Na,K-ATPase     

Characterization of "High-Affinity"[3H]Ouabain Binding in the Rat Central Nervous System

The characteristics of [3H]ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of "high-affinity" binding sites with an apparent binding affinity (KD) of 10.4 +/- 0.9 nM and an estimated binding capacity (Bmax) of 7.6 +/- 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. [3H]Ouabain binding to rat brain was sodium- and ATP-dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific [3H]ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K-ATPase. To assess whether the high-affinity binding sites for [3H]ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal [3H]ouabain binding was examined. Kainic acid lesions of the striatum reduced [3H]ouabain binding to striatal homogenates by 79.6 +/- 1.6%. This suggests that the "high-affinity" [3H]ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high-affinity binding of [3H]ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K-ATPase.     

Different sensitivity of ATP + Mg + Na (I) and Pi + Mg (II) dependent types of ouabain binding to phospholipase A2

Summary The effect of phospholipase A₂ and of related agents on ouabain binding and Na,K-ATPase activity were studied in intact and detergent-treated membrane preparations of rat brain cortex and pig kidney medulla. It was found that phospholipase A₂ (PLA₂) may distinguish or dissociate ouabain binding complexes I (ATP+Mg+Na) and II (P_i+Mg), stimulating the former and inhibiting the latter. Procedures which break the permeability barriers of vesicular membrane preparations, such as repeated freezing-thawing, sonication or hypoosmotic shock failed to mimic the effect of PLA₂, indicating that it was not acting primarily by opening the inside-out oriented vesicles. The detergent digitonin exhibited similar effects on ouabain binding in both ATP+Mg+Na and P_i+Mg media. Other detergents were ineffective.The ability of PLA₂ to distinguish between ouabain binding type I and II can be manifested even in SDS-treated, purified preparations of Na,K-ATPase. The number of ATP+Mg+Na-dependent sites is unchanged, while the P_i+Mg-dependent sites are decreased in number in a manner similar to that seen in original membranes. This inhibition is completely lost in the reconstituted Na,K-ATPase system, where the ATP- as well as P_i-oriented ouabain sites are inhibited by PLA₂.     

Stimulation of brain Na,K-ATPase by norepinephrine but not taurine

The effect of taurine on rat and hamster brain Na,K-ATPase was examined and compared to norepinephrine (NE) stimulation of the enzyme. Although NE stimulation of microsomal Na,K-ATPase was observed in the presence of the cell cytosolic fraction, taurine was without effect in the presence and absence of this fraction. Taurine also failed to modulate pubescent and mature hamster brain Na,K-ATPase. Presence or absence of ion chelators did not change taurine's effect. These results are discussed in relation to previous reports of taurine and catecholamine stimulation of Na,K-ATPase.     

Subchronic treatment with vanadate does not potentiate the toxicity of cardiac glycosides

Since it has been claimed that vanadate is an endogenous regulator of Na/K-ATPase activity and that it potentiates the toxicity of cardiac glycosides, we were alarmed to discover that certain Finnish physicians were prescribing vanadate in combination with other trace minerals to elderly patients for many different chronic diseases (e.g., cancer, rheumatism). To study the interaction of vanadate and cardiac glycosides, we fed vanadate in the drinking water (25 μg/mL) to guinea pigs for 20 d, and studied either their sensitivity to the acute toxicity of the cardiac glycoside ouabain or whether the vanadate would influence the subacute toxicity of ouabain. Vanadate had no influence on the toxicity of ouabain either acute or subchronically administered, nor was there any sign of inhibition of Na/K-ATPase activity as measured by⁸⁶Rb-uptake into intact erythrocytes (RBCs), RBC content of sodium or potassium or Na/K-ATPase activity in RBC membranes prepared from the vanadate-treated guinea pigs. Vanadate had been absorbed in substantial quantities from the gastrointestinal tract, since serum, heart, liver, and especially kidney contained measurable amounts of vanadium in contrast to controls, but it is concluded that this vanadate is not in a biologically active form.     

Ouabain resistance of the epithelial cell line (Ma104) is not due to lack of affinity of its pumps for the drug

Na⁺, K⁺-pumps of most eukaryotic animal cells bind ouabain with high affinity, stop pumping, and consequently loose K⁺, detach from each other and from the substrate, and die. Lack of affinity for the drug results in ouabain resistance. In this work, we report that Ma104 cells (epithelial from Rhesus monkey kidney) have a novel form of ouabain-resistance: they bind the drug with high affinity (Km about 4×10^–8 m), they loose their K⁺ and stop proliferating but, in spite of these, up to 100% of the cells remain attached in 1.0 m ouabain, and 53% in 1.0 mm. When 4 days later ouabain is removed from the culture medium, cells regain K⁺ and resume proliferation. Strophanthidin, a drug that attaches less firmly than ouabain, produces a similar phenomenon, but allows a considerably faster recovery. This reversal may be associated to the fact that, while in ouabain-sensitive MDCK cells Na⁺, K⁺-ATPases blocked by the drug are retrieved from the plasma membrane, those in Ma104 cells remain at the cell-cell border, as if they were cell-cell attaching molecules. Cycloheximide (10 g/ml) and chloroquine (10 m) impair this recovery, suggesting that it also depends on the synthesis and insertion of a crucial protein component, that may be different from the pump itself. Therefore ouabain resistance of Ma104 cells is not due to a lack of affinity for the drug, but to a failure of its Na⁺, K⁺-ATPases to detach from the plasma membrane in spite of being blocked by ouabain.We wish to thank Dr. E. Rodríguez-Boulán for the generous supply of Ma104 cells, as well as acknowledge the generous economic support of the National Research Council (CONACYT) of Mexico. Confocal experiments were performed in the Confocal Microscopy Unit of the Physiology Department, CINVESTAV.     

Characterization of [3H]Ouabain Binding Sites in Human Brain, Platelet, and Erythrocyte

[3H]Ouabain binding was investigated in membranes prepared from human brain, erythrocyte, and platelet. Scatchard analysis of [3H]ouabain binding to human hypothalamic membranes revealed a single class of noninteracting binding sites with an apparent affinity constant (KD) of 21 nM. Though the number of [3H]ouabain binding sites was lower in human platelets than in erythrocytes, both tissues exhibited a single class of high-affinity binding sites with an apparent KD similar to that found in human brain. Specific [3H]ouabain binding in basal ganglia tissue from patients with Huntington's disease was more than 50% lower than in tissue from age- and sex-matched controls. These results, along with previous findings in rat brain, suggest that high-affinity [3H]ouabain binding labels the neuronal form of Na, K-ATPase in human brain, and may prove useful in quantitating this enzyme in postmortem brain samples.     

Ouabain,endogenous ouabain and ouabain-like factors: The Na+ pump/ouabain receptor,its linkage to NCX,and its myriad functions

In this brief review we discuss some aspects of the Na⁺ pump and its roles in mediating the effects of ouabain and endogenous ouabain (EO): i) in regulating the cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT) via Na/Ca exchange (NCX), and ii) in activating a number of protein kinase (PK) signaling cascades that control a myriad of cell functions. Importantly, [Ca²⁺]_CYT and the other signaling pathways intersect at numerous points because of the influence of Ca²⁺ and calmodulin in modulating some steps in those other pathways. While both mechanisms operate in virtually all cells and tissues, this article focuses primarily on their functions in the cardiovascular system, the central nervous system (CNS) and the kidneys.     

Assignment of the gene governing cellular ouabain resistance to Mus musculus chromosome 3 using human/mouse microcell hybrids

Human/mouse microcell hybrids were used to establish the assignment of the gene governing resistance to the cardiac glycoside ouabain (Oua-1) to Mus musculus chromosome 3. Microcells were prepared from primary mouse embryo fibroblasts and fused with HeLa S3 cells, and microcell hybrids were isolated and maintained in medium containing 10^–6 m ouabain. Resistance to ouabain was not expressed concordantly with any of 26 murine isozyme markers. Karyotypic analysis of five primary clones showed that one to five murine chromosomes had been transferred from donor to recipient in these experiments. Only mouse chromosome 3 was common to all ouabain-resistant primary clones. Both ouabain-resistant and -sensitive subclones were isolated from hybrids grown in the absence of selective pressure, and karyotyping showed that loss of resistance to ouabain was concordant with the loss of murine chromosome 3.These studies were supported by Grant GM9966 from the National Institutes of Health.     

Novel metabolic aspects related to adenosine deaminase inhibition in a human astrocytoma cell line

Adenosine deaminase, which catalyzes the deamination of adenosine and deoxyadenosine, plays a central role in purine metabolism. Indeed, its deficiency is associated with severe immunodeficiency and abnormalities in the functioning of many organs, including nervous system. We have mimicked an adenosine deaminase-deficient situation by incubating a human astrocytoma cell line in the presence of deoxycoformycin, a strong adenosine deaminase inhibitor, and deoxyadenosine, which accumulates in vivo when the enzyme is deficient, and have monitored the effect of the combination on cell viability, mitochondrial functions, and other metabolic features. Astrocytomas are the most common neoplastic transformations occurring in glial cell types, often characterized by a poor prognosis. Our experimental approach may provide evidence both for the response to a treatment affecting purine metabolism of a tumor reported to be particularly resistant to chemotherapeutic approaches and for the understanding of the molecular basis of neurological manifestations related to errors in purine metabolism. Cells incubated in the presence of the combination, but not those incubated with deoxyadenosine or deoxycoformycin alone, underwent apoptotic death, which appears to proceed through a mitochondrial pathway, since release of cytochrome c has been observed. The inhibition of adenosine deaminase increases both mitochondrial reactive oxygen species level and mitochondrial mass. A surprising effect of the combination is the significant reduction in lactate production, suggestive of a reduced glycolytic capacity, not ascribable to alterations in NAD⁺/NADH ratio, nor to a consumption of inorganic phosphate. This is a hitherto unknown effect presenting early during the incubation with deoxyadenosine and deoxycoformycin, which precedes their effect on cell viability.     

The occurrence of polysialogangliosides in a human trophoblast cell line

Several lines of evidence establish that the crystallizable aspartyl—tRNA synthetase from Baker's yeast contains some covalently bound glucose: (i) a positive staining of the enzyme was obtained after polyacrylamide gel electrophoresis followed by the concanavalin A-peroxidase test which is specific for glucose and mannose containing proteins; (ii) thin-layer chromatography and gas-liquid chromatography revealed the presence of glucose in enzyme hydrolysates; (iii) immunoaffinoelectrophoresis in agarose gels containing concanavalin A and antibodies raised against aspartyl—tRNA synthetase showed that the enzyme was able to precipitate entirely in the lectin. Finally incubation of the enzyme with [¹⁴C]glucose or [¹⁴C]glucose 6-phosphate led to the incorporation of radioactivity into trichloroacetic acid-precipitable protein. Indeed immunoprecipitation of [¹⁴C]glucose-labelled aspartyl-tRNA synthetase with specific antibodies using the rocket method followed by autoradiography gave a radioactive peak. This last result also demonstrates the possibility of in vitro glycosylation of yeast aspartyl—tRNA synthetase.     

Chromosome-mediated transfer of murine alleles for hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and ouabain resistance into human cell lines

Genetic drug-resistance markers were transferred via purified metaphase chromosomes from mouse L cells into the human fibrosarcoma line HT1080 and HeLa S3 cells. Interspecific chromosome-mediated transfer of hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8) from mouse L cells into HGPRT^– HT1080 cells occurred at a frequency of approximately 1×10^–7. The presence of the mouse allele for HGPRT in transferent isolates was confirmed by isoelectric focusing. Transfer of ouabain resistance from mouse L cells to HT1080 and HeLa S3 cells occurred at an average frequency of approximately 4×10^–7. Expression of the mouse trait in transferent isolates was confirmed by their ability to withstand doses of ouabain which would be lethal to spontaneous ouabain-resistant mutants of the human cells but not to mouse L cells. Ouabain-resistant transferents of human cells showed 10⁴- to >10⁵-fold enhanced drug resistance, characteristic of either wild-type or mutant alleles, respectively, from ouabain-resistant donor L cells. Unstable expression of the transferred phenotypes in the absence of selection was seen in some isolates, but expression was lost at slow rates.This work was supported by National Institutes of Health Grant GM30383/21665 to RMB, Core Grants CA14051 to S. E. Luria and CA24538 to E. Mihich, and institutional predoctoral Training Grant GM07287.     

Isolation of functional human trophoblast cells and their partial characterization in primary cell culture

Summary Human trophoblast isolation and cell culture procedures were examined to identify variables that enhance secretion of chorionic gonadotropin (HCG) in primary culture. Brief exposure of unminced first-trimester placental specimens to a solution of trypsin-EDTA-DNAse, and isolation of the dispersed cells after Ficoll-hypaque centrifugation yielded primary cultures that were high in HCG secretion and content of epithelial-like cells. The gradual decline in HCG level with time in monolayer culture in these presumptive trophoblast cells was retarded by treatment with theophylline and cyclic adenosine monophosphate. Exposure to methotrexate (MTX) did not increase HCG secretion in normal trophoblast cells, in contrast to a 5-fold stimulation by MTX in the JAR line of choriocarcinoma cells. Clusters of polygonal cells in primary culture progressively lost their capacity to secrete HCG and their epithelial-like morphology. However, they could be maintained as cell strains through approximately 15 passages over a period of 13 to 16 weeks.     

Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals

Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the β-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC₅₀ for the single mutations of N122H (61.0 μM) or T797A (63.3 μM) was increased roughly 250-fold relative to the wild-type (0.24 μM), the double mutations of Q111V-N122H (IC₅₀ 550 μM) and Q111T-N122H (IC₅₀ 583 μM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely.     

人胚胎干细胞向滋养层分化的研究进展

哺乳动物胚胎发育产生的第一个细胞系的分离是内细胞团和滋养层的分离,不同哺乳动物之间胚胎干细胞向滋养层细胞分化不同,滋养层细胞对胚胎的植入、促进胚胎在子宫内的生存和生长至关重要.人胚胎干细胞为研究人类胚胎发育及向滋养层分化提供了一个独特的模型.人胚胎干细胞可以在实验室条件下保持无限期稳定的培养,用于最初胚胎和滋养外胚层发生的机制研究.目前人胚胎干细胞分化为滋养层细胞在体外可以通过自发分化、基因敲除、分离EB小体和BMP4诱导等几种途径实现.不同哺乳动物之间胚胎干细胞向滋养层分化机制,主要通过信号通路如BMP4,LIF等以及某些标志基因如OCT4,CDX2,Eomes等的变化调节.人胚胎干细胞向滋养层分化的研究为临床应用提供了一定的基础.     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.