Translocation of Na(+),K(+)-ATPase is induced by Rho small GTPase in renal epithelial cells

The distribution of transmembrane proteins is considered to be crucial for their activities because these proteins mediate the information coming from outside of cells. A small GTPase Rho participates in many cellular functions through its downstream effectors. In this study, we examined the effects of RhoA on the distribution of Na(+),K(+)-ATPase, one of the transmembrane proteins. In polarized renal epithelium, Na(+),K(+)-ATPase is known to be localized at the basolateral membrane. By microinjection of the constitutively active mutant of RhoA (RhoA(Val14)) into cultured renal epithelial cells, Na(+),K(+)-ATPase was translocated to the spike-like protrusions over the apical surfaces. Microinjection of the constitutively active mutant of other Rho family GTPases, Rac1 or Cdcd42, did not induce the translocation. The translocation induced by RhoA(Val14) was inhibited by treatment with Y-27632, a Rho-kinase specific inhibitor, or by coinjection of the dominant negative mutant of Rho-kinase. These results indicate that Rho and Rho-kinase are involved in the regulation of the localization of Na(+),K(+)-ATPase. We also found that Na(+),K(+)-ATPase seemed to be colocalized with ERM proteins phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in cells microinjected with RhoA(Val14).     

Ouabain activates signaling pathways associated with cell death in human neuroblastoma

Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na⁺,K⁺-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 μM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca²⁺] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid-induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 μM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).     

A novel type of cell-cell cooperation between epithelial cells

Ma104 cells (renal, epithelial) have a peculiar way of resisting ouabain: their Na⁺,K⁺-pumps bind the drug with high affinity, cellular K⁺ is lost and cell division arrested, but cells do not detach as most cell types do. Then, if up to 4 days later the drug is removed, Ma104 cells recover K⁺ and resume proliferation (Contreras et al., 1994). In the present work, we investigate whether Ma104 cells are able to protect ouabain-sensitive MDCK cells in co-culture. The main finding is that they do, but in this case protection is not elicited by the usual mechanism of maintaining the K⁺ content of neighboring cells through cell-cell communications. Ma104 cells treated with ouabain simply remain attached to the substrate and to their MDCK neighbors, and both cells lose K⁺. This attachment includes tight junctions, because the transepithelial electrical resistance of the monolayers is not abolished by ouabain. Although the -subunit of the Na⁺,K⁺-ATPase is known to possess molecular characteristics of cell-cell attachment molecules, attachment between Ma104-MDCK cells does not seem to be mediated by this enzyme, as immunofluorescence analysis reveals that Na⁺,K⁺-ATPase is only inserted in the plasma membrane facing a neighboring cell of the same type.We wish to thank Dr. Enrique Rodríguez-Boulan (Cornell University Medical College) for the generous supply of Ma104 cells, as well as the generous economic support of COSBEL, SA de CV and the CONACYT of Mexico and the National Institutes of Health. Confocal experiments were performed in the Physiology Department's confocal microscopy unit, CINVESTAV.     

The digitalis-like steroid hormones: new mechanisms of action and biological significance

Digitalis-like compounds (DLC) are a family of steroid hormones synthesized in and released from the adrenal gland. DLC, the structure of which resembles that of plant cardiac glycosides, bind to and inhibit the activity of the ubiquitous cell surface enzyme Na(+), K(+)-ATPase. However, there is a large body of evidence suggesting that the regulation of ion transport by Na(+), K(+)-ATPase is not the only physiological role of DLC. The binding of DLC to Na(+), K(+)-ATPase induces the activation of various signal transduction cascades that activate changes in intracellular Ca(++) homeostasis, and in specific gene expression. These, in turn, stimulate endocytosis and affect cell growth and proliferation. At the systemic level, DLC were shown to be involved in the regulation of major physiological parameters including water and salt homeostasis, cardiac contractility and rhythm, systemic blood pressure and behavior. Furthermore, the DLC system has been implicated in several pathological conditions, including cardiac arrhythmias, hypertension, cancer and depressive disorders. This review evaluates the evidence for the different aspects of DLC action and delineates open questions in the field.     

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.     

Signaling pathways involving the sodium pump stimulate NO production in endothelial cells

The cardiac steroid ouabain, a known inhibitor of the sodium pump (Na⁺,K⁺-ATPase), has been shown to release endothelin from endothelial cells when used at concentrations below those that inhibit the pump. The present study addresses the question of which signaling pathways are activated by ouabain in endothelial cells. Our findings indicate that ouabain, applied at low concentrations to human umbilical cord endothelial cells (HUAECs), induces a reaction cascade that leads to translocation of endothelial nitric oxide synthase (eNOS) and to activation of phosphatidylinositol 3-kinase (PI3K). These events are followed by phosphorylation of Akt (also known as protein kinase B, or PKB) and activation of eNOS by phosphorylation. This signaling pathway, which results in increased nitric oxide (NO) production in HUAECs, is inhibited by the PI3K-specific inhibitor LY294002. Activation of the reaction cascade is not due to endothelin-1 (ET-1) binding to the ET-1 receptor B (ET_B), since application of the ET_B-specific antagonist BQ-788 did not have any effect on Akt or eNOS phosphorylation. The results shown here indicate that ouabain binding to the sodium pump results in the activation of the proliferation and survival pathways involving PI3K, Akt activation, stimulation of eNOS, and production of NO in HUAECs. Together with results from previous publications, the current investigation implies that the sodium pump is involved in vascular tone regulation.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

The Identification of the Sodium Pump

The identification of the sodium potassium pump as a Na⁺, K⁺-ATPase is described.     

Mechanisms of liquid flux across pulmonary alveolar epithelial cell monolayers

Summary Active transport of sodium by pulmonary alveolar epithelial cells (AEC) is believed to be an important component of edema clearance in the normal and injured lung. Data supporting this premise have come from measurements of sodium movement across AEC monolayers or from perfused lung model systems. However, direct measurement of fluid flux across AEC monolayers has not been reported. In the present work, AEC were studied with an experimental system for the measurement of fluid flux (Jv) across functionally intact cell monolayers. Primary adult rat type II alveolar epithelial cells were cultured on 0.8 μm nuleopore filters previously coated with gelatin and fibronectin. Intact monolayers were verified by high electrical resistance (> 1000 Θ) at 4–5 d of primary culture. At the same time interval, transmission electron microscopy revealed cells with type I cell-like morphology throughout the monolayer. These were characterized by both adherens and tight junctional attachments. Fluid flux across the monolayers was measured volumetrically over a period of 2 h in the presence of HEPES-buffered DMEM containing 3% fatty acid-free bovine serum albumin. Flux (Jv) was inhibited 39% by 1 × 10⁻⁴ M ouabain (P < 0.01) and 27% by 5 × 10⁻⁴ M amiloride (P < 0.05). These data support the concept that AEC Na⁺/K⁺-ATPase and Na⁺ transport systems are important determinants of AEC transepithelial fluid movement in vitro.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Digitalis-induced cell signaling by the sodium pump: On the relation of Src to Na/K-ATPase

In addition to performing its essential transport function, the sodium pump also activates multiple cell signaling pathways in response to digitalis drugs such as ouabain. Based mainly on cell-free studies with mixtures of purified Src kinase and Na⁺/K⁺-ATPase, a well-advocated hypothesis on how ouabain initiates the activation of signaling pathways is that there is a preexisting physiological complex of inactive Src bound to the α-subunit of Na⁺/K⁺-ATPase, and that ouabain binding to this subunit disrupts the bound Src and activates it. Because of the published disagreements of the results of such cell-free experiments of two other laboratories, our aim was to attempt the resolution of these discrepancies. We reexamined the effects of ouabain, vanadate, and oligomycin on mixtures of Src, Na⁺/K⁺-ATPase, Mg²⁺, and ATP as specified in prior studies; and assayed for Src-418 autophosphorylation as the measure of Src activation. In contrast to the findings of the proponents of the above hypothesis, our results showed similar effects of the three inhibitors of Na⁺/K⁺-ATPase; indicating that Src activation in such experiments is primarily due to the ATP-sparing effect of the ATPase inhibitor on the mixture of two enzymes competing for ATP. We conclude that there is no solid evidence for direct molecular interaction of Src with Na⁺/K⁺-ATPase under physiological conditions.     

Intracerebroventricular Administration of Ouabain to Rats Changes the Expression of NMDA Receptor Subunits in Cerebral Cortex and Hippocampus

Ouabain exerts neurotoxic action and activates the population of NMDA receptors. Herein the effect of ouabain on the expression of NMDA subunits was evaluated. Adult Wistar rats were administered intracerebroventricularly with 0.1, 10 and 100 nmol ouabain or saline solution (control). Two days later, membranes of cerebral cortex and hippocampus were isolated. Western blots with antibodies for the NMDA receptor subunits: NR1; NR2A; NR2B; NR2C and NR2D were carried out. In cerebral cortex, NR2D subunit increased 30% with 10 nmol ouabain dose. With 100 nmol ouabain, NR1 and NR2D subunits enhanced 40 and 20%, respectively. In hippocampus, with the dose of 0.1 nmol ouabain, NR1 subunit enhanced roughly 50% whereas NR2B subunit decreased 30%. After administration of 10 nmol ouabain dose, NR2A, NR2B and NR2C subunits decreased 40, 50 and 30%, respectively. With the dose of 100 nmol of ouabain, NR1, NR2A and NR2B subunits diminished 10–20%. It is concluded that ouabain administration led to a differential regulation in the expression of NMDA subunits. These results may be correlated with the modulatory action of ouabain on NMDA receptor.     

Immunocytochemical characterization of two types of microvillar cells in rodent olfactory epithelium

Microvillar cells (MCs) have been identified in the olfactory epithelium of various mammalian species from rodents to humans. Studies on properties and functions of MCs to date have yielded partially controversial results, supporting alternatively an epithelial or a neuronal nature of these cells. In the present study, single and double immunolabeling investigations were carried out using antibodies against cytoskeletal and integral membrane proteins in order to further characterize MCs in rat and mouse olfactory epithelium. Application of antibodies against ankyrin (ANK), a protein that links integral membrane proteins to the submembrane cytoskeleton, led to intense labeling of the basolateral membranes of numerous cells with characteristic MC morphology. ANK-immunoreactive (ir) cells bore an apical tuft of -actin-ir microvilli, were filled with cytokeratin 18 (CK18)-ir filamentous network, and extended a basal process that appeared to end above the basal membrane. Immunoreactions for villin, an actin-crosslinking protein particularly prominently expressed in brush cells in the gastrointestinal and respiratory tract epithelia, and for the -subunit of sodium-potassium ATPase (Na⁺, K⁺-ATPase), revealed that ANK-ir MCs fall into two subpopulations. The less frequent type I MCs displayed villin immunoreactivity in their apical microvilli and underneath the basolateral membranes; the more numerous type II MCs were negative for villin but possessed intense basolateral immunoreactivity for Na⁺, K⁺-ATPase. Strong reactivity for the epithelial-type integral membrane protein of adherens junctions, E-Cadherin, was localized in basolateral membranes of both types of MCs. Our results support an epithelial nature of ANK-ir MCs in rat and mouse olfactory epithelium. Type I MCs strongly resemble brush cells in their immunocytochemical characteristics, namely, their ANK reactivity, CK18 reactivity, and villin reactivity. The intense Na⁺, K⁺-ATPase reactivity of type II MCs implicates these cells in transport processes.     

Effect of taurine on the isolated retinal pigment epithelium of the frog: Electrophysiologic evidence for stimulation of an apical,electrogenic Na+−K+ pump

Summary The apical surface of the retinal pigment epithelium (RPE) faces the neural retina whereas its basal surface faces the choroid. Taurine, which is necessary for normal vision, is released from the retina following light exposure and is actively transported from retina to choroid by the RPE. In these experiments, we have studied the effects of taurine on the electrical properties of the isolated RPE of the bullfrog, with a particular focus on the effects of taurine on the apical Na⁺–K⁺ pump.Acute exposure of the apical, but not basal, membrane of the RPE to taurine decreased the normally apical positive transepithelial potential (TEP). This TEP decrease was generated by a depolarization of the RPE apical membrane and did not occur when the apical bath contained sodium-free medium. With continued taurine exposure, the initial TEP decrease was sometimes followed by a recovery of the TEP toward baseline. This recovery was abolished by strophanthidin or ouabain, indicating involvement of the apical Na⁺–K⁺ pump.To further explore the effects of taurine on the Na⁺–K⁺ pump, barium was used to block apical K⁺ conductance and unmask a stimulation of the pump that is produced by increasing apical [K⁺] ₀ . Under these conditions, increasing [K⁺] ₀ hyperpolarized the apical membrane and increased TEP. Taurine reversibly doubled these responses, but did not change total epithelial resistance or the ratio of apical-to-basal membrane resistance, and ouabain abolished these responses.Collectively, these findings indicate the presence of an electrogenic Na⁺/taurine cotransport mechanism in the apical membrane of the bullfrog RPE. They also provide direct evidence that taurine produces a sodium-dependent increase in electrogenic pumping by the apical Na⁺–K⁺ pump.     

The inhibitory activity of a brain extract on synaptosomal Na+,K+-ATPase is sensitive to carboxypeptidase A and to chelating agents

In the present study some properties of an inhibitory extract of synaptosomal membrane Na⁺,K⁺-ATPase were investigated. This extract (peak II) was prepared by gel filtration in Sephadex G-50 of a soluble fraction of the rat cerebral cortex. Ultrafiltration of peak II through Amicon membranes indicated that the inhibitor has a low MW (<1000). The inhibitory activity was not modified by heating in neutral pH at 95°C for 20 min but it was destroyed by charring in acid pH at 200°C for 120 min. The inhibitory activity decreased by incubation of peak II with carboxypeptidase A. These findings suggest that the factor responsible for the inhibition of Na⁺,K⁺-ATPase activity is probably a polypeptide. On the other hand, the inhibition was reverted by the chelators EDTA and EGTA, indicating the participation of an ionic compound as well. The increase of Mg²⁺ concentration during the enzyme assay did not increase the inhibition, indicating that the ion involved might not be vanadate. It is suggested that both a polypeptide and an ionic compound coparticipate in the inhibitory effect of peak II on Na⁺,K⁺-ATPase activity.     

Vanadate inhibition of rat cerebral cortex Na+, K+-ATPase during postnatal development

The Na⁺, K⁺-ATPase activity and its response to vanadate inhibition was investigated in cerebral cortex homogenates of 7-, 12- and 18-day-old rats. The enzyme was inhibited by vanadate in a dose-dependent manner in all these age groups. Furthermore, there was a different sensitivity towards vanadate during postnatal development; the concentration of V⁺⁵ needed for 50% inhibiton of Na⁺, K⁺-ATPase was 1.1×10^–6M, 2×10^–7M and 4.4×10^–7M for 7-, 12- and 18-day-old rats, respectively. It is suggested that the different sensitivity of Na⁺, K⁺-ATPase towards vanadate inhibition during postnatal development might be due to age-dependent changes in the ratio of various cell types.Special Issue dedicated to Dr. O. H. Lowry.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

Endobain E, a brain Na+, K+ -ATPase inhibitor, decreases norepinephrine uptake in rat hypothalamus

The ability of an endogenous brain Na+, K+ -ATPase inhibitor, termed endobain E, to increase [3H]norepinephrine release in rat hypothalamus was previously reported. Endobain E effect on neurotransmitter uptake was studied by assaying [3H]norepinephrine uptake in rat hypothalamus preparations, to observe uptake inhibition, which reached 60% with endobain E equivalent to 100 mg fresh cerebral cortex, an effect achieved with 40 or 400 microM ouabain. Results support the proposal that endobain E behaves as an ouabain-like substance. Taken jointly results obtained on neurotransmitter release and uptake, the suggestion that endobain E may enhance norepinephrine availability in the synaptic gap and thus lead to an increase in noradrenergic activity is advanced.     

Formation Mechanism of the Free Radical Product and Its Precursor by the Reaction of Dehydro-L-ascorbic Acid with Amino Acid

During the formation of radical A (2) and its precursor (tris(2-deoxy-2-L-ascorbyl)amine, 1) by the reaction of dehydroascorbic acid (DHA) with amino acid, ascorbic acid (AsA) and the reduced red pigment (3) were newly identified, in addition to scorbamic acid (SCA) and the red pigment (4), as intermediate products. The addition of AsA to the DHA-amino acid reaction, as well as to the DHA-SCA reaction, greatly increased the formation of 3 and 1. The reaction of AsA with 4 gave rapidly 3, followed by the gradual production of 1. From these results, a reaction pathway is proposed that 3 formed by the reduction of 4 with AsA is a key intermediate and its condensation with DHA followed by reduction with AsA might produce 2 and 1.