Overexpression of Na+/K+-ATPase Parallels the Increase in Sodium Transport and Potassium Recycling in an In Vitro Model of Proximal Tubule Cellular Ageing

Na⁺/K⁺-ATPase plays a key role in the transport of Na⁺ throughout the nephron, but ageing appears to be accompanied by changes in the regulation and localization of the pump. In the present study, we examined the effect of in vitro cell ageing on the transport of Na⁺ and K⁺ ions in opossum kidney (OK) cells in culture. Cells were aged by repeated passing, and Na⁺/K⁺-ATPase activity and K⁺ conductance were evaluated using electrophysiological methods. Na⁺K⁺-ATPase α₁– and β₁-subunit expression was quantified by Western blot techniques. Na⁺/H⁺ exchanger activity, changes in membrane potential, cell viability, hydrogen peroxide production and cellular proliferation were determined using fluorimetric assays. In vitro cell ageing is accompanied by an increase in transepithelial Na⁺ transport, which results from an increase in the number of Na⁺/K⁺-ATPase α₁- and β₁-subunits, in the membrane. Increases in Na⁺/K⁺-ATPase activity were accompanied by increases in K⁺ conductance as a result of functional coupling between Na⁺/K⁺-ATPase and basolateral K⁺ channels. Cell depolarization induced by both KCl and ouabain was more pronounced in aged cells. No changes in Na⁺/H⁺ exchanger activity were observed. H₂O₂ production was increased in aged cells, but exposure for 5 days to 1 and 10 μM of H₂O₂ had no effect on Na⁺/K⁺-ATPase expression. Ouabain (100 nM) increased α₁-subunit, but not β₁-subunit, Na⁺/K⁺-ATPase expression in aged cells only. These cells constitute an interesting model for the study of renal epithelial cell ageing.     

Effect of metabolic depletion on the furosemide-sensitive Na and K fluxes in human red cells

Summary We report in this paper the effect of metabolic depletion on several modes of furosemide-sensitive (FS) Na and K transport in human red blood cells. The reduction of ATP content below 100 mol/liter cells produced a marked decrease in the maximal activation (V _max) of the outward. FS transport of Na and K into choline medium in the presence of ouabain (0.1 mM) and 1 mM MgCl₂. TheK _0.5 for internal Na to activate the FS Na efflux was not altered by metabolic depletion. However, metabolic depletion markedly decreased the K _i for external K (K _o ) to inhibit the FS Na efflux into choline medium (from 25 to 11 mM). Repletion of ATP content by incubation of cells in a substraterich medium recovered control levels ofV _max of the FS Na and K fluxes and of K _i for external K to inhibit FS Na efflux. TheV _max of FS Na and K influxes was also markedly decreased when the ATP content dropped below 100 mol/liter cells. This was mainly due to a decrease in the inward-coupled transport of K and Na (Na _o -stimulated K influx and the K _o -stimulated Na influx). The FS K _i /K _o exchange pathway of the Na–K cotransport, estimated from the FS K influx from choline-20 mM K _o medium into cells containing 22 mmol Na/liter cells, was also reduced by starvation. Starvation did not inhibit the FS Na _i /Na _o exchange pathway, estimated as FS Na influx from a medium containing 130 mM NaCl into cells containing 22 mmol Na/liter cells. The unidirectional FS²²Na efflux and influx were also measured in control and starved cells containing 22 mmol Na/liter cells, incubated in a Na medium (130 mM) at varying external K (0 to 20 mM). In substrate-fed cells, incubated in the absence of external K, FS Na efflux was larger than Na influx. This FS net Na extrusion (400 to 500 mol/liter cells·hr) decreased when external K was increased, approaching zero around 15 mM K _o . In starved cells the net Na extrusion was markedly decreased and it approached zero at lower K _o than in substrate-fed cells. Our results indicate that the FS Na and K fluxes, and their major component, the gradient driven Na–K–Cl cotransport system, are dependent on the metabolic integrity of the cells.     

转铁蛋白在低钾刺激Madin Darby狗肾细胞钠-钾ATP酶中的作用

体外低钾培养肾细胞能刺激细胞膜钠-钾ATP酶。本研究利用Madin Darby狗肾细胞能在无血清培养液中健康生存48h这一特征,研究体外低钾刺激细胞膜钠-钾ATP酶所依赖的血清中的活性因子,观察了表皮生长因子(EGF)、胰岛素样生长因子(IGF1)、前列腺素1(PGE1)和转铁蛋白(tranderrin)在这一过程中的作用。结果表明,在无血清培养液中低钾并不能刺激细胞膜钠—钾ATP酶,而添加转铁蛋白可模拟血清的作用。转铁蛋白能剂量依赖性地增加ouabain结合位点,对细胞膜钠-钾ATP酶作用呈良好的时间效应关系。在低钾无血清培养液中,细胞膜钠-钾ATP酶α1亚基启动子活性增强,α1与β1亚基蛋白质表达的增加依赖于转铁蛋白的存在。进一步研究结果表明,低钾在转铁蛋白的无血清培养液环境中能增加细胞对铁的摄取(^59Fe),该作用可被铁螯合剂(deferoxamine,DFO;35 μmol/L)所阻断。DFO也可阻断转铁蛋白依赖性低钾刺激细胞膜钠-钾ATP酶数目的增多,α1亚基启动子活性增强,α1与β1亚基蛋白质表达增加。以上结果表明,低钾对细胞膜钠-钾ATP酶活性的刺激作用依赖于转铁蛋白所调节的铁的摄取。     

Effects of pH,potential, chloride and furosemide on passive Na+ and K+ effluxes from human red blood cells

Summary Ouabain-resistant effluxes from pretreated cells containing K⁺/Na⁺=1.5 into K⁺ and Na⁺ free media were measured.Furosemide-sensitive cation effluxes from cells with nearly normal membrane potential and pH were lower in NO ₃ ^– media than in Cl^– media; they were reduced when pH was lowered in Cl^– media. When the membrane potential was positive inside furosemide increased the effluxes of Na⁺ and K⁺ (7 experiments). With inside-positive membrane potential thefurosemideinsensitive effluxes were markedly increased, they decreased with decreasing pH at constant internal Cl^– and also when internal Cl^– was reduced at constant pH. The correlation between cation flux and the membrane potential was different for cells with high or low internal chloride concentrations. The data with chloride47mm showed a better fit with the single-barrier model than with the infinite number-of-barriers model. With low chloride no significant correlation between flux and membrane potential was found. The data are not compatible with pure independent diffusion of Na⁺ and K⁺ in the presence of ouabain and furosemide.     

Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations

This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K⁺) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca²⁺-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca²⁺ concentration, or increase of extracellular K⁺ to a depolarizing level that opens astrocytic L-channels for Ca²⁺ and thereby also increase intra-astrocytic Ca²⁺ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K⁺ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K⁺ concentrations.     

Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca²⁺ activation site was modified using N-bromoacetamide (NBA). 1mm NBA in the bath solution, at 2.5 m Ca²⁺ reduces the open probability,P _o , of the channel to <0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca²⁺-sensitive, requiring 25mm Ca²⁺ to raiseP _o to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca²⁺ (1mm) protects the channels from modification. Also presented is a second class of Ca²⁺-activated K⁺ channels which are normally present in MTAL cells which open infrequently at 10 m Ca²⁺ (P _o =0.01) but have aP _o of 0.08 at 1mm Ca²⁺. We can conclude (i) that NBA modifies the channel by shifting Ca²⁺-sensitivity to very high Ca²⁺, (ii) that NBA acts on a site involved in Ca²⁺ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.     

Ca2+ and cAMP activate K+ channels in the basolateral membrane of crypt cells isolated from rabbit distal colon

Summary Using patch-clamp techniques, we have studied Ca²⁺-activated K⁺ channels in the basolateral membrane of freshly isolated epithelial cells from rabbit distal colon. Epithelial cell clusters were obtained from distal colon by gentle mechanical disruption of isolated crypts. Gigaohm seals were obtained on the basolateral surface of the cell clusters. At the resting potential (approximately –45 mV), with NaCl Ringer's bathing the cell, the predominant channels had a conductance of 131±25 pS. Channel activity depended on voltage as depolarization of the membrane increased the open probability. In excised inside-out patches, channels were found to be selective for K⁺ over Na⁺. Channel activity correlated directly with bath Ca²⁺ concentration in the excised patches. Channel currents were blocked by 5mm TEA⁺ and 1mm Ba²⁺. In cell-attached patches, after addition of the Ca²⁺ ionophore A23187, which increases intracellular Ca²⁺, open probability was markedly increased. Channel activity was also regulated by cAMP as addition of 1mm dibutyryl-cAMP in the bath solution in cell-attached patches increased channel open probability over 20-fold. Channels that had been activated by cAMP were further activated by Ca²⁺. We conclude that the basolateral membrane of epithelial cells from descending colon contains a class of potassium channels, which are regulated by intracellular Ca²⁺ and cAMP.     

Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face

Summary In the first part of the paper, evidence has been presented that electrochromic styryl dyes, such as RH 421, incorporate into Na, K-ATPase membranes isolated from mammalian kidney and respond to changes of local electric field strength. In this second part of the paper, fluorescence studies with RH-421-labeled membranes are described, which were carried out to obtain information on the nature of charge-translocating reaction steps in the pumping cycle. Experiments with normal and chymotrypsin-modified membranes show that phosphorylation by ATP and occlusion of Na⁺ are electroneutral steps, and that release of Na⁺ from the occluded state to the extracellular side is associated with translocation of charge. Fluorescence signals observed in the presence of K⁺ indicate that binding and occlusion of K⁺ at the extracellular face of the pump is another major electrogenic reaction step. The finding that the fluorescence signals are insensitive to changes of ionic strength leads to the conclusion that the binding pocket accommodating Na⁺ or K⁺ is buried in the membrane dielectric. This corresponds to the notion that the binding sites are connected with the extracellular medium by a narrow access channel (ion well). This notion is further supported by experiments with lipophilic ions, such as tetraphenylphosphonium (TPP⁺) or tetraphenylborate (TPB^–), which are known to bind to lipid bilayers and to change the electrostatic potential inside the membrane. Addition of TPP⁺ leads to a decrease of binding affinity for Na⁺ and K⁺, which is thought to result from the TPP^–-induced change of electric field strength in the access channel.Deceased (September 13, 1990).     

The cytosolic Na+ : K+ ratio does not explain salinity-induced growth impairment in barley: a dual-tracer study using 42K+ and 24Na+

It has long been believed that maintenance of low Na+ : K+ ratios in the cytosol of plant cells is critical to the plant's ability to tolerate salinity stress. Direct measurements of such ratios, however, have been few. Here we apply the non-invasive technique of compartmental analysis, using the short-lived radiotracers 42K+ and 22Na+, in intact seedlings of barley (Hordeum vulgare L.), to evaluate unidirectional plasma membrane fluxes and cytosolic concentrations of K+ and Na+ in root tissues, under eight nutritional conditions varying in levels of salinity and K+ supply. We show that Na+ : K+ ratios in the cytosol of root cells adjust significantly across the conditions tested, and that these ratios are poor predictors of the plant's growth response to salinity. Our study further demonstrates that Na+ is subject to rapid and futile cycling at the plasma membrane at all levels of Na+ supply, independently of external K+, while K+ influx is reduced by Na+, from a similar baseline, and to a similar extent, at both low and high K+ supply. We compare our results to those of other groups, and conclude that the maintenance of the cytosolic Na+ : K+ ratio is not central to plant survival under NaCl stress. We offer alternative explanations for sodium sensitivity in relation to the primary acquisition mechanisms of Na+ and K+.     

Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis

To investigate the involvement of K⁺ efflux in apoptotic cell shrinkage, we monitored efflux of the K⁺ congener,⁸⁶ Rb⁺, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in ⁸⁶Rb⁺ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed ⁸⁶Rb⁺ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K⁺ channels could not inhibit CD95-mediated efflux of⁸⁶ Rb⁺, however, the uptake of⁸⁶ Rb⁺ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of⁸⁶ Rb⁺ in Jurkat cells was sensitive to ouabain (a specific Na⁺/K⁺-ATPase inhibitor), demonstrating Na⁺/K⁺-ATPase dependent K⁺ uptake. Ouabain induced significant⁸⁶ Rb⁺ efflux in untreated cells, as well as it seemed to compete with⁸⁶ Rb⁺ efflux induced by the anti-CD95 antibody, supporting a role for Na⁺/K⁺-ATPase in the CD95-mediated⁸⁶ Rb⁺ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na⁺+/K⁺-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.     

Expression and allocation of proteins of the exo-endocytotic machinery in U373 glioma cells: similarities to long-term cultured astrocytes

1. Cultured astrocytes cells release a variety of low and high molecular weight messenger substances and express proteins of the exocytotic pathway including synaptic SNARE proteins. For analyzing the molecular mechanisms of astrocytic messenger release, permanent cell lines with astrocytic properties would provide useful tools.2. We analyzed the potential of the human malignant astrocytoma-derived cell line U373 MG to express proteins involved in regulated exo- and endocytosis. An immunoblot analysis identified the astrocyte marker glial fibrillary acidic protein, microtubule-associated protein 2, the v-SNAREs VAMP I, VAMP II, and cellubrevin and the t-SN AREs syntaxin I, SNAP-23, and SNAP-25.3. The cells also express the secretory granule protein secretogranin II. Although secretogranin II immunofluorescence reveals larger fluorescence spots, the majority of the SNARE proteins is associated with smaller organelles. The immunofluorescence is distributed throughout the cytoplasm and accumulates at processes and the growing edges of cells.4. The organellar association of SNARE proteins was confirmed by heterologous expression of recombinant fusion proteins. Following subcellular fractionation organelles of lower buoyant density carried the majority of VAMP II. Secretogranin II was associated with organelles of high buoyant density containing a small contribution of VAMP II.5. The results suggest that U373 MG cells have in common a considerable number of properties with long-term cultured astrocytes rather than with cultured oligodendrocytes or neurons. They contain two types of organelles that can be physically separated and may be employed in the differential release of messengers.     

Voltage dependence of transient and steady-state Na/K pump currents in myocytes

Experiments are reviewed here in which Na/K pump current was determined as strophanthidin-sensitive current in guinea-pig ventricular myocytes, voltage-clamped and internally-dialyzed via wide-tipped pipettes. In the presence of 150 mM extracellular [Na], both outward and inward pump current, during forward and reverse Na/K exchange respectively, were strongly voltage dependent. But reduction of external [Na] to 1.5 mM severely attenuated the voltage sensitivity of outward Na/K pump current. Voltage jumps elicited large transient pump currents during forward or reverse Na/K exchange, or when pump activity was restricted to Na translocation steps, but not when pumps were presumably engaged in K/K exchange. These findings indicate that Na translocation, but not K translocation, involves net charge movement through the membrane field, and that both forward and reverse Na/K transport cycles are rate-limited not by that voltage-sensitive step but by a subsequent voltage-insensitive step.     

Iron-Induced Inhibition of Na+, K+-ATPase and Na+/Ca2+ Exchanger in Synaptosomes: Protection by the Pyridoindole Stobadine

The effect of oxidative stress, induced by Fe²⁺-EDTA system, on Na⁺,K⁺-ATPase, Na⁺/Ca²⁺ exchanger and membrane fluidity of synaptosomes was investigated. Synaptosomes isolated from gerbil whole forebrain were incubated in the presence of 200 M FeSO₄-EDTA per mg of protein at 37°C for 30 min. The oxidative insult reduced Na⁺,K⁺-ATPase activity by 50.7 ± 5.0 % and Na⁺/Ca²⁺ exchanger activity measured in potassium and choline media by 47.1 ± 7.2 % and 46.7 ± 8.6 %, respectively. Membrane fluidity was also significantly reduced as observed with the 1,6-diphenyl-1,3,5-hexatriene probe. Stobadine, a pyridoindole derivative, prevented the decrease in membrane fluidity and in Na⁺/Ca²⁺ exchanger activity. The Na⁺,K⁺-ATPase activity was only partially protected by this lipid antioxidant, indicating a more complex mechanism of inhibition of this protein. The results of the present study suggest that the Na⁺/Ca²⁺ exchanger and the Na⁺,K⁺-ATPase are involved in oxidation stress-mediated disturbances of intracellular ion homeostasis and may contribute to cell injury.     

Interleukin-2-dependent regulation of Na/K pump in human lymphocytes

The present study provides the first evidence that the abundance of catalytic alpha1-subunit of Na,K-ATPase increases in the course of T cell blast transformation. Immunodepressant cyclosporin A at anti-proliferative doses diminished the induction of alpha1 protein in activated lymphocytes. Furthermore, in competent T cells, IL-2 increases both the transport activity of Na/K pump and the content of Na,K-ATPase alpha1 protein in a time-dependent manner. A correlation was found between the long-term elevation in ouabain-sensitive Rb influxes and the increase in alpha1 protein content in late activated T cells. These results suggest that (1) the increased expression of Na,K-ATPase proteins underlie the cell cycle-dependent upregulation of ion pump during T cell transformation, and (2) IL-2 is involved in the regulated expression of Na,K-ATPase in human lymphocytes.     

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     

Fast charge translocations associated with partial reactions of the Na,K-pump: II. Microscopic analysis of transient currents

Summary Nonstationary pump currents which have been observed in K⁺-free Na⁺ media after activation of the Na,K-ATPase by an ATP-concentration jump (see the preceding paper) are analyzed on the basis of microscopic reaction models. It is shown that the behavior of the current signal at short times is governed by electrically silent reactions preceding phosphorylation of the protein; accordingly, the main information on charge-translocating processes is contained in the declining phase of the pump current. The experimental results support the Albers-Post reaction scheme of the Na,K-pump, in which the translocation of Na⁺ precedes translocation of K⁺. The transient pump current is represented as the sum of contributions of the individual transitions in the reaction cycle. Each term in the sum is the product of a net transition rate times a dielectric coefficient describing the amount of charge translocated in a given reaction step. Charge translocation may result from the motion of ion-binding sites in the course of conformational changes, as well as from movement of ions in access channels connecting the binding sites to the aqueous media. A likely interpretation of the observed nonstationary currents consists in the assumption that the principal electrogenic step is the E₁-P/P-E₂ conformational transition of the protein, followed by a release of Na⁺ to the extracellular side. This conclusion is supported by kinetic data from the literature, as well as on the finding that chymotrypsin treatment which is known to block the E₁-P/P-E₂ transition abolishes the current transient. By numerical simulation of the Albers-Post reaction cycle, the proposed mechanism of charge translocation has been shown to reproduce the experimentally observed time behavior of pump currents.