K+-Na+ Exchange and Selectivity in Barley Root Cells: Effect of Na+ on the Na+ Fluxes

Using the compartmental analysis the unidirectional Na⁺ fluxesin cortical cells of barley roots, the cytoplasmic and vacuolarNa⁺ contents Q_c and Q_v, and the trans-root Na⁺ transport R'have been studied as a function of the external Na⁺ concentration.Using the re-elution technique the effect of low K⁺ concentrationson the plasmalemma efflux _co of Na⁺ (K+-Na+ exchange) and onR' was investigated at different Na+ concentrations and correspondinglydifferent values of the cytoplasmic sodium content Qc. The relationof the K+-dependent Na+ efflux coK+-dep to Qc or to the cytoplasmicNa+ concentration obeyed Michaelis-Menten kinetics. This isconsistent with a linkage of co, K+-dep to K+ influx by a K⁺-Na⁺exchange system. The apparent Km corresponded to a cytoplasmicNa⁺ concentration of 28 mM at 0·2 mM K⁺ and about 0·2mM Na⁺ in the external solution. 0·2 mM K⁺ stimulatedthe plasma-lemma efflux of Na⁺ and inhibited Na⁺ transport selectivelyeven in the presence of 10 mM Na⁺ in the external medium showingthe high efficiency of the K⁺-Na⁺ exchange system. However,_co, K⁺-dep was inhibited at 10 mM Na¹ compared to lower Na¹concentrations suggesting some competition of Na¹ with K¹ atthe external site of the exchange system. The effect of theNa+ concentration on Na¹ influx _oc is discussed with respectto kinetic models of uuptake.     

The effect of Na+ and K+ on the thermal denaturation of Na+ and + K+-dependent ATPase.

To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy.     

Na+ Sensitivity of ROMK1 K+ Channel: Role of the Na+/H+ Antiporter

To examine the extracellular Na⁺ sensitivity of a renal inwardly rectifying K⁺ channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K⁺ channel, ROMK1 (Kir1.1). When extracellular Na⁺ was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na⁺ from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na⁺ removal from the bath solution. A blocker of Na⁺/H⁺ antiporters, amiloride, largely inhibited the Na⁺ removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much less sensitive to Na⁺ removal. Na⁺ removal was found to induce a significant decrease in intracellular pH in the oocytes using H⁺-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na⁺/H⁺ antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na⁺ in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular Na⁺, and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na⁺ sensitivity of ROMK1 channel via regulating intracellular pH. Received: 13 April 1999/Revised: 15 July 1999     

K+Nutrition and Na+Toxicity: The Basis of Cellular K+/Na+Ratios

The capacity of plants to maintain a high cytosolic K⁺/Na⁺ratiois likely to be one of the key determinants of plant salt tolerance.Important progress has been made in recent years regarding theidentification and characterization of genes and transportersthat contribute to the cytosolic K⁺/Na⁺ratio. For K⁺uptake,K⁺efflux and K⁺translocation to the shoot, genes have been isolatedthat encode K⁺uptake and K⁺release ion channels and K⁺carriersthat are coupled to either a H⁺or Na⁺gradient. Although thepicture is less clear for the movement of Na⁺, one pathway,in the form of non-selective ion channels, is likely to playa role in Na⁺uptake, whereas Na⁺efflux and compartmentationare likely to be mediated by H⁺-coupled antiport. In addition,several proteins have been characterized that play prominentroles in the regulation of K⁺and/or Na⁺fluxes. In this BotanicalBriefing we will discuss the functions and interactions of thesegenes and transporters in the broader context of K⁺nutritionand Na⁺toxicity. Copyright 1999 Annals of Botany Company Salinty, K⁺/N⁺ratio, transporter, membrane.     

Na+, K+ and Cl- in Xylem Sap Flowing to Shoots of NaCl-Treated Barley

Munns, R. 1985. Na⁺, K⁺ and Cl^– in xylem sap flowing toshoots of NaCl-treated barley.—J. exp. Bot. 36: 1032–1042. Na⁺, Cl^– and K⁺ concentrations were measured in xylemsap obtained by applying pressure to the roots of decapitatedbarley plants grown at external [NaCl] of 0, 25, 50, 100, 150and 200 mol m^–3. For any given NaCl treatment, ion concentrationsin the xylem sap were hyperbolically related to the flux ofwater. Ion concentrations in sap collected at very low volumefluxes (without applied pressure) were 5–10 times higherthan in sap collected at moderate fluxes (under pressure). Fora given moderate volume flux, Na+ concentration in the xylemsap, [Na⁺]_x, was only 4.0 mol m^–3 at external [NaCl] of25–150 mol m^–3, and increased to 7.0 mol m^–3at 200 mol m^–3. [Cl-]x showed a similar pattern. Thisshows there would be little difference in the rate of uptaketo the shoot of plants at 25–150 mol m^–3 externalNaCl and indicates little change even at 200 mol m^-3 NaCl becausetranspiration rates would be much lower. Thus the reduced growthof the shoot of plants at high NaCl concentrations is not dueto higher uptake rates of Na⁺ or Cl^–. The fluxes of Na⁺, Cl^– and K^– increased non-linearlywith increasing volume flux indicating little movement of saltin the apoplast. The flux of K⁺ increased even when [K⁺]_x wasgreater than external [K⁺], indicating that membrane transportprocesses modify the K⁺ concentration in the transpiration streamas it flows through the root system. Key words: -Xylem sap, Na⁺, K⁺, Cl^– fluxes, salinity, barley     

The Role of Na+/K+-ATPase during Chick Skeletal Myogenesis

The formation of a vertebrate skeletal muscle fiber involves a series of sequential and interdependent events that occurs during embryogenesis. One of these events is myoblast fusion which has been widely studied, yet not completely understood. It was previously shown that during myoblast fusion there is an increase in the expression of Na⁺/K⁺-ATPase. This fact prompted us to search for a role of the enzyme during chick in vitro skeletal myogenesis. Chick myogenic cells were treated with the Na⁺/K⁺-ATPase inhibitor ouabain in four different concentrations (0.01-10 μM) and analyzed. Our results show that 0.01, 0.1 and 1 μM ouabain did not induce changes in cell viability, whereas 10 μM induced a 45% decrease. We also observed a reduction in the number and thickness of multinucleated myotubes and a decrease in the number of myoblasts after 10 μM ouabain treatment. We tested the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects during myogenesis, since both pathways have been associated with Na⁺/K⁺-ATPase. The MEK-ERK inhibitor U0126 alone did not alter cell viability and did not change ouabain effect. The p38 inhibitor SB202190 alone or together with 10 μM ouabain did not alter cell viability. Our results show that the 10 μM ouabain effects in myofiber formation do not involve the MEK-ERK or the p38 signaling pathways, and therefore are probably related to the pump activity function of the Na⁺/K⁺-ATPase.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

Abscisic Acid Decreases Leaf Na+ Exclusion in Salt-Treated Phaseolus vulgaris L.

Previous results showed that in short-term NaCl-treated beans increased leaf abscisic acid (ABA) concentration was triggered by Na⁺ but not by Cl^-. In this work, the specificity of ABA signaling for Na⁺ homeostasis was studied by comparing the plant’s responses to solutions that modified accumulation of ABA and/or Na⁺ uptake and distribution, such as supplemental Ca²⁺, increased nutrient strength, different isosmotic composition, application of exogenous ABA, fluridone (an ABA inhibitor) and aminooxiacetic acid (AOA, an ethylene inhibitor). After fluridone pretreatment, salt-treated beans had lower Na⁺ uptake and higher leaf Na⁺ exclusion capacity than non-pretreated plants. Moreover, Na⁺ uptake was increased and leaf Na⁺ exclusion was decreased by AOA and ABA. NaCl and KCl similarly increased leaf ABA and decreased transpiration rates, whereas supplemental Ca²⁺ and increased strength nutrient solution decreased leaf ABA and leaf Na⁺. These results show (1) a non-ion-specific increase in ABA that probably signaled the osmotic component of salt, and (2) increased ABA levels that resulted in higher leaf Na⁺ concentrations due to lower Na⁺ exclusion or increased root-shoot Na⁺ translocation.     

Inward-directed current generated by the Na+,K+ pump in Na+- and K+-free medium

In Na⁺- and K⁺-free solution, an inward-directed current can be detected in Xenopus oocytes, which is inhibited by cardic glycosides and activated by ATP. Therefore, it is assumed to be generated by the Na⁺, K⁺ pump. At negative membrane potentials, the pump current increases with more negative potentials and with increasing [H⁺] in the external medium. This current is not observed when Mg²⁺ instead of Ba²⁺ is the only divalent cation present in the bath medium, and it does not depend on whether Na⁺ or K⁺ is present internally. At 5 to 10 mM Na⁺ externally, maximum pump-generated current is obtained while no current can be detected in presence of physiological [Na⁺]. It is suggested that in low-Na⁺ and K⁺-free medium the Na⁺, K⁺ pump molecule can either form a conductive pathway that is permeable to Ba²⁺ or protons or operate in its conventional transport mode accepting Ba²⁺ as a K⁺ congener. A reversed pump mode or an electrogenic uncoupled Na⁺-efflux mode is excluded.     

K+-independent active transport of Na+ by the (Na+ and K+)-stimulated adenosine triphosphatase

The (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from canine kidney reconstituted into phospholipid vesicles showed an ATP-dependent, ouabain-inhibited uptake of 22Na+ in the absence of added K+. This transport occurred against a Na+ concentration gradient, was not affected by increasing the K+ concentration to 10 microM (four times the endogenous level), and could not be explained in terms of Na+in in equilibrium Na+out exchange. K+-independent transport occurred with a stoichiometry of 0.5 mol of Na+ per mol of ATP hydrolyzed as compared with 2.9 mol of Na+ per mol of ATP for K+-dependent transport.     

Interaction between Tubulin and Na2+,K2+-ATPase in Brain Stem Neurons

Functionally active Na²⁺,K²⁺-ATPase isozymes containing three types of the catalytic subunits (1, 2, and 3) were obtained from calf brain by two methods: selective removal of contaminating proteins according to Jorgensen (1974) and selective solubilization of the enzyme with subsequent reformation of the membrane structure according to Esmann (1988). All preparations were characterized with respect to ouabain-inhibition constants. The presence of the cytoskeleton protein tubulin (3 isoform) in the high-molecular-weight complex of Na²⁺,K²⁺-ATPase 31 isozyme from brain stem axolemma and the junction between Na²⁺,K²⁺-ATPase 3 subunit and tubulin 3 subunit are shown for the first time.     

Inhibitors of the Na+ K+-atpase

1. The major ionmotive ATPase, in animal cells, is the Na⁺, K⁺-ATPase or sodium pump.2. This membrane bound enzyme is responsible for the translocation of Na⁺ ions and K⁺ ions across the plasma membrane, an active transport mechanism that requires the expenditure of the metabolic energy stored within the ATP molecule.3. This ubiquitous enzyme controls directly or indirectly many essential cellular functions, such as, cell volume, free calcium concentration and membrane potential.4. It is, therefore, apparent that alterations in its regulation may play key roles in pathological processes.     

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Ouabain binding toNa⁺/K⁺-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca²⁺ concentration([Ca²⁺]_i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na⁺/K⁺-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of₁- and ₂-isoforms ofNa⁺/K⁺-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar ₁-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and ₂-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na⁺/K⁺-ATPase to ERK1/2 and[Ca²⁺]_i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and ₂-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.

     

周期性张应变对面颌部肌细胞Na+/K+-ATPaseα亚单位蛋白表达的影响

目的:研究周期性张应变对面颌肌细胞Na+/K+-ATPaseα亚单位蛋白表达的影响以确定其作用及机制.方法:在建立面颌肌细胞的力学刺激-细胞体外培养模型的基础上,采用Western blot法分析周期性张应变对Na+/K+-ATPase α1和α2亚单位蛋白表达的影响,加力组分别给予1、2、12、24和48h的力学刺激,施加力值为15％的细胞形变,频率为10cycles/min.以静态组为对照组.对照组及实验组各包含4个实验样本.Western blot检测Na+/K+-ATPaseα1和α2亚单位蛋白的表达.结果:α1亚单位的蛋白表达量除加力1h组与对照组之间、加力24 h与48 h之间无统计学差异外,其余各组之间以及各组与对照组之间均有显著的统计学意义.α2亚单位蛋白表达量除加力24 h与48 h组之间无统计学差异外,其余各组之间以及各组与对照组之间均有显著的统计学意义.结论:在一定时间范围内,周期性张应变可刺激α1和α2亚单位蛋白表达增加,随作用时间的延长蛋白表达受抑制.提示在肌能力的刺激下,面颌肌细胞的相关酶蛋白的功能及表达将发生适应性改建,但其功能亚基的调控机制可能不同.这为选择不同的方法和手段进行临床干预提供了理论依据,因而具有重要的参考意义.     

The effects of several ligands on the potassium-vanadate interaction in the inhibition of the (Na+ + K+)-ATPase and the Na+, K+ pump

Inhibition by vanadate of the K+-dependent p-nitrophenylphosphatase activity catalyzed by the (Na+ + K+)-ATPase partially purified from pig kidney showed competitive behavior with the substrate, K+ and Mg2+ acted as cofactors in promoting that inhibition. Ligands which inhibited the K+-dependent p-nitrophenyl phosphate hydrolysis (Na+, nucleotide polyphosphates, inorganic phosphate) protected against inhibition by vanadate. The magnitude of that protection was proportional to the inhibition produced in the absence of vanadate. In the presence of only p-nitrophenyl phosphate and Mg2+, or when the protective ligands were tested alone, the activation of p-nitrophenyl phosphate hydrolysis by K+ followed a sigmoid curve in the presence as well in the absence of vanadate. However, the combination of 100 mM NaCl and 3 mM ATP resulted in a biphasic effect of K+ on the p-nitrophenyl phosphate hydrolysis in the presence of vanadate. After an initial rise at low K+ concentration, the p-nitrophenylphosphatase activity declined at high K+ concentrations; this decline became more pronounced as the vanadate concentration was increased. This biphasic response was not seen when a nonphosphorylating ATP analog was combined with Na+ (which favors the nucleotide binding) or with inorganic phosphate (a requirement for K+ - K+ exchange). Experiments with inside-out resealed vesicles from human red cells showed that in the absence of Na+ plus ATP, K+ promoted vanadate inhibition of p-nitrophenylphosphatase activity in a nonbiphasic manner, acting at cytoplasmic sites. On the other hand, in the presence of Na+ plus ATP, the biphasic response of p-nitrophenyl phosphate hydrolysis is due to K+ acting on extracellular sites. In vanadate-poisoned intact red blood cells, the biphasic response of the ouabain-sensitive Rb+ influx as a function of the external Rb+ concentration failed to develop when there was no Na+ in the extracellular media. In addition, in the absence of extracellular Na+, external Rb+ did not influence the magnitude of inhibition. The present findings indicate that external K+ favors vanadate inhibition by displacing Na+ from unspecified extracellular membrane sites.     

The Physiological Relevance of Na+-Coupled K+-Transport

Plant roots utilize at least two distinct pathways with high and low affinities to accumulate K+. The system for high-affinity K+ uptake, which takes place against the electrochemical K+ gradient, requires direct energization. Energization of K+ uptake via Na+ coupling has been observed in algae and was recently proposed as a mechanism for K+ uptake in wheat (Triticum aestivum L.). To investigate whether Na+ coupling has general physiological relevance in energizing K+ transport, we screened a number of species, including Arabidopsis thaliana L. Heynh. ecotype Columbia, wheat, and barley (Hordeum vulgare L.), for the presence of Na+-coupled K+ uptake. Rb+-flux analysis and electrophysiological K+-transport assays were performed in the presence and absence of Na+ and provided evidence for a coupling between K+ and Na+ transport in several aquatic species. However, all investigated terrestrial species were able to sustain growth and K+ uptake in the absence of Na+. Furthermore, the addition of Na+ was either without effect or inhibited K+ absorption. The latter characteristic was independent of growth conditions with respect to Na+ status and pH. Our results suggest that in terrestrial species Na+-coupled K+ transport has no or limited physiological relevance, whereas in certain aquatic angiosperms and algae this type of secondary transport energization plays a significant role.