Extrusion of Sodium Ions by Barley Roots I. Characteristics of the Extrusion Mechanism

Evidence is presented for an outwardly directed sodium ion pumpin excised barley roots. The efflux has a Q₁₀ of about 2 andis inhibited by ouabain at concentrations of the inhibitor downto 10^–5 M. A threefold stimulation of the sodium losswas observed both with ATP and inorganic phosphate at concentrationsof 10^–3 M. A stimulatory effect of different concentrationsof DNP on the extrusion of sodium has been observed and is attributedto permeability changes resulting from the use of this inhibitor.It is suggested that the sodium extrusion mechanism reportedhere for excised barley roots is similar to the sodium pumpsfound in animal tissues and certain algal cells.     

Sodium Recirculation and Loss from Phaseolus vulgaris L.

In a split-root experiment, ²²Na was supplied to Phaseolus vulgarisL. roots emerging from the stem, 2.5 cm above the main roots.Sodium exported from these upper roots was translocated a shortdistance upward in the stem and downward to the main roots.Most of the ²²Na arriving in the main roots was lost to themedium. Sodium loss from P. vulgaris roots into KCI or NaCl was similarand was not affected by oligomycin. The results confirm a previous hypothesis regarding the mechanismof sodium exclusion from the tops of sodium non-accumulatorplants. Phaseolus vulgaris L., bean, sodium transport     

Role of synaptosomal Na-accumulation in transmitter release

The mechanism whereby Na⁺, K⁺-ATPase inhibitors such as ouabain trigger transmitter release in a calcium-independent manner remains obscure. We have examined the possible role of intra-synaptosomal sodium ion accumulation in ouabain-induced acetylcholine (ACh) release by: 1) Measuring²²Na accumulation in cat cortical synaptosomes in the presence of ouabain, A23187, veratridine, or strophanthidin over the same time course in which we previously determined their effects on ACh release; and 2) measuring synaptosomal²²Na accumulation and ACh-release in the presence of ouabain plus tetrodotoxin in normal or calcium-free buffer. Our results indicate that tetrodotoxin-dependent²²Na accumulation is at least partially responsible for ouabain-induced ACh release in normal and calcium-free media, but that this ion-accumulation per se is not sufficient to elicit release with other secretogogues.Dedicated to Henry McIlwain.     

Involvement of Reactive Oxygen Species in a Feed-forward Mechanism of Na/K-ATPase-mediated Signaling Transduction

Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-l-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial ²²Na⁺ transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, ouabain stimulated direct carbonylation of two amino acid residues in the actuator domain of the Na/K-ATPase α1 subunit. Taken together, the data indicate that carbonylation modification of the Na/K-ATPase α1 subunit is involved in a feed-forward mechanism of regulation of ouabain-mediated renal proximal tubule Na/K-ATPase signal transduction and subsequent sodium transport.     

The Effect of Increased Internal Ion Concentration upon the Ion Uptake Isotherms of Excised Maize Root Segments

The influence of salt status of root tissue of Zea mays on influxof ⁸⁴Rb and ²²Na and net accumulation of K⁺ and Na⁺ was studied.Low-salt roots were grown in 0.5 mM CaCl₂, and high-salt rootsin 2.5 mM KC1 + 7.5 mM NaCl + 0.5 mM CaCl₂. High-salt statusgreatly reduced (approx. 90 per cent inhibition) both ²²Na and⁸⁶Rb influxes in the low concentration range isotherm (i.e.at external concentrations below 1 mM). A less marked inhibitionwas observed in the higher concentration range isotherm (1–30mM), indicating that the uptake in this range is less affectedby the salt status of the tissue. During transition from low- to high-salt status there was anet accumulation of K⁺ but not of Na⁺ despite the presence ofa measurable ²²Na⁺ influx at all times. The presence of a continuous²²Na influx but no net accumulation implies an Na⁺ efflux frommaize root tissue. The results differ significantly from thosepreviously published for barley and a possible explanation ofthese differences is discussed.     

The Extrusion of Sodium Ions from Presynaptic Nerve Endings of Rat Cerebral Cortex

(Na⁺-K⁺) ATPase is present in synaptosomal preparations and it is assumed to represent the sodium-potassium pump. 10 μm -noradrenaline activates (Na⁺-K⁺) ATPase approximately 100%, but 50 μm -noradrenaline does not stimulate the rate of ²²Na extrusion from synaptosomes. The results suggest that it is unlikely that the noradrenaline stimulation of (Na⁺-K⁺) ATPase is part of a feedback mechanism whereby released noradrenaline can influence the activity of the presynaptic sodium pump.     

Sodium currents in muscles incubated in potassium-free Ringer's solution. Effect of ouabain on unidirectional sodium currents

Frog sartorius muscles subjected to loading with Na in K-free Ringer solution in the cold were subsequently labelled with 22Na. The uptake of 22Na is not sensitive to ouabain (10(-4) M) while sodium efflux is decreased by oubain. It is concluded that ouabain-sensitive Na-for Na interchange is not present in this condition. Possibly ouabain-sensitive sodium efflux is partly or completely potassium-requiring fraction since some K (approximately 10 microM) is inevitably present in K-free solution. The increase in the rate constant for potassium loss in the presence of ouabain favours this supposition.     

An Analysis of the Leakages of Sodium Ions into and Potassium Ions out of Striated Muscle Cells

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na]_i, and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na]_i and a component that increased linearly with increasing [Na]_i. Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na]_o, and a steady-state balance of the sodium ion fluxes occurred at [Na]_o = 40 mM. When solutions were K-free and contained 10^-4 M ouabain, net sodium influx varied linearly with [Na]_o and a steady state for the intracellular sodium was observed at [Na]_o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na]_i but dependent on [Na]_o. Replacing external NaCl with MgCl₂ brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na]_o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

Sodium movements in the human red blood cell

Measurements were made of the sodium outflux rate constant, ^o k _Na, and sodium influx rate constant, ⁱ k _Na, at varying concentrations of extracellular (Na_o) and intracellular (Na_c) sodium. ^o k _Na increases with increasing [Na_o] in the presence of extracellular potassium (K_o) and in solutions containing ouabain. In K-free solutions which do not contain ouabain, ^o k _Na falls as [Na_o] rises from 0 to 6 mM; above 6 mM, ^o k _Na increases with increasing [Na_o]. Part of the Na outflux which occurs in solutions free of Na and K disappears when the cells are starved or when the measurements are made in solutions containing ouabain. As [Na_o] increases from 0 to 6 mM, ⁱ k _Na decreases, suggesting that sites involved in the sodium influx are becoming saturated. As [Na_c] increases, ^o k _Na at first increases and then decreases; this relation between ^o k _Na and [Na_c] is found when the measurements are made in high Na, high K solutions; high Na, K-free solutions; and in (Na + K)-free solutions. The relation may be the consequence of the requirement that more than one Na ion must react with the transport mechanism at the inner surface of the membrane before transport occurs. Further evidence has been obtained that the ouabain-inhibited Na outflux and Na influx in K-free solutions represent an exchange of Na_c for Na_o via the Na-K pump mechanism.     

Effect on Membrane Potential and Electrical Activity of Adding Sodium to Sodium-Depleted Cardiac Purkinje Fibers

Canine cardiac Purkinje fibers exposed to Na-free solutions containing 128 mM TEA and 16 mM Ca show resting potentials in the range -50 to -90 mV; if the concentration of Na in the perfusate is raised from 0 to 4 to 24 mM, hyperpolarization follows. If the initial resting potential is low, the hyperpolarization tends to be greater; the average increase in the presence of 8 mM Na is 14 mV. Such hyperpolarization is not induced by adding Na to K-free solutions, is not seen in cooled fibers, or in fibers exposed to 10^-3 M ouabain, nor is it induced by adding Li and thus may result from electrogenic sodium extrusion. Fibers exposed to Na-free solutions are often spontaneously active; if they are quiescent they often show repetitive activity during depolarizing pulses. Such spontaneous or repetitive activity is suppressed by the addition of Na. This suppression may or may not be related to the hyperpolarization.     

Effect of K+, its Counter Anion, and pH on Sodium Efflux from Barley Root Tips

Sodium efflux from ²²Na⁺-loaded root tips root tips of Hordeumvulgare L. was markedly increased by replacing 10mM Na₂SO₄ inthe washing solution by K₂SO₄ with the same electrical conductivity.This increase was inhibited by both an uncoupler and an inhibitorof oxidative phosphorylation but not by ouabain. Potassium ionsdid not enhance Na⁺ efflux in the presence of a rapidly absorbedcounter anion, such as Cl^–, instead of . Efflux of ²²Na⁺ could also be enhanced by a low pH in theabsence of K⁺; this was prevented by uncouplers, but not byan inhibitor of the mitochondrial ATPase. It seems that K⁺ indirectly enhances Na⁺ efflux. It is suggestedthat metabolic K⁺ uptake in excess of the counter anion resultsin a proton gradient across the plasmalemma (acid outside) inducingH⁺/Na⁺ antiport.     

Determination of Unidirectional Sodium Fluxes in Roots of Intact Sunflower Seedlings

The method of compartmental analysis was applied to study sodiumfluxes in roots of intact seedlings of Helianthus annuus L.By measuring sodium uptake and transport to the shoots of theseedlings in parallel experiments, transport of tracer sodiumto shoots and net accumulation of Na⁺ in the roots during theflux measurements was accounted for. The steady-state sodiumfluxes in the intact sunflower roots were similar in size tothose in excised roots but in general they were somewhat higher.This indicates more metabolic activity in the intact tissues.Using whole plants it is possible to study the response of ionfluxes in roots to ecophysiological stimuli received by theshoots, and in the present experiments the effect of continuouslight versus long-day growth conditions was investigated. Potassium,when continually present, depressed all fluxes and the cytoplasmiccontent of sodium but tended to increase the vacuolar sodiumcontent, in particular when this was related to the cytoplasmiccontent. When added to sodium-loaded roots, potassium stimulatedthe plasmalemma sodium efflux but slightly, suggesting a lowefficiency of K⁺-Na⁺ exchange across the plasmalemma in intactas well as excised sunflower roots. Subsequently, however, potassiuminduced a transient decrease in the ²²Na efflux that was followedby oscillations in tracer efflux. These changes were attributedto potassium-induced transfer of sodium to vacuoles. Moreover,the oscillations seem to indicate the operation of negativefeedback control of sodium fluxes.     

Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway

Endogenous cardiotonic glycosides bind to the inhibitory binding site of the plasma membrane sodium pump (Na⁺/K⁺-ATPase). Plasma levels of endogenous cardiotonic glycosides increase in several disease states, such as essential hypertension and uremia. Low concentrations of ouabain, which do not inhibit Na⁺/K⁺-ATPase, induce cell proliferation. The mechanisms of ouabain-mediated response remain unclear. Recently, we demonstrated that in opossum kidney (OK) proximal tubular cells, low concentrations of ouabain induce cell proliferation through phosphorylation of protein kinase B (Akt) in a calcium-dependent manner. In the present study, we identified ERK as an upstream kinase regulating Akt activation in ouabain-stimulated cells. Furthermore, we provide evidence that low concentrations of ouabain stimulate Na⁺/K⁺-ATPase-mediated ⁸⁶Rb uptake in an Akt-, ERK-, and Src kinase-dependent manner. Ouabain-mediated ERK phosphorylation was inhibited by blockade of intracellular calcium release, calcium entry, tyrosine kinases, and phospholipase C. Pharmacological inhibition of phosphoinositide-3 kinase and Akt failed to inhibit ouabain-stimulated ERK phosphorylation. Ouabain-mediated Akt phosphorylation was inhibited by U0126, a MEK/ERK inhibitor, suggesting that ouabain-mediated Akt phosphorylation is dependent on ERK. In an in vitro kinase assay, active recombinant ERK phosphorylated recombinant Akt on Ser⁴⁷³. Moreover, transient transfection with constitutively active MEK1, an upstream regulator of ERK, increased Akt phosphorylation and activation, whereas overexpression of constitutively active Akt failed to stimulate ERK phosphorylation. Ouabain at low concentrations also promoted cell proliferation in an ERK-dependent manner. These findings suggest that ouabain-stimulated ERK phosphorylation is required for Akt phosphorylation on Ser⁴⁷³, cell proliferation, and stimulation of Na⁺/K⁺-ATPase-mediated ⁸⁶Rb uptake in OK cells. opossum kidney cells; sodium/potassium adenosine triphosphatase; extracellular signal-regulated kinase; cell proliferation     

Amiloride-dependent transport is the main mechanism implicated in sodium iinflux regulation in rat mast cells

Mast cell sodium regulation is a largely unknown field. In our effort to study the mechanisms by which mast cells regulate sodium levels, we have examined the effect of amiloride and ouabain on ²²Na entry in rat mast cells in isotonic and hypertonic conditions. Ouabain (0.5 mM) enhances sodium uptake by 32% in isotonic conditions. Hypertonicity increases by 400% the uptake of sodium through an amiloride (1 mM) dependent mechanism. Ouabain has no appreciable effect on the entry of ²²Na in hypertonic conditions. © 1993 Wiley-Liss, Inc.     

Sodium flux in the smooth muscle of frog stomach

Sodium efflux from rings of frog stomach muscle was measured at 5° and 15°C in three different steady states. After incubation in normal, K-free, or ouabain (10^-4 M) solutions, intracellular cations stabilized at markedly differing levels. At 5°C, inhibition of Na extrusion was shown in the rate coefficients for ²²Na efflux, which were slightly smaller in K-free than in normal solutions, and much smaller in ouabain. Due to the intracellular Na concentration differences, total Na efflux was similar in K-free and ouabain solutions, and only ⅕ as large in normal solution. At 15°C, normal total Na flux was only 1/7;–1/10 inhibitors, and may be underestimated. The total flux differences may involve dependence of the Na pump and Na permeation on internal Na concentration. The Q ₁₀ of the steady-state fluxes was 3.7 in ouabain, 2.8 in K-free solution, and 1.9 in normal solution. The high temperature dependence of influx as well as efflux suggests transport mechanisms other than simple diffusion. Sodium turnover in the cell water was 46–66 mM/hr in inhibitors at 15°C, and a high rate of Na extrusion in normal muscle is suggested. However, cell volume:surface ratio is only 1.6 µ and all estimates of Na flux were under 3 pmoles/cm² per sec, indicating low Na permeability.     

Sodium Transport in Turtle Erythrocytes : Apparent stimulation of exchange diffusion by anaerobiosis

Studies were performed on Na and K transport by red blood cells of the freshwater turtle under anaerobic and aerobic conditions. Although it had previously been assumed that cation transport in turtle red blood cells was dependent on respiration, the present data show greater Na efflux rates in N₂ than in O₂. However, ouabain inhibited Na transport by the same amount quantitatively in O₂ and N₂ gas phases. Thus there was no difference in ouabain-sensitive or "pump" Na transport rates. Na influx rates were higher in nitrogen than in air and potassium influx rates were not significantly different under aerobic and anaerobic conditions. Moreover in the absence of sodium in the bathing medium no difference between air and nitrogen could be discovered. Finally with ethacrynic acid plus ouabain there was an additional decrease in Na efflux but there was a persisting difference between air and nitrogen. These studies do not rule out the existence of a ouabain-insensitive ethacrynic acid-inhibitable flux; however, they suggest that at least part of the activation of Na efflux observed in N₂ was due to increased exchange diffusion.     

Sodium Exclusion from the Shoots by Roots of Zea mays (cv. LG 11) and its Breakdown with Oxygen Deficiency

The effects of sodium chloride salinity and root oxygen deficiency(anoxia) were studied in 11-12d old maize plants (Zea mays L.cv. LG 11) in nutrient solution culture. Transport of ²²Na bythe roots to the shoot in 24 h was markedly increased by anoxiawhen the external concentration of NaCl was in the range 0·1-10·9mol m^–3. Anoxia severely inhibited uptake of ⁴²K by rootsand its transport to the shoot, so that the ratio of Na⁺/K⁺moving into the shoot was increased by a factor of approximately10. When the external concentration of NaCl was increased to2.4 mol m^–3, the roots showed much less ability to excludeNa⁺ under aerobic conditions, and anoxia caused no further increasein the movement of Na⁺ to the shoot. It is concluded that atthe higher concentration the ability of the roots to excludeNa⁺, presumably through an active mechanism in the xylem parenchymacells or in the root cortex and transporting Na⁺ to the outersolution, is saturated by excessive inward diffusion of Na⁺.The ratio of Na⁺/K⁺ transported to the shoot increased by afactor of 600 when the concentration of NaCl was increased from2·4 mol m^–3 to 40 mol m^–3 and roots weremade anoxic. Such imbalances in the supply of cations to theshoot, particularly when roots are oxygen-deficient, may contributeto salinity damage. Key words: Anaerobic, Anoxic, Oxygen deficiency, Roots, Salinity, Salt stress, Sodium chloride, Zea mays     

Vacuolar Na/K Exchange, its Occurrence in Root Cells of Hordeum, Atriplex and Zea and its Significance for K/Na Discrimination in Roots

Using excised low-salt roots of barley and Atriplex hortenslsthe transport of endogenous potassium through the xylem vesselswas studied It was enhanced by nitrate and additionally by sodiumions which apparently replaced vacuolar potassium which wasthen available in the symplasm of root cells for transport tothe shoot Vacuolar Na/K exchange also has been investigatedby measurements of longitudinal ion profiles in single rootsof both species. In Atriplex roots a change in the externalsolution from K⁺ to Na⁺ induced an exchange of vacuolar K⁺ forNa⁺, in particular in the subapical root tissues and led toincreased K⁺ transport and loss of K⁺ from the cortex. In inverseexperiments a change from Na⁺ to K⁺ did not induce an exchangeof vacuolar Na⁺; merely in meristematic tissues Na⁺—apparentlyfrom the cytoplasm—was extruded in exchange for K⁺. Inroots of barley seedlings without caryopsis, as in excised roots,a massive exchange of K⁺ for Na⁺ was observed in the continuouspresence of external 1.0 mM Na and 0.2 mM K. This exchange alsowas attributed to the vacuole and was most pronounced in theyoung subapical tissues. It did not occur, however, in the correspondingtissues in roots of fully intact barley seedlings. In these,the young tissues retained a relatively high K/Na ratio alsoin their vacuoles. Similarly, contrasting results were obtainedwith intact and excised roots of Zea mays L. Based on theseresults a scheme of the events that lead to selective cationuptake in intact barley roots is proposed. In this scheme acrucial factor of selectivity is sufficient phloem recirculationof K⁺ by the aid of which K⁺ rich cortical cells are formednear the root tip. When matured these cells are suggested tomaintain a high cytoplasmic K/Na ratio due to K⁺ dependent sodiumextrusion at the plasmalemma and due to recovery of vacuolarK⁺ by Na/K exchange across the tonoplast. Key words: Potassium/Sodium selectivity, Vacuolar exchange, Xylem transport, Hordeum, Zea, Atriplex     

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells

Cardiotonic glycosides, like ouabain, inhibit Na⁺-K⁺-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser⁴⁷³ phosphorylation, as evidenced by an increase in phospho-Akt Ser⁴⁷³ band density. Ouabain-stimulated Akt Ser⁴⁷³ phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser⁴⁷³ phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser⁴⁷³ phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in ⁸⁶Rb uptake but did not significantly alter Na⁺-K⁺-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na⁺-K⁺-ATPase-mediated ion transport. Na⁺-K⁺-ATPase; opossum kidney cells     

Reappraisal of the Role of Sodium in the Light-Dependent Active Transport of Pyruvate into Mesophyll Chloroplasts of C4 Plants

The mechanism of light-dependent active transport of pyruvatein C₄ mesophyll chloroplasts has not been clarified, particularlyin Na⁺-type C₄ species, in which the pyruvate uptake into mesophyllchloroplasts is enhanced by illumination or by making a Na⁺gradient (Na⁺-jump) across the envelope in the dark. We re-investigatedhere the effect of Na⁺ on the active transport of pyruvate inmesophyll chloroplasts of Panicum miliaceum, a Na⁺-type C₄ species,by comparing the rate of pyruvate uptake at various externalpHs under four conditions; in the light and dark together with/withoutNa⁺-jump: (1) At neutral pH, the rate of pyruvate uptake inthe dark was enhanced by Na⁺-jump but scarcely by illumination.(2) While the enhancement effect by Na⁺-jump was independentof external pH, that by illumination increased greatly at pHover 7.4, and the effects of light and Na⁺ at the alkaline pHwere synergistic. (3) The light-enhanced pyruvate uptake wasrelated to stromal alkalization induced by illumination. Infact, pyruvate uptake was induced by H⁺-jump in the medium frompH 8.0 to 6.7. (4) Stromal pH was lowered by the addition ofK⁺-pyruvate and more by Na⁺-pyruvate into the medium at pH 7.8in the light. (5) However, the pH and ATP levels in the stromawere not affected by Na⁺-jump. Thus, we discussed possibility that besides pyruvate/Na⁺ cotransportat neutral pH in the medium, pyruvate/H⁺ cotransport enhancedby the presence of Na+ operates in mesophyll chloroplasts ofNa⁺-type C4 species at alkaline medium. ¹Present address: Biological Resources Division, Japan InternationalResearch Center for Agricultural Sciences (JIRCAS), Ministryof Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba,305 Japan