Early changes of Cl− efflux and H+ extrusion induced by osmotic stress in Arabidopsis thaliana cells

In various plant materials changes in turgor pressure, following hyper- or hypo-osmotic stress, were associated with the activation or inactivation of the plasma membrane H⁺-ATPase, respectively. To see if the turgor changes might indirectly influence H⁺-ATPase activity by regulating ion fluxes through plasma membrane, we investigated, in cultured cells of Arabidopsis thaliana (L.) Heynh., the early effects of hyper- and hypo-osmotic stress on Cl^? fluxes in comparison, in the case of hyper-osmotic treatment, with its effect on net H⁺ extrusion. The results obtained showed that hyper-osmotic stress (200 mM mannitol) quickly reduced Cl^? efflux (?70%) from cells preloaded with ³⁶C1^? for 18 h. This inhibiting effect was independent of the simultaneous mannitol-induced stimulation of Cl^? influx and rapidly reversible after removal of the hyper-osmotic treatment. The inhibition of Cl^? efflux was associated with a stimulation of net H⁺ extrusion, and these two effects showed the same dependence on the external mannitol concentration. Fusicoccin (FC, 20 µM), which stimulated H⁺ extrusion to about the same extent as 200 mM mannitol, did not affect Cl^? efflux. When cells preloaded with ³⁶C1^? for 18 h in the presence of mannitol (from 25 up to 200 mM) were eluted in a mannitol-free medium an early and strong increase in Cl^? efflux was found. The increase of Cl- efflux was already detectable for a small hypo-osmotic jump (25 mM), and was reduced (?50%) by the anion channel inhibitor A9C (300 µM). These results lead to exclude a direct causal relationship mediated by E_m changes between the effects of osmoticum on Cl^? efflux and net H⁺ extrusion, and favour the view that the changes in turgor pressure induced by hyper/hypo-osmotic stress may respectively induce an early inactivation/activation of stretch-sensitive anion channels.     

Inhibition of fusicoccin-stimulated K+/H+ transport in root tips from maize seedlings pretreated with Chlorsulfuron

Abstract Fusicoccin (FC)-stimulated K⁺ (₈₆Rb) uptake and proton extrusion of maize (Zea mays) root apical segments were inhibited by pretreatment of 4-day-old seedlings with the herbicide Chlorsulfuron. In the range of Chlorsulfuron concentrations 0.01-10 mmol m^?3, the percentage of inhibition was 15% at 0.01 mmol m^?3 and progressively increased with Chlorsulfuron concentration up to 60% at 10 mmol m^?3. At the maximum concentration tested (10 mmol m^?3), the inhibition was evident after 1.5 h of pre-treatment. The binding of FC to microsomal fractions of root segments from Chlorsulfuron-pretreated seedlings was inhibited by 30%. It is suggested that Chlorsulfuron causes an alteration at the plasmalemma level involving the FC binding sites. The ineffectiveness of Chlorsulfuron in inhibiting FC-stimulaled K⁺ uptake when administered to excised segments, while inhibiting the enzyme acetolactate synthase, pointed out by Ray (1984) as the site of action of Chlorsulfuron in pea plants, suggests that the observed inhibition of K⁺ uptake and H⁺ extrusion is not induced by Chlorsulfuron inhibition of this enzyme. An alternative site of action of Chlorsulfuron is hypothesized in maize plants.     

A mutant of Arabidopsis thaliana partially resistant to fusicoccin has reduced plasma membrane H+-ATPase

5-2 is a mutant of Arabidopsis thaliana which is partially resistant to fusicoccin in vivo. We have analysed fusicoccin binding and the activity and amount of H⁺-ATPase in plasma membrane isolated from mature leaves of the wild type and of mutant 5-2. Fusicoccin binding was similar in plasma membrane from the two genotypes, while H⁺-ATPase activity was markedly (c. 50%) lower in plasma membrane from mutant 5-2 than in that from the wild type. The H⁺-ATPase of mutant 5-2 was activated by fusicoccin as much as that of the wild type. In plasma membrane from mutant 5-2, the amount of immunodetectable H⁺-ATPase, quantified by densitometry of Western blots, was about half that in the wild type. These results indicate that the major defect of mutant 5-2 detectable at the plasma membrane level is a reduction in the amount of H⁺-ATPase.     

Synergistic activation of plasma membrane H+– ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin

The regulation of the H⁺-ATPase of plasma membrane is a crucial point in the integration of transport processes at this membrane. In this work the regulation of H⁺-ATPase activity induced by changes in turgor pressure was investigated and compared with the stimulating effect of fusicoccin (FC). The exposure of cultured cells of Arabidopsis thaliana L. (ecotype Landsberg 310–14-2) to media containing mannitol (0. 15 or 0. 3 M ) or polyethylene glycol 6000 (PEG) (15. 6% or 22% w/v) resulted in a decrease in the turgor pressure of the cells and in a strong stimulation of H⁺ extrusion in the incubation medium. The osmotica-induced H⁺ extrusion was (1) inhibited by the inhibitor of plasma membrane H⁺-ATPase, erythrosin B (EB), (2) dependent on the external K⁺ concentration, (3) associated with a net K⁺ influx, and (4) lead to an increase of cellular malate content. These results show that the reduction of external osmotic potential stimulates the activity of plasma membrane H⁺-ATPase
The effect of mannitol was only partially inhibited by treatments with cycloheximide (CH) and cordycepin, which block protein and mRNA synthesis, respectively. All the effects of osmotica were qualitatively and quantitatively similar to those induced by 5 μ M FC. However, when FC and mannitol (or PEG) were fed together, their effects on H⁺ extrusion appeared synergistic, irrespective of whether FC was present at suboptimal or optimal concentrations. This behaviour suggests that the modes of action of FC and of the osmotica on H⁺-ATPase activity differ at least in some step(s)     

A Phosphopeptide Corresponding to the Cytosolic Stretch Connecting Transmembrane Segments 8 and 9 of the Plasma Membrane H+-ATPase Binds 14-3-3 Proteins and Inhibits Fusicoccin-Induced Activation of the H+-ATPase

Abstract: A putative consensus domain for binding of 14-3-3 proteins to the plasma membrane (PM) H⁺-ATPase was identified in the highly-conserved sequence RSR(p)SWSF [where (p)S is Ser776 of the maize isoform MHA2], localized in the cytosolic stretch connecting transmembrane segments 8 and 9. A 15 amino acid biotinylated phosphopeptide comprising this motif: i) bound a recombinant 14-3-3 protein, ii) inhibited fusicoccin-induced stimulation of the PM H⁺-ATPase activity both in PM isolated from germinating radish ( Raphanus sativus L.) seedlings and in ER isolated from Saccharomyces cerevisiae expressing AHA1 (an isoform of Arabidopsis thaliana PM H⁺-ATPase), and iii) inhibited fusicoccin binding to PM isolated from germinating radish seedlings. The corresponding non-phosphorylated peptide was inactive in all the performed assays. Together, these results suggest that the cytosolic strand connecting transmembrane segments 8 and 9 of the PM H⁺-ATPase is a 14-3-3 binding site which might cooperate with the C-terminal domain of the'enzyme in generating a stable association between the H⁺-ATPase and 14-3-3 protein.     

Long-term effects of abscisic acid on K+ transport in young wheat plants of different K+ status

The effects of abscisic acid (ABA) on growth, uptake and translocation of potassium ions, K⁺,Mg²⁺-ATPase activity and transpiration were investigated in young wheat ( Triticum aestivum L. cv. Martonvásári-8) plants grown at different K⁺ supplies. Long-term treatment with ABA (10 μ M ) reduced growth in high-K⁺ plants, but had less effect under low-K⁺ conditions. K⁺(⁸⁶Rb) uptake was inhibited by about 70 and 40% in low- and high-K⁺ plants, respectively. The stimulation by K⁺ of the Mg²⁺-ATPase activity in the root microsomal fraction was lost with ABA treatment. It is suggested that the inhibitory effect of ABA on K⁺ uptake may be related to this effects on the K⁺,Mg²⁺-ATPase. Translocation of K⁺ to the shoot was inhibited in low-K⁺ plants only, and it was not affected in high-K⁺ plants. In parallel to this, ABA treatment reduced transpiration by about 50% in low-K⁺ plants, whereas a much smaller effect was seen in high-K⁺ plants. These observations suggest that the regulation by ABA of the stomatal movements is strongly counteracted by high-K⁺ status.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.     

Erythrosin B as an effective inhibitor of electrogenic H+ extrusion

Abstract In 24-h-genninaled radish seedlings erythrosin B (EB), an effective inhibitor of microsomal as well as of partially purified vanadate-sensitive ATPase markedly inhibited the basal and the FC-stimulated proton extrusion, and induced a rapid depolarization of FC-hyperpolarized trans-membrane electric potential (PD) without causing any significant change of ATP level. The effects of EB on H⁺ extrusion were partially additive with those of vanadatc, another inhibitor of plasma membrane H⁺-ATPase. These effects are interpreted as due to a direct inhibition by EB on plasma membrane H⁺-ATPase involved in H⁺ electrogenic transport in the higher plants.     

Demonstration of a K+/H+ exchange activity in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Abstract Washed cells of Rhodopseudomonas sphaeroides forma sp. denitrificans , grown under photodenitrifying conditions, exhibited K⁺ uptake dependent on the transmembrane proton gradient (Δ pH). These cells also acidified the suspension medium in response to K⁺ pulses both aerobically and anaerobically in light and in the dark. The results indicate that the photodenitrifier has a reversible K⁺/H⁺ exchange activity which reflects its role in regulating the intracellular K⁺ concentration, as well as intracellular pH. The acidification of the external medium resulting from K⁺ pulses was inhibited by carbonyl cyanide- m -chlorophenylhydrazone (CCCP) indicating that the antiporter is energy-dependent. Addition of KCl to washed cells depolarized the membrane potential (Δψ) with a concomitant increase in ΔpH, indicating that the K⁺/H⁺ antiporter was electrogenic.     

Ouabain Binding, ATP Hydrolysis, and Na+,K+-Pump Activity During Chemical Modification of Brain and Muscle Na+,K+-ATPase

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.     

Inhibition of Rat Brain Microsomal Na+,K+-ATPase by S-Adenosylmethionine

Abstract: Rat brain microsomes were preincubated with S -adenosylmethionine (SAM), MgCl₂, and CaCl₂, then re-isolated, and the activity of Na⁺,K⁺-ATPase determined. SAM inhibited the Na⁺,K⁺-ATPase activity compared with microsomes subjected to similar treatment in the absence of SAM. A biphasic inhibitory effect was observed with a 50% decrease at a SAM concentration range of 0.4 μ M -3.2 μ M and a 70% reduction at a concentration range above 100 μ M . Inclusion of either S- adenosylhomocysteine or 3-deazaadenosine in the preincubations prevented the SAM inhibition of Na⁺,K⁺-ATPase activity. The inhibition by SAM appeared to be Mg²⁺- or Ca²⁺-dependent.     

Effect of potassium deficiency on the plasma membrane H+-ATPase of the wood ray parenchyma in poplar

The effect of K⁺ deficiency on the plasma membrane (PM) H⁺‐ATPase was studied in young stems of poplar plants (Populus tremula × tremuloides) grown with low or full‐strength K⁺ supply. Immunological assays using different antibodies were applied to test if K⁺ deficiency affects the amount of immunodetectable PM H⁺‐ATPases in the stem tissue. The monoclonal antibody clone 46 E5 B11 revealed an increased abundance of PM H⁺‐ATPases under conditions of low K⁺ supply, and immunolabelling experiments showed that this increase was restricted to vessel‐associated cells (VACs) of the wood ray parenchyma. Replacement of the monoclonal antibody by a polyclonal antibody against PM H⁺‐ATPase gave a specific immunoreactivity on blots as well as tissue sections too, but the labelling intensity showed no difference between plants with low or full‐strength K⁺ supply. Measurements of extracellular H⁺ concentrations using non‐invasive, H⁺‐selective microelectrodes revealed a lowering of the pH at the surface of VACs and an enhancement of net efflux of H⁺ in plants grown with low K⁺ supply. The present results indicate an up‐regulation of specific isoforms of the PM H⁺‐ATPase in VACs under K⁺‐deficient conditions and suggest a key role for these PM H⁺‐ATPases in unloading K⁺ from the xylem stream.     

Two Isoenzymes of Na+, K+-ATPase Have Different Kinetics of K+ Dephosphorylation in Normal Cat and Human Brain Cortex

Analysis of purified Na+,K+-ATPase from cat and human cortex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two large catalytic subunits called alpha (-) (lower molecular weight) and alpha (+) (higher molecular weight). Differences in K+ dephosphorylation of these two molecular forms have been investigated by measuring the phosphorylation level of each protein after their separation on sodium dodecyl sulfate gels. In the presence of Na+, Mg2+, and ATP, both subunits are phosphorylated. Increasing concentrations (from 0 to 3 mM) of K+ induce progressive dephosphorylation of both alpha-subunits, although the phosphoprotein content of alpha (-) is decreased significantly less than that of alpha (+). Ka values of alpha (-) for K+ are 40% and 50% greater in cat and human cortex, respectively, than values of alpha (+). alpha (-) and alpha (+) are thought to be localized in specific cell types of the brain: alpha (-) is the exclusive form of nonneuronal cells (astrocytes), whereas alpha (+) is the only form of axolemma. Our results support the hypothesis that glial and neuronal Na+,K+-ATPases are different molecular entities differing at least by their K+ sensitivity. Results are discussed in relation to the role of glial cells in the regulation of extracellular K+ in brain.     

The Na+,K+ -ATPase: A Plausible Trigger for Voltage-Independent Release of Cytoplasmic Neurotransmitters

A comparison was made between the releasability of eight neurotransmitters from eight regions of mouse brain in response to either 60 mM-K+ or 20 microM-ouabain, a specific inhibitor of the Na+,K+-ATPase. With few exceptions, all transmitters were released by either or both agents from each brain region examined. Potassium was superior in releasing the biogenic amines and acetylcholine, while the putative amino acid transmitters were generally releasable by both agents. Measurements of tissue depolarization using [3H]-tetraphenylphosphonium uptake indicated that 60 mM-K+ is capable of depolarizing brain tissue above the threshold necessary for initiating an action potential, but 20 microM-ouabain is not. The pattern of release by ouabain coupled with its failure to depolarize brain tissue at 20 microM suggests that inhibition of the Na+,K+-ATPase is capable of releasing cytoplasmic neurotransmitters in a voltage-independent manner.     

Nerve Growth Factor Induces Na+, K+-ATPase in a Nerve Cell Line

The effects of nerve growth factor (NGF) on induction of Na+,K+-ATPase were examined in a rat pheochromocytoma cell line, PC12h. Na+,K+-ATPase activity in a crude particulate fraction from the cells increased from 0.37 +/- 0.02 (n = 19) to 0.55 +/- 0.02 (n = 20) (means +/- SEM, mumol Pi/min/mg of protein) when cultured with NGF for 5-11 days. The increase caused by NGF was prevented by addition of specific anti-NGF antibodies. Epidermal growth factor and insulin had only a small effect on induction of Na+,K+-ATPase. A concentration of basic fibroblast growth factor three times higher than that of NGF showed a similar potency to NGF. The molecular form of the enzyme was judged as only the alpha form in both the untreated and the NGF-treated cells by a simple pattern of low-affinity interaction with cardiotonic steroids: inhibition of enzyme activity by strophanthidin (Ki approximately 1 mM) and inhibition of Rb+ uptake by ouabain (Ki approximately 100 microM). As a consequence, during differentiation of PC12h cells to neuron-like cells, NGF increases the alpha form of Na+,K+-ATPase, but does not induce the alpha(+) form of the enzyme, which has a high sensitivity for cardiotonic steroid and is a characteristic form in neurons.     

K+ influx by Kup in Escherichia coli is accompanied by a decrease in H+ efflux

Escherichia coli accumulates K+ by means of multiple uptake systems of which Kup is the major transport system at acidic pH. In cells grown under fermentative conditions at pH 5.5, K+ influx by a wild-type strain upon hyper-osmotic stress at pH 5.5 was accompanied by a marked decrease in H+ efflux, with a 1:1 ratio of K+ to H+ fluxes. This was observed with cells treated with N,N'-dicyclohexylcarbodiimide. Similar results with a mutant defective in Kdp and TrkA but with a functional Kup system but not in a mutant defective in Kdp and Kup but having an active TrkA system suggest that Kup operates as a H+ -K+ -symporter.     

Quantification of Rat Cerebral Cortex Na+,K+-ATPase: Effect of Age and Potassium Depletion

Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles.     

Distribution of Na+, K+-ATPase α-Subunit Isoforms in Rat Pituitary

The distributions of alpha-subunit isoforms of the Na+,K(+)-ATPase in rat pituitary were determined by immunoblotting and immunohistochemistry. Immunoreactivity for all three forms is present in the neural lobe, whereas the anterior lobe contains only alpha 1 and alpha 2. Most areas of the intermediate lobe exhibit faint immunoreactivity for only alpha 1, but thin strands of cells which stain strongly for all three isoforms are also present in this lobe. The previously reported ouabain inhibitable Na+,K(+)-ATPase activity in the neural lobe is consistent with the presence of both alpha 2 and alpha 3 subunits.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

Interactions between K+ and NH4+: effects on ion uptake by rice roots

Interactive effects of K⁺ and N (principally NH₄⁺) on plant growth and ion uptake were investigated using hydroponically grown rice (Oryza sativa L. cv. M202) seedlings by varying the availability of NH₄⁺ or NO₃^? and K⁺ during an 18d growth period, a 3d pretreatment period and during flux measurements. Plants grew best in media containing 100 mmol m^?3 NH₄⁺ and 200mmolm^?3 K⁺ (N100/K200), followed by N2/K200 < N100/K2 < N2/K2. ⁸⁶Rb⁺(K⁺) fluxes were increased by exposure to N during the 18 d growth period and the 3 d of pretreatment, but decreased by the presence of NH₄⁺ during flux measurements. This inhibition was a function of prior N/K provision and the [NH₄⁺]₀ present during flux determinations. NH₄⁺ was least inhibitory to 86Rb⁺(K⁺) influx in high-N/low-K plants. Pretreatments with K⁺ failed to stimulate NH₄⁺ uptake, and the presence of K⁺ in the uptake solutions reduced NH₄⁺ fluxes only in high-N/low-K plants.