Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.     

Enhanced Net K Uptake Capacity of NaCl-Adapted Cells

Maintenance of intracellular K⁺ concentrations that are not growth-limiting, in an environment of high Na⁺, is characteristic of NaCl-adapted cells of the glycophyte, tobacco (Nicotiana tabacum/gossii). These cells exhibited a substantially greater uptake of ⁸⁶Rb⁺ (i.e. an indicator of K⁺) relative to unadapted cells. Potassium uptake into NaCl-adapted cells was 1.5-fold greater than unadapted cells at 0 NaCl and 3.5-fold greater when cells were exposed to 160 millimolar NaCl. The difference in net K⁺ uptake between unadapted and NaCl-adapted cells was due primarily to higher rates of entry rather than to reduced K⁺ leakage. Presumably, enhanced K⁺ uptake into adapted cells is a result of electrophoretic flux, and a component of uptake may be linked to vanadate-sensitive H⁺ extrusion.     

Potassium-induced inhibition of photosynthesis and associated electron transport chain of Microcystis: Implication for controlling cyanobacterial blooms

Potassium toxicity to survival and growth of Microcystis has been investigated for the first time by taking photosynthetic parameters and change in internal pH of Microcystis. The concentration of potassium reducing 50% population of Microcystis was found to be 6 mM. At this concentration, the internal pH of cells increased from 7.2 to 9.8 in comparison to control. 6.0 mM concentration of potassium reduced protein content by 44% and generated Na⁺ efflux of 55% as compared to control. O₂ evolution, ATP content and CO₂ fixation were found to be very sensitive to above K⁺ concentration and registered a respective decline of 38, 32 and 36%. PS II was the primary site of action depicting about 35% inhibition at above K⁺ concentration. PS I and whole electron transport chain were also inhibited but the extent was less pronounced in comparison to PS II. A definite correlation between requirement of Na⁺ for growth and maintenance of cytoplasmic pH was observed. K⁺-induced loss of Na⁺ from cells of Microcystis could result in increase in internal pH, which in turn affects survival, growth, and other physiological parameters of Microcystis. Thus, K⁺ appears to hold excellent potential for the control of Microcystis blooms in fresh water ponds and lakes.     

Validation of the use of the lipophilic thiocyanate anion for the determination of membrane potential in Ehrlich ascites tumor cells

Summary The utility of the lipophilic anion thiocyanate (SCN⁺) as a probe for the indirect estimation of the cell membrane potential (V _m ) in Ehrlich ascites tumor cells has been evaluated by comparison to direct electrophysiological measurements. SCN accumulation is consisten with first-order uptake into a single kinetically-identifiable cellular compartement, achieving steadystate distribution in 20–30 min at 22°C. The steady-state distribution ratio ([SCN^–] _c /[SCN^–] _e ) in physiological saline is 0.44±0.02. Treatment of the cells with proparanolol (0.13 mM), an activator of Ca²⁺ dependent K⁺ channels, reduces the steady-state distribution ratio to 0.19±0.02. Conversely, treatmetn with BACl₂ (10 mM), an antagonist of the pathway, increases the SCN^– distribution ratio to 0.62±0.01. The equilibrium potentials (V _SCN ) calculated under these conditions are virtually identical to direct electrophysiological measurements of theV _m made under the same conditions. The effect of varing extracellular [K⁺]([K⁺] _e ) in the presence of constant [Na⁺] _e =100 mM has also been tested. In control cells, elevation of [K⁺] _e from 6 to 60 mM reducesV _SCN from –20.6±1.0 to –13.2±1.2 mV. Again, microelectrode measurements give excellent quantitative agreement. Propranolol increases the sensitivity of the cells to varying [K⁺] _e , so that a 10-fold elevation reducesV _SCN by approximately 31 mV. BaCl₂ greatly reduces this reponse: a 10-fold elevation in [K⁺] _e yielding only a 4-mV rediction inV _SCN . It is concluded that the membrane potential of Ehrlich cells can be estimated accurately from SCN^– distribution measurements.     

Cation co-tolerance phenomenon in cell cultures of Oryza sativa adapted to LiCl and NaCl

Cell lines of Oryza sativa L. (cv. Taipei-309) were adapted to 30 mM LiCl and 150 mM NaCl. Both adapted lines were considerably more tolerant than non adapted line when grown on 200, 250 and 300 mM NaCl and 30 mM LiCl stresses. The tolerance of LiCl-adapted line to NaCl (150 to 300 mM) and the tolerance of NaCl-adapted cells line to LiCl (30 mM) indicated that there was a cross-adaptation towards alkali metals (Na⁺ and Li⁺) not the Cl^–. Na⁺ and K⁺ contents of all lines which increased with increasing medium salinity but to a different degree. The increase in Na⁺ and K⁺ content in NaCl-adapted and non-adapted lines were comparable, while LiCl-adapted line accumulated significantly lower Na⁺and higher K⁺ content. Proline content of all lines increased with the increase in NaCl-stress but the magnitude of increase was much higher in the LiCl-adapted than other lines. The differential response of adapted lines to NaCl stress in accumulating proline and maintaining the ionic contents reveals that adapted lines have evolved different features of adaptation to cope with NaCl stress.     

Resting potential of the mouse neuroblastoma cells: I. The presence of K+ channels activated at high K+ concentration but closed at low K+ concentration including the physiological concentration

The effects of changes in extracellular K⁺ concentration ([K⁺]_o) on the resting membrane potential, the input resistance and ⁸⁶Rb efflux (as a marker of K⁺ efflux) were examined with use of the cultured mouse neuroblastoma cells (N-18 clone). The results obtained are as follows. (1) The membrane potential was depolarized, with an increase in [K⁺]_o at concentrations above 10–20 mM at a rate of 55–58 mV per 10-fold change in [K⁺]_o, but practically unchanged with varying [K⁺]_o below this concentration. (2) Above the critical [K⁺]_o of 10–20 mM, the input membrane resistance decreased sharply by a factor of $\text{1}\text{4}\text{?}\text{1}\text{5}$ with an increase in [K⁺]_o. A similar decrease in the resistance occurred even under the conditions that the membrane potential was held at control level (about ?55 mV) by a steady-state current passage. (3) Elimination of Na⁺ and Cl^? from the external solution brought about practically no change in the membrane potential. (4) A fractional escape rate of ⁸⁶Rb from N-18 cells remained constant at relatively low level (0.125%/min on average) in the low [K⁺]_o range, but increased sharply with increasing [K⁺]_o above 15 mM (e.g., approx. 3.4- and 4.5-fold at 30 and 100 mM [K⁺]_o, respectively). (5) The high K⁺-induced ⁸⁶Rb efflux was not practically inhibited by 1 mM tetraethylammonium or 0.1 mM 4-aminopyridine, indicating that the K⁺ channels activated by an elevation of [K⁺]_o are not the delayed (voltage-dependent) K⁺ channels. The present results favoured the conclusion that N-18 cells carry K⁺ channels which open at high [K⁺]_o but are closed at low [K⁺]_o including the physiological range for the mouse neuroblastoma cells (around 5.4 mM). This conclusion leads to the notion that in the mouse neuroblastoma N-18 cells the K⁺ permeability does not mainly contribute to determining the resting membrane potential under physiological conditions.     

Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K⁺]_o was increased from 1.2 to 10 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. As [K⁺]_o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K⁺]_o was increased from 60 to 122 mM, an increase in [K⁺]_i and [Cl^–]_i and a decrease in [Na⁺]_i were observed.Inneurons, as [K⁺]_o was increased from 1.2 to 2.8 mM, [Na⁺]_i and [Cl^–]_i were decreased, whereas [K⁺]_i was increased. As [K⁺]_o was increased from 2.8 to 30 mM, [K⁺]_i, [Na⁺]_i and [Cl^–]_i showed no significant change. When [K⁺]_o was increased from 30 to 122 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. Inastrocytes, pH_i increased when [K⁺]_o was increased. Inneurons, there was a biphasic change in pH_i. In lower [K⁺]_o (1.2–2.8 mM) pH_i decreased as [K⁺]_o increased, whereas in higher [K⁺]_o (2.8–122 mM) pH_i was directly related to [K⁺]_o. In bothastrocytes andneurons, changes in [K⁺]_o did not affect the extracellular water content, whereas the intracellular water content increased as the [K⁺]_o increased. Transmembrane potential (E_m) as measured with Tl-204 was inversely related to [K⁺]_o between 1.2 and 90 mM, a ten-fold increase in [K⁺]_o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The E_m values measured with Tl-204 were close to the potassium equilibrium potential (E_k) except those in neurons at lower [K⁺]_o. However, they were not equal to the chloride equilibrium potential (E_Cl) at [K⁺]_o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K⁺]_o produced different changes in [K⁺]_i, [Na⁺]_i, [Cl^–]_i, and pH_i in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K⁺]_o, in such a way to maintain a more suitable environment for neurons.     

The effect of potassium depletion on the initial kinetics of glycolysis in ascites tumor cells

Ehrlich ascites carcinoma cells depleted of K⁺ and provided with 5.5 mM K⁺ in isosmotic 50 mM tris(hydroxymethyl)methylglycine buffer at pH 7.4 and 38 °C take up K⁺ from the medium at a rate of 6 μmoles/ml intracellular fluid per min. Depleted cells exposed to K⁺ for 2 min prior to glucose addition exhibit a higher initial rate of glycolysis, a lower glycose-6-P accumulation, and a higher fructose-1,6-P₂ accumulation than depleted cells incubated in a K⁺-free medium. Both the K⁺ transport and the effect of K⁺ on glycolysis are blocked by 2 mM oubain.Calculation of thein vitro velocities of glycolytic enzymes from the rates of accumulation of lactate and glycolytic intermediates shows that the presence of K⁺ accelerates the velocities of fructose-6-phosphate kinase and lactate dehydrogenase about 2-fold and the velocity of hexokinase about 1.5-fold during the first 15 s. In either the presence or absence of K⁺, the hexokinase velocity is highest immediately after glucose addition and declines sharply with time; this decline is greater than would be predicted by product inhibition by the accumulated glucose-6-P. The maximal stimulation of fructose-6-phosphate kinase attibutable to the increasing intarcellular K⁺ concentration is only 1.25-fold. These observations indicate that the initial acceleration in glycolysis is not simply mediated through a direct K⁺ activation of fructose-6-phosphate kinase.The calculated theoretical rate of ATP generation by glycolysis shows that glycolysis is an ATP-utilizing system for the first 5–10 s both in the presence and in the absence of K⁺. Hence, the initial stimulation of glycolysis by K⁺ is not a consequence of an increased rate of ATP hydrolysis associated with K⁺ transport, although this mechanism may be responsible for the stimulation of steady-state glycolysis.The initial rate of phosphate ester (hexose and triose phosphates) accumulation corresponds to be rate of ATP generation by the “tail-end” of glycolysis, or twice the rate of lactate accumulation, in either the absence or presence of K⁺, but both the rate and the maximal level of ester accumulated are higher in the presence of K⁺. This implies that the oxidatively generated pool of ATP which is diverted from endogenous reactions to hexokinase and fructose-6-phosphate kinase on the introduction of glucose is larger in the presence of K⁺.Valinomycin (0.27 μM) under certain conditions can produce effects on the glycolysis of non-depleted cells which superficially resemble the effects of K⁺ on depleted cells. However, unlike K⁺, valinomycin stimulates the initial rate of glycolytic ATP generation, and abolishes the initial correspondence between the ATP generation by the “tail-end” of glycolysis and phosphate ester accumulation. These observations are interpreted to mean that valinomycin introduces an ATPase activity effective on glycolytically generated ATP.Comparison of the theoretical ATP generation in the presence and absence of K⁺ indicates that approximately one ATP is hydrolyzed for each K⁺ transported.     

Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum

Summary Anti-L serum prepared by immunization of a high-potassium-type (HK) (blood type MM) sheep with blood from a low-potassium-type (LK) (blood type ML) sheep contained an antibody which stimulated four- to sixfold K⁺-pump influx in LK (LL) sheep red cells. In long-termin vitro incubation experiments, LK sheep red cells sensitized with anti-L showed a net increase in K⁺ after two days of incubation at 37°C, whereas HK-nonimmune (NI)-serum-treated control cells lost K⁺. The antibody could be absorbed by LK (LL) sheep red cells but not by HK sheep red cells. Kinetic experiments showed that the concentration of external K⁺ ([K⁺]₀) required to produce halfmaximum stimulation of the pump ([Na⁺]₀=0, replaced by Mg⁺⁺) was the same (0.25 mM) in L-antiserum-treated or untreated LK cells. LK cells with different [K⁺]_i (Na⁺ replacement) were prepared by the p-chloromercuribenzene sulfonate (PCMBS) method. At [K⁺]₀=5 mM, pump influx decreased as [K⁺]_i increased from 1 to 70 mM in L-antiserum-treated LK cells, whereas LK cells treated with HK-NI-serum ceased to pump at [K⁺]_i=35 mM. Exposure to anti-L serum produced an almost twofold increase in the number of pump sites of LK cells as measured by the binding of tritiated ouabain by LK sheep red cells. These findings indicate that the formation of a complex between the L-antigen and its antibody stimulates active transport in LK sheep red cells both by changing the kinetics of the pump and by increasing the number of pump sites.     

Cloning and functional expression in <Emphasis Type="Italic">Saccharomyces cereviae</Emphasis> of a K<Superscript>+</Superscript> transporter,AlHAK, from the graminaceous halophyte, <Emphasis Type="Italic">Aeluropus littoralis</Emphasis>

High-affinity K⁺ transporters play an important role in K⁺ absorption of plants. We isolated a HAK gene from Aeluropus littoralis, a graminaceous halophyte. The amino acid sequence of AlHAK showed high homology with HAK transporters obtained from Oryza sativa (82%) and Hordeum vulgare (82%). When expressed in Saccharomyces cereviae WΔ3, AlHAK performed high-affinity K⁺ uptake with a K_m value of 8 μM, and the growth of transformants was dramatically inhibited by 150 mM Rb⁺ and 150 mM Cs⁺ but less affected by 300 mM Na⁺. AlHAK may thus improve the capacity of plants to maintain a high cytosolic K⁺/Na⁺ ratio at high salinity.     

Cell growth and ouabain-sensitive Rb uptake and (Na + K)-ATPase activity in 3T3 and SV40 transformed 3T3 fibroblasts

The uptake of ouabain-sensitive ⁸⁶Rb⁺ uptake measured at 5 min and the uptake measured at 60 min was 4.5- and 2.7-fold greater respectively for SV40 transformed 3T3 cells compared to 3T3 cells during the late log phase of growth. This uptake, however, varied markedly with cell growth. Ouabain-sensitive ⁸⁶Rb⁺ uptake was found to be a sensitive indicator of protein synthesis as measured by total protein content. Cessation of cell growth as measured by total protein content was associated with a decline in ouabain-sensitive ⁸⁶Rb⁺ uptake in both cell types. This increased ouabain-sensitive cation transport was reflected in increased levels of (Na⁺ + K⁺)-ATPase activity for SV40 3T3 cells, which showed a 2.5-fold increase V but the same K_rmm as 3T3 cells.These results are compared with the results of related work. Possible mechanisms for these effects are discussed and how changes in cation transport might be related to alterations in cell growth.     

Solutes contributing to osmotic adjustment in cultured plant cells adapted to water stress

Osmotic adjustment was studied in cultured cells of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) adapted to different levels of external water potential ranging from −4 bar to −28 bar. The intracellular concentrations of reducing sugars, total free amino acids, proline, malate, citrate, quaternary ammonium compounds, K⁺, NO₃⁻, Na⁺, and Cl⁻ increased with decreasing external water potential. At any given level of adaptation, the maximum contribution to osmotic potential was from reducing sugars followed by potassium ions. The sucrose levels in the cells were 3- to 8-fold lower than reducing sugar levels and did not increase beyond those observed in cells adapted to −16 bar water potential. Concentrations of total free amino acids were 4- to 5-fold higher in adapted cells. Soluble protein levels declined in the adapted cell lines, but the total reduced nitrogen was not significantly different after adaptation. Uptake of nitrogen (as NH₄⁺ or NO₃⁻) from the media was similar for adapted and unadapted cells. Although the level of quaternary ammonium compounds was higher in the nonadapted cells than that of free proline, free proline increased as much as 500-fold compared to only a 2- to 3-fold increase observed for quaternary ammonium compounds. Although osmotic adjustment after adaptation was substantial (up to −36 bar), fresh weight (volume increase) was restricted by as much as 50% in the adapted cells. Altered metabolite partitioning was evidenced by an increase in the soluble sugars and soluble nitrogen in adapted cells which occurred at the expense of incorporation of sugar into cell walls and nitrogen into protein. Data indicate that the relative importance of a given solute to osmotic adjustment may change depending on the level of adaptation.     

Effect of external K+, Ca2+, and Ba2+ on membrane potential and ionic conductance in rat astrocytes

Summary 1. The purpose of this study was (a) to identify if astrocytes show a similar non-Nernstian depolarization in low K⁺ or low Ca²⁺ solutions as previously found in human glial and glioma cells, and (b) to analyze the influence of the K⁺ conductance on the membrane potential of astrocytes.2. The membrane potential (E_m) and the ionic conductance were studied with whole-cell patch-clamp technique in neonatal rat astrocytes (5–9 days in culture) and in human glioma cells (U-251MG).3. In 3.0 mM K⁺, E_m was –75 ± 1.0 mV (mean ± SEM,n=39) in rat astrocytes and –79 ± 0.7 mV (n=5) in U-251MG cells. In both cell types E_m changed linearly to the logarithm of [K⁺]₀ between 3.0 and 160 mM K⁺. K⁺ free medium caused astrocytes to hyperpolarize to –93 ± 2.7 mV (n=21) and U-251MG cells to depolarize to –27 ± 2.1 mV (n=3).4. The I-E curve did not show inward rectification in astrocytes at this developmental stage. The slope conductance (g) exhibited only a small decrease (–19%) in K⁺ free solution and no significant change in 160 mM K⁺.5. Ba²⁺ (1.0 mM) depolarized astrocytes to –45 ± 2.9 mV (n=11), decreasing the slope conductance (g) by 42.4 ± 8.3% (n=11). Ca²⁺ free solution depolarized astrocytes to –53 ± 3.4 mV (n=12) and resulted in a positive shift of the I-E curve, increasing g by 15.3 ± 8.2% (n=8).6. Calculations indicated that a block of K⁺ channels explains the depolarizing effect of Ba²⁺. The effects of K⁺ free or Ca²⁺ free solutions on E_m can be explained by a transformation of K⁺ channels to non-specific leakage channels. That astrocytes show a different reaction to low K⁺ than glioma cells can be related to the lack of inwardly rectifying K⁺ channels in astrocytes at this developmental stage.     

K+ Uptake by Fermenting Escherichia coli Cells: pH Dependent Mode of the TrkA System Operating

Escherichia coli accumulates K⁺ by means of multiple transportsystems, of which TrkA is the most prominent at neutral and alkalinepH while Kup is major at acidic pH. In the present study, K⁺ uptakewas observed with cells grown under fermentative conditions at an initialpH of 9.0 and 7.3 (the medium pH decreased to 8.4 and 6.8, respectively,during the mid-logarithmic growth phase), washed with distilled water andresuspended in a K⁺ containing medium at pH 7.5 in the presence ofglucose. The kinetics for this K⁺ uptake and the amount of K⁺accumulated by the wild type and mutants having a functional TrkA orKup could confirm that K⁺ uptake by E. coli grown either at pH 9.0or pH 7.3 occurs mainly through TrkA. The following results distinguishpH dependent mode of TrkA operating: (1) K⁺ uptake was inhibited byDCCD in cells grown either at pH 9.0 or pH 7.3, although the stoichiometryof K⁺ influx to DCCD-inhibited H⁺ efflux for bacteria grownat pH 9.0 varied with external K⁺ concentration, but remained constantfor cells grown at pH 7.3; (2) K⁺ uptake was observed with an atpDmutant grown at pH 9.0 but not at pH 7.3; (3) The DCCD-inhibited H⁺efflux was increased 8-fold less by 5 mM K⁺ added into a K⁺ freemedium for bacteria grown at pH 9.0 than that for cells grown at pH 7.3;(4) the DCCD-inhibited ATPase activity of membrane vesicles from bacteriagrown at pH 9.0 was reduced a little in the presence of 100 mM K⁺,but stimulated more than 2.4-fold at pH 7.3.     

Studies on Electrogenesis under High K+ Concentrations in Internodal Cells of Chara corallina

+ concentration ([K⁺]_o) on the membrane potential (E_m) of Chara corallina was studied. E_m more negative than -100 mV was maintained even at 100 mM [K⁺]_o. Addition of Ca²⁺ to the external medium further increased this tendency. However, E_m responded sensitively to the increase in [K⁺]_o, when the electrogenic proton pump of the plasma membrane was inhibited by treating cells with dicyclohexylcarbodiimide, an inhibitor of proton pump. Analysis using equivalent circuit model of the plasma membrane suggested that the electrogenic proton pump was activated by the increase in [K⁺]_o. In the presence of 100 mM K⁺, action potentials were generated by electric stimuli. The ionic mechanism of generation of action potentials in the presence of K⁺ at high concentration was discussed. Received 3 October 2000/ Accepted in revised form 6 January 2001     

Variations in the Ability of Ruminal Gram-Negative Prevotella Species to Resist Monensin

Gram-negative, ruminal Prevotella strains (n = 15) differed greatly in their sensitivity to the feed additive monensin. Strains that were repeatedly transferred with sublethal doses tolerated more monensin than those that were unadapted, but growth experiments indicated that the sensitivity range was as great as 2000-fold. Prevotella bryantii B₁4 grew with monensin concentrations as high as 20 μM, but P. ruminicola H15a, D31d, 20-63, E40a, and D42f never initiated growth if monensin was greater than 0.01 μM. Washed cell preparations that were energized with glucose lost intracellular potassium when monensin was added, and potassium depletion could also be used as an index of monensin sensitivity. Adapted cells of P. bryantii B₁4 had a half-maximal potassium depletion constant (K _d) of 3.2 μM, but the K _d values of P. ruminicola strains H15a, D31d, 20-63, E40a, and D42f were less than 0.04 μM. Maximal potassium depletion (K _max) values range from 90% to 40%, and monensin-adapted cells always had lower K _max values than unadapted cells. A linear regression of log K _d/K _max versus percentage decrease in optical density divided by monensin concentration had an r² of 0.75, and this regression indicated that potassium depletion from washed cells closely correlated with growth inhibition. P. bryantii B₁4 had a K _d/K _max ratio that was sevenfold greater than other Prevotella strains, and this result indicated that P. bryantii may be unusual in its ability to grow with very high concentrations of monensin. Received: 13 August 1999 / Accepted: 5 October 1999     

Potassium channel currents in intact stomatal guard cells: rapid enhancement by abscisic acid

Evidence of a role for abscisic acid (ABA) in signalling conditions of water stress and promoting stomatal closure is convincing, but past studies have left few clues as to its molecular mechanism(s) of action; arguments centred on changes in H⁺-pump activity and membrane potential, especially, remain ambiguous without the fundamental support of a rigorous electrophysiological analysis. The present study explores the response to ABA of K⁺ channels at the membrane of intact guard cells ofVicia faba L. Membrane potentials were recorded before and during exposures to ABA, and whole-cell currents were measured at intervals throughout to quantitate the steady-state and time-dependent characteristics of the K⁺ channels. On adding 10 M ABA in the presence of 0.1, 3 or 10 mM extracellular K⁺, the free-running membrane potential (V _m) shifted negative-going (–)4–7 mV in the first 5 min of exposure, with no consistent effect thereafter. Voltage-clamp measurements, however, revealed that the K⁺-channel current rose to between 1.84- and 3.41-fold of the controls in the steady-state with a mean halftime of 1.1 ± 0.1 min. Comparable changes in current return via the leak were also evident and accounted for the minimal response inV _m. Calculated atV _m, the K⁺ currents translated to an average 2.65-fold rise in K⁺ efflux with ABA. Abscisic acid was not observed to alter either K⁺-current activation or deactivation.These results are consistent with an ABA-evoked mobilization of K⁺ channels or channel conductance, rather than a direct effect of the phytohormone on K⁺-channel gating. The data discount notions that large swings in membrane voltage are a prerequisite to controlling guard-cell K⁺ flux. Instead, thev highlight a rise in membranecapacity for K⁺ flux, dependent on concerted modulations of K⁺-channel and leak currents, and sufficiently rapid to account generally for the onset of K⁺ loss from guard cells and stomatal closure in ABA.     

Sodium/potassium selectivity and pleiotropy in stl2, a highly salt-tolerant mutation of Ceratopteris richardii

The roles of Na⁺ and K⁺ (Rb⁺) uptake were further studied in a NaCl-tolerant strain of Ceratopteris richardii containing the stl2 mutation by direct comparison with the wild-type strain. In addition to Na⁺ tolerance, stl2 also confers tolerance to Mg²⁺ and sensitivity to K⁺. In addition to higher K⁺ (Rb⁺) uptake at concentrations commonly associated with low-affinity K⁺ transport, stl2 maintained higher uptake down to 0·1 mol m^–3 Rb⁺. Up to a 25-fold excess of Na⁺ had little effect in either genotype on K⁺ (Rb⁺) uptake at low concentrations, i.e. 0·2 and 0·5 mol m^–3 RbCl. Pretreatment with K⁺ (20 mol m^–3) inhibited uptake of K⁺ (Rb⁺) in the wild type, whereas concurrent inclusion of K⁺ inhibited uptake of Rb⁺ more in stl2. In the absence of K⁺, Na⁺ uptake (0·01–60 mol m^–3) was nearly identical in the wild type and stl2. K⁺ inhibited Na⁺ uptake more effectively in stl2 than the wild type, especially at 60 mol m^–3 Na⁺. Greater inhibition of K⁺ uptake in stl2 occurred with MgCl₂ or TEA (tetraethylammonium chloride) preincubation or with simultaneous inclusion of Al³⁺ (Al₂SO₄). The higher effective velocity of K⁺ uptake at a wide range of concentrations and the enhanced selectivity for K⁺ and against Na⁺ contribute to the preservation of higher cytosolic K⁺ and lower Na⁺ under salinity stress.     

Enhanced H Transport Capacity and ATP Hydrolysis Activity of the Tonoplast H-ATPase after NaCl Adaptation

Tonoplast enriched membrane vesicle fractions were isolated from unadapted and NaCl (428 millimolar) adapted tobacco cells (Nicotiana tabacum L. var Wisconsin 38). Polypeptides from the tonoplast enriched vesicle fractions were separated by SDS-PAGE and analyzed by Western blots using polyclonal antibodies to the 70 kilodalton subunit of the red beet tonoplast H⁺-ATPase. These antibodies cross-reacted exclusively to a tobacco polypeptide of an apparent molecular weight of 69 kilodaltons. The antibodies inhibited ATP-dependent, NO₃⁻ sensitive H⁺ transport into vesicles in tonoplast enriched membrane fractions from both unadapted and NaCl adapted cells. The relative H⁺ transport capacity per unit of 69 kilodalton subunit of the tonoplast ATPase of vesicles from NaCl adapted cells was fourfold greater than that observed for vesicles from unadapted cells. The increase in specific H⁺ transport capacity after adaptation was also observed for ATP hydrolysis.