Control of K+ (Rb+) influx and transport with changed internal K+ concentration and age in two varieties of barley

The influx of Rb⁺ into the roots of two barley varieties (Hordeum vulgare L. cv. Salve and cv. Ingrid) from a K⁺-free ⁸⁶Rb-labelled nutrient solution with 2.0 mM Rb⁺, was checked at intervals from day 6 to day 18. The control plants were continuously grown in complete nutrient solution containing 5.0 mM K⁺, while two other groups of plants were grown in K⁺-free nutrient solution starting on day 6 and between day 6 and day 9, respectively. The pattern of Rb⁺ influx was similar for both varieties, although their efficiencies in absorbing Rb⁺ were different. The relationship between Rb⁺ influx and K⁺ concentration of the root could be interpreted in terms of negative feedback through allosteric control of uptake across the plasmalemma of the root cells. Hill plots were bimodal, but in the opposite direction. The Hill coefficients, reflecting the minimum number of interacting allosteric binding sites for K⁺ (Rb⁺), were low (≤–3.0). It is discussed whether the threshold value, that is the breaking point in the Hill plot, is indicative of a changed efficiency of transporting units for K⁺ (Rb⁺) transport to the xylem. Moreover, feedback regulation might be involved in transport of K⁺ between root and shoot. The variation in K⁺ concentrations in the roots and shoots of control plants were cyclic but in phase opposition despite an exponential growth. The average K⁺ concentration varied only slightly with age.     

Influence of low temperature on regulation of Rb+ and Ca2+ influx in roots of winter wheat

Influx of Rb⁺(⁸⁶Rb⁺) and Ca²⁺(⁴⁵Ca²⁺) was determined in roots of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) after 14 days at 16°C/16 h light, after 1 and 8 weeks of cold acclimation (2°C/8 h light) and at intervals after deacclimation (16°C/16 h light) for up to 14 days. The plants were cultivated at 3 ionic strengths: 100, 10 and 1% of a full strength nutrient solution, containing 3.0 mM K⁺ and 1.0 mM Ca²⁺. K⁺ concentrations in roots and shoots increased during cold treatment, while Ca²⁺ in the roots decreased. In the shoots Ca²⁺ concentrations remained the same. Influx of Rb⁺ as a function of average K⁺ concentration in the roots of 14-day-old, non-cold-treated plants was high at a certain K⁺ level in the root and decreased at higher root K⁺ levels (negative feedback). The pattern for Ca²⁺ influx versus average concentration of Ca²⁺ in the root was the reverse. Independent of duration of treatment (1–8 weeks), cold acclimation partly changed the regulation of Rb⁺ influx, so that it became less dependent upon negative feedback and more dependent on the ionic strength of the cultivation solution. After exposure to 2°C, Ca²⁺ influx increased at high Ca²⁺ concentrations in the root as compared with influx in roots of 14-day-old non-cold-treated plants. Under deacclimation, Ca²⁺ influx gradually decreased again, and reached the level observed before cold treatment within 7–14 days at 16°C; the number of days depending on the exposure time at 2°C. It is suggested that Rb⁺(K⁺) influx became adjusted to low temperature and that abscisic acid (ABA) may be involved in this mechanism. It is also suggested that extrusion of Ca²⁺ was impaired and/or Ca²⁺ channels were activated at 2°C in roots of plants grown in the full-strength solution and that extrusion was gradually restored and/or Ca²⁺ channels were closed under deacclimation conditions.     

Differential effects of Na+, Mg2+, K+, Ca2+ and osmotic stress on the wild type and the NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii

In order to identify physiological components that contribute to salinity tolerance, we compared the effects of Na⁺, Mg²⁺ and K⁺ salts (NaCl, Na₂SO₄, MgCl₂, MgSO₄, KCl and K₂SO₄), Ca₂₊ (CaSO₄), mannitol and melibiose on the wild type and the single-gene NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii. Compared with gametophytic growth of the wild type, stl2 showed a low level of tolerance that was restricted to Na⁺ salts and osmotic stress. stl2 exhibited high tolerance to both Na⁺ and Mg²⁺ salts, as well as to osmotic stress. In response to short-term exposure (3 d) to NaCl, accumulation of K⁺ and Na⁺ was similar in the wild type and stl1. In contrast, stl2 accumulated higher levels of K⁺ and lower levels of Na⁺. Ca²⁺ supplementation (1.0 mol m^?3) ameliorated growth inhibition by Na⁺ and Mg²⁺ stress in wild type and stll, but not in stl2. In addition, under Na⁺ stress (175 mol m^?3) wild-type, stll and stl2 gametopbytes maintained higher tissue levels of K⁺ and lower levels of Na⁺ when supplemented with Ca²⁺ (1.0 mol m^?3). stl2 gametophytes were extremely sensitive to K⁺ supplementation. Growth of stl2 was greater than or equal to that of the wild type at trace concentrations of K⁺ but decreased substantially with increasing K⁺ concentration. Supplementation with K⁺ from 0 to 1.85 mol m^?3 alleviated some of the inhibition by 75 mol m^?3 NaCl in the wild type and in stl1. In stl2, growth at 75 mol m^?3 NaCl was similar at 0 and 1.85 mol m^?3 K⁺ supplementation. Although K⁺ supplementation above 1.85 mol m^?3 did not alleviate inhibition of growth by Na⁺ in any genotype, stl2 maintained greater relative tolerance to NaCl at all K⁺ concentrations tested.     

Mechanism for Cd2+ inhibition of (K++ Mg2+)ATPase activity and K+ (86Rb+) uptake join roots of sugar beet (Beta vulgaris)

Steady state kinetics were used to examine the influence of Cd²⁺ both on K⁺ stimulation of a membrane-bound ATPase from sugar beet roots (Beta vulgaris L. cv. Monohill) and on K⁺(⁸⁶Rb⁺) uptake in intact or excised beet roots. The in vitro effect of Cd²⁺ was studied both on a 12000–25000 g root fraction of the (Na⁺+K⁺+Mg²⁺)ATPase and on the ATPase when further purified by an aqueous polymer two-phase system. The observed data can be summarized as follows: 1) Cd²⁺ at high concentrations (>100 μM) inhibits the MgATPase activity in a competitive way, probably by forming a complex with ATP. 2) Cd²⁺ at concentrations <100 μM inhibits the specific K⁺ activation at both high and low affinity sites for K⁺. The inhibition pattern appears to be the same in the two ATPase preparations of different purity. In the presence of the substrate MgATP, and at K⁺ <5 mM, the inhibition by Cd²⁺ with respect to K⁺ is uncompetitive. In the presence of MgATP and K⁺ >10 μM, the inhibition by Cd²⁺ is competitive. 3) At the low concentrations of K⁺, Cd²⁺ also inhibits the 2,4-dinitrophenol(DNP)-sensitive (metabolic) K⁺(⁸⁶Rb⁺) uptake uncompetitively both in excised roots and in roots of intact plants. 4) The DNP-insensitive (non metabolic) K⁺(⁸⁶Rb⁺) uptake is little influenced by Cd²⁺. As Cd²⁺ inhibits the metabolic uptake of K⁺(⁸⁶Rb⁺) and the K⁺ activation of the ATPase in the same way at low concentrations of K⁺, the same binding site is probably involved. Therefore, under field conditions, when the concentration of K⁺ is low, the presence of Cd²⁺ could be disadvantageous.     

Effects of cadmium and lead on the accumulation of Ca2+ and K+ and on the influx and translocation of K+ in wheat of low and high K+ status

The effects of cadmium and lead on the internal concentrations of Ca²⁺ and K⁺, as well as on the uptake and translocation of K(⁸⁶Rb⁺) were studied in winter wheat (Triticum aestivum L. a. MV-8) grown hydroponically at 2 levels of K⁺ (100 uM and 10 mM). Cd²⁺ and Pb²⁺ were applied in the nutrient solution in the range of 0.3 to 1000 u.M. Growth was more severely inhibited by Cd²⁺ and in the high-K⁺ plants as compared to Pb^z+ and low-K⁺ plants. Ions of both heavy metals accumulated in the roots and shoots, but the K⁺ status influenced their levels. Ca²⁺ accumulation was increased by low concentrations of Cd²⁺ mainly in low-K⁺ shoots, whereas it was less influenced by Pb²⁺. The distribution of Cd²⁺ and Ca²⁺ in the plant and in the growth media indicated high selectivity for Cd²⁺ in the root uptake, while Ca²⁺ was preferred in the radial and/or xylem transport. Cd²⁺ strongly inhibited net K⁺ accumulation in high-K⁺ plants but caused stimulation at low K⁺ supply. In contrast, the metabolis-dependent influx of K⁺(⁸⁶Rb⁺) was inhibited in low-K⁺ plants, while the passive influx in high-K⁺ plants was stimulated. Translocation of K⁺ from the roots to the shoots was inhibited by Cd²⁺ but less influenced in Pb²⁺-treated plants. It is concluded that the effects of heavy metals depend upon the K⁺-status of the plants.     

Kinetic studies of a (Na++ K++ Mg2+)ATPase in sugar beet roots. III. A proposed model for the (Na++ K+) activation and its significance for field properties

A microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase preparation from sugar beet roots was used. The activation by simultaneous addition of Na⁺ and K⁺ at different levels was examined in terms of steady state kinetics. The observed data can be summarized in the following way: 1. The apparent affinity between the enzyme and the substrate MgATP depends on the ratio between Na⁺ and K⁺. At low Na⁺ concentration (below 5 mM), the apparent K_m decreases with increasing concentrations of K⁺ (1–20 mM). At 5 mM Na⁺, the K⁺ level does not change the apparent K_m, while at Na⁺ levels above 10 mM, the apparent K_m between enzyme and substrate increases with increasing concentration of K⁺. 2. When the MgATP concentration is kept constant, homotropic cooperativity (concerning one type of ligand) and heterotropic cooperativity (concerning different types of ligands) exist in the activation by Na⁺ and K⁺. The Na⁺ binding is cooperative with different K_m values and Hill coefficients (n) in the presence of low and high concentration of K⁺. At low Na⁺ level (< 5 mM). a negative cooperativity exists for Na⁺ (n_Na < 1) which is more pronounced in the presence of high [K⁺]. When the concentration of Na⁺ is raised the negative cooperativity disappears and turns into a positive one (n_Na > 1). Only K⁺ binding in the presence of low [Na⁺] shows cooperativity with a Hill coefficient that reflects changes from negative to positive homotropic cooperativity with increasing concentrations of K⁺ (n_K < 1 → n_K > 1). In the presence of [Na⁺] > 10 mM, the changes in n_k are insignificant. 3. A model is proposed in which one or two different K sites and one or two Na sites control the catalytic activity, with multiple interactions between Na⁺, K⁺ and MgATP. 4. In the presence of Na⁺ (< 10 mM), K⁺ is probably bound to two K sites, one of which translocates K⁺ through the membrane by an antiport Na⁺/K⁺ mechanism. This could be connected with an elevated K⁺ uptake in the presence of Na⁺ and could therefore explain some field properties of sugar beets.     

Hormonal Regulation of Ca2+ Stimulated K+ Influx and Ca2+, K+- ATPase in Rice Roots: in vivo and in vitro Effects of Auxins and Reconstitution of the ATPase

The role of natural and synthetic auxins in regulation of ion transport and ATPase activity was studied in rice roots (Oryza sativa L. cv. Dunghan Shah). In vivo treatment of seedlings with 2,4-dichlorophenoxyacetic acid at 2 × 10^?6M for a short period enhanced subsequent Ca²⁺ stimulated K⁺ influx and ATPase activity, while a longer treatment diminished both K⁺ influx and ATPase activity. Indoleacetic acid at 10^?10–10^?8M induced ATPase activity. In in vitro experiments both 2,4-dichloro phenoxyacetic acid and indoleacetic acid (10^?10–10^?8M) stimulated Ca²⁺, K⁺-ATPase activity of a plasmalemma rich micro somal fraction from the roots. Acetone extracted ATPase preparations lost their activity. The enzyme regained its activity and its sensitivity towards ions (Ca²⁺+ K⁺) when reconstituted with phosphatidyl choline. Addition of auxins also indicated that the presence of the lipid was necessary in the interaction between the ATPase and auxins. Auxins and ions probably interact with the intact ATPase lipoprotein complex, which may possess a receptor site for the auxins, possibly as a sub unit.     

Effects of Cd2+ and EDTA on young sugar beets (Beta vulgaris). II. Net uptake and distribution of Mg2+, Ca2+ and Fe2+/Fe3+

Levels of Mg²⁺, Ca²⁺ and Fe²⁺/Fe³⁺ were determined in roots and shoots of sugar beet seedlings (Beta vulgaris L. cv. Monohill) cultured for 5 weeks in a complete nutrient solution to which either Cd²⁺ (0, 5 or 50 μM), EDTA (0, 10 or 100 μM) or a combination of both was added. The plants subjected to the various treatments showed a variety of deficiency symptoms. Leaves of the Cd²⁺-treated plants became thin and chlorotic (Mg- and Fe-deficiency symptoms). The plants showed reduced growth and developed only a few brownish roots with short laterals (Ca-deficiency symptoms). EDTA treatment resulted in green, stunted, hard leaves and reduced growth (Ca-deficiency symptoms). The deficiency symptoms observed correspond well with the observed uptake rates and distributions of Mg²⁺, Ca²⁺ and Fe²⁺/Fe³⁺. Increases in either Cd²⁺, EDTA or a combination of both in the growth medium, were correlated with increasing Mg²⁺ levels in the roots and with decreasing Mg²⁺ levels in the shoots. Cd²⁺ alone or in combination with EDTA had little influence on Ca²⁺ levels in the shoots but decreased Ca²⁺ levels in the roots. Thus, Cd²⁺ affects Mg²⁺ and Ca²⁺ transport in opposite ways: Mg²⁺ transport to the shoots is inhibited while that of Ca²⁺ is facilitated. Treatment with EDTA alone did not affect Ca²⁺ concentrations in either the shoots or the roots. Treatment with Cd²⁺ lowered Fe²⁺ concentrations in both roots and shoots.     

Kinetics of NH4+ and K+ uptake by ectomycorrhizal fungi. Effect of NH4+ on K+ uptake

NH₄⁺ and K⁺ uptake experiments have been conducted with 3 ectomycorrhizal fungi, originating from Douglas fir (Pseudotsuga menziesii (Mirb.] Franco) stands. At concentrations up to 250 μM, uptake of both NH₄⁺ and K⁺ follow Michaelis-Menten kinetics. Laccaria bicolor (Maire) P. D. Orton, Lactarius rufus (Scop.) Fr. and Lactarius hepaticus Plowr. ap. Boud. exhibit K_m values for NH₄⁺ uptake of 6, 35, and 55 μM, respectively, and K_m values for K⁺ uptake of 24, 18, and 96 μM, respectively. Addition of 100 μM NH₄⁺ raises the K_m of K⁺ uptake by L. bicolor to 35 μM, while the V_max remains unchanged. It is argued that the increase of K_m is possibly caused by depolarization of the plasma membrane. It is not due to a competitive inhibition of K⁺ by NH₄⁺ since the apparent inhibitor constant is much higher than the K_m, for NH₄⁺ uptake. The possibility that NH₄⁺ and K⁺ are taken up by the same carrier can be excluded. The K_m, values for K⁺ uptake in the two other fungi are not significantly affected by 100 μM NH₄⁺. Except for a direct effect of NH₄⁺ on influx of K⁺ into the cells, there may also be an indirect effect after prolonged incubation of the cells in the presence of 100 μM NH₄⁺.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Antagonistic Effect of Na+ and Mg2+ on P2z Purinoceptor-Associated Pores in Dibutyryl Cyclic AMP-Differentiated NG108-15 Cells

Abstract: The effect of replacement of extracellular Na⁺ with N-methyl-d -glucamine (NMG) on P₂ receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15 cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) with an EC₅₀ value of 230 µM. Replacement of Na⁺ with NMG as well as removal of Mg²⁺ from the bathing buffer potentiated ethidium bromide uptake, [Ca²⁺]_i increase, and ⁴⁵Ca²⁺ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg²⁺ to limit the amount of ATP^4?, replacement of Na⁺ with NMG had no effect on the ATP-induced [Ca²⁺]_i increase but caused a markedly larger [Ca²⁺]_i increase when the calculated concentration of ATP^4? was >10 µM. The calculated EC₅₀ value for ATP^4? stimulation of the [Ca²⁺]_i increase was 23 µM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca²⁺ release was the major pathway for the ATP-induced [Ca²⁺]_i increase; both removal of Mg²⁺ and replacement of Na⁺ with NMG did not affect the action of ATP. These data suggest that ATP^4?-promoted pores are antagonized by Na⁺ and Mg²⁺ in dibutyryl cyclic AMP-differentiated NG108-15 cells.     

Effects of Ca2+ and Temperature on K+ Content Distribution along Roots of Wheat, Rice and Cucumber

The effects of Ca²⁺ and temperature on the K⁺ contents of root segments of wheat (Triticum aestivum L. cv. Aurora), rice (Oryza sativa L. cv. Dunghan Shali) and cucumber (Cucumis sativus L. cv. Csemege Fürtös) were investigated with special regard to the low-temperature anomaly of the K⁺ uptake of thermophilic plants. The anomaly occurred in those root segments where the K⁺ contents turned out to be highest. The K⁺ contents of the apical root sections of thermophilic species responded in general to Ca²⁺ in more pronounced ways than those of wheat, at both 0 and 25°C. The concerted actions of a purely physical process with negative temperature coefficient and of the special thermotropic properties of the membranes are suggested to be responsible for the low-temperature anomaly. A strict discrimination between the Viets-effect on the content (classical Viets-effect) and on the uptake is proposed. The classical Viets-effect seems to be related to alterations in both active transport and exchange processes.     

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

K+-Dependent Stimulation of Dopamine Synthesis in Striatal Synaptosomes Is Mediated by Protein Kinase C

Dopamine synthesis rate was measured in striatal synaptosomes. Removal of Na⁺ increased synthesis rate; this was blocked in Ca²⁺-free medium and by addition of the Ca²⁺/calmodulin inhibitor N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W7). The increase in dopamine synthesis rate caused by the addition of the phorbol ester 12-O-tetradecanoylphorboI-13-acetate (TPA) was blocked by the protein kinase C inhibitor polymyxin B. K⁺-stimulated synthesis was unchanged in Ca²⁺-free medium or by addition of W7; it was blocked by polymyxin B. The effect of 50 mM K⁺ was additive with that of 8-Br cyclic AMP and of Na⁺ removal; the combined effect of 50 mM K⁺ and TPA was no greater than that of either alone. These results suggest that stimulation of dopamine synthesis in striatal synaptosomes by 50 mM K⁺ is mediated by protein kinase C.     

High salinity tolerance in the stl2 mutation of Ceratopteris richardii is associated with enhanced K+ influx and loss

The roles of K⁺ uptake and loss in the salinity response of the wild type and the salt-tolerant mutant stl2 of Ceratopteris richardii were studied by measuring Rb⁺ influx and loss and the effects of Na⁺, Mg²⁺, Ca²⁺ and K⁺-transport inhibitors. In addition, electrophysiological responses were measured for both K⁺ and Rb⁺ and for the effects of Na⁺ and NH₄⁺ on subsequent K⁺-induced depolarizations. stl2 had a 26–40% higher uptake rate for Rb⁺ than the wild type at 0.5–10 mol m^?3 RbCl. Similarly, membrane depolarizations induced by both RbCl and KCl were consistently greater in stl2. In the presence of 0–180 mol m^?3 NaCl, stl2 maintained a consistently greater Rb⁺ influx than the wild type. stl2 retained a greater capacity for subsequent KCl-induced depolarization following exposure to NaCl. Five mol m^?3 Mg²⁺ decreased Rb⁺ uptake in stl2; however, additional Mg²⁺ up to 40 mol m^?3 did not affect Rb⁺ uptake further. Ca²⁺ supplementation resulted in a very minor decrease of Rb⁺ uptake that was similar in the two genotypes. Tetraethylammonium chloride and CsCl gave similar inhibition of Rb⁺ uptake in both genotypes, but NH₄Cl gave substantially greater inhibition in the wild type than in stl2. NH₄Cl resulted in a greater membrane depolarization in the wild type and the capacity for subsequent depolarization by KCl was markedly reduced. stl2 exhibited a higher Independent loss of Rb⁺ than the wild type, but, in the absence of external K⁺, loss of Rb⁺ was equivalent in the two genotypes. Since constitutive K⁺ contents are nearly identical, we conclude that high K⁺ influx and loss exact a metabolic cost that is reflected in the inhibition of gametophytic growth. Growth inhibition can be alleviated by reduced supplemental K⁺ or by treatments that slightly reduce K⁺ influx, such as moderate concentrations of Na⁺ or Mg²⁺. We propose that high throughput of K⁺ allows maintenance of cytosolic K⁺ under salt stress and that a high uptake rate for K⁺ results in a reduced capacity for the entrance and accumulation of alternative cations such as Na⁺ in the cytosol.     

Kinetic Studies of a (Na++ K++ Mg2+) ATPase in Sugar Beet Roots

Kinetic studies of a dithiothreitol treated membrane ATPase fraction from sugar beet roots led to the following conclusions: 1) In the presence of MgATP, Na⁺ and K⁺ stimulate the ATPase activity in different ways following simple Michaelis-Menten kinetics. Thus separate sites for Na⁺ and K⁺ are suggested. 2) In the absence of K⁺, Na⁺ acts as an uncompetitive modifier raising the apparent K_m and V_max for MgATP. 3) In the absence of Na⁺, K⁺ activates non-competitively with respect to MgATP. Thus K⁺ increases V_max but does not affect the apparent affinity constant. 4) K⁺ and Na⁺ double the rate constants. 5) In the presence of Na⁺ or K⁺, Mg²⁺ in excess acts as a weak inhibitor to Na⁺ and/or K⁺ activity. 6) The temperature-activity dependence in the 5–40°C interval shows biphasic Arrhenius plots with the transition point between 15–18°C. The activation energy is lowered at temperatures > 18°C.     

Relationship Between Ca2+ Uptake and Catecholamine Secretion in Primary Dissociated Cultures of Adrenal Medulla

Abstract: Carbachol or elevated K⁺ stimulated ⁴⁵Ca²⁺ uptake into chromaffin cells two- to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca²⁺ uptake and catecholamine secretion induced by the mixed nicotinic-muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca²⁺ uptake occurred within 15 s of stimulation by carbachol or elevated K⁺ at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K⁺ was similar to that of Ca²⁺ uptake. There was a close correlation between Ca²⁺ uptake and catecholamine secretion at various concentrations of Ca²⁺. The concentration dependencies for inhibition of both processes by Mg²⁺ or Cd²⁺ were similar. Ca²⁺ uptake saturated with increasing Ca²⁺ concentrations, with an apparent K_m for both carbachol-induced and elevated K⁺-induced Ca²⁺ uptake of approximately 2 mM. The Ca²⁺ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca²⁺ entry and a presumed increase in cytosolic Ca²⁺ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca²⁺- dependent processes associated with catecholamine secretion. Ca²⁺ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage-sensitive Ca²⁺ channels.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Aluminium interactions with K+(86Rb+) and 45Ca2+ fluxes in three cultivars of sugar beet (Beta vulgaris)

Three cultivars of sugar beet (Beta vulgaris L.), which are sensitive to aluminium (Al) in the order Primahill > Monohill > Regina, were grown in water culture for 2 weeks. Nutrients were supplied at 15% increase of amounts daily, corresponding to the nutrient demand for maximal growth. The 2.4-dinitrophenol (DNP)-sensitive (metabolic) and DNP-insensitive (non-metabolic) uptake of aluminium, phosphate. ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in roots were measured as well as transport to shoots of intact plants. All 3 cultivars absorbed more aluminium if DNP was present during the aluminium treatment than in its absence. It is suggested that sugar beets are able to extrude aluminium activity or that they possess an active mechanism to keep Al outside the cell. The presence of Al in the medium during the 1-h experiment affected the metabolic and non-metabolic fluxes of ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in different ways. In the presence of DNP, the influx of both ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) and the efflux of ⁴⁵Ca²⁺ were inhibited by Al in a competitive way. At inhibition of ⁴⁵Ca²⁺ influx, 2 Al ions are probably bound per Ca²⁺ uptake site in cv. Regina (Al-tolerant), but in cvs Primahill and Monohill only one Al ion is bound (more Al sensitive). Aluminium competitively inhibited the active efflux of ⁴⁵Ca²⁺ (absence of DNP) in almost the same way in the 3 cultivars. In contrast, aluminium stimulated the influx of K⁺(⁸⁶Rb⁺) in cvs Primahill, Monohill and Regina in the absence of DNP. Thus, the Al effects on active and passive K⁺(⁸⁶Rb⁺) influx are different. The total influx of K⁺(⁸⁶Rb⁺) increased in the presence of Al and might be connected to an active exclusion of Al. Regina is the least Al-sensitive cultivar, probably because Al interferes less with the Ca²⁺ fluxes and because this cultivar actively excludes phosphate in the presence of Al. Thus Al-phosphate precipitation within the plant could be avoided.