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.     

Characterization of Exchange Inhibitory Peptide Effects on Na+/Ca2+ Exchange in Rat and Human Brain Plasma Membrane Vesicles

Abstract: The inhibitory effects of Na⁺/Ca²⁺ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na⁺/Ca²⁺ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na⁺-dependent Ca²⁺ uptake in both rat brain [IC₅₀ = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC₅₀ = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ～80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na⁺-dependent Ca²⁺ release, and the inhibitory effect was enhanced by increasing the extravesicular Na⁺ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na⁺. When XIP was added at steady state (5 min after the initiation of intravesicular Na⁺-dependent Ca²⁺ uptake), it was found that the intravesicular Ca²⁺ content declined with time. Analysis of unidirectional fluxes for Ca²⁺ at steady state showed that 50 µM XIP inhibited Ca²⁺ influx and efflux ～85 and 70%, respectively. This result suggested that XIP inhibited both Na⁺/Ca²⁺ exchange and Ca²⁺/Ca²⁺ exchange but had no effect on the passive release pathway for Ca²⁺. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na⁺ or other monovalent cations, e.g., K⁺, is required for XIP to have its inhibitory effect on Ca²⁺ transport.     

The role of plasma membrane H+‐ATPase in jasmonate‐induced ion fluxes and stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H⁺, Ca²⁺ and K⁺ in guard cells of wild‐type (Col‐0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1‐1 and the PM H⁺‐ATPase mutants aha1‐6 and aha1‐7, using a non‐invasive micro‐test technique. We showed that MeJA induced transmembrane H⁺ efflux, Ca²⁺ influx and K⁺ efflux across the PM of Col‐0 guard cells. However, this ion transport was abolished in coi1‐1 guard cells, suggesting that MeJA‐induced transmembrane ion flux requires COI1. Furthermore, the H⁺ efflux and Ca²⁺ influx in Col‐0 guard cells was impaired by vanadate pre‐treatment or PM H⁺‐ATPase mutation, suggesting that the rapid H⁺ efflux mediated by PM H⁺‐ATPases could function upstream of the Ca²⁺ flux. After the rapid H⁺ efflux, the Col‐0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H⁺‐ATPase was reduced. Finally, the elevation of cytosolic Ca²⁺ concentration and the depolarized PM drive the efflux of K⁺ from the cell, resulting in loss of turgor and closure of the stomata.     

The modulating effects of pH and benzyladenine on the Ca2+– and Mg2+-ATPases in the plasmalemma of wheat root cells

Plant cells frequently and rapidly have to respond to environmental changes for survival. Regulation of transport and other energy-requiring processes in the plasmalemma of root cells is therefore one important aspect of the ecological adaptation of plants. Wheat (Triticum aestivum L. cv. Drabant) was grown hydroponically, with or without 50 nM benzyladenine in the medium, and plasma membranes from root cells of 8-day-old plants were prepared by aqueous polymer two-phase partitioning. The influence of Ca²⁺ and Mg²⁺ on the plasmalemma ATPase activities was investigated. The presence of benzyladenine during growth increased the ATPase activity, that dependent upon Ca²⁺ more than that elicited by Mg²⁺. As a general characteristic, ATP was the preferred substrate, but all nucleotide tri- and diphosphates could be accepted with activities in plasma membranes from control plants of 7-36% (Mg²⁺) and 40-86% (Ca²⁺) and in plasma membranes from benzyladenine-treated plants of 12-47% (Mg²⁺) and 53-102% (Ca²⁺) as compared with activities obtained with ATP. Nucleotidemonophosphates were not hydrolyzed by the preparations. In preparations from benzyladenine-treated plants one peak of Ca²⁺-ATPase at pH 5.2–5.6, with a tail from pH 6 and upwards, and one peak of Mg²⁺-ATPase at pH 6.0–6.5 were observed in the presence of EDTA in the assay media. In preparations from control plants, the addition of EDTA to the assays resulted in a wide optimum between pH 6 and 7 for Mg²⁺-ATPase and low Ca²⁺-ATPase activity with no influence of pH in the range 4.5 to 8. Analysis of the pH dependence in the presence of both Ca²⁺ and Mg²⁺ indicates that the control plants mainly contain Mg²⁺-ATPase corresponding to the proton pump. Preparations from benzyladenine-treated wheat roots show, in addition, activation by Ca²⁺, which, in the slightly alkaline pH range may correspond to a Ca²⁺-extruding (Ca²⁺+ Mg²⁺)-ATPase. In the acidic range, the responses are more complicated: the Mg²⁺-ATPase is inhibited by vanadate, while the Ca²⁺-ATPase is insensitive, and benzyladenine added during growth influences the interaction between Ca²⁺ and Mg²⁺ in a way that parallels the effect of high salt medium.     

ATP-dependent Ca2+ transport in wheat root plasma membrane vesicles

Plasma membrane preparations of high purity were obtained from roots of dark-grown wheat (Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. These preparations mainly contained sealed, right-side-out vesicles (ca 90% exposing the original outside out). By subjecting the preparations to 4 freeze/thaw cycles the proportion of sealed, inside-out (cytoplasmic side out) vesicles increased to ca 30%. Inside-out and right-side-out plasma membrane vesicles were then separated by partitioning the freeze/thawed plasma membranes in another aqueous polymer two-phase system. In this way, highly purified, sealed, inside-out (>60% inside-out) vesicles were isolated and subsequently used for characterization of the Ca²⁺ transport system in the wheat plasma membrane. The capacity for ⁴⁵Ca²⁺ accumulation, nonlatent ATPase activity and proton pumping (the latter two markers for inside-out plasma membrane vesicles) were all enriched in the inside-out vesicle fraction as compared to the right-side-out fraction. This confirms that the ATP-binding site of the ⁴⁵Ca²⁺ transport system, similar to the H⁺-ATPase, is located on the inner cytoplasmic surface of the plant plasma membrane. The ⁴⁵Ca²⁺ uptake was MgATP-dependent with an apparent K_m for ATP of 0.1 mM and a high affinity for Ca²⁺ [K_m(Ca²⁺/EGTA) = 3 μM]. The pH optimum was at 7.4–7.8. ATP was the preferred nucleotide substrate with ITP and GTP giving activities of 30–40% of the ⁴⁵Ca²⁺ uptake seen with ATP. The ⁴⁵Ca²⁺ uptake was stimulated by monovalent cations; K^? and Na⁺ being equally efficient. Vanadate inhibited the ⁴⁵Ca²⁺ accumulation with half-maximal inhibitions at 72, 57 and 2 μM for basal, total (with KCI) and net K⁺-stimulated uptake, respectively. The system was also highly sensitive to erythrosin B with half-maximal inhibition at 25 nM and total inhibition at 1μM. Our results demonstrate the presence of a primary Ca²⁺ transport ATPase in the plasma membrane of wheat roots. The enzyme is likely to be involved in mediating active efflux (ATP-binding sites on the cytoplasmic side) to the plant cell exterior to maintain resting levels of cytoplasmic free Ca²⁺ within the cell.     

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.     

Transmembrane potential-mediated coupling between H+ pump operation and K+ fluxes in Elodea densa leaves hyperpolarized by fusicoccin, light or acid load

In isolated Elodea densa leaves, the relationships between H⁺ extrusion (-ΔH⁺), K⁺ fluxes and membrane potential (E_m) were investigated for two different conditions of activation of the ATP-dependent H⁺ pump. The ‘basal condition’ (darkness, no pump activator present) was characterized by low values of-ΔH⁺ and K⁺ uptake (ΔK⁺), wide variability of the ?ΔH⁺/ΔK⁺ ratio, relatively low membrane polarization and E_m values more positive than EK for external K⁺ concentrations (|K⁺]_o of up to 2mol m^?3. A net K⁺ uptake was seen already at [K⁺]_o below 1 mol m^?3, suggesting that K⁺ influx in this condition was a thermodynamically uphill process involving an active mechanism. When the H⁺ pump was stimulated by fusicoccin (FC), by cytosol acidification, or by light (the ‘high polarization condition’), K⁺ influx largely dominated K⁺ and C^? efflux, and the ?ΔH⁺/ΔK⁺ ratio approached unity. In the range 50 mmol m^?3?5 mol m^?3 [K⁺]₀, E_m was consistently more negative than E_K. The curve of K⁺ influx at [K⁺]₀ ranging from 50 to 5000mmol m^?3 fitted a monophasic, hyperbolic curve, with an apparent half saturation value = 0–2 mol m^?3. Increasing |K⁺]₀ progressively depolarized E_m, counteracting the strong hyperpolarizing effect of FC. The effects of K⁺ in depolarizing E_m were well correlated with the effects on both K⁺ influx and ?ΔH⁺, suggesting a cause-effect chain: K⁺₀ influx → depolarization → activation of H⁺ extrusion. Cs⁺ competitively inhibited K⁺ influx much more strongly in the ‘high polarization’ than in the ‘basal’ condition (50% inhibition at [Cs⁺]/[K⁺]₀ ratios of 1:14 and 1:2, respectively) thus confirming the involvement of different K⁺ uptake systems in the two conditions. These results suggest that in E. densa leaves two distinct modes of interactions rule the relationships between H⁺ pump, membrane polarization and K⁺ transport. At low membrane polarization, corresponding to a low state of activation of the PM H⁺-ATP^ase and to E_m values more positive than E_K, K⁺ influx would mainly     

Changes of membrane-associated Mg2+-activated ATPase of cucumber roots during calcium starvation

The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca²⁺ (Ca²⁺-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca²⁺ starvation, while Mg²⁺-activation of the enzyme decreased and even resulted in inhibition by high Mg²⁺ concentration at the late stage of the Ca²⁺ starvation. The complete enzyme had low basal activity and showed a Mg²⁺-activated hyperbolic reaction curve in relation to ATP concentration. Ca²⁺-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg²⁺-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca²⁺ starvation. The basal ATPase activity of Ca²⁺-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg²⁺-activation was found only for ADP and ATP in both the complete and Ca²⁺-deficient enzymes, but the activation for ATP was greatly reduced by Ca²⁺ starvation. The heat inactivation curves for basal and Mg²⁺-activated ATPase did not differ much between the complete and Ca²⁺-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg²⁺-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca²⁺-deficient enzyme.     

ATP-driven Ca2+ transport in sealed plasma membrane vesicles prepared by aqueous two-phase partitioning from leaves of Commelina communis

Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U₃+U₃′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven ⁴⁵Ca²⁺ transport [? 15 pkat (mg protein)⁻¹]. Ca²⁺ uptake into the vesicles had a pH optimum of 7.2 and apparent K_m values for Ca²⁺ of 4.4 μM and for Mg-ATP of 300 μM. Ca²⁺ uptake, K⁺, Mg²⁺-ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm⁻³) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca²⁺ transport into the vesicles, excluding a Ca²⁺/H⁺ exchange driven by a proton gradient. ATP-dependent Ca²⁺ uptake was inhibited by erythrosin B (I₅₀= 0.1 μM), ruthenium red (I₅₀= 30 μM), La³⁺ (I₅₀= 10 μM) and vanadate (I₅₀= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I₅₀= 70 μM) and W-7 (I₅₀= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca²⁺ efflux from the vesicles rather than inhibit Ca²⁺ uptake. Our results demonstrate the presence of a Ca²⁺-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca²⁺ out of the cell. Its high affinity for Ca²⁺ makes it a likely component involved in adjusting low cytoplasmic Ca²⁺ levels. No indications for a secondary active Ca²⁺/H⁺ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca²⁺-ATPase from the H⁺-translocating K⁺. Mg²⁺-ATPase in C. communis plasma membranes.     

Role of Na+-Ca2+ Exchanger in Agonist-Induced Ca2+ Signaling in Cultured Rat Astrocytes

Abstract: We have previously demonstrated that activation of the Na⁺-Ca²⁺ exchanger in the reverse mode causes Ca²⁺ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na⁺-Ca²⁺ exchanger is involved in agonist-induced Ca²⁺ signaling in cultured rat astrocytes. The astrocytic intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased by l -glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca²⁺]_i. The Na-induced Ca²⁺ signal was also attenuated by S-nitroso-l -cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na⁺-Ca²⁺ exchanger enhanced the ionomycin-induced increase in [Ca²⁺]_i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca²⁺ signal. Furthermore, the ionomycin-induced Ca²⁺ signal was enhanced by removal of extracellular Na⁺ and pretreatment with ascorbic acid. These findings indicate that the Na⁺-Ca²⁺ exchanger is a target for NO modulation of elevated [Ca²⁺]_i and that the exchanger plays a role in Ca²⁺ efflux when [Ca²⁺]_i is raised above basal levels in astrocytes.     

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 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.     

Influence of abscisic acid on unidirectional fluxes and intracellular compartmentation of K+ and Na+ in excised barley root segments

Using compartmental analysis, unidirectional fluxes of K⁺ and Na⁺ and their intracellular compartmentation in excised barley (Hordeum distichon L. cv. Kocher-perle) root segments have been measured during a steady state in the presence or absence of ABA. Almost all flux rates were altered in the presence of external ABA, in particular the xylem transport R’ and the plasmalemma influx Ø_oc (see below) were strongly inhibited in the steady state. At the same time the presence of ABA induced a strong increase in the vacuolar K⁺ and Na⁺ content Q_v and a decrease in the cytoplasmic one (Q_c). Since the fluxes of an ion and its vacuolar or, in particular, cytoplasmic concentrations are interrelated, the ratios of fluxes originating from the cytoplasm and the cytoplasmic ion content were taken into account. On this basis ABA had the following effects: a) the secretion of K⁺ or Na⁺ to the xylem vessels was drastically inhibited; b) the plasmalemma K⁺ or Na⁺ efflux Ø_co was moderately stimulated and c) the tonoplast influx Ø_cv of Na⁺ was stimulated, while the tonoplast influx of K⁺ appeared to be unchanged (the decrease in Ø_cv being due to the decreased cytoplasmic K⁺ content). By a similar argument, also the apparent inhibition of the plasmalemma influx Ø_oc of K⁺ and Na⁺ in the steady state merely is an indirect effect of ABA. It only reflects the strong ABA-induced decrease in the xylem transport, that governs the magnitude of Ø_oc in the steady state. The results are discussed with reference to possible regulatory functions of ABA. In this respect it is suggested that – in particular under conditions of stress – ABA might regulate cellular metabolic processes by changing the cytoplasmic K⁺ level.     

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K+ channels in Arabidopsis guard cells

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca²⁺] ([Ca²⁺]_i), and also on mechanisms that are independent of [Ca²⁺]_i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)‐induced stomatal closure response in the [Ca²⁺]_i‐elevation‐independent pathway? (ii) How do ABA‐insensitive mutants affect the [Ca²⁺]_i‐elevation‐independent pathway? (iii) Does ABA enhance (prime) the Ca²⁺ sensitivity of anion and inward‐rectifying K⁺ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca²⁺]_i elevations and clamping [Ca²⁺]_i to resting levels. The absence of [Ca²⁺]_i elevations was confirmed by ratiometric [Ca²⁺]_i imaging experiments. ABA‐induced stomatal closure in the absence of [Ca²⁺]_i elevations above the physiological resting [Ca²⁺]_i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca²⁺]_i elevations. The ABA‐insensitive mutants ost1‐2, abi2‐1 and gca2 showed partial stomatal closure responses that correlate with [Ca²⁺]_i‐dependent ABA signaling. Interestingly, patch‐clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca²⁺ to activate S‐type anion channels and down‐regulate inward‐rectifying K⁺ channels, providing strong evidence for a Ca²⁺ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca²⁺]_i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca²⁺]_i sensitivity of stomatal closure mechanisms.     

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.     

The use of reconstitution and inhibitor/ion interaction assays to distinguish between Ca2+/H+and Cd2+/H+ antiporter activities of oat and tobacco tonoplast vesicles

Tonoplast, ion antiport activities are critical to ion homeostasis and sequestration in plants. The biochemical properties of these activities, and the enzymes that catalyse them, are little characterized. Here we applied biochemical approaches to study some characteristics and to distinguish between Ca²⁺/H⁺ and Cd²⁺/H⁺ antiporter activities of tonoplast vesicles from non‐transformed, wild‐type plants. Solubilization and reconstitution of oat‐seedling (Avena sativa L.) root tonoplast vesicles resulted in about a 6‐fold loss of protein, about a 6‐fold enhancement of Cd²⁺/H⁺ antiport specific activity (at 10 µM Cd²⁺), and almost complete loss of Ca²⁺/H⁺ antiport activity. Similar results were found for vesicles from mature tobacco (Nicotiana tabacum) roots. Cd²⁺ concentration‐dependent proton efflux was similar and linear with both oat vesicles and proteoliposomes. In contrast, Ca²⁺ concentration‐dependent proton efflux of oat vesicles was easily observed while that with proteoliposomes was minimal and non‐linear. Cd²⁺ pre‐treatment of oat vesicles reduced verapamil inhibition of Cd²⁺/H⁺ activity and verapamil binding to vesicles, while Ca²⁺ pre‐treatment was much less protective of Ca^2+/H⁺ activity and verapamil binding. Results show the usefulness of reconstitution, and also inhibitor/ion interaction assays for distinguishing between transporter activities in vitro, but they do not resolve the question of whether there are separate enzymes for Cd²⁺/H⁺ and Ca²⁺/H⁺. Our observation that solubilization and reconstitution have similar effects on both Cd²⁺/H⁺ and Ca²⁺/H⁺ activities of root tonoplast vesicles from immature oat and mature tobacco roots suggests that the transporters involved are similar in young and mature roots, and in roots of different species.     

Changes in Rb+ and Ca2+ influx in winter wheat roots before, during and after exposure to low temperature and short days

Influx of Rb⁺(⁸⁶Rb⁺) and Ca²⁺ (⁴⁵Ca²⁺) in roots of intact winter wheat (Triticum aestivum L. cv. Weibulls Starke II) was determined at intervals before, during and after exposure to cold acclimation conditions (2°C and 8 h light period). The plants were grown in nutrient medium of two ionic strengths. During the initial two weeks of growth at 16°C and 16 h light period, Rb⁺ influx into roots decreased with increasing age, probably as a consequence of a decreasing proportion of metabolically active roots. The presence of 10^?4M 2,4-dinitrophenol (DNP) reduced Rb⁺ influx to a low and constant level, indicating that metabolic influx was the dominant process. In contrast, Ca²⁺ influx in plants grown in full strength nutrient solution was higher in the presence than in the absence of DNP. This effect may have been due to an active extrusion mechanism mediating re-export of absorbed Ca²⁺(⁴⁵Ca²⁺) during the uptake experiment. With the metabolic uncoupler inhibiting such extrusion the Ca²⁺(⁴⁵Ca²⁺) influx mesured would increase. During cold treatment, Rb⁺ influx remained at a low level, and was further decreased when DNP was present in the uptake solution. This effect may have been due to inhibition of residual active influx of Rb⁺ at 2°C by the uncoupler and/or to a decrease in membrane permeability. In contrast to Rb⁺, Ca²⁺ influx increased during cold treatment, which could again be explained as inhibition of re-export. The presence of DNP reduced Ca²⁺ influx at 2°C, indicating decreased membrane permeability by DNP at low temperature. After transfer of plants from cold acclimation conditions to 16°C, Rb⁺ and Ca²⁺ influx increased in plants grown at both ionic strengths. Influx levels were independent of the length of the cold acclimation period (1, 6 and 8 weeks), but the patterns were different for the two ions. After each of the cold acclimation periods, Rb⁺ influx increased during the first week and decreased or remained at the same level during the second week, while Ca²⁺ influx always decreased during the second week of post-cold treatment.     

Effects of W-7, W-5, Verapamil and Diltiazem on vacuolar proton transport. Comparison of vacuolar H+-ATPase and H+-PPase from roots of Zea mays

The vacuolar membrane of plant cells is characterized by two proton pumps: the vacuolar H⁺-ATPase (V-ATPase; EC 3.6.1.3) and the vacuolar H⁺-PPase (V-PPase; EC 3.6.1.1). Recently, Du Pont and Morrissey reported that Ca²⁺ stimulates hydrolytic activity of purified V-ATPase (Arch. Biochim. Biophys., 1992. 294: 341–346). Since this effect may be due to degradation during purification further investigation of Ca²⁺ regulation of native V-ATPase was done. However, native tonoplast membranes contain a Ca²⁺/H⁺ antiport activity, which interferes with effects of calcium ions on proton transport activity of vacuolar ATPase. Therefore, the effects of anti-calmodulin drugs (W-7, W-5, calmidazolium), and calcium channel antagonists (Verapamil, Diltiazem) on proton transport activities of the vacuolar-type H⁺-ATPase and H⁺-PPase in tonoplast enriched membrane vesicle preparations from roots of Zea mays L. were studied. The concentrations for half maximal inhibition of vacuolar H⁺-ATPase (H⁺-PPase) were: 71 (191) μM W-7, 470 (> 800) μM W-5, 26 (24) μM calmidazolium (= compound R 24571). 398 (700) μM Verapamil, and 500 (1 330) μM Diltiazem. Estimation of Hill coefficients (n_H) for the inhibition by Verapamil showed a further difference between the two vacuolar proton pumps (H⁺-ATPase, n_H= 2.02; H⁺-PPase, n_n= 0.96). The data indicate that the vacuolar H⁺-ATPase itself is affected by these chemicals. It is suggested that some biological activities of W-7, W-5, Verapamil, and Diltiazem are due to their effects on proton translocation by the vacuolar-type H⁺-ATPase.     

Abscisic-acid-induced turion formation in Spirodela polyrrhiza L. IV. Comparative ion flux characteristics of the turion and the vegetative frond and the effect of ABA during early turion development

Abstract Using the method of compartmental analysis, the ion fluxes and compartment concentrations of Ca²⁺, K⁺ and Cl^- have been compared in the untreated vegetative frond and the abscisic acid (ABA) induced turion of Spirodela polyrrhiza. The ABA-induced turion is characterized by reduced Ca²⁺ exchange across the tonoplast and low vacuolar Ca²⁺ concentration relative to the vegetative frond. In addition the turion exhibits a higher plasmalemma flux with a correspondingly high Ca²⁺ concentration in the cytoplasm. The concentration of K⁺ and Cl^- is much lower in the cytoplasm of the ABA-induced turion than in the vegetative frond with the influx/efflux ratio at both the plasmalemma and the tonoplast being less than 1, a finding exhibited also in dormant storage tissue. Treatment of vegetative fronds with ABA for 18 h resulted in a reduced K⁺ plasmalemma efflux relative to untreated vegetative fronds and a concomitant increase in the cytoplasmic concentration. There was no rapid effect of ABA on Ca²⁺, K⁺ or Cl^- fluxes through either membrane. These results are consistent with the notion that drastic changes in ion fluxes and concentrations in the turion are a secondary consequence of ABA-induced development, possibly due to prior regulation by ABA of enzymes inherent to processes involved in membrane transport.     

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.