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 effect of inorganic phosphate on sodium fluxes in dog red blood cells

The effect of extracellular inorganic phosphate on Na⁺ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na⁺ efflux increases by 2- to 3-fold and Na⁺ influx increases approximately 2-fold. This enhancement of Na⁺ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4′-iso-thiocyantostilbene-2,2′-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na⁺ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na⁺ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.     

Cation-dependent uptake of zinc in human fibroblasts

The influence of K⁺ and Ca²⁺ on Zn²⁺ transport into cultured human fibroblasts was investigated. Zn²⁺ uptake was markedly reduced in the presence of both valinomycin and nigericin (electrogenic and electroneutral K ⁺ ionophores, respectively), and by reduction in the transmembrane K⁺ gradient produced by replacement of extracellular K⁺ with Na⁺, suggesting that Zn²⁺ may be driven by a Zn²⁺/K⁺ counter-transport system. To test the counter-transport hypothesis, we used ⁸⁶Rb as an analog of K ⁺ for efflux studies. The rate of Rb⁺ efflux was 3760 times that of Zn²⁺ uptake, thus the component of K⁺ involved in the Zn²⁺ counter-transport system was only a small proportion of the total K⁺ efflux. In investigating the effect of Ca²⁺ on Zn²⁺ uptake, we identified two components: (1) a basal Zn²⁺ uptake pathway, independent of hormonal or growth factors which does not require extracellular Ca²⁺ and (2) a Ca²⁺-dependent mechanism. The absence of Ca²⁺ decreased Zn²⁺ uptake, while increasing extracellular C+a²⁺ stimulated Zn²⁺ uptake. The effect was mediated by Ca²⁺ influx as the ionophores A23187 and ionomycin also stimulated Zn²⁺ uptake. We could not ascribe the Ca²⁺ effect to known Ca²⁺ influx pathways. We conclude that Zn²⁺ uptake occurs by a K⁺-dependent process, possibly by Zn²⁺/K⁺ counter-transport and that a component of this is also Ca²⁺-dependent.     

On the Protection by Inorganic Phosphate of Calcium-Induced Membrane Permeability Transition

The role of inorganic phosphate as inhibitor of mitochondrial membrane permeability transition was studied. It is shown that in mitochondria containing a high phosphate concentration, i.e., 68 nmol/mg, Ca²⁺ did not activate the pore opening. Conversely, at lower levels of matrix phosphate, i.e., 38 nmol/mg, Ca²⁺ was able to induce subsequent pore opening. The inhibitory effect of phosphate was apparent in sucrose-based media, but it was not achieved in KCl media. The matrix free Ca²⁺ concentration and matrix pH were lowered by phosphate, but they were always higher in K⁺-media. In the absence of ADP, phosphate strengthened the inhibitory effect of cyclosporin A on carboxyatractyloside-induced Ca²⁺ efflux. Acetate was unable to replace phosphate in the induction of the aforementioned effects. It is concluded that phosphate preserves selective membrane permeability by diminishing the matrix free Ca²⁺ concentration.     

Release of endogenous dopamine from tuberoinfundibular neurons

Release of endogenous dopamine (DA) from arcuate-periventricular nucleus-median eminence fragments has been analyzed in an $\text{in vitro}$ static incubation system.Exposure of these hypothalamic fragments to increasing concentrations of K⁺ ions produced a dose-dependent release of endogenous DA. The highest rate of K⁺-stimulated DA efflux occurred in the first 10 minutes, thereafter it progressively decline reaching prestimulated levels at 30 minutes. If two consecutive depolarizing stimuli of 40 mM KCl were applied to the same hypothalamic fragment, after a 40 minutes rest period, an equivalent release of endogenous DA occurred. Removal of Ca⁺⁺ ions from the incubation medium containing the Ca⁺⁺ chelator EGTA caused a decrease of basal DA efflux and completely prevented the K⁺-induced release of DA.Furthermore when verapamil, a blocker of Ca⁺⁺ entrance, was added to the incubation medium in a concentration of 50 μM, the K⁺-induced DA efflux was completely counteracted, whereas spontaneous release was unmodified.Finally nomifensine, a potent blocker of DA uptake, added $\text{in vitro}$ in a final concentration of 10 μM, significantly reinforced K⁺-induced release of endogenous DA. Since nomifensine did not modify basal DA release, this study confirmed its prevalent uptake blocking property rather than its releasing action on DA.     

Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina

The uptake of K⁺ and Ca²⁺ in Dunaliella salina is mediated by two distinct carriers: a K⁺ carrier with a high selectivity against Na⁺, Li⁺, and choline⁺ but not towards Rb⁺, K⁺, Cs⁺, or NH₄⁺, and a Ca²⁺ carrier with a high selectivity against Mg²⁺. The latter is specifically blocked by La³⁺ and by Cd²⁺. Apparent K_m values for K⁺ and Ca²⁺ uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K⁺ and Ca²⁺ uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H⁺-ATPases and protonionophores inhibit K⁺ and Ca²⁺ uptake and accelerate K⁺ efflux. The results suggest that an H⁺-ATPase in the cell membrane provides the driving force for K⁺ and Ca²⁺ uptake. Efflux measurements from ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ loaded cells suggest that part of the intracellular K⁺ and most of the intracellular Ca²⁺ is nonexchangeable with the extracellular pool. Correlations between phosphate and K⁺ contents and the effect of phosphate on K⁺ efflux suggest intracellular associations between K⁺ and polyphosphates. On the basis of these results, it is suggested that: (a) K⁺ and Ca²⁺ uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H⁺-ATPase of the plasma membrane. (b) Part of the intracellular K⁺ is associated with polyphosphate bodies, while most of the intracellular Ca²⁺ is accumulated in intracellular organelles in the algal cells.     

Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

Sulfhydryl-reactive heavy metals increase cell membrane K+ and Ca2+ transport in renal proximal tubule

Summary The cellular mechanisms by which nephrotoxic heavy metals injure the proximal tubule are incompletely defined. We used extracellular electrodes to measure the early effects of heavy metals and other sulfhydryl reagents on net K⁺ and Ca²⁺ transport and respiration (QO₂) of proximal tubule suspensions. Hg²⁺, Cu²⁺, and Au³⁺ (10^–4 m) each caused a rapid net K⁺ efflux and a delayed inhibition of QO₂. The Hg²⁺-induced net K⁺ release represented passive K⁺ transport and was not inhibited by barium, tetraethylammonium, or furosemide. Both Hg²⁺ and Ag⁺ promoted a net Ca²⁺ uptake that was nearly coincident with the onset of the net K⁺ efflux. A delayed inhibition of ouabainsensitive QO₂ and nystatin-stimulated QO₂, indicative of Na⁺, K⁺-ATPase inhibition, was observed after 30 sec of exposure to Hg²⁺. More prolonged treatment (2 min) of the tubules with Hg²⁺ resulted in a 40% reduction in the CCCP-uncoupled QO₂, indicating delayed injury to the mitochondria. The net K⁺ efflux was mimicked by the sulfhydryl reagents pCMBS and N-ethylmaleimide (10^–4 m) and prevented by dithiothreitol (DTT) or reduced glutathione (GSH) (10^–4 m). In addition, both DTT and GSH immediately reversed the Ag⁺-induced net Ca²⁺ uptake. Thus, sulfhydryl-reactive heavy metals cause rapid, dramatic changes in the membrane ionic permeability of the proximal tubule before disrupting Na⁺, K⁺-ATPase activity or mitochondrial function. These alterations appear to be the result of an interaction of the metal ions with sulfhydryl groups of cell membrane proteins responsible for the modulation of cation permeability.     

Use of an extracellular,ion-selective,vibrating microelectrode system for the quantification of K+, H+, and Ca2+ fluxes in maize roots and maize suspension cells

An ion-selective vibrating-microelectrode system, which was originally used to measure extracellular Ca²⁺ gradients generated by Ca²⁺ currents, was used to study K⁺, H⁺ and Ca²⁺ transport in intact maize (Zea mays L.) roots and individual maize suspension cells. Comparisons were made between the vibrating ion-selective microelectrode, and a technique using stationary ion-selective microelectrodes to measure ionic gradients in the unstirred layer at the surface of plant roots. The vibrating-microelectrode system was shown to be a major improvement over stationary ion-selective microelectrodes, in terms of sensitivity and temporal resolution. With the vibrating ion microelectrode, it was easy to monitor K⁺ influxes into maize roots in a background K⁺ concentration of 10 mM or more, while stationary K⁺ electrodes were limited to measurements in a background K⁺ concentration of 0.3 mM or less. Also, with this system it was possible to conduct a detailed study of root Ca²⁺ transport, which was previously not possible because of the small fluxes involved. For example, we were able to investigate the effect of the excision of maize roots on Ca²⁺ influx. When an intact maize root was excised from the seedling at a position 3 cm from the site of measurement of Ca²⁺ transport, a rapid fourfold stimulation of Ca²⁺ influx was observed followed by dramatic oscillations in Ca²⁺ flux, oscillating between Ca²⁺ influx and efflux. These results clearly demonstrate that wound or perturbation responses of plant organs involve transient alterations in Ca²⁺ transport, which had previously been inferred by demonstrations of touch-induced changes in cytoplasmic calcium. The sensitivity of this system allows for the measurement of ion fluxes in individual plant cells. Using vibrating K⁺ and H⁺electrodes, it was possible to measure H⁺efflux and both K⁺ influx and efflux in individual maize suspension cells under different conditions. The availability of this technique will greatly improve our ability to study ion transport at the cellular level, in intact plant tissues and organs, and in specialized cells, such as root hairs or guard cells.Symbol X amplitude of vibration The authors would like to thank Richard Sanger for his invaluable work on the design and improvement of the ion-selective vibratingmicroelectrode system. The research presented here was supported in part by U.S. Department of Agriculture Competitive Grant No. 90-37261-5411 to Leon Kochian and William Lucas.     

Silver ions induce a rapid Ca2+ release from isolated intact bovine rod outer segments by a cooperative mechanism

Summary Micromolar concentrations of silver ion activate large Ca²⁺ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag⁺-induced Ca²⁺ efflux from intact ROS depended on the Ag⁺ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag⁺ the rate of Ca²⁺ efflux was 7×10⁶ Ca²⁺/outer segment/sec; this represents a change in total intracellular Ca²⁺ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag⁺-induced Ca²⁺ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag⁺-induced Ca²⁺ efflux when gramicidin was present. In the presence of gramicidin, Ag⁺-induced Ca²⁺ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca²⁺ flux across ROS disk membranes. The ion selectivity of the Ag⁺-induced pathway proved to be broad with little discrimination between the alkali cations Li⁺, Na⁺, K⁺, and Cs⁺ or between Ca²⁺ and Mg²⁺. The properties of the Ag⁺-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.     

A microdialysis study in rat brain of dihydrokainate,a glutamate uptake inhibitor

Microdialysis in neostriatum of anaesthetized rats was performed to study effects on amino acid efflux of the glutamate uptake-inhibitor dihydrokainate (DHK). Both basal and K⁺-evoked (100 mM) efflux of glutamate increased in the presence of DHK. The increase in the basal glutamate efflux occurred at lower DHK concentrations than during K⁺-depolarization (when the extracellular glutamate concentration was several-fold higher), confirming that DHK is a competitive inhibitor. The increase in basal efflux caused by DHK did not exhibit Ca²⁺-dependency, whereas ∼50% of the increase in glutamate efflux during K⁺-depolarization was Ca²⁺-dependent. The Ca²⁺-dependent efflux is related to transmitter release, whereas the Ca²⁺-independent efflux is probably due to metabolic events and/or transport of DHK into cells in exchange for glutamate. Taurine efflux in response to DHK increased both during basal conditions and K⁺-depolarization, probably secondary to the increase in glutamate concentration, whereas aspartate, GABA, glutamine and alanine effluxes did not change.     

Further studies on the effect of phosphoenolpyruvate on respiration-dependent calcium transport by rat heart mitochondria

Phosphoenolpyruvate was found to depress extra oxygen consumption associated with Ca²⁺-induced respiratory jump by rat heart mitochondria. Addition of phosphoenolpyruvate to mitochondria which have accumulated Ca²⁺ in the presence of glutamate and inorganic phosphate causes the release of Ca²⁺ from mitochondria. The phosphoenolpyruvate-stimulated Ca²⁺ efflux can be observed with mitochondria loaded with low initial Ca²⁺ concentration (0.12 mM) in the incubation medium. Measurements of mitochondrial H⁺ translocation produced by addition of Ca²⁺ to respiring mitochondria show that phosphoenolpyruvate depresses H⁺ ejection and enhances H⁺ uptake by mitochondria. The Ca²⁺-releasing effect of phosphoenolpyruvate was found to be significantly stronger than that produced by rotenone when added to mitochondria loaded with Ca²⁺ in the presence of glutamate and inorganic phosphate. Dithiothreitol cannot overcome the effect of phosphoenolpyruvate on mitochondrial Ca²⁺ transport.     

Potassium fluxes across the blood brain barrier of the cockroach, Periplaneta americana

Potassium fluxes across the blood-brain barrier of the cockroach Periplaneta americana were measured using the scanning ion-selective microelectrode technique. In salines containing 15 mM or 25 mM K⁺, an efflux of K⁺ from the ganglia of isolated nerve cords was counterbalanced by an influx across the connectives. Metabolic inhibition with CN⁻ resulted in an increase in K⁺ efflux across both the ganglia and the connectives. Depletion of K⁺ by chilling the nerve cords in K⁺-free saline was associated with subsequent K⁺ influx across the connectives in K⁺-replete saline at room temperature. There were dramatic increases in K⁺ efflux across both ganglia and connectives when the nerve cords were exposed to the pore-forming antibiotic amphotericin B. K⁺ fluxes across the ventral nerve cord were also altered when paracellular leakage was augmented by transient exposure to 3 M urea. K⁺ efflux was reduced by the K⁺ channel blockers Ba²⁺ and tetraethylammonium or by exposure to Ca²⁺-free saline and K⁺ efflux from the ganglia was increased by addition of ouabain to the bathing saline. The results provide direct support for a model proposing that K⁺ is cycled through a current loop between the ganglia and the connectives and that both the Na⁺/K⁺-ATPase and K⁺ channels are implicated in extracellular K⁺ homeostasis within the central nervous system.     

Time and charge/pH-dependent activation of K+ channel-mediated K+ influx and K+/H+ exchange in guinea pig heart isolated mitochondria; role in bioenergetic stability

Mitochondria play an important role not only in producing energy for the cell but also for regulating mitochondrial and cell function depending on the cell's needs and environment. Uptake of cations, anions, and substrates requires a stable, polarized transmembrane charge potential (ΔΨ_m). Chemiosmosis requires ion exchangers to remove Na⁺, K⁺, Ca²⁺, PO₄^3?, and other charged species that enter mitochondria. Knowledge of the kinetics of mitochondrial (m) cation channels and exchangers is important in understanding their roles in regulating mitochondrial chemiosmosis and bioenergetics. The influx/efflux of K⁺, the most abundant mitochondrial cation, alters mitochondrial volume and shape by bringing in anions and H₂O by osmosis. The effects of K⁺ uptake through ligand-specific mK⁺ channels stimulated/inhibited by agonists/antagonists on mitochondrial volume (swelling/contraction) are well known. However, a more important role for K⁺ influx is likely its effects on H⁺ cycling and bioenergetics facilitated by mitochondrial (m) K⁺/H⁺ exchange (mKHE), though the kinetics and consequences of K⁺ efflux by KHE are not well described. We hypothesized that a major role of K⁺ influx/efflux is stimulation of respiration via the influx of H⁺ by KHE. We proposed to modulate KHE activity by energizing guinea pig heart isolated mitochondria and by altering the mK⁺ cycle to capture changes in mitochondrial volume, pH_m, ΔΨ_m, and respiration that would reflect a role for H⁺ influx via KHE to regulate bioenergetics. To test this, mitochondria were suspended in a 150 mM K⁺ buffer at pH 6.9, or in a 140 mM Cs⁺ buffer at pH 7.6 or 6.9 with added 10 mM K⁺, minimal Ca²⁺ and free of Na⁺. O₂ content was measured by a Clark electrode, and pH_m, ΔΨ_m, and volume, were measured by fluorescence spectrophotometry and light-scattering. Adding pyruvic acid (PA) alone caused increases in volume and respiration and a rapid decrease in the transmembrane pH gradient (ΔpH_m = pH_in–pH_ext) at pH_ext 6.9> > 7.6, so that ΔΨ_m was charged and maintained. BK_Ca agonist NS1619 and antagonist paxilline modified these effects, and KHE inhibitor quinine and K⁺ ionophore valinomycin depolarized ΔΨ_m. We postulate that K⁺ efflux-induced H⁺ influx via KHE causes an inward H⁺ leak that stimulates respiration, but at buffer pH 6.9 also utilizes the energy of ΔpH_m, the smaller component of the overall proton motive force, ΔμH⁺. Thus ΔpH_m establishes and maintains the ΔΨ_m required for utilization of substrates, entry of all cations, and for oxidative phosphorylation. Thus, K⁺ influx/efflux appears to play a pivotal role in regulating energetics while maintaining mitochondrial ionic balance and volume homeostasis.     

Calcium-dependent K efflux from rat submandibular gland: The effects of trifluoperazine and quinidine

The Ca²⁺-dependent K⁺ efflux from rat submandibular gland was studied using a K⁺-sensitive electrode. A K⁺ efflux was induced by either adrenalin or by using the divalent cation ionophore A23187 plus added Ca²⁺ to bypass the receptor mechanism. Trifluoperazine, which was used to investigate the role of calmodulin, was found to block the adrenalin-induced K⁺ efflux but not the A23187/Ca²⁺-induced K⁺ efflux. The adrenalin-induced K⁺ efflux was abolished by quinidine and the A23187/Ca²⁺-induced K⁺ efflux was significantly reduced by quinidine. In other experiments, the presence of indomethacin did not inhibit the adrenalin-induced K⁺ efflux, and exogenously added arachidonic acid did not induce a K⁺ efflux. It is concluded that neither prostaglandin synthesis, nor a cytosolic Ca²⁺-calmodulin complex is involved in the agonist-induced K⁺ efflux from rat submandibular gland. A similarity between the Ca²⁺-dependent K⁺ efflux mechanism of erythrocyte ghosts and submandibular tissue is indicated by their common response to quinidine.     

Neutral Red as a Redox Dye Induces K+ Efflux and Current-Voltage Changes in Eremosphaera,Lemna, and Guard Cells*

Membrane effects of the redox and pH indicator neutral red were studied with the chlorococcal alga Eremosphaera viridis, with Lemna gibba, and with “isolated” guard cells in epidermal peels of Valerianella locusta. Neutral red was extracellularly reduced and caused transmembrane current-voltage changes, an increase in membrane conductance by about 14 nS, an apparent K⁺ net efflux of up to 120 μmol g^?1 FW in 5 min, and an intracellular acidification by up to 0.7 pH units. Neutral red-triggered K⁺ net efflux was most pronounced at low pH, at an E_o more positive than ?200 mV, and without extracellular Ca²⁺. From the experimental data it is concluded that, due to the redox function of the phenazine molecule, extracellular neutral red triggers a trans-plasmalemma e^? transfer, leading to strong membrane depolarization and charge compensating K⁺ net efflux, in addition to some unspecific ion release. As a consequence the intracellular concentration of strong cations relative to strong anions (SID) decreases, resulting in intracellular acidification.     

Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone     

Role of calcium in reactive oxygen species-induced apoptosis in Candida albicans: an antifungal mechanism of antimicrobial peptide,PMAP-23

PMAP-23 (RIIDLLWRVRRPQKPKFVTVWVR-NH₂) is an antimicrobial peptide (AMP) derived from porcine myeloid. Membrane disruption is thought to underpin the anticandidal activity of PMAP-23. However, many AMPs act via mechanisms other than simple membrane permeabilisation. Here, we investigated the anticandidal mechanism of PMAP-23 at low concentrations. Membrane disruption and depolarisation and rapid K⁺ efflux were observed in Candida albicans cells treated with 5?µM PMAP-23. In contrast, 2.5?µM PMAP-23 caused membrane depolarisation and K⁺ efflux without membrane disruption. The lower PMAP-23 concentration increased cytosolic and mitochondrial Ca²⁺ levels. Disruption of Ca²⁺ homeostasis altered the NAD⁺/NADH ratio and resulted in reactive oxygen species (ROS) accumulation and glutathione oxidation. PMAP-23 treatment also stimulated apoptosis, as evidenced by metacaspase activation, DNA fragmentation, and phosphatidylserine externalisation. Pretreatment with the mitochondrial Ca²⁺ uptake inhibitor (ruthenium red) or ROS scavenger (N-acetylcysteine) attenuated these apoptotic events. Our results suggest that PMAP-23 induces apoptosis as antifungal mechanism, and mitochondrial Ca²⁺-induced ROS is major factor to trigger the apoptosis. Thus, the anticandidal activity of PMAP-23 is not based solely on disruption of biological membranes but also involves induction of apoptosis via mitochondrial Ca²⁺-dependent ROS. PMAP-23 mode of action sheds new light on the antifungal mechanism of antimicrobial peptides, supporting the role of Ca²⁺ and ROS in apoptosis regulation.     

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.     

Effect of Insulin on Potassium Flux and Water and Electrolyte Content of Muscles from Normal and from Hypophysectomized Rats

It was reported previously that insulin hyperpolarized rat skeletal muscle and decreased K⁺ flux in both directions. The observations on K⁺ flux are now extended to take advantage of the greater sensitivity to insulin of hyperphysectomized rats. Insulin caused a shift of water from extracellular to intracellular space if glucose was present, but not in its absence. Insulin caused net gain of muscle fiber K⁺, though not necessarily an increase in K⁺ concentration in fiber water. It probably also decreased intrafiber Na⁺ and Cl^-. Insulin decreased K⁺ efflux. The effect was dose-dependent. Muscles from hypophysectomized rats were more sensitive to the action of insulin on K⁺ flux than were those from normal rats. The effect was demonstrable within the time resolution of the system, suggesting that insulin's action is on cell surfaces. K⁺ influx was also decreased by insulin. Bookkeeping suggests that some K⁺ influx be called active. Insulin seemed to decrease active K⁺ influx and passive K⁺ efflux. It is not resolved whether insulin has a true dual effect or whether it acts only on passive fluxes in both directions (the apparent action on active K⁺ influx being an artefact of incomplete definition of passive flux) or whether a single alteration in the membrane may affect both active and passive fluxes.