Changes in the permeability of ascospores of Neurospora tetra-sperma during germination

The respiration and germination of activated ascospores of Neurospora tetrasperma have been shown to be almost completely inhibited by concentrations of ethylene diaminetetraacetic acid (EDTA) as low as 0.0035 M. In contrast, however, dormant ascospores are insensitive to this chelating agent. At any time up to about 150 minutes after activation Ca(++) or Mg(++) can completely reverse this toxicity but Cu(++), Co(++), and Mn(++) only partially reverse it. After this time, the minerals of the Neurospora "minimal" medium taken singly, or in various combinations cannot reverse this effect. Adding EDTA at 120 minutes after activation eliminates the lag period associated with its effect upon respiration. Inhibition occurs even though the cells seem to be impermeable to EDTA. Cationic exchange resins, as another example of a non-penetrating metal-binding agent, gave effects similar to those noted with EDTA. Of the resins used the H(+) form of IR-120 and the Na(+) and K(+) forms of amberlite IRC-50 were the most toxic to activated ascospores. On the other hand, dormant ascospores were entirely unaffected by the resins. The release of Ca(++) from activated ascospores coincided with the period of maximum sensitivity to EDTA. More than 60 per cent of the cell's content of K(+) is released by EDTA-inhibited ascospores. A low pH decreased the effectiveness of EDTA as a poison. The data are consistent with the possibility that non-penetrating metal-binding agents are toxic because of the irreversible removal of essential cations from the cell. The kinetic data for the inhibitory effects, and for the release of Ca(++) establish that the permeability of germinating ascospores to minerals changes drastically as a result of activation.     

Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of V-type ATPase from Arabidopsis

Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.     

Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ(m)) and pH gradient (ΔpH(m)) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH(m), whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH(m) in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (～7.6) and ΔpH(m) (～0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH(m) decreased during cytosolic Ca(2+) elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca(2+)-ATPases and transmitted to mitochondria by P(i)/H(+) symport and K(+)/H(+) exchange, whereas the decrease in ΔpH(m) reflected the low H(+)-buffering power of mitochondria (～5 mm, pH 7.8) compared with the cytosol (～20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca(2+) and pH, mitochondria alkalinized and ΔpH(m) increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca(2+) caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH(m) are regulated by opposing Ca(2+)-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.     

Regulation of Na+/H+ and Cl-/HCO3- antiports in Vero cells

The effect of serum, phorbol-12-myristate-13-acetate (TPA), and forskolin on the activity Na+/H+ antiport and the Na(+)-coupled and Na(+)-independent Cl-/HCO3- antiport was studied in Vero cells by measuring 22Na+ and 36Cl- fluxes and changes in cytosolic pH (pHi). The Na(+)-independent Cl-/HCO3- antiport, which acts as an acidifying mechanism, is strongly pH-sensitive. In serum-starved cells it is activated at alkaline cytosolic pH, with a half-maximal activity at pHi approximately 7.20. Incubation with serum increased the activity of the Na(+)-independent Cl-/HCO3- antiport at pHi values from 6.8 to 7.2. Thus serum appeared to alter the pHi sensitivity of this antiporter such that the threshold value for activation of the antiport was shifted to a more acidic value. Na+/H+ antiport was somewhat stimulated initially by addition of serum, but further incubation with serum (greater than 45 min) decreased its activity. The activity of the Na(+)-coupled Cl-/HCO3- antiport, which is the major alkalinizing antiport in Vero cells, was not altered by short-term incubation with serum (less than 10 min) but decreased after prolonged incubation (greater than 45 min). Our findings with TPA and forskolin indicate that the effect of serum is partly mediated by the protein kinase C pathway, whereas the cyclic adenosine monophosphate pathway does not appear to play an important role. The net effect of serum on the pHi-regulating antiports was a slight decrease in intracellular pH.     

Molecular cloning,expression, and functional characterization of a novel zebrafish cytosolic sulfotransferase

By searching the zebrafish expressed sequence tag (EST) database, we have identified a cDNA clone encoding a putative zebrafish cytosolic sulfotransferase (ST). This cDNA was isolated and subjected to nucleotide sequencing. Analysis of the sequence data revealed that this novel zebrafish ST displays 32-35% amino acid sequence identity to members of all major cytosolic ST gene families. Therefore, this zebrafish ST, while belonging to the cytosolic ST gene superfamily, appears to be independent from all known constituent ST gene families. Recombinant zebrafish ST, expressed using the pET23c prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 34-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified zebrafish ST displayed sulfating activities toward dopamine and thyroid hormones (T(3) and T(4)), with a pH optimum spanning 7-9. The enzyme also exhibited activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 48 degrees C. Among 10 divalent metal cations tested, Fe(++), Hg(++), Co(++), Zn(++), Cu(++), and Cd(++) exhibited dramatic inhibitory effects on the activity of the enzyme. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic ST.     

The intracellular dissipation of cytosolic calcium following glucose re-addition to carbohydrate depleted Saccharomyces cerevisiae

Glucose re-addition to carbohydrate starved yeast cells leads to a transient elevation of eytosolic calcium (TECC). Concomitantly, a cytosolic proton extrusion occurs through the activation of the vacuolar H(+)-ATPase and the plasma membrane H(+)-ATPases. This study addressed the dissipation of the TECC through intracellular compartmentalization and the possible affects of the H(+)-ATPases on this process. Both the vacuole and the Golgi-ER apparatus were found to play important roles in distributing calcium to internal stores. Additionally, the inhibition of cytosolic proton extrusion augmented cytosolic calcium responses. A model where pH dependent cytosolic calcium buffering plays an important role in the dissipation of the TECC in Saccharomyces cerevisiae is proposed.     

In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHluorin

Peroxisomes are metabolically active organelles that participate in the oxidation of long-chain fatty acids and in the biosynthesis of bile acids, cholesterol, and ether phospholipids. Even though maintenance of a stable acid-base milieu is essential for proper peroxisomal function, the determination of the peroxisomal pH (pH(p)) remains inconclusive, and little is known about its regulation. To measure the pH of intact peroxisomes in situ, we used the peroxisome-specific carboxyl-terminal targeting sequence, SKL, to deliver a pH-sensitive mutant of the green fluorescent protein (pHluorin-SKL) selectively into peroxisomes. Proper targeting was verified by colocalization with the peroxisomal marker catalase. Peroxisomes were visualized by imaging fluorescence microscopy, and ratiometric measurements were combined with calibration using ionophores or a null-point method to estimate pH(p). The pH(p) was between 6.9 and 7.1, resembling the cytosolic pH. Manipulation of the cytosolic pH in intact cells or after permeabilization of the plasmalemma with streptolysin O revealed that pH(p) changed in parallel, suggesting that the peroxisomal membrane is highly permeable to H(+) (equivalents). We conclude that peroxisomes do not regulate their pH independently, but instead their large H(+) permeability effectively connects them with the buffer reservoir of the cytoplasm and with the homeostatic mechanisms that control cytosolic pH.     

Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor

NADH is a key metabolic cofactor whose sensitive and specific detection in the cytosol of live cells has been difficult. We constructed a fluorescent biosensor of the cytosolic NADH-NAD(+) redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex. Although the initial construct reported [NADH] × [H(+)] / [NAD(+)], its pH sensitivity was eliminated by mutagenesis. The engineered biosensor Peredox reports cytosolic NADH:NAD(+) ratios and can be calibrated with exogenous lactate and pyruvate. We demonstrated its utility in several cultured and primary cell types. We found that glycolysis opposed the lactate dehydrogenase equilibrium to produce a reduced cytosolic NADH-NAD(+) redox state. We also observed different redox states in primary mouse astrocytes and neurons, consistent with hypothesized metabolic differences. Furthermore, using high-content image analysis, we monitored NADH responses to PI3K pathway inhibition in hundreds of live cells. As an NADH reporter, Peredox should enable better understanding of bioenergetics.     

Identification and partial purification of a cytosolic activator of vacuolar H(+)-ATPases from mammalian kidney.

A factor that activates affinity-purified vacuolar H(+)-ATPase from bovine kidney microsomes was identified and partially purified from bovine kidney cytosol. The activator is a heat-stable, trypsin-sensitive acidic protein with a Mr by gel filtration of approximately 35,000. The activator increased the activity of renal microsomal and brush border H(+)-ATPase by over 60% but stimulated lysosomal H(+)-ATPase activity by only 28%; it had little or no activity against the remaining N-ethylmaleimide-insensitive ATPase in kidney microsomes and other transport ATPases. Stimulation of ATPase activity appeared to result from binding of the activator to the H(+)-ATPase. Activation was saturable, with a Hill coefficient of 1 at low protein concentrations. Both activator binding and stimulation of H(+)-ATPase activity were enhanced at pH values less than or equal to 6.5. The activator has selective effects on different H(+)-ATPases and is poised to activate the enzyme at low physiologic values of cytosolic pH; this newly identified cytosolic proteins may participate in the physiologic regulation of the vacuolar H(+)-ATPase.     

Evidence of differential pH regulation of the Arabidopsis vacuolar Ca2+/H+ antiporters CAX1 and CAX2.

The Arabidopsis Ca(2+)/H(+) antiporters cation exchanger (CAX) 1 and 2 utilise an electrochemical gradient to transport Ca(2+) into the vacuole to help mediate Ca(2+) homeostasis. Previous whole plant studies indicate that activity of Ca(2+)/H(+) antiporters is regulated by pH. However, the pH regulation of individual Ca(2+)/H(+) antiporters has not been examined. To determine whether CAX1 and CAX2 activity is affected by pH, Ca(2+)/H(+) antiport activity was measured in vacuolar membrane vesicles isolated from yeast heterologously expressing either transporter. Ca(2+) transport by CAX1 and CAX2 was regulated by cytosolic pH and each transporter had a distinct cytosolic pH profile. Screening of CAX1/CAX2 chimeras identified an amino acid domain within CAX2 that altered the pH-dependent Ca(2+) transport profile so that it was almost identical to the pH profile of CAX1. Results from mutagenesis of a specific His residue within this domain suggests a role for this residue in pH regulation.     

Vesicular ATPase inserted into the plasma membrane of motor terminals by exocytosis alkalinizes cytosolic pH and facilitates endocytosis

Key components of vesicular neurotransmitter release, such as Ca(2+) influx and membrane recycling, are affected by cytosolic pH. We measured the pH-sensitive fluorescence of Yellow Fluorescent Protein transgenically expressed in mouse motor nerve terminals, and report that Ca(2+) influx elicited by action potential trains (12.5-100 Hz) evokes a biphasic pH change: a brief acidification (～ 13 nM average peak increase in [H(+)]), followed by a prolonged alkalinization (～ 30 nM peak decrease in [H(+)]) that outlasts the stimulation train. The alkalinization is selectively eliminated by blocking vesicular exocytosis with botulinum neurotoxins, and is prolonged by the endocytosis-inhibitor dynasore. Blocking H(+) pumping by vesicular H(+)-ATPase (with folimycin or bafilomycin) suppresses stimulation-induced alkalinization and reduces endocytotic uptake of FM1-43. These results suggest that H(+)-ATPase, known to transfer cytosolic H(+) into prefused vesicles, continues to extrude cytosolic H(+) after being exocytotically incorporated into the plasma membrane. The resulting cytosolic alkalinization may facilitate vesicular endocytosis.     

Interrelationship between growth factor-induced activation of the Na+/H(+)-antiporter and mobilization of intracellular Ca2+ in NIH3T3-fibroblasts

Addition of serum growth factors or bombesin to quiescent NIH3T3-fibroblasts leads to a simultaneous mobilization of intracellular Ca2+ and an increase in cytosolic pH which is inhibitable by dimethylamiloride. The mobilization of intracellular Ca2+ is a pH-dependent process with an optimum at pH 7.1. In quiescent cells with a pHi greater than or equal to 6.8, inhibition of the Na+/H(+)-antiporter by dimethylamiloride or reduction of extracellular Na+ attenuates the growth factor induced Ca2(+)-response. It is concluded that the growth factor induced activation of the Na+/H(+)-antiporter facilitates the mobilization of Ca2+ by shifting the internal pH towards the optimum for the Ca2(+)-release.     

Photosensory transduction in unicellular eukaryote Blepharisma

In the ciliated protozoan Blepharisma, step-up photophobic response is mediated by a novel type of photosensory complex of pink-colored pigment "blepharismins" and 200-kDa membrane protein contained in the pigment granules located just beneath the plasma membrane. We found that the fluorescence intensity of isolated blepharismins decreased prominently with a decrease of H(+) concentration in the surrounding medium. In the present study, therefore, we utilized the endogenous pigment blepharismins as the pH indicator. Light stimulation evoked a sudden decrease in fluorescence intensity in a photosensitive anterior portion of the cell, suggesting that a drop in H(+) concentration occurred in the anterior region. The result indicates that the photosignal is transduced into cytoplasmic signaling of H(+) translocation across the outer membrane surrounding the pigment granules, so that cytosolic H(+) concentration in the vicinity of plasma membrane might be increased.     

Effect of azathioprine on Na(+)/H(+) exchanger activity in dendritic cells

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 μg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 μM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.     

pH regulation in the intracellular malaria parasite, Plasmodium falciparum. H(+) extrusion via a V-type H(+)-ATPase

The mechanism by which the intra-erythrocytic form of the human malaria parasite, Plasmodium falciparum, extrudes H(+) ions and thereby regulates its cytosolic pH (pH(i)), was investigated using saponin-permeabilized parasitized erythrocytes. The parasite was able both to maintain its resting pH(i) and to recover from an imposed intracellular acidification in the absence of extracellular Na(+), thus ruling out the involvement of a Na(+)/H(+) exchanger in both processes. Both phenomena were ATP-dependent. Amiloride and the related compound ethylisopropylamiloride caused a substantial reduction in the resting pH(i) of the parasite, whereas EMD 96785, a potent and allegedly selective inhibitor of Na(+)/H(+) exchange, had relatively little effect. The resting pH(i) of the parasite was also reduced by the sulfhydryl reagent N-ethylmaleimide, by the carboxyl group blocker N,N'-dicyclohexylcarbodiimide, and by bafilomycin A(1), a potent inhibitor of V-type H(+)-ATPases. Bafilomycin A(1) blocked pH(i) recovery in parasites subjected to an intracellular acidification and reduced the rate of acidification of a weakly buffered solution by parasites under resting conditions. The data are consistent with the hypothesis that the malaria parasite, like other parasitic protozoa, has in its plasma membrane a V-type H(+)-ATPase, which serves as the major route for the efflux of H(+) ions.     

Determinants of the pH of the Golgi complex

The factors contributing to the establishment of the steady state Golgi pH (pH(G)) were studied in intact and permeabilized mammalian cells by fluorescence ratio imaging. Retrograde transport of the nontoxic B subunit of verotoxin 1 was used to deliver pH-sensitive probes to the Golgi complex. To evaluate whether counter-ion permeability limited the activity of the electrogenic V-ATPase, we determined the concentration of K(+) in the lumen of the Golgi using a null point titration method. The [K(+)] inside the Golgi was found to be close to that of the cytosol, and increasing its permeability had no effect on pH(G). Moreover, the capacity of the endogenous counter-ion permeability exceeded the rate of H(+) pumping, implying that the potential across the Golgi membrane is negligible and has little influence on pH(G). The V-ATPase does not reach thermodynamic equilibrium nor does it seem to be allosterically inactivated at the steady state pH(G). In fact, active H(+) pumping was detectable even below the resting pH(G). A steady state pH was attained when the rate of pumping was matched by the passive backflux of H(+) (equivalents) or "leak." The nature of this leak pathway was investigated in detail. Neither vesicular traffic nor H(+)/cation antiporters or symporters were found to contribute to the net loss of H(+) from the Golgi. Instead, the leak was sensitive to voltage changes and was inhibited by Zn(2+), resembling the H(+) conductive pathway of the plasma membrane. We conclude that a balance between an endogenous leak, which includes a conductive component, and the H(+) pump determines the pH at which the Golgi lumen attains a steady state.     

Membrane surface charge dictates the structure and function of the epithelial Na+/H+ exchanger

The Na(+)/H(+) exchanger NHE3 plays a central role in intravascular volume and acid-base homeostasis. Ion exchange activity is conferred by its transmembrane domain, while regulation of the rate of transport by a variety of stimuli is dependent on its cytosolic C-terminal region. Liposome- and cell-based assays employing synthetic or recombinant segments of the cytosolic tail demonstrated preferential association with anionic membranes, which was abrogated by perturbations that interfere with electrostatic interactions. Resonance energy transfer measurements indicated that segments of the C-terminal domain approach the bilayer. In intact cells, neutralization of basic residues in the cytosolic tail by mutagenesis or disruption of electrostatic interactions inhibited Na(+)/H(+) exchange activity. An electrostatic switch model is proposed to account for multiple aspects of the regulation of NHE3 activity.     

Importance of bicarbonate transport for protection of cardiomyocytes against reoxygenation injury

Isolated cardiomyocytes from adult rats were incubated in anoxic bicarbonate-buffered media at extracellular pH (pH(o)) 6.4 until a cytosolic Ca(2+) overload and intracellular pH (pH(i)) of 6.4 were reached. On reoxygenation, the pH of the medium was changed to 7.4 to activate the Na(+)/H(+)exchanger (NHE) and the Na(+)-HCO(-)(3) symporter (NBS). The reoxygenation was performed in the absence or presence of the NHE inhibitor HOE-642 (3 micromol/l) and/or the NBS inhibitor DIDS (0.5 mmol/l), as in bicarbonate-free media. In reoxygenated control cells pH(i) rapidly recovered to the preanoxic level, and a burst of spontaneous oscillations of cytosolic Ca(2+) occurred, accompanied by the development of hypercontracture. When NBS and NHE were simultaneously inhibited during reoxygenation, pH(i) recovery was prevented, Ca(2+) oscillations were attenuated, and hypercontracture was abolished. Sole inhibition of NBS or NHE showed no protection against hypercontracture. In the absence of cytosolic acidosis, HOE-642 or DIDS did not prevent hypercontracture induced by Ca(2+) overload. The results demonstrate that simultaneous inhibition of NHE and NBS is needed to protect myocardial cells against reoxygenation-induced hypercontracture.     

23Na NMR study of the interaction between hyaluronan and the bications Ca(++), Mg(++) and Cu(++)

The relaxation rate R = pi Delta nu(1/2) of the quadrupolar (23)Na nucleus was measured at pH approximately 7 using a 200 MHz NMR spectrometer with a view to observe the interaction between hyaluronan and its natural counterion Na(+) and the bications Ca(++), Mg(++) and Cu(++). An interpretation of our results, by means of the "entropy of fluctuations" concept of Na(+), is presented. We show that Cu(++) ions are more effective than Ca(++) and Mg(++). A possible model of complexation of Cu(++) in a cage formed by the 1-4 glycosidic bond, the carboxylate side-chain and the acetoamide side-chain is proposed, according to electrostatic potential computations using the ZINDO1 quantum semi empirical method.     

Cation-binding Capacity of Membranes Isolated from Micrococcus lysodeikticus

A study was made of H(+), Na(+), K(+), Ca(++), and Mg(++) binding and ion-exchange properties of the plasma-mesosome membrane system isolated from Micrococcus lysodeikticus strain NCTC 2665. Titration curves were obtained on membranes prepared according to the method of M. R. J. Salton and further exposed to pH 4 for 4 hr (membranes-H). The dissociation coefficients and binding capacities were obtained by applying the mass law equation and the plot of G. Schatchard to the data. The membranes-H possess four kinds of dissociable groups with pK 4.96, 4.18, 3.60, and 3.09, respectively, and a total binding capacity of 0.65 meq/g (dry weight). Potentiometric titrations of cations in the presence and in the absence of membranes-H show that cations (Na(+), K(+), Ca(++), and Mg(++)) are bound by the dissociated groups of the membrane. The fall in pH value for bivalent cations is greater than that for monovalent cations. Cations of the same valency produce equal diminutions on pH. Furthermore, ion-exchange tests carried out on membranes saturated with Mg(++) or Na(+) and suspended in a medium containing (45)Ca show that the cations are reversibly bound.