Effect of divalent cations on the potassium contracture of slow muscle fibers ofRana temporaria

Summary K contractures of single slow muscle fibers ofRana temporaria were measured isometrically in the presence of normal, reduced, and increased Ca²⁺ concentrations at 18 to 20°C. At normal Ca²⁺ concentration (1.8mm) contracture tension decreased from its peak value of 35.4±8.2 N/cm² to 59.4±23.9% within one minute, and to 48.3±27% within two minutes (30 fibers). Peak tension was virtually unaffected by changes of the Ca²⁺ concentration, but maintenance of tension was impaired by low (0.2mm), and improved by high (10mm) Ca²⁺ concentrations. When Ca²⁺ was added during the K contracture, there was practically no restoration of lost tension. Effects similar to those of Ca²⁺ were observed upon addition of foreign divalent cations to the medium. Co²⁺, Ni²⁺, and Cd²⁺ were slightly more effective than Ca²⁺ and Mn²⁺; the smallest effects were obtained with Mg²⁺, Sr²⁺, and Ba²⁺. The effects of foreign divalent cations were independent of the presence of Ca²⁺. It is concluded that in slow fibers ofRana temporaria maintenance of contracture tension is not due to an influx of Ca²⁺ ions. Instead, binding of divalent cations to superficial sites seems to be essential.     

Toxicity of chromium and tin toAnabaena doliolum Interaction with bivalent cations

Summary The toxicity of chromium and tin on growth, photosynthetic carbon-fixation, oxygen evolution, heterocyst differentiation and nitrogenase activity ofAnabaena doliolum and its interaction with bivalent cations has been studied. Some interacting cations, viz. Ca²⁺, Mg²⁺ and Mn²⁺, substantially antagonised the toxic effects of chromium and tin with reference to growth, heterocyst differentiation and nitrogenase activity in the following hierarchal sequence: Ca²⁺ > Mg²⁺ > Mn²⁺. However, the sequence of hierarchy was Mg²⁺ > Ca²⁺ > Mn²⁺ for carbon fixation and Mn²⁺ > Mg²⁺ > Ca²⁺ for photosynthetic oxygen evolution. Synergistically inhibitory patterns were noticed for all the parameters, viz. growth,¹⁴CO₂ uptake, oxygen evolution, heterocyst differentiation and nitrogenase activity ofA. doliolum when Ni²⁺, Co²⁺ and Zn²⁺ were combined with the test metals in the growth medium. These cations followed the following sequence of synergistic inhibition: Ni²⁺ > Co²⁺ > Zn²⁺. Among all the interacting cations, Ca²⁺, Mg²⁺ and Mn²⁺ exhibited antagonistic effects which relieved the test cyanobacterium from metal toxicity. In contrast to this, Ni²⁺, CO²⁺ and Zn²⁺ showed synergistic inhibition which potentiating the toxicity of test metals in the N₂-fixing cyanobacteriumA. doliolum. It is evident from the present study that bivalent cations, viz. Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Co²⁺ and Zn²⁺, may appreciably regulate the toxicity of heavy metals in N₂-fixing cyanobacteria if present in aquatic media.     

Effects of Ca2+ and other divalent cations on K+-evoked force production of slow muscle fibers from Rana esculenta and Rana pipiens

Summary Slow muscle fibers were dissected from cruralis muscles of Rana esculenta and Rana pipiens. Isometric contractures were evoked by application of K⁺-rich Ringer's containing Ca²⁺, Ni²⁺, Co²⁺, Mn²⁺ or Mg²⁺. High (7.2 mmol/liter) external Ca²⁺ concentration raised, 0 Ca²⁺ lowered the K⁺ threshold. Replacing Ca²⁺ by Ni²⁺ or Co²⁺ had an effect similar to that of high Ca²⁺ Ringer's. In Mg²⁺ Ringer's the K⁺ concentration-response curve was flattened. These effects were observed already after short exposure times in both species of slow fibers. When Ca²⁺ was removed for long periods of time the slow fibers of R. esculenta lost their contractile response to application of high K⁺ concentrations much more quickly than those of R. pipiens, while the response to caffeine (20 mmol/liter) was maintained. Upon readmission of Ca²⁺ contractile ability was quickly restored in the slow fibers of both R. esculenta and R. pipiens, but the effects of Ni²⁺ (or Co²⁺, Mn²⁺ and Mg²⁺) were much larger in R. esculenta than in R. pipiens slow fibers. It is concluded that divalent cations have two different sites of action in slow muscle fibers. K⁺ threshold seems to be affected through binding to sites at the membrane surface; these sites bind Ni²⁺ and Co²⁺ more firmly than Ca²⁺. The second site is presumably the voltage sensor in the transverse tubular membrane, which controls force production, and where Ca²⁺ is the most effective species of the divalent cations examined.We are grateful to Mrs. S. Pelvay for technical assistance.     

Divalent cation-induced interaction of phospholipid vesicle and monolayer membranes

The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg²⁺, Ca²⁺, Mn²⁺, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time.When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg²⁺, 7 mM for Sr²⁺, 6 mM for Ca²⁺, 3.5 mM for Ba²⁺ and 1.8 mM for Mn²⁺. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn²⁺ > Ca²⁺ > Mg²⁺ and their critical concentrations were in between the former two cases. The threshold concentrations also depended upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1 : 1) vesicles, above the critical concentrations of Mn²⁺ and Ca²⁺, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h.This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The     

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase ofRhodospirillum rubrum: Effects of pH and divalent cations

The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores ofRhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 while the Pi-PPi exchange reaction was at 7.5; the pH affects mainly theK _m of Mg²⁺ or Pi for the enzyme; Mn²⁺ and Co²⁺ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg²⁺; other divalent cations like Zn²⁺ or Ca²⁺ do not support the exchange reaction. In the hydrolytic reaction, Zn²⁺, at low concentration, substitutes for Mg²⁺ as substrate, and Co²⁺ also substitutes in limited amount (50%). Other cations (Ca²⁺, Cu²⁺, Fe²⁺, etc.) do not act as substrates in complex with PPi. The Zn²⁺ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn²⁺. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn²⁺>Mn²⁺>Ca²⁺Co²⁺>Fe²⁺>Cu²⁺>Mg²⁺. The data in this work suggest that H⁺ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.     

Regulation of smooth muscle phosphatase-II by divalent cations

Summary Smooth Muscle Phosphatases II (SMP-I1) which has been purified from turkey gizzards and previously classified as protein phosphatase 2C, is inactive in the absence of divalent cations. Study of the activation of SMP-II by Mg²⁺ and Mn²⁺ revealed differences in the modes of activation by these cations. The maximal activation elicited by Mg²⁺ is 1.5–2.5-fold higher than the maximal Mn²⁺ activation. However, the latter is achieved at a lower concentration than the maximal Mg²⁺-activation. Furthermore, at low cation concentrations ( 2 mM), the Mn²⁺-activated activity is higher than the Mg²⁺-activated activity. In the presence of both cations, the effect of Mn²⁺ predominates suggesting that the affinity of the enzyme for Mn²⁺ is greater than for Mg²⁺. In contrast to Mg²⁺ and Mn²⁺, Ca²⁺ does not activate SMP-II but it was observed to antagonize the effects of Mg²⁺ and Mn²⁺. Ca²⁺ acts as a competitive inhibitor of Mg²⁺. However, the inhibitory effect at high Ca²⁺ concentrations is not completely reversed by increasing the Mg²⁺ concentration. Mn²⁺ activation is also inhibited by Ca²⁺ but to a lesser extent. Ca²⁺ cannot completely inhibit Mn²⁺-activation suggesting that SMP-I1 has greater affinity for Mn²⁺ than for Ca²⁺. The finding that Ca²⁺ inhibits the activation of SMP-II raises the possibility that Ca²⁺ may be a regulator of SMP-II in vivo.Abbreviations SMP-II Smooth Muscle Phosphatase-II - MOPS 3-[N-Morpholine]propane Sulfonic Acid - PLC Phosphorylated Myosin Light Chains     

Dual Control by Divalent Cations and Mitogenic Cytokines of α4β1 and α5β1 Integrin Avidity Expressed by Human Hemopoietic Cells

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function α4β1 and α5β1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by “inside-out” mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn²⁺, Mg²⁺ and Ca²⁺ for α4β1 and α5β1 activation by “inside-out” mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-β₁ integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of β₁ integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, α4β1 and α5β1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca²⁺, Mg²⁺ or Mn²⁺ were able to restore adhesive functions of α4β1 and α5β1 when activated by 8A2, only Mg²⁺ and Mn²⁺ were able to support activation of α5β1 and α5β1 by cytokines. Furthermore, high concentrations of Ca²⁺ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn²⁺ and cytokines but not by 8A2. On the contrary, in the presence of both Ca²⁺ and Mg²⁺, Mn²⁺ had an additive effect on the activation of α5β1 and α5β1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca²⁺, Mg²⁺ and Mn²⁺ may modulate in vivo α4β1 and α5β1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

A transient outward current dependent on external calcium in rat cerebellar granule cells

Summary The outward potassium current of rat cerebellar granule cells in culture was studied with the whole-cell patch-clamp method. Two voltage-dependent components were identified: a slow current, resembling the classical delayed rectifier current, and a fast component, similar to anI _A-type current. The slow current was insensitive to 4-aminopyridine and independent of external Ca²⁺, but significantly inhibited by 3mM tetraethylammonium. The fast current was depressed by external 4-aminopyridine, with an ED₅₀=0.7mM, and it was abolished by removal of divalent cations from the external medium. The sensitivity of the transient outward current to different divalent cations was investigated by equimolar substitution of Ca²⁺, Mn²⁺ and Mg²⁺. In 2.8mM Mn²⁺, the transient potassium conductance was comparable to that in 2.8mM Ca²⁺, while in 2.8mM Mg²⁺ the transient component was drastically reduced, as in the absence of any divalent cations. However, when Ca²⁺ was present, Mg²⁺ up to 5mM had no effect. The transient current increased with increasing concentrations of external Ca²⁺, [Ca²⁺] _o , and the maximum conductancevs. [Ca²⁺] _o curve could be approximated by a one-site model. In addition, the current recorded with 5.5mM BAPTA in the intracellular solution was not different from that recorded in the absence of any Ca²⁺ buffer. These results suggest that divalent cations modulate the potassium channel interacting with a site on the external side of the cell membrane.     

Identification of brain ecto-apyrase as a phosphoprotein

The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca²⁺ 0.25, Mg²⁺ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 M) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe²⁺, Zn²⁺ or Cu²⁺. Ca²⁺ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg²⁺ or Mn²⁺ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn²⁺ or Ca²⁺ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba²⁺, Zn²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ca²⁺), as well as La³⁺, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca²⁺, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca²⁺.     

Kinetic characterization of barley root plasma membrane-bound Ca2+- and Mg2+-dependent adenosine triphosphatase activities

A plasma membrane-rich microsome fraction isolated from barley (Hordeum vulgare L. cv. Conquest) roots contained considerable divalent cation-dependent ATPase activity when assayed at 16°C. The maximal divalent cation-stimulation of the apparent basal ATPase activity varied as Ca²⁺ > Mg²⁺ > Mn²⁺= Zn²⁺ > Co²⁺ > Ni²⁺, with all other divalent cations tested being inhibitory. Double reciprocal plots of the Ca²⁺- and Mg²⁺-dependent ATPase velocities as a function of substance concentration were nonlinear, suggesting the presence of multiple catalytic sites. Both MgATP^2- and CaATP^2- served as the true substrates and apparently bind to the same catalytic sites. Free ATP and Ca²⁺ could inhibitit the Ca²⁺- and Mg²⁺-dependent ATPase. Increasing free Mg²⁺ levels enhanced the affinity of the Mg²⁺-dependent ATPase for MgATP^2-, while slightly inhibiting the V_max values. Other divalent cation-nucleoside triphosphate complexes produced maximal enzyme velocities equal to or greater than those generated by CaATP^2- and MgATP^2-. However, the ATPase had significantly higher affinities for CaATP^2- and MgATP^2-, than for the alternative substrates. The high and low affinity components of the Ca²⁺- and Mg²⁺-dependent ATPase exhibited optimal V_max values at pH 5 and 6, respectively. Analysis of the pH-dependence of the enzyme K_m values indicated enzyme-substrate binding with charge neutralization at neutral and alkaline pH's. Nonlinear double reciprocal plots were obtained at all assay temperatures. However, the complexity of the enzyme kinetics became less apparent at the higher assay temperatures. The kinetics of the barley root divalent cation-dependent ATPase activities are discussed in terms of the kinetics of ATPases from other plants and the methods used to obtain them, and compared to the kinetics of ion transport ATPases from animal membranes.     

Biochemical characterization of glutamine synthetase from the diazotrophic cyanobacterium,Anabaena doliolum

In cyanobacteria, the glutamine synthetase-L-glutamine-2-oxoglutarate aminotransferase (GS-GOGAT) pathway is the major ammonia-assimilating route. The GS ofAnabaena doliolum was synthesized more under N₂-fixing conditions, followed by ammonium, nitrate, and nitrite as nitrogen sources. The activities of both the glutamine synthetase, Mg²⁺-dependent biosynthetic and Mn²⁺-dependent -glutamyl transferase were optimum at pH 7. The active site of the enzyme bears sulfhydryl (-SH) groups; this was confirmed with the-SH group inhibitors, para-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM). The biosynthetic and -glutamyl transferase activities showed specificity for the divalent cations, Mg²⁺ and Mn²⁺, respectively. The other divalent cations Co²⁺, Cu²⁺, and Ni²⁺ were poor substitutes. This enzyme also required these divalent cations to stabilize its structure and function under extreme conditions such as high and low temperatures and urea denaturation. The glutamate analogl-methionine-d,l-sulfoximine, inactivated the enzyme, whereas the GOGAT inhibitor, azaserine, had no effect on the enzyme activity. The GS enzyme required de novo protein synthesis.     

Quantification of single-stranded nucleic acid and oligonucleotide interactions with metal ions by affinity capillary electrophoresis: part I

The interactions between oligonucleotides and inorganic cations have been measured by capillary zone electrophoresis. With increasing concentrations of divalent cations (Ca²⁺, Mg²⁺, Mn²⁺ and Ni²⁺) in the running buffer, the migration behavior was evaluated by calculation of the binding constants. Besides these fundamental studies of binding equilibria, different buffer components, tris(hydroxymethyl)aminomethane and 3-(N-morpholino)propanesulfonic acid, have been investigated and their effects on metal ion binding quantified.     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Bidirectional Modulation of GABA-Gated Chloride Channels by Divalent Cations: Inhibition by Ca2+ and Enhancement by Mg2+

Abstract: The effects of the divalent cations Ca²⁺, Sr²⁺, Ba²⁺, Mg²⁺, Mn²⁺, and Cd²⁺ were studied on γ-aminobutyric acid_A (GABA_A) responses in rat cerebral cortical synaptoneurosomes. The divalent cations produced bidirectional modulation of muscimol-induced ³⁶Cl^? uptake consistent with their ability to permeate and block Ca²⁺channels. The order of potency for inhibition of muscimol responses was Ca²⁺ > Sr²⁺ > Ba²⁺, similar to the order for permeation of Ca²⁺ channels in neurons. The order of potency for enhancement of muscimol responses was Cd²⁺> Mn²⁺ > Mg²⁺, similar to the order for blockade of Ca²⁺channels in neurons. Neither Ca²⁺ nor Mg²⁺ caused accumulation of GABA in the extravesicular space due to increased GABA release or decreased reuptake of GABA by the synaptoneurosomes. The inhibition of muscimol responses by Ca²⁺ was most likely via an intracellular site of action because additional inhibition could be obtained in the presence of the Ca²⁺ ionophore, A23187. This confirms electrophysiologic findings in cultured neurons from several species. In contrast, the effects of Cd²⁺, Mn²⁺, and Mg²⁺ may be mediated via blockade of Ca²⁺ channels or by intracellular sites, although the results of these studies do not distinguish between the two loci. The effects of Zn²⁺ were also studied, because this divalent cation is reported to have widely divergent effects on GABA_A responses. In contrast to other studies, we demonstrate that Zn²⁺ inhibits GABA_A responses in an adult neuronal preparation. Zn²⁺ produced a concentration-dependent inhibition (limited to 40%) of muscimol responses with an EC₅₀ of 60 μM. The inhibition of muscimol-induced ³⁸Cl^? uptake by Zn²⁺ was noncompetitive. The effect of Zn²⁺was reduced in the presence of Mg²⁺ in a competitive or allosteric manner. The portion of GABA_A receptors sensitive to Zn²⁺ may reflect a specific subunit composition in cerebral cortex as previously observed for recombinant GABA_A receptors in several expression systems. The modulation of GABA_A receptor function by Ca²⁺ and other divalent cations may play an important role in the development and/or attenuation of neuronal excitability associated with pathologic conditions such as seizure activity and cerebral ischemia.     

The effect of divalent metal ions on the activity of Mg++ depleted ribulose-1,5-bisphosphate oxygenase

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg⁺⁺. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO₂. Incubation with Mg⁺⁺ and CO₂ restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn⁺⁺, Co⁺⁺, and Ni⁺⁺) and CO₂ fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO₂-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn⁺⁺ ions were present. Other divalent cations such as Ca⁺⁺ and Zn⁺⁺, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn⁺⁺ to the Mg⁺⁺ and CO₂ preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.Abbreviation RuBP ribulose-1,5-bisphosphate. The enyme unit for RuBP carboxylase is defined as mol CO₂ fixed·min^-1 and for the RuBP oxygenase as mol O₂ consumed · min^-1     

Some characteristics of Ca2+ uptake by yeast cells

Summary Experiments were performed to obtain information on: (i) the specific properties of Ca²⁺ binding and transport in yeast (ii) the relationship between both parameters; (iii) similarities to or differences from other biological systems as measured by the effects of inhibitors; and (iv) the effects of mono and divalent cations, in order to get some insight on the specificity and some characteristics of the mechanism of the transport system for divalent cations in yeast.The results obtained gave some kinetic parameters for a high affinity system involved in the transport of Ca²⁺ in yeast. These were obtained mainly by considering actual concentrations of Ca²⁺ in the medium after substracting the amounts bound to the cell. Ak _m of 1.9 m and aV _max of 1.2 nmol (100 mg·3 min)^–1 were calculated.The effects of some inhibitors and other cations on Ca²⁺ uptake allow one to postulate some independence between binding and transport for this divalent cation.Of the inhibitors tested, only lanthanum seems to be a potent inhibitor of Ca²⁺ uptake in yeast.The effects of Mg²⁺ on the uptake of Ca²⁺ agree with the existence of a single transport system for both divalent cations.The actions of Na⁺ and K⁺ on the transport of Ca²⁺ offer interesting possibilities to study further some of the mechanistic properties of this transport system for divalent cations.     

A ROLE FOR DIVALENT CATIONS IN THE UPTAKE OF NORADRENALINE BY SYNAPTOSOMES

–The effects of divalent cations on the initial rates of noradrenaline uptake by synaptosomes were determined using Millipore filtration to terminate the reaction. The removal of either Ca²⁺ or Mg²⁺ from the incubation medium had no effect on uptake, but when both Ca²⁺ and Mg²⁺ were removed, uptake was reduced. Uptake was also diminished when Ca²⁺ was absent and 1 mm -EGTA added to the medium. It appeared that Ca²⁺ was required for optimal uptake but that Mg²⁺ could partially substitute for Ca²⁺ in this regard. The reduction in the rate of uptake when both Ca²⁺⁰ and Mg²⁺ were absent could be rapidly and completely reversed by restoring Ca²⁺, Mg²⁺, or both Ca²⁺ and Mg²⁺ to the incubation medium. Of the divalent cations tested, Ca²⁺ and Mg²⁺, but not Mn²⁺, supported noradrenaline uptake. When the kinetics of uptake were examined, it was found that removing both Ca²⁺ and Mg²⁺ from the medium resulted in a reduction of the V_max for noradrenaline uptake. It is apparent from these results that, in addition to facilitating the release of noradrenaline from noradrenergic terminals, Ca²⁺ may also play a role in the uptake of noradrenaline by presynaptic nerve-endings in the CNS.     

Activation of the Ca2+ “receptor” on the osteoclast by Ni2+ elicits cytosolic Ca2+ signals: Evidence for receptor activation and inactivation,intracellular Ca2+ redistribution,and divalent cation modulation

Earlier studies have demonstrated that a high (mM) extracellular Ca²⁺ concentration triggers intracellular [Ca²⁺] signals with a consequent inhibition of bone resorptive activity. We now report that micromolar concentrations of the divalent cation, Ni²⁺, elicited rapid and concentration-dependent elevations of cytosolic [Ca²⁺]. The peak change in cytosolic [Ca²⁺] increased monotonically with the application of [Ni²⁺] in the 50–5,000 μM range in solutions containing 1.25 mM-[Ca²⁺] and 0.8 mM-[Mg²⁺]. The resulting concentration-response function suggested Ni²⁺-induced activation of a single class of binding site (Hill coefficient = 1). The triggering process also exhibited a concentration-dependent inactivation in which conditioning Ni²⁺ applications in the range 5–1,500 μM-[Ni²⁺] inhibited subsequent responses to a maximally effective [Ni²⁺] of 5,000 μM. Ni²⁺-induced cytosolic [Ca²⁺] responses were not dependent on extracellular [Ca²⁺]. Thus, when 5,000 μM-[Ni²⁺] was applied to osteoclasts in Ca²⁺-free, ethylene glycol bis-(aminoethyl ether) tetraacetic acid (EGTA)-containing medium (≤5 nM-[Ca²⁺] and 0.8 mM-[Mg²⁺]), cytosolic [Ca²⁺] responses resembled those obtained in the presence of 1.25 mM-[Ca²⁺]. Prior depletion of intracellular Ca²⁺ stores by ionomycin prevented Ni²⁺-induced cytosolic [Ca²⁺] responses, suggesting a major role for intracellular Ca²⁺ redistribution in the response to Ni²⁺. The effects of Ni²⁺ were also modulated by the extracellular concentration of the divalent cations, Ca²⁺ and Mg²⁺. When these cations were not added to the culture medium (0 μM-[Ca²⁺] and [Mg²⁺]), even low [Ni²⁺] ranging between 5 pM and 50 μM elicited progressively larger cytosolic [Ca²⁺] transients. However, the response magnitude decreased at higher, 250–5,000 μM-[Ni²⁺], resulting in a “hooked” concentration-response curve. Furthermore, increasing extracellular [Mg²⁺] or [Ca²⁺] (0–1 mM) diminished the response to 50 μM-[Ni²⁺], a concentration on the rising phase of the “hook.” Similar increases (0–10 mM) in extracellular [Mg²⁺] or [Ca²⁺] increased the response to 5,000 μM-[Ni²⁺], a concentration on the falling phase of the “hook”. These findings are consistent with the existence of a membrane receptor strongly sensitive to Ni²⁺ as well as the divalent cations, Ca²⁺ and Mg²⁺. Receptor occupancy apparently activates intracellular Ca²⁺ release followed by inactivation. Furthermore, repriming is independent of intracellular Ca²⁺ stores, suggesting that such inactivation operates at a transduction step between receptor occupancy and intracellular Ca²⁺ release. © 1993 Wiley-Liss, Inc.