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PubMed2000

The effect of divalent cations on bovine retinal NOS activity.

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 microM) 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+.

Animals↗

PubMed1988

Effect of divalent cation on the structure of skeletal muscle G-actin molecule.

Trypsin and chymotrypsin were used as probes of structure-divalent cation relationships in G-actin molecule. The pattern of fragments produced has been analyzed by sodium dodecyl sulfate gel electrophoresis. The tryptic product of G-actin, 33 kDa is a protease-resistant fragment in the presence of divalent cations. However, once divalent cations are eliminated from the solution during the digestion, the 33 kDa fragment starts to degrade into smaller peptides via a 30 kDa fragment. On the other hand the chymotryptic product of G-actin, 35 kDa (precursor of 33 kDa) is rather stable even in the absence of divalent cations. In addition it is observed that the presence of divalent cation is necessary for the degradation of G-actin to the 33 kDa fragment by trypsin. The ultra violet and intrinsic tryptophan fluorescence spectra of G-actin are changed after the elimination of divalent cations. These results suggest that the structure of G-actin molecule depends on the presence or absence of divalent cations, and that the divalent cation-dependency of G-actin structure is still conserved even after the tryptic digestion.

Actins↗

PubMed1983

Simple shifts in the voltage dependence of sodium channel gating caused by divalent cations.

The effect of elevated divalent cation concentration on the kinetics of sodium ionic and gating currents was studied in voltage-clamped frog skeletal muscle fibers. Raising the Ca concentration from 2 to 40 mM resulted in nearly identical 30-mV shifts in the time courses of activation, inactivation, tail current decay, and ON and OFF gating currents, and in the steady state levels of inactivation, charge immobilization, and charge vs. voltage. Adding 38 mM Mg to the 2 mM Ca bathing a fiber produced a smaller shift of approximately 20 mV in gating current kinetics and the charge vs. voltage relationship. The results with both Ca and Mg are consistent with the hypothesis that elevated concentrations of these alkali earth cations alter Na channel gating by changing the membrane surface potential. The different shifts produced by Ca and Mg are consistent with the hypothesis that the two ions bind to fixed membrane surface charges with different affinities, in addition to possible screening.

Animals↗

PubMed1992

Chemical properties of the divalent cation binding site on potassium channels.

The actions of divalent cations on voltage-gated ion channels suggest that these cations bind to specific sites and directly influence gating kinetics. We have examined some chemical properties of the external divalent cation binding sites on neuronal potassium channels. Patch clamp techniques were used to measure the electrophysiological properties of these channels and Zn ions were used to probe the divalent cation binding site. The channel activation kinetics were greatly (three- to fourfold) slowed by low (2-5 mM) concentrations of Zn; deactivation kinetics were only slightly affected. These effects of Zn were inhibited by low solution pH in a manner consistent with competition between Zn and H ions for a single site. The apparent inhibitory pK for this site was near 7.2. Treatment of the neurons with specific amino acid reagents implicated amino, but no histidyl or sulfhydryl, residues in divalent cation binding.

Animals↗

PubMed1994

Dual role of divalent cations in the bile acid:CoA ligase catalyzed reaction.

The role of divalent cations in the bile acid:CoA ligase catalyzed reaction of cholic acid, CoA and ATP to yield cholyl-CoA was investigated using guinea pig liver microsomes as the source of enzyme. EDTA treatment completely eliminated activity indicating an absolute requirement for divalent cation for enzyme activity. Analysis of this requirement revealed that it was twofold. First, the data suggested that ATP which was not complexed with a divalent cation did not appreciably bind to the enzyme and thus a divalent cation complex of ATP is the form of ATP that is the substrate for the enzyme. Further, this was shown to be the basis for the absolute requirement for divalent cation in the reaction. In addition, analysis revealed that there is a secondary site which binds divalent cations with relatively low affinity, and results in a rate enhancement. Binding at this secondary site is estimated to increase the rate by greater than 60%.

Adenosine Triphosphate↗

PubMed1978

Ionization and divalent cation dissociation constants of nalidixic and oxolinic acids.

The ionization constants and some divalent cation dissociation constants of nalidixic and oxolinic acids, both specific inhibitors of bacterial DNA replication, have been determined. The carboxylic pKa' values are 6.1 and 6.9 at 25 degrees for nalidixic and oxolinic acids, respectively. These values indicate that intramolecular hydrogen-bonding stabilizes the un-ionized form of these compounds in aqueous solution. Both compounds bind divalent cations; the divalent cation dissociation constants for oxolinic acid are somewhat smaller that those for nalidixic acid. We suggest that both compounds may act by forming a complex in situ with a divalent cation in a metalloprotein involved in DNA replication. The evidence that both drugs inhibit at the same target site is briefly reviewed.

Cations, Divalent↗

PubMed1983

Localization of neuronal Ca2+ buffering near plasma membrane studied with different divalent cations.

Absorbance changes associated with divalent cation binding to arsenazo III were used to measure changes in Ca2+, Sr2+, and Ba2+ concentrations under a variety of experimental conditions. The rate of the falling phase of an absorbance change signal, measured in nerve cell bodies injected with arsenazo III and under membrane potential control, was taken as an index of divalent cation buffering. With influx of ions through the membrane or with ionophoretic injection, we found the buffering, i.e., the dye-absorbance signal's falling rate, to be greatest for Ca2+ ions: the sequence was Ca2+ greater than Sr2+ much greater than Ba2+. Injecting Ca2+ or Sr2+ into the center of a nerve cell produced a significantly greater amplitude of arsenazo III signal than the same injection near the cell membrane. We did not find this to be the case for Ba2+ or Mg2+ injections. We conclude that the Ca2+ regulatory system binds Ca2+ most strongly compared to the other ions tested, and there is a variable distribution of buffering machinery within the nerve soma, with increased buffer capacity near the plasma membrane of the cell. A preliminary report of some of the results presented in this paper has appeared previously ( Tillotson and Gorman, 1980).

Animals↗

PubMed1996

Injection of rat hepatocyte poly(A)+ RNA to Xenopus laevis oocytes leads to expression of a constitutively-active divalent cation channel distinguishable from endogenous receptor-activated channels.

The expression of hepatocyte plasma membrane receptor-activated divalent cation channels in immature (stages V and VI) Xenopus laevis oocytes and the properties which allow these channels to be distinguished from endogenous receptor-activated divalent cation channels were investigated. Divalent cation inflow to oocytes housed in a multiwell plate was measured using the fluorescent dyes Fluo-3 and Fura-2. In control oocytes, ionomycin, cholera toxin, thapsigargin, 3-fluoro-inositol 1,4,5-trisphosphate (InsP3F) and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulated Ca2+ and Mn2+ inflow following addition of these ions to the oocytes. Ionomycin-, cholera-toxin-, thapsigargin- and InsP3F-stimulated Ca2+ inflow was inhibited by Gd3+ (half maximal inhibition at less thari 5 microM Gd3+ for InsP3F-stimulated Ca2+ inflow). GTP gamma S-stimulated Ca2+ inflow was insensitive to 50 microM Gd3+ and to SK&F 96365. These results indicate that at least three types of endogenous receptor-activated Ca2+ channels can be detected in Xenopus oocytes using Ca(2+)-sensitive fluorescent dyes: lanthanide-sensitive divalent cation channels activated by intracellular Ca2+ store depletion, lanthanide-sensitive divalent cation channels activated by cholera toxin, and lanthanide-insensitive divalent cation channels activated by an unknown trimeric G-protein. Oocytes microinjected with rat hepatocyte poly(A)+ RNA exhibited greater rates of Ca2+ and Mn2+ inflow in the basal (no agonist) state, greater rates of Ca2+ inflow in the presence of vasopressin or InsP3F and greater rates of Ba2+ inflow in the presence of InsP3F, when compared with "mock"-injected oocytes. In poly(A)+ RNA-injected oocytes, vasopressin- and InsP3F-stimulated Ca2+ inflow, but not basal Ca2+ inflow, was inhibited by Gd3+. It is concluded that at least one type of hepatocyte plasma membrane divalent cation channel, which admits Mn2+ as well as Ca2+ and is lanthanide-insensitive, can be expressed and detected in Xenopus oocytes.

Animals↗

PubMed2005

Action of extracellular divalent cations on native alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors.

The effects of divalent cations on Ca2+-impermeable containing (GluR2 subunit) MPA receptors of hippocampal pyramidal neurones isolated from rat brain was studied using patch-clamping. Ca2+, Mg2+, Mn2+, Co2+, Ni2+ and Zn2+ inhibited currents induced by kainate and glutamate. Inhibition was fast, reversible and voltage independent. The rank order of activities was Ni2+ > Zn2+ > Co2+ > Ca2+ > Mn2+ > Mg2+. Cyclothiazide (0.1 mm) significantly reduced inhibition by divalent cations and 6, 7 dinitroquinoxaline-2.3-dione (DNQX). However, high concentrations of Ni2+ and DNQX inhibited AMPA receptors even in the presence of cyclothiazide. The inhibitory effect of divalent cations as well as DNQX was counteracted by an increase in agonist concentration. In the presence of divalent cations the EC50 values of kainate and glutamate were increased, but the maximal response was not changed. An increase in agonist concentration induced a parallel shift in the concentration-inhibition curve for a divalent cation. These data suggest a competitive-like type of inhibition. However, an increase in agonist concentration reduced the inhibitory action of Ni2+ less than that of DNQX. This gave evidence against direct competition between divalent cations and AMPA receptor agonists. A 'complex-competition' hypothesis was proposed to explain the inhibitory action of divalent cations; it is suggested that divalent cations form ion-agonist complexes, which compete with free agonist for agonist-binding sites on AMPA receptors.

Algorithms↗

PubMed1991

Effects of internal divalent cations on the gating of rat brain Na+ channels reconstituted in planar lipid bilayers.

The effects of different intracellular divalent cations on the gating of single batrachotoxin-activated Na+ channels were investigated in planar lipid bilayers. Intracellular divalent cations increased the open probability (Po) of Na+ channels; the gating curve [Po versus membrane potential (Vm) relationship] shifted to more negative potentials. The relative ability of different intracellular divalent cations in shifting the gating curve decreased in the sequence: Mg2+, Ca2+, Ba2+, Sr2+. The cations Ca2+, Ba2+, and Sr2+ induced a larger voltage shift when applied to the extracellular than to the intracellular side of the Na+ channel, whereas, Mg2+ induced the same voltage shift from both sides. The increase in Po induced by intracellular divalent cations was the result of a simultaneous decrease in the closing rate and increase in the opening rate constant, however, the effect of intracellular divalent cations on the closing rate was larger than on the opening rate. These results suggest that there are both differences in surface charge densities between the intracellular and extracellular surfaces of the Na+ channel and differences in chemical affinities of those charges for different divalent cations. The effects of internal divalent cations on Na+ channel gating cannot be explained solely by surface charge reduction, which predicts that the opening and closing rates should be affected equally, but rather are consistent with a mechanism that involves screening and binding of surface charges present on the channel, plus a specific modulatory effect that accounts for the preferential effect of intracellular divalent cations on the closing rate constant.

Animals↗

PubMed1992

Modulation of (+)-[3H]pentazocine binding to guinea pig cerebellum by divalent cations.

The ability of cations to modulate the binding of the sigma 1 receptor-selective ligand (+)-[3H]pentazocine to guinea pig cerebellum was investigated. Di- and trivalent cations biphasically inhibited (+)-[3H]pentazocine binding, revealing multiple affinity states. The rank order of potency of these cations (based on the high affinity component of inhibition) was Zn2+ > Co2+ >> La3+ = Ni2+ = Cd2+ = Mn2+ = Gd2+ > Ba2+ = Sr2+ >> Mg2+ > Ca2+. The inhibition of 1,3-[3H]di(2-tolyl)guanidine binding to the sigma 2 receptor by these cations differed qualitatively and quantitatively from their effects on (+)-[3H]pentazocine binding. Although monovalent cations decreased the Kd for (+)-[3H]pentazocine binding, divalent cations split (+)-[3H]pentazocine binding into low and high affinity components. The Bmax of the high affinity component decreased with increasing divalent cation concentrations. Both mono- and divalent cations significantly reduced the rate of association of (+)-[3H]pentazocine with the sigma 1 receptor without altering the dissociation rate. (+)-[3H]Pentazocine binding was not altered by guanine nucleotides or by treatment with cholera or pertussis toxins. However, nonselective cation channel blockers (cinnarizine, hydroxyzine, prenylamine, amiodarone, and proadifen) potently inhibited (+)-[3H]pentazocine binding. These results indicate that physiologically relevant concentrations of divalent cations allosterically modulate (+)-[3H]pentazocine binding to the sigma 1 receptor, to reveal multiple affinity states. These sites do not represent sigma 1 to sigma 2 subtype interconversion or ternary complex formation with guanine nucleotide-binding proteins. However, the rank order of cation potency and the inhibition of binding by cation channel blockers is consistent with a potential role for sigma receptors as constituents of cation channels.

Animals↗

PubMed1988

High calcium and other divalent cations increase inositol trisphosphate in bovine parathyroid cells.

Calcium and other divalent cations rapidly increase intracellular free Ca2+ ([Ca2+]i) in bovine parathyroid cells and inhibit PTH release. In other secretory cells, agonist-dependent generation of inositol trisphosphate (InsP3) through polyphosphoinositide turnover initiates the rise in [Ca2+]i by mobilizing Ca2+ from intracellular stores. To determine whether polyphosphoinositide breakdown is involved in mediating the response to Ca2+ and the divalent cations Ba2+, Mn2+, and Sr2+, we measured the production of inositol polyphosphates in parathyroid cells. Within 120 sec of increasing extracellular Ca2+ to 2.0 mM, InsP3, inositol bisphosphate (InsP2), and inositol monophosphate (InsP1) rose 95 +/- 37%, 87 +/- 17%, and 96 +/- 29%, respectively, vs. values in cells at 0.5 mM Ca2+ (n = 5). Raising extracellular Ca2+ from 0.5-3.0 mM produced even greater peak increments of 134 +/- 13%, 179 +/- 35%, and 313 +/- 65% in InsP3, InsP2, and InsP1, respectively, by 120 sec (n = 4). Similarly, within 10 sec of their addition, BaCl2 (2 mM), MnCl2 (2 mM), and SrCl2 (4 mM) stimulated the production of InsP3 56 +/- 2%, 152 +/- 31%, and 160 +/- 25%, respectively, vs. that in untreated cells at 0.5 mM Ca2+. At later time points, InsP2 and InsP1 were increased. The Ca2+ ionophore ionomycin at concentrations up to 500 nM had no effect on inositol phosphates, although it inhibited PTH release in a dose-dependent manner. Since high Ca2+ and other divalent cations depolarize parathyroid cells, we assessed the effect of high extracellular K+ on inositol polyphosphates. The addition of depolarizing concentrations of K+ (40 mM) did not change inositol phosphates. Thus, Ca2+ and other divalent cations increase the production of InsP3, InsP2, and InsP1 in parathyroid cells by a mechanism independent of increases in [Ca2+]i and of membrane depolarization. We conclude that parathyroid cells express membrane receptors or sensors for Ca2+ and other divalent cations linked to polyphosphoinositide turnover.

Animals↗

PubMed1986

Analysis of oxygen binding to Panulirus japonicus hemocyanin. The effect of divalent cations on the allosteric transition.

The effects of H+ and divalent cations on the O2 equilibrium of hexameric hemocyanin from a spiny lobster, Panulirus japonicus, were examined. The hemocyanin showed the normal Bohr effect. When divalent cations were removed by EDTA treatment, the protein showed a fivefold increase in the O2 affinity and a considerable decrease in the cooperativity. Several cooperativity models were tested for the conformity with the observed O2-binding isotherms by the least-square curve fitting. Among the models examined, the three-state concerted model was found to be most consistent with the results. It was postulated that in the absence of divalent cations deoxyhemocyanin is mainly in the intermediate-affinity state. The arthropod hemocyanins were found to be classifiable into two groups according to their functional responses to the divalent cations. It was suggested that the cations act differently on the allosteric transitions of the two groups of hemocyanins.

Allosteric Site↗

PubMed1962

Glass electrodes sensitive to divalent cations.

Glass electrodes suitable for measurement of divalent cations have been made and tested. Empirical and theoretical electrode equations have been presented to describe electrode behavior in a variety of aqueous solutions. Most electrodes show response interpretable as showing nearly ideal solid-solution behavior of the cations in the glass surface. The electrodes should be useful in the measurement of divalent-cation activities in natural waters and biological fluids, and useful in general analytical chemistry.

Cations↗

PubMed1984

Divalent cation effects on acetylcholine-activated channels at the frog neuromuscular junction.

The effects of the divalent cations Ca and Mg on the properties of ACh-activated channels at the frog neuromuscular junction were studied using a two-microelectrode voltage clamp. The divalent cation concentration was varied from 2 to 40 mM in solutions containing 50% normal Na. The reversal potential was determined by interpolation of the acetylcholine (ACh)-induced current versus voltage relationship. The single-channel conductance and the mean channel lifetime were calculated from fluctuation analysis of the ACh-induced end-plate current. Extracellular Na and/or divalent cations affected the reversal potential of endplate channels in a way that cannot be described by the Goldman-Hodgkin-Katz equation or by a simple two-barrier, one-binding site model of the channel if the assumption was made that permeability ratios were constant and not a function of ion concentrations. Increasing the divalent cation concentration decreased the single-channel conductance to approximately 10 pS in solutions with 50% Na and 40 mM divalent cation concentrations. The effect of the divalent cations Ca and Mg on the mean channel lifetime was complex and dependent on whether the divalent cation was Ca or Mg. The mean channel lifetime was not significantly changed in most solutions with increased Ca concentration, while it was slightly prolonged by increased Mg concentration.

Acetylcholine↗

PubMed1979

Effects of divalent cation ionophores on the neuron membrane of the crayfish.

The effects of divalent cation ionophores, A23187 and X-537A, on the electrical membrane properties were investigated by using the soma membrane of the X-organ of the crayfish. They reduced the amplitude and maximum rate of rise of Ca-action potential in lower concentration. As the concentration increased, a reduction of membrane resistance and hyperpolarization occurred simultaneously. Further increase resulted in membrane depolarization with a further decrease in resistance. The threshold concentration of X537A was 100 times higher than that of A23187. These effects were reversible only when the application period was relatively short, while a longer application resulted in an incomplete reversibility or in no reversibility at all. The ionophore effect was facilitated in high Ca medium and diminished in low Ca medium. In Sr medium, the same effects on the resistance and the membrane potential were barely observable. TEA reduced the effects of A23187 but did not completely inhibit the effects. The Na-cation potential was also reduced by the higher concentration of the ionophore. From these results it is concluded that the divalent cation ionophores. A23187 and X537A, carry divalent cations, Ca ions in a physiological medium, into the neuron soma through the membrane and the consequent increase of the intracellular divalent cations induces K conductance increase and that higher concentration of the ionophore induces the increase in the conductance of the other ion species, such as Na.

Action Potentials↗

PubMed1985

Properties of action potentials carried by divalent cations in identified leech neurons.

Properties of divalent cation potentials carried by either Sr2+ or Ca2+ ions in Na+-free, TEA-Ringer solution were characterized in identified neurons of two species of leeches (Macrobdella and Haementeria). In Macrobdella, the overshoot of the potentials varied logarithmically with [Sr2+]0 (28.5 mV per 10-fold change). The overshoot, Vmax, and duration of the potentials increased with increasing divalent cation concentration and saturated at about 20 to 30 mM [Sr2+]0. The Vmax, amplitude, and duration of the potentials were reversibly blocked by Co2+ and Mn2+. The block by Mn2+ could be well-fitted by a reverse Langmuir-curve with an apparent KI of 100 micromolar. The local anesthetic procaine also reversibly inhibited the Vmax and duration of the potentials. The inhibition was greater at alkaline pH suggesting that procaine blocks the calcium channel from inside the membrane. The identified leech neurons examined in Macrobdella varied considerably in their ability to sustain somatic divalent cation potentials. Stimulation of T cells and most motoneurons produced no or only weak potentials, whereas stimulation of Retzius, N, Nut, and AP cells evoked overshooting potentials of several seconds' duration. Stimulation of the ALG cell of Haementeria in normal Ringer solution evoked a slowly-rising, purely Ca2+-dependent potential of approximately 100 ms duration. This response was TTX-resistant, unaffected by complete removal of Na+ from the Ringer solution, and abolished by 1 mM Mn2+. The overshoot varied logarithmically with a slope of 28 mV/decade change in [Ca2+]0.

Action Potentials↗

PubMed1989

Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons.

We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins.

Animals↗