Leucocyte locomotion and chemotaxis : The influence of divalent cations and cation ionophores

Human blood neutrophil leucocytes and monocytes incubated in the absence of Ca²⁺ and Mg²⁺ showed reduced, but still substantial migration into micropore filters towards chemotactic agents, compared with cells migrating in a divalent cation-rich medium. This reduction in migration could be reversed by adding low doses of divalent cation ionophores (X537A or A23187) to the Ca²⁺- and Mg²⁺-free medium which suggests that migrating leucocytes in media depleted of extracellular divalent cations can make use of intracellular divalent cations and that the intracellular cation exchange necessary for locomotion is facilitated by the ionophores. At higher doses, the ionophores inhibited locomotion, as did procaine which reduces membrane permeability to cations. Little effect of K⁺ depletion or of ouabain on leucocyte locomotion was noted.     

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.     

Calcium-sensitive univalent cation channel formed by lysotriphosphoinositide in bilayer lipid membranes

A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to univalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb⁺ > Cs⁺ > Na⁺ > K⁺ > Li⁺. The conductance of the membrane was increased up to a value of about 10⁻² ohm⁻¹/cm² with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn²⁺ > Cd²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺.     

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.     

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

In the E₁ state of the Na,K-ATPase all cations present in the cytoplasm compete for the ion binding sites. The mutual effects of mono-, di- and trivalent cations were investigated by experiments with the electrochromic fluorescent dye RH421. Three sites with significantly different properties could be identified. The most unspecific binding site is able to bind all cations, independent of their valence and size. The large organic cation Br₂-Titu³⁺ is bound with the highest affinity (<μm), among the tested divalent cations Ca²⁺ binds the strongest, and Na⁺ binds with about the same equilibrium dissociation constant as Mg²⁺ (∼0.8 mm). For alkali ions it exhibits binding affinities following the order of Rb⁺≃ K⁺ > Na⁺ > Cs⁺ > Li⁺. The second type of binding site is specific for monovalent cations, its binding affinity is higher than that of the first type, for Na⁺ ions the equilibrium dissociation constant is < 0.01 mm. Since binding to that site is not electrogenic it has to be close to the cytoplasmic surface. The third site is specific for Na⁺, no other ions were found to bind, the binding is electrogenic and the equilibrium dissociation constant is 0.2 mm. Received: 7 August 2000/Revised: 14 November 2000     

Effect of inotropic agents on the calcium binding to isolated cardiac sarcolemma

Ca²⁺ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 · 10^?5 and 1.2 · 10^?3 M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca²⁺ binding were studied. All cations tested inhibited Ca²⁺ binding with the following order of potency: trivalent > divalent > monovalent cations. The order of potency for the monovalent ions was: Na⁺ > K⁺ > Li⁺ ? Cs⁺ and for the divalent and trivalent ions: La³⁺ ? Mn²⁺ > Sr²⁺ ? Ba²⁺ > Mg²⁺. 1 · 10^?3 M caffeine and 1 · 10^?8 M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 · 10^?7 M verapamil, acidosis (pH 6.4), 1^?10^?5 M Mn²⁺ and 1 · 10^?4 M ouabain depressed the capacity of the low-affinity sites to a range of 154–291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents.     

Electrophoresis, Zeta potential and Surface Charges of Barley Mesophyll Protoplasts

The electrophoretic mobility of barley mesophyll protoplastswas measured by the micro-electrophoresis technique to determinethe surface charge density in medium that contained variouscations and anions at neutral pH. The surface charge was stronglyinfluenced by the presence of di- and trivalent cations in themedium, suggesting that certain di- and trivalent cations arespecifically adsorbed on or bound to the plasma membrane ofthe protoplasts. The order of ad-sorbability was Mg²⁺ < Ca²⁺,Mn²⁺, Ba²⁺ for divalent cations, and La³⁺ < < Sm³⁺ <Tm³⁺, Yb³⁺ for trivalent cations. The adsorption of Ca²⁺ andLa³⁺ ions on the surface of the membrane was analyzed usingthe Langmuir adsorption isotherm. The maximum amount of adsorptionand the adsorption coefficient were found to be 3.17 mC/m² and1.16 mM^–1 for Ca²⁺ ions, and 7.12 mC/m² and 8.26 mM^–1for La³⁺ ions, respectively. (Received August 26, 1988; Accepted November 21, 1988)     

Non-covalent (iso)guanosine-based ionophores for alkali(ne earth) cations

Different (iso)guanosine-based self-assembled ionophores give distinctly different results in extraction experiments with alkali(ne earth) cations. A lipophilic guanosine derivative gives good extraction results for K⁺, Rb⁺, Ca²⁺, Sr²⁺, and Ba²⁺ and in competition experiments it clearly favors the divalent Sr²⁺ (and Ba²⁺) cations. 1,3-Alternate calix[4]arene tetraguanosine hardly shows any improvement in the extraction percentages compared to its reference compound 1,3-alternate calix[4]arene tetraamide. This indicates that one G-quartet does not provide efficient cation complexation under these conditions. In the case of the lipophilic isoguanosine derivative there is a cation size dependent affinity for the monovalent cations (Cs⁺ ? Rb⁺ ? K⁺), but not for the divalent cations (Ca²⁺ > Ba²⁺ > Sr²⁺ > Mg²⁺). In competition experiments the isoguanosine derivative, unlike guanosine, does not discriminate between monovalent and divalent cations, giving an almost equal extraction of Cs⁺ and Ba²⁺.     

Divalent Cations Modulate TMEM16A Calcium-Activated Chloride Channels by a Common Mechanism

The gating of Ca²⁺-activated Cl^? channels is controlled by a complex interplay among [Ca²⁺]_i, membrane potential and permeant anions. Besides Ca²⁺, Ba²⁺ also can activate both TMEM16A and TMEM16B. This study reports the effects of several divalent cations as regulators of TMEM16A channels stably expressed in HEK293T cells. Among the divalent cations that activate TMEM16A, Ca²⁺ is most effective, followed by Sr²⁺ and Ni²⁺, which have similar affinity, while Mg²⁺ is ineffective. Zn²⁺ does not activate TMEM16A but inhibits the Ca²⁺-activated chloride currents. Maximally effective concentrations of Sr²⁺ and Ni²⁺ occluded activation of the TMEM16A current by Ca²⁺, which suggests that Ca²⁺, Sr²⁺ and Ni²⁺ all regulate the channel by the same mechanism.     

Characterization of store-operated Ca channels in pancreatic duct epithelia

Store-operated Ca²⁺ channels (SOCs) are activated by depletion of intracellular Ca²⁺ stores following agonist-mediated Ca²⁺ release. Previously we demonstrated that Ca²⁺ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca²⁺ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca²⁺ influx as Ca²⁺ was released from intracellular stores. Direct measurement of [Ca²⁺] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP₃ or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La³⁺ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca²⁺ entry through SOCs fills internal Ca²⁺ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca²⁺ such as salt and mucin secretion from the exocrine pancreatic ducts.     

Common action of certain viruses,toxins, and activated complement: pore formation and its prevention by extracellular Ca2+

Haemolysis by Sendal virus, -toxin, and activated complement is inhibited by high concentrations of divalent cations. In Daudi cells, sublytic amounts of these agents induce the following changes: collapse of surface membrane potential, uptake of Na⁺ and loss of K⁺ from cells, and leakage of phosphorylated metabo-tites from cells. The changes induced by Sendal virus and complement are sensitive to physiological concentrations of extracellular Ca²⁺. It is concluded that fluctuations in plasma Ca²⁺ concentration may affect the damaging action of certain pore-forming agents on susceptible cells.     

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     

Regulation of divalent cations of the membrane-bound pyrophosphatase of Rhodospirillum rubrum,as shown by the hydrolysis of tripositive-pyrophosphate complexes

Tripositive-pyrophosphate [M(III)-PPi] complexes were used to investigate the role of free divalent cations on the membrane-bound pyrophosphatase. Divalent cations remain free and the M(III)-PPi complexes were employed as substrates. Formation of a La-PPi complex was studied by fluorescence, and the fact that Zn²⁺ and Mg²⁺ remain free in the solution was validated. Hydrolysis of La-PPi is stimulated by the presence of fixed concentrations of free Mg²⁺ or Zn²⁺ and this stimulation depends on the concentration of the cations when the La-PPi complex is fixed. The divalent cation stimulation order is Zn²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Ca²⁺ (at 0.5 mm of free cation). With different M(III)-PPi complexes, Zn²⁺ produces the same K _m, for all the complexes and Mg²⁺ stimulates with a different K _m. The results suggest that both Mg²⁺ and Zn²⁺ activate the membrane-bound pyrophosphatase but through different mechanisms.     

Physical properties of biological membranes determined by the fluorescence of the calcium ionophore A23187

Interactions between the divalent cation ionophore, A23187, and the divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ were studied in sarcoplasmic reticulum and mitochondria. Conductance measurements suggest that A23187 facilitates the movement of divalent cations across bilayer membranes via a primarily electroneutral process, although a cationic form of A23187 does carry some current.On the basis of fluorescence excitation spectra, A23187 can form either a 1:1 or 2:1 complex with Ca²⁺ in organic solvents. However, in biological membranes, only the 1:1 complexes with Ca²⁺, Mg²⁺, or Mn²⁺ are detected. A23187 produces fluorescent transients under conditions of Ca²⁺ uptake in sarcoplasmic reticulum, which appear to represent changes in intramembrane Ca²⁺ content. Changes in A23187 fluorescence due to mitochondrial Ca²⁺ accumulation are much smaller by comparison and fluorescence transients are not detected.Studies of A23187 fluorescence polarization and lifetimes in biological membranes allow a determination of the rotational correlation time (ρh) of the ionophore. In mitochondria at 22 °C, ρh is 11 nsec in the presence of Ca²⁺ and Mg²⁺, and less than 2 nsec in the presence of excess EDTA.The present results are consistent with a model of ionophore-mediated cation transport in which free M²⁺ binds with A23187 at the membrane surface to form the complex M(A23187)⁺. Reaction of this complex with another molecule of A23187 at the membrane surfaces results in the formation of electrically neutral M(A23187)₂, which carries the divalent cation through the membrane.These results are discussed in terms of physical properties of biological membranes in regions in which divalent cation transport occurs.     

Effects of divalent cations and glucose on mitotic-like events in fused interphase-metaphase cells

The effects of Ca²⁺, Mg²⁺ and glucose on the mitotic-like events of prophasing and telophasing were studied in Sendai virus-fused interphase-metaphase (I-M) Chinese hamster binucleate cells. At normal extracellular ion concentrations and neutral pH, about 80–90% of I-M binucleates show prophasing (nuclear envelope dissolution and chromatin condensation) of the I nucleus and 10–15% show telophasing (nuclear envelope reformation and chromatin decondensation) of the M nucleus. To study the effects of cellular divalent cations, cells, depleted of about 77 % of exchangeable cell Ca²⁺ as determined by ⁴⁵Ca²⁺ studies, were incubated in different concentrations of Ca²⁺ or Mg²⁺ for 30 min prior to cell fusion. We found that relatively high concentrations of Ca²⁺ or Mg²⁺ (0.84 mM) were essential for prophasing and that in the presence of 10-fold less Ca²⁺ or Mg²⁺ (0.084 mM) the majority of binucleates showed telophasing. In contrast to a differential effect of divalent cations on the nuclear changes, we found that glucose metabolism was required for both prophasing and telophasing. Additionally, interruption of glucose metabolism in the M cell, but not in the I cell, prior to cell fusion depressed the prophasing frequency about 70%. Although we do not know how divalent cations and glucose function in prophasing and telophasing, we will discuss evidence which suggests that the effects are not mediated through secondary effects on membrane potential, by changes in intracellular concentrations of Na⁺ or K⁺, by simple osmotic changes, or through inhibition of protein synthesis.     

Intracellular calcium release channels mediate their own countercurrent: the ryanodine receptor case study

Intracellular calcium release channels like ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP₃Rs) mediate large Ca²⁺ release events from Ca²⁺ storage organelles lasting >5 ms. To have such long-lasting Ca²⁺ efflux, a countercurrent of other ions is necessary to prevent the membrane potential from becoming the Ca²⁺ Nernst potential in <1 ms. A recent model of ion permeation through a single, open RyR channel is used here to show that the vast majority of this countercurrent is conducted by the RyR itself. Consequently, changes in membrane potential are minimized locally and instantly, assuring maintenance of a Ca²⁺-driving force. This RyR autocountercurrent is possible because of the poor Ca²⁺ selectivity and high conductance for both monovalent and divalent cations of these channels. The model shows that, under physiological conditions, the autocountercurrent clamps the membrane potential near 0 mV within ∼150 μs. Consistent with experiments, the model shows how RyR unit Ca²⁺ current is defined by luminal [Ca²⁺], permeable ion composition and concentration, and pore selectivity and conductance. This very likely is true of the highly homologous pore of the IP₃R channel.     

Ca2+ and the interaction of pore-formers with membranes

The interaction of pore-forming agents, such as Sendai virus, influenza virus (at pH 5 3), activated complement,Staphylococcus aureus α-toxin, melittin and polylysine, with the surface membrane of cells has been studied. In each case the following changes are initiated: collapse of membrane potential, leakage of ions, and leakage of phosphorylated metabolites. The changes can be inihibited by extracellular Ca²⁺ at physiological concentration; Mg²⁺ is less effective, and Zn²⁺ is more effective, than Ca²⁺ Ca²⁺ appears to act at a stage subsequent to the binding of pore-forming agent to cells. It is concluded that divalent cations are able to protect cells against the damaging effects of certain viruses, toxins or the components of activated complement in a manner that is worthy of further investigation.     

Requirement of divalent cations for photoactivation of the laten water-oxidation system in intact chloroplasts from flashed leaves

The effects of divalent cations on photoactivation of the latent water-oxidation system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination were investigated by using A23187, an ionophore for divalent cations, and the following results were obtained. (a) Photoactivation in the intact chloroplasts was inhibited by A23187, but was restored on addition of a low concentration of Mn²⁺ (10 μM). (b) A high concentration of Mn²⁺ (70 μM) was inhibitory, in contrast, for photoactivation, but the inhibition was restored by the coexistence of a suitable concentration of Ca²⁺ (5 mM). (c) The Ca²⁺-dependent restoration was inhibited by a high concentration of Mg²⁺ or Sr²⁺, but the inhibition was restored by the coexistence of Ca²⁺. (d) Kinetic analyses of these competitive effects between divalent cations revealed that: (i) High concentration of Ca²⁺ inhibits photoactivation in competition with Mn²⁺. (ii) High concentration of Mn²⁺ inhibits photoactivation in competition with Ca²⁺. (iii) High concentration of Mg²⁺ affects photoactivation by inhibiting Ca²⁺-dependent restoration in competition with Ca²⁺. Based on these results, we propose that the latent water-oxidation center has two binding sites, each specific for Mn²⁺ and Ca²⁺, and that photoactivation takes place in the center having both Mn²⁺ and Ca²⁺ on their respective binding sites.     

Effect of an anti-tumor platinum complex,Pt(II)diaminotoluene,on mitochondrial membrane properties

The effects of platinum complexes, selected for their potent anti-tumor activities, have been studied on rat liver mitochondria. Among the mitochondrial properties which have been studied, the most marked effects of platinum complexes were obtained on functions linked to the inner membrane.cis-Pt(II)(3,4-diaminotoluene) dichloride is shown to stimulate state 4 respiration. It inhibits the phosphate transport into mitochondria, decreases the accumulation of Ca²⁺, and induces a more rapid release of the accumulated Ca²⁺. A release of Mg²⁺ from mitochondria incubated in the absence of added divalent cations, and an efflux of divalent cations from mitochondrial membranes are also observed.All these results indicate a profound modification of the permeability of mitochondrial membrane.