Calcium-induced oscillations in K+ conductance and membrane potential of human erythrocytes mediated by the ionophore A23187

The time-dependence of ionophore A23187-induced changes in the conductance of the Ca2+-sensitive K+ channels of the human red cell has been monitored with ion-specific electrodes. The membrane potential was reflected in CCCP-mediated pH changes in a buffer-free extracellular medium, and changes in extracellular K+ activity and electrode potential of an extracellular Ca2+-electrode were recorded. Within a narrow range of ionophore-mediated Ca2+ influx, the above-mentioned parameters were found to oscillate when ionophore was added to a suspension of glucose-fed cells. The period of oscillation was about 2 min/cycle depending on ionophore concentration, and the amplitude of hyperpolarization was about 60 mV, corresponding to a maximal gK+ of the same magnitude as gCl-. Without CCCP present no oscillation in K+ conductance was observed. The Ca2+ affinity for the opening process was in the micromolar range. The closing of the K+ channels was a spontaneous process in that the depolarization was well under way before the Ca2+-ATPase-mediated Ca2+ net efflux started. Below the Ca2+ influx range for oscillations, no response was observed for up to 20 min after the addition of ionophore. Above the upper limit, a permanent hyperpolarization resulted with an extracellular K+ activity increasing monotonically as a function of time. In experiments with ATP-depleted cells, responses of the latter type ensued at all ionophore concentrations above the lower limit. Addition of surplus EGTA to suspensions of hyperpolarized cells restores the normal membrane potential in the case of glucose-fed cells, whereas the K+-channels in ATP-depleted cells remained open.     

Calcium and ionophore A23187 induce the sickle cell membrane phosphorylation pattern in normal erythrocytes

Pre-treatment of normal erythrocytes with micromolar Ca²⁺ and ionophore A23187 induces abnormal phosphorylation of membrane polypeptides, as determined by labeling with exogenous ³²P_i. The Ca²⁺-induced effects, which include increased incorporation of ³²P into acid-stable linkages and increased labeling in the Band 3 and 4.5–4.9 regions of SDS gels, are similar to those seen in untreated sickle erythrocytes. Part of the abnormal phosphorylation of sickle cells may be caused by their elevated intracellular Ca²⁺ levels.     

The effect of calcium ionophore A23187 on the ATP level of human erythrocytes

Incubation of red cells at 37° with the ionophore A23187 results in a loss of ATP that is dependent on the concentrations of A23187 and Ca²⁺ in the medium. ATP hydrolysis is greatest at micromolar concentrations of Ca²⁺ and decreases as Ca²⁺ in the medium is raised to millimolar levels. The ATP depletion is due to stimulation of calcium ATPase by A23187-mediated Ca²⁺ influx into the cell. The biphasic nature of Ca²⁺-stimulated ATP depletion in whole cells reflects the activity of Ca²⁺-ATPase in membrane preparations at varying Ca²⁺ concentrations. The ionophore can be removed by washing the cells with plasma or bovine serum albumin-containing medium and the ATP levels restored to normal by reincubating with 5 mM adenosine for 1 hr.     

Effect of ionophore A23187 on the membrane permeability in mouse fibroblasts.

Addition of the divalent cation ionophore A23187 to transformed mouse fibroblasts (3T6) resulted in an increase in the cell membrane permeability to normally impermeant solutes (e.g., nucleotides). The membrane permeability was assessed by following the efflux of prelabeled adenine nucleotides, the influx of p-nitrophenyl phosphate in cells attached to plastic dishes and reconstitution of intracellular protein synthesis in the presence of exogenously added normally impermeant factors required for macromolecular synthesis. The permeability change of 3T6 cells was found to be dependent on the specific presence of external calcium ion. The permeabilization was found to occur preferably in alkaline pH and specific to certain transformed cells. It is preceded by rapid efflux of K+, influx of Na+ and partial hydrolysis of cellular nucleotides in 3T6 cells. Similar ion fluxes were previously found to precede cell permeabilization by electrogenic ionophores for monovalent ions and by exogenous ATP. Our data suggest that a calcium dependent process caused the K+ release and excess Na+ entry, causing dissipation of the membrane potential and subsequent formation of aqueous channels.     

Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187.

1. When the ionophore A23187 and Ca2+ were added to normal human erythrocytes, the incorporation of 32P into phosphatidate was enhanced within 1 min, but there was only slight labelling of other phospholipids. 2. Labelling of phosphatidate in these cells did not continue to increase after about 20min at 37 degrees C; by this time, radioactivity in phosphatidate was about ten times higher inionophore A23187-treated cells than in controls. A net synthesis of phosphatidate was measured in response to the increase in intracellular Ca2+ concentration; the content of this phospholipid in the cell was increased by approximately 50%. 3. In the presence of 2.5 mM-Ca2+ a maximum effect was seen with about 0.5 mug of ionophore/ml. 4. The concentration of Ca2+ giving half-maximal labelling of phosphatidate in the presence of 10 mug of ionophore A23187/ml was about 10 muM. 5. A rapid decrease of ATP content in the cell occurred in ionophore-treated cells. 6. Labelling of phosphatidate appeared to be secondary to the production of 1,2-diacylglycerol in the cells; accumulation of 1,2-diacylglycerol was only seen after about 15 min. After 60 min, the 1,2-diacylglycerol content of the cells was five to seven times that of untreated control cells. 7. The change in the shape of erythrocytes treated with Ca2+ and ionophore appeared to be related to accumulation of 1,2-diacylglycerol. 8. The source of 1,2-diacylglycerol has not been definitely identified, but its fatty acid compositon was similar to that of phosphatidylcholine. However, it has an unusually high content of hexadecenoic acid, a fatty acid not common in the major erythrocyte phospholipids. 9. Accumulation of 1,2-diacyglycerol also occurred in energy-starved cells, even in the absence of calcium; in this case it appeared to be produced by phosphatidate breakdown.     

Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187

Challenge of human peripheral blood leukocytes with ionophore A23187 resulted in leukotriene (LT) synthesis, a decrease in total cellular 5-lipoxygenase activity, and a change in the subcellular localization of the enzyme. In homogenates from control cells, greater than 90% of the 5-lipoxygenase activity and protein was localized in the cytosol (100,000 X g supernatant). Ionophore challenge (2 microM) resulted in a loss of approximately 55% of the enzymatic activity and 35% of the enzyme protein from the cytosol. Concomitantly, there was an accumulation of inactive 5-lipoxygenase in the membrane (100,000 X g pellets) which accounted for at least 45% of the lost cytosolic protein. There was a good correlation between the quantities of LT synthesized and 5-lipoxygenase recovered in the membrane over an ionophore concentration range of 0.1-6 microM. The time course of the membrane association was similar to that of LT synthesis. Furthermore, although the pellet-associated enzyme recovered from ionophore-treated leukocytes was inactive, an irreversible, Ca2+-dependent membrane association of active 5-lipoxygenase could be demonstrated in cell-free systems. To determine whether ionophore treatment induced proteolytic degradation of 5-lipoxygenase, the total activity and protein content of 10,000 X g supernatants from control and ionophore-treated cells were examined. These supernatants, which included both cytosolic and membrane-associated enzyme, showed a 35% loss of 5-lipoxygenase activity but only an 8% loss of enzyme protein as a result of ionophore challenge (2 microM). Therefore, the majority of the loss of 5-lipoxygenase activity was most likely due to suicide inactivation during the LT synthesis, rather than to proteolytic degradation. Together these results are consistent with the hypothesis that ionophore treatment results in a Ca2+-dependent translocation of 5-lipoxygenase from the cytosol to a membrane-bound site, that the membrane-associated enzyme is preferentially utilized for LT synthesis, and that it is consequently inactivated. Thus, membrane translocation of 5-lipoxygenase may be an important initial step in the chain of events leading to full activation of this enzyme in the intact leukocyte.     

Rapid clearance of Plasmodium yoelii-infected erythrocytes after exposure to the ionophore A23187

1. The effects of Ca2+ and the calcium ionophore A23187 on the intraerythrocytic development of the asexual forms of Plasmodium yoelii were examined. 2. Erythrocyte-free parasites obtained by saponin lysis of infected cells remained viable after exposure to 1 mM Ca2+. 3. A23187 inhibited the growth of P. yoelii and the inhibition was augmented by Ca2+ in cells infected with parasites at young stage of development. 4. A23187-treated infected cells disappeared from the circulation shortly after intravenous injection and this disappearance was profound in infected cells treated with the ionophore in the presence of Ca2+.     

Calcium ionophore A23187 calcium-dependent cytolytic degranulation in human eosinophils

The divalent cation ionophore A23187 is frequently used for studies of eosinophil degranulation. Nonetheless, the mechanism whereby A23187 induces degranulation in human eosinophils is still unclear. In the present experiments, A23187 caused human eosinophils to release a granule protein, eosinophil-derived neurotoxin (EDN) and a membrane-associated protein, Charcot-Leyden crystal (CLC) protein in a calcium and a concentration-dependent manner. However, A23187 at a concentration (1 microgram/ml) that caused 15% EDN release and 30% CLC protein release also produced release of the cytoplasmic enzyme lactic dehydrogenase (LDH) and loss of cell viability, both of which were calcium dependent. CLC protein release preceded EDN release and was detectable even at 15 min after the addition of 1 microgram/ml A23187, whereas EDN release occurred after a lag period of 30 min, and coincided with LDH release. At 1 microgram/ml A23187, neither the release of LDH nor the loss of viability occurred with purified neutrophils obtained in the same blood sample as a by-product of eosinophil purification. Electron microscopic examination demonstrated that exposure to A23187 for 15 min resulted in an increase and elongation of microridges on the cell surface, and exposure for 45 min caused cell disruption followed by extrusion of membrane-bound granules through breaks in the plasma membrane. Only once was granule exocytosis observed. These results indicate that A23187 treatment of eosinophils causes an initial release of membrane-associated CLC protein by a noncytolytic mechanism, and causes degranulation as a result of eosinophil lysis.     

Effect of membrane association on the stability of complexes between ionophore A23187 and monovalent cations

The monovalent cation complexation properties of ionophore A23187 in methanol-water (65-95% w/w) and bound to unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) are contrasted. In both solution and vesicle-containing systems, 1:1 complexes between the ionophore and Li+ or Na+ predominate. The analogous complexes with K+, Rb+, and Cs+, which exist in methanol, are not detected on DMPC vesicles by changes in the absorption or fluorescence emission spectra of the ionophore. In solution, the logarithms of stability constants (log KMA) for both the LiA and NaA complexes increase by 1.5 units over the range of solvent polarity encompassed by 65% methanol-water to methanol. Selectivity for Li+ vs. Na+ is constant at a ratio of 5 in these solutions. On DMPC vesicles, selectivity for Li+ vs. Na+ is improved 15-fold with log KbLiA (3.23 +/- 0.03, T = 25 degrees C, mu = 0.05 M) being comparable to the value obtained in 80% methanol-water. In the latter solvent, increasing ionic strength (0.005-0.085 M) has little effect on log KLiA or log KHA but increases these constants by 0.4-0.5 unit in the DMPC vesicle system. Transition from the vesicle liquid-crystalline to gel-phase state reduces log KbLiA and log KbNaA by approximately 0.6 unit but has no effect on log KbHA. Thermodynamic parameters for formation of HA, LiA, and NaA in 80% methanol-water and on DMPC vesicles are reported. Analysis of these data and related considerations suggests that differences in the membrane interaction energies of particular ionophore species dominate in establishing the observed difference in complexation properties between the solution and vesicle-containing systems.     

Effects of divalent cation ionophore A23187 on potassium permeability of rat erythrocytes.

A23187 transports calcium rapidly into rat erythrocytes, apparently by an electroneutral exchange for intracellular magnesium and protons. When red cells are incubated in the absence of any added divalent cations, A23187 transports internal magnesium out of the cells, in exchange for extracellular protons. Magnesium uptake into erythrocytes is produced by A23187, providing the extracellular concentration of this cation exceeds intracellular levels, and the ionophore also transports strontium, but not barium, into red cells. A23187 produces a rapid and extensive loss of intracellular potassium from erythrocytes during uptake of calcium or strontium, but not magnesium. When red cells are incubated in the absence of any exogenous divalent cations, A23187 still produces a potassium efflux and this is inhibited completely by small amounts of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and restored by the addition of calcium in excess of the chelator. Although EDTA enhances the extent of magnesium release from erythrocytes incubated with A23187, it prevents the potassium efflux. Dipyridamole and 4-acetamid-4'-isothiocyano-stilbene-2,5'-disulfonic acid, which decrease chloride premeability of erythrocytes, inhibit the A23187-induced potassium loss from red cells. Rutamycin, peliomycin, venturicidin, and A23668B also inhibit potassium efflux from intact cells incubated with A23187, but this effect is not correlated with their abilities to inhibit various ATPases in red cell membrane preparations. It is concluded that A23187 does not transport potassium directly across the erythrocyte plasma membrane, but permits small amounts of endogenous calcium to interact with some membrane component to enhance potassium permeability of the cell.     

Electron paramagnetic resonance of copper ion and manganese ion complexes with the ionophore A23187.

The binding of Cu2+ and Mn2+ to the ionophore A23187 in chloroform, 90% ethanol, and sonicated phospholipid dispersions in aqueous mediums has been investigated with electron paramagnetic resonance (epr). The spectra indicated axial symmetry for the Cu2+ complexes and distorted octahedral for the Mn2+ complexes. The coordination between metal ion and its ligands is predominantly ionic in character. The stoichiometry, at the concentrations employed, was found to be 1:2 M2+/ionophore except in 90% ethanol where evidence existed for the 1:1 Cu-A23187 complex, as well. Through competition with Mn2+, the sequence of relative affinities in 90% ethanol was measured to be: Mn2+ greater than La3+ greater than Cu2+ greater than Ca2+ greater than Mg2+ greater than Sr2+. The K A of Mn-A23187 binding is greater than 10 10 M-2. In phospholipid dispersions the spectral characteristics of the Cu complex, particularly g, were observed to be a sensitive function of the hydrocarbon chain mobility. This allowed a calculation of the rotational correlation time of the complex to be made. In sonicated dipalmitoyllecithin was computed to be 10-9 sec, reflecting a local viscosity similar to that sensed by the nitroxide spin-label 2,2,6,6-tetramethylpiperidin-1-oxyl. In a (1:1) lecithin-cholesterol dispersion the complex was significantly more immobilized.     

Activation of human platelet phospholipase C by ionophore A23187 is totally dependent upon cyclo-oxygenase products and ADP.

Human platelets exposed to the Ca2+ ionophore A23187 form cyclo-oxygenase metabolites from liberated arachidonic acid and secrete dense granule substituents such as ADP. I have shown previously that A23187 causes activation of phospholipase A2 and some stimulation of phospholipase C. I now report that, in contrast to the case for thrombin, the activation of phospholipase C in response to ionophore is completely dependent upon the formation of cyclo-oxygenase products and the presence of ADP. The addition of A23187 to human platelets induces a transient drop in the amount of phosphatidylinositol 4,5-bisphosphate, a decrease in the amount of phosphatidylinositol, and the formation of diacylglycerol and phosphatidic acid. In addition, lysophosphatidylinositol and free arachidonic acid are produced. The presence of cyclo-oxygenase inhibitors or agents which remove ADP partially impairs these changes. When both types of inhibitor are present, the changes in phosphatidylinositol 4,5-bisphosphate and the formation of diacylglycerol and phosphatidic acid are blocked entirely, whereas formation of lysophosphatidylinositol and free arachidonic acid are relatively unaffected. The prostaglandin H2 analogue U46619 activates phospholipase C. This stimulation is inhibited partially by competitors for ADP. I conclude that phospholipase C is not activated by Ca2+ in the platelet, and suggest that stimulation is totally dependent upon a receptor coupled event.     

Effect of the calcium ionophore A23187 on superoxide generation in phorbol ester stimulated human neutrophils

The calcium ionophore, A23187, and the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), interacted synergistically to elicit an accelerated superoxide production response in human neutrophils. The lag period preceding PMA-induced superoxide generation was decreased in a dose-dependent manner by A23187 at a concentration range from 1.0 X 10(-8) to 1.0 X 10(-5) M. Superoxide production rate, however, was subject to biphasic effects. While the rate was potentiated in a dose-dependent manner at A23187 concentrations below 1.0 X 10(-6) M, inhibitory influences became manifest at higher concentrations. Total superoxide production was subject to inhibitory effects, characterized by a mean inhibitory dose of 1.3 X 10(-6) M. The synergistic interaction of A23187 with PMA is consistent with a role for protein kinase C in neutrophil activation. Inhibition at high A23187 concentrations appeared to result from the effects of elevated intracellular Ca2+ levels on either NADPH oxidase itself, or some step in the transduction process linking protein kinase C to the oxidase complex.     

Action of insulin and cell calcium: Effect of ionophore A23187

Summary We have measured the effects of the carboxylic Ca⁺⁺ ionophore A23187 on muscle tension, resting potential and 3-O-methylglucose efflux. The ionophore produces an increase in tension that is dependent on external Ca⁺⁺ concentration since (a) the contracture was blocked by removing external Ca⁺⁺ and (b) its size was increased by raising outside Ca⁺⁺. Neither resting potential nor resting and insulin-stimulated sugar efflux were modified by the ionophore. These data imply that the action of insulin is not mediated by increasing cytoplasmic [Ca⁺⁺]. Additional support for this conclusion was obtained by testing the effects of caffeine on sugar efflux. This agent, which releases Ca⁺⁺ from the reticulum, did not increase resting sugar efflux and inhibited the insulin-stimulated efflux. Incubation in solutions containing butyrated derivatives of cyclic AMP or cyclic GMP plus theophylline did not modify the effects of insulin on sugar efflux. Evidence suggesting that our experimental conditions increased the cytoplasmic cyclic AMP activity was obtained.     

Erythrocyte membrane in protein-energy malnutrition: A23187-induced changes in osmotic fragility of human and rat erythrocytes

Erythrocytes from protein-energy malnourished children have been shown to have increased resistance to osmotic lysis (4). Osmotic fragility studies were carried out in protein-energy malnourished rats and A23187-induced changes in osmotic fragility were studied in rat and human erythrocytes. Rat erythrocytes were found to be much more sensitive to A23187 effect on osmotic fragility as compared to the human erythrocytes. Erythrocytes from protein-deficient rats but not from the energy-restricted rats showed increased resistance to osmotic lysis. A23187 (+Ca)-induced changes in osmotic fragility were not different between control and experimental erythrocytes, either for humans or rats. There was, however, a difference in the extent to which Na accumulation and K depletion occurred in erythrocytes from control and experimental animals after A23187 + Ca²⁺ treatment.     

The effects of ionophore A23187 and concanavalin A on the membrane potential of human peripheral blood lymphocytes and rat thymocytes

Effects of the Ca2+-ionophore A23187 and concanavalin A on the membrane potential of human lymphocytes and rat thymocytes have been studied using the fluorescent potential probe diS-C3-(5). At concentrations of 10(-8) to 10(-6) M A23187 changes the membrane potential, inducing both hyper- and depolarization. Depending on concentrations of A23187 and the external Ca2+, and on the type of lymphocytes, one of these effects predominates. The hyperpolarization induced by A23187 is caused by activation of Ca2+-dependent K+ channels. It is blocked by quinine and high concentrations of extracellular K+. The dependence of Ca2+-activated K+ transport on extracellular Ca2+ and its sensitivity to calmodulin antagonists is different for human lymphocytes and for thymocytes. As distinct from lymphocytes, in thymocytes calmodulin is not involved in activation of Ca2+-dependent K+ transport. The depolarization induced in lymphocytes by A23187 is caused by an increase in Na+ permeability of the lymphocyte plasma membrane: it is eliminated in a low-Na+ medium. At mitogenic concentrations concanavalin A does not change the membrane potential of the lymphocytes. The results obtained permit elucidation of the relationship between two early events in lymphocyte activation, namely the increase in intracellular Ca2+ concentration and the increase in lymphocyte plasma membrane permeabilities to monovalent cations.     

The effects of ionophore A23187 on erythrocytes relationship of ATP and 2,3-diphosphoglycerate to calcium-binding capacity

The divalent cation ionophore, A23187, was employed as a means to load fresh human erythrocytes with calcium, and the capacity for accumulation was characterized. Erythrocytes exposed to A23187 in calcium-containing media rapidly accumulated calcium in millimolar quantities. The final cellular concentration was dependent upon medium calcium concentration and the size of the cellular organophosphate pool. When ATP and 2,3-diphosphoglycerate contents were depleted or repleted, the cellular calcium content changed proportionally. Calcium loading of fresh erythrocytes produced no discernible change in the cellular concentrations of ATP or 2,3-diphosphoglycerate. Calcium accumulation was also accompanied by loss of cellular potassium and H₂O, deterioration of cell filterability, and spheroechinocytic transformation.