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.     

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.     

Equilibria between ionophore A23187 and divalent cations: stability of 1:1 complexes in solutions of 80% methanol/water

The cation complexation equilibria between ionophore A23187 and several alkaline earth and first transition series divalent cations have been investigated. Absorption and fluorescence spectroscopy were used to monitor the reactions which were studied in solutions of 80% methanol/water, at 25 degrees C, and under conditions of controlled ionic strength and pH. Titration of the ionophore with divalent cations results first in formation of the dimeric species MA2 and subsequently in the formation of MA+ by disproportionation of the first product. With Zn2+, Ni2+, and Co2+ (above pH approximately 6), a third species is detected which is postulated to be MA.OH. The existence of this species with Mn2+ and alkaline earth cations is uncertain. For formation of MA2, the second stepwise stability constant is similar to or exceeds the first value with all cations studied. However, it is possible to isolate the first reaction and determine accurate stability constants by working at an ionophore concentration of 3 X 10(-8) M or less and by employing pH values which preclude interference by the mixed ionophore/hydroxide species. Under these conditions, the relationship between log KMA' and pH is linear and displays a slope of 1.0. pH-independent stability constants were calculated by using pH-dependent stability constants and the known value of the ionophore's protonation constant in this solvent. The logarithms of the values obtained ranged from 7.54 +/- 0.06 for Ni2+ to 3.60 +/- 0.06 for Ba2+. The selectivity sequence and relative affinities (in parentheses) for the species MA+ are as follows: Ni2+ (977) greater than Co2+ (331) greater than Zn2+ (174) greater than Mn2+ (34) greater than Mg2+ (1.00) approximately equal to Ca2+ (0.89) greater than Sr2+ (0.20) greater than Ba2+ (0.11). Data are discussed in comparison to other studies on the complexation properties of A23187 and in terms of their significance to interpreting the transport properties of this ionophore.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opzonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B₄ and C₄. After 3–4 min, the levels of LTB₄ and LTC₄ were 93 and 35 pmol/310⁷ cells, respectively. These amounts were 2–4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC₄ were 8 and 20 times lower than those of LTB₄ after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opsonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B4 and C4. After 3-4 min, the levels of LTB4 were 93 and 35 pmol/3*10(7) cells, respectively [corrected]. These amounts were 2-4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC4 were 8 and 20 times lower than those of LTB4 after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

Solution conformation of the ionophore A23187 and its magnesium salt

The analysis of proton nmr spectra at 300 MHz in C₆D₆ and in CDCl₃ of the divalent cation ionophore A23187 and its magnesium salt are reported and discussed in terms of configurational and conformational behaviour. The conformation of the free acid in solution was found to be almost identical to that described previously (M. O. Chaney, P. V. Demarco, N. D. Jones, and J. L. Occolowitz, J. Amer. Chem. Soc.96, 1932 (1974)) for the solid state, except that in apolar solvents no head-to-tail interaction is observed, in that free rotation around certain bonds (e.g., C₁₅–C₁₉, structures 1 and 2) substantially prevents such quasi-cyclic forms. For the conformation of the magnesium salt, two monovalent units aggregate around the divalent cation, the two substructures displaying equivalent atoms in each of their corresponding positions. A compact sphere-like form having a C₂ axis and a cavity of about 3.5 Å is proposed, the cation being held in the middle by two carboxylate ions, carbonyl and N-benzoxazole units as the ligands. These conclusions result from consideration of the extracted parameters in the proton nmr spectrum and from model building. Thus, the most probable form of the Mg-salt in solution corresponds almost exactly to that of the Ca-salt in the solid state, as very recently revealed by the X-ray analysis of M. O. Chaney, N. D. Jones, and M. Debono (J. Antibiot, in press).     

Calcium-dependent irreversible effect of ionophore A23187 on melanophores

The calcium ionophore, A231187, induces a Ca2+ -dependent movement (dispersion) of melanosomes within skin melanophores of the lizard, Anolis carolinensis, in vitro. The effects of A23187 are irreversible, since after repeated rinsing of the skins in the absence of the ionophore they will always darken in Ringer containing Ca2+ but will immediately lighten when transferred to Ca2+ -free Ringer. These results suggest that the ionophore is irreversibly localized to the melanophore membrane and that its melanosome-dispersing effect is continuously dependent upon extracellular calcium.     

Calcium-dependent interactions of an ionophore A23187 with calmodulin

We found that ionophore A23187 interacted reversibly with calmodulin (CaM), in a calcium-dependent fashion. It was found that A23187 interacts selectively with CaM, among calcium binding proteins (such as troponin C and S-100 protein) and other proteins. However, apparently differing from W-7, A23187 did not suppress CaM-dependent enzyme activity such as myosin light chain kinase and Ca2+-dependent cyclic nucleotide phosphodiesterase. Our observations suggest that there are novel calcium-dependent regions of CaM which can be monitored using ionophore A23187 and may not be related to enzyme activation.     

Dual action of ionophore A23187 on intact chloroplasts

1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O₂ evolution by envelope-free chloroplasts and oxaloacetate-dependent O₂ evolution by intact chloroplasts. The half maximal concentration ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml^?1.2. Ionophore A23187 also induces inhibition of CO₂ and 3-phosphoglycerate-dependent O₂ evolution by intact chloroplasts in the presence of 3 mM MgCl₂. The half maximal concentrations ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for inhibition of O₂ evolution are 3 μM and 5 μM respectively · 100 μg^?1 chlorophyll · ml^?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg^?1 chlorophyll · ml^?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.     

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.     

Aggregation of calcium ionophore (A23187) in phospholipid vesicles

The circular dichroism studies on calcium ionophore, A23187, incorporated in Dipalmitoyl phosphatidyl choline (DPPC) vesicle showed interesting time dependent changes in the CD spectra. Analysis of the data indicated the possible aggregation of the observed dimeric structure of this molecule in non-polar solvents into a stacked dimeric pore in the phospholipid vesicle.     

Cytokine- and calcium ionophore A23187-mediated arachidonic acid metabolism in neutrophils

Arachidonic acid (AA) metabolism is implicated as an intracellular and/or intercellular second messenger system for the transmission of cytokine-initiated signals that affect neutrophils and mediate systemic toxicity. The purpose of the present study is to ascertain if cytokines that are known to affect neutrophil function in vivo and in vitro directly stimulate neutrophil AA metabolism in vitro. The recombinant human cytokines multi-colony stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1, tumor necrosis factor (TNF), and interleukin 6 and the calcium ionophore A23187 were incubated with purified 14C-AA radiolabeled human peripheral blood neutrophils and the effects were assayed by one- and two-dimensional thin layer lipid chromatography. None of the cytokines appeared to induce the release of cell-incorporated AA or to increase the level of radiolabeled phosphatidic acid. TNF induces severe systemic toxicity that is inhibited by cyclooxygenase inhibitors, which suggests a role for AA metabolites in the pathophysiologic effects of TNF; we have confirmed that TNF and endotoxin act synergistically to induce indomethacin-inhibitable fatal shock in rats. However, when in 3H-AA radiolabeled human neutrophils were incubated with TNF in kinetic, cold-chase, and TNF preincubation experiments, TNF was not found to increase AA metabolism, although changes in the intracellular neutral lipid content were noted. GM-CSF, which has been reported by previous investigators to directly induce the release of AA, did not release neutrophil-associated 3H-AA. In conclusion, the direct release of AA from membrane-associated phospholipids does not appear to be a major second messenger pathway for cytokine-initiated activation of neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The effects of the ionophore A23187 on pattern formation in the alga Micrasterias

We have used the divalent cation ionophore A23187 to investigate the hypothesis that cytoplasmic localization of Ca2+ is responsible for localized growth in the alga Micrasterias. In a preliminary study we found that, of the major salts contained in the cell's medium, only CaCl2 was needed for normal development. In cells developing in the presence of A23187 and extracellular Ca2+, we postulated that the ionophore would induce a spatially uniform influx of Ca2+ that would overwhelm endogenous Ca2+ gradients. When developing cells were treated with A23187 and 2 mM CaCl2, we observed a delocalization of the cell's normal pattern of wall deposition. This effect was less pronounced when cells were exposed to A23187 and 2 mM MgCl2. These results support the hypothesis that localized regions of high Ca2+ concentration normally mediate localized expansion of tip-growing lobes in Micrasterias.     

Enhancement of the muscarinic synaptosomal phospholipid labeling effect by the ionophore A23187

Addition of either acetylcholine (ACh) or the ionophore A23187 to synaptopsomes resulted in a selective stimulation of 32Pi incorporation into phosphatidate (PhA) and phosphatidylinositol (PhI), while the labeling of phosphatidylinositol phosphate (PhIP) and phosphatidylinositol diphosphate (PHIP2) was reduced. The inclusion of both ACh and A23187 resulted in a synergistic increase in PhA and PhI labeling, and a synergistic decrease in the labeling of the polyphosphoinositides. Added calcium was not required, although inclusion of EGTA prevented the alterations in lipid labeling. The enhanced labeling of PhA and PhI by ACh or A23187 was not the result of either an increase in the radioactivity of the precursor [32P]ATP pool, or increased de novo synthesis of these lipids as judged from the incorporation of [3H]glycerol, [3H]glucose or [3H]myo-inositol. The synergistic alterations in PhA, PhI, and polyphosphoinositide labeling were observed with ionophore only in the presence of selected muscarinic agonists, and with the inclusion of atropine or scopolamine the labeling reverted to a value which approximated that seen with the ionophore alone. Synergistic effects on phospholipid labeling with muscarinic agonists were also obtained with the calcium ionophore, ionomycin, but not with X537A, monensin, or valinomycin. Neither the apparent number of muscarinic receptors present, nor their affinity for the ligand were altered by the presence of A23187. In prelabeling experiments, A23187 accelerated the loss of [32P]label from PhIP and PhIP2, and the rate of loss was further augmented by the addition of ACh. Neither agent produced comparable effects on the breakdown of prelabeled PhA or PhI. It is suggested that phosphodiesteratic cleavage of the polyphosphoinositides might account for both the decrease in labeled PhIP and PhIP2 and increased labeling of PhA and PhI via the availability of resultant diglyceride. In any event, the results demonstrate that the turnover of polyphosphoinositides, in addition to that of PhA and PhI, is linked to the activation of muscarinic receptors.     

Activation of respiration in sea urchin spermatozoa by calcium ionophore A23187

The respiratory rate in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, in Na+-free seawater, where sperm are immotile and their respiration remains inactive, was stimulated by calcium ionophore A23187. Addition of ionophore A23187 to Na+-free seawater induced swimming as well as activating energy metabolism in sea urchin sperm. The increase of respiratory rate and the initiation of motility in sperm were independent of external Ca2+.     

The calcium ionophore A23187 increases the tight-junctional permeability in rat liver.

The effect of an increase in intracellular Ca2+ concentration on tight-junctional permeability in rat liver was studied by using the calcium ionophore A23187. Infusion of 100 microliters of dimethyl sulphoxide containing various amounts of A23187 over 30 min into isolated perfused livers was followed by a pulse of horseradish peroxidase (HRP) under single-pass conditions. The first biliary HRP peak, a measure of junctional permeability, was increased 4-fold with 100 micrograms of A23187. There were, however, no significant effects on bile flow or on aspartate aminotransferase leakage as compared with the control at this dosage, and thus the increase in junctional permeability was occurring without evidence of appreciable cholestatic or hepatocellular damage. Higher dosages of A23187, however, caused not only an increase in HRP peak height but also changes in bile flow and increases in aminotransferase leakage, indicating more extensive effects at these higher dosages. A second peak of HRP secretion, occurring 20-25 min after the HRP pulse, was also elevated approx. 3.5-fold; this may indicate that pinocytosis and transcellular movement of HRP are also increased under these conditions.