Effects of ionophores A23187 and X537A on brain calcium,catecholamines and excitability

The ionophores A23187 and X537A inhibit ⁴⁵Ca uptake by rabbit brain mitochondria and synaptosomes and also stimulate the release of accumulated ⁴⁵Ca from these preparations, but have no effect on ⁴⁵Ca binding by synaptic membranes or on total brain Ca in mice. Both agents inhibit uptake and stimulate release of ³H-norepinephrine by rabbit P₂ synaptosomal preparations, while the NE and serotonin levels of mouse brain are depressed by X537A. The changes in Ca activities may be related both to the elevated thresholds for cortical after-discharge produced in cats by these ionophores, and to the ionophore induced reduction of pentylenetetrazol seizures in mice.     

Mechanism of release of norepinephrine from peripheral adrenergic neurones by the calcium ionophores X 537A and A 23187

The effects of the calcium ionophores, X 537A and A 23187, in causing release of norepinephrine (NE) were examined in isolated guinea-pig vas deferens and rat atrial segments. Examination of calcium dependence, dopamine-β-hydroxylase (DBH) release and release of deaminated metabolites of NE suggest that while A 23187 releases by both an exocytotic and non-exocytotic mechanism, X 537A releases NE predominantly through a non-exocytotic mechanism. The relative impotence of A 23187 in effecting release of NE may explain its inactivity in alteration of cardiac responses.     

The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A

The divalent ionophores A23187 and X-537A induce parthenogenesis in sea urchin eggs. This results from their ability to mobilize intracellular Ca²⁺, which is implicated in both artificial parthenogenesis as well as the natural fertilization process. A23187 causes expulsion of cortical granules and elevation of the fertilization membrane within 0.5–9 min followed by an initiation of cell cleavage. The broader spectrum ionophore X-537A is less potent, but the production of cytoplasmic aberrations are more apparent. In contrast to the sperm-activated egg, the initial phase of ionophore induced activation is accompanied either by relatively insignificant changes in membrane resistance, or an increase.     

The release of dopamine from synaptosomes from rat striatum by the ionophores X 537A and A 23187

The antibiotics X 537A and A 23187 are negatively charged divalent cation ionophores. X 537A may, in addition, be an ionophore for amines including catecholamines. The effects of these ionophores were examined on the uptake and release of dopamine by synaptosomes prepared from rat corpus striatum. Both X 537A and A 23187, at concentrations less than 0.5 μM, release both endogenous and [³H]-dopamine from synaptosomes. They had virtually no effect on the uptake of exogenous dopamine. These compounds act by different mechanisms. X 537A causes divalent ion-independent release in which a large fraction of the effluent consists of deaminated products. X 537A, in addition, releases [³H]dopamine from rat adrenal medullary chromaffin granules. The results suggest that X 537A causes release of dopamine from intrasynaptosomal storage vesicles and perhaps is acting as a catecholamine carrier across the vesicular membrane. A 23187, on the other hand, causes a Ca²⁺-dependent release in which only a small fraction of the catechol in the effluent is deaminated. A 23187 has little effect on the release of [³H]dopamine from chromaffin granules. These results suggest that A 23187 carries Ca²⁺ into the synaptosomes and thereby initiates exocytotic release.     

Effects of the ionophores X537A and A23187 on GABA,glycine, and taurine release from chick retinal subcellular fractions

The ionophore X537A at concentrations of 5–20 M stimulated the release of [³H]GABA and [³⁵S]taurine, from retinal subcellular crude nuclear (P₁) and crude synaptosomal (P₂) fractions. The release of [³H]GABA increased 114% and 136% over control values in P₁ and P₂ fractions, respectively. The efflux of [³⁵S]taurine from P₁ was increased by 45% and that from P₂ by 21%. X537A increased⁴⁵Ca²⁺ uptake in the P₂ fraction but not in the P₁ fraction. The effect of X537A on the amino acid release was not dependent on the presence of exogenous calcium. X537A did not affect [³H]GABA or [³⁵S]taurine uptake by the retinal fractions. A23187 enhanced [³H]GABA release from P₁ and P₂ by 52% and 105%, respectively. The ionophore also increased [¹⁴C]glycine liberation in both P₁ (35%) and P₂ (50%) but failed to stimulate [³⁵S]taurine release. A23187 produced a transient increase of⁴⁵Ca²⁺ uptake of 38% in P₁ and 30% in P₂. The effects of A23187 on the release of amino acids were calcium dependent. The amino acid uptake was not affected by the ionophore. These results are consisent with the suggested neurotransmitter role for GABA at the outer synaptic layer and for GABA and glycine at the inner synaptic layer of the retina. A neurotransmitter role for taurine is not supported by the present results.     

Ultrastructural changes in neurohaemal tissue of the stick insect,Carausius morosus,induced by the ionophores Br-X-537 A and A-23187

Summary The antibiotic ionophores Br-X-537A and A-23187 alter the ultrastructure of neurohaemal tissue on the transverse nerve of the stick insect, Carausius morosus. Br-X-537A induces dramatic changes in the ultrastructural appearance of all three types of neurosecretory fibres present in the neurohaemal tissue. The neurosecretory granules become more electron-lucent and the mitochondria become more electron-opaque. The bounding membrane of the granules is frequently ruptured. A-23187, on the other hand, has no effect on two of the three types of fibres, but does produce an increase in the number of exocytotic profiles in the third.The two ionophores therefore have different effects on the same tissue. The results are discussed in the light of previous work with the use of these ionophores.We wish to thank Mrs. J. Birch for assistance with the electron micrographs, and Roche Products Ltd. and Lilly Research Centre Ltd. for gifts of the ionophores Br-X-537A and A-23187. The work was supported by the Science Research Council     

Calcium ionophores A23187 and X537A affect cell agglutination by lectins and capping of lymphocyte surface immunoglobulins

The microtubule-disruptive drugs colchicine and vinblastine alter ligand-induced redistribution of cell surface immunoglobulins and lectin receptors. These effects can be duplicated by treatment of cells with the divalent cation ionophores A23187 and X537A. Ionophore activity was dependent upon the presence of Ca²⁺ (1.8·10^?3?4·10^?4 M) in the culture medium. The K⁺-selective ionophore valinomycin had no effect on ligand-induced redistribution of surface receptors. It is suggested that A23187 and X537A impair membrane-associated microtubules involved in transmembrane control of receptor mobility and topography. In contrast to the action of colchicine and vinblastine that bind directly to microtubules, it is proposed that ionophores indirectly affect microtubules by raising the concentration of Ca²⁺ in the cytoplasm to levels that favor microtubule depolymerization and inhibit microtubule assembly.     

Ionophores X537A and A23187. Effects on the permeability of lipid bimolecular membranes to dopamine and calcium

X537A carries dopamine across lipid bimolecular membranes. The rate of transport increases linearly with the X537A concentration and is independent of an electric field across the membrane. The evidence suggests that the permeating species is a neutral 1:1 complex between dopamine and X537A. A23187 does not transport dopamine. The permeability of the membrane to calcium increases as the square of the X537A concentration; the transport of calcium is also increased by A23187. With both ionophores, calcium is probably transported as an uncharged complex. Neither desmethylimipramine nor cocaine alters the transport of dopamine with X537A.     

Effects of glucagon, insulin and cyclic-AMP on mitochondrial calcium uptake in the liver.

The effects of glucagon and insulin administration $\text{in vivo}$ on hepatic mitochondrial Ca²⁺ uptake were compared with the effects of these hormones when they were added directly to the perfused liver. Glucagon administration increased mitochondrial calcium uptake both $\text{in vivo}$ and in the perfused liver. In contrast, while injection of insulin into rats stimulated, addition of insulin to the perfusate, inhibited Ca²⁺ uptake. Cyclic AMP, when added to the perfusate, also increased the uptake of Ca²⁺ by mitochondria, subsequently isolated. The possible implications of the results are discussed.     

The stoichiometry of A23187- and X537A-mediated calcium ion transport across lipid bilayers

Initial rates of ionophore-mediated Ca2+ transport across egg phosphatidylcholine bilayers of large unilamellar vesicles were measured using the absorbance change of arsenazo III at 650 nm as an indicator of Ca2+ translocation. A23187 induced the movement of Ca2+ in a 2:1 ionophore: Ca2+ complex, whereas its methyl ester (CH3A23187) and X537A mediated Ca2+ movement in a 1:1 ionophore: Ca2+ complex. The relative potencies of these ionophores in transporting Ca2+ across lipid membranes were A23187 much greater than X537A greater than CH3A23187.     

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.     

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta: III. Large bleb formation caused by Sr2+, ionophores X537A and A23187, and compound 4880

Fertilized eggs of Ilyanassa obsoleta form a protuberance which resembles a normal polar lobe when injected with Sr²⁺ or Ca²⁺ by microiontophoresis. Eggs also form a lobe-like protuberance when exposed to any of three drugs: compound $\text{48}\text{80}$, ionophore X537A, and ionophore A23187. Protuberances form more quickly and at lower drug concentrations if additional exogenous Ca²⁺ is added, whereas higher concentrations of Mg²⁺ do not have such an effect. When eggs are exposed to these drugs in Ca²⁺-, Mg²⁺-“free” seawater, with or without 10 mM EDTA, the eggs are still able to undergo extensive shape changes and form protuberances. Drug-induced shape changes are prevented by cytochalasin B, but will still occur in the presence of colchicine. Approximately 75% of Ilyanassa eggs are capable of forming and resorbing their third polar lobe and undergoing cytokinesis in Ca²⁺-, Mg²⁺-“free” artificial seawater (even containing 10 mM EDTA), solutions which by atomic absorption spectroscopy are shown to contain low concentrations of Ca²⁺ (3–5 μM) and Mg²⁺ (1.0–3.5 μM). The data suggest that if Ca²⁺ is required for normal polar lobe formation and cytokinesis, it is derived from intracellular sources or is required in only very low exogenous concentrations (i.e., less than 10^?2 μM free Ca²⁺, in the presence of 10 mM EDTA).     

Cation ionophores A23187 and valinomycin enhance protein-mediated transfer of rat liver microsomal phosphatidylinositol to liposomes

Summary A standard reaction mixture has been established in which partially purified rat liver phosphatidylinositol exchange proteins sustain a maximal rate of phosphatidylinositol transfer from rat liver microsomes to liposomes. Determination of the transfer kinetics confirms the findings of Brophy et al. (Biochem J. 174:413–420, 1918) that under such conditions a maximum 70–80% of the homogenously radiolabeled, microsomal phosphatidylinositol is exchanged with biphasic kinetics. The phosphatidylinositol exchange proteins thus indicate the presence of three microsomal phosphatidylinositol pools: One pool is not subject to protein-mediated exchange; the other two pools are both exchangeable but are exchanged with significantly different half-lives. Both the divalent cation ionophore, A23187, and the monovalent cation ionophore, valinomycin, significantly enhance phosphatidylinositol transfer in the standard reaction mixture at concentrations 1 to 2 orders of magnitude greater than those sufficient for the ionophores to facilitate cation transport across membranes. The stimulatory effect of each ionophore, however, is not a function of the ionophore/microsome mass ratio in the reaction miture. Although both ionophores increase the relative amount of exchangeable phosphatidylinositol, neither ionophore results in all of the exchangeable phosphatidylinositol being transferred with singlestate kinetics. The evidence demonstrates that A23187 and valinomycin are the first substances found to markedly enhance the reactivity of a microsomal phospholipid class with phospholipid exchange proteins.     

Inhibition of antigen and calcium ionophore A23187 induced contractions of guinea pig airways by isoprenaline and forskolin

Isoprenaline and forskolin both inhibit contractions induced by antigen or by the calcium ionophore A23187 of guinea pig tracheal spirals and parenchymal strips. Antigen-induced airway contraction is considerably more sensitive to the inhibitory effects of isoprenaline than is A23187-induced contraction. In contrast, forskolin is equiactive as an inhibitor of antigenic and ionophoric contractions. Forskolin is a more effective inhibitor of the prolonged phase of antigen-induced tracheal contraction than of the initial peak phase, which may suggest selectivity for the lipoxygenase pathway of arachidonic acid metabolism. Isoprenaline inhibits the mechanisms of the primary peak phase and of the prolonged phase equally. Although there were little, if any, differences between normal and sensitized tissues in the modulation of A23187-induced contractions of parenchyma, distinct differences were observed in trachea. Low concentrations (10(-8)-10(-7) M) of isoprenaline and forskolin enhanced A23187-induced contraction of sensitized, but not normal trachea. Higher concentrations were inhibitory. The results demonstrate that sensitization affects the modulation by isoprenaline and forskolin of A23187-induced contraction of guinea pig trachea.     

Ionophore A23187, calcium and contractility in frog eggs

The livers of adult rats were dissociated after collagenase-hyaluronidase perfusion. A high yield of viable cells was obtained and these regularly gave rise to continuous lines of polygonal epithelial cell types.     

Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria

A23187, a new antibiotic with ionophore properties, uncoupled oxidative phosphorylation in mitochondria which oxidized either malate plus glutamate or succinate. Ca²⁺, but not Mg²⁺, enhanced the uncoupling effect. Fluorescence of ANS¹ was increased by A23187 suggesting the mitochondrial membranes were de-energized. This de-energization was presumably by activation of the energy-dependent uptake of Ca²⁺. The steady-state measurements of murexide-divalent cation complexes showed that A23187 caused mitochondria to release the accumulated Ca²⁺ to the medium. This reduced the transmembrane Ca²⁺ gradient even though normal active Ca²⁺ uptake could take place. A23187 inhibited activity of ATPase induced by 2,4-dinitrophenol, valinomycin, and Ca²⁺. The addition of Mg²⁺ could prevent this inhibition presumably by maintaining the endogenous Mg²⁺ concentration. The above metabolic events could be explained by the fact that molecules of A23187 function in the mitochondrial inner membrane as mobile carriers for divalent cations.