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.     

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.     

Induced acetylcholine release from active purely cholinergic Torpedo synaptosomes.

Viablse, purely cholinergic synaptosomes were prepared from the electric organ of Torpedo ocellata and partially purified by differential and sucrose density centrifugation. The synaptosomes contain acetylcholine (ACh), synaptic vesicles, cytoplasmic markers and mitochondria. No adherent postsynaptic membranes were detected. K⁺ depolarization as well as the ionophore A23187 mediate Ca²⁺ permeation into the synaptosomes and the consequent release of ACh. Mg²⁺ does not evoke ACh release whereas Sr²⁺ and Ba²⁺ can replace Ca²⁺ in evoking K⁺ depolarization induced ACh secretion. In accordance with the calcium hypothesis of stimulus–secretion coupling, both K⁺ depolarization and the ionophore A23187 seem to mediate the release of the same population of ACh molecules. The mode of action of the ionophore X537A differs from that of A23187. X537A acts independently of Ca²⁺ and induces the release of a larger fraction of the ACh contained in the fractionated nerve terminals. These results demonstrate that the Torpedo synaptosomes contain the neurosecretion apparatus in a functional active state. This preparation extends the utility of synaptosomes for structural and functional biochemical studies of neurotransmission, as it uniquely contains only one neurosecretion system (cholinergic).     

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.     

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.     

Effects of the ionophores,X-537A and A-23187 on catecholamine release from the in vitro frog adrenal

1. The ionophore X-537A increases the rate of catecholamine release from the $\text{in vitro}$ frog adrenal.2. The ratio of epinephrine/norepinephrine measured during X-537A stimulation was the same as that during spontaneous release.3. Even when Ca⁺⁺ was removed from the Ringer, X-537A stimulated catecholamine release, but depolarization by elevated extra-cellular K⁺ was no longer effective.4. X-537A also increases the release of dopamine β-hydroxylase, suggesting that the ionophore acts, at least in part, by stimulating the exocytosis of the chrommaffin granule contents.5. Therefore, it is questionable whether the release of catecholamines by X-537A is owing to its action as a Ca⁺⁺- ionophore.6. The divalent cation ionophore, A-23187 (50μM), did not affect the rate of catecholamine release.     

Influence of calcium on phosphorylation of a synaptosomal protein

Synaptosomal proteins isolated from rat cerebral cortex were phosphorylated endogeneously in the presence of [γ-³²P]ATP. The phosphorylated proteins were found to be membrane bound by differential and density gradient centrifugation. In contrast to the phosphorylation of all synaptosomal proteins, phosphorylation of one protein (C), 41 000–43 000 daltons, was inhibited by Mg²⁺ and stimulated by Ca²⁺. In addition, the ionophores X537A and A23187, as well as papaverine, selectively enhanced phosphorylation of protein C without affecting phosphorylation of the other proteins. Cyclic AMP did not influence the phosphorylation of protein C but markedly affected the phosphorylation of other synaptosomal proteins. It appears that the phosphorylation of protein C is stimulated by agents which trigger the release of neurotransmitters (Ca²⁺, X537A, A23187 and papaverine), and is inhibited by Mg²⁺, which inhibits release. It is proposed that the phosphorylation of protein C is related to membranal events underlying the release of neurotransmitters.     

Influence of ionophores which bind calcium on the release of norepinephrine from synaptosomes.

A preparation of synaptosomes isolated from rat brain was used as a model of nerve to study affects of drugs on uptake and release of biogenic amines. The influence of ionophores, which bind calcium, on the release of noripinephrine from synaptosomes was examined to determine their effect on the release of the amine. A23187 induced release of norepinephrine mainly as the amine and this action was enhanced by calcium and depressed by magnetism. X-537A however, released norepinephrine mostly as deaminated metabolites but acted independently of calcium or magnetism. A23187, therefore is thought to be associated at least in part, with exocytotic amine release, possibly by enhancing entry of calcium across the plasma membrane. X-537A on the other hand may act as a carrier of the amine across the vesicular membrane and expose the amine to intrasynaptosomal monoamine oxidase.     

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.     

The action of ionophores at the frog neuromuscular junction

The ionophores A23187 and X537A have markedly different actions on the MEPP frequency recorded at the frog neuromuscular junction. A23187 has no significant effect at 9–17°C, but causes a small increase in MEPP frequency at 6°C. At 25°C, on the other hand, A23187 causes a marked and progressive rise in MEPP rate. It is suggested that, in spite of increased Ca²⁺ influx associated with application of the ionophore, the presynaptic terminals can maintain [Ca²⁺]_i constant at 9–17°C, although [Ca²⁺]_i rises at higher and lower temperatures, causing an increase in frequency of MEPPs. As previously reported by Kita and Van der Kloot (5) X537A causes a dramatic increase in MEPP frequency, but it is suggested that its action is more complex and probably involves an increase in Na⁺ permeability.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

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.     

Studies on growth hormone secretion IX. Prostaglandins do not act like ionophores

To gain further insight on the mechanism of GH secretion in general and on the stimulation of this process by prostaglandins in particular, we compared the effects of PGE₁ and PGE₂ on hormone release and cyclic nucleotide levels with those of the ionophores A23187 and X537A under a variety of experimental conditions. All these substances (in the presence but not in the absence of calcium) enhanced GH release in incubated rat anterior pituitaries , prostaglandins being considerably more potent than ionophores. However, while PGE₂ caused a dose-dependent rise in pituitary cyclic AMP levels (from doubling at 10⁻⁷ M to a two-hundred fold increase at 10⁻⁵ M), the ionophores had no effect on the concentrations of this nucleotide. Neither PGE₂ nor the ionophores had any measurable effect on cyclic GMP levels. Exposure of tissues to ionophores for 60 minutes rendered them refractory to subsequent stimulation by PGE₁ or to ionophores themselves, whereas preincubation with PGE₁ did not diminish GH responses during a second incubation period. On the other hand, 60-minute preincubation of hemipituitaries in the presence of ionophores (10⁻⁵ M) did not suppress subsequent PGE₁-promoted cyclic AMP accumulation. Metabolic blockers inhibited PGE₂ and A23187-promoted GH-release but failed to suppress GH-response to X537A. Verapamil partially inhibited PGE₂ but not ionophore induced GH secretion. Ionophores particularly X537A, accelerated 45Ca efflux while PGE₁ did not influence this. Electronmicroscopy revealed extensive vacuolization localized chiefly at the Golgi apparatus when tissues were incubated with X537A. PGE₁ and A23187 had no such morphological effect. It is concluded that prostaglandins E and ionophores promote GH secretion by different mechanisms.     

Quercetin: A novel inhibitors of Ca2+ influx and exocytosis in rat peritoneal mast cells

The effect of the transport ATPase inhibitor, quercetin on histamine secretion from antigen sensitized mast cells was examined. At micromolar concentrations, quercetin had an immediate inhibitory effect on histamine secretion mediated by antigen, concanavalin A and ATP but it had little effect on release induced by the ionophores A23187 and X537A. Quercetin exerts its effect after the binding of the releasing ligands and the distinction between its effect on ligand induced and A23187 induced secretion suggests that it affects the normal path of Ca²⁺ entry into the cell.The inhibitory effects of quercetin were compared with those of the structurally related anti-allergic drugs cromoglycate and AH7725.     

Some effects of ionophores for divalent cations on blood platelets Comparison with the effects of thrombin

The ionophores A 23187 and X-537 A induce an uptake of ⁴⁵Ca by human blood platelets. They induce the release of adenine nucleotides and of serotonin. A 23187 also induces platelet aggregation and the retraction of a clot formed in platelet-rich plasma by reptilase. These results suggest that an increase of the concentration of Ca²⁺ in the cytoplasm plays a role in the activation of blood platelets.     

Intrastriatal kainic acid elicits functional supersensitivity of muscarinic receptors regulating dopamine release

Acetylcholine enhanced in a concentration-dependent way the K⁺ (15 mM)-evoked release of [³H]dopamine from synaptosomes isolated from rat corpus striatum and prelabeled with the radioactive catecholamine. The concentration-effect curve of ACh obtained in presence of 1.2 mM Ca²⁺ was progressively shifted to the left when [Ca²⁺] was lowered to 0.4 and to 0.2 mM. Intrastriatal injections of kainic acid reduced (70%) the uptake of [³H]choline in synaptosomes prepared 8 days after the lesion but did not affect significantly the uptake of [³H]dopamine. Also the release of [³H]dopamine evoked by K⁺ was minimally affected by kainic acid treatment. In contrast, acetylcholine (tested in presence of 1.2 or 0.2 mM Ca²⁺) was much more effective in enhancing [³H]dopamine release in synaptosomes from kainic acid-lesioned than from unlesioned striata. The results suggest that muscarinic receptors located on dopamine nerve terminals undergo supersensitivity following intrastriatal kainic acid injection.     

Presynaptic muscarinic receptor activation enhances striatal dopamine release evoked by depolarization but not that induced by non-depolarizing stimuli

The release of [³H]dopamine stimulated by depolarization with 15 mM KCl of superfused rat striatal synaptosomes was potentiated by acetylcholine through the activation of presynaptic muscarinic receptors. In contrast, acetylcholine did not potentiate the release of [³H]dopamine elicited by d-amphetamine nor that caused by the calcium ionophore A23187. The dopamine carrier blocker nomifensine prevented the releasing action of amphetamine but not that of acetylcholine. The results suggest that the activation of muscarinic receptors on dopamine terminals in the rat corpus striatum selectively affects the calcium-dependent depolarization-induced release of the [³H]catecholamine. Moreover, the [³H]dopamine release caused by acetylcholine seems to occur independently of the membrane dopamine carrier.     

Inhibition by Quinacrine of Depolarization-Induced Acetylcholine Release and Calcium Influx in Rat Brain Cortical Synaptosomes

The effects of quinacrine on depolarization-induced [³H]acetylcholine (ACh) release and ⁴⁵Ca²⁺ influx were examined in rat brain cortical synaptosomes. Quinacrine significantly reduced the stimulated release of [³H]ACh by high K⁺ and veratridine without affecting the spontaneous efflux from the preloaded synaptosomes. Quinacrine had no effect on ionophore A23187-induced release of [³H]ACh from the synaptosomes. Quinacrine (100 μM) markedly diminished the stimulated Ca²⁺ influx by veratridine and high K⁺ but not that by “Na⁺-free.” Trifluoperazine, a potent calmodulin antagonist, inhibited both Ca²⁺ influx and ACh release induced by the depolarizing agents. Inhibitory potencies of the two drugs on ACh release and Ca²⁺ influx were compared with the antagonism of calmodulin by two drugs, suggesting that the inhibition of depolarization-induced Ca²⁺ influx and ACh release by these drugs could not be explained by the antagonism of calmodulin.     

The relationship between secretion and intracellular free calcium in bovine adrenal chromaffin cells

The effect of carbamylcholine and the calcium ionophore A23187 on catecholamine release and intracellular free calcium, [Ca²⁺]i, in bovine adrenal chromaffin cells was determined. At 10^–4M carbamylcholine maximal release occurred with an accompanying increase i n [Ca²⁺]i from a basal level of 168 nM to less than 300 nM. An increase in [Ca²⁺]i of a similar magnitude was found following challenge with 40 nM A23187. However, in this case, no catecholamine release occurred. These results suggest that stimulation of secretion from chromaffin cells by carbamylcholine may involve additional triggers which stimulate secretion at low [Ca²⁺]i.