Stepwise activation of T cells. Role of the calcium ionophore A23187

The calcium ionophore A23187, at a concentration of 1 microgram/ml, is able to stimulate proliferation of freshly isolated peripheral blood lymphocytes, CD4+-enriched cells, or CD8+-enriched cells as measured by [3H]thymidine incorporation. This proliferation is accompanied by an increase in interleukin 2 (IL-2) receptor expression but not by a detectable up-regulation in (IL-2) production or the development of cytotoxicity. Proliferation can be blocked by anti-CD3, CD4, or CD8 monoclonal antibodies, but not by anti-Tac. If CD8+-enriched cells are activated for 3 days with A23187 and the blasts present on day 3 are sorted and returned to culture, they rapidly develop cytolytic activity in the presence of recombinant IL-2 but not recombinant interferon-gamma. CD4+ enriched cells, after activation with A23187, do not become cytotoxic in the presence of either recombinant IL-2 or recombinant interferon-gamma. These findings permit study of the stepwise maturation of T cells in this alternative pathway by using "minimal signals" that do not, by themselves and as used in these studies, stimulate precursor Tc to mature to full effector cytotoxic function. These findings are consistent with the model that A23187 drives T cells only part way along a pathway of maturation and that an additional second signal must be given to effect maturation of cytotoxic status.     

Effect of calcium ionophore A23187 on the sensitivity of early sea urchin embryos to cytotoxic neuropharmacological drugs

The ability of cytotoxic neurochemicals (indole and amphetamine derivatives) to block first cleavage division in the embryos of the sea urchin Arbacia lixula abruptly increases when the embryos are incubated in calcium-free seawater and decreases when the external Ca concentration is raised up to 46.4 mM. Sensitivity of the embryos to these drugs decreases also in the presence of the Ca-ionophore A23187. It is suggested that Ca ions are involved in the realization of physiological effects of "prenervous" neurotransmitters whose presence in early sea urchin embryos was shown by us earlier.     

Morphological and functional changes in Lewis lung carcinoma cells after treatment with A23187 calcium ionophore

Cultured Lewis Lung carcinoma cells (3LL) treated with A23187 calcium ionophore were studied by transmission electron microscopy and HPLC analysis. Results showed that A23187 calcium ionophore induces on osmiophilic inclusion bodies of 3LL cells similar process of lamellar bodies formation to those reported in the development of the osmiophilic inclusions bodies within the granular pneumocyte of normal lungs. HPLC analysis shows that calcium ionophore generates quantitative changes in the 3LL cytoplasmic protein content expressed by an increase of 18-22 kDa and 4-9 kDa proteins.     

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.     

Role of lipid metabolism in cell killing by calcium plus ionophore A23187

Cultured fibroblasts treated with divalent cation ionophore A23187 in the presence of extracellular calcium provide a useful model system for studying mechanisms of cell death associated with elevated intracellular calcium concentrations. Cell death induced by A23187 plus calcium can be conveniently monitored as membrane permeabilization to Trypan blue dye. Because lipids are a major component of cell membranes and play an important role in determining membrane permeability, the present study was initiated to identify changes in cell lipid composition that occur during membrane permeabilization induced by calcium plus A23187. The percent label in each of the major structural lipids in biosynthetically labeled NIH3T3 fibroblasts changed < 10% during the time course of membrane permeabilization. During the course of membrane permeabilization there was significantly increased label in lysophosphatidylinositol and lysophosphatidylcholine and reduced label in phosphatidylinositol 4,5-bisphosphate. The time course of these changes corresponded to that of the arachidonic acid release response stimulated by calcium plus A23187, not to the time course of membrane permeabilization, which occurs later. These observations are consistent with lipid metabolism induced by A23187 plus calcium playing only a possible regulatory or intermediatory role in membrane permeabilization, rather than causing direct permeabilization of the lipid phase of the membrane.     

Uniform ionophore A23187 distribution and cytoplasmic calcium buffering in intact human red cells

The divalent cation-selective ionophore A23187 has been used to characterize cytoplasmic Ca and Mg buffering, Ca2+-pump parameters and the properties of a Ca2+-activated K+-channel in intact red cells. A critical assumption in these studies has been that the ionophore causes a uniform increase in divalent cation-permeability in all the cells. This has now been tested directly in ATP-depleted human red cells by analysing the kinetics of ionophore-induced 45Ca-tracer and net Ca2+ fluxes. The experimental curves were all adequately fitted by single-exponentials at all ionophore concentrations tested. Moreover, statistical analysis of 61 individual tracer influx curves and of pooled data showed no trend towards fast second exponential components. These results demonstrate uniformity of ionophore distribution, ionophore-induced Ca2+-permeability, and cytoplasmic Ca-buffering among all the cells. Experiments involving mixing of cell suspensions with high and low original ionophore content, and involving ionophore extraction by albumin, demonstrate a rapid redistribution of ionophore among the cells, indicating that homogeneity of ionophoric effects is achieved through dynamic ionophore redistribution.     

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.     

Calcium ultrastructural localization in Xenopus laevis eggs following activation by pricking or by calcium ionophore A 23187

In the egg of Xenopus laevis a cortical network of smooth endoplasmic reticulum (SER) surrounds and interconnects each cortical granule (CG) (Campanella and Andreuccetti, '77). This network is a possible intracellular site of calcium storage to be called into action for CG exocytosis. In our experiments, Xenopus eggs, unfertilized or activated by pricking or by calcium ionophore A 23187, have been fixed in osmium-pyroantimonate for calcium localization. Our data show that deposits can be detected only in activated eggs. The calcium chelator edetate (EGTA) and x-ray microprobe analysis demonstrate that they contain calcium. Deposits are found on liposomes and on all intraovular cytomembranes, which therefore appear to be possible sites of calcium sequestration. In the case of ionophore-activated eggs, deposits are detectable independently of the presence of extracellular calcium. These data show that in Xenopus at activation an intracellular liberation of calcium occurs similar to that described in other species. Furthermore, the fact that antimony deposits are observed only after activation makes Xenopus eggs appropriate material in which to follow the temporal and spatial sequence of appearance of the deposits during the early stages of activation. Our results show that antimony deposits appear first in SER vesicles between the plasma membrane and CGs and then spread to the rest of the egg cytomembranes. These data corroborate our hypothesis that in Xenopus the cortical SER network is the first intracellular site where calcium is released at activation. The possible mechanism of calcium release and propagation along the egg cortex is discussed.     

Role of phospholipid hydrolysis in the mechanism of toxic cell death by calcium and ionophore A23187

Manganese chloride inhibits the hydrolysis of arachidonate-containing phospholipids stimulated in 3T3 mouse fibroblasts by ionophore A23187 in the presence of extracellular calcium. The inhibition is reduced by increasing extracellular calcium concentrations. Stimulation by A23187 of this phospholipid hydrolysis and cell killing are inhibited at similiar concentrations by (i) manganese chloride or (ii) reduced extracellular calcium. These results indicate an important role for the phospholipid hydrolysis in the mechanism of cell killing by A23187 plus calcium. Analysis of the rates of the two processes indicates that phospholipid hydrolysis triggers cell killing, but it is not itself the membrane permeabilizing step.     

Role of calcium in the regulation of sugar transport in the avian erythrocyte: Effects of the calcium ionophore,A23187

The tissue/medium distribution of the nonmetabolized glucose analog [¹⁴C]-3-0-methyl-D-glucose was measured in pigeon erythrocytes and related to changes in ⁴⁵Ca uptake and efflux, total calcium content and ATP levels. Sugar transport was not affected by changes in external Ca²⁺. However, both sugar and ⁴⁵Ca influx were increased by the Ca-ionophore A23187. In the absence of external Ca²⁺, the ionophore caused a delayed increase in sugar transport and net loss of calcium, probably through releasing Ca²⁺ from internal storage sites into the cytoplasm. Increasing internal Na⁺ through Na⁺ pump inhibition or using the sodium ionophore monensin did not alter influx of sugar or ⁴⁵Ca, indicating Na⁺-Ca²⁺ exchange was absent in these cells. The results are consistent with A23187 causing increased Ca²⁺ influx or release from mitochondrial storage and the resulting rise in cytoplasmic Ca²⁺ stimulating hexose transport. Experiments with low Mg⁺⁺ and high K⁺ media and measurements of ATP levels exclude alternative explanations for the action of A23187. We conclude that sugar transport regulation in avian erythrocytes is Ca²⁺-dependent and resembles that in muscle in its basic mechanism. It differs in the response to some modulating agents, largely because of a different pattern of Ca²⁺ fluxes in these cells.     

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.     

Action of insulin and cell calcium: effect of ionophore A23187.

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.     

Role of TSC-22 during early embryogenesis in Xenopus laevis

Transforming growth factor-beta1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved. During mouse embryogenesis, TSC-22 is expressed at the site of epithelial-mesenchymal interaction. Here, we isolated Xenopus laevis TSC-22 (XTSC-22) and analyzed its function in early development. XTSC-22 mRNA was first detected in the ectoderm of late blastulae. Translational knockdown using XTSC-22 antisense morpholino oligonucleotides (XTSC-22-MO) caused a severe delay in blastopore closure in gastrulating embryos. This was not due to mesoderm induction or convergent-extension, as confirmed by whole-mount in situ hybridization and animal cap assay. Cell lineage tracing revealed that migration of ectoderm cells toward blastopore was disrupted in XTSC-22-depleted embryos, and these embryos had a marked increase in the number of dividing cells. In contrast, cell division was suppressed in XTSC-22 mRNA-injected embryos. Co-injection of XTSC-22-MO and mRNA encoding p27Xic1, which inhibits cell cycle promotion by binding cyclin/Cdk complexes, reversed aberrant cell division. This was accompanied by rescue of the delay in blastopore closure and cell migration. These results indicate that XTSC-22 is required for cell movement during gastrulation though cell cycle regulation.     

Changes in the distribution of intramembranous particles in hen erythrocytes during cell fusion induced by the bivalent-cation ionophore A23187.

Incubation of hen erythrocytes with Ca2+ and the bivalent-cation ionophore A23187 induced slight cell fusion in 1 h at 37 degrees C, and extensive fusion during a subsequent 15 min at 47 degrees C. Redistributions of intramembranous particles were observed, possibly involving interactions between Ca2+ and phospholipids, which are discussed in relation to molecular mechanimss of cell fusion.     

Morphological changes in cultured mammalian cells: prevention by the calcium ionophore A23187.

The morphological changes induced by butyrate in HeLa cells and by monobutyryl or dibutyryl cAMP in CHO cells are prevented by micromolar concentrations of the divalent cation ionophore A23187. The ionophore is unable to prevent such changes in medium from which calcium is omitted. At slightly higher (but nontoxic) concentrations, the ionophore inhibits the butyrate-mediated induction of the ganglioside biosynthetic enzyme, sialyltransferase, in HeLa. In CHO, sialyltransferase activity is normally high and not altered by any of the compounds tested.     

Inhibition of proteolytic cleavage of the hemagglutinin of influenza virus by the calcium-specific ionophore A23187.

At calcium-specific ionophore A23187 concentrations of approximately 0.25 microM [which still allow assembly and release of fowl plague virus (FPV) particles] post-translational proteolytic cleavage of the viral hemagglutinin precursor HA into the fragments HA1 and HA2 is inhibited. The resulting virus particles with uncleaved hemagglutinin, that cannot be obtained under normal conditions, provide a suitable substrate for in vitro assays of the protease sensitivity of the FPV hemagglutinin. Proteolytic activation is accomplished with trypsin. Treatment with cathepsin B at low pH yields aberrant cleavage products suggesting that the cellular cleavage enzyme is not of lysosomal origin. A protease that cleaves the FPV hemagglutinin in the correct place can be detected in lysates of MDBK cells. This enzyme is calcium dependent and has a neutral pH optimum.     

Interaction of the calcium ionophore A.23187 (Calcimycin) with Bacillus cereus and Escherichia coli

Ionophores isolated from bacterial strains, and especially A.23187, are efficient antibiotics against Gram-positive bacteria and devoid of activity on Gram-negative species. This difference in activity was attributed to the outer membrane of Gramnegative bacteria which is presumably impermeable to these very hydrophobic compounds. In this context, the partition of the calcium ionophore A.23187 between bacteria and the medium was studied on Escherichia coli (Gram-negative) and Bacillus cereus (Gram-positive) using, on the one hand, fluorimetric measurements and, on the other hand, radioautographic analysis of bacteria incubated with the [³H]-labelled ionophore. Although the first method did not give a definitive answer, the second one clearly showed that the tritiated metabolite was only incorporated into B. cereus.