Bilayers containing calcium ionophore A23187 form channels.

For the first time, based on bilayer membrane conductance experiments, it has been shown that A23187, a carboxylic calcium ionophore, incorporated in lipid bilayers gives single channel currents similar to the well known gramicidin channel. The current characteristics indicate the possibility that the transmembrane ion transport by this important calcium ionophore is initially by a carrier mechanism but with time is by a channel or pore mechanism due to the aggregation of the molecule in a lipid matrix.     

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+.     

Structural and functional changes in root cells induced by calcium ionophore A23187

The shifts of Ca²⁺, K⁺ and proton homeostasis of wheat (Triticum aestivum L. M. cv Ljuba) root cells induced by the Ca²⁺-ionophore A23187 caused different responses, depending on the time of exposure to the ionophore. Oxygen consumption and heat production by roots were increased when the Ca²⁺-specific effect of A23187 was expressed. Ultrastructural re-organization of cell organelles was found to follow the ion shifts. The endoplasmic reticulum, Golgi apparatus and mitochondria rearranged their membranes following treatment. The increased ion permeability of root cell membranes is proposed to cause an excessive energy expenditure for the restoration of ion homeostasis.     

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.     

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 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.     

Hyperactivated motility induced in mouse sperm by calcium ionophore A23187 is reversible

The reversibility of hyperactivated motility was tested in caudal epididymal mouse sperm by treating them with 1 microM calcium ionophore A23187 in dimethyl sulfoxide (DMSO), followed 2 min later by the addition of medium containing high levels of bovine serum albumin (BSA) (final concentrations: 0.5 microM A23187, 22 mg/ml BSA). Controls received DMSO alone, followed by BSA. Immediately following treatment with A23187, motility was weak and vibratory. Two minutes after the addition of high levels of BSA, motility was hyperactivated, as determined by videotape analysis of linearity of trajectory and acuteness of flagellar bending. Ten minutes after the addition, the movement pattern returned to that of fresh, uncapacitated epididymal sperm. Control sperm retained the linear swimming pattern of fresh caudal epididymal sperm during the 10 min of observation. Ninety minutes later, however, both control and treated sperm became hyperactivated. The percentage of motile sperm was not affected by treatment or time. Thus, ionophore-induced hyperactivation is reversible and does not interfere with the normal development of hyperactivation during incubation under capacitating conditions in vitro.     

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.     

The mechanism of basophil histamine release induced by antigen and by the calcium ionophore A23187.

The mechasism of human basophil histamine release by the calcium ionophore A23187 has been compared to that induced by the interaction of antigen with cell bound IgE antibody. Ionophore induced histamine release (Ion. H.R.) occurs with the leukocytes of both normal and allergic donors. It is completely calcium dependent; LaCl3 inhibits both Ion. H.R. and antigen induced histamine release (Ag. H.R.) at about 10-minus 7 M. The kinetics of Ion. H.R. suggest that this process has no "desensitization" phase as does Ag. H.R. and the ionophore is fully active on antigen-desensitized cells. Pharmacologic studies indicate that dibutyryl cyclic AMP and agents which increase endogenous cyclic AMP levels do not inhibit Ion. H.R. as they inhibit the early stages of Ag. H.R. Of the agents which affect microtubules, colchicine inhibits and D2O enhances Ion. H.R. in a manner which is qualitatively similar but quantitatively less marked than their effects on Ag. H.R. The metabolic antagonist 2-deoxyglucose inhibits both Ion. H.R. and Ag. H.R. in a similar fashion. Based on these data and the observation that cells pretreated with ionophore show a marked (synergistic) enhancement of Ag. H.R. we conclude that Ion. H.R. has a similar or identical mechanism to the later stages if Ag. H.R. but "short circuits" the cyclic AMP-associated events of Ag. H.R.     

Regulation of acetylcholinesterase expression by calcium signaling during calcium ionophore A23187- and thapsigargin-induced apoptosis

We have recently reported that acetylcholinesterase expression was induced during apoptosis in various cell types. In the current study we provide evidence to suggest that the induction of acetylcholinesterase expression during apoptosis is regulated by the mobilization of intracellular Ca(2+). During apoptosis, treatment of HeLa and MDA-MB-435s cells with the calcium ionophore A23187 resulted in a significant increase in acetylcholinesterase mRNA and protein levels. Chelation of intracellular Ca(2+) by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester), an intracellular Ca(2+) chelator, inhibited acetylcholinesterase expression. A23187 also enhanced the stability of acetylcholinesterase mRNA and increased the activity of acetylcholinesterase promoter, effects that were blocked by BAPTA-AM. Perturbations of cellular Ca(2+) homeostasis by thapsigargin resulted in the increase of acetylcholinesterase expression as well as acetylcholinesterase promoter activity during thapsigargin induced apoptosis in HeLa and MDA-MB-435s cells, effects that were also inhibited by BAPTA-AM. We further demonstrated that the transactivation of the human acetylcholinesterase promoter by A23187 and thapsigargin was partially mediated by a CCAAT motif within the -1270 to -1248 fragment of the human acetylcholinesterase promoter. This motif was able to bind to CCAAT binding factor (CBF/NF-Y). These results strongly suggest that cytosolic Ca(2+) plays a key role in acetylcholinesterase regulation during apoptosis induced by A23187 and thapsigargin.     

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)     

Evidence on the role of three calcium pools in Ca-ionophore A23187-stimulated rat blood platelet aggregation.

The effect of Ca-ionophore A23187 on activation of rat blood platelets was investigated to elucidate the involvement of extracellular and intracellular Ca2+ ions. Platelet aggregation induced by 10 concentrations of the stimulus was studied in Ca-free medium as well as in the presence of EGTA and/or calcium. In Ca-free medium, A23187 induced platelet aggregation in a dose-dependent way; the mean effective concentration was 1.43 +/- 0.08 mumol/l. The stimulatory effect of ionophore was potentiated by addition of 0.01 and 0.1 mM calcium and inhibited when the calcium concentration was increased to 1 mmol/l. In the presence of EGTA, A23187-stimulated aggregation of isolated rat platelets was recorded only at a 10-times higher ionophore concentration and was then reduced to 30% in comparison with aggregation in Ca-free medium. The inhibitory effect of 1 mM EGTA was abolished by addition of 2 mM calcium. We suggest the participation of at least three calcium pools in the stimulation of rat platelets by A23187, i.e. the extracellular pool, the membrane-associated pool and the pool displacing calcium intracellularly.     

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.     

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.     

Inhibition of tyrosinase activity and protein synthesis in melanoma cells by calcium ionophore A23187

Calcium ionophore A23187 lowers basal levels of tyrosinase and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. Ionophore at a concentration of 10(-6) g/ml causes a 50% reduction in basal levels of tyrosinase and inhibits the MSH stimulated level of enzyme. Ionophore A23187 also inhibits the PGE1 mediated stimulation of tyrosinase, as well as the rise in enzyme activity observed in cells exposed to either theophylline (1 mM) or dbcAMP (10(-4)M). Ionophore does not affect basal levels of cyclic AMP nor the elevated levels produced by either MSH or PGE1, suggesting then, that the antagonistic activity of A23187 is localized to a point in the pathway of tyrosinase activation distal to the formation of cAMP. Ionophore causes a rapid and marked (greater than 50%) inhibition of cellular protein synthesis and it is possible that this calcium mobilizing compound may exert its inhibitory effects on tyrosinase activity by causing a general reduction in cellular translation. Since the inhibition of protein synthesis occurs in cells exposed to ionophore in either the presence or absence of calcium in the medium, it seems, likely that the ionophore may exert its effects by causing the release of calcium from intracellular sites.     

The role of calcium in eicosanoid production induced by ricinoleic acid or the calcium ionophore A23187

Rat isolated intestine incubated in Krebs solution converted exogenous [14C]-arachidonic acid into products that chromatographed with prostaglandins, leukotriene B4 and 5-hydroxy-eicosatetraenoic acid. Accumulation of these products was increased by the laxative ricinoleic acid (0.34 mM) or the calcium ionophore A23187 (7.6 microM). In the presence of the calcium antagonists TMB-8 (0.43 microM) or verapamil (0.2 microM) the mean effects of ricinoleic acid or the calcium ionophore were smaller. Stimulation of arachidonic acid metabolism by ricinoleic acid therefore seems likely to involve a calcium-dependent mechanism.     

Activation of (arachidonyl) phosphatidylinositol turnover in rabbit neutrophils by the calcium ionophore A23187.

The addition of the Ca2+ ionophore A23187 to rabbit neutrophils stimulated [14C]arachidonic acid incorporation into phosphatidylinositol and lysosomal enzyme secretion. A significant increase in phosphatidylinositol labelling was observed after a 2 min exposure to 0.1 microM-ionophore A23187. Maximum increases in rate of labelling were obtained with 1 microM-ionophore A23187 within 1 min, declining to basal rates after 15 min. Similarly, maximum rate of enzyme release occurred during the first 2 min of exposure to ionophore and release was essentially complete by 15 min. Threshold and peak ionophore A23187 concentrations for stimulating both processes were identical. In contrast with the specificity of phosphatidylinositol labelling induced by 1 microM-ionophore A23187 in the absence of cytochalasin B, ionophore also significantly stimulated labelling of phosphatidylserine and phosphatidylethanolamine in the presence of cytochalasin B. With a threshold ionophore concentration (0.1 microM), the enhanced incorporation of arachidonate was relatively specific for phosphatidylinositol in cytochalasin-treated cells. Ionophore A23187 did not accelerate labelling of phosphatidylinositol by [14C]acetate or [14C]glycerol, indicating that ionophore A23187 does not stimulate phosphatidylinositol synthesis de novo, although it did promote [14C]palmitate and [32P]Pi incorporation into neutrophil phosphatidylinositol. However, the increase in phosphatidylinositol labelling with these latter precursors was generally slower in onset and much more modest in magnitude than that observed with arachidonic acid. These results support the hypothesis that a Ca2+-dependent phospholipase, which acts on the arachidonate moiety of phosphatidylinositol, is responsible for initiating at least certain of the membrane events coupled to the release of secretory product from the neutrophil.     

Hypoxia potentiates killing of hepatocyte monolayers by leukotrienes, hydroperoxyeicosatetraenoic acids, or calcium ionophore A23187

Potentiation of chemical toxicity by hypoxia was studied in confluent hepatocyte monolayers. Addition of either hydroperoxyarachidonic acid (50 micrograms), leukotriene C4 (10 micrograms), or calcium ionophore A23187 (1.8 micrograms) to hepatocyte monolayers followed by incubation in 2% oxygen for 24 h killed 95% of the hypoxic cells, but was without effect on the normoxic cells. The greater than 10-fold increase in toxicity of A23187 suggests that hypoxic cells are less able to regulate intracellular calcium. The increased toxicity of hydroperoxyarachidonic acid and leukotriene C4 may be due to a related reduction in activity of protective enzymes.