Effect of alpha-tocopherol and its derivatives on actinomycin D induced apoptosis of rat thymocytes

The induction of rat thymocyte apoptosis by actinomycin D was associated with the increased caspase-3 activity and DNA fragmentation, the both effects were attenuated by alpha-tocopherol. Apoptosis was also decreased by alpha-tocopheryl acetate, but these suppressive effects were less than those of alpha-tocopherol. Tocopheryl quinone had no pronounced antiapoptotic effect. It was proposed that the difference in antiapoptotic effects of alpha-tocopherol derivatives is attributed to structure properties of the chroman head group and to the ability for scavenging reactive oxygen species but it is not excluded that antiapoptotic activity of alpha-tocopherol may exceed that of a mere antioxidant.     

Effects of calpain inhibitor on the apoptosis of hepatic stellate cells induced by calcium ionophore A23187

We previously showed that changes in calcium concentrations were related to cell apoptosis in vitro. The endoplasmic reticulum (ER) is the main component of calcium storage and signal transduction, and disrupting the balance of intracellular Ca²⁺ can cause endoplasmic reticulum stress (ERS). In this process, the ER releases stored Ca ²⁺ into the cytoplasm and activates calpain-2. To further investigate the effect of calpain in hepatic stellate cells (HSCs), in the current study, we examine the effect of N-acetyl-leu-leu-norleucinal (ALLN) on apoptosis resulting from calcium ionophore A23187–induced ERS. Our findings indicate that calpain inhibition reduces calcium ionophore A23187–induced apoptosis of HSCs and decreases the expression of ER stress proteins that may be related to the calpain/caspase signaling pathway.     

A postulated mechanism for the coordinate effects of ionophore A23187 on calcium uptake and cell viability in rat thymocytes

Human beta-thromboglobulin, low affinity platelet factor 4 and platelet basic protein have been purified to homogeneity from the material released by thrombin-stimulated platelets. Purification steps included isoelectric focusing and heparin-agarose chromatography. Antibodies against each of these proteins have been raised in rabbits. Antigenic identity of the proteins has been demonstrated in radioimmunoassay using 125I-labelled platelet basic protein or 125I-labelled low affinity platelet factor 4 and a variety of antibodies. The molecular weight of platelet basic protein estimated by gel filtration in 6 M guanidine hydrochloride using Sepharose 6B corresponded to approx. 10 000 daltons, slightly higher than that of beta-thromboglobulin (8851 daltons) and low affinity platelet factor 4 (9278 daltons). These findings raise the possibility that the formation of low affinity platelet factor 4 beta-thromboglobulin may be a consequence of the action of proteolytic enzymes on platelet basic protein.     

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.     

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.     

Effect of calcium ionophore A23187 on pregnenolone metabolism to progesterone in rat granulosa cells

The effect of calcium ionophore A23187 on the metabolism of pregnenolone to progesterone was examined in rat granulosa cells during a 24-h culture period. Granulosa cells harvested from pregnant mare's serum gonadotropin treated immature rats were incubated in the presence and absence of the divalent cation ionophore A23187. The ionophore induced progesterone synthesis from both endogenous sterol substrate and exogenous pregnenolone in a time- and concentration-dependent manner. Pregnenolone metabolism was examined in the presence of aminoglutethimide phosphate, an inhibitor of endogenous pregnenolone production. Steroid secretion resulting from metabolism of endogenous substrate was more sensitive to A23187 in that a lower concentration of the ionophore was required to induce a significant increase than that noted for exogenous pregnenolone metabolism. In addition, progesterone production from endogenous sterol occurred 6 h earlier than the observed increase in the conversion of pregnenolone to progesterone. These results indicate that A23187 and therefore possibly enhanced calcium influx may play a significant role in the regulation of pregnenolone metabolism in granulosa cells depending on the duration of incubation. The earlier steroidogenic response from endogenous substrate may be a reflection of an acute effect of A23187 on certain steroidogenic steps proximal to pregnenolone production.     

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.     

Influence of calcium ionophore A23187 on neurotransmitter release in rat brain synaptosomes

The effect of calcium ionophore A23187 on the release of nonmetabolizable glutamate analogues [3H]D-aspartate and the exocytosis registered by fluorescent dyes in synaptosomes was investigated. It was shown that A23187 is able to induce neurotransmitter release both in calcium-containing and calcium-free medium, the effect in the latter case being more pronounced. Calcium ionophore is able to induce exocytosis registered by acridine orange and FM 2-10. The influence of A23187 on the fluorescence of acridine orange was mainly calcium-independent, whereas the change in the fluorescence of FM 2-10 was calcium-dependent. It was suggested that the calcium-independent increase in acridine orange fluorescence is related to the dissipation of pH gradient in synaptic vesicles. Probably, the calcium-independent release of D-aspartate is also associated with the dissipation of pH gradient and subsequent leakage of neurotransmitters.     

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.     

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 (7.6μM). In the presence of the calcium antagonists TMB-8 (0.43μM). or verapamil (0.2μM) 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.     

Influence of the calcium ionophore A23187 on rat egg behavior and cortical F-actin

Rat eggs treated with the calcium ionophore A23187 and subjected to long-term observation by phase microscopy were found to undergo many developmental changes that are normally associated with fertilization. These included cortical granule exocytosis and the abstriction of the second polar body. In addition, time-lapse video microscopy revealed that, unlike untreated eggs, whose surfaces remained relatively immotile, the ionophore-treated eggs underwent a lengthy period of surface undulatory activity. Since all of these events were remarkably similar in timing and morphology to those seen in fertilized eggs, we conclude that A23187 is capable of activating rat eggs. Using NBD-phallacidin, the distribution of F-actin in ionophore-activated eggs was determined. During most of the postactivation period the eggs possessed an uninterrupted, uniform band of polymerized actin encompassing the entire cortex of the egg. However, during a discrete 1.5-h period after the formation of the second polar body, an area adjacent to the region of polar body abstriction exhibited more intense staining than the rest of the cortex. Cytochalasin B treatment caused a dramatic reduction and/or rearrangement in cortical NBD-phallacidin staining in activated eggs as compared to activated controls not exposed to the drug. We observed that all the developmental changes described above could be produced in the absence of exogenous calcium, suggesting that the rat egg possesses internal stores of calcium sufficient to elicit an activational response. We conclude that the ionophore-induced release of free calcium ions into the cytosol stimulates many of the developmental changes that are normally seen during fertilization. These results indicate that calcium influx and cytoskeletal activity are correlated during the activation of this animal egg.     

Differential effects of prostaglandins on macrophage activation induced by calcium ionophore A23187 or IFN-gamma.

Calcium ionophore A23187 can mimic IFN-gamma-induced macrophage activation for intracellular Leishmania killing and secretion of L-arginine-derived nitrite. Because the effects of ionophore are not restricted to calcium mobilization but also involve alterations of phospholipid metabolism, we have examined the role of PGE2 in the activation process. Macrophages exposed to A23187 or IFN-gamma in the presence of LPS and FCS secreted significant amounts of PGE2 independently of the presence of L-arginine in the incubation medium. The addition of the cyclooxygenase inhibitor indomethacin or omission of FCS abrogated PGE2 secretion but had little effect on nitrite production or intracellular killing. The addition of exogenous PGE2, of agents increasing PGE2 production such as arachidonic acid and colchicine, or of an analogue of cAMP, dibutyryl cAMP inhibited A23187 + LPS-induced activation whereas that mediated by IFN-gamma + LPS remained unimpaired. Our results indicate that PGE2 can modulate activation induced by A23187 but not by IFN-gamma, probably by a process involving cAMP. Conceivably, ionophore can mimic IFN-gamma for the induction of activation but lacks the capacity to help maintain the activated state because of its inability to desensitize macrophages to negative regulation by PGE2, as suggested previously for IFN-gamma-dependent activation.     

Alterations in the vesicular pattern and wall growth ofPhycomyces induced by the calcium ionophore A 23187

Summary Hyphal elongation, chitin synthesis in vivo, and invertase secretion inPhycomyces blakesleeanus were all inhibited almost instantly by the addition of 5–10 M calcium ionophore A 23187. Protein biosynthesis was inhibited in these conditions by 30–50%. The ionophore did not affect cell respiration for at least 40 min. Effect on chitin biosynthesis was not due to alterations of the chitin synthetase levels or its activity; nor to impairement in GlcNAc metabolism. In drug-treated cells the number of apical vesicles was severely reduced even at very short periods of incubation, and these low numbers remained constant for at least 60 min of incubation with the ionophore. We suggest that the ionophore collapses the cellular calcium gradient and/or interferes with the normal electrical transhyphal current. As a consequence, formation and migration of apical vesicles are inhibited. These results are further evidence of the role of vesicles in fungal tip growth and exhibit the fact that active chitin synthetase is short-lived in vivo demanding its continuous supply by chitosomes to the cell surface.Abbreviations GlcNAc N-acetylglucosamine - TCA trichloroacetic acid - UDPGIcNAc uridine diphosphate-N-acetylglucosamine - DMSO dimethylsulfoxide     

Action of insulin and cell calcium: Effect of ionophore A23187

Summary 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.     

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.     

Morphological changes induced by the calcium ionophore A23187 in rat basophilic leukemia (2H3) cells.

RBL-2H3 cells have been widely used to study histamine release in vitro. It was previously shown that these cells undergo striking morphological changes after IgE-mediated secretion. The present study was undertaken to examine if the morphological changes were dependent on activation of the Fc epsilon receptor. Therefore, the cells were stimulated to release histamine by two different mechanisms: activation of the Fc epsilon receptor by antigen and treatment with the calcium ionophore A23187. Cell surface and cytoskeletal changes were examined by fluorescence microscopy and scanning electron microscopy after either IgE- or ionophore-mediated histamine release. After exposure of the cells to either secretagogue, the cells spread over the surface of the culture dish and underwent rearrangement of the cytoskeleton. In addition, scanning electron microscopy revealed that deep ruffles developed on the surface of the cells undergoing IgE-mediated release. The surface changes were not as pronounced with the ionophore. The distribution of the cytoskeletal elements was examined by immunofluorescence using FITC-phalloidin and antibodies against vimentin and tubulin. In unstimulated cells actin was localized at the cell periphery, just under the plasma membrane. In the stimulated cells it was associated with the cell periphery and concentrated in the surface ruffles. As the stimulated cells spread, intermediate filaments and microtubules became distributed throughout the cell body, but there was no obvious association with the membrane ruffles. These morphological changes were dependent on the presence of extracellular calcium and on the concentration of ionophore or antigen, and were also correlated with the amount of histamine released. Additionally, IgE-mediated stimulation led to increased uptake of the soluble-phase tracer Lucifer yellow, whereas stimulation with the ionophore A23187 showed no increase in Lucifer yellow internalization. Ionophore A23187 produced changes similar but not identical to those seen in the RBL-2H3 cells after IgE-mediated histamine release. The differences may be owing to the involvement of the Fc epsilon receptor in IgE-mediated secretion.     

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.