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Results for “Ionomycin”

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PubMed1984

Characterization of the response of human thymocytes and blood lymphocytes to the synergistic mitogenicity of 12-O-tetradecanoylphorbol-13-acetate (TPA)-ionomycin.

The combination of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore ionomycin is synergistically mitogenic for human fetal and infant thymocytes as well as peripheral blood lymphocytes. Optimal mitogenic stimulation is achieved when TPA and ionomycin are used at doses of 0.5-1 ng/ml and 0.5-1 microgram/ml, respectively. Phenotypic analysis and cell sorting show that the thymocytes responsive to the mitogen have a mature or medullary phenotype (T1+, T3+, T11+, T6-, HLA-A,B++, [TdT]-); similarly in blood the T cell subsets (T11+, T4+ and T11+, T8+) are selectively responsive to TPA-ionomycin. Both activated lymphocytes and thymocytes express HLA-DR antigens as well as activation antigens such as T9, T10 and T cell activation antigen. T cells activated by TPA-ionomycin can be grown for periods of up to 50 days without addition of exogeneous interleukin 2. The observations may have implications for the membrane-associated signals involved in T cell growth and proliferation.

Cell Division↗

PubMed2002

Activation of stress response by ionomycin in rat hepatoma cells.

All living systems respond to a variety of stress conditions by inducing the synthesis of stress or heat shock proteins (HSPs), which transiently protect cells. HSP synthesis was preceded by an increase in intracellular free calcium concentration [(Ca(2+))i]. In this study, we show that Ca(2+) ionophore, ionomycin, induced an immediate increase in intracellular free Ca(2+) and examined how this increase affects heat shock response in rat hepatoma cell line H4II-E-C3. Results indicate that incubating H4II-E-C3 cells with 0.3 microM ionomycin at 37 degrees C for 15 min results in the induction of HSP 70 in both Ca(2+)-containing and Ca(2+)-free medium. Associated with this increase in free Ca(2+) is an in vivo change in membrane organization and activation of signaling molecules like ERKS and SAPKs/JNK. In Ca(2+) containing medium HSP 70 induction mediated by HSF-HSE interaction was faster upon ionomycin treatment as compared to heat shock. Our results show that ionomycin, at sub lethal concentration, increases intracellular free Ca(2+) concentration, activates SAPK/JNK and HSF-HSE interaction, and induces HSP 70 synthesis.

Animals↗

PubMed1996

Effect of ionomycin on cell pH in isolated renal proximal tubules.

In isolated rabbit proximal tubules the addition of 2.0 microM but not 0.2 microM ionomycin induced a sustained increase in cell pH ([pH]i). This [pH]i response to 2.0 microM ionomycin was shown to be independent of several transporters such as Na+/H+ exchanger, Na(+)-HCO3- cotransporter, Cl-/HCO3- exchanger, or H(+)-ATPase. On the other hand, the removal of extracellular Ca2+ abolished the [pH]i increase or even induced a transient [pH]i decrease in the presence of ionomycin. These results are consistent with the induction of Ca2+/H+ exchange by ionomycin. Therefore Ca2+ ionophores should be used with caution as probes to estimate renal tubule functions.

Animals↗

PubMed2000

Expression of the Na(+)/Ca(2+) exchanger ameliorates ionomycin-induced cell death.

PC12 cells were stably transfected with cDNA encoding the Na(+)/Ca(2+) exchanger (NCX1.4). A robust Na(+)-dependent Ca(2+) uptake confirmed the functional expression of the protein. When NCX1. 4 expressing cells (NO) and vector transfected control cells (VC) were exposed to 0.5-20 microM ionomycin for 6 h, a dose-dependent increase in LDH release was observed. LDH release was significantly reduced in NO when compared with VC. When either VC and NO were treated with 3 microM ionomycin and 1.1 mM EGTA, the increase in LDH release was nearly abolished. However, when VC and NO were treated with ionomycin and then EGTA was added 2 min later, LDH release remained elevated. These data suggest ionomycin-induced cell death was Ca(2+) dependent and expressing NCX1.4 may have ameliorated cell death by reducing elevated [Ca(2+)](I).

Animals↗

PubMed1992

The effect of taurine on the age-related decline of the immune response in mice: the restorative effect on the T cell proliferative response to costimulation with ionomycin and phorbol myristate acetate.

1. Proliferative responses to the costimulation with phorbol-12-myristate-13-acetate (PMA) and suboptimal doses of ionomycin in the purified T and B cells from old mice were lower than those from young mice. 2. The degree of the age-related decline was more significant in T cells than in B cells. 3. Taurine, a sulfur containing amino acid, augmented the proliferative responses of T cells from both young and old mice. 4. The augmentation of the proliferative response by taurine was more marked in old T cells than in young T cells. 5. The concentration of intracellular free calcium ion ([Ca2+]i) was significantly lower in old T cells when stimulated with PMA and ionomycin than observed in young T cells. 6. In the presence of taurine, the concentration of [Ca2+]i in the old T cells significantly increased under stimulation by PMA and ionomycin. 7. The results indicate that taurine improved the proliferative response in old T cells by restoration of the increment of the concentration of [Ca2+]i under the stimulation by PMA and ionomycin.

Aging↗

PubMedDate not supplied

An Sp1 binding site involves the transcription of the Fas ligand gene induced by PMA and ionomycin in Jurkat cells.

The transcriptional regulation of the Fas ligand (FasL) gene in Jurkat cells was investigated. We demonstrated that an Sp1 binding site, located between -280 and -275 bp relative to the translational start site (+1) of the FasL gene, was important for the transcription of the FasL gene by deletion and mutation analysis in Jurkat cells after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. Nuclear extract of Jurkat cells formed complexes with the oligonucleotides bearing the Sp1 site within -280 to -275 of the FasL promoter. Apart from the constitutive complexes, a new complex was observed after PMA and ionomycin stimulation. Plasmid containing the Sp1 site sequence with site-directed mutation reduced the FasL promoter activity in driving the expression of reporter luciferase gene expression in transfected Jurkat cells after PMA and ionomycin stimulation. The binding of activated Jurkat cell nuclear extract to the mutated Sp1 binding site of the FasL promoter was ablated. In addition, the oligomer containing the Sp1 site of the FasL promoter could compete with oligomer with conserved Sp1 binding sequence in nuclear protein binding of activated Jurkat cells. The data presented in this study suggest that the transactivation of the FasL promoter via the Sp1 binding sequence (-280 to -275) involves the PMA- and ionomycin-induced expression of the FasL gene.

Binding Sites↗

PubMedDate not supplied

Absence of acetylcholine- and ionomycin-activated Cl- currents in submandibular cells of early postnatal rats.

Using the whole-cell patch-clamp technique, we investigated developmental changes in the expression of an acetylcholine- (Ach-) activated Cl- conductance in rat submandibular acinar cells. ACh induced an oscillatory inward current in cells isolated from animals older than 5 weeks, but not in animals less than 2-3 weeks of age. The current/voltage (I/V) relationship of the ACh-induced current was that of an outward rectifier, and the current was inhibited by intracellular BAPTA, a Ca2+ buffer, indicating the current was Ca2+ activated. The ACh-induced current was also blocked in the presence of DPC and SITS, two Cl- current inhibitors in other tissues. Ionomycin mimicked the effect of ACh but in a nonoscillatory fashion. The appearance of the ionomycin-induced currents was also age related, as the current was not observed to occur in animals less than 2-3 weeks old. Since both ACh and ionomycin significantly increase cytosolic [Ca2+] in the acinar cells of young animals, the correlation between the age dependence of the ACh-activated Cl- current and the ionomycin-activated Cl- current responses suggests that the lack of responsiveness observed in the young animals is due to the absence of Ca2+-activated Cl- channels, rather than to a deficiency of a cellular mediator.

Acetylcholine↗

PubMed1994

Effect of N,N-diethyldithiocarbamate on ionomycin-induced increase in oxidation of cellular 2',7'-dichlorofluorescin in dissociated cerebellar neurons.

Effect of N,N-diethyldithiocarbamate (DDC), an inhibitor for cytosolic superoxide dismutase, on an ionomycin-induced increase in oxidative metabolism was examined in cerebellar neurons dissociated from the rats, using a flow cytometer and 2',7'-dichlorofluorescin diacetate and fluo-3-AM, fluorescent dyes for intracellular hydrogen peroxide and Ca2+, respectively. DDC reduced the ionomycin-induced augmentation of 2',7'-dichlorofluorescin fluorescence in a dose-dependent manner. DDC did not affect cellular content of 2',7'-dichlorofluorescin and ionomycin-induced increase in intracellular Ca2+ concentration. Results indicate that ionomycin increases the formation of superoxide anion in brain neuron.

Aniline Compounds↗

PubMed1990

Ionomycin, a Ca++ ionophore, increases platelet volume independently of the Na+/H+ exchanger.

A Ca++-ionophore, ionomycin, increased the volume of human platelets suspended in a Ca++-containing buffer. This change in cell volume was dependent upon ionomycin and extracellular Ca++ concentrations, suggesting that the volume change occurs when the intracellular Ca++ reaches a certain level (greater than uM as determined by aequorin method). The ionomycin-induced volume increase was suppressed by replacement of extracellular Na+ with membrane-impermeable N-methyl-D-glucamine or Cs+, but not with Li+, K+, or Rb+. Ethylisopropylamiloride, a potent inhibitor of the Na+/H+ exchanger, had only weak inhibitory effect, and the apparent Km for Na+ was approximately 350 mM, which is much larger than that of the Na+/H+ exchanger. It is suggested that certain mechanisms other than the Na+/H+ exchanger are responsible for ionomycin-induced volume increase.

Blood Platelets↗

PubMed1992

Production of interleukin-2 mRNA by bovine lymph node lymphocytes in response to concanavalin A, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin.

Interleukin-2 (IL-2) is a lymphokine which, upon binding to its receptor, leads to the proliferation and differentiation of T-cells (helper, suppressor, and cytotoxic) and B-cells. While human and murine IL-2 have been extensively studied, less is known about bovine IL-2. In order to understand the induction of bovine IL-2 at the molecular level, we have examined IL-2 mRNA induction. The dose-responses and time courses of the production of IL-2 mRNA in response to Concanavalin A (ConA), 12-O-tetradecanoylphorbol-13-acetate (TPA), and ionomycin in lymph node lymphocytes (LNC) were determined. We found that high levels of IL-2 mRNA were produced in response to 1 microgram ml-1 ConA plus 10(-8) M TPA, but that even higher levels were produced in response to 1 microM ionomycin plus 10(-8) M TPA. We also found that LNC stimulated with ConA displayed two phases of IL-2 mRNA production, one occurring approximately 2-4 h after stimulation and one occurring approximately 10 h after stimulation. However, in the presence of ConA plus TPA or ionomycin plus TPA the response was monophasic. IL-2 mRNA was detected within 2 h of addition of ConA plus TPA (the earliest time examined), reached maximum levels within 6 h, and declined to low levels after 12 h. IL-2 mRNA from LNC incubated with ionomycin plus TPA appeared within 2 h, and reached maximum levels at about 9 h. In contrast to the decrease seen after 12 h with ConA plus TPA, IL-2 mRNA from these cells remained high for 18 h and declined to low levels after 24 h.

Animals↗

PubMed1996

Promoter elements and second messenger pathways involved in transcriptional activation of tyrosine hydroxylase by ionomycin.

Membrane depolarization, or agents which increase intracellular calcium, elicit transcriptional activation of tyrosine hydroxylase (TH). In this study we analyze the factors involved in the regulation of the TH promoter by a calcium ionophore. PC12 cells were transiently transfected with plasmids containing wild type or mutated 5' flanking sequences of the rat TH gene, fused to bacterial chloramphenicol acetyl transferase (CAT). Point mutations introduced into the consensus cAMP-regulatory element (CRE) abolished the induction of CAT by ionomycin indicating that it is essential for mediating the calcium response. An intact and functional AP1 site did not confer calcium inducibility when the CRE/CaRE sequence was mutated. The extent and kinetics of the increase in intracellular calcium as well as the induction of CAT activity under the control of TH promoter by ionomycin were similar in PC12 cells and in the A123.7, protein kinase A (PKA) deficient cell line. In both cell lines addition of ionomycin rapidly increased the phosphorylation of transcription factor CREB at Ser-133. These results suggest that the activation of TH transcription by ionomycin does not require PKA. However, KN62 an inhibitor of Ca2+/calmodulin dependent (CaM) kinases prevented the induction indicating possible involvement of CAM kinases in the calcium response.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Unexpected up-regulation of gene expression by cyclosporin A and FK-506 in a T-cell lymphoma: both immunosuppressants augment Ly-6E antigen induction by interferon-gamma in the presence of ionomycin.

Cyclosporin A (CsA) and FK-506 inhibit lymphokine gene activation in T-cells. In the present study, we investigated the effects of these immunosuppressants on the regulation of a non-lymphokine molecule, the Ly-6E surface antigen, in the YAC-1 T-cell lymphoma. These cells do not normally express Ly-6E mRNA or Ly-6E surface molecules but are induced to do so upon treatment with IFN-gamma. At submicromolar concentrations, CsA or FK-506 did not alter this induction. However, at higher concentrations (1-12 microM), they both increased the induction of Ly-6E mRNA expression. Cyclosporin A or FK-506 also markedly affected Ly-6E induction when the cultures were co-treated with the calcium ionophore, ionomycin. In the absence of CsA or FK-506, ionomycin suppressed Ly-6E induction by IFN-gamma. Both immunosuppressants reversed this inhibitory effect and increased Ly-6E mRNA and Ly-6E surface expression to levels that were 2- to 3-fold higher than in cells induced with IFN-gamma alone. In this system, the two immunosuppressants were active at pharmacologically relevant concentrations, similar to those inhibiting normal T-cell activation, with FK-506 being 30- to 50-fold more potent than CsA. The ability of CsA analogs to enhance Ly-6E induction in the presence of ionomycin also correlated with their immunosuppressive activity. Therefore, through mechanisms apparently related to those involved in their immunosuppressive action, both CsA and FK-506 convert the negative effect of ionomycin on IFN-gamma-mediated Ly-6E induction into an overall positive effect. The YAC-1 cell model, described here, provides a unique example of upregulation of gene expression by these two immunosuppressants.

Animals↗

PubMed1993

A digitized fluorescence imaging study of intracellular free calcium, mitochondrial integrity and cytotoxicity in rat renal cells exposed to ionomycin, a calcium ionophore.

The objective of this study was to explore the role of extracellular Ca2+ and mitochondrial integrity in ionomycin-induced cytotoxicity in primary cultures of rat kidney cortical epithelial cells using digitized fluorescence imaging (DFI), which is a powerful tool for continuously observing the dynamic intracellular biochemistry of single living cells. Using DFI, intracellular free calcium ion concentration ([Ca2+]i), mitochondrial membrane potential and loss of cell viability in individual rat renal cortical epithelial cells were examined temporally by fura-2, rhodamine 123 (Rh-123) and propidium iodide (PI), respectively. Images were taken within 10 min after exposure to 5 and 10 microM ionomycin. These three parameters, [Ca2+]i, mitochondrial membrane potential and cell viability, were also measured in populations of cells by a multiwell fluorescence scanner with fluo-3, Rh-123 and PI, respectively. Cytotoxicity was also assessed by two colorimetric cytotoxicity tests (LDH leakage and mitochondrial MTT reduction). Using DFI, the fluorescence scanner and the colorimetric cytotoxicity tests, we found that exposure of primary cultures of rat kidney cortical epithelial cells to high concentrations of ionomycin (5 and 10 microM) caused a rapid and sustained rise in [Ca2+]i, which preceded dissipation of the mitochondrial membrane potential and loss of cell viability and that chelation of extracellular Ca2+ with EGTA attenuated these responses. We demonstrated the value of using DFI to continuously observe the dynamic intracellular biochemistry of single living cells by establishing a sequence of elevated [Ca2+]i, dissipation of mitochondrial membrane potential and cytotoxicity. We conclude that a combination of the influx of extracellular Ca2+ and loss of mitochondrial integrity may be responsible for the cytotoxicity observed in individual renal cells and populations of renal cells after treatment with ionomycin.

Aniline Compounds↗

PubMed1998

Mechanism and specificity of lanthanide series cation transport by ionophores A23187, 4-BrA23187, and ionomycin.

A23187, 4-BrA23187, and ionomycin transport several lanthanide series trivalent cations at efficiencies similar to Ca2+, when compared at cation concentrations of approximately 10(-5) M, ionophore concentrations of approximately 10(-6) M, and a pH of 7.00. Selectivity sequences and the range of relative rates are as follows: A23187, Nd3+ > La3+ > Eu3+ > Gd3+ > Er3+ > Yb3+ > Lu3+ (approximately 34-fold); 4-BrA23187, Nd3+ > Eu3+ > Gd3+ > La3+ > Er3+ > Yb3+ > Lu3+ (approximately 34-fold); ionomycin, La3+ > Yb3+ > Nd3+ > Lu3+ > Er3+ > Eu3+ > Gd3+ (approximately 4-fold). At concentrations between 9 and 250 microM, La3+ is transported by an electroneutral mechanism, predominately through mixed complexes of the type (ionophore)2La-OH (A23187 and 4-BrA23187) or (ionophore)La-OH (ionomycin), when no membrane potential is present. For all three ionophores, an induced potential of approximately 160 mV accelerates transport by approximately 50-100%. However, measured values of H+/La3+ exchange indicate that only 4-BrA23187 displays a significant electrogenic activity under these conditions. At a La3+ concentration of 17 mM, transport by all three ionophores is electroneutral and apparently occurs through complexes of type (ionophore)3La (A23187 and 4-BrA23187) or (ionophore)La-OH (ionomycin). Analysis of these patterns in a context of comproportionation equilibria involving the transporting species and free La3+ indicates that the species containing three ionophore molecules are formed on the membrane when aqueous phase solution conditions would strongly favor a 1:1 complex, based upon previous studies in solution. The implications of this and other findings are discussed.

Aminoquinolines↗

PubMed1996

Ionomycin induced changes in intracellular free calcium in SH-SY5Y human neuroblastoma cells: sources of calcium and effects on [3H]-noradrenaline release.

In adherent SH-SY5Y human neuroblastoma cells cultured for 14 days to promote uptake and release of [3H]-noradrenaline, ionomycin induced a biphasic elevation of the intracellular [Ca2+] ([Ca2+]i). This consisted of a rapid transient elevation followed by a marked, persistent secondary elevation. Further study indicated that the peak [Ca2+]i elevation was dependent upon intracellular Ca2+ whilst the secondary elevation was dependent upon extracellular Ca2+. This profile of response and dependence upon intracellular and extracellular sources of Ca2+ was similar to that evoked by the muscarinic agonist, methacholine but was independent of inositol 1,4,5-trisphosphate generation. Ionomycin also stimulated the release of [3H]-noradrenaline from preloaded cells. Both intracellular and extracellular sources of Ca2+ were needed for the full response and synergised to effect release. Thus, in adherent SH-SY5Y cells, ionomycin elevates [Ca2+]i in a complex way in a manner partly analogous to the elevation of [Ca2+]i by agonists of phosphoinositidase C-linked receptors. Furthermore the effects of [Ca2+]i elevation on [3H]-noradrenaline release by these two processes are similar. Such functional consequences may, however, differ under circumstances where the profile and source of Ca2+ for ionomycin-mediated changes differs to that of receptor agonists.

Calcium↗

PubMed2002

Regulation of ionomycin-mediated granule release from rat basophil leukemia cells.

Cross-linking the high affinity IgE receptor on the rat basophil leukemia clone 2H3 (RBL-2H3) cell line, an vitro model for mast cell signaling, results in granule release. A great deal of research has focused on the earliest steps in this signaling cascade resulting in models which include the participation of lyn, syk, phospholipase C (PLC), protein kinase C (PKC) and intracellular calcium mobilization. In an effort to look at pathways downstream of calcium mobilization, ionomycin-mediated granule release was studied. The kinase inhibitors PP1 (src family), GF109203X (PKC), PD98059 (MEK1/2), and U0126 (MEK1/2) substantially inhibited ionomycin-mediated granule release, while the p38 kinase inhibitor SB203580 did not. Both p38 and erk were phosphorylated upon ionomycin treatment, but only extracellular regulated kinase (erk) activation was completely inhibited by PP1 treatment and partially inhibited by the MEK inhibitors, thus, correlating with the granule release data. Interestingly, while GF109203X alone had no affect on erk activation, combining it with U0126 completely blocked this response. This suggests the existence an alternate pathway for erk activation that is MEK independent and PKC dependent. Experiments in which ionomycin and PP1 were titrated (independently) demonstrated a correlation between erk phosphorylation and granule release, implicating erk in a PP1-inhibitable pathway operating downstream of calcium and controlling mast cell degranulation.

Animals↗

PubMed2001

Ionomycin and 2,5'-di(tertbutyl)-1,4,-benzohydroquinone elicit Ca2+-induced Ca2+ release from intracellular pools in Physarum polycephalum.

Calcium level in organelles of the slime mold Physarum polycephalum was monitored by chlortetracycline, a low-affinity calcium indicator. It was found that 2,5'-di(tertbutyl)-1,4,-benzohydroquinone (BHQ) at a concentration of 100 microM, but not the highly specific inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), thapsigargin (1-10 microM), elicited calcium release from the CTC-stained intracellular calcium pool. Ionomycin also caused a calcium release (23.7+/-5.1%), which was less than that induced by BHQ (30.1+/-6.0%). Procaine (10 mM), a blocker of ryanodine receptor, completely abolished the responses to BHQ and ionomycin. Another blocker, ryanodine (100 microM), only slightly diminished the responses to ionomycin and BHQ. Apparently, BHQ and ionomycin acting as a Ca2+-ATPase inhibitor and an ionophore, respectively, elicit an increase in [Ca2+]i, which in turn triggers a calcium-induced calcium release (CICR) via the ryanodine receptor. Caffeine, an activator of ryanodine receptor, at a concentration of 25-50 mM produced a Ca2+-release (5.6-16.0%), which was not similar in magnitude to CICR. The response to 25 mM caffeine was only moderately inhibited by 25 mM procaine, and almost completely abolished by 50 mM procaine and 100 microM ryanodine.

Animals↗

PubMed2004

Cysteamine, glutathione and ionomycin treatments improve in vitro fertilization of prepubertal goat oocytes.

The aim of this study was to improve in vitro embryo development of prepubertal goat oocytes by studying the effect of adding cysteamine to in vitro maturation medium, glutathione (GSH) to in vitro fertilization medium and ionomycin to the sperm capacitation medium. In experiment 1, we analysed the effect of 1 mM GSH added to fertilization medium of oocytes matured with 400 microM cysteamine. The control group were oocytes without cysteamine and GSH. In experiment 2, oocytes matured and fertilized in the presence of 400 microM cysteamine and 1 mM GSH, respectively, were inseminated with spermatozoa treated with ionomycin or heparin. In experiment 1, the percentages of total and normal fertilized oocytes were significantly higher for oocytes supplemented with cysteamine and GSH (40.26% and 30.20%, respectively) than for oocytes from the control group (16.66%, and 10.61%, respectively). The percentage of total embryos obtained after 7 days of culture was significantly higher in the group supplemented with cysteamine and GSH (30.62%) than in the control group (8.09%). In experiment 2, percentages of total and normal fertilized oocytes were significantly higher for the group of spermatozoa capacitated with ionomycin (52.21% and 37.17%, respectively) than with heparin (38.62% and 28.35%, respectively). After 7 days of culture, total embryo rate was significantly higher in the group of sperm capacitated with ionomycin (44.91%) than with heparin (38.69%). However, the percentage of embryos developed to the blastocyst stage was not affected by any of the treatments studied.

Animals↗