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PubMed1999

Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin.

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.

Animals↗
PubMed1984

Phorbol 12-myristate 13-acetate, ionomycin or ouabain, and raised extracellular magnesium induce proliferation of chicken heart mesenchymal cells.

Cultured chicken heart mesenchymal cells are proliferatively quiescent at low densities in medium containing plasma at 10%. Mitogenic hormones like epidermal growth factor and insulin-like growth factors cause these cells to proliferate very actively, as does infection with avian sarcoma viruses, erythroblastosis virus, or myelocytomatosis virus. We have found that the combination of phorbol 12-myristate 13-acetate (PMA), ionomycin or ouabain, and raised extracellular magnesium, likewise, causes these cells to proliferate very actively. Although these agents have no significant effect when acting singly, the combination of PMA at 100 ng/ml and 0.5 microM ionomycin induces a 6-fold increase in cell number at 4 days, and the combination of PMA, ionomycin, and 5.6 mM magnesium induces 12-fold multiplication. Likewise, PMA plus 1 microM ouabain induces 3-fold multiplication, whereas the combination of PMA, ouabain, and magnesium induces 6-fold multiplication. The tumor promoter PMA, like diacylglycerol released by breakdown of plasma membrane phosphatidylinositol diphosphate, is known to activate the serine- and threonine-specific intracellular enzyme kinase C. The divalent cation ionophore ionomycin is known to carry calcium into cells down an electrochemical gradient, and the Na+,K+-ATPase inhibitor ouabain appears to elevate intracellular calcium by means of a sodium-mediated exchange mechanism. Magnesium, like calcium, is known to enter cells passively down an electrochemical gradient and to be involved in the regulation of many key intracellular reactions. Our findings with PMA, ionotropes, and magnesium support a hypothesis that diacylglycerol-mediated activation of kinase C plus cellular divalent cation influx and/or mobilization, caused by the action of mitogenic hormones or the protein products of onc genes, are key events in the initiation of cell replication.

Animals↗
PubMed2002

Ionomycin-induced neutrophil NADPH oxidase activity is selectively inhibited by the serine protease inhibitor diisopropyl fluorophosphate.

The calcium-specific ionophore ionomycin triggers neutrophils to activate their NADPH oxidase and generate reactive oxygen species. This activation is restricted to intracellular sites and involves the neutrophil granules. Cells that have experienced an ionomycin-induced rise in intracellular calcium will also mobilize their intracellular granules and are primed to subsequent challenge with the chemoattractant formylmethionyl-leucyl-phenylalanine (fMLF), but have lost their ability to become desensitized to the same agonist. We have investigated the involvement of serine proteases in the calcium-induced effector functions using the inhibitor diisopropyl fluorophosphate (DFP). The ionomycin-induced NADPH oxidase activity was abrogated by the protease inhibitor, whereas the activity induced by fMLF was unaffected. The DFP-dependent inhibition was restricted to the NADPH oxidase activity, as all other ionomycin-induced cellular activities were largely unaffected. We thus suggest that a serine protease is of importance for the calcium ionophore-induced signal(s) to reach and activate the dormant NADPH oxidase in the neutrophil granules.

Cell-Free System↗
PubMed2001

Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation.

The assessment of intracellular cytokines at the single-cell level by flow cytometry has recently become a potent tool in many areas of cell biology and in defining the role of cytokines in various human diseases. Three-color flow cytometry for detection of intracellular cytokines combined with simultaneous determination of lymphocytes (CD3(+) and CD4(+)) or monocytes (CD33(+) and CD14(+)) was used for comparison of phytohemagglutinin (PHA)-and phorbol myristate acetate (PMA)-ionomycin-induced production of intracellular cytokines in peripheral blood mononuclear cells (PBMCs) of healthy donors. We found that the number of PBMCs stained for tumor necrosis factor alpha and gamma interferon after 6 h of activation was higher when PMA-ionomycin was used for stimulation, while the frequencies of cells positive for interleukin 4 (IL-4) were similar for both stimulators. However, PMA-ionomycin stimulation caused prominent alterations of cell morphology and membrane expression of CD4 and CD14. In contrast, PHA did not cause downregulation of surface markers and resulted in less pronounced alterations in both forward and side scatter signals during flow cytometry analysis. Moreover, during 48 h of culture PHA stimulated tumor necrosis factor beta and IL-10 production, which was not observed when PMA-ionomycin was used. We conclude that the use of PHA for cell activation may limit in vitro artifacts and allow more precise analysis of intracellular cytokine production in various disease states.

Antigens, CD↗
PubMed2002

Nuclear translocation of p90Rsk and phosphorylation of CREB is induced by ionomycin in a Ras-independent manner in PC12 cells.

In the present study we examined the possible role of p90Rsk in pathways leading to neuronal differentiation of PC12 cells induced by nerve growth factor (NGF) and the calcium ionophore ionomycin. PC12-M17 cells, expressing a dominant inhibitory Ras protein, do not undergo neuronal differentiation in response to NGF like wild-type PC12 cells, but exhibit neurite outgrowth when treated with NGF in combination with ionomycin. However, the blockade of Ras in these cells results in failure of activation of mitogen-activated protein kinase (MAPK)/extracellular signal regulation kinase (ERK) (MEK) and ERK activation as well, therefore kinases other than those of the ERK pathway might play a role in the induction of neuronal differentiation in this case. Here we show that p90Rsk translocates to the nucleus in response to ionomycin in both wild-type PC12 and PC12-M17 cells, and this spatial distribution is followed by increased phosphorylation of the cAMP response element binding protein (CREB). Since CREB is believed to be the transcription factor that can integrate Ca2+, growth factor and cAMP-induced signals, we suggest that p90Rsk may be one of the kinases which is able to replace ERKs under certain circumstances, thereby participating in Ras-independent neuronal differentiation induced by NGF plus ionomycin.

Animals↗
PubMed2001

Activation of p42/p44 mitogen-activated protein kinase and contraction by prostaglandin F2alpha, ionomycin, and thapsigargin in cat iris sphincter smooth muscle: inhibition by PD98059, KN-93, and isoproterenol.

In the present study we investigated the cross talk between the Ca2+ mobilization pathway and the mitogen-activated protein (MAP) kinase pathway and contraction in the cat iris sphincter smooth muscle. Three Ca2+-mobilizing agonists, namely, prostaglandin F2alpha (PGF2alpha), ionomycin, and thapsigargin, and three specific inhibitors, PD98059, a p42/p44 MAP kinase inhibitor; KN-93, a Ca2+-calmodulin-dependent protein kinase II (CaMKII) blocker; and isoproterenol, a cAMP-elevating agent, were used. Changes in tension in response to the agonists were recorded isometrically and MAP kinase phosphorylation and activation were monitored by Western blotting and by in situ myelin basic protein phosphorylation, respectively. We found that 1) stimulation of the sphincter muscle with PGF2alpha, ionomycin, or thapsigargin resulted in rapid phosphorylation and activation of p42/p44 MAP kinase and contraction; and 2) treatment of the muscles with PD98059, KN-93, or isoproterenol resulted in inhibition of the Ca2+-mobilizing agonist-induced responses. The contractile responses induced by PGF2alpha, ionomycin, and thapsigargin were (mg of tension/mg of wet weight tissue) 15.2, 15.4, and 16.2, respectively; the increases in MAP kinase phosphorylation by these agonists were 228, 203, and 190%, respectively; and the increases in MAP kinase activation by the agonists were 212, 191, and 162%, respectively. The stimulatory effects of the agonists on contraction and on MAP kinase phosphorylation and activation were blocked by preincubation of the muscle with PD98059, KN-93, or isoproterenol. These data demonstrate that in the iris sphincter phosphorylation and activation of p42/p44 MAP kinases by PGF2alpha, ionomycin, or thapsigargin require intracellular Ca2+ either from extracellular sources or from internal stores, that CaMKII plays an important role in the regulation of contraction, that CaMKII acts upstream of MAP kinase to control its activation, and that the MAP kinase signaling pathway can play a significant role in mediating the cellular effects of these Ca2+-mobilizing agonists.

Adrenergic beta-Agonists↗
PubMed2002

Role of CD80, CD86, and CTLA4 on mouse CD4(+) T lymphocytes in enhancing cell-cycle progression and survival after activation with PMA and ionomycin.

Cell surface interactions between the T cell costimulatory receptors, CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA4), with their cognate ligands, CD80 and CD86, on antigen-presenting cells play an important role in T cell activation. Although CD80 and CD86 are induced on T cells after activation, not much is known about their role in modulating T cell function. We show that CD80, CD86, and CTLA4 are induced on purified CD4(+) T cells after in vitro activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and they play an essential role for proliferation and survival. Blockade of CTLA4-CD80/CD86 interactions greatly reduces PMA and ionomycin-mediated mouse CD4(+) T cell activation. The three key features of this inhibition of activation are: First, late events in T cell activation (after 18 h) are affected; second, these cells do not undergo anergy; and third, CD4(+)CD25(+) regulatory T cells are not responsible. Activation of T cells with PMA and ionomycin together with CTLA4-CD80/CD86 blockade results in decreased induction of CD25 and Bcl-X(L), reduced interleukin (IL)-2, and enhanced transforming growth factor-beta (TGF-beta) production. Furthermore, extended CTLA4-CD80/CD86 blockade results in decreased cell-cycle progression and enhanced apoptosis in a large proportion of cells. This inhibition of T cell proliferation can be rescued completely with anti-CD28 or IL-2 and partially with TGF-beta antagonists. This study reveals a functional role for CD80, CD86, and CTLA4 on CD4(+) T lymphocytes and sheds light on the mechanisms by which these molecules enhance activation and survival with PMA and ionomycin.

Abatacept↗
PubMed2003

[Phenotype characteristic of the ionomycin-resistant CD4+ subset of the peripheral blood T-lymphocytes].

The differential sensitivity of peripheral blood (PB) CD4+ T lymphocytes to the calcium ionophore ionomycin was investigated. Effect of ionomycin exerted on T cells was time- and dose-dependent. We have shown that resistant cells belonged to some distinct T cell subsets. The resting naive CD4+CD45RA+ T cells showed a little, if any, resistance to ionomycin treatment. The primed CD4+CD45R0+ memory T cells behaved similarly as did ionomycin-resistant (IR) cells. Although IR CD4+ T cells had a typical "memory" phenotype, some quantitative differences were found in expression of CD11a, CD28, CD29, CD62L and CD243 markers between PB CD4+CD45R0+ T cells and corresponding IR cells.

Antigens, CD↗
PubMed1991

The formation constants of ionomycin with divalent cations in 80% methanol/water.

The protonation constants and complex formation constants of ionomycin have been determined in 80% methanol/water (w/w) at 25.0 degrees C and mu = 0.050 (tetraethylammonium perchlorate). Potentiometric and spectrometric titration techniques give the following values for the mixed-mode protonation constants of ionomycin: log KH1 = 11.94 +/- 0.02 and log KH2 = 6.80 +/- 0.03. Comparison of these values with those for model compounds indicates that KH1 and KH2 refer to equilibria involving the beta-diketone and carboxylic acid moieties, respectively. Titrations of ionomycin with metal ion at fixed values of pH produced changes in the UV-visual absorbance spectra which were analyzed to give conditional complex formation constants, KMI'. The pH dependence of the values of KMI' indicated that 1:1 divalent metal ion-ionomycin (MI) complexes and protonated MHI+ complexes were formed in the pH range studied. The values of log KMI ranged from 5.30 +/- 0.11 for Sr2+ to 10.25 +/- 0.03 for Ni2+. The selectivity pattern and relative affinities (in parentheses) for the formation of the species MI are as follows: Ni2+ (2000) greater than Zn2+ (600) greater than CO2+ (440) greater than Mn2+ (47) greater than Mg2+ (1.00) greater than Ca2+ (0.21) greater than Sr2+ (0.022). Logarithmic values of KMHI, for the reaction MI + H+ in equilibrium MHI+, ranged from 5.9 (Ni2+) to 8.4 (Sr2+). Calculations using the values of the equilibrium constants determined indicate that an appreciable fraction of the complexed ionophore exists as the protonated complex, MHI+, in the pH range of 6.5-8.5.

Cations↗
PubMed1978

Characterization of ionomycin as a calcium ionophore.

The ionophorous properties of a new antibiotic, ionomycin, have been studied. It was found that the antibiotic is capable of extracting calcium ion from the bulk of an aqueous phase into an organic phase. The antibiotic also acts as a mobile ion carrier to transport the cation across a solvent barrier. The divalent cation selectivity order for ionomycin as determined by ion competition experiments was found to be: Ca greater than Mg greater than Sr = Ba, where the binding of strontium and barium by the antibiotic is insignificant. The antibiotic also binds La3+ to some extent, but its complexation with monovalent alkali metal ions is negligible. Measurement of the binding of ionomycin with Ca2+ indicates that ionomycin complexes and transports calcium ion in a one to one stoichiometry.

Anti-Bacterial Agents↗
PubMed2004

Oxidative stress inhibits ionomycin-mediated cell death in cortical neurons.

Thiol-proteases play important roles in many cellular processes, including maintenance of protein homeostasis and execution of cell death. Therefore, determining how this family of enzymes is regulated is critical for our understanding of both physiological and pathological conditions. Because these proteases require a reduced cysteine residue for activity, the cellular redox state plays a crucial role in regulating the activity of thiol proteases. Importantly, increased oxidative stress can result in the direct modification of the active site cysteine, leading to enzyme inactivation. This would suggest that oxidative stress that occurs during pathological insults could prolong cell survival by preventing the execution of thiol-protease-dependent cell death pathways. To test this hypothesis, cultured rat cortical neurons were treated with the oxidizing agent diamide or doxorubicin in the presence or absence of the calcium ionophore ionomycin. Under normoxic conditions, ionomycin treatment resulted in approximately 70% cell death, which was prevented by addition of the calpain-selective inhibitor benyzloxycarbonyl-Leu-Leu-Tyr fluoromethylketone. Similarly, pretreatment of neurons with either oxidant was also protective. Protection resulting from oxidative stress was not due to new protein synthesis, insofar as cycloheximide did not affect oxidant-mediated protection. Interestingly, treatment with the antioxidant Trolox to reverse or prevent oxidative stress blocked the protective effects of both oxidants against ionomycin-induced cell death. We interpret these findings to suggest that, in diseases or conditions in which oxidative stress is increased, the ability of thiol-proteases to execute cell death pathways fully is decreased and may prolong cell survival.

Animals↗
PubMed1995

The intracellular Ca2+ concentration optimal for T cell activation is quite different after ionomycin or CD3 stimulation.

The relationship between the initial increase of intracellular Ca2+ concentration ([Ca2+]i) (measured at the single-cell level with an imaging system) and the ensuing proliferation was examined in a human T cell clone stimulated by a phorbol ester in combination with ionomycin, thapsigargin or an anti-CD3 mAb (monoclonal antibody against the CD3 molecule, UCHT1). From the responses to various ionomycin concentrations, one can define a range of [Ca2+]i values (400-900 nM) which appears optimal for T cell proliferation; lower [Ca2+]i values are suboptimal, higher values are cytotoxic. It was then examined if the [Ca2+]i requirements were similar following anti-CD3 stimulation. [Ca2+]i oscillations elicited by a concentration of UCHT1 (1/1,000) optimal for mitogenicity fall precisely within the 400-900 nM range. However, very low concentrations of UCHT1 (1/100,000) which evoke barely detectable [Ca2+]i responses still cause the cells to proliferate. The possibility that the lower [Ca2+]i requirements observed following anti-CD3 stimulation was due to [Ca2+]i oscillations was tested under conditions which prevented the appearance of these oscillations. It turns out that an oscillatory Ca2+ signal is not more mitogenic than a sustained augmentation of [Ca2+]i. Finally, it was examined if overstimulation via CD3 could have toxic consequences similar to those elicited after ionomycin overstimulation. Large transient [Ca2+]i responses can be observed following anti-CD3 stimulation in appropriate conditions, and namely in T cells pretreated with interleukin-2. These [Ca2+]i augmentations are not cytotoxic. A role for the plasmalemmal Ca2+ pump in the prevention of cytotoxicity can be demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1999

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (delta F508) cystic fibrosis transmembrane conductance regulator.

The Ca2+ ionophore ionomycin induced cytosolic [Ca2+ ]i elevation as well as strong activation of Cl- efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl- efflux was abolished by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, whereas both activators and inhibitors of phospholipase A2 had no effect, indicating the involvement of Ca2+-dependent Cl- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A2. These results indicate that phospholipase A2 activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Measurement of antigen-dependent interleukin-4 production by human peripheral blood mononuclear cells. Introduction of an amplification step using ionomycin and phorbol myristate acetate.

The study of T cell responses in parasitic disease and allergy in humans has been limited by difficulties in the measurement of interleukin-4 (IL-4) in supernatants from antigen-stimulated peripheral blood mononuclear cells (PBMC). To obtain measurable amounts of IL-4 in vitro, we have added an amplification step to the antigen-specific response. Human PBMC were stimulated by tetanus toxoid (TT) or tuberculin (PPD) for 6 days and then pulsed with ionomycin and phorbol myristate acetate (PMA) for 24 h. TT-stimulated cells from nine revaccinated donors but not from seven unvaccinated donors and four that had only received childhood vaccinations against tetanus produced high levels of IL-4 (median (range) 1500 (300-3800), 316 (0-1600), and 270 (100-410) pg/ml, respectively, as measured by an enzyme linked immunosorbent assay, P = 0.005). PPD did not increase IL-4 production above the background level, although the majority of PPD-stimulated PBMC proliferated and produced interferon-gamma (IFN-gamma) in cultures without ionomycin and PMA. TT-induced IL-4 production correlated positively with proliferation. Culture supernatants did not interfere with IL-4 immunoreactivity and failed to affect ionomycin and PMA induced IL-4 production. The findings suggest that proliferating antigen-specific T cells were the source of IL-4 in these experiments. The method should prove useful for comparing the IL-4 producing ability of antigen-specific T cells from different individuals.

Antigens↗
PubMed1992

Ionomycin produces an improved volume recovery by an increased efflux of taurine from hypoosmotically stressed molluscan red blood cells.

Nucleated erythrocytes of the blood clam, Noetia ponderosa, recover cell volume after a hypoosmotic stress by an efflux of K+, Cl- and taurine. When the cells are exposed to ionomycin followed by hypoosmotic stress, swelling is less and volume recovery is both faster and more complete than in control cells without the ionophore. The improved volume recovery is caused by a large increase in the efflux of taurine. The taurine efflux is altered by changing Ca2+ concentrations in the presence of the ionophore. Potassium regulation by the osmotically stressed erythrocytes is also increased in the presence of ionomycin, but only by a small amount, perhaps accounting for the initial decrease in swelling. Variation of Ca2+ in the presence of ionomycin without osmotic stress produces no change in the regulation of either osmolyte. These results indicate that both the osmotic stress and an increase in [Ca2+]i are required for the permeability change that produces taurine efflux.

Animals↗
PubMed1991

Platelet activation by diacylglycerol or ionomycin is inhibited by nitroprusside.

Experiments were performed to elucidate the role of cyclic guanosine monophosphate (cGMP) on platelet activation induced by protein kinase C (PKC) activators and calcium ionophore. Human platelets were pretreated with acetylsalicylic acid and with hirudin and apyrase. Aggregation and ATP secretion in response to the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetylglycerol (OAG) were inhibited by the nitrovasodilator sodium nitroprusside (SNP), an activator of guanylate cyclase, and by 8-bromo-cyclic GMP (8-Br-cGMP). The experiments were performed in the presence of M&B 22948, an inhibitor of cGMP phosphodiesterase. SNP and 8-Br-cGMP also inhibited platelet aggregation and secretion evoked by the ionophore ionomycin. In fura-2 loaded platelets SNP did not affect basal cytosolic Ca2+ level nor the rise induced by low concentrations of ionomycin, both in the presence and absence of extracellular Ca2+. The phosphorylation of the 47 and 20 kDa protein induced by ionomycin or PMA were not significantly decreased by SNP or 8-Br-cGMP. The present results suggest that cGMP is able to inhibit both the PKC and the Ca(2+)-dependent pathways leading to platelet activation by interfering, similarly to cAMP, with processes following protein phosphorylation, close to the effector systems.

Adenosine Triphosphate↗
PubMed1991

Comparison of the role of protein kinase C in platelet functional responses induced by three different mechanisms, PAF, ionomycin and arachidonic acid.

The role of protein kinase C (PKC) in modulating platelet activation has been examined in platelets pre-incubated with either the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the non-specific protein kinase inhibitor, staurosporine. In order to determine where in the signal transduction pathway PKC is exerting its effect platelets were activated either with a receptor-operated stimulus platelet activating factor (PAF) or by direct elevation of [Ca2+]i (ionomycin) or with arachidonic acid which is converted into thromboxane B2 (TxB2). In PAF-stimulated platelets activation of PKC inhibited both [Ca2+]i elevation and TxB2 generation but had no effect on 5-hydroxytryptamine (5-HT) release whilst staurosporine increased the duration of [Ca2+]i elevation and potentiated TxB2 generation but inhibited 5-HT release. In ionomycin-stimulated platelets modulation of PKC had no effect on [Ca2+]i elevation but in contrast to PAF-stimulated platelets PKC activation caused potentiation of TxB2 generation and 5-HT release whilst inhibition of PKC caused inhibition of TxB2 generation and 5-HT release. Modulation of PKC did not affect arachidonic acid-induced TxB2 generation. These findings suggest that in receptor activated platelets endogenously activated PKC is exerting a negative feedback role, however, when [Ca2+]i elevation is not modified by PKC activation or inhibition (such as in ionomycin stimulated platelets) the relationship between the state of PKC activation and subsequent platelet functional responses corresponds more closely. The findings from this study suggest a different relationship between PKC and TxB2 generation than between PKC and dense granule release in PAF-stimulated platelets.

Alkaloids↗
PubMed1996

Effect of intracellular acidity and ionomycin on apoptosis in HL-60 cells.

The aim was to investigate in detail the influence of intracellular pH (pHi) and intracellular Ca2+ concentration ([Ca2+]i) on apoptosis in HL-60 human promyelocytic leukaemia cells. The pHi was controlled by changing the pH of media as well as by interfering with the pHi regulatory mechanisms with 3-amino-6-chloro-5-(1-homopiperidyl)-N-(diaminomethylene) pyrazincarboxamide (HMA; an inhibitor of Na+/H+ antiport), 4-diiosothiocyanatostilbene-2,2'disulfonic acid, (DIDS; an inhibitor of Na(+)-dependent HCO3-/Cl- exchange) and nigericin (a K+ ionophore). The [Ca2+]i was increased with ionomycin, a Ca2+ ionophore. The apoptosis of HL-60 cells was measured with conventional agarose gel electrophoresis for DNA fragmentation and also with the release of 3H from 3H-thymidine-labelled DNA. Based on the magnitude of DNA fragmentation and 3H release at different pHi, it was shown that apoptosis occurred in HL-60 cells when the pHi was lowered from normal pHi of 7.4 to about 7.2-6.7 with a peak increase at pHi 6.8-6.9. Addition of 4 microM ionomycin to RPMI 1640 medium, which contained 615 microM Ca2+, elevated the apoptosis in the cells. Such an increase in apoptosis by ionomycin in HL-60 cells appeared to result from both an increase in [Ca2+]i and from a decline in pHi. The results indicate that the acidic intratumour environment may greatly affect the response of neoplastic tissues to hyperthermia, radiation and chemotherapeutic drugs which cause apoptosis.

Apoptosis↗