thapsigargin and Leukemia--Erythroblastic--Acute

In the present experiments in HEL cells, we have investigated the requirement for a hyperpolarised resting membrane potential for the initial activation of the Ca(2+) activated K(+) channel, KCa3.1, following activation of the Ca(2+) release activated Ca(2+) (CRAC) entry pathway. In intact cells, fluorimetric measurements of [Ca(2+)]i following thapsigargin-mediated activation of CRAC entry revealed a sustained increase in [Ca(2+)]i. Block of KCa3.1 by application of charybdotoxin resulted in a 50% reduction in the steady-state [Ca(2+)]i, consistent with the well established role for KCa3.1-mediated hyperpolarisation in augmenting CRAC entry. Interestingly, subsequent depolarisation to 0mV by application of gramicidin resulted in a fall in steady-state Ca(2+) levels to values theoretically below that required for activation of KCa3.1. Whole cell patch clamp experiments confirmed the lack of KCa3.1 activation at 0mV following activation of the CRAC entry pathway, indicating an absolute requirement for a hyperpolarised resting membrane potential for the initial activation of KCa3.1 leading to hyperpolarisation and augmented Ca(2+) entry. Current clamp experiments confirmed the requirement for a hyperpolarised resting membrane potential in KCa3.1 activation by CRAC entry. Given the critical role played by KCa3.1 and membrane potential in general in the control of CRAC-mediated [Ca(2+)]i changes, we investigated the hypothesis that inhibition of the CRAC-mediated changes in [Ca(2+)]i observed following 2-APB addition may in part arise from direct inhibition of KCa3.1 by 2-APB. Under whole cell patch clamp, 2-APB, at concentrations typically used to block the CRAC channel, potently inhibited KCa3.1 in a reversible manner (half maximal inhibition 14.2 μM). This block was accompanied by a marked shift in the reversal potential to depolarised values approaching that set by endogenous membrane conductances. At the single channel level, 2-APB applied to the cytosolic face resulted in a significant reduction in open channel probability and a fall in the mean open time of the residual channel activity. Our data highlight the absolute requirement for a hyperpolarising resting membrane conductance for the initial activation of KCa3.1 by CRAC entry. Additionally, our results document direct inhibition of KCa3.1 by 2-APB, thus highlighting the need for caution when ascribing the site of inhibition of 2-APB exclusively to the CRAC entry pathway in experiments where membra

Topics: Aniline Compounds; Boron Compounds; Calcium; Calcium Channels; Cell Line, Tumor; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Ion Channel Gating; Leukemia, Erythroblastic, Acute; Membrane Potentials; Patch-Clamp Techniques; Thapsigargin; Xanthenes

Even though the erythroleukemia cell lines K562 and HEL do not express α1-adrenoceptors, some α1-adrenergic drugs influence both survival and differentiation of these cell lines. Since Ca2+ is closely related to cellular homeostasis, we examined the capacity of α1-adrenergic drugs to modulate the intracellular Ca2+ content in K562 cells. Because of morphological alterations of mitochondria following α1-adrenergic agonist treatment, we also scrutinized mitochondrial functions. In order to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells, we evaluated the application of the fluorescent α1-adrenergic antagonist BODIPY® FL-Prazosin. We discovered that the α1-adrenergic agonists naphazoline, oxymetazoline and also the α1-adrenergic antagonist benoxathian are able to raise the intracellular Ca2+-content in K562 cells. Furthermore, we demonstrate that naphazoline treatment induces ROS-formation as well as an increase in Δψm in K562 cells. Using BODIPY® FL-Prazosin we were able to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells. Interestingly, the SERCA-inhibitor thapsigargin appears to interfere with the binding of BODIPY® FL-Prazosin. Our data suggest that the effects of α1-adrenergic drugs on erythroleukemia cells are mediated by a thapsigargin sensitive binding site, which controls the fate of erythroleukemia cells towards differentiation, senescence and cell death through modulation of intracellular Ca2+.

Topics: Adrenergic Agents; Adrenergic alpha-1 Receptor Agonists; Adrenergic alpha-1 Receptor Antagonists; Aging; Binding Sites; Calcium; Cell Death; Cell Differentiation; Cell Line, Tumor; Cell Survival; Homeostasis; Humans; K562 Cells; Leukemia, Erythroblastic, Acute; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Naphazoline; Reactive Oxygen Species; Receptors, Adrenergic, alpha-1; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Hexamethylene bisacetamide (HMBA) stimulates Ca(2+) signals in murine erythroleukemia (MEL) cells serving as an important component of the HMBA-induced pathway that promotes differentiation to the erythroid phenotype. We observed that 1,6-diaminohexane (DAH) triggered a more rapid and robust increase in MEL cell Ca(2+) levels compared to HMBA and the monodeacetylated N-acetyl-1,6-diaminohexane (NADAH), and that polyamine deacetylase inhibition completely abolished the ability of HMBA and NADAH to induce Ca(2+) signals in MEL cells. Our work indicates that DAH mediates Ca(2+) signal propagation via its ability to activate inositol 1,4,5-trisphosphate (IP(3)) receptors, as we observed similar Ca(2+) release characteristics and heparin sensitivity of DAH and IP(3) in permeabilized MEL cells. Finally, we observed that the DAH-induced Ca(2+) release pathway robustly coupled to a Ca(2+) influx pathway that could be distinguished from thapsigargin-induced Ca(2+) influx by its unusual insensitivity to 2-aminoethoxydiphenyl borate.

Topics: Acetamides; Acetylation; Animals; Boron Compounds; Calcium; Cell Differentiation; Cell Line, Tumor; Diamines; Erythroid Cells; Inositol 1,4,5-Trisphosphate; Leukemia, Erythroblastic, Acute; Mice; Signal Transduction; Thapsigargin

To assess the role of G16, a trimeric G protein exclusively expressed in hematopoietic cells, Galpha16 antisense RNA was stably expressed in human erythroleukemia (HEL) cells. Western blot analysis showed that in transfected cell lines, the expression of endogenous Galpha16 protein was suppressed, but the expression of Galphaq/11, Galphai2, and Galphai3 remained unaffected. Suppression of Galpha16 in transfected HEL cells did not interfere with transient elevations of intracellular free Ca2+ concentrations induced by prostaglandin E1 (PGE1), platelet-activating factor, or thrombin. In parental HEL cells, UTP and ATP mobilized Ca2+ from intracellular stores with half-maximum effective concentrations of 3. 6 +/- 0.7 and 4.7 +/- 1.6 microM, respectively, apparently by stimulating P2U purinoceptors. By contrast, Ca2+ mobilization by UTP or ATP was completely abrogated in Galpha16-suppressed cells, indicating specific coupling of G16 to P2U purinoceptors. Pertussis toxin inhibited the effect of UTP in parental HEL cells by 57.6 +/- 4.9%. These data indicate that signaling by the P2U purinoceptor obligatorily requires G16 but may be modulated further by activation of Gi. Priming of HEL cells with UTP or ATP prior to stimulation with PGE1 markedly enhanced the PGE1-induced intracellular Ca2+ release. This indirect, potentiating effect of UTP and ATP was not impaired in Galpha16-suppressed cells but was inhibited by pertussis toxin, indicating that functional P2U purinoceptors are present on these cells and that the potentiating effect primarily depends on Gi. The data demonstrate (i) that Galpha16 antisense RNA selectively inhibits endogenous Galpha16 protein expression in HEL cells; (ii) that stimulation of endogenous P2U (P2Y2) purinoceptors leads to the mobilization of intracellular Ca2+ by a mechanism that strictly depends on Galpha16; and (iii) that P2U purinoceptors in HEL cells can communicate with two distinct signaling pathways diverging at the G protein level.

Topics: Adenosine Triphosphate; Calcium; Enzyme Inhibitors; GTP-Binding Proteins; Hematopoietic Stem Cells; Humans; Leukemia, Erythroblastic, Acute; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; RNA, Antisense; Signal Transduction; Thapsigargin; Tumor Cells, Cultured; Uridine Triphosphate

1. A combination of single cell fluorescence and patch clamp techniques were used to study the mechanisms underlying thrombin-evoked Ca2+ signals in human erythroleukaemia (HEL) cells, a leukaemic cell line of platelet-megakaryocyte lineage. 2. Thrombin caused a transient increase in intracellular Ca2+ ([Ca2+]i), consisting of both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Mn2+ quench studies indicated that the thrombin-evoked divalent cation-permeable pathway was activated during, but not prior to, release from internal stores. 3. Thapsigargin (1 microM) irreversibly released internal Ca2+ from the same store as that released by thrombin and continuously activated a Ca(2+)-influx mechanism. The amplitude of the thrombin- and thapsigargin-induced Ca2+ influx displayed a marked single cell heterogeneity which showed no correlation with the size of the store Ca2+ transient. 4. In whole-cell patch clamp recordings, both thrombin and thapsigargin evoked an inwardly rectifying Ca2+ current which developed with little or no increase in current noise, showed no reversal in the voltage range -110 to +60 mV and was blocked by 1 mM Zn2+. The apparent divalent cation permeability sequence of this pathway was Ca2+ > > Ba2+ > Mn2+, Mg2+. The thapsigargin-evoked current density at -100 mV varied between 0.42 and 2.1 pA pF-1 in different cells. Thrombin failed to activate additional Ca2+ current if it was added after the thapsigargin-induced inward current had fully developed. 5. These studies indicate that thrombin activates Ca2+ influx in HEL cells entirely via a Ca(2+)-store-release-activated Ca2+ current (Icrac) rather than via receptor-operated or second messenger-dependent Ca2+ channels. The level of expression of Icrac appears to be a major factor in determining the duration of the thrombin-evoked [Ca2+]i response and therefore represents a means by which cells can exert control over [Ca2+]i-dependent events.

Topics: Calcium; Cations, Divalent; Electrophysiology; Fluorescence; Fluorescent Dyes; Fura-2; Humans; Ion Transport; Leukemia, Erythroblastic, Acute; Manganese; Patch-Clamp Techniques; Signal Transduction; Thapsigargin; Thrombin; Tumor Cells, Cultured

Heat shock proteins (HSPs) are induced in mammalian cells in a variety of pathophysiological states and have an important role in cytoprotection in vitro and in vivo. In this study, we report that the calcium ionophore A23187, a glucose-regulated protein (GRP) inducer, dramatically inhibits HSP70 synthesis and HSP70 mRNA transcription after induction by heat shock, sodium arsenite, or prostaglandin A1 treatment in human K562 cells. A23187 does not suppress, and it actually prolongs, the DNA-binding activity of the human heat shock transcription factor (HSF), while it alters HSF1 phosphorylation in heat shock-treated cells. To inhibit HSP70 expression, A23187 needs to be present during heat shock, while treatment before or after heat shock does not affect HSP70 mRNA transcription. The GRP inducer thapsigargin, which specifically inhibits the endoplasmic reticulum Ca2+-ATPase, has no effect on heat-induced HSP70 synthesis, indicating that A23187 inhibitory activity is not due to depletion of intracellular calcium stores and is independent of the concomitant induction of GRP genes. Inhibition of HSP70 expression is correlated with alterations in HSF1 phosphorylation in heat-shocked cells, but not in sodium arsenite-treated cells, indicating that different mechanisms may be involved in mediating A23187 inhibitory activity.

Topics: Calcimycin; Calcium-Transporting ATPases; Cell Line; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression; Heat Shock Transcription Factors; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Ionophores; Leukemia, Erythroblastic, Acute; Membrane Proteins; Methionine; Molecular Chaperones; Phosphorylation; Protein Biosynthesis; RNA, Messenger; Terpenes; Thapsigargin; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

This study was conducted on human K562 lymphocytes to investigate the mechanisms implicated in the regulation of the serotonin transport process. The uptake of serotonin in these cells was saturable (Km, 3.37 microM; Vmax, 2.03 nmol/10(6) cells) and Na+ dependent; isoosmotic replacement of Na+ with choline chloride in the assay medium resulted in the decreased uptake process. Augmentation of intracellular free calcium, [Ca2+]i, by thapsigargin decreased the uptake of serotonin in these cells. Similarly, addition of calcium ionophores (A23187) and ionomycin also inhibited serotonin transport. In Fura-2-loaded cells, these agents increased the [Ca2+]i contents. These results suggest that an increase in [Ca2+]i is implicated with a decrease in serotonin transport. Since an increase in [Ca2+]i is known to activate calmodulin (CaM), we employed CaM antagonists. Calmodulin antagonists W-7 (N-[6-aminohexyl]-5-chloro-1-naphthalene-sulfonamide) and mellitin inhibited serotonin uptake in K562 cells, suggesting that CaM is involved in serotonin transport regulation. Furthermore, acute exposure of K562 cells to known protein kinase C (PKC) activators, phorbol-12-myristate-13-acetate (PMA) and sn-1,2-dioctanoylglycerol (DiC8), curtailed serotonin uptake by these cells. However, staurosporine (a PKC inhibitor) failed to abolish the inhibitory effects of PMA on serotonin transport in these cells, indicating that the target of PMA is not PKC. Nonetheless, the PMA-induced inhibitory effects are specific as 4 alpha-phorbol-12,13,didecanoate (a phorbol ester known not to activate PKC) failed to mimic PMA-like actions on serotonin transport in K562 cells. DiC8 not only exerted higher inhibitory effects than PMA but also had additive effects in the presence of the latter on serotonin transport. These results suggest that in addition to PKC, there are other cellular targets (of PMA) implicated in serotonin transport regulation.

Topics: Biological Transport; Calcimycin; Calcium; Calmodulin; Enzyme Activation; Humans; Ionomycin; Kinetics; Leukemia, Erythroblastic, Acute; Lymphocytes; Protein Kinase C; Serotonin; Serotonin Antagonists; Thapsigargin; Tumor Cells, Cultured

Cyclopiazonic acid and thapsigargin, inhibitors of the endoplasmic reticulum Ca2+ pump were shown to elevate [Ca2+]i in Friend erythroleukemia cells, line F4-6, at concentrations of 1-5 microM and 0.5-2 nM, respectively. At the same concentrations, these agents induced a strong suppression of c-myb mRNA levels within 3 h, whereas c-myc expression remained unaffected. The c-myb expression recovered and approached pretreatment levels at 9-12 h of incubation. The decrease in c-myb mRNA was prevented in Ca(2+)-free medium. Treatment of F4-6 cells with EGTA led to a transient increase in c-myb mRNA with the same kinetics as the Ca2+ pump inhibitor-induced suppression, indicating that c-myb expression is bidirectionally regulated by changes in [Ca2+]i. Studies on the differentiation status of F4-6 cells following cyclopiazonic acid or thapsigargin exposure demonstrated a marked increase in beta-globin mRNA synthesis at 60h and in hemoglobin production at 96 h. These results provide further evidence that a rise in the cytosolic Ca2+ concentration is capable, in Friend erythroleukemia cells, of inducing an early transient suppression of c-myb mRNA levels, which is followed by terminal erythroid differentiation.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cell Differentiation; Dimethyl Sulfoxide; Gene Expression; Genes, myc; Indoles; Leukemia, Erythroblastic, Acute; Mice; Oncogenes; RNA, Messenger; Terpenes; Thapsigargin; Time Factors; Tumor Cells, Cultured

Human erythroleukemic (HEL) cells, loaded with fura-2, respond to neuropeptide Y (NPY) with a fast and transient increase in intracellular calcium. The Y1 receptor-specific agonist (Leu-31,Pro-34)-NPY is 4-fold more potent and the carboxyl-terminal fragment NPY13-36 is 150-fold less potent than NPY. Thus, it is concluded that the response is mediated through the activation of a Y1 type of NPY receptor. HEL cells do not respond to a second addition of NPY but do respond to a further addition of alpha-thrombin (alpha-T). However, in a calcium-free medium, prior stimulation with NPY largely inhibits a subsequent response to alpha-T. Moreover, prior stimulation with alpha-T in the absence of external calcium completely prevents the response to the addition of NPY, indicating a common effector pathway. The latter is further reinforced by using thapsigargin (TG), which has been shown to deplete the Inositol 1,4,5-trisphosphate-dependent calcium pool in other systems. HEL cells preincubated with TG in calcium-free medium fail to respond to either NPY or alpha-T. Likewise, prior stimulation with NPY or alpha-T in calcium-free medium significantly inhibits the response to TG. Preincubation of cells with phorbol esters strongly inhibits the NPY-induced release of intracellular Ca2+ in HEL cells, an effect that is partially prevented by preincubation of the cells with H7, a protein kinase C inhibitor. However, neither the homologous nor the apparent heterologous desensitization of the NPY receptor can be prevented by H7. It is concluded that NPY releases intracellular Ca2+ from an inositol 1,4,5-trisphosphate-sensitive calcium pool, which is restored by external calcium, and that NPY receptor desensitization is protein kinase C independent.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcium; Enzyme Activation; Erythrocytes; Fura-2; Humans; Inositol 1,4,5-Trisphosphate; Isoquinolines; Leukemia, Erythroblastic, Acute; Neuropeptide Y; Piperazines; Protein Kinase C; Spectrometry, Fluorescence; Terpenes; Thapsigargin; Tumor Cells, Cultured