thapsigargin and Leukemia--Basophilic--Acute

Recent breakthroughs in the store-operated calcium (Ca(2+)) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore forming subunit of the highly Ca(2+)-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca(2+) release-activated Ca(2+) current (I(CRAC)), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na(+) currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I(CRAC) in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I(CRAC). Ectopic expression of Stim1 in ECs increased their I(CRAC) to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.

Topics: Animals; Calcium; Calcium Channels; Cell Division; DNA Primers; Endothelium, Vascular; Humans; Leukemia, Basophilic, Acute; Membrane Glycoproteins; Membrane Proteins; Neoplasm Proteins; ORAI1 Protein; Patch-Clamp Techniques; Rats; RNA, Messenger; RNA, Small Interfering; Stromal Interaction Molecule 1; Thapsigargin; TRPC Cation Channels; Umbilical Veins

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.

Topics: Animals; Biological Transport; Calcium; Calcium Channels; Calcium-Transporting ATPases; Calmodulin; Cell Line, Tumor; Cell Membrane; Kinetics; Leukemia, Basophilic, Acute; Patch-Clamp Techniques; Rats; Thapsigargin

1. Agonist interaction with phospholipase C-linked receptors at the plasma membrane can elicit both Ca2+ and Na+ influxes in lymphocytes. While Ca2+ influx is mediated by Ca2+ release-activated Ca2+ (CRAC) channels, the pathway responsible for Na+ influx is largely unknown. 2. We show that thapsigargin, ionomycin, ADP-ribose and IP3 activated a nonselective cation channel in lymphocytes that had a slightly outwardly rectifying I-V relationship, and a single channel conductance of 23.1 pS. We termed this channel a Ca2+ release-activated nonselective cation (CRANC) channel. 3. On activation in cell-attached configuration, switching to an inside-out configuration abolished CRANC channel activity. 4. Transfection of Jurkat T cells with antisense oligonucleotides for LTRPC2 reduced capacitative Ca2+ entry. 5. These results suggest that CRANC channels are responsible for the Na+ influx as well as a portion of the Ca2+ influx in lymphocytes induced by store depletion, that sustained activation of CRANC channels requires some property of the environment of a cell depleted of its Ca2+ stores; and that LTRPC2 protein is a likely component of the CRANC channel.

Topics: Animals; Biological Transport; Calcium; Cations; Enzyme Inhibitors; Humans; Ion Channels; Jurkat Cells; Leukemia, Basophilic, Acute; Lymphocytes; Membrane Potentials; Membrane Proteins; Rats; Sodium; Thapsigargin; Transfection; TRPM Cation Channels

Using a combination of fluorescence measurements of intracellular Ca(2+) ion concentration ([Ca(2+)](i)) and membrane potential we have investigated the sensitivity to serine/threonine phosphatase inhibition of Ca(2+) entry stimulated by activation of the Ca(2+) release-activated Ca(2+) (CRAC) entry pathway in rat basophilic leukemia cells. In both suspension and adherent cells, addition of the type 1/2A phosphatase inhibitor calyculin A, during activation of CRAC uptake, resulted in a fall in [Ca(2+)](i) to near preactivation levels. Pre-treatment with calyculin A abolished the component of the Ca(2+) rise associated with activation of CRAC uptake and inhibited Mn(2+) entry, consistent with a requirement of phosphatase activity for activation of the pathway. Depletion of intracellular Ca(2+) stores is accompanied by a large depolarisation which is absolutely dependent upon Ca(2+) entry via the CRAC uptake pathway. Application of calyculin A or okadaic acid, a structurally unrelated phosphatase antagonist inhibits this depolarisation. Taken in concert, these data demonstrate a marked sensitivity of the CRAC entry pathway to inhibition by calyculin A and okadaic acid.

Topics: Animals; Boron Compounds; Calcium; Calcium Signaling; Cations, Divalent; Cells, Cultured; Enzyme Inhibitors; Ion Transport; Leukemia, Basophilic, Acute; Manganese; Marine Toxins; Membrane Potentials; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Protein Phosphatase 1; Rats; Thapsigargin

Indirect evidence for the existence of a specific protein-mediated process for the cellular uptake of endocannabinoids has been reported, but recent results suggested that such a process, at least for AEA [ N -arachidonoylethanolamine (anandamide)], is facilitated uniquely by its intracellular hydrolysis by FAAH (fatty acid amide hydrolase) [Glaser, Abumrad, Fatade, Kaczocha, Studholme and Deutsch (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 4269-4274]. In the present study, we show that FAAH alone cannot account for the facilitated diffusion of AEA across the cell membrane. In particular, (i) using a short incubation time (90 s) to avoid AEA hydrolysis by FAAH, AEA accumulation into rat basophilic leukaemia or C6 cells was saturable at low microM concentrations of substrate and non-saturable at higher concentrations; (ii) time-dependent and, at low microM concentrations of substrate, saturable AEA accumulation was observed also using mouse brain synaptosomes; (iii) using synaptosomes prepared from FAAH-deficient mice, saturable AEA accumulation was still observed, although with a lower efficacy; (iv) when 36 AEA and N -oleoylethanolamine analogues, most of which with phenyl rings in the polar head group region, were tested as inhibitors of AEA cellular uptake, strict structural and stereochemical requirements were needed to observe significant inhibition, and in no case the inhibition of FAAH overlapped with the inhibition of AEA uptake; and (v) AEA biosynthesis by cells and sensory neurons was followed by AEA release, and this latter process, which cannot be facilitated by FAAH, was still blocked by an inhibitor of AEA uptake. We suggest that at least one protein different from FAAH is required to facilitate AEA transport across the plasma membrane in a selective and bi-directional way.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Biological Transport; Brain Chemistry; Carrier Proteins; Cell Line, Tumor; Cell Membrane; Cells, Cultured; Endocannabinoids; Enzyme Inhibitors; Ganglia, Spinal; Humans; Kidney; Leukemia, Basophilic, Acute; Male; Mice; Mice, Knockout; Neoplasm Proteins; Organophosphonates; Polyunsaturated Alkamides; Rats; Synaptosomes; Thapsigargin

In many cell types, the emptying of intracellular Ca2+ stores results in the opening of store-operated Ca2+ channels in the plasma membrane. However, the nature of the signal that couples store content to the opening of these Ca2+ channels is unclear. One model proposes that the Ca2+ channels are initially stored in cytoplasmic vesicles but inserted into the plasma membrane upon store depletion via a regulated exocytoytic mechanism (vesicular fusion model). Using the whole-cell patch-clamp technique to measure the store-operated Ca2+ current ICRAC and the capacitance method to monitor vesicular fusion, an indicator of exocytosis, we have investigated the effects of interfering with regulated exocytosis on the ability of ICRAC to activate. We find that the recombinant protein alpha-SNAP1-285, an inhibitor of exocytosis in many systems, suppresses such fusion but has no impact on the activation of ICRAC. A variety of other manoeuvres that interfere with vesicle trafficking and exocytosis were also without effect on ICRAC. Impairing constitutive exocytosis with brefeldin A reduced the extent of ICRAC, but this effect was less pronounced when current density was considered instead. Activation of ICRAC can therefore be clearly dissociated from an exocytotic mechanism, a finding that is not easily reconcilable with the vesicular fusion model.

Topics: Animals; Brefeldin A; Calcium; Calcium Channels; Carrier Proteins; Cell Line, Tumor; Electric Capacitance; Ethylmaleimide; Exocytosis; Leukemia, Basophilic, Acute; Membrane Potentials; Membrane Proteins; Oligopeptides; Patch-Clamp Techniques; Protein Transport; ras Proteins; Rats; Recombinant Proteins; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; Tetanus Toxin; Thapsigargin; Vesicular Transport Proteins

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells and basophils results in FcepsilonRI beta and gamma subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FcepsilonRI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FcepsilonRI beta and gamma chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FcepsilonRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FcepsilonRI and activated Syk to the proteasome for degradation.

Topics: Animals; Enzyme Precursors; Intracellular Signaling Peptides and Proteins; Kinetics; Leukemia, Basophilic, Acute; Ligands; Oligopeptides; Phosphorylation; Protein Subunits; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Rats; Receptors, IgE; Syk Kinase; Thapsigargin; Tumor Cells, Cultured; Ubiquitin; Ubiquitin-Protein Ligases; Vaccinia virus

We previously showed that silver stimulates degranulation and leukotriene (LT) C(4) production in rat basophilic leukemia mast cells and now show that silver induces these events by a mechanism that differs from the FcepsilonRI-mediated response. In common with FcepsilonRI cross-linking, silver induced tyrosine phosphorylation of extracellular signal-regulated kinases and furthermore, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinase dose-dependently inhibited the silver-induced LTC(4) production. In contrast to FcepsilonRI cross-linking, silver had no effect on the production of IL-4 and TNF-alpha, indicating that different mechanisms are involved in the activation by these two stimuli. In line with this, silver had no or only marginal effect on the tyrosine phosphorylation of FcepsilonRIbeta, Lyn, Syk, and linker for activation of T cells, the early and crucial events in FcepsilonRI signaling. Silver induced calcium signals that were involved in the metal-induced degranulation, but not LTC(4) production. Unlike Ag, the silver-induced calcium signals were resistant to the depletion of thapsigargin-sensitive calcium stores and the inhibition of tyrosine kinases and phospholipase Cgamma. These findings indicate that silver activates mast cells by bypassing the early signaling events required for the induction of calcium influx. Our data strongly suggest the existence of an alternative pathway bypassing the early signaling events in mast cell activation and indicate that silver may be useful for analyses of such alternative mechanisms.

Topics: Animals; Calcium; Calcium Signaling; Carrier Proteins; Cell Degranulation; Enzyme Precursors; Immunity, Innate; Interleukin-4; Intracellular Signaling Peptides and Proteins; Isoenzymes; Leukemia, Basophilic, Acute; Leukotriene C4; Mast Cells; Mitogen-Activated Protein Kinases; Phospholipase C gamma; Phosphoproteins; Protein-Tyrosine Kinases; Rats; Receptors, IgE; Silver; Silver Nitrate; src-Family Kinases; Syk Kinase; Thapsigargin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Type C Phospholipases; Tyrosine

To detect low levels of histamine, we developed a histamine microsensor using recombinant histamine oxidase. Histamine oxidase with a histidine tag was readily purified using a histidine affinity column. The enzyme showed higher catalytic activity on histamine than diamines (e.g., putrescine and cadaverine) or N(tau)-methylhistamine. The sensor had three carbon film electrodes modified with osmium-polyvinylpyridine-based gel containing horseradish peroxidase, histamine oxidase, and Ag. When a standard solution of histamine was aspirated at a flow rate of 2 microl/min, the detected current was proportional to the histamine concentration and the lower detection limit was 11.3 nM. When rat basophilic leukemia cells (1 x 10(6)) were stimulated by various concentrations of antigen (2, 20, and 200 ng/ml), the histamine concentrations were 0.32, 2.7, and 1.3 microM, respectively, and 20 ng/ml of antigen was found to be the optimal concentration for the antigen-antibody reaction. In contrast, when thapsigargin, an inhibitor of Ca-ATPase in the endoplasmic reticulum, was added (50, 100, and 500 nM), the detected current increased with thapsigargin concentrations and the measured histamine concentrations were 28 nM, 1.3 microM, and 2.7 microM, respectively. These results indicate that the microsensor is useful for the analysis of histamine release from mast cells.

Topics: Amine Oxidase (Copper-Containing); Animals; Biosensing Techniques; Calcium-Transporting ATPases; Catalysis; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Histamine; Histamine Release; Hydrogen Peroxide; Leukemia, Basophilic, Acute; Monitoring, Physiologic; Polymerase Chain Reaction; Rats; Recombinant Proteins; Substrate Specificity; Thapsigargin; Tumor Cells, Cultured

We investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP(3)) in capacitative calcium entry and calcium release-activated calcium current (I(crac)) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with blocked calcium entry and I(crac) activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and I(crac). The membrane-permeant IP(3) receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and I(crac). However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP(3) receptor because the drug was equally effective in wild-type DT40 B-cells and in DT40 B-cells whose genes for all three IP(3) receptors had been disrupted. Intracellular application of another potent IP(3) receptor antagonist, heparin, failed to inhibit activation of I(crac). Finally, the inhibition of I(crac) activation by or wortmannin was not reversed or prevented by direct intracellular application of IP(3). These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP(3) and IP(3) receptor in this mechanism, at least in these particular cell types.

Topics: Androstadienes; Animals; B-Lymphocytes; Boron Compounds; Calcium; Calcium Channels; Cell Line, Transformed; Cells, Cultured; Chickens; Enzyme Inhibitors; Estrenes; Heparin; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Kinetics; Lacrimal Apparatus; Leukemia, Basophilic, Acute; Mice; Phosphatidylinositol Phosphates; Pyrrolidinones; Rats; Receptors, Cytoplasmic and Nuclear; Thapsigargin; Tumor Cells, Cultured; Type C Phospholipases; Wortmannin

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.

Topics: Animals; Calcium; Cell Membrane; Diglycerides; Female; Humans; Ion Channels; Ionomycin; Jurkat Cells; Kinetics; Leukemia, Basophilic, Acute; Lymphocytes; Membrane Potentials; Oocytes; Patch-Clamp Techniques; Rats; Saccharomyces cerevisiae; Second Messenger Systems; T-Lymphocytes; Thapsigargin; Tumor Cells, Cultured; Xenopus laevis

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.

Topics: Animals; Animals, Newborn; Astrocytes; Biological Transport; Calcium; Calcium Channels; Calcium-Transporting ATPases; Cells, Cultured; Cerebral Cortex; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Fluorescent Dyes; Glutamic Acid; Indoles; Lanthanum; Leukemia, Basophilic, Acute; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thapsigargin; TRPC Cation Channels; Tumor Cells, Cultured

In many electrically non-excitable cells, Ca2+ entry is mediated predominantly by the store-operated Ca2+ influx pathway. The best-characterised store-operated Ca2+ current is the Ca2+ release-activated Ca2+ current (ICRAC). It is generally believed that high concentrations of intracellular Ca2+ buffer are required to measure ICRAC, due to Ca2+-dependent inactivation of the channels. Recently, we have recorded robust ICRAC in rat basophilic leukaemia (RBL-1) cells at physiological levels of Ca2+ buffering when stores were depleted by inhibition of the sarcoplasmic/ endoplasmic reticulum Ca2+-activated adenosine triphosphatase (SERCA) pumps. However, the second messenger inositol 1,4,5-trisphosphate (InsP3) was not able to evoke the current under such conditions, despite inducing substantial Ca2+ release. We have therefore suggested that a threshold exists within the Ca2+ stores which has to be overcome for macroscopic ICRAC to activate. To establish whether this is a specific feature of ICRAC in RBL-1 cells or whether it is a more general phenomenon, we investigated whether a threshold is also seen in other cell-types used to study store-operated Ca2+ entry. In Jurkat-T lymphocytes, ICRAC is activated weakly by InsP3 in the presence of low concentrations of Ca2+ buffer, whereas the current is large when SERCA pumps are blocked simultaneously, as in RBL-1 cells. Although the electrophysiological properties of ICRAC in the Jurkat cell are very similar to those of RBL-1 cells, the Na+ conductance in the absence of external divalent cations is quite different. Unexpectedly, we failed consistently to record any store-operated Ca2+ current in macrovascular pulmonary artery endothelia whereas robust ICRAC was seen under the same conditions in RBL-1 cells. Our results show that ICRAC has a similar profile of activation in the presence of physiological levels of Ca2+ buffering for Jurkat T-lymphocytes and RBL-1 cells, indicating that the threshold mechanism may be a general feature of ICRAC activation. Because ICRAC in pulmonary artery endothelia is, at best, very small, additional Ca2+ influx pathways may also contribute to agonist-induced Ca2+ entry.

Topics: Animals; Calcium; Calcium Channels; Calcium-Transporting ATPases; Electric Conductivity; Endothelium, Vascular; Enzyme Inhibitors; Humans; Inositol 1,4,5-Trisphosphate; Jurkat Cells; Leukemia, Basophilic, Acute; Pulmonary Artery; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases; T-Lymphocytes; Temperature; Thapsigargin; Tumor Cells, Cultured

Using whole-cell current-clamp measurements we have found that thapsigargin-mediated activation of store-regulated Ca2+ entry in rat basophilic leukemia cells is accompanied by complex changes in membrane potential. These changes consisted of: (i) an initial slow, small depolarization, (ii) a transitional change in potential to a depolarized value and (iii) transitional changes between a hyperpolarized and a depolarized potential. These complex changes in potential can be explained by the interaction between the endogenous inwardly rectifying K+ conductance and the generation of a small inward current. To investigate the possible influence of these changes of potential on [Ca2+]i, single cell measurements of fura2 fluorescence were undertaken alone or in combination with current-clamp measurements. Thapsigargin-mediated activation of the store-regulated Ca2+ entry pathway was accompanied by a marked increase of [Ca2+]i. During this increase, transient, abrupt declines in [Ca2+]i were detected in approximately 60% of the cells investigated. These changes of [Ca2+]i are consistent with the observed changes of membrane potential recorded under current-clamp.

Topics: Animals; Calcium; Calcium Signaling; Electric Conductivity; Electrophysiology; Fluorescent Dyes; Fura-2; Leukemia, Basophilic, Acute; Membrane Potentials; Patch-Clamp Techniques; Potassium; Rats; Thapsigargin; Tumor Cells, Cultured

The influence of cooling on the intracellular concentration of Ca2+ ([Ca2+]i) was tested in cell lines expressing chemical receptors. First, when ATP was externally added to rat basophilic leukemia (RBL-2H3) cells, cooling from 37 degrees C to 27 degrees C induced a transient rapid increase in [Ca2+]i. In the absence of extracellular Ca2+, the [Ca2+]i response was induced whereas an inhibitor of microsomal Ca2+ ATPase, thapsigargin, largely abolished the [Ca2+]i response, suggesting that the internal Ca2+ store liberate the Ca2+. A purinergic receptor antagonist, suramin, completely inhibited the [Ca2+]i response to the cooling. Secondly, when serotonin (5-HT) was added to rat glioma C6BU-1 cells, the cooling induced a transient increase in [Ca2+]. This [Ca2+]i response was induced in the absence of external Ca2+, suggesting that the internal Ca2+ stores liberate the Ca2+. These results raise the possibility that some G protein-coupled receptors are sensitive to cooling in the presence of agonist for the receptor.

Topics: Adenosine Triphosphate; Aniline Compounds; Animals; Calcium; Calcium-Transporting ATPases; Cold Temperature; Fluorescent Dyes; Glioma; GTP-Binding Proteins; Kinetics; Leukemia, Basophilic, Acute; Microsomes; Rats; Receptors, Cell Surface; Serotonin; Signal Transduction; Suramin; Thapsigargin; Time Factors; Tumor Cells, Cultured; Xanthenes

The antiallergic drugs astemizole and norastemizole inhibit exocytosis in mast cells, which might be relevant for their therapeutic action. From previous studies, it appeared that the drugs inhibited 45Ca2+ influx. Here, we present a more detailed study on the effects of astemizole and norastemizole on Ca2+ fluxes. Fura-2-loaded rat basophilic leukemia (RBL-2H3) cells were activated through the high-affinity receptor for IgE (FcepsilonRI) with antigen or by the endoplasmatic reticulum ATPase inhibitor thapsigargin, bypassing direct FcepsilonRI-related events. It appeared that astemizole (>15 microM), in contrast to norastemizole, showed a dual effect on intracellular calcium concentration ([Ca2+]i): a rise in intracellular calcium concentration was induced, which originated in the release of intracellular Ca2+ stores, whereas Ca2+ influx via store-operated Ca2+ (SOC) channels was inhibited. Ca2+ influx was further characterized using Ba2+ influx, whereas processes in the absence of Ca2+ influx were studied using Ni2+ or EGTA. It was concluded that the drugs most likely affect the store-operated Ca2+ channels in RBL cells directly. The two effects of astemizole on Ca2+ fluxes had opposing influences on exocytosis, thereby accounting for the biphasic effect of increasing astemizole concentration on mediator release in RBL cells.

Topics: Animals; Anti-Allergic Agents; Astemizole; Barium; Calcium; Calcium Channels; Exocytosis; Leukemia, Basophilic, Acute; Nickel; Rats; Receptors, IgE; Thapsigargin; Tumor Cells, Cultured

The antiallergic drug oxatomide and analogs inhibit mediator release from a rat basophilic leukemia (RBL-2H3) cell line, which is frequently used as a mast cell model. By investigating a series of derivatives of oxatomide with different inhibiting activities on exocytosis, we aimed to evaluate the role of their effects on the early steps of the signal transduction cascade in the inhibition of exocytosis. The active compounds induced hyperphosphorylation of tyrosine residues both in stimulated as well as in resting cells. Furthermore, some elevation of the inositol 1,4,5-trisphosphate (IP3) formation upon antigen activation was observed for the active derivatives. Ca2+ fluxes were also studied. The inhibition of the antigen-induced 45Ca2+ influx correlated with the effects of the drugs on exocytosis. Furthermore, the inhibitory activity on antigen- and thapsigargin-mediated exocytosis correlated well. Adherence of the cells to fibronectin, stimulating cellular integrin receptors, was synergistic to antigen activation of the RBL cells. However, oxatomide did lack any effect on integrin-mediated processes, as the IC50 value for exocytosis was identical for fibronectin-adhered cells and standard cultured cells. We conclude that oxatomide and its analogs inhibit exocytosis, mainly by inhibiting Ca2+ influx over store-operated Ca2+ (SOC) channels. The drugs have a direct effect on the store-operated Ca2+ channels or affect the direct regulation of these channels.

Topics: Animals; Anti-Allergic Agents; Calcium; Calcium Radioisotopes; Cell Adhesion; Enzyme Inhibitors; Exocytosis; Extracellular Space; Fibronectins; Inositol 1,4,5-Trisphosphate; Leukemia, Basophilic, Acute; Neoplasm Proteins; Phosphorylation; Piperazines; Rats; Receptors, IgE; Signal Transduction; Thapsigargin; Tumor Cells, Cultured; Tyrosine

To investigate the effect of Ca2+-ATPase inhibitors on the production of TNF-alpha in rat basophilic leukemia (RBL-2H3) cells.. Two Ca2+-ATPase inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), and three hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ), 2,5-di-(tert/amyl)-1,4-hydroquinone (DTAHQ), 2-(tertbutyl)-1,4-hydroquinone (MTBHQ) were used.. Cells were treated with TG, CPA, DTBHQ, DTAHQ and MTBHQ for 3 h in the presence of 12-Otetradecanoylphorbol-13-acetate (TPA) and released TNF-alpha from the cells was measured (n > or = 4).. All Ca2--ATPase inhibitors (TG, CPA, DTBHQ and DTAHQ) induced TNF-alpha release in a dose-dependent manner. TNF-alpha release was inhibited by treatment with protein kinase C inhibitors (staurosporine, Ro31-8220, calophostin C) (p < or = 0.05). In contrast, MTBHQ, which does not induce increases in [Ca2+]i, did not induce the release of TNF-alpha. TNF-alpha release induced by DTBHQ and CPA was inhibited by treatment with actinomycin-D, the immunosuppressant FK506 and the glucocorticoid dexamethasone (p < or = 0.01).. These results suggest 1) that [Ca2+]i increase and subsequent activation of protein kinase C is necessary for the release of TNF-alpha, and they work synergistically, 2) that the TNF-alpha release induced by Ca2+-ATPase inhibitors can be regulated at the transcriptional level.

Topics: Animals; Calcium-Transporting ATPases; Dactinomycin; Dexamethasone; Enzyme Activation; Enzyme Inhibitors; Hydroquinones; Indoles; Leukemia, Basophilic, Acute; Protein Kinase C; Rats; Tacrolimus; Thapsigargin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Ca2+-dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 microM free Ca2+ and 300 microM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) resulted in prominent capacitance increases. However, dialysis with either Ca2+ (225 nM to 10 microM) or GTP[gamma-S] alone failed to induce a capacitance change. Under conditions of weak Ca2+ buffering (0.1 mM EGTA), activation of Ca2+-release-activated Ca2+ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP3) failed to induce a capacitance increase even in the presence of GTP[gamma-S]. However, when Ca2+ATPases were inhibited by thapsigargin, InsP3 and GTP[gamma-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca2+ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca2+ influx because it was effectively suppressed when external Ca2+ was removed. Our results demonstrate that ICRAC represents an important source of Ca2+ for triggering a secretory response.

Topics: Animals; Calcium; Calcium Channels; Calcium-Transporting ATPases; Chelating Agents; Dialysis; Egtazic Acid; Electric Conductivity; Enzyme Inhibitors; Guanosine 5'-O-(3-Thiotriphosphate); Inositol 1,4,5-Trisphosphate; Leukemia, Basophilic, Acute; Rats; Thapsigargin; Tumor Cells, Cultured

Patch-clamp experiments aimed at determining the relationship between intracellular Ca2+ release and activation of store-operated calcium current I(CRAC) reveal that both agonist and InsP3-mediated activation of I(CRAC) are highly nonlinear, occurring over a narrow concentration range. Ca2+ release and Ca2+ influx can be dissociated, as they possess differential sensitivities to InsP3: low concentrations induce substantial Ca2+ release without any activation of I(CRAC), whereas micromolar concentrations of InsP3 are required to activate Ca2+ influx. This suggests functionally distinct stores controlling Ca2+ release and influx and enables cells to switch between sources of Ca2+ to fit best their current needs.

Topics: Animals; Calcium; Calcium Channels; Enzyme Inhibitors; Inositol 1,4,5-Trisphosphate; Ion Channel Gating; Leukemia, Basophilic, Acute; Nonlinear Dynamics; Patch-Clamp Techniques; Rats; Thapsigargin; Tumor Cells, Cultured

Antigen stimulation of mast cells via the IgE receptor, Fc epsilon RI, results in recruitment of the cytosolic tyrosine kinases, Lyn and Syk, and the phosphorylation of proteins. We examined the effects of the glucocorticoid dexamethasone on these events in a cultured (RBL-2H3) mast cell line. Nanomolar concentrations of dexamethasone suppressed phosphorylation of proteins that were associated with the activation of the mitogen-activated protein (MAP) kinase/phospholipase A2 pathway without inhibiting initial events. For example, tyrosine phosphorylation of the subunits of Fc epsilon RI, Lyn, or Syk or of the Ras-guanine nucleotide exchange factor, Vav, was not suppressed in cells treated with up to 1 microM dexamethasone. In contrast, phosphorylation of Raf1, MEK1, p42mapk, and cytosolic phospholipase A2, as well as the associated increase in MAP kinase activity and release of arachidonic acid, were markedly inhibited in cells treated with as little as 10 nM dexamethasone--a concentration that only partially inhibited hydrolysis of inositol phospholipids or release of secretory granules. Prolonged exposure to dexamethasone also resulted in a partial decrease in expression of MEK1, p42mapk, and cytosolic phospholipase A2, which may contribute further to the effects of dexamethasone on this pathway. Activation of the MAP kinase/phospholipase A2 pathway by the calcium-mobilizing agent thapsigargin was similarly suppressed in dexamethasone-treated cells. These findings suggested that an early step in the pathway, possibly a step immediately before the activation of Raf1, was suppressed by low concentrations of dexamethasone.

Topics: Animals; Antigens; Arachidonic Acids; Calcium-Calmodulin-Dependent Protein Kinases; Dexamethasone; Dose-Response Relationship, Immunologic; Enzyme Activation; Immunosuppressive Agents; Leukemia, Basophilic, Acute; MAP Kinase Kinase 1; Mast Cells; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase Kinases; Phospholipases A; Phospholipases A2; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Rats; Receptors, IgE; src-Family Kinases; Thapsigargin; Tumor Cells, Cultured; Tyrosine

The antigen induced stimulation of mast cells by aggregation of Fc epsilon RI receptors activates a signal transduction cascade leading to release of mediators of inflammation like histamine, arachidonic acid metabolites and cytokines. In this study we investigated a series of structurally related anti-allergic drugs, containing a common lipophilic diphenylmethyl piperazinyl tail and head groups that differ in lipophilicity. Effects of these drugs on various steps of the signal transduction cascade was investigated to gain insight into the mechanism of action of these drugs. It appeared that addition of the drugs to resting cells induced changes in the tyrosine phosphorylation of cellular proteins. The most active anti-allergics in inhibiting exocytosis, AL3264 and oxatomide, also induced the largest changes in phosphorylation. The effects of the drugs on tyrosine phosphorylation after cell activation was complex. Additionally, Ca2+ fluxes were investigated. Ca2+ efflux from the cells was negligibly influenced by the active drugs. However, the drugs inhibited influx from extracellular Ca2+, which was correlated with the effects of the drugs on inhibition of exocytosis and on membrane stabilization induced by the drugs, measured as haemolysis of erythrocytes. It is concluded that inhibition of Ca2+ influx is the major mechanism with which these drugs inhibit exocytosis and that for this effect drug-membrane interactions, possibly affecting the function of membrane embedded proteins, are of importance. Possible mechanisms including drug-membrane interactions, phosphorylation and inhibition of Ca2+ influx are discussed.

Topics: Animals; Anti-Allergic Agents; Calcium; Calcium Channels; Erythrocyte Membrane; Exocytosis; Extracellular Space; Intracellular Fluid; Leukemia, Basophilic, Acute; Phosphorylation; Phosphotyrosine; Rats; Receptors, IgE; Signal Transduction; Thapsigargin; Tumor Cells, Cultured

The effects of three Ca(2+)-ATPase inhibitors, thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), on the Ca2+ response, degranulation, and leukotriene C4 (LTC4) release in RBL-2H3 cells were investigated. All three compounds elevated the intracellular free Ca2+ concentration ([Ca2+]i), and caused degranulation in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator. The dose-dependency of each compound in the Ca2+ response was in good agreement with that in degranulation. TG and CPA also caused the release of LTC4 in a dose-dependent manner, and this effect was unaffected by TPA or calphostin C, a selective PKC inhibitor. DTBHQ, however, did not induce LTC4 release, and rather inhibited the antigen-induced release of LTC4. These results suggest [1] that both degranulation and LTC4 release caused by these compounds are dependent on their [Ca2+]i increasing effect, [2] that degranulation and LTC4 release are mediated via independent pathways following the Ca2+ response, and [3] that DTBHQ additionally prevents the synthesis of LTC4 possibly by inhibition of 5-lipoxygenase.

Topics: Animals; Basophils; Calcium; Calcium-Transporting ATPases; Cell Degranulation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydroquinones; Indoles; Leukemia, Basophilic, Acute; Leukotriene C4; Lipoxygenase Inhibitors; Mice; Naphthalenes; Spectrometry, Fluorescence; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

Antigenic stimulation of rat basophilic leukemia cells releases Ca2+ from internal stores and increases membrane permeability to Ca2+. The delta isomer of hexachlorocyclohexane (delta-HCH) is structurally similar to myo-inositol-1,4,5-trisphosphate (IP3) and is a potent releaser of stored Ca2+ from permeabilized cells. This release of Ca2+ is not mediated by a competitive interaction with the IP3 receptor on the Ca2+ release channel on the endoplasmic reticulum. In intact cells, delta-HCH and, to a lesser extent, lindane (gamma-hexachlorocyclohexane) transiently increase the intracellular Ca2+ concentration. The return to basal concentrations is mediated by the plasma membrane Ca2+ pumps and not by resequestration of Ca2+ into intracellular stores. Treatment of cells with delta-HCH (25-100 microM), but not lindane, leads to a progressive inhibition of the antigen- and thapsigargin-stimulated Ca2+ signal. Caffeine, a modulator of the ryanodine receptor Ca2+ channel, attenuates the rise in intracellular Ca2+ induced by delta-HCH, suggesting that ryanodine receptor-like Ca2+ channels may be present in RBL cells. At 25 microM delta-HCH, a concentration that does not inhibit the antigen-stimulated Ca2+ signal, the release of [3H]serotonin from antigen-stimulated cells is enhanced as is secretion of [3H]serotonin from cells pretreated with 25-100 microM lindane. The depletion of Ca2+ from intracellular stores by delta-HCH should evoke Ca2+ entry into the cells by a capacitative mechanism; however; divalent cation permeability across the plasma membrane (Mn2+ influx) is not increased but rather is decreased by delta-HCH. An understanding of the mechanism of action of delta-HCH in releasing stored Ca2+ and blocking Ca2+ influx across the plasma membrane may provide insights into the regulation of capacitative Ca2+ entry in nonexcitable cells.

Topics: Adenosine Triphosphate; Animals; Antigens; Binding, Competitive; Caffeine; Calcium; Calcium-Transporting ATPases; Cell Membrane Permeability; Endoplasmic Reticulum; Enzyme Inhibitors; Hexachlorocyclohexane; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Isomerism; Leukemia, Basophilic, Acute; Manganese; Rats; Sensitivity and Specificity; Signal Transduction; Stimulation, Chemical; Terpenes; Thapsigargin; Tritium; Tumor Cells, Cultured

Effects of cycloheximide, an inhibitor of protein synthesis, on histamine release from RBL-2H3 cells were examined. RBL-2H3 cells sensitized by rat antiserum to ascaris extract were challenged by the antigen, and histamine release during a period of 30 min was measured. Pretreatment with cycloheximide (1 microgram/ml) for 1 h significantly inhibited the antigen-induced histamine release (36% inhibition). The cycloheximide-induced inhibition of histamine release was abolished when the cells were further incubated in the absence of cycloheximide for 2 h. Pretreatment with puromycin (3 and 10 micrograms/ml), an inhibitor of protein synthesis, or actinomycin D (0.1-1 microgram/ml), an inhibitor of DNA-dependent RNA synthesis, also inhibited the antigen-induced histamine release in a concentration-dependent manner. Both ionomycin- and thapsigargin-induced histamine release were also inhibited by pretreatment with cycloheximide. Measurement of intracellular Ca2+ levels using quin 2 revealed that cycloheximide inhibits the increase in Ca2+ levels induced by the antigen, ionomycin or thapsigargin. These results suggest that histamine release induced by the antigen, ionomycin and thapsigargin in RBL-2H3 cells is mediated by protein(s) which is newly synthesized and inactivated rapidly, and the newly synthesized protein(s) is involved in the increase of intracellular Ca2+ levels induced by these stimulants.

Topics: Animals; Antigens; Calcium; Carcinogens; Culture Media, Conditioned; Cycloheximide; Dactinomycin; Histamine Release; Ionomycin; Leukemia, Basophilic, Acute; Protein Biosynthesis; Proteins; Puromycin; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

The anti-inflammatory agent tenidap has previously been shown to inhibit antigen-induced secretion in tumor mast cells. We have investigated the possibility that this effect is due to modulation of the Ca2+ response in mast cells and in particular that tenidap might be an inhibitor of the Ca2+ influx pathway or channel in these and other non-excitable cells. Tenidap inhibited the antigen-induced increase in intracellular Ca2+ measured both in cell suspensions and at the single cell level using digital imaging of Fura-2 fluorescence. Tenidap also inhibited both antigen- and thapsigargin-induced 45Ca influx across the plasma membrane at concentrations similar to those required for the inhibition of secretion. Somewhat unexpectedly, the compound itself caused some release of calcium from intracellular stores; however, this effect did not appear to be related to the inhibition of calcium influx or secretion. In mouse pituitary tumour (AtT-20) cells, tenidap inhibited depolarization-induced increases in intracellular Ca2+ suggesting that this compound also inhibits Ca2+ influx through voltage-sensitive calcium channels. We conclude that tenidap has a number of interesting effects on calcium handling which makes it a potentially valuable tool for the study of calcium movements particularly in non-excitable cells.

Topics: Animals; Antigens; Calcium; Calcium Channels; Indoles; Inositol Phosphates; Leukemia, Basophilic, Acute; Mast Cells; Mice; Oxindoles; Phosphatidylinositols; Pituitary Neoplasms; Rats; Signal Transduction; Terpenes; Thapsigargin; Tumor Cells, Cultured

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arachidonate 5-Lipoxygenase; Basophils; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Compartmentation; Cell Membrane; Egtazic Acid; In Vitro Techniques; Inositol Phosphates; Ionomycin; Leukemia, Basophilic, Acute; Leukotrienes; Rats; Receptors, Immunologic; Receptors, Leukotriene; SRS-A; Terpenes; Thapsigargin; Tumor Cells, Cultured

In RBL-2H3 rat basophilic leukemia cells, cholera toxin does not per se stimulate secretion but it enhances secretion stimulated by antigens that crosslink IgE receptors, by the Ca2+ ionophore, ionomycin, and by thapsigargin, a tumor promoter that releases cytoplasmic Ca2+ stores. Calmodulin inhibitors reduce both the basal and cholera toxin-enhanced secretory responses to antigen and Ca2(+)-mobilizing agents. These synergistic effects suggest that the activation of a Gs-like GTP-binding protein, together with a (probably calmodulin-dependent) event activated by an increase in cytoplasmic Ca2+ levels, may jointly provide a sufficient signal for secretion. Antigen-stimulated secretion is inhibited by depleting cells of GTP with mycophenolic acid but is maximal in cells treated with mycophenolic acid plus cholera toxin. The simplest explanation is that cholera toxin selectively reactivates the Gs-coupled pathway leading to secretion in GTP-depleted cells without restoring the activity of a separate GTP-binding protein(s) that constrains antigen-stimulated secretion.

Topics: Animals; Antigens, Differentiation, B-Lymphocyte; Calcium; Calmodulin; Cell Line; Cholera Toxin; GTP-Binding Proteins; Immunoglobulin E; Kinetics; Leukemia, Basophilic, Acute; Leukemia, Experimental; Mast Cells; Mycophenolic Acid; Rats; Receptors, Fc; Receptors, IgE; Serotonin; Sulfonamides; Terpenes; Thapsigargin; Trifluoperazine