Rat Basophilic Leukemia 2H3 Cells Research Articles

The secretion-coupled endocytosis correlates with membrane tension changes in RBL 2H3 cells.

Stimulated secretion in endocrine cells and neuronal synapses causes a rise in endocytosis rates to recover the added membrane. The endocytic process involves the mechanical deformation of the membrane to produce an invagination. Studies of osmotic swelling effects on endocytosis indicate that the increased surface tension is tightly correlated to a significant decrease of endocytosis. When rat basophilic leukemia (RBL) cells are stimulated to secrete, there is a dramatic drop in the membrane tension and only small changes in membrane bending stiffness. Neither the shape change that normally accompanies secretion nor the binding of ligand without secretion causes a drop in tension. Further, tension decreases within 6 s, preceding shape change and measurable changes in endocytosis. After secretion stops, tension recovers. On the basis of these results we suggest that the physical parameter of membrane tension is a major regulator of endocytic rate in RBL cells. Low tensions would stimulate endocytosis and high tensions would stall the endocytic machinery.

The Chinese herbal medicine, Shinpi-To, inhibits IgE-mediated leukotriene synthesis in rat basophilic leukemia-2H3 cells

We examined the action of Shinpi-To (Formula divinita; TJ-85), a granular extract of seven Chinese medicinal herbs that is used in treating childhood asthma, on the leukotriene synthesis in rat basophilic leukemia-2H3 cells (RBL-2H3 cells). IgE-loaded cells were stimulated with anti-IgE serum in the presence or absence of Shinpi-To. Released LTC 4 and LTB 4 were measured by radioimmunoassay (RIA). Shinpi-To significantly inhibited IgE-mediated synthesis of leukotriene (LT)C 4 and LTB 4. To identify the inhibitory sites, we investigated the action of this extract on four synthetic enzymes, phospholipase A 2 (PLA 2), 5-lipoxygenase (5-LO), LTC 4 synthase, and LTA 4 hydrolase. Shinpi-To inhibited the A23187-stimulated release of [ 3H]arachidonic acid (AA) from the cell membrane, reflecting an effect on PLA 2 activity. It also suppressed production of LTC 4 and LTB 4 when cell lysates were incubated with AA as substrate. It did not inhibit the production of LTC 4 and LTB 4 when LTA 4-free acid was used as the substrate. Shinpi-To did not inhibit the IgE-mediated increase of intracellular Ca 2+ ([Ca 2+] i) concentration. Results indicate that Shinpi-To inhibits LT synthesis by inhibiting PLA 2 and 5-LO activities without affecting the mobilization of [Ca 2+] i.

Anti-ganglioside monoclonal antibody AA4 selectively inhibits IgE-induced signal transduction pathways in rat basophilic leukemia cells

In rat basophilic leukemia 2H3 (RBL-2H3) cells, mAb AA4 binds to two derivatives of ganglioside GD 1b that associate with the Src family kinase p53 56 lyn and a serine kinase. Pre-incubation of cells with mAb AA4 blocks immunoglobulin E (IgE) mediated histamine release. In the present study we investigated the effect of incubation with mAb AA4 on signal transduction events. In addition to stimulation of the high affinity IgE receptor (FcεRI), cells were also activated by the calcium ionophore A23187 and the acetylcholine agonist carbachol in RBL-2H3 cells transfected with the G protein-coupled m3 muscarinic receptor. Incubation of cells with mAb AA4 in a dose-dependent manner inhibited the following FcεRI-induced signal transduction events: the increase of intracellular free calcium, phosphoinositol breakdown, tyrosine phosphorylation of proteins including the β- of FcεRI and secretion. However, there was no inhibition of degranulation or of these biochemical events when cells were stimulated with calcium ionophore or activated via a G protein-coupled pathway. Our results demonstrate that mAb AA4 selectively blocks FcεRI-induced cell activation at a very early step upstream of receptor tyrosine phosphorylation. As mAb AA4 has previously been found to bind to gangliosides associated with FcεRI, inhibition of very early biochemical events may be due to interaction of mAb AA4 with the FcεRI induced signal transduction cascade at the receptor level.

Aggregation of IgE Receptors in Rat Basophilic Leukemia 2H3 Cells Induces Tyrosine Phosphorylation of the Cytosolic Protein-tyrosine Phosphatase HePTP

The cDNA encoding the rat equivalent of the human hematopoietic tyrosine phosphatase, also known as leukocyte phosphatase, was isolated from a rat basophilic leukemia mast cell cDNA library. By two-dimensional electrophoresis, the protein expressed in the mast cells was of a size (40 kDa) and pI (6.9) predicted from the deduced amino acid sequence. Thus, although previously shown to be preferentially expressed in T cells and B cells, the phosphatase is also found in mast cells. By immunofluorescence microscopy, rat hematopoietic tyrosine phosphatase localized to discrete, globular compartments within the cytoplasm and was not found either in the nucleus or associated with the cell surface membrane. Aggregation of high affinity IgE receptors in the mast cells induced tyrosine phosphorylation of the phosphatase. The tyrosine phosphorylation was mimicked by stimulation with calcium ionophore A23187 but not by direct activation of protein kinase C. Since phosphorylation of the phosphatase was dramatically reduced when the cells were activated in Ca(2+)-free media, it is dependent on a rise in intracellular Ca2+. These data strongly suggest that hematopoietic tyrosine phosphatase may be involved in the IgE receptor-mediated signaling cascade.

Desensitization of the m3-muscarinic acetylcholine-receptor in the smooth muscle and the intracellular signal transduction

Desensitization of the m3-muscarinic acetylcholine-receptor in the smooth muscle of the digestive tract is discussed together with the changes in intracellular signal transduction. Isolated single cells that show an all-or-none contractile response to acetylcholine were desensitized by treatment with 0.1 mM acetylcholine for 10 min, resulting in an increase in the threshold concentration of acetylcholine for contraction, but without changing any of the binding characteristics. Permeabilized cells showed that the desensitization is via uncoupling between the receptor and G-protein. Secretory cells (rat basophil leukemia-2H3 cells) transfected with human m3-receptor showed desensitization when treated with 0.1 mM carbachol for 30 min. The coupling between the receptor and G-protein was not impaired, but some unknown Ca(2+)-independent mechanism may be involved. Smooth muscle tissue was tested for its time-course of desensitization, and a novel transient resensitization was found at 1 min of incubation with 0.1 mM carbachol. This resensitization, and the desensitization prior to it, were accompanied with changes in binding affinity. However, the affinity was not changed, in parallel with desensitization afterwards, but the positive feedback loop of Ca(2+)-influx caused by alkalization via receptor-stimulation was suppressed. After a 30-min treatment, a Ca(2+)-independent mechanism caused the uncoupling and affinity decrease. Treatment for 3 hr increased the number of binding sites without recovery of the response. The desensitizing process is very diverse to achieve selectivity, but its purpose is in unity.

Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells.

The surface engagement of high affinity immunoglobulin E receptor (Fc epsilon RI) of rat basophilic leukemia 2H3 (RBL-2H3) cells induced histamine secretion and leukotriene release following activation of the tyrosine kinase Lyn together with phosphatidylinositol 3-kinase (PI3-kinase). Wortmannin inhibited the activity of partially purified PI3-kinase from calf thymus, as well as the PI3-kinase activity in anti-PI3-kinase p85 immunoprecipitates from RBL-2H3 cells, at a concentration as low as 1.0 nM and with IC50 values of 3.0 nM, but did not inhibit PI4-kinase activity. The inhibition of PI3-kinase by wortmannin was irreversible. Wortmannin inhibited both Fc epsilon RI-mediated histamine secretion and leukotriene release up to 80% with IC50 values of 2.0 and 3.0 nM, respectively. Wortmannin inhibited PI3-kinase activity in intact cells up to 80% with an IC50 value of 2.0 nM, which is almost equal to those for PI3-kinase in vitro and for histamine secretion and leukotriene release. With anti-wortmannin antibody, we have shown that wortmannin binds to the 110-kDa protein, but not to PI3-kinase 85-kDa regulatory subunit both in vitro and in whole cells. Furthermore, there was a positive correlation between the potencies of wortmannin derivatives as inhibitors of PI3-kinase and as inhibitors of histamine secretion. Wortmannin had no effect on the activation of the tyrosine kinase Lyn. These results suggest that PI3-kinase is involved in the signal transduction pathway responsible for histamine secretion following stimulation of Fc epsilon RI and that wortmannin blocks these responses through direct interaction with the catalytic subunit of this enzyme.

Measurement of murine ovalbumin-specific IgE by a rat basophil leukemia cell serotonin release assay: Comparison to the rat passive cutaneous anaphylaxis assay

A modified in vitro method to measure murine ovalbumin-specific IgE antibody as an alternative to the in vivo rat passive cutaneous anaphylaxis assay is presented in this report. This assay uses rat basophil leukemia (RBL) 2H3 cells that have been loaded with [ 3H]serotonin. Exposure of RBL-2H3 cells to mouse antisera and subsequent antigen challenge results in the release of [ 3H]serotonin. Using a monoclonal mouse anti-DNP IgE antibody and DNP-human serum albumin as the antigen, various steps of this assay were optimized to decrease the amount of time and reagents needed for the assay. The percent [ 3H]serotonin released was used to calculate antibody titer of sera from ovalbumin-immunized mice. Mouse anti-ovalbumin IgE titers determined by the RBL release method and passive cutaneous anaphylaxis assay were found to correlate very well over a wide range of antibody titers (correlation coefficient, r 2 = 0.94).

O-339 - Imidazole derivatives, econazole and SK&F 96365 inhibit Ca entry in rat basophilic leukemia(2H3) cells.

O-339 - Imidazole derivatives, econazole and SK&F 96365 inhibit Ca entry in rat basophilic leukemia(2H3) cells.

O-457 - Inhibitory effect of SK&F 96365 on Ca entry and histamine secretion in rat basophilic leukemia (2H3) cells

O-457 - Inhibitory effect of SK&F 96365 on Ca entry and histamine secretion in rat basophilic leukemia (2H3) cells

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

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

Characterization of a new type of variant of rat basophilic leukemia 2H3 cells presenting a different pattern of calcium signal

We selected a new type of variant (designated 3C7) derived spontaneously from parental RBL-2H3 cells. 3C7 cells showed lower contact inhibition, anchorage dependency, and serotonin release activity than those of RBL-2H3 cells. We conclude that 3C7 cells are a transformant of RBL-2H3 cell with greater malignancy. The production of inositol bisphosphate and the release of Ca 2+ from intracellular stores induced by IgE-antigen stimulation were enhanced in 3C7. Oscillation of [Ca 2+] i in individual 3C7 cells was observed by a digital imaging microscopic technique. We propose that 3C7 cells are a useful model system for studies on the mechanisms of stimulus-secretion coupling and the relationships between malignant alterations and disorders of signal transduction.

Release and Synthesis of Histamine in Rat Basophilic Leukemia (2H3) Cells

On the stimulation of rat basophilic leukemia (2H3) cells with an IgE oligomer, the intracellular Ca2+ concentration, which was measured with fura-2 as a fluorescent probe, began to increase after 1 min lag period, reached its maximum at 4 min, and gradually decreased to the original level. The histamine release occurred slightly more slowly than the increase in the intracellular Ca2+. It started 2-3 min after the stimulation, reaching a peak at 6 min and thereafter decreasing slowly. To supplement the released histamine, the activity of histidine decarboxylase, a histamine-forming enzyme, increased 5-fold several hours after the stimulation by an IgE oligomer, and this increase may be mediated by protein kinase C system.

Cardiotoxin from cobra venom increases the level of phosphatidylinositol 4-monophosphate and phosphatidylinositol kinase activity in two cell lines

In rat basophilic leukemia-2H3 (RBL-2H3) and Madin-Darby canine kidney (MDCK) cells, cardiotoxin from cobra venom induced a marked decrease in the level of [ 3H]phosphatidylinositol and a corresponding increase in the level of [ 3H]phosphatidylinositol 4-monophosphate over the course of 20 min as demonstrated in cells that had been labeled to equilibrium with [ 3H]inositol. The effect was dependent on the concentration (5–30 μg/ml) of the toxin. In plasma membrane-enriched fractions isolated from the two cell lines, the cardiotoxin enhanced the endogenous activity of phosphatidylinositol kinase especially at temperatures above 14°C. In RBL-2H3 cells, cardiotoxin also induced release of substantial amounts of histamine and lactate dehydrogenase. The release of histamine, but not of lactate dehydrogenase, was totally dependent on external calcium and this release probably represented an exocytotic response of the cells to cardiotoxin. Although, initially, treatment with the toxin did not impair antigen-induced hydrolysis of inositol phospholipids or prevent the antigen-induced rise in the concentration of cytosol Ca 2+, prolonged exposure to the toxin did result in a progressive loss of responsiveness of RBL-2H3 cells to antigen.

Induction of histidine decarboxylase of rat basophilic leukemia (2H3) cells stimulated by higher oligomeric IgE or phorbol myristate acetate

When rat basophilic leukemia (2H3) cells were stimulated by higher oligomer, the chemically cross-linked oligomers of IgE, in the presence of calcium the activity of histidine decarboxylase (HDC, L-histidine carboxylase, E.C.4.1.1.22), a histamine-forming enzyme, was increased by 1 hr, reaching maximum activity by 2 hr, and returning to the original level by 8 hr. A similar increase in enzyme activity was observed in cells treated with phorbol myristate acetate (PMA) or oleoyl-acetylglycerol (OAG), which are known activators of protein kinase C. Removal of calcium from medium abolished the increase in HDC activity in response to higher oligomer but not that induced by PMA or OAG, suggesting that the increase in HDC activity may be mediated by protein kinase C. The increase in the HDC activity probably required induction of enzyme synthesis, because it was prevented by cycloheximide.

Loss of secretory response of rat basophilic leukemia (2H3) cells at 40 degrees C is associated with reversible suppression of inositol phospholipid breakdown and calcium signals.

Antigen-induced stimulatory signals as well as histamine secretion from the RBL-2H3 cells were found to be highly temperature dependent. There was no hydrolysis of inositol phospholipids, increase in cytosol calcium concentration (calcium signal), or secretion upon antigen stimulation at temperatures below 20 degrees C. At higher temperatures (i.e., 20 to 37 degrees C), all responses increased in extent with increase in temperature. Temperatures of 38 degrees C or higher, however, resulted in a marked decline in all responses, until no responses were observed at 40 to 42 degrees C. As indicated by the decay in calcium signal, the duration of response was also temperature dependent. The response was of long duration at 30 to 32 degrees C, but it became progressively more transient as the temperature was increased from 32 to 40 degrees C. The effects of low or high temperature were fully reversible. For example, in the presence of antigen, stimulatory signals immediately appeared once the temperature was decreased from 40 to 37 degrees C. Although the diminished responses could be explained, in part, by a reduction in rates of IgE receptor aggregation and phospholipase C activity, the reductions were insufficient to account for complete loss of activity at 40 degrees C. We conclude that generation of intracellular signals in 2H3 cells is blocked by quite small elevations in temperature above 37 degrees C, possibly as consequence of changes in membrane fluidity.

Quantitative relationships between aggregation of IgE receptors, generation of intracellular signals, and histamine secretion in rat basophilic leukemia (2H3) cells. Enhanced responses with heavy water.

RBL-2H3 cells (a tumor analog of rat mast cells) have plasma-membrane receptors that bind immunoglobulin E, which when aggregated, initiate degranulation. As in other systems, secretion is preceeded by enhanced hydrolysis of inositol phospholipids and by a rise in intracellular Ca2+. Unlike the responses of many other cells, however, both of these earlier events require extracellular Ca2+. The relationship of these events to each other and to the subsequent secretory process is thus unclear. By exposing cells to covalent oligomers of IgE one can demonstrate substantial increases in secretion of histamine by increasing the concentration and size of the oligomers or by using heavy water (D2O) in the medium. We have used such maneuvers to examine the quantitative relationships between aggregation of the receptors and the breakdown of inositol phospholipids, the increase in cytosolic Ca2+ and secretion. Our principal findings were: all treatments that increased secretion, correspondingly increased the changes that precede degranulation. These early events correlated with the degree of aggregation of the receptors even when the stimulatory conditions resulted in maximal secretion. Although the results were insufficient to prove that the hydrolysis of inositol phospholipids is required for the rise in cytosolic Ca2+, the studies with D2O and other observations supported this view. Since a plasma-membrane ion channel for Ca2+ has been implicated in the IgE-mediated rise in cytosolic Ca2+ in RBL 2H3 cells, this in turn suggests a heretofore undescribed role for hydrolysis of inositol phospholipids.

Effect of bleaching light on measurements of lateral diffusion in cell membranes by the fluorescence photobleaching recovery method.

The Fc receptors for IgE on rat basophil leukemia 2H3 cells were labeled with either rhodamine- or fluorescein-conjugated IgE and then with Fab fragments of anti-IgE conjugated with the other dye. Fluorescence photobleaching recovery measurements of lateral diffusion were performed with one of the dyes before and after extensive bleaching of the other dye over an entire cell. Bleaching one dye did not affect subsequent measurements made with the other dye. We thus detect no evidence for photoinduced artifacts in fluorescence photobleaching recovery experiments.