IgE receptor-activated calcium permeability pathway in rat basophilic leukemia cells: measurement of the unidirectional influx of calcium using quin2-buffered cells

The intracellular calcium indicator and buffer quin2 has been used to generate a large calcium buffering capacity in the cytoplasm of rat basophilic leukemia cells. Above 3 mM intracellular quin2, there is no further increase in the initial rate of antigen-induced 45Ca uptake, suggesting that 45Ca buffering by quin2 is now sufficient to prevent the immediate efflux of 45Ca from the cells. Thus, the initial rate of 45Ca uptake should reflect the true unidirectional influx of calcium that occurs when immunoglobulin E (IgE) receptors are aggregated by antigen. The antigen-induced calcium permeability pathway appears to be saturable, with a Km of about 0.7 mM and a Vmax of 0.9 nmol of calcium (10(6) cells)-1 min-1. Although net 45Ca uptake reaches a plateau a few minutes after antigen stimulation, the increase in plasma membrane permeability is maintained for at least an hour, provided that receptors for IgE remain aggregated. The initial rate of 45Ca influx correlates well with the subsequent secretion of [3H]serotonin in response to different concentrations of antigen. Both 45Ca uptake and [3H]serotonin secretion are maximal when only 10% of the receptors are occupied with antigen-specific IgE. Thus, 45Ca influx correlates more closely with secretion than with the number of IgE receptors aggregated by antigen.     

Microfilaments regulate the rate of exocytosis in rat basophilic leukemia cells

Disruption of microfilaments in rat basophilic leukemia (RBL) cells by exposure to cytochalasin B is observed to potentiate the rate of antigen-stimulated secretion from these cells. Under these conditions, cytochalasin B is without effect on the antigen-stimulated production of inositol phosphates or 45Ca2(+)-influx. In streptolysin-O-permeabilized RBL cells, cytochalasin B is observed to potentiate the rate of secretion in response both to guanosine 5'-(2-thio)-O-triphosphate (GTP gamma S) and to Ca2+ (buffered between 0.1 and 10 microM). However, under these conditions, cytochalasin B does not affect to antigen-stimulated production of inositol phosphates. Consistent with these data, microfilaments are proposed to regulate a terminal step in exocytosis, in a physiologically relevant manner.     

Activation of rat basophilic leukemia cells. Temporal identification of the signal calcium influx mediated by the receptor-operated channel pathway

Antigen-induced 45calcium influx into rat basophilic leukemia (RBL) cells was examined with emphasis on the early time domain under conditions that exclude loss of the cation during the subsequent washing step. Such preparations demonstrate a distinct, temporally separate influx peaking at 3 min, followed by a substantial efflux. This internalized 45Ca2+ approaches millimolar total intracellular concentration and is therefore either sequestered or becomes bound to intracellular components (proteins). The amplitude of this influx is linearly proportional to IgE-receptor occupancy at low receptor occupancies, and is sensitive to the anti-allergic drug cromolyn. Furthermore, the timing of both the maximal uptake and the maximal susceptibility to cromolyn correlates with the Quin-2 signal in these cells, and the initial degranulation pattern bears some resemblance to trends in the 45Ca2+ uptake curve. These qualities suggest that the early peak at 2-3 min, rather than any later 45Ca2+ uptake, comprises the initial signalling Ca2+ pool. Maximal apparent inhibition by cromolyn for 45Ca2+ uptake was about 65% and required a 10-15-min preincubation with the cells. The inhibitory effect was limited to the peak at 3 min, suggesting that tracer incorporation beyond 5-6 min largely involves other pools or pathways, triggered by receptor aggregation, yet only indirectly related to channel activity or to the signal proper. A quantitative similarity was found between the early peak measured on intact cells and the single channel conductance measured on reconstituted planar bilayers containing the purified receptor for IgE and the purified cromolyn-binding protein [Corcia, A. et al. (1986) EM BO J. 5, 849-854]. This, as well as the effects of cromolyn, support the assumption that cromolyn-binding protein is a major constituent involved in this early influx, or that influx is a principal pathway for the signaling calcium mass.     

Depolarization of rat basophilic leukemia cells inhibits calcium uptake and exocytosis

We have investigated the unusual observation that depolarization of rat basophilic leukemia cells in high potassium not only fails to induce secretion, but also inhibits the secretion induced when receptors for IgE are aggregated by antigen. Antigen-stimulated 45Ca uptake and the rise in cytoplasmic free ionized calcium measured with the fluorescent indicator quin2 were both inhibited in depolarized cells. 45Ca efflux, on the other hand, was unaffected, which confirms that IgE receptor activation was not impaired in high potassium. Unlike the large increase in total cell calcium seen when cells in normal saline solution were stimulated with antigen, there was a decrease in total cell calcium when depolarized cells were stimulated. This is consistent with our finding that 45Ca uptake was inhibited while 45Ca efflux was unaffected. Inhibition of 45Ca uptake and secretion closely paralleled the decrease in membrane potential, and could be overcome by increasing the extracellular calcium concentration. We conclude that changes in the electrochemical gradient for calcium are important in determining calcium influx and the magnitude of antigen-stimulated secretion from rat basophilic leukemia cells, while the release of calcium from intracellular stores is unaffected.     

Endogenous somatostatin-like peptides of rat basophilic leukemia cells

The tetradecapeptide somatostatin (SOM 14) and a 28-amino acid biosynthetic precursor (SOM 28) are constituents of diverse neuroendocrine tissues that are released by noxious stimuli from a subset of sensory nerve endings, and substantially modify the functions of basophils and mast cells. SOM-like factors were detected initially in the fluid phase of suspensions of immunologically challenged rat basophilic leukemia cells (RBL), and were purified from ethanol/0.2 M acetic acid (3/1, v/v) extracts of replicate portions of 3 X 10(9) RBL. Sephadex G-25 columns resolved factors of over 10,000, 2000 to 4000, and 300 to 1200 daltons that are antigenically related to SOM 14, as assessed by a radioimmunoassay specific for SOM 14. Only the two larger factors were detected by a radioimmunoassay for SOM 28(1-14), which binds to prepro-SOM and SOM 28 but not SOM 14. Reverse-phase high performance liquid chromatography distinguished the two smaller SOM peptides of RBL from SOM 28 and SOM 14, respectively. Amino acid analyses showed major differences in composition between the 2000 to 4000 dalton SOM of RBL and SOM 28. Picomolar to nanomolar concentrations of both of the smaller SOM peptides of RBL inhibited the IgE-dependent release of histamine from basophils to the same extent as SOM 14. The finding of 3 to 5 ng of structurally unique SOM-like peptides per 10(8) RBL suggests that endogenous mechanisms analogous to those of specialized sensory neurons may regulate the expression of hypersensitivity.     

Surface functions during mitosis in rat basophilic leukemia cells

At the entry into mitosis, cells abruptly lose membrane activities such as phagocytosis, pinocytosis, and capping. The present studies test if mitotic cells also resist functional responses to cell surface ligand-receptor interactions. The IgE receptors of RBL-2H3 rat basophilic leukemia cells were labeled with anti-dinitrophenol IgE (anti-DNP-IgE) and then cross-linked with multivalent ligands (DNP-bovine serum albumin [BSA]; DNP-B-phycoerythrin; DNP-BSA-gold). IgE-receptor cross-linking modulates cell surface organization and function and releases serotonin and other mediators of allergic and asthmatic reactions from interphase cells (Pfeiffer, J. R., JC. Seagrave, B. H. Davis, G. G. Deanin, and J. M. Oliver, 1985, J. Cell Biol., 101:2145-2155). It was found that anti-DNP-IgE-receptor complexes are preserved on the cell surface throughout mitosis; they continue to bind DNP-proteins, and the resulting antigen-IgE-receptor complexes can redistribute to coated pits on the cell surface. Furthermore, there is no loss of [3H]serotonin through mitosis. Nevertheless, antigen-stimulated [3H]-serotonin release is strongly impaired in mitotic-enriched as compared with mixed interphase or G1-enriched cell populations. In addition, antigen binding transforms the surface of interphase cells from a microvillous to a plicated topography and stimulates the uptake of fluorescein isothiocyanate-conjugated dextran by fluid pinocytosis. Mitotic cells maintain a microvillous surface topography after antigen treatment, and fluid pinocytosis virtually ceases from prometaphase to telophase. Phorbol myristate acetate, a tumor promoter that activates protein kinase C, restores surface ruffling activity to mitotic cells. Thus, the mitosis-specific freezing of membrane and secretory responses is most likely due to the failure of transmembrane signaling.     

Lanthanum increases the rat thymocyte cytoplasmic free calcium concentration by enhancing calcium influx

In the present study, I have examined the effect of lanthanum (La3+) on cytoplasmic free calcium concentration in isolated rat thymocytes employing the quin2 technique. As with its effect on 15Ca accumulation in rat thymocytes (Segal, J. and Ingbar, S.H. (1984) Endocrinology, 115, 160-166), La3+ produced a concentration-related increase in thymocyte cytoplasmic free calcium concentration. This effect of La3+ was very prompt in onset, evident within about 30 s from the time of addition of La3+. The lowest effective concentration of La3+ was 6 microM (+22.7% above control), and it increased progressively to reach maximal values at 25 microM (+100% above control). La3+ added to quin2-loaded thymocytes suspended in a calcium-free medium was without effect. In addition, La3+ had no significant effect on 45Ca efflux, and La3+ did not inhibit calcium-ATPase activity in the rat thymocytes. These results demonstrate that in rat thymocytes La3+ increases cytoplasmic free calcium concentration by increasing the extracellular calcium influx into the cell rather than the release of calcium from an intracellular pool.     

Effect of cholera enterotoxin on calcium uptake and cyclic AMP accumulation in rat basophilic leukemia cells

Cholera enterotoxin (CT), at an optimal concentration of 2.38 X 10(-10) M, stimulated calcium uptake (P less than 0.01) and cyclic AMP accumulation (P less than 0.02) in cultured rat basophilic leukemia cells. No significant effect of CT on calcium release or cyclic GMP accumulation was detected. Pharmacologic and chemical agents which block calcium uptake or prostaglandin synthesis antagonized the effect of CT.     

Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells

Ion channels, activated upon IgE-Fc epsilon receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc epsilon receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc epsilon receptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 microM Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 +/- 11 mV in the presence of sodium, 125 +/- 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of calcium from intracellular stores in rat basophilic leukemia cells monitored with the fluorescent probe chlortetracycline.

Release of calcium from intracellular stores of rat basophilic leukemia cells was monitored using the fluorescent probe chlortetracycline. The ability of chlortetracycline to indicate release from intracellular calcium stores was initially validated. The decrease of chlortetracycline fluorescence upon antigen-stimulation was not the result of secretion of granule-associated dye or of changes in the properties of the membranes. The chlortetracycline fluorescence signal was not influenced by Ca2+ influx across the plasma membrane. Results obtained from these chlortetracycline fluorescence measurements corresponded well with 45Ca efflux data, an indirect measurement of release of calcium from stores. Chlortetracycline was used to examine the rate of antigen-induced release of calcium from stores, the depletion of intracellular calcium stores by EGTA, and the relationship between the antigen-stimulated release of stored calcium and exocytosis. Chlortetracycline was shown to be a useful qualitative indicator for the release of intracellular calcium with a relatively rapid response time.     

Cytosolic and intranuclear calcium signals in rat basophilic leukemia cells as revealed by a confocal fluorescence microscope.

A confocal fluorescence microscope with an argon-ion laser (488 nm) and a He-Cd laser (325 nm) was used to study spatial heterogeneity of the calcium signals in rat basophilic leukemia 2H3 cloned cell line (RBL-2H3). After stimulation with antigen (2,4-dinitrophenol-conjugated bovine serum albumin), fluo-3-fluorescence intensities increased in individual RBL-2H3 cells with different lag times. Time-dependent profiles of the fluo-3-fluorescence intensities resembled closely the patterns of the sequential fluorescence-ratio images of fura-2, which were used to measure the intracellular free-calcium concentration ([Ca2+]i) in individual RBL-2H3 cells using a conventional fluorescence microscope. The present results obtained using the confocal fluorescence microscope showed spatial heterogeneities of fluo-3-fluorescence intensities, suggesting the existence of spatial heterogeneity of [Ca2+]i in RBL-2H3 cells. That is, the results showed that calcium signals first occurred transiently at pseudopodia in RBL-2H3 cells, then the signals transferred to the central parts of the cells. In addition, from the fluorescence images of co-loaded Hoechst 33342 (bisbenzimide H 33342, a DNA-specific probe) which were produced by excitation with a He-Cd laser, it was found that the fluorescence images of the nucleus were quite similar to those of the calcium signals mentioned above. This suggested that the receptor-mediated calcium signals were transferred not only to the cytoplasm but also to the nucleus.     

Immunoglobulin E mediated membrane conductance changes in rat basophilic leukemia cells

Electrophysiological effects of anaphylactic stimulation of rat basophilic leukemia cells (RBL-2H3) were studied using conventional microelectrodes. Stimulation of passively sensitized cells by anti-immunoglobulin E resulted in hyperpolarization followed by depolarization. These changes in membrane polarization were associated with a decrease in input membrane resistance. No effect of anaphylactic stimulation was seen in Ca2+-free solution or when Ca2+ influx was blocked by Co2+, but it was mimicked by the Ca2+ ionophore A-23187. This suggests that the changes in ionic conductances were associated with calcium influx. These results support the hypothesis that membrane conductance changes are involved in the stimulus-secretion process of the RBL-2H3 cells.     

Structurally distinctive vasoactive intestinal peptides from rat basophilic leukemia cells

Peptides recognized by rabbit antibodies to vasoactive intestinal peptide (VIP) were extracted from diisopropyl fluorophosphate-treated rat basophilic leukemia (RBL) cells and resolved by filtration on Sephadex G-25 in 50 mM acetic acid. The immunoreactive VIPs of RBL cells eluted from Sephadex G-25 at 35-41%, 53-60%, and 69-73% bed volume, but not at 63-68% as for the neuropeptide VIP1-28. The two forms of immunoreactive VIP larger than VIP1-28 reacted with antibodies to both VIP1-9 and VIP10-28, but the smallest was bound only by antibodies to VIP10-28. The smallest immunoreactive VIP was purified by ion-exchange and reverse-phase high-performance liquid chromatography, and the amino acid sequence was determined to be that of VIP10-28 with asparagine-free acid at the carboxyl terminus rather than the amide of VIP neuropeptide. Challenge of RBL cells with 1 microM ionophore A23187 at 37 degrees C released VIP10-28 rapidly to a mean of 75% at 5 min and 77% at 30 min. The VIP generated and released by mast cells thus consists of a mixture of peptides that all differ structurally from the neuropeptide VIP.     

Calcium influx in a single rat basophilic leukemia cell as revealed with a digital imaging fluorescence microscope

Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.     

Transport of 5-hydroxytryptamine in membrane vesicles from rat basophilic leukemia cells

Rat basophilic leukemia (RBL) cells were grown as tumors. Membrane vesicles were isolated from them and serotonin transport was measured. Two types of transport were identified. One was sensitive to imipramine and sodium-dependent, while the other was sensitive to reserpine and ATP-dependent. The transport systems exhibit different affinities for serotonin (sodium-dependent, Km 0.22 microM; ATP-dependent, Km 2.6 microM) and are different in their substrate specificity, the former being much more specific. The 5-hydroxytryptamine transport by the reserpine-sensitive system was strongly inhibited by other biogenic amines, like norepinephrine, epinephrine and dopamine, whereas that by the imipramine-sensitive system was not. Upon Ficoll gradient centrifugation the two transport systems were separated. The reserpine-sensitive activity was found much further into the gradient than the imipramine-sensitive one. The latter co-migrated with the receptor of IgE, which is located in the plasma membrane. Characterization of latter showed that in addition to the dependence of 5-hydroxytryptamine influx on external sodium it was also absolutely dependent on external chloride and was strongly stimulated by internal potassium. On the other hand, efflux required external potassium. An alternative potassium independent way of loss of labelled 5-hydroxytryptamine was by exchange. A small but consistent stimulation of influx was observed in the presence of valinomycin, indicating that the process is electrogenic. The reserpine-sensitive system could also be driven in the absence of ATP. This required the imposition of pH gradient (acid inside) and was stimulated by an artificially imposed membrane potential (positive inside).     

The fate of IgE bound to rat basophilic leukemia cells.

The present study investigates the fate of the cell-bound IgE by using a well-characterized rat basophilic leukemia cell line and a purifed IgE myeloma protein. Both histamine-releasing and nonreleasing cell lines were examined. In both cases, no evidence for cell-mediated IgE catabolism could be elicited. Both the dissociated IgE and the receptors remained intact for prolonged periods of time, as demonstrated by binding assays. Internalization and/or recycling of membrane-bound IgE could not be demonstrated by E. M. autoradiography. We found only limited time-dependent changes in accessibility to anti-IgE antibody, trypsin, or elution at low pH (2.9 to 3.1). A biphasic dissociation of cell-bound 125I-IgE during incubation in the presence of excess unlabeled IgE was reproducibly observed; the more slowly dissociated IgE was also less readily dissociated at pH 3.4. These studies lead us to conclude that, in vitro, IgE resides in a functional orientation on the surface of RBL-1 cells, for prolonged periods of time.     

Purification of a mammalian 5-lipoxygenase from rat basophilic leukemia cells

5-Lipoxygenase (5-lipox) has been purified to homogeneity from the 20,000 xg supernatant of sonicated rat basophilic leukemia (RBL-1) cells using a 4-step procedure. Purification was achieved primarily through the use of anion-exchange HPLC on two different media. Using the supernatant from 1 × 10 ⁹ cells, approximately 33 μg of the enzyme can be routinely isolated with an estimated net yield of 5–10%. Purified 5-lipox consists of a single Mr 73,000 band on SDS gels (reduced or unreduced). When the purified enzyme was incubated with radiolabeled arachidonic acid and products analyzed by both straight phase and reversed phase HPLC, 5-hydroperoxyeicosate-traenoic acid (5-HPETE) was the only enzymatic product detected. The purified enzyme exhibits the same characteristic lag phase and premature cessation of reaction as does the 5-lipox activity seen in crude cell homogenates.     

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.     

Purification of a mammalian 5-lipoxygenase from rat basophilic leukemia cells

5-Lipoxygenase (5-lipox) has been purified to homogeneity from the 20,000 xg supernatant of sonicated rat basophilic leukemia (RBL-1) cells using a 4-step procedure. Purification was achieved primarily through the use of anion-exchange HPLC on two different media. Using the supernatant from 1 X 10(9) cells, approximately 33 micrograms of the enzyme can be routinely isolated with an estimated net yield of 5-10%. Purified 5-lipox consists of a single Mr 73,000 band on SDS gels (reduced or unreduced). When the purified enzyme was incubated with radiolabeled arachidonic acid and products analyzed by both straight phase and reversed phase HPLC, 5-hydroperoxyeicosatetraenoic acid (5-HPETE) was the only enzymatic product detected. The purified enzyme exhibits the same characteristic lag phase and premature cessation of reaction as does the 5-lipox activity seen in crude cell homogenates.     

Chalcone glycosides isolated from aerial parts of Brassica rapa L. ‘hidabeni’ suppress antigen-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells

We isolated three chalcone glycosides along with other glycoside constituents from the aerial parts of Brassica rapa L. ‘hidabeni’ and examined the effects of these compounds on the antigen-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells. Treatments with both 4′-O-β-d-glucopyranosyl-4-hydroxy-3′-methoxychalcone (C1) and 4′-O-β-d-glucopyranosyl-3′,4-dimethoxychalcone (C2) markedly inhibited antigen (Ag)-stimulated degranulation. To gain further insight into the inhibitory mechanisms by C1 and C2, we examined early intracellular signaling events, Ca²⁺ mobilization and intracellular reactive oxygen species (ROS) production. Both C1 and C2 did not affect early intracellular signaling events but exhibited the suppression of intracellular ROS production through NADPH oxidase (NOX) inactivation. From these results, we proposed that the inhibitory effects of C1 and C2 on Ag-stimulated degranulation were mainly due to suppression of intracellular Ca²⁺ elevation by suppression of intracellular ROS production through NOX inactivation. Our findings suggest that C1 and C2 would be beneficial to alleviate symptoms of type I allergy.