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C Fewtrell

Publications and source records attributed to C Fewtrell.

At least 37 records · Page 2Linked to original sources
PubMed1988

Cholera toxin increases the rate of antigen-stimulated calcium influx in rat basophilic leukemia cells.

Cholera toxin pretreatment has been found to cause a 3-fold increase in the initial rate of antigen-stimulated secretion of serotonin from rat basophilic leukemia (RBL) cells. Under similar conditions, cholera toxin enhances the antigen-stimulated rise in cytoplasmic free ionized calcium levels and causes a 2-3-fold increase in the rate of antigen-stimulated influx of 45Ca. In intact RBL cells cholera toxin pretreatment potentiates the antigen-stimulated production of inositol phosphates, but in permeabilized cells, with strongly buffered free calcium levels, no effect of cholera toxin pretreatment on the antigen-stimulated activation of cellular phospholipase activities is observed. In addition, pretreatment of cells with tetradecanoylphorbol acetate inhibits antigen-stimulated production of inositol phosphates by greater than 95%, while the stimulated influx of 45Ca remains unaffected. These data indicate that the antigen-stimulated influx of calcium into RBL cells can be dissociated from the production of inositol phosphates in these cells. The observed effects of cholera toxin on exocytosis and Ca2+ influx in RBL cells are not due to the elevation of cellular cyclic AMP levels since a variety of agents capable of elevating cellular cyclic AMP levels do not mimic these effects. Together, these data suggest that a cholera toxin-sensitive guanine nucleotide-binding protein is involved in the pathway responsible for the antigen-stimulated influx of calcium into RBL cells.

Animals↗
PubMed1988

Imaging asynchronous changes in intracellular Ca2+ in individual stimulated tumor mast cells.

Changes in the level of intracellular free calcium ([Ca2+]i) are associated with the secretion of mediators of immediate hypersensitivity by rat basophilic leukemia (RBL) cells, a mast cell line. Digital fluorescence ratio imaging of fura-2 was used to measure [Ca2+]i in individual RBL cells. Changes in [Ca2+]i that occurred in response to crosslinking of IgE receptors on the cell surface or to application of the Ca2+ ionophore ionomycin were studied. Stimulation of RBL cells with antigen resulted in rapid increases in [Ca2+]i following lag times that varied widely from cell to cell. Simple averaging of the Ca2+ responses of many cells yielded a gradual response profile that closely resembled that of suspensions of cells measured in the fluorometer. The results show that single cells can respond much more rapidly to antigen than has previously been suggested by studies on populations of cells. The lag time between addition of antigen and initiation of the increase in [Ca2+]i varied considerably between cells in the same field of view. Both the rise time and the variability and average duration of the lag time increased with decreasing antigen concentration.

Animals↗
PubMed1987

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.

Aminoquinolines↗
PubMed1987

The relative contributions of extracellular and intracellular calcium to secretion from tumor mast cells. Multiple effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone.

The proton ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited antigen-stimulated secretion and calcium influx in rat basophilic leukemia cells. In a glucose-free solution the inhibitory effects of CCCP were due to a decrease in the intracellular ATP concentration; however, when glucose was present there was no decrease in ATP. Instead, we found that in a glucose-containing saline solution, CCCP inhibited antigen-stimulated calcium uptake because it depolarized the plasma membrane, which in rat basophilic leukemia cells inhibits antigen-stimulated calcium uptake. In the presence of glucose, relatively low concentrations of CCCP inhibited calcium uptake while higher concentrations were required to inhibit secretion. In contrast, the initial antigen-stimulated rise in cytoplasmic calcium, measured with the fluorescent calcium indicator quin2, was not inhibited by CCCP. This suggests that the release of calcium from intracellular stores might, in some cases, be sufficient to support antigen-stimulated secretion. In the presence of CCCP the pH gradient becomes important for regulating the membrane potential across the plasma membrane. When cells were depolarized with CCCP and the external pH was increased, the membrane potential returned to resting levels and antigen-stimulated calcium uptake was restored. Inhibition of antigen-stimulated secretion by higher concentrations of CCCP could also be reversed by increasing the external pH.

Adenosine Triphosphate↗
PubMed1987

IgE receptor-mediated depolarization of rat basophilic leukemia cells measured with the fluorescent probe bis-oxonol.

Receptor-mediated changes in plasma membrane potential were recorded in rat basophilic leukemia (RBL) cells with the potential-sensitive fluorescent indicator bis-oxonol. Depolarization of the mitochondria with metabolic inhibitors was not detected by bis-oxonol, suggesting that only potential changes across the plasma membrane were being measured. The resting membrane potential of RBL cells was largely generated by the equilibrium distribution of K+ and not through electrogenic activity of the sodium pump. Depolarization was maintained as long as IgE receptors remained aggregated. We believe that at physiologic calcium concentrations a large portion of the measured potential change may be due to calcium influx across the plasma membrane. Prevention of calcium influx by lanthanum, disruption of aggregated receptors, or prior depolarization in a high K+ saline solution completely inhibited the antigen-induced depolarization. The time course of the antigen-stimulated increase in bis-oxonol fluorescence was similar, but not identical, to the antigen-stimulated rise in cytoplasmic free ionized calcium measured with fura-2. Antigen-stimulated depolarization was inhibited by removing both calcium and sodium and could be restored by the addition of either ion. Reduction of total cellular adenosine triphosphate inhibited depolarization in response to antigen stimulation.

Adenosine Triphosphate↗
PubMed1987

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.

Animals↗
PubMed1986

Cross-linking of IgE-receptor complexes at the cell surface: synthesis and characterization of a long bivalent hapten that is capable of triggering mast cells and rat basophilic leukemia cells.

The ability of a series of bivalent haptens to bind and cross-link immunoglobulin E (IgE) in solution and on the surface of cells was examined. Several short (less than 30 A) dinitrophenyl (DNP) haptens were found to bind tightly to and cross-link a monoclonal anti-DNP IgE in solution, but these failed to trigger substantial release of 3H-serotonin from sensitized rat basophilic leukemia (RBL) cells or rat peritoneal mast cells. A longer bivalent hapten, approximately 50 A in length, consisting of two DNP-aminocaproyl-L-tyrosine (DCT) groups coupled to the alpha-amino groups of L-cystine was synthesized and characterized. This bivalent hapten [(DCT)2-cystine], binds very tightly to the same monoclonal anti-DNP IgE in solution and cross-links these antibodies to form higher mol. wt aggregates as judged by gel filtration and binding studies. It also stimulates degranulation of both RBL and mast cells sensitized with two different monoclonal anti-DNP IgE antibodies, with the mast cells exhibiting generally greater responsiveness to this ligand. The (DCT)2-cystine bivalent hapten appears to have the structural features necessary for carrying out detailed binding studies with receptor-bound IgE on the cell surface.

Animals↗
PubMed1983

Changes in the receptor for immunoglobulin E coincident with receptor-mediated stimulation of basophilic leukemia cells.

Aggregation of the receptor for immunoglobulin E on mast cells and related tumor cells initiates exocytosis. We examined tumor cells that had incorporated [3H]leucine and 32P to see if stimulating them produced modifications in the receptors themselves. No changes were observed in the yield of receptors or in the relative proportion and the molecular weights of their alpha, beta, and gamma subunits. In addition, no new "receptor-associated" components were observed. However, after the cells were stimulated, the gamma chains of the receptors showed an average 35% decrease in their associated 32P. Changes in the beta subunits were more variable but on the average showed a similar-sized increase in 32P. Using a novel protocol that permitted examination of aggregated and unaggregated receptors from the same cell, we found that changes in the unaggregated receptors were quantitatively indistinguishable from those exhibited by the aggregated receptors. These findings raise the possibility that the changes are related to one of the inactivation reactions thought to accompany the activation sequence.

Animals↗
PubMed1982

Phosphorylation of the receptor of immunoglobulin E.

Specific immune precipitation of immunoglobulin E(IgE)-receptor complexes from detergent extracts of 32P-labeled rat basophilic leukemia cells yielded a phosphoprotein of Mr approximately 35,000 on gel electrophoresis in sodium dodecyl sulfate. This phosphoprotein was shown by several criteria to be the beta chain of the receptor for IgE. Phosphorylation occurs at a serine residue (or residues) in a region (beta 2) of the beta chain that is thought to be exposed on the cytoplasmic face of the plasma membrane. Our results suggest that phosphorylation probably takes place after the insertion of the beta chain into the membrane. The IgE-binding alpha chain of the receptor and the IgE associated with it are not phosphorylated. We have so far been unable to detect any changes in the state of phosphorylation of either chain of the receptor or of IgE itself after IgE-mediated triggering of the cells.

Animals↗
PubMed1982

Structure of the high-affinity mast cell receptor for IgE.

Mast cells and related rat tumor basophils have a surface receptor that binds monomeric IgE with high avidity. The receptor in situ is unclustered, mobile, and univalent, and its aggregation into dimers and higher oligomers triggers degranulation. It has two subunits, alpha and beta. The alpha chain has a molecular weight of about 50,000, of which 30% is carbohydrate. Heterogeneity in the latter accounts at least in part and perhaps fully for the heterogeneity of the alpha chain. Studies with proteases have delineated two domains: alpha 1 appears slightly larger on sizing gels, and incorporation studies suggest it has more carbohydrate than alpha 2. The alpha 2 domain and a 24,000-dalton subfragment of it contain the principal site whose surface labeling is blocked by the presence of IgE. The alpha subunit is firmly but noncovalently bound to beta in a 1:1 complex in situ and in detergent extracts. Nevertheless, during thorough washing of IgE-receptor complexes with detergent, beta dissociates. A portion of beta, beta 1, is integrated in the membrane; it is this domain that is labeled when the receptor is isolated from cells reacted with the intramembranous probe iodonapthylnitrene and that interacts with the alpha chain. These results and others have been used to draw a provisional model of the receptor.

Animals↗
PubMed1981

Unexpected findings from target analysis of immunoglobulin E and its receptor.

The membrane receptor for immunoglobulin E (IgE) and its ligand, IgE, were irradiated with high-energy electrons. Loss of binding activity was measured for each, and the size of the functional targets was assessed. In both cases, the target size was substantially smaller than the covalent structure of the molecule. The direction of this discrepancy is unprecedented on the basis of experience with loss of enzymatic activity by irradiation; indeed, two enzymes which were present in the receptor preparations gave expected values when measured simultaneously. We suggest that in instances where a function such as ligand binding resides in a conformationally stable domain, radiation inactivation may be capable of revealing this.

Acetylglucosaminidase↗
PubMed1981

Secretion from rat basophilic leukaemia cells induced by calcium ionophores. Effect of pH and metabolic inhibition.

Previous experiments on the functional properties of rat basophilic leukaemia cells showed a major anomaly when compared to normal mast cells: though IgE-mediated secretion was dependent on external Ca2+ with both types of cells, substantial non-cytotoxic release with ionophore A23187 could be demonstrated with the normal cells but not with the tumour cells. We now show that when the pH of the incubation medium is increased to 8 it is possible to obtain excellent Ca-dependent, non-cytotoxic secretion from tumour basophils with the ionophores A23187 and ionomycin. These results provide further evidence that secretion from the tumour cells occurs via a mechanism similar to that used by normal mast cells and basophils. Experiments with metabolically inhibited tumour cells suggest that their unusual sensitivity to the cytotoxic effects of Ca2+ ionophores may be related to their ability to sequester intracellular calcium. Changes in the conditions of cell culture appeared to produce substantial and at least partially reversible changes in responsiveness to IgE-mediated triggering and ionophores.

Animals↗