Clare Fewtrell

Calcium Influx through L-type Channels Is Required for Selective Activation of Extracellular Signal-regulated Kinase by Gonadotropin-releasing Hormone

The hypothalamic decapeptide gonadotropin-releasing hormone stimulates mobilization of two discrete pools of calcium in clonal (αT3-1) and primary pituitary gonadotropes. A multidisciplinary approach was implemented to investigate the effects of discrete calcium fluctuations on the signaling pathways linking the gonadotropin-releasing hormone receptor to activation of mitogen-activated protein kinases and immediate early genes. Blockade of calcium influx through nifedipine-sensitive voltage-gated calcium channels reduced buserelin-induced activation of extracellular signal-regulated kinase (ERK) and c-Fos while activation of c-Jun N-terminal kinase and c-Jun was unaffected. Inhibition of buserelin-stimulated ERK activity by nifedipine was also observed in rat pituitary cells in primary culture. Direct activation of αT3-1 cell L-type calcium channels with the agonist Bay-K 8644 resulted in phosphorylation of ERK and induction of c-Fos. However, simple voltage-induced channel activation did not produce a sufficient calcium signal, since depolarization with 35 mm KCl failed to induce activation of ERK. Depletion of intracellular calcium stores with thapsigargin did not affect buserelin-induced ERK activation. An inhibitor of protein kinase C decreased calcium influx through nifedipine-sensitive calcium channels and phosphorylation of ERK induced by buserelin. Pharmacological inhibition of protein kinase C did not block Bay-K 8644-induced ERK activation. These observations suggest that calcium influx through L-type channels is required for GnRH-induced activation of ERK and c-Fos and that the influence of calcium lies downstream of protein kinase C.

Correlating Ca2+ Responses and Secretion in Individual RBL-2H3 Mucosal Mast Cells

The role of Ca2+ in stimulus-response coupling in nonexcitable cells is still not well understood. The Ca2+ responses of individual cells are extremely diverse, often displaying marked oscillations, and almost nothing is known about the specific features of these Ca2+signals that are important for the functional response of a cell. Using the RBL-2H3 mucosal mast cell as a model, we have studied the temporal relationship between changes in intracellular Ca2+ and serotonin secretion at the single-cell level using simultaneous indo-1 photometry and constant potential amperometry. Secretion in response to antigen never occurs until intracellular Ca2+ is elevated, nor is it seen during the first few oscillations in Ca2+. Exocytotic events tend to be clustered around the peaks of oscillations, but excellent secretion is also seen in cells with sustained elevations in Ca2+. Ca2+ release from stores in the absence of influx fails to elicit secretion. If refilling and continued release of Ca2+ from stores is prevented with thapsigargin, Ca2+ influx can still trigger secretion, suggesting that store-associated microdomains of Ca2+ are not required for exocytosis. Our findings demonstrate the importance of an amplitude-encoded Ca2+ signal and Ca2+influx for stimulus-secretion coupling in these nonexcitable cells.

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.

Tenidap: a novel inhibitor of calcium influx in a mast cell line

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.

Subcloning the RBL-2H3 mucosal mast cell line reduces Ca2+ response heterogeneity at the single-cell level

Ca2+ imaging experiments have revealed that for a wide variety of cell types, including RBL‐2H3 mucosal mast cells, there are considerable cell‐to‐cell differences of the Ca2+ responses of individual cells. This heterogeneity is evident in both the shape and latency of the responses. Mast cells within a single microscopic field of view, which have experienced identical culture conditions and experimental preparation, display a wide variety of responses upon antigen stimulation. We have subcloned the RBL‐2H3 mucosal mast cell line to test the hypothesis that genetic heterogeneity within the population is the cause of the Ca2+ response heterogeneity. We found that cell‐to‐cell variability was significantly reduced in four of five clonal lines. The response heterogeneity remaining within the clones was not an experimental artifact caused by differences in the amount of fura‐2 loaded by individual cells. Factors other than genetic heterogeneity must partly account for Ca2+ response heterogeneity. It is possible that the complex shapes and variability of the Ca2+ responses are reflections of the fact that there are multiple factors underlying the Ca2+ response to antigen stimulation. Small differences from cell to cell in one or more of these factors could be a cause of the remaining Ca2+ response heterogeneity.

Activation and Desensitization of Receptors for IgE on Tumor Basophils

The aggregation of receptors for IgE on the surface of mast cells and basophils leads to secretion of a variety of mediators of immediate hypersensitivity. Many of these mediators, including histamine and serotonin, are stored in granules within the cytoplasm, while others such as arachidonic acid and its metabolites (e. g., prostaglandins and leukotrienes) are newly formed when cells are stimulated to secrete. In common with other secretory systems, mast cell granule exocytosis is presumed to occur as a result of a rise in cytoplasmic free Ca2+ and is also dependent on a source of metabolic energy. The mechanism of IgE receptor activation and the events leading to exocytosis are beginning to be unraveled [1] and we now know a considerable amount about the molecular structure of the first component in the sequence, namely the receptor for IgE [2].

Mediation of effector functions by antibodies: report of a workshop.

Abstract On 25–27 October 1978 a workshop on the subject “Mediation of Effector Functions by Antibodies” was held in Bethesda, MD, U.S.A. The conference was organized by Henry Metzger, William Paul and David Segal in order to bring together workers from diverse disciplines whose work contributes to an understanding of two principal problems: (1) the role of various parts of antibody molecules in interactions with effector systems such as complement and Fc-receptor bearing cells and (2) the molecular mechanism by which antigens promote antibody-effector system interaction. What follows is a summary of the proceedings prepared by the undersigned. The purpose of the summary is to give an overall view of the spirit of the meeting and the direction in which this field is moving.

Protein kinase C activator PMA reduces the Ca2+ response to antigen stimulation of adherent RBL‐2H3 mucosal mast cells by inhibiting depletion of intracellular Ca2+ stores

Activation of protein kinase C has been shown to reduce the Ca2+ responses of a variety of cell types. In most cases, the reduction is due to inhibition of Ca2+ influx, but acceleration of Ca2+ efflux and inhibition of Ca2+ store depletion by protein kinase C activation have also been described. For adherent RBL‐2H3 mucosal mast cells, results from whole‐cell patch clamp experiments suggest that protein kinase C activation reduces Ca2+ influx, while experiments with intact, fura‐2‐loaded cells suggest that Ca2+ influx is not affected. Here we present single‐cell data from Ca2+ imaging experiments with adherent RBL‐2H3 cells, showing that antigen‐stimulated Ca2+ responses of phorbol 12‐myristate 13‐acetate (PMA)‐treated cells are more transient than those of control cells. PMA also reduced the response to antigen in the absence of extracellular Ca2+, indicating that depletion of intracellular Ca2+ stores is inhibited. If PMA was added after stores had been depleted by thapsigargin, a small decrease in [Ca2+]i was observed, consistent with a slight inhibition of Ca2+ influx. However, the major effect of PMA on the antigen‐stimulated Ca2+ response is to inhibit depletion of intracellular Ca2+ stores. We also show that inhibition of protein kinase C did not enhance the Ca2+ response to antigen, suggesting that inhibition of the Ca2+ response by activation of protein kinase C does not contribute to the physiological response to antigen. J. Cell. Physiol. 181:113–123, 1999.

The Receptor for Immunoglobulin E on Mast Cells and Basophils

The methods and approaches now outlined and later amplified are related to phenomena [l] that are summarized overleaf.