SUMMARY Crosslinking of IgE-bound FcεRI triggers mast cell degranulation. Previous FRAP and phosphorescent anisotropy studies suggested that FcεRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods are used to redefine relationships between receptor mobility and signaling. QD-IgE-FcεRI aggregates of at least three receptors remain highly mobile over extended times at low concentrations of antigen that induce Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remains mobile at low doses that support secretion. FcεRI immobilization is marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocks secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates, is a trigger for receptor internalization, and is not required for tyrosine kinase activation leading to secretion.

Antigen-mediated activation of mast cells results in Ca 2+ -dependent exocytosis of preformed mediators of the inflammatory response. To investigate the role of secretory vesicle motility in this response, we have performed time-lapse confocal microscopy on RBL-2H3 cells transfected with a green fluorescent protein-Fas ligand fusion protein (GFP-FasL). Green fluorescent protein-labeled vesicles exhibit rapid, bidirectional movement in both resting and activated cells and can be localized adjacent to microtubules. Colchicine treatment inhibits the motility of secretory vesicles as measured by fluorescence recovery after photobleaching (FRAP). Colchicine also inhibits both the extent and the rate of exocytosis triggered by receptor activation or by Ca 2+ ionophore, demonstrating that microtubule-dependent movement of secretory vesicles plays an important role in the exocytic response.

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (FcER1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, FcER1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of

In RBL-2H3 rat tumor mast cells, cross-linking the high affinity IgE receptor, Fc epsilon R1, activates the protein-tyrosine kinases Lyn and Syk and initiates a series of responses including protein-tyrosine phosphorylation, inositol 1,4,5-trisphosphate synthesis, Ca2+ mobilization, secretion, membrane ruffling, and actin plaque assembly. The development of chimeric receptors containing cytoplasmic domains of individual subunits of the heterotrimeric (alpha beta gamma 2) Fc epsilon R1 has simplified analyses of early signaling events in RBL-2H3 cells. Here, RBL-2H3 cells were transfected with cDNAs encoding the extracellular and transmembrane domains of the interleukin-2 receptor alpha subunit (the Tac antigen) joined to the C-terminal cytoplasmic domains of the Fc epsilon R1 gamma and beta subunits (TT gamma and TT beta). Both sequences contain tyrosine activation motifs implicated in antigen receptor signal transduction. TT gamma and TT beta are expressed independently of the native Fc epsilon R1, as demonstrated by the ability of Tac cross-linking agents to trigger the clustering and internalization through coated pits of both chimeric receptors without co-clustering the Fc epsilon R1. A full range of signaling activities is induced by TT gamma cross-linking; the TT gamma-induced responses are slower and, except for Lyn activation, smaller than the Fc epsilon R1-induced responses. In striking contrast, TT beta cross-linking elicits no tyrosine phosphorylation or signaling responses, it impairs basal activities measured in secretion and anti-PY (anti-phosphotyrosine antibody) immune complex kinase assays, and it antagonizes Fc epsilon R1-induced Lyn and Syk activation, protein-tyrosine phosphorylation, and signaling responses. We hypothesize that the isolated beta subunit binds a specific kinase or coupling protein(s) required for signaling activity, sequestering it from the signal-transducing gamma subunit. Binding the same kinase or coupling protein to the beta subunit of the intact Fc epsilon R1 may serve instead to present it to the adjacent gamma subunit, resulting in enhanced kinase activation and signaling responses.

No abstract

Summary .isoproterenol (8). In the second model, we have demonstratedThe chronically reserpinized rat has been proposed as an animal model for cystic fibrosis on the basis of morphologic and secretory alterations in the submaxillary gland and of abnormalities in pulmonary secretions. In this investigation, the volume and composition of pancreatic juice from reserpine treated rats (0.5 mg/kg/day) have been compared to those of untreated controls after stimulation with purified cholecystokinin (0.1 pg/ kg body wt) and with crude and purified preparations of secretin (6p/100 g body wt) infused iv for 30-min periods. The results demonstrate that the treated animals secreted a significantly lower volume of pancreatic juice after stimulation with these secretagogues. Fldw rates were also significantly reduced after stimulation with cholecvstokinin and crude secretin. Protein and amylase outputs in response to cholecystokinin were smaller than in control animals after unrestricted feedings, but greater after a 24-hr fast. Total bicarbonate output was also reduced after stimulation with either crude or purified secretin and the normal excretion patterns for bicarbonate and chloride were either absent or reversed in the secretin-stimulated pancreatic juice of the treated animals. Whole pancreas homogenates from the treated animals showed significant increases in Ca++ and protein content. These results indicate that chronic administration of reserpine alters the secretion of water, protein, and bicarbonate from the rat pancreas and that it affects several of the exocrine glands involved in cystic fibrosis. These findings lend support to the concept of an animal model for the human disease. SpeculationThe chronic administration of reserpine to rats has been shown to induce alterations in salivary and pulmonary secretions which resemble those seen in patients with cystic fibrosis. The concept of an animal model has been proposed on the basis of these observations. The pancreas is another exocrine gland prominently involved in cystic fibrosis and if it can be demonstrated that reserpine administration also induces alterations in pancreatic secretion, the concept of the animal model would be strengthened considerably.The generalized exocrine gland dysfunction which is characteristic of cystic fibrosis (3) has been partially reproduced in experimental animal models induced by treating rats in a chronic fashion with certain drugs that influence the level of autonomic input into these tissues. In the first of these models, changes in the excretion of monovalent ions and of basic proteins from the rat parotid gland were observed after chronic administration of that administration of reserpine for several days induces morphologic and secretory changes in the rat submaxillary gland which resemble those of cystic fibrosis (CF) (11,12). In addition, we have shown that this procedure increases the secretion of total protein and of a specific type of glycoprotein from the lungs of the treated animals, in a manner that parallels the abn...

Intravenous infusions of the undecapeptide substance P caused a moderate but transient secretory response from the rat submandibular gland. The electrolyte composition of the saliva elicited by this peptide was qualitatively similar to that of the secretion induced by parasympathomimetic agents. Substance P inhibited the secretory response to acetylcholine and isoproterenol, but did not modify the binding of specific ligands to glandular autonomic receptors. It is concluded that this naturally occurring peptide plays a role in the physiologic regulation of salivary secretions, which involves both direct stimulatory effects and a modulating action on the effect of autonomic neurotransmitters on the salivary gland cells. The latter may involve similar effects of substance P and the neurotransmitters on a metabolic pathway localized beyond receptor activation.

Clustering the tetrameric (αβγ2) IgE receptor, FcεRI, on basophils and mast cells activates the Src-family tyrosine kinase, Lyn, which phosphorylates FcεRI β and γ subunit tyrosines, creating binding sites for the recruitment and activation of Syk. We reported previously that FcεRI dimers formed by a particular anti-FcεRI α mAb (H10) initiate signaling through Lyn activation and FcεRI subunit phosphorylation, but cause only modest activation of Syk and little Ca2+ mobilization and secretion. Curtailed signaling was linked to the formation of unusual, detergent-resistant complexes between Lyn and phosphorylated receptor subunits. Here, we show that H10-FcεRI multimers, induced by adding F(ab′)2 of goat anti-mouse IgG to H10-treated cells, support strong Ca2+ mobilization and secretion. Accompanying the recovery of signaling, H10-FcεRI multimers do not form stable complexes with Lyn and do support the phosphorylation of Syk and phospholipase Cγ2. Immunogold electron microscopy showed that H10-FcεRI dimers colocalize preferentially with Lyn and are rarely within the osmiophilic “signaling domains” that accumulate FcεRI and Syk in Ag-treated cells. In contrast, H10-FcεRI multimers frequently colocalize with Syk within osmiophilic patches. In sucrose gradient centrifugation analyses of detergent-extracted cells, H10-treated cells show a more complete redistribution of FcεRI β from heavy (detergent-soluble) to light (Lyn-enriched, detergent-resistant) fractions than cells activated with FcεRI multimers. We hypothesize that restraints imposed by the particular orientation of H10-FcεRI dimers traps them in signal-initiating Lyn microdomains, and that converting the dimers to multimers permits receptors to dissociate from Lyn and redistribute to separate membrane domains that support Syk-dependent signal propagation.