SUMMARY1. Substance P (SP) induces histamine release from isolated rat peritoneal mast cells at concentrations of 0-1-10 /tM.2. Inhibitors of glycolysis and oxidative phosphorylation prevent the release of histamine induced by SP. 3. Cells heated to 47 'C for 20 min release histamine when treated with an agent causing cell lysis but fail to release histamine in response to SP.4. SP does not release histamine by interacting with cell-bound IgE.5. Histamine release by SP is rapid, with more than 90 % of the response occurring within 1 min of the addition of the peptide to mast cells at 37 'C. 6. Substance P, unlike antigen-antibody or compound 48/80, does not show enhanced release of histamine when calcium (0 1-1 mM) is present in the extracellular medium but calcium increases the response to SP when the ion is added after the peptide. Extracellular calcium (01-1 mM), magnesium (1-10 mM) and cobalt (0-01-0-1 mM) all inhibit SP-induced histamine release when added before the peptide.Pre-treatment of the cells with EDTA (10 mM) and washing in calcium-free medium inhibits the histamine release induced by SP.7. Histamine release induced by SP was optimum at an extracellular pH of 7-2. 8. A number of peptides structurally related to SP were examined for histaminereleasing activity. At the concentrations tested, the N-terminal dipeptides Lys-Pro and Arg-Pro, tuftsin, physalaemin, eledoisin, SP31,, Phe7]-SP6 -1 were all found to be inactive. The relative activities of the other peptides were:
We have used thin section and freeze-fracture electron microscopy to study membrane changes occurring during exocytosis in rat peritoneal mast cells. By labeling degranulating mast cells with ferritin-conjugated lectins and anti-immunoglobulin antibodies, we demonstrate that these ligands do not bind to areas of plasma membrane or granule membrane which have fused with, or are interacting with, granule membrane. Moreover, intramembrane particles are also largely absent from both protoplasmic and external fracture faces of plasma and granule membranes in regions where these membranes appear to be interacting. Both the externally applied ligands and intramembrane particles are sometimes concentrated at the edges of fusion sites. These results indicate that membrane proteins are displaced laterally into adjacent membrane regions before the fusion process and that fusion occurs between protein-depleted lipid bilayers. The finding of protein-depleted blebs in regions of plasma and granule membrane interaction raises the interesting possibility that blebbing may be a process for exposing the granule contents to the extracellular space and for the elimination of excess lipid while conserving membrane proteins.In most secretory cells, the triggered release of specific materials stored in secretory granules occurs by exocytosis (2,18,37,43). This process involves the fusion of granule membrane with plasma membrane, and results in the exposure of the granule contents to the extracellular space (2,18,37,43). The molecular events in exocytosis, and in membrane fusion in general, are unknown. We have studied the behavior of membrane lipids and proteins during and after the fusion events occurring in the process of degranulation of rat peritoneal mast cells. The extensive membrane interactions occurring all around the circumference and inside degranulating mast cells make these cells particularly attractive for studying membrane fusion.Mast cells can easily be obtained in homogeneous cell suspension (9). Having high affinity surface receptors for the Fc region of immunoglobulin E (IgE) (so-called Fc receptors) (5), mast cells obtained from appropriately immunized rats have cytophilic IgE antibodies on their surface which serve as receptors for the specific antigen (6). Such sensitized cells can be stimulated to secrete histamine (and other substances) by exposure to the specific antigen (4, 6), or to anti-Ig antibody (6,
Abstract. 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.
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