Exocytosis In Mast Cells Research Articles

Edema formation and degranulation of mast cells by Trimeresurus mucrosquamatus snake venom

Trimeresurus mucrosquamatus venom, carrageenin, compound 48/80, trypsin and bovine serum albumin were injected s.c. into the plantar muscle to induce edema formation in the hind paw of rats. The venom was the most potent, and it and compound 48/80 induced the maximum swelling rate of edema within 15–30 min after injection. The edema volume induced by the venom was dose-dependent between 2.5 and 10 μg. Hydrocortisone, phenylbutazone, indomethacin and diphenhydramine inhibited edema induced by the venom and other inflammatory agents. Diphenhydramine was the most effective inhibitor of edema and increased vascular permeability induced by the venom. Injection of the venom i.p. caused exocytosis and degranulation of mesentery mast cells with a decreased electron density of released granules. Systemic administration of diphenhydramine inhibited the venom-induced exocytosis. Diphenhydramine and pyrilamine inhibited the contraction of guinea-pig ileum caused by venom or compound 48/80. It is concluded that histamine released from mast cells plays an important role in the causation of the edema induced by Trimeresurus mucrosquamatus snakebites.

Calmodulin, phospholipase, and exocytosis. p-Bromophenacyl bromide inhibits but mepacrine stimulates secretion in rat mast cells.

p-Bromophenacyl bromide (10-50 microM), a reagent that reacts with and inhibits phospholipase A2, completely suppressed exocytotic responses in rat serosal mast cells elicited by A23187. In contrast, mepacrine (0.01-0.1 mM), an antimalarial drug which has likewise been reported to inhibit phospholipase and additionally calmodulin, did not inhibit exocytosis elicited by concanavalin A (con A) or ionophore A23187. Rather, mepacrine (0.1-0.5 mM) alone increased histamine release up to 70%. Light microscopic observations following ruthenium red staining demonstrated the mepacrine-induced response to be exocytosis. Like the response to compound 48/80, that elicited by mepacrine was maximal within 40 s and was inhibited by phosphatidylserine. In the absence of extracellular calcium, secretion elicited by mepacrine and by con A was inhibited by 87 and 90%, respectively, whereas that elicited by 48/80 was unaffected. Incubation of mast cells in the presence of 2 mM EDTA for 2 h inhibited responses to 48/809 by 76% and nearly abolished those to mepacrine and to con A. 5,8,11,14-Eicosatetraynoic acid (50 and 100 microM) inhibited secretion elicited by con A but not that evoked by mepacrine or polymyxin B. The phenothiazines thioridazine (10-50 microM) and chlorpromazine (25-125 microM) inhibited secretory responses to mepacrine, to 48/80 and to con A. Both phenothiazines inhibited secretion elicited by con A more effectively than that elicited by mepacrine or 48/80. The results indicate that mepacrine, like con A, elicits exocytosis in mast cells by drawing on extracellular sources of calcium, but the early events of secretion initiated by these two secretagogues are pharmacologically distinct.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoxygenase inhibitors exert secretagogue-specific effects on mast cell exocytosis

The lipoxygenase inhibitors HTYA (5,8,11,14-henicosatetraynoic acid, 25–150 μM) and ITYA (4,7,10,13-icosatetraynoic acid, 25–75 μM), inhibited histamine secretion from rat mast cells evoked by concanavalin A but not that elicited by compound 48/80 or polymyxin B. Arachidonic acid (100 μM) did not inhibit concanavalin A-induced histamine secretion. The results are consistent with the notion that lipoxygenase inhibitors affect an early stage of stimulus-secretion coupling in the mast cell, peculiar to antibody-directed secretagogues, perhaps that involving calcium influx.

Reorganization of Membrane Cholesterol at anEarly Stage of Exocytosis (Degranulation) in Rat Mast Cells: Inferred from the Ditribution of Filipin-Cholesterol Comlexes

Using filipin, a sterol-specific polyene antibiotic, we examined the redistribution of cholesterol molecules present at an early stage of degranu-lation (release of heparin-protein complexes) in rat peritoneal mast cells with freeze-fracture electron microscopy. Isolated mast cells first were stimulated to undergo degranulation with compound 48/80 (1 μg/ml) for 3 sec, then were treated with a glutaraldehyde solution containing filipin (50 μg/ml) for 30 min. Freeze-fractures of the plasma membrane of these cells showed small depressions which lacked filipin-cholesterol complexes. These are the first recognizable membrane alterations and are suggested to correspond to the initial sites of fusion between the plasma and granule membranes. Subsequent-ly, these depressions became bulges. Filipin-cholesterol complexes are absent from the portion of the bulges in close contact with the plasma membrane, probably because the bulges have not yet fused with the underlying granule membrane. These complexes also were absent from the periphery of the two apposed membranes at exocytotic sites during fusion. Even after fusion was complete, no complexes appeared in the circumferential zone where the plasma membrane and the secretory granule membrane had fused. Our results suggest that membrane cholesterol is reorganized at the fusion sites between the plasma and granule membranes during mast cell secretion, and that this alteration in the organization of cholesterol in the plasma membrane represents an initial event during mast cell secretion. A hypothetical model for the sequential changes in the plasma membrane during degranulation is proposed.

Differential inhibitory effects of the arachidonic acid analog ETYA on rat mast cell exocytosis evoked by secretagogues utilizing cellular or extracellular calcium

ETYA (5,8,11,14-eicosatetraynoic acid; 50–100 μM), which inhibits both cyclo-oxygenase and lipoxidase, inhibited histamine release evoked by secretagogues dependent on extracellular calcium (antigen, dextran, and concanavalin A) but failed to inhibit secretion elicited by secretogogues capable of mobilizing calcium from intracellular cellular sites (48/80, polymyxin B, protamine sulfate and poly-L-lysine). Responses to these latter secretagogues were inhibited only by higher concentrations of ETYA (100–200 μM) that were cytotoxic. Secretion evoked by the calcium ionophore A23187 (0.1 μg/ml) was inhibited at much lower concentrations of ETYA (1–10μM) but this inhalation could not be overcome by increasing the concentration of calcium. Responses to higher concentrations of ionophore were not inhibited by ETYA except in amounts affecting cell viability. Like ETYA, each of several fatty acids, including arachidonic acid, were inhibitory towards histamine release evoked by A23187 or 48/80. The results indicate that ETYA acts at some early stage of stimulus-secretion coupling rather than on the final, common, calcium-activated steps of exocytosis. Moreover, this action may be unrelated to inhibition of lipoxidase or cyclooxygenase.

Arrest of membrane fusion events in mast cells by quick-freezing.

We have used quick-freezing and freeze-fracture to study early stages of exocytosis in rat peritoneal mast cells. Mast cells briefly stimulated with 48/80 (a synthetic polycation and well-known histamine-releasing agent) at 22 degrees C displayed single, narrow-necked pores (some as small as 0.05 micrometer in diameter) joining single granules with the plasma membrane. Pores that had become as large as 0.1 micrometer in diameter were clearly etchable and thus represented aqueous channels connecting the granule interior with the extracellular space. Granules exhibiting pores usually did not have wide areas of contact with the plasma membrane, and clearings of intramembrane particles, seen in chemically fixed mast cells undergoing exocytosis, were not present on either plasma or granule membranes. Fusion of interior granules later in the secretory process also appeared to involve pores; granules were often joined by one pore or a group of 2-4 pores. Also found were groups of extremely small, etchable pores on granule membranes that may represent the earliest aqueous communication between fusing granules.

The effects of calcium on morphology and histamine content in isolated intact mast cell granules [proceedings

These studies on isolated intact mast cell granules, indicate that changes in the concentration of the divalent cations (Ca2+, Mg2+)alone cannot explain the membrane fusions and histamine release during sequential exocytosis in mast cells.

Effects of microfilament-active drugs, phalloidin and the cytochalasins A and B, on exocytosis in mast cells evoked by 48/80 or A23187

Rat peritoneal mast cells were used as a model system to study the effect, on exocytosis, of three agents known to interact with microfilaments. Mast cell secretion was evaluated by fluorimetric assay of histamine and by ruthenium red staining, the latter method allowing a direct visualization and quantitation of exocytosis at the light microscopic level. Phalloidin, in concentrations up to 300 microgram/ml, was without effect on either spontaneous or 48/80-evoked secretion, even after cells were exposed to the drug for 28 h. The failure of even high doses of phalloidin to influence cellular morphology and exocytosis in the mast cell may reflect the absence of a specific membrane receptor. Cytochalasin B was likewise without effect on the response to 48/80 in normally respiring cells; but inhibited this response in the presence of Antimycin A. This inhibitory effect probably reflects the ability of cytochalasin B to block glucose transport. In normally respiring cells, neither phalloidin nor cytochalasin B affected the active expulsion of granules from exocytotic pits. Cytochalasin A, without concomitant treatment with Antimycin A, completely inhibited secretion in response to both 48/80 and A23187, and did so in low concentration. Whether this striking inhibitory effect results from an interaction with microfilaments is uncertain for the inhibition could be mimicked by nonpenetrating thiol-oxidizing agents and prevented by impermeant thiol-protecting agents suggesting that cytochalasin A may inhibit histamine release by thiol-oxidation at the cell surface. Possible surface sulfhydryls are important for membrane rearrangements accompanying exocytosis.

Quercetin: A novel inhibitors of Ca 2+ influx and exocytosis in rat peritoneal mast cells

The effect of the transport ATPase inhibitor, quercetin on histamine secretion from antigen sensitized mast cells was examined. At micromolar concentrations, quercetin had an immediate inhibitory effect on histamine secretion mediated by antigen, concanavalin A and ATP but it had little effect on release induced by the ionophores A23187 and X537A. Quercetin exerts its effect after the binding of the releasing ligands and the distinction between its effect on ligand induced and A23187 induced secretion suggests that it affects the normal path of Ca 2+ entry into the cell. The inhibitory effects of quercetin were compared with those of the structurally related anti-allergic drugs cromoglycate and AH7725.

Molecular events during membrane fusion. A study of exocytosis in rat peritoneal mast cells.

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. The 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.

Calcium-induced extrusion of secretory granules (exocytosis) in mast cells exposed to 48-80 or the ionophores A-23187 and X-537A.

Isolated peritoneal mast cells from rats were observed, by phase contrast microscopy, to extrude secretory granules when exposed to 48/80 or the ionophores A-23187 and X-537A, which are known to facilitate transmembrane fluxes of calcium. These effects were abolished when the cells were treated with EDTA and suspended in a Ca-free environment. Ca-deprived cells exposed to any one of the three drugs promptly extruded granules when calcium, but not magnesium, was added to the incubation medium. Such Ca-evoked or Ca-dependent responses persisted when Na was omitted from the incubation medium and replaced with sucrose, choline, or K. The responses thus seem independent of possible shifts in the alkali metal ions. The results are considered support for the view that Ca influx mediates stimulus-secretion coupling and does so by initiating exocytosis.