IgE-mediated histamine release in rat basophilic leukemia cells: receptor activation, phospholipid methylation, Ca2+ flux, and release of arachidonic acid

Stimulation of IgE receptors on rat basophilic leukemia cells causes a transient rise and fall of methylated phopholipids, Ca²⁺ influx, and release of arachidonic acid previously incorporated into phosphatidylcholine and liberation of histamine. Inhibition of phospholipid methylation by methyltransferase inhibitors, 3-deazaadenosine and homocysteine thiolactone, almost completely blocks the influx of Ca²⁺, and release of arachidonic acid and histamine. Stimulation of immunoglobulin E receptors by antigen releases only [¹⁴C]arachidonic acid but not [¹⁴C]linoleic acid, [¹⁴C]oleic acid and [¹⁴C]stearic acid, all of which were previously incorporated into phospholipids. [¹⁴C]Arachidonate was found to be incorporated mainly into phosphatidylcholine. The phosphatidycholine rich in arachidonate appeared to be synthesized to a considerable extent by the transmethylation pathway. These findings suggest that in rat basophilic leukemia cells, immunoglobulin E receptors, phospholipid methyltransferases, Ca²⁺ ion channel, and phospholipase(s) that cause release of arachidonic acid and the discharge of histamine are associated.     

Dexamethasone inhibits receptor-activated phosphoinositide breakdown in rat basophilic leukemia (RBL-2H3) cells

The activation of rat basophilic leukemia cells for histamine release is accompanied by Ca2+ influx and arachidonic acid release. IgE receptor but not A23187 ionophore stimulation of these cells also resulted in phosphoinositide breakdown. In these experiments, the culture of these cells with dexamethasone inhibited IgE- and ionophore-mediated histamine release. The concentration for 50% of maximal inhibition was 12 nM, and prolonged exposure to the drug was required, with maximal effect observed in 8 to 15 hr. The inhibitory effect of dexamethasone was reversible (t1/2 for recovery was 16 hr). Dexamethasone blocked the IgE-mediated 45Ca2+ influx and the release of [14C]-arachidonic acid (IC50 of 1 nM and 10 nM respectively). Dexamethasone inhibited the IgE receptor-mediated phosphoinositide breakdown (IC50 of 5 nM). It also decreased arachidonic acid release after A23187 stimulation demonstrating an effect on phospholipase A2. Therefore, exposure of the cells to dexamethasone results in the inhibition of both phospholipase A2 and phospholipase C pathways of arachidonic acid generation.     

Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation

The biochemical events that lead to thrombin-stimulated release of von Willebrand factor and prostacyclin synthesis in cultured endothelial cells are examined. Treatment of human umbilical vein endothelial cells with thrombin results in an instantaneous increase in phospholipid methylation which can be blocked by 3-deazaadenosine, a methyltransferase inhibitor. 3-Deazaadenosine also blocks the thrombin-induced Ca2+ influx into endothelial cells and the release of von Willebrand factor, indicating that these processes are coupled. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) and the Ca2+ ionophore A23187 both bypass the phospholipid methylation and directly stimulate Ca2+ influx and von Willebrand factor release. In contrast to the stimulus-induced von Willebrand factor release, the thrombin-induced prostacyclin synthesis cannot be blocked by 3-deazaadenosine. Similarly, incubation of endothelial cells with EDTA has no influence on the thrombin-induced prostacyclin synthesis, and PMA has no stimulatory effect on prostacyclin synthesis. These observations indicate that thrombin induces different metabolic responses in endothelial cells: phospholipid methylation followed by a Ca2+ influx, which subsequently leads to release of von Willebrand factor, and liberation of arachidonic acid from phospholipids for prostacyclin formation, which is independent of phospholipid methylation and Ca2+ influx.     

Phospholipid methylation affects immunoglobulin E-mediated histamine and arachidonic acid release in rat leukemia basophils

Antigenic stimulation of rat basophilic leukemia cells sensitized with immunoglobulin E causes the release of histamine as well as arachidonic acid and its metabolites. The release of these substances is preceded by an increase in phospholipid methylation. Inhibition of phospholipid methylation is correlated to the inhibition of histamine release. Inhibition of methylation also reduces arachidonate release. Phospholipid methylation appears to be associated with both histamine secretion and the release of arachidonate and its metabolites.     

Phospholipid metabolism, calcium flux, and the receptor-mediated induction of chemotaxis in rabbit neutrophils

Rabbit neutrophils were stimulated with the chemotactic peptide fMet-Leu-Phe in the presence of the methyltransferase inhibitors homocysteine (HCYS) and 3-deazaadenosine (3-DZA). HCYS and 3-DZA inhibited chemotaxis, phospholipid methylation, and protein carboxymethylation in a dose-dependent manner. The chemotactic peptide-stimulated release of [14C]arachidonic acid previously incorporated into phospholipid was also partially blocked by the methyltransferase inhibitors. Stimulation by fMet-Leu-Phe or the calcium ionophore A23187 caused release of arachidonic acid but not of previously incorporated [14C]-labeled linoleic, oleic, or stearic acids. Unlike the arachidonic acid release caused by fMet-Leu-Phe, release stimulated by the ionophore could not be inhibited by HCYS and 3-DZA, suggesting that the release was caused by a different mechanism or by stimulating a step after methylation in the pathway from receptor activation to arachidonic acid release. Extracellular calcium was required for arachidonic acid release, and methyltransferase inhibitors were found to partially inhibit chemotactic peptide-stimulated calcium influx. These results suggest that methylation pathways may be associated with the chemotactic peptide receptor stimulation of calcium influx and activation of a phospholipase A2 specific for cleaving arachidonic acid from phospholipids.     

Biochemical analysis of triggering signals induced by bridging of IgE receptors

Bridging of IgE receptors on rat mast cell plasma membranes induces phospholipid methylation and a monophasic increase in cyclic AMP. The stimulation of phospholipid methylation in the plasma membrane appears to be intrinsic to the processes leading to Ca2+ influx and histamine release. Evidence was obtained that IgE receptors are closely associated with methyltransferases and adenylate cyclase in the plasma membranes. The activation of one enzyme is regulated by the other. An increase in the cyclic AMP level before receptor bridging suppressed phospholipid methylation. On the other hand, inhibition of phospholipid methylation may affect the initial rise in cyclic AMP. Our experiments also indicated that bridging the receptor activates a membrane-associated proteolytic enzyme. Inasmuch as the inhibition of the enzyme activation results in the suppression of both phospholipid methylation and initial rise in cyclic AMP induced by receptor bridging, the proteolytic enzyme may be involved in the activation of methyltransferases and adenylate cyclase.     

Release of histamine and arachidonate from mouse mast cells induced by glycosylation-enhancing factor and bradykinin

Stimulation of normal rat splenic T cells with pertussigen (lymphocytosis-promoting factor from Bordetella pertussis) resulted in the release of a soluble factor that enhanced the assembly of N-linked oligosaccharides to IgE-binding factors during their biosynthesis. The glycosylation-enhancing factor (GEF) is a kallikrein-like enzyme and is purified by absorption to p-aminobenzamidine-Agarose followed by elution with benzamidine. Incubation of normal mouse mast cells with affinity-purified GEF or bradykinin, a product of cleavage of kininogen by kallikrein, resulted in the release of histamine and arachidonate from the cells. Passive sensitization of mast cells with mouse IgE antibody, followed by pretreatment of the cells with a suboptimal concentration of GEF, resulted in an enhancement of antigen-induced histamine release. It was found that GEF and bradykinin induced the same biochemical events in mast cells as those induced by bridging of IgE receptors. Both GEF and bradykinin induced phospholipid methylation and an increase in intracellular cyclic AMP (cAMP). Incorporation of 3H-methyl groups into phospholipids and intracellular cAMP levels both reached a maximum 30 sec after challenge with GEF or bradykinin, and then declined to base-line levels within 2 to 3 min. These biochemical events were followed by 45Ca influx and histamine release; 45Ca uptake reached a plateau value at 2 min, and histamine release reached a maximum at 5 to 8 min. The initial rise in cAMP induced by GEF (or bradykinin) was not inhibited by indomethacin, indicating that the activation of adenylate cyclase is not the result of prostaglandin synthesis. In both IgE-mediated and GEF-induced histamine release, inhibitors of methyltransferases, such as 3-deaza adenosine and L-homocysteine thiolactone, inhibited not only phospholipid methylation but also the cAMP rise and subsequent Ca2+ uptake and histamine release. The results indicate that GEF induces activation of methyltransferases and that phospholipid methylation is involved in the cAMP rise, Ca2+ uptake, and histamine release. The induction of the same biochemical events in the same sequence by bridging of IgE receptors and by GEF (bradykinin) supports the hypothesis that receptor bridging induces the activation of serine protease(s) and cleavage products of this enzyme in turn activate methyltransferases in mast cells.     

Biochemical analysis of glucocorticoid-induced inhibition of IgE-mediated histamine release from mouse mast cells

Pretreatment of mouse mast cells with 10(-7) to 10(-6) M dexamethasone (DM) during overnight sensitization with mouse IgE antibody resulted in inhibition of antigen-induced histamine release and degranulation. The inhibition of both degranulation and histamine release increased linearly with the duration of the treatment; maximal inhibition was obtained after approximately 16 hr with DM. The addition of DM to sensitized mast cells immediately before antigen challenge did not affect the antigen-induced histamine release. DM interacted directly with mast cells by binding to DM-specific cytoplasmic receptors. The treatment of mast cells with DM did not affect the binding of IgE to mast cells or intracellular cAMP levels. Bridging of cell-bound IgE anti-DNP antibody on mouse mast cells either by multivalent DNP-HSA or by anti-IgE induced phospholipid methylation at the plasma membrane and Ca++ influx into the cells. Pretreatment of mast cells with DM inhibited the antigen-induced phospholipid methylation and Ca++ uptake but failed to affect histamine release by Ca++ ionophore A23187. The results suggest that DM treatment inhibits histamine release by the inhibition of the early stage of biochemical processes leading to opening Ca++ channels but does not affect the process distal to Ca++ influx or the binding of IgE molecules to IgE receptors.     

Stimulated release of histamine by a rat mast cell line is inhibited during mitosis

Stimulated histamine release was depressed at least tenfold in mitotic 2H3 rat basophilic cells when compared with interphase cells even though both contained comparable amounts of histamine. Antigen stimulation of IgE-sensitized interphase cells initiated an influx of Ca2+ that preceded secretion of histamine and a similar Ca2+ influx occurred in stimulated mitotic cells. This strongly suggests that during mitosis there is a dramatic inhibition of one or more of the steps on the pathway leading from elevated intracellular Ca2+ to the fusion of secretory granules with the plasma membrane.     

Phospholipase A2 stimulation during cell secretion in rat basophilic leukemia cells

The bridging of IgE receptors on rat basophilic leukemia cells (RBL-2H3) results in a number of biochemical events that accompany histamine secretion. Prominent among these is the release of arachidonic acid from cellular phospholipids, which could be due to the activation of phospholipase enzymes. In the present experiments we studied the intracellular activation of phospholipase A2 (PLA2) during histamine release. RBL-2H3 cells were stimulated through the IgE receptor, and the homogenates were prepared and tested for phospholipase A2 activity on 1-stearoyl-2-[14C]arachidonyl-sn-3-phosphatidylcholine. The amount of activity in the homogenates was dependent on the concentration of secretagogue used to activate the cells. Under optimal conditions there was a 1.86 +/- 0.12-fold (mean +/- SEM, N = 44) increase in the activity found in homogenates of stimulated cells. Activity was present in homogenates prepared 30 sec after cell activation, was optimal between 5 and 10 min, and decreased later. In time course experiments the PLA2 activation preceded histamine release. The activation of the enzyme in the cell occurred in the presence of 10 microM EGTA in the extracellular medium, which completely inhibited release of arachidonic acid and histamine. However, the activity of the enzyme required Ca2+. The PLA2 activity in the homogenates and the extent of cell stimulation for histamine release were maximal at the same concentration of antigen, and both were blocked by the addition of a monovalent hapten. The enzyme in the homogenates was capable of cleaving arachidonic acid from different phospholipids. The production of lysophospholipids could play a critical role in histamine release from cells. These results demonstrate the activation of PLA2 enzyme in cellular homogenates during the secretory process.     

Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.     

Phospholipase activation in the IgE-mediated and Ca2+ ionophore A23187-induced release of histamine from rat basophilic leukemia cells

The importance of phospholipase(s) activation in the IgE-mediated and ionophoreinduced histamine release from the rat basophilic leukemia cell line has been examined. The activation of phospholipase(s) as measured by [¹⁴C]arachidonic acid release and the release of histamine both required Ca²⁺ and were temporally parallel. Inhibition of phospholipase(s) activity by the inhibitors mepacrine and α-parabromoacetophenone also correlated with the inhibition of histamine release. [¹⁴C]Arachidonic acid released by the phospholipase(s) was mainly metabolized to prostaglandin D₂. The inhibition of the cyclooxygenase pathway by indomethacin did not affect histamine release. 5,8,11,14-Eicosatetraynoic acid inhibited both histamine and [¹⁴C]arachidonic acid release suggesting an effect not only on the cyclooxygenase and lipoxygenase pathways but also on the phospholipase(s). These results suggest that activation of phospholipase appears to be necessary for histamine release in the rat bosophilic leukemia cells.     

Biochemical analysis of desensitization of mouse mast cells

Biochemical mechanisms of desensitization were explored by using peritoneal mouse mast cells saturated with monoclonal mouse IgE anti-DNP antibody. It was found that a 1-min incubation of the sensitized cells with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ was sufficient to desensitize the cells completely. The treated cells failed to release a detectable amount of histamine upon incubation with an optimal concentration (0.1 to 1.0 micrograms/ml) of DNP-HSA and Ca2+. Determination of the number of antigen molecules bound to mast cells revealed that only a small (less than 10%) fraction of cell-bound IgE antibody molecules reacted with desensitizing antigen, and that desensitized cells and untreated (sensitized) cells could bind comparable amounts of antigen upon incubation with rechallenging antigen. However, the binding of antigen molecules to desensitized cells failed to induce any of the early biochemical events, i.e., phospholipid methylation, cAMP rise, and 45Ca uptake, as well as histamine release. It was also found that intracellular cAMP levels in desensitized cells were comparable to those in sensitized cells. Desensitization by a suboptimal concentration of DNP-HSA was prevented by inhibitors of methyltransferases, such as 3-deaza adenosine plus L-homocysteine thiolactone. Sensitized cells pretreated with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ and in the presence of the methyltransferase inhibitors responded to an optimal concentration of antigen for histamine release when they were rechallenged in the presence of Ca2+. Inhibition of desensitization by methyltransferase inhibitors was reversed by the addition of S-adenosyl-L-methionine to the system. The results indicated that the activation of methyltransferases, induced by receptor bridging, is involved in the process of desensitization. Desensitization was inhibited by reversible inhibitors of serine proteases, such as p-aminobenzamidine, indole, and synthesized substrates of rat mast cell proteases. It was also found that diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, completely blocked desensitization at the concentration of 10 to 40 nM. This concentration of DFP did not affect the antigen-induced histamine release, whereas 100- to 1000-fold higher concentrations of DFP did inhibit histamine release. The results suggest that serine proteases are involved in both the induction of histamine release and desensitization, and that the protease involved in desensitization is distinct from that involved in triggering histamine release.     

Ionophore A-23187 induced histamine release from rat mast cells and rat basophil leukemia (RBL-1) cells.

Ionophore A-23187 releases histamine from normal mast cells apparently by promoting Ca++ influx (Foreman et al, Nature 245: 249, 1973). In our hands at concentrations of greater than 0.2 mug/ml release occurs in 1 to 2 min, is blocked by metabolic inhibitors, and is unaccompanied by cytotoxicity (trypan-blue uptake, lactic dehydrogenase (LDH) release). At higher doses (0.5 mug/ml) histamine release is followed by significant cytotoxicity, but again Ca++ is required. In parallel studies, we examined cultured rat basophilic leukemia (RBL-1) cells. These cells, which apparently have normal surface receptors for IgE, contained approximately 700 ng histamine/10(6) cells but did not release histamine when IgE-mediated release was looked for. They do not respond to doses of ionophore which would be expected to give non-cytotoxic histamine release. At higher doses histamine release is preceded by progressive LDH release: LDH release is 75% complete at 5 min whereas 10 min are required for 75% maximal histamine release. This reaction requires Ca++ and is temperature dependent but is not inhibited by metabolic poisons (2-deoxyglucose, dinitrophenol, CN-). These studies suggest that either Ca++ does not enter into these cells normally or that one or more mechanisms which are ordinarily triggered by the changes in Ca++ flow are unresponsive in the RBL-1 cells. These studies also underline the importance of ruling out cytotoxicity in ionophore-induced phenomena.     

Role of isoprenoid metabolism in IgE receptor-mediated signal transduction.

In the 2H3 subline of rat basophilic leukemia cells (RBL-2H3), IgE receptor cross-linking stimulates a signal transduction pathway that leads to the secretion of histamine, serotonin, and other inflammatory mediators; the assembly of F-actin; and the transformation of the cell surface from a microvillous to a lamellar or ruffled architecture. We report here that 20 h incubation of RBL-2H3 cells with 10 microM lovastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG CoA reductase), inhibits both the secretory and morphologic responses to IgE receptor cross-linking. Ag-induced Ca2+ mobilization, determined from the influx and efflux of 45Ca2+, and Ag-induced 1,4,5-inositol trisphosphate production are also inhibited in lovastatin-treated RBL-2H3 cells. Under the same conditions, lovastatin does not alter cell proliferation or IgE receptor expression, and it causes only a small impairment of responses initiated by drugs that bypass the earliest steps in the receptor-activated transduction pathway (ionomycin-induced secretion and PMA-induced membrane ruffling). Receptor-mediated Ca2+ mobilization, secretion, and ruffling are all restored by 0.5- to 4-h incubation of lovastatin-treated cells with mevalonic acid, the product of HMG CoA reductase and the first committed intermediate of the isoprenoid biosynthetic pathway. In contrast, dolichol and cholesterol, which are synthesized from products of the isoprenoid pathway, do not restore receptor-activated responses. These data implicate an isoprenoid pathway intermediate in an early step in the IgE receptor-activated signal-transduction sequence. We postulate that this intermediate is required for a newly described post-translational modification of proteins, their post-synthetic isoprenylation. The substrates for this modification include the ras family of GTP-binding proteins and the gamma subunits of the heterotrimeric guanine nucleotide-binding protein.     

Depletion of guanine nucleotides with mycophenolic acid suppresses IgE receptor-mediated degranulation in rat basophilic leukemia cells

In RBL-2H3 rat basophilic leukemia cells, Ag that crosslink IgE-receptor complexes stimulate the turnover of inositol phospholipids, the mobilization of Ca2+ from intra- and extracellular sources, the release of serotonin and other substances from granules and the transformation of the cell surface from a microvillous to a lamellar architecture. This study explores the role of GTP-binding proteins (G proteins) in the control of these biochemical and functional responses. We report that incubating RBL-2H3 cells for 4 h with 10 microM mycophenolic acid (MPA), an inhibitor of de novo GTP synthesis, reduces GTP levels by over 60% and causes an average reduction of 50% in Ag-stimulated serotonin release. This inhibition of secretion is associated with a 50% decrease in the rate of 45Ca2+ influx in MPA-treated cells. In contrast, Ag-stimulated inositol trisphosphate production is only slightly reduced, indicating that the phosphatidylinositol-specific phospholipase C can be activated by Ag in GTP-depleted cells. The membrane responses to IgE receptor cross-linking are unaffected by incubating cells with MPA. Exogenous guanine or guanosine protects the GTP pools in MPA-treated cells and permits normal ion transport and secretory responses to Ag; adenine does not. These results implicate a guanine nucleotide-binding protein in the control of IgE receptor-dependent signal transduction in RBL-2H3 cells. This protein may particularly control the Ca2+ influx pathway that is essential for secretion.     

Inhibition of IgE-mediated histamine release by myosin light chain kinase inhibitors.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK) blocked IgE mediated histamine release from rat basophilic leukemia cell (RBL-2H3) and human basophils dose-dependently. Its IC50 was 20 nM for RBL-2H3 cells and 30 nM for human basophils. There was complete inhibition at the concentration of 1 microM. Wortmannin inhibited partially the A23187 induced histamine release from RBL-2H3 cells (40% inhibition at 1 microM). This inhibition was not accompanied by any significant effect on cytosolic free calcium concentration [( Ca2+]i). KT5926, another MLCK inhibitor, inhibited histamine release comparably with wortmannin and blocked to some degree the increase of [Ca2+]i in RBL-2H3 cells. Thus, the phosphorylation of myosin seems to be involved in signal transduction through Fc epsilon RI.     

Inhibitory effect of xanthones isolated from the pericarp of Garcinia mangostana L. on rat basophilic leukemia RBL-2H3 cell degranulation

Mangostin, Garcinia mangostana L. is used as a traditional medicine in southeast Asia for inflammatory and septic ailments. Hitherto we indicated the anticancer activity induced by xanthones such as alpha-, beta-, and gamma-mangostin which were major constituents of the pericarp of mangosteen fruits. In this study, we examined the effect of xanthones on cell degranulation in rat basophilic leukemia RBL-2H3 cells. Antigen (Ag)-mediated stimulation of high affinity IgE receptor (FcepsilonRI) activates intracellular signal transductions resulting in the release of biologically active mediators such as histamine. The release of histamine and other inflammatory mediators from mast cell or basophils is the primary event in several allergic responses. These xanthones suppressed the release of histamine from IgE-sensitized RBL-2H3 cells. In order to reveal the inhibitory mechanism of degranulation by xanthones, we examined the activation of intracellular signaling molecules such as Lyn, Syk, and PLCgammas. All the xanthones tested significantly suppressed the signaling involving Syk and PLCgammas. In Ag-mediated activation of FcepsilonRI on mast cells, three major subfamilies of mitogen-activated protein kinases were activated. The xanthones decreased the level of phospho-ERKs. Furthermore, the levels of phospho-ERKs were observed to be regulated by Syk/LAT/Ras/ERK pathway rather than PKC/Raf/ERK pathway, suggesting that the inhibitory mechanism of xanthones was mainly due to suppression of the Syk/PLCgammas/PKC pathway. Although intracellular free Ca(2+) concentration ([Ca(2+)](i)) was elevated by FcepsilonRI activation, it was found that alpha- or gamma-mangostin treatment was reduced the [Ca(2+)](i) elevation by suppressed Ca(2+) influx.     

A new model for the mechanism of stimulus-secretion coupling

By combining ultrastructural techniques with a biochemical approach to study the mechanism of mast cell stimulus-secretion coupling and by using purified secretory granules to confirm those early biochemical events which originate from within the secretory granule, a new model for the mechanism of secretory granule exocytosis has emerged. This model not only provides the mechanism by which an activated granule can achieve fusion with the plasma membrane, but it also provides the rationale for the linking of the various early biochemical events to the process of granule activation and thus to exocytosis. Although we still do not understand how the 'activating signal', which results from the stimulation of cell surface receptors, can be conveyed to the granule to cause its activation, we are certain that this 'signal' must cause an influx of water into the matrix of the target granule. This influx of water is what initiates the granule activation process. The major intragranular events which are triggered by this water influx include: (i) de novo membrane assembly; (ii) protein proteolysis; (iii) release of arachidonic acid from matrix-bound phospholipid by phospholipase A2; (iv) initiation of the arachidonic acid cascade and the synthesis of eicosanoids; (v) rapid phospholipid turnover; and (vi) the discharge of matrix materials into the cytoplasm of the activated cell via the fusion of de novo generated vesicles with the perigranular membrane. The ejection of some matrix contents which may include histamine, Ca2+, calmodulin, protease, the products of the arachidonic acid cascade and the products of phospholipid turnover into the cytosole, may serve to turn on the various metabolic machineries needed to initiate a cellular recovery phase.     

Establishment of four mouse mastocytoma cell lines

Four mastocytoma cell lines were isolated from four different mouse mastocytoma tumors. The tumors had been induced in mice treated with tetramethylpentadecane (pristane) and infected with Abelson murine leukemia virus. The cell lines have been carried in culture for over a year and can induce tumors when injected into the mouse strain in which the tumor originated. The cells contain histamine, have high affinity IgE receptors and release histamine by IgE, immune complex or ionophore A23187-induced reactions. This histamine release reaction requires Ca2+, is optimal at 37 degrees C, and is blocked by a number of metabolic inhibitors. There is no requirement for phosphatidylserine. Cloned sublines have been obtained which will be useful for Fc epsilon R, Fc gamma R; and histamine release studies.