Inhibition of compound 48--80 induced histamine liberation from mast cells by triton X-100]

Triton X-100 at concentrations preceding those which liberated histamine, produced dose-dependent inhibition of compound 48/80-induced histamine release from rat mast cells. Triton X-100 (0.00002 1/1) depleted ATP content in the mast cells and blocked compound 48/80-induced histamine release. The inhibition of compound 48/80-induced histamine release and depletion of the ATP content in the mast cells was reversed by glucose (10 mmole). It is concluded that inhibition by Triton X-100 of histamine release induced by compound 48/80 is dependent on inhibition of energy production.     

The isolation and some properties of guinea pig mesenteric mast cells

A technique is described for obtaining isolated mast cells from guinea-pig mesentery by an enzymatic digestion process using hyaluronidase and collagenase. One to 4 × 10⁶ mast cells were obtained from the mesentery of each animal. Isolated mast cells from guinea-pigs of about 400 g were approximately spherical with a mean diameter of 6.1 μm and a mean histamine content of 8.8 pg. Studies on isolated mast cells from sensitised animals showed that the cells were still capable of an anaphylactic release of histamine when challenged with the appropriate antigen. Isolated mast cells did not sensitise when incubated with antibody dissolved in physiological saline but sometimes became weakly sensitised when incubated with the same antibody in isotonic-buffered glucose. Mast cells were found to survive in culture but they were no longer capable of an antigen-induced histamine release.     

Effect of NCDC, a protease inhibitor, on histamine release from rat peritoneal mast cells induced by anti-IgE.

NCDC dose-dependently inhibited histamine release from rat peritoneal mast cells induced by anti-IgE. Moreover, NCDC inhibited Ca(2+)-mobilization from intracellular Ca(2+)-stores as well as histamine release in mast cells activated by anti IgE, the effect on both of these phenomena being closely correlated. Anti-IgE induced a rapid increase in IP3 production from phosphoinositides in mast cells, with its production in 15 sec, followed to baseline levels within 1 min. Anti-IgE stimulated PLC activity on mast cells membrane preparation. NCDC dose-dependently inhibited the generation of IP3. These results suggest that the inhibitory effect of NCDC on the release of histamine induced by anti-IgE is due to, in part at least, the inhibition of PI-specific PLC and that the inhibitory effects of NCDC are involved in intracellular calcium store.     

Concerted stimulation of PI-turnover, Ca2+-influx and histamine release in antigen-activated rat mast cells

Phospholipid metabolism in rat mast cells activated by antigen was examined with reference to phosphatidylinositol (PI) turnover. Upon antigen stimulation, histamine release from passively sensitized mast cells with IgE was potentiated by adding phosphatidylserine (PS). The addition of antigen to [3H]glycerol-prelabeled and sensitized mast cells induced a marked loss of radioactivity of PI and a concurrent accumulation of 1,2-diacylglycerol (DG) and phosphatidic acid (PA) within 5 to 60 sec. Furthermore, this antigen-induced PI breakdown was enhanced in the presence of Mg2+. Histamine release occurred in parallel with PI breakdown. On the other hand, the transient Ca2+ influx into mast cells, as measured by uptake of 45Ca2+, was found to occur quickly after cells were activated by antigen, which was concerted with PI breakdown. These results suggest that enhanced PI turnover may be an important step in the biochemical sequence of events leading to release of histamine, and that not only Ca2+ but also Mg2+ appears to take a part in stimulus-response coupling in rat mast cells.     

SG-HQ2 inhibits mast cell-mediated allergic inflammation through suppression of histamine release and pro-inflammatory cytokines

In this study, we investigated the effect of 3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide) (SG-HQ2), a synthetic analogue of gallic acid (3,4,5-trihydroxybenzoic acid), on the mast cell-mediated allergic inflammation and the possible mechanism of action. Mast cells play major roles in immunoglobulin E-mediated allergic responses by the release of histamine, lipid-derived mediators, and pro-inflammatory cytokines. We previously reported the potential effects of gallic acid using allergic inflammation models. For incremental research, we synthesized the SG-HQ2 by the modification of functional groups from gallic acid. SG-HQ2 attenuated histamine release by the reduction of intracellular calcium in human mast cells and primary peritoneal mast cells. The inhibitory efficacy of SG-HQ2 was similar with gallic acid. Enhanced expression of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, interleukin-4, and interleukin-6 in activated mast cells was significantly diminished by SG-HQ2 100 times lower concentration of gallic acid. This inhibitory effect was mediated by the reduction of nuclear factor-κB. In animal models, SG-HQ2 inhibited compound 48/80-induced serum histamine release and immunoglobulin E-mediated local allergic reaction, passive cutaneous anaphylaxis. Our results indicate that SG-HQ2, an analogue of gallic acid, might be a possible therapeutic candidate for mast cell-mediated allergic inflammatory diseases through suppression of histamine release and pro-inflammatory cytokines.     

Effects of ADP-ribosylation of GTP-binding protein by pertussis toxin on immunoglobulin E-dependent and -independent histamine release from mast cells and basophils

Pretreatment of rat peritoneal mast cells, human basophils, bone marrow-derived mouse mast cells (BMMC) and mouse mast cell line PT-18 cells with 1 microgram/ml pertussis toxin (PT) failed to inhibit immunoglobulin E (IgE)-dependent histamine release from the cells. In BMMC and PT-18 cells, even 20-hr incubation of the cells with 1 microgram/ml PT, which ADP-ribosylates more than 97% of 41 kDa, alpha-subunit of Ni in the cells, failed to affect the IgE-dependent release of histamine or arachidonate. The results indicate that GTP-binding protein, Ni, is not involved in the transduction of triggering signals induced by cross-linking of IgE receptors. In contrast, pretreatment of rat mast cells with 1 ng/ml to 0.1 microgram/ml PT for 2 hr inhibited histamine release induced by compound 48/80 in a dose-dependent manner. A similar pretreatment with PT inhibited thrombin-induced histamine release from BMMC and N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced histamine release from human basophils in a similar dose-dependent fashion. However, even 20 hr of incubation of sensitized BMMC with 1 microgram/ml PT failed to inhibit either thrombin-induced or antigen-induced breakdown of phosphatidylinositides (PI), i.e., the formation of inositol triphosphate and diacylglycerol, Quin-2 signal, and the release of arachidonic acid. The results indicate that the inhibition of thrombin-induced histamine release by PT-treatment is not due to the inhibition of PI-turnover, and that Ni is not involved in thrombin-induced or antigen-induced (IgE-dependent) hydrolysis of phosphatidylinositides in mast cells.     

Influence of the tyrosine kinase inhibitors STI571 (Glivec), lavendustin A and genistein on human mast cell line (HMC-1(560)) activation

The human mast cell line (HMC-1(560)) was used to study the effects of tyrosine kinase (TyrK) inhibition on histamine release in consequence of intracellular Ca2+ or pH changes. This is important since the TyrK inhibitor STI571 (Glivec) inhibits proliferation and induces apoptosis in HMC-1(560). HMC-1(560) cells have a mutation in c-kit, which leads to a permanent phosphorylation of the KIT protein and their ligand-independent proliferation. The TyrK inhibitors STI571, lavendustin A and genistein decrease spontaneous histamine release in 24-h pre-incubated cells. Results are compared with those of the mast cell stabiliser cromoglycic acid, which also drops spontaneous histamine release. When exocytosis is stimulated by alkalinisation, STI571 pre-incubated cells release more histamine than non-pre-incubated cells. Alkalinisation-induced histamine release reaches still higher levels in STI571 cells with activated protein kinase C (PKC) by PMA. We do not observe modifications on histamine release in cells, treated with PKC inhibitors (rottlerin, Gf109203 or G?6976). Lavendustin A- and genistein 24-h incubated cells behave similar to STI571 cells, whereas cromoglycic acid does not show effects after stimulation with alkalinisation. Stimulation of exocytosis with the Ca2+ ionophore ionomycin does not modify histamine response in TyrK inhibited cells. Ca2+ and pH changes are observed after long-time incubation with STI571. Results show that pH is still higher in STI571 pre-incubated cells after alkalinisation with NH4Cl, whereas intracellular Ca2+ concentration remains stable. This work further strength the importance of pHi as a cell signal and suggest that STI571 has transduction pathways in common with other TyrKs.     

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.     

Human skin mast cells: their dispersion, purification, and secretory characterization

Digestion of human foreskin with collagenase and hyaluronidase disperses approximately 3.4 X 10(7) nucleated cells per gram of tissue, of which mast cells constitute 4.7%. These may be purified to 80% by use of density gradient centrifugation. The majority of mast cells (79%) measured between 9 and 13 micron in diameter, and the mean histamine content was 4.6 pg/cell. Viability was demonstrated by trypan blue exclusion by 93% of the cells and the low spontaneous histamine secretion of less than 7% in functional studies. Anti-IgE released up to 17.5% of cell-associated histamine within 5 to 7 min. Calcium ionophore-induced release was optimal with 0.3 microM A23187 when 28.6% histamine was released. Unlike human lung mast cells, skin mast cells released histamine in response to compound 48/80 and poly-L-lysine. This release, which was complete within 20 sec, was totally dependent on intact glycolysis and oxidative phosphorylation and partially dependent on extracellular calcium. The same characteristics were observed with secretion induced by substance P and morphine. The weak activity of eledoisin and physalaemin suggests that the substance P receptor, like that of the rat mast cell, is not of the classical types described for smooth muscle. Morphine-induced secretion was partially blocked by naloxone in a manner not compatible with competitive antagonism at a classical opioid receptor. The sensitivity of skin mast cells to nonimmunologic stimulation clearly distinguishes them from mast cells of the lung and lymphoid tissues and provides evidence of functional heterogeneity within human mast cells.     

Histamine release induced by dendroaspis natriuretic peptide from rat mast cells

Dendroaspis natriuretic peptide (DNP), recently isolated from the venom of the green Mamba snake Dendroaspis angusticeps, is a 38 amino acid peptide containing a 17 amino acid disulfide ring structure similar to that of the natriuretic peptide family. The natriuretic peptide family is known to induce histamine release from human and rat mast cells, but there are no published data concerning the effects of DNP on histamine release from mast cells. The purpose of this study is to investigate whether DNP induces the histamine release from rat peritoneal mast cells (RMPCs) and to determine the mechanism of DNP-induced histamine release from RPMCs. After treatment of RPMC with DNP, mast cell degranulation was observed, and calcium uptake and histamine release were measured. DNP released the histamine, induced the mast cell degranulation, and increased the calcium uptake of RPMCs, in a dose-dependent manner. The results indicate that DNP can increase Ca-uptake and induce histamine release.     

Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology.

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.     

INHIBITION OF RAT PERITONEAL MAST CELL EXOCYTOSIS BY FRUSEMIDE:: A COMPARISON WITH DISODIUM CROMOGLYCATE

Among the loop diuretics, frusemide possesses unique airway protective activities which may be due to the inhibition of airway inflammatory cells such as the mast cell. We previously reported that frusemide and disodium cromoglycate (DSCG) demonstrated a similar profile of inhibitory activities against histamine release from rat peritoneal mast cells activated by various stimuli which increased intracellular calcium via different routes. Furthermore, the inhibitory activities of both compounds demonstrated marked tachyphylaxis and we hence postulated that frusemide and DSCG might share the same mechanism of action which involves the prevention of extracellular calcium influx into the mast cell cytoplasm. The present study confirmed the postulation by (a) demonstrating that cross-tachyphylaxis exists between the two compounds and (b) extending the observations on histamine release to the influx of extracellular calcium (⁴⁵Ca) into rat peritoneal mast cells.© 1997 Elsevier Science Inc.     

Mechanisms of mouse mast cell activation and inactivation for IgE-mediated histamine release.

The IgE-mediated histamine release from mouse mast cells requires Ca++, is optimal at 37 degrees C, and is enhanced by phosphatidylserine. The rate of release is relatively slow. The mast cells can be activated to release histamine by either anti-IgE or anti-Fab antibodies and, in the case of cells from sensitized mice, by the immunizing antigen. The incubation of mast cells with antigen in the absence of Ca++ or phosphatidylserine fails to release histamine. Such cells are desensitized to the further addition under optimal conditions of the same antigen. Desensitization is antigen specific, requires optimal levels of antigen, and occurs at both 30 degrees and 37 degrees C. In contrast, anti-IgE desensitizes all IgE-mediated histamine release reactions.     

The involvement of protein kinase C in exocytosis in mast cells.

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.     

Inhibition by adenosine 3':5'-monophosphate of eicosanoid and platelet-activating factor biosynthesis in the mouse PT-18 mast cell.

A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor.     

Activation and inhibition of mast cells degranulation affect their morphometric parameters

Activation of mast cells, the key cells of allergic inflammation, causes typical morphological changes associated with an increase in volume, that is a function of area and perimeter. The purpose of this study was to evaluate the effect of mast cell activation to degranulate, carried out by the secretagogue Compound 48/80, and of inhibition of this activation carried out by Nedocromil sodium, a mast cell stabilizing drug, on mast cell area, perimeter and shape factor by a computerized image analyzer. Mast cells were isolated and purified by peritoneal lavage of rats (purity >98%) and co-cultured with mouse 3T3 fibroblasts to which they adhere. Cultures were incubated for 10 min at 37 degrees C with culture medium alone (Enriched Medium) or Enriched Medium containing either Nedocromil (10(-4) M) or Compound 48/80 (0.3 microg/ml) or Compound 48/80 and Nedocromil (0.3 microg/ml and 10(-4) M respectively). Supernatants were then assessed for histamine release, as a marker of mast cell activation and the cell monolayers were fixed and stained with an alcoholic-acidic toluidine blue solution and examined with a computerized image analyzer connected with a light microscope. Mast cells incubated in Enriched Medium or Nedocromil possessed similar morphometric parameters. Mast cells activated with Compound 48/80 (70% histamine release) had a significant increase in area and perimeter and a decrease in shape factor in comparison to mast cells in Enriched Medium alone. Simultaneous incubation of mast cells with Compound 48/80 and Nedocromil significantly inhibited their histamine release (36% histamine release) and the increase in area and perimeter, but did not affect significantly their shape factor, in comparison with mast cells incubated with Compound 48/80 alone. These data clearly show that there is a relationship between mast cell activation, consequent histamine release and changes in cell area, perimeter and shape factor and that Nedocromil not only inhibits mast cell histamine release but also the activation induced morphometric changes in mast cells.     

Stimulation of histamine release and arachidonic acid metabolism in rat peritoneal mast cells by thapsigargin, a non-TPA-type tumor promoter

Thapsigargin, a non-TPA-type tumor promoter, releases histamine and stimulates arachidonic acid metabolism in rat peritoneal mast cells. In order to clarify the relationship between the histamine-releasing activity and the arachidonic acid metabolism-stimulating activity of thapsigargin in mast cells, the effects of cyclooxygenase inhibitors, indomethacin and ibuprofen, a lipoxygenase inhibitor, AA861, and dual inhibitors for cyclooxygenase and lipoxygenase, nordihydroguaiaretic acid and BW755C, on histamine release and arachidonic acid metabolism were examined. High-performance liquid chromatography analysis revealed that the peritoneal mast cells preferentially produce prostaglandin D2 by thapsigargin treatment. These inhibitors suppressed thapsigargin-induced prostaglandin D2 production in a dose-dependent manner, but failed to inhibit histamine release, suggesting that the mechanisms for stimulation of histamine release by thapsigargin is not dependent on increased arachidonic acid metabolism. Time-course experiments of histamine release and the release of radioactivity from [3H]arachidonic acid-labeled mast cells also provide evidence for a difference in mechanism.     

Catestatin (CgA344-364) stimulates rat mast cell release of histamine in a manner comparable to mastoparan and other cationic charged neuropeptides

Catestatin (bovine CgA(344-364)) is a cationic peptide, which besides reducing catecholamine secretion from chromaffin cells in vitro also acts a potent vasodilator in the rat in vivo. The alleged histamine releasing effect of catestatin was tested in vitro in rat mast cells. The most active domain of catestatin (bovine CgA(344-358): RSMRLSFRARGYGFR) caused concentration-dependent (0.01-5 microM) release of histamine from peritoneal and pleural mast cells. The potency and efficacy of catestatin was higher than for the wasp venom peptide, mastoparan. Only in the pleural cells was neurotensin (NT) more potent than catestatin, mastoparan and substance P (SP), consistent with a receptor-mediated histamine release by neurotensin. Amongst these cationic peptides, substance P was least effective. The acidic CgA peptide (WE-14, bovine CgA (324-337)) neither stimulated nor modulated histamine release by the cationic peptides. The catestatin and neurotensin evoked histamine release were suppressed by pertussis toxin (PTX), suggesting involvement of a G(i) subunit. Electron micrographs of rat pleural mast cells responding to catestatin revealed a concentration-dependent discharge of granular material. We propose that catestatin activates histamine release from rat mast cells by a mechanism analogous to that already established for mastoparan and other amphiphilic cationic neuropeptides (the peptidergic pathway) and distinct from the mechanism of inhibition of catecholamine release from chromaffin cells.     

Antiallergic mechanisms of beta-adrenergic stimulants in rats.

Antiallergic mechanisms of beta-adrenergic stimulants were investigated in rats. Isoproterenol administered intravenously inhibited IgE antibody-mediated homologous passive cutaneous anaphylaxis (PCA) and histamine-induced cutaneous reaction (HCR) elicited at the same time in the same rats significantly. The inhibition of PCA was more potent than that of HCR, suggesting that PCA is inhibited by at least 2 mechanisms. One is the inhibition of vascular permeability increase. In vivo histamine release in the rat peritoneal cavity caused by intravenous antigen was inhibited by the intravenous administration of isoproterenol or salbutamol dose-dependently. On the contrary, when the histamine release in the peritoneal cavity was caused by intraperitoneal antigen, isoproterenol or salbutamol administered simultaneously with antigen failed to inhibit the reaction. Furthermore, antigen-induced histamine release from sensitized peritoneal exudate cells in vitro was not inhibited by isoproterenol or salbutamol. These results indicate that the primary target of beta-adrenergic stimulants is the vascular endothelium, and that the direct inhibition of chemical mediator release from mast cells does not play an important role for the inhibition of PCA and in vivo histamine release in the peritoneal cavity in rats. Beta-adrenergic stimulants therefore may prevent intravenously administered antigen from activating sensitized mast cells through affecting endothelial cells.     

Rat mast cell phospholipase A2: activity toward exogenous phosphatidylserine and inhibiton by N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylserine

The presence of phospholipase A2 in intact rat peritoneal mast cells was investigated by using two synthetic radiolabeled phosphatidylserine (PS) substrates. Incubation of intact cells with 1-oleoyl-2-[3H]oleoyl-PS resulted in the release of a considerable quantity of [3H]oleic acid from the substrate. To establish that [3H]oleic acid release was mediated via direct enzymatic attack at the sn-2 position, we measured release of the [3H]serine moiety from the glycerol backbone of 1,2-dimyristoylphosphatidyl[3H]serine. This activity, which represents the combined actions of phospholipases C and D, was 10-fold lower than [3H]oleic acid release, indicating that neither of these enzymes is required for the release of the preponderance of [3H]oleic acid. These results establish the existence in intact rat mast cells of a phospholipase A2 active toward exogenous PS. Over the concentration range at which exogenous PS activates mast cell secretion, intact mast cells and broken cells possessed nearly equal levels of phospholipase A2 activity, and enzyme activity was 3--4-fold higher toward PS than phosphatidylcholine. Several agents were tested for their ability to inhibit phospholipase A2 in intact mast cells. Of the agents tested, an N-substituted derivative of PS previously identified as an inhibitor of mast cell secretion was shown to be a particularly potent and efficacious inhibitor of mast cell phospholipase A2. The concentration dependence of enzyme inhibition paralleled inhibition of histamine secretion, providing a strong positive correlation between the level of phospholipase A2 in mast cells and the capacity for secretion.