Modulation of chymase-mediated rat serosal mast cell degranulation by trypsin or diisopropyl fluorophosphate.

Exposure of rat serosal mast cells (RSMC) to chymase, an endogenous secretory granule serine protease, at 37 degrees results in exocytosis, as determined by beta-hexosaminidase release. As the number of RSMC is increased with a set amount of chymase, the net percentage beta-hexosaminidase release decreases linearly, implying a finite set of cellular interactions per chymase unit. Pretreatment of RSMC with trypsin at 37 degrees renders them refractory to subsequent exocytosis mediated by chymase in a dose- and time-dependent fashion, with complete refractiveness occurring by 15 min at 37 degrees with 2.5 micrograms trypsin/ml. Anti-IgE-mediated coupled activation-secretion of RSMC is not affected by the same trypsin pretreatment. When RSMC are pretreated with trypsin (2.5 micrograms/ml) for 0-120 min at 1 degree a progressive loss of sensitivity to activation by chymase at 37 degrees occurs. RSMC susceptibility to chymase-mediated degranulation after trypsin pretreatment can be partially regenerated by culturing the RSMC for about 24 hr in medium at 37 degrees. These findings suggest that a trypsin-sensitive constituent, possibly a receptor or substrate, is necessary for the functional interaction of chymase with RSMC. When added with diisopropyl fluorophosphate (DFP), chymase does not induce RSMC degranulation at 37 degrees. However, if the DFP is removed before addition of chymase at 37 degrees or is added after the chymase-priming event occurs at 1 degree, subsequent degranulation at 37 degrees is not inhibited. Thus, the induction and not the secretion phase is DFP-inhibitable in chymase-induced activation-secretion. In addition, the priming but not the exocytosis phase of chymase-initiated RSMC activation-secretion, which is not dependent on temperature and calcium ion concentration, involves a cellular trypsin-sensitive protein.     

Inhibition of mediator and cytokine release from dispersed nasal polyp cells by mizolastine

BACKGROUND: Mizolastine is a potent and selective H1-receptor antagonist with antiallergic properties; in in-vitro animal models, mizolastine was shown to inhibit 5-lipoxygenase activity and to decrease the release of leukotrienes (LT) and tumor necrosis factor-alpha (TNF-alpha). This study investigated the effects of three concentrations of mizolastine (0.1, 1.0, 10 microM) on the release of LT (LTB4 and LTC4/D4) and prostaglandin D2 (PGD2) after stimulation by anti-IgE, and on the spontaneous release of cytokines (TNF-alpha and granulocyte/macrophage-colony-stimulating factor [GM-CSF]), from dispersed cells obtained from surgically resected nasal polyps of patients with nasal polyposis. METHODS: Cells from nasal polyps were obtained using enzymatic dispersion. For experiments involving the measurement of LT and PGD2, the cells were preincubated with mizolastine or its dissolution vehicle for 20 min prior to challenge with 10 microg/ml epsilon-chain specific anti-IgE for 45 min at 37 degrees C; for the cytokine release, cells were incubated with mizolastine or its dissolution vehicle for 24 h. LT and PGD2 were measured by enzyme immunoassay (EIA) and cytokines by enzyme-linked immunosorbent assay (ELISA) using commercially available kits. RESULTS: Mizolastine inhibited significantly and in a dose-dependent manner the release of LTB4 and TNF-alpha at all concentrations, LTC4/D4 at 10 microM, and GM-CSF from 1 microM; no effect was observed on the release of PGD2. CONCLUSION: Mizolastine inhibits the release of LT, TNF-alpha and GM-CSF in this in vitro model, which mimics closely the inflammatory cells of allergic rhinitis.     

Olopatadine inhibits TNFalpha release from human conjunctival mast cells.

BACKGROUND: Tumor necrosis factor-alpha (TNFalpha) release likely plays a crucial role in allergic ocular inflammation via increasing ICAM-1 on epithelial cells and triggering other proinflammatory events. The immediate and prolonged release of TNFalpha from human conjunctival mast cells in response to allergen challenge is potentially an important target for therapeutic intervention, yet the effect of ocular anti-allergic agents on this process has not been examined. Olopatadine (Patanol) is a clinically effective dual-action ophthalmic anti-allergic agent that has been shown to inhibit mast cell histamine, tryptase, and PGD2 release in vitro and promote decreased H1 receptor binding activity in vitro and functional H1 receptor antagonism in vivo. OBJECTIVE: To investigate the effect of olopatadine on TNFalpha release from anti-IgE antibody challenged purified human conjunctival mast cells. METHODS: Human conjunctival mast cells were purified (>95%) from cadaveric tissues using a procedure combining enzymatic digestion and Percoll gradient centrifugation. These cells were incubated with olopatadine for 30 minutes then challenged with anti-IgE antibody for 90 minutes. Supernatants were analyzed for TNFalpha. RESULTS: Purified human conjunctival mast cells responded to anti-IgE antibody challenge with TNFalpha release in a concentration dependent manner (optimum concentration was 10 microg/mL). Olopatadine pre-incubation resulted in a dose-dependent decrease in anti-IgE antibody mediated TNFalpha release (IC50 = 13.1 microM). At a concentration of 3 mM olopatadine reduced TNFalpha release to the level of unchallenged controls. CONCLUSION: Olopatadine inhibited anti-IgE antibody-mediated release of TNFalpha from human conjunctival mast cells. This effect could contribute to the long duration of anti-allergic activity reported for the drug.     

Cleavage of a rat serosal mast cell membrane component during degranulation mediated by chymase, a secretory granule protease

Exogenous addition of purified chymase, a rat serosal mast cell (RSMC) chymotryptic enzyme, results in RSMC degranulation at 37 degrees, but not at 1 degree. Chymase can cause an active site-dependent inducing event at 1 degree such that RSMC degranulation occurs if the cells are later incubated at 37 degrees. RSMC exposed to chymase or other stimuli were surface radiolabelled using 125I and Iodo-Gen, solubilized with 1% Nonidet-40, and the resulting 25,000 g supernatants analysed by SDS-PAGE and autoradiography. A 125I-labelled RSMC membrane protein of approximate 90,000 MW decreased upon exposure to either chymase or alpha-chymotrypsin (alpha-CT) for 5 min at 37 degrees or to chymase for 60 min at 1 degree. Exposure of RSMC to the secretagogues ionophore A23187, compound 48/80, and anti-IgE for 5 min at 37 degrees resulted in beta-hexosaminidase (a secretory granule enzyme) release, but did not cause a detectable change in the 90,000 MW surface-labelled protein. Lima bean trypsin inhibitor, which inhibits both the esterase and RSMC degranulation activities of chymase and alpha-CT, prevented the disappearance of the 125I-labelled 90,000 MW band when added with chymase or alpha-CT. Exposure of RSMC to chymase at 1 degree for 0-10 min, prior to addition of LBTI, led to a progressive disappearance of the 90,000 MW band, which corresponded to the kinetics of priming for subsequent RSMC degranulation at 37 degrees. When RSMC were exposed to trypsin (2.5 micrograms/ml) for 0-120 min at 1 degree, a progressive disappearance of the 90,000 MW band occurred, in association with a loss of sensitivity to subsequent activation by chymase at 37 degrees. The disappearance of the 90,000 MW determinant in association with chymase-mediated priming for degranulation and the inability of chymase to mediate degranulation of trypsin-treated RSMC, which lack this membrane protein, suggests that it is involved in chymase-mediated RSMC degranulation.     

Antiallergic activity profile in vitro of RHC 2963 and related compounds

RHC 2963 (7-methyl-pyrido (3',2':4,5)-thieno (3,2-d)-1,2,3 triazine-4(3H)-one and 20 related compounds have been investigated for their antiallergic activities in 3 in vitro models of anaphylaxis and for their effects on cyclic nucleotide phosphodiesterases (cNUC-PDE) from purified rat mast cells (RMC). Nine compounds were potent (I50 less than or equal to 80 microM) inhibitors of antigen-induced release of histamine (AIR) from RMC, 2 compounds inhibited anti-IgE-induced release of histamine from human basophils (I50 less than or equal to 60 microM) and one compound inhibited AIR from guinea pig lung slices (I50 = 55 microM). RHC 2963 was 18 times more potent than disodium cromoglycate (DSCG) as inhibitor of AIR from RMC and had an activity profile identical to that of DSCG in the following respects: loss of inhibitory activity with increasing preincubation time, tachyphylactic properties and inability to inhibit non-immunologic release of histamine induced by compound 48/80. Neither RHC 2963 nor DSCG had any effect on anti-IgE-induced release of histamine from human basophils or IgG1-mediated release of histamine from guinea pig lung. Twelve of the compounds in this chemical series were more potent than theophylline as inhibitors of cyclic AMP and/or cyclic GMP phosphodiesterase (PDE) from RMC. Paired regression analysis of the I50 values for inhibition of AIR and cNUC-PDE from RMC revealed no statistically significant correlation between the inhibition of AIR and inhibition of cAMP- or cGMP-PDE. We conclude: (1) RHC 2963 and some of the related compounds are potent inhibitors of immunologic release of histamine from RMC with a mechanism of action similar to that of DSCG, and (2) inhibition of cAMP- or cGMP-PDE by these compounds is not the biochemical mechanism by which they inhibit AIR from RMC.     

The effect of heliox treatment in a rat model of focal transient cerebral ischemia

At nerve terminals G protein coupled receptors modulate neurotransmitter release probability. We recently showed that prolonged activation of metabotropic glutamate receptor 7, mGlu7 receptor, potentiates glutamate release. This signalling involves phospholipase C activation via a pertussis toxin insensitive G protein, the hydrolysis of phosphatidylinositol (4,5)-bisphosphate, and the subsequent activation of the non-kinase diacylglycerol binding protein Munc13-1 which primes synaptic vesicle for exocytosis at the active zone. Here we found that inhibitors of diacylglycerol metabolism (diacylglycerol kinase inhibitor II and diacylglycerol lipase inhibitor RHC80267) remarkably reduce the time of mGlu7 receptor stimulation required for glutamate release potentiation in mice cerebrocortical nerve terminals. We conclude that changes in diacylglycerol levels at nerve terminals control the efficiency of the exocytotic release machinery.     

Effect of adenosine analogues and cAMP-raising agents on TNF-, GM-CSF-, and chemotactic peptide-induced degranulation in single adherent neutrophils.

Adenosine and adenosine analogues are potent inhibitors of the respiratory burst in neutrophils. Most investigators, however, have found little or no effect of these compounds on neutrophil degranulation from cytochalasin B-treated neutrophils in suspension. We have instead investigated the effect of adenosine and 2-chloroadenosine on degranulation in adherent neutrophils in the absence of cytochalasin B. Both adenosine and 2-chloroadenosine were effective inhibitors of lactoferrin secretion induced by the chemotactic peptide N-formyl-methionine-leucyl-phenylalanine (fMLP) [50% inhibitory concentration (IC50) of less than 10(-6) M]. Secretion induced by tumor necrosis factor (TNF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was inhibited only at high concentrations (IC50 of approximately 10(-4) M). In the presence of cytochalasin B no inhibitory effect of 2-chloroadenosine was seen. The effect of cAMP-raising agents on secretion from adherent neutrophils was also investigated. Dibutyryl cAMP at 0.2 mM reduced secretion in response to fMLP by 50% but did not inhibit TNF- and GM-CSF-induced degranulation. At a concentration of 2.0 mM dibutyryl cAMP also inhibited exocytosis in response to the two cytokines. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 300 microM reduced fMLP-induced degranulation, whereas a concentration of 1 mM was required to inhibit TNF- and GM-CSF-mediated secretion. The adenylate cyclase activator forskolin (50 microM) alone did not inhibit secretion in response to TNF or fMLP. However, in combination with IBMX (300 microM), forskolin (50 microM) reduced both TNF- and fMLP-induced secretion to less than 10%. PMA-induced exocytosis was unaffected by all these agents. In conclusion, adenosine appears to be an effective inhibitor of neutrophil granule protein secretion induced by fMLP but only a weak inhibitor of exocytosis in response to TNF or GM-CSF. Secretion in response to fMLP was also found to be more susceptible to a rise in cAMP than degranulation induced by TNF and GM-CSF.     

Effects of thiazinamium chloride on histamine release from rat peritoneal mast cells and on phosphodiesterase activity in guinea pig lung

The effect of thiazinamium Cl (TCl) on histamine release from rat peritoneal mast cells (RPMC) was investigated. Although TCl inhibited compound 48/80-induced histamine release moderately (IC50 value 40 microM), the drug was a weaker inhibitor of ovalbumin-induced histamine release (100 microM, -21%). In contrast, promethazine HCl (PHCl) was more effective against antigen-induced histamine release (IC50 value 13 microM) than against compound 48/80-induced histamine release (100 microM, -53%). Disodium cromoglycate (DSCG) was effective against both antigen and compound 48/80-induced release of histamine with IC50 values of 7 and 1 microM, respectively. Neither TCl nor DSCG at 1 mM increased spontaneous release of histamine from RPMC, whereas PHCl induced spontaneous release by over 50% at 1 mM. TCl did not inhibit phosphodiesterase (PDE) activity in guinea pig lung at 1 mM, whereas theophylline and DSCG inhibited PDE with IC50 values of 1.1 and 0.32 mM, respectively. These data suggest that high local concentrations of TCl may reduce histamine release during an asthmatic attack and improve its effectiveness as a bronchoprotectant.     

Effect of chronic hypoxia on voltage-independent calcium influx activated by 5-HT in rat intrapulmonary arteries

5-Hydroxytryptamine (5-HT) is a potent pulmonary vasoconstrictor and mitogenic agent whose concentration increases in pulmonary hypertensive patients. Chronic hypoxia induces selective pulmonary arterial hypertension; therefore, we investigated chronic hypoxia effect on the calcium and contractile responses to 5-HT focusing on voltage-independent calcium influx in rat intrapulmonary arteries. Chronic hypoxia, induced by introducing rats in a hypobaric chamber for 3 weeks, potentiated the contraction to 5-HT and this effect was insensitive to nitrendipine. Calcium signal to 5-HT was characterized by a transient followed by a sustained phase in both normoxia and chronic hypoxia. The sustained phase was dependent on extracellular calcium and inhibited by lanthanum. RHC 80267, a specific inhibitor of diacylglycerol lipase, reduced the 5-HT-induced calcium influx in chronic hypoxia but not in normoxia. Furthermore, unlike gadolinium, RHC 80267 inhibited more the contraction to 5-HT in chronic hypoxia. Despite the apparent role of voltage-independent calcium channels in chronic hypoxia, Western blot and flow cytometry analyses demonstrated no variations in TRPC₆ expression. This study shows for the first time that the 5-HT-induced calcium and contractile signals in chronic hypoxia are more dependent on a voltage-independent, RHC 80267-sensitive calcium influx and the hyperreactivity to 5-HT may thus be explained by this influx.     

Role of protein kinase C in histamine release from human basophils

In this study, we investigated the role of calcium and phospholipid-dependent protein kinase (protein kinase C, PKC) in the modulation of histamine release from human basophils. A novel and potent inhibitor of PKC, K-252a, inhibited the release of histamine induced by anti-IgE in a dose-dependent manner with ID50 (the dose required for 50% inhibition of histamine release) of 2.2 x 10(-8) M. Histamine release stimulated with 12-0-tetradecanoyl-phorbol-13-acetate(TPA) was also suppressed by K-252a with maximal inhibition of 48.0 +/- 9.3% at 10(-7) M. In contrast, K-252a did not inhibit the release of histamine in response to FMLP and ionophore A23187. Another inhibitor of PKC, H-7, exhibited a dose-dependent inhibition of anti-IgE-induced histamine release with ID50 of 8.6 x 10(-4) M. H-8 and HA1004, which closely resemble H-7 in chemical structure but are less potent in inhibiting PKC, did not inhibit histamine release stimulated with anti-IgE, but rather enhanced the release at higher concentrations. These results strongly suggest that PKC activation plays a crucial role in the mediation of IgE-mediated histamine release from human basophils.     

Prostaglandin D2 generation in mouse bone marrow-derived mast cells exposed to dexamethasone is associated with endogenous peroxidase activity

The appearance of fixative-sensitive peroxidase activity in the nuclear envelope and endoplasmic reticulum of bone marrow-derived mast cells (BMMC) cultured in the presence of 1 microM dexamethasone (DM) for up to 14 days and its relationship with immunologic release of prostaglandin D2 (PGD2) by these cells were studied. Endogenous peroxidase activity, previously shown as a marker of arachidonic acid metabolism in various cell types, was visualized by cell incubation in 3,3' diaminobenzidine-containing solution before glutaraldehyde fixation. PGD2 release was induced by passive sensitization of BMMC with an optimal dose of monoclonal IgE and subsequent challenge with specific a antigen. We found that 4-week-old BMMC, used as the starting population of the present study, exhibited immature morphologic features, did not present peroxidase activity when cytochemically processed, and released minute amounts of PGD2 in response to IgE-dependent stimulation. When such BMMC were exposed to DM during 24 hours, they showed aldehyde-inhibited peroxidase activity in the perinuclear envelope and a few endoplasmic reticulum segments. As compared with untreated cells, 24-hour DM-exposed BMMC released higher amounts of PGD2 upon immunologic stimulation. After an additional 14-day period of DM exposure, an intense peroxidase activity was detected in the perinuclear envelope and the endoplasmic reticulum of BMMC, which, under immunologic stimulation, released as much as 42.4 +/- 14.7 ng of PGD2/1 x 10(6) cells. Aminotriazole (20 and 50 mM) extinguished both peroxidase activity and PGD2 release from BMMC whereas indomethacin (1 microM) suppressed PGD2 production, but did not alter endogenous peroxidase activity. Previous cell fixation with glutaraldehyde totally inhibited endogenous peroxidase reaction in DM-exposed BMMC. Moreover, 14-day DM-exposed BMMC exhibited morphologic characteristics of mature mast cells and possessed alcian blue+/safranin+ granules. Therefore, the present data suggest that appearance of peroxidase activity in the nuclear envelope and the endoplasmic reticulum of DM-exposed BMMC is associated with the ability of the cells to synthetize PGD2 and appears as a cytochemical marker of the in vitro maturation of mouse bone marrow-derived mast cells.     

Differential effect of hypertonicity on FMLP-, anti-IgE- and Ca2+ ionophore A23187-induced histamine release from human basophil leukocytes.

Effects of different extracellular Na+ and K+ concentrations (respectively, 135, 155, 220, 260 mM NaCl, and 2.7, 20, 50, 100 mM KCl) on IgE-dependent and IgE-independent histamine release from human basophils were examined. High extracellular Na+ and K+ concentrations were shown to reduce N-formyl-methionyl-leucyl-phenyl-alanine- (FMLP), but not anti-IgE- or Ca2+ ionophore A23187-induced histamine release. A high extracellular Ca2+ (7.2 mM CaCl2) concentration increased basophil response to anti-IgE and FMLP. The enhancement of FMLP- but not of anti-IgE-induced histamine release was antagonized by high extracellular Na+ and K+ concentrations. When leukocytes were suspended in isotonic choline chloride solutions (choline is a nonpermeant monovalent cation), an enhancement of anti-IgE- and FMLP-induced histamine release was observed. This suggests that monovalent cations, namely Na+ ions, at physiological concentrations, downregulate histamine release from human basophils. At high choline chloride concentrations, FMLP-, but not anti-IgE-induced histamine release was inhibited. Thus, the reduction of FMLP-evoked histamine secretion from human basophils seems to be due to hypertonicity and not to the type of monovalent cation, either permeant or nonpermeant, contained in extracellular milieu. The different effects of a hypertonic solution on anti-IgE and FMLP-induced histamine release are probably related to the different cell activation pathways triggered by the two stimuli.     

PGE suppresses excessive anti-IgE induced cysteinyl leucotrienes production in mast cells of patients with aspirin exacerbated respiratory disease

BACKGROUND: Aspirin causes bronchospasm in patients with aspirin exacerbated respiratory disease (AERD). The contribution of mast cells to the increased cysteinyl-leucotrienes (cys-LTs) detected in AERD patients is however not defined. AIMS OF THE STUDY: Effects of prostaglandin (PG) E(2) and inhibitors of cyclooxygenase (COX) and lipoxygenase (LO) pathways on mediator release from cultured mast cells of normal subjects, aspirin tolerant asthma (ATA) and AERD patients were compared to better define the role of mast cells in AERD. METHODS: Mast cells were cultured from peripheral blood progenitors and were activated by anti-IgE. Histamine, PGD(2) and cys-LTs released were then determined. RESULTS: Basal release of all three mediators was similar in all subjects. Although the release of all three mediators was increased by anti-IgE, mast cells from AERD patients produced significantly more cys-LTs (6.9 +/- 2.0 ng/10(6) cells) than normal and ATA subjects (2.3 +/- 0.8 and 1.7 +/- 0.5 ng/10(6) cells, respectively). While COX and LO pathway inhibitors did not affect anti-IgE induced histamine release, they significantly suppressed the production of PGD(2) and cys-LTs, respectively, in all patients. PGE(2) significantly enhanced anti-IgE induced histamine and PGD(2) release from mast cells of normal subjects but not those of ATA and AERD patients. In contrast, PGE(2) suppressed only anti-IgE induced cys-LTs release from mast cells of AERD patients. CONCLUSION: We speculate that overproduction of cys-LTs is unique to mast cells of AERD patients and is particularly sensitive to suppression by PGE(2). Consequently reduction of PGE(2) production by aspirin removes this endogenous control of cys-LTs overproduction, resulting in asthma attack.     

"Low" concentrations of sodium fluoride inhibit neurotransmitter release from the guinea-pig superior cervical ganglion

The role of G proteins and related second messenger system on the modulation of acetylcholine release from [3H]choline-preloaded guinea-pig superior cervical ganglion was investigated using the potent general activator NaF. The electrically evoked (1 Hz, 5 min) [3H] release was inhibited by "low" F- concentrations (1-2.5 mM), by the adenylyl cyclase blocker MDL 12330A (10 microM), alone and in combination with 1 mM NaF, and increased by 0.5 mM 8Br-cAMP, 100 microM forskolin and 0.5 mM 3-isobutyl-1-methylxantine. No effect of 1 mM F- was observed on spontaneous release. Fluoride-induced inhibition was counteracted by the G protein blocker sulmazole (1 mM), forskolin and alteration of calcium influx by increasing [Ca2+]out from 2.2 to 6 mM, raising the rate of stimulation (10 Hz, 30 s), or broadening the presynaptic action potential with 10 microM 4-aminopyridine and 50 microM tetraethylammonium chloride. Thus a NaF-sensitive G protein, linked to cAMP synthesis, is determinant for the inhibition of neurosecretion in this cholinergic synapse, involving Ca2+-dependent mechanisms.     

Effect of loop diuretics on rat peritoneal and human lung mast cells

The effects of furosemide and bumetanide on immunologically stimulated rat peritoneal and human lung mast cells were compared. Furosemide and bumetanide had different modulatory actions on the rat peritoneal mast cell. Furosemide inhibited anti-IgE-induced histamine release. Preincubation of the cells with the drug, prior to anti-IgE stimulation, significantly reduced furosemide's inhibitory effect. In contrast, bumetanide potentiated anti-IgE-induced histamine secretion from the rat peritoneal mast cell. Both diuretics were modest inhibitors of anti-IgE-mediated histamine release from human lung mast cells. For furosemide, inhibition decreased with preincubation, while preincubation increased bumetanide's inhibitory action.     

Inhibition of cAMP increase by an anti-allergic agent, NCO-650, during histamine release

Antigen and concanavalin A (Con A) induced an increase in cAMP and histamine release from rat peritoneal mast cells. In a dose-dependent manner, the compound, NCO-650, significantly inhibited both the initial and secondary increases in cAMP stimulated by antigen, anti-IgE and Con A in rat peritoneal mast cells. IC50 values of NCO-650 for cAMP increase stimulated by antigen, anti-IgE and Con A were 3.8, 3.4 and 2.8 microM, respectively.     

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on substance P-induced histamine release from rat peritoneal mast cells

12-O-tetradecanoylphorbol-13-acetate (TPA, 1 to 30 ng/ml) produced a dose-related inhibition of substance P (SP)-induced histamine release from rat peritoneal mast cells. TPA itself induced some histamine release over this concentration range (maximum release about 20% of total). Maximum inhibition of SP-induced release by TPA required preincubation with TPA for at least 10 min. The inhibitory action of TPA was observed in the absence as well as in the presence of extracellular calcium (0.4 mM). Inhibition of diacylglycerol kinase by R 59022 or of diacylglycerol lipase by RHC 80267 reduced SP-induced histamine release. Oleolylacetylglycerol (OAG, 50 M) inhibited histamine release induced by SP but was less potent than TPA. It is concluded that protein kinase C activation in rat peritoneal mast cells is associated with inhibition of SP-induced histamine release.     

CI-922--a novel, potent antiallergic compound--I. Inhibition of mediator release in vitro

CI-922 (3,7-dimethoxy-4-phenyl-N-1H-tetrazol-5-yl-4H-furo[3,2-b]-indole- 2-carboxamide, L-arginine salt) is a novel antiallergy compound which inhibits the release of the inflammatory mediators histamine and leukotriene (LT) from stimulated cells. CI-922 showed potent, effective inhibition of antigen-induced mediator release from human basophils and isolated guinea pig lung. The drug inhibited ragweed or housedust-induced histamine release from basophils of allergic human donors (IC50 = 8.6 microM). The antiallergy agents proxicromil (IC50 = 80 microM) and cromolyn (100 microM) were less potent than CI-922 or inactive, respectively. In fragmented lung from actively sensitized guinea pigs, CI-922 (IC50 = 1.5 microM), blocked the antigen-induced production of LT and was a more potent inhibitor of histamine release (IC50 = 13.4 microM) than proxicromil (IC50 = 72.9 microM), or cromolyn (inactive at 1 mM). CI-922 (IC50 = 0.9 microM) completely inhibited repeated contractions of guinea pig lung strips that were induced by low antigen concentration in the presence of antihistamine (H1). Nordihydroguaiaretic acid (NDGA) (IC50 = 2.8 microM), proxicromil (IC50 = 6.2 microM) and the LT antagonist FPL-55712 (IC50 = 3.3 microM) also were fully effective, but cromolyn (300 microM) was inactive. In other experiments, CI-922 (IC50 = 7.0 microM) inhibited a strong, nonrepeatable lung contraction induced with high antigen concentration (histamine responses blocked), and was six times more potent than FPL-55712. Other investigations in isolated tissue preparations showed CI-922 to be a weak inhibitor of LT or histamine-induced effects with no anticholinergic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide-, chemotactic peptide- and phorbol ester-induced exocytosis in HL-60 leukemic cells

Undifferentiated and differentiated HL-60 leukemic cells possess nucleotide receptors which functionally couple to phospholipase C via pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins). We investigated the role of extracellular nucleotides in the regulation of beta-glucuronidase release in HL-60 cells. In dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells, the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), the phosphorothioate analogue of ATP, adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]), and UTP increased cytosolic Ca2+ from 100 nM up to 1.2 microM with EC50 values of 4 nM, 1 microM and 100 nM, respectively. In these cells, ATP[gamma S] induced exocytosis with an EC50 of 4 microM and an effectiveness amounting to 50-70% of that of fMet-Leu-Phe. ATP, ITP, UTP, CTP, and uridine 5'-O-[2-thio]diphosphate activated exocytosis as well. Phorbol myristate acetate (PMA) induced exocytosis with an EC50 of 115 ng/ml and an effectiveness similar to that of ATP[gamma S]. Cytochalasin B (CB) differently potentiated exocytosis induced by ATP[gamma S], fMet-Leu-Phe and PMA. Treatment of Bt2cAMP-differentiated HL-60 cells with pertussis toxin (500 ng/ml) for 24 h resulted in ADP-ribosylation of more than 97.5% of the G-proteins. Under these conditions, pertussis toxin almost completely inhibited the increase in cytosolic Ca2+ and beta-glucuronidase release induced by fMet-Leu-Phe but only partially inhibited the effects of ATP[gamma S] and UTP. fMet-Leu-Phe at a non-stimulatory concentration (1 nM) potentiated ATP[gamma S]-induced beta-glucuronidase release in the presence but not in the absence of CB. In contrast, ATP[gamma S] and fMet-Leu-Phe synergistically activated superoxide formation in the absence of CB. PMA potentiated superoxide formation induced by ATP[gamma S] or fMet-Leu-Phe and did not affect exocytosis induced by ATP[gamma S] or fMet-Leu-Phe. In undifferentiated HL-60 cells, fMet-Leu-Phe, ATP[gamma S], UTP and PMA did not induce beta-glucuronidase release. fMet-Leu-Phe did not increase cytosolic Ca2+ in undifferentiated HL-60 cells, whereas ATP[gamma S] and UTP were similarly potent and effective as in Bt2cAMP-differentiated cells. In differentiated HL-60 cells, fMet-Leu-Phe induced aggregation, and ATP[gamma S] induced a transient shape change. Our results show (I) that exocytosis in HL-60 cells does not obligatorily depend on CB. (II) Purine and pyrimidine nucleotides activate exocytosis via pertussis toxin-sensitive and -insensitive signal transduction pathways.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of luteolin, quercetin and baicalein on immunoglobulin E-mediated mediator release from human cultured mast cells

BACKGROUND: Flavonoids have a variety of activities including anti-allergic activities, and are known to inhibit histamine release from human basophils and murine mast cells. OBJECTIVE: The effects of luteolin, a flavone, on the immunoglobulin (Ig) E-mediated allergic mediator release from human cultured mast cells (HCMCs) were investigated and compared with those of baicalein and quercetin. METHODS: HCMCs were sensitized with IgE, and then treated with flavonoids before challenge with antihuman IgE. The amount of released mediators was determined as was mobilization of intracellular Ca2+ concentration, protein kinase C (PKC) translocation and phosphorylation of intracellular proteins were detected after anti-IgE stimulation. RESULTS: Luteolin, baicalein and quercetin inhibited the release of histamine, leukotrienes (LTs), prostaglandin D2 (PGD2), and granulocyte macrophage-colony stimulating factor (GM-CSF) from HCMC in a concentration-dependent manner. Additionally, the three flavonoids inhibited A23187-induced histamine release. As concerns Ca2+ signalling, luteolin and quercetin inhibited Ca2+ influx strongly, although baicalein did slightly. With regard to PKC signalling, luteolin and quercetin inhibited PKC translocation and PKC activity strongly, although baicalein did slightly. The suppression of Ca2+ and PKC signallings might contribute to the inhibition of mediator release. The activation of extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinase (JNK), that were activated just before the release of LTs and PGD2 and GM-CSF mRNA expression in IgE-mediated signal transduction events, were clearly suppressed by luteolin and quercetin. In contrast, the flavonoids did not affect the activation of p38 mitogen-activated protein kinase (p38 MAPK) pathway. CONCLUSION: These results indicate that luteolin is a potent inhibitor of human mast cell activation through the inhibition of Ca2+ influx and PKC activation.