Arachidonic acid metabolism in purified human lung mast cells

Arachidonic acid metabolism has been explored in preparations of purified human lung mast cells prelabeled with arachidonic acid (AA). Cells were of 83 to greater than 96% purity, and each experiment was performed with four to six different preparations of mast cells. After overnight culture of the purified cells in the presence of 3H-AA, followed by extensive washing in buffer, mast cell uptake of labeled AA was 61.4 +/- 14.8 pmol/10(6) cells with 21 +/- 2.4% of the label in phospholipids, 73 +/- 2.1% in neutral lipids, and 3.6 +/- 0.8% as free AA. Analysis of the distribution of radioactivity in phospholipid classes revealed 51.4 +/- 5.5% of the label in phosphatidylcholine, 14.5 +/- 1.6% in phosphatidylinositol, 12.0 +/- 3.0% in phosphatidylethanolamine, and 9.1 +/- 2.4% in sphingomyelin, with the rest in other phospholipid classes. Challenge of these cells with an optimal concentration of anti-IgE led to the release of 20 +/- 4.0% of cellular histamine and to a reduction in labeled phosphatidylcholine and phosphatidylinositol to 75.5 +/- 8.8% and 84.2 +/- 4.5% of the control levels, respectively, (p less than 0.05); anti-IgE challenge produced no statistically significant change in the quantities of other labeled phospholipids. Activation of human lung mast cells with anti-IgE led to the release of 3.4 +/- 1.3% of the cellular 3H as AA and AA metabolites (1.5 +/- 0.6% as unmetabolized AA) in conjunction with 16 +/- 4.3% of the cellular histamine. Although activation of human lung mast cells with ionophore A23187 caused 70 +/- 1.1% histamine release, a similar quantity of AA and AA metabolites was released (a total of 4.0 +/- 0.8% with 2.3 +/- 1.5% as unmetabolized AA). Analysis of the released metabolites by liquid scintillation spectrometry after high performance liquid chromatography separation showed that approximately equal amounts of metabolites were produced after mast cell activation with anti-IgE and ionophore A23187. In this series of experiments approximately equal amounts of cyclooxygenase and lipoxygenase products were generated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the anti-inflammatory effect of FK-506 on human mast cells.

We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187-induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK-506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP).     

Histamine release by pharmacological agents in the absence of external free Ca2+

The involvement of extracellular free Ca2+ in histamine release was investigated in rat peritoneal mast cells. Incubation of non-antigenized cells in a media with high extracellular potassium did not increase histamine release. Secretion induced by A23187 and compound 48/80 in the presence of Ca2+ requires metabolic energy. In the absence of external free Ca2+ (2.5 microM) histamine release induced by A23187 is reduced but not abolished. Secretion induced by compound 48/80 is independent of extracellular Ca2+. These results lead us to suggest that mast cell plasma membranes probably lack voltage-gated Ca2+ channels and that external Ca2+ may not be an absolute requisite for histamine secretion.     

Ouabain-induced enhancement of rat mast cells response. Modulation by protein phosphorylation and intracellular pH

The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in response to several stimuli, which is mediated by Na+/Ca2+ exchanger. In this work, we studied the effect of ouabain on cytosolic calcium, intracellular pH and histamine release with Ca2+ ionophore A23187 in conditions designed to maximize ouabain-induced potentiation of rat mast cells response. The effect of protein kinase C (PKC), cAMP and phosphatase inhibition was also tested. Ouabain induced an enhancement in histamine release, cytosolic calcium and intracellular pH. The adenylate cyclase activator forskolin reduced the effect of ouabain on histamine release and intracellular pH, but enhanced the effect on cytosolic calcium. PKC activator PMA enhanced the effect of ouabain on histamine release and cytosolic calcium, without affecting intracellular pH. A PKC inhibitor, GF-109203X, reduced ouabain-induced enhancement of histamine release and intracellular pH, but increased the enhancement on cytosolic calcium. Finally, inhibition of protein phosphatases 1 and 2A with okadaic acid, increased the effect of ouabain on histamine release and intracellular pH, but reduced cytosolic calcium in presence of ouabain. This result suggest that ouabain-induced potentiation of rat mast cell histamine release with A23187 is modulated by kinases, and this modulation may be carried out by changes in intracellular alkalinization. However, the mechanism underlying cellular alkalinization remains to be elucidated.     

Low density lipoprotein (LDL) inhibits histamine release from human mast cells

We examined the effect of low density lipoprotein (LDL) on histamine release from purified human lung mast cells. LDL inhibited anti-IgE- induced histamine release in a dose-dependent manner, with 100 micrograms/ml LDL-protein inhibiting histamine release by 53 +/- 8% (mean +/- SEM); half-maximal inhibition occurred at 40-80 micrograms/ml. LDL also inhibited calcium ionophore A23187-induced histamine release in a dose-dependent manner, with 1 mg/ml of LDL inhibiting histamine release by 83 +/- 9%; half maximal inhibition occurred at 220-280 micrograms/ml. Inhibition by LDL was time-dependent: half-maximal inhibition of anti-IgE- induced histamine release by LDL occurred at 30-50 minutes of incubation. The inhibitory effect of LDL was independent of buffer calcium concentrations (0-5 mM) or temperature (0-37 degrees C). These data are consistent with a newly defined immunoregulatory role for LDL.     

Dysfunctional inhibitory muscarinic receptors mediate enhanced histamine release in isolated human bronchi

In human airways mucosal mast cells are under the control of inhibitory muscarinic receptors. The described experiments tested, whether the inhibitory potency of two muscarinic receptor agonists (oxotremorine, acetylcholine) becomes impaired in advanced chronic obstructive pulmonary disease (COPD). Isolated human bronchi obtained from 26 patients with lung cancer were separated into two groups. Group 1 patients suffered from moderate COPD (mean FEV1 56%; range 34-71%; mean pack years of cigarette smoking 50, range 20-96; one non-smoker). Group 2 patients had no or only a mild form of COPD; mean FEV1 was 82% (62-97%) and the number of pack years 22 (6-45; 3 non-smoker). The calcium ionophore A23187 induced a maximal histamine release of 4100+/-870 pmol/g/5 min in group 1 bronchi, in contrast to only 1730+/-240 pmol/g/5 min in group 2 bronchi (p<0.02). Oxotremorine (1 nmol/L) reduced the stimulated histamine release by 81+/-5% in group 2 bronchi, but did not produce a significant effect in group 1 bronchi (11+/-14%). In conclusion, the present experiments show an enhanced histamine release in advanced COPD, which can be explained by a dysfunction of inhibitory muscarinic receptors.     

Influence of hypoxia on histamine and leukotriene release from dispersed porcine lung cells

The ability of hypoxia (PO2 57 Torr) and anoxia (PO2 0 Torr) to induce the release of histamine or sulfidopeptide leukotrienes from dispersed porcine parenchymal lung cells was examined. Spontaneous release of histamine (9.2 +/- 1.3%) was not significantly increased during hypoxia or anoxia, and spontaneous leukotriene release was not detected under any conditions. The release of leukotriene induced by A23187 (78 +/- 11 pmol leukotriene D4 equivalent/10(7) parenchymal lung cells) was unchanged during hypoxia and was significantly reduced (55.4 +/- 7.7% control leukotriene release) during anoxia, whereas A23187-induced histamine release (63.2 +/- 4.2% total cell histamine) was unaffected by reduced oxygenation. Reduction of final buffer pH from 7.4 to 7.0 did not affect mediator release. High-pressure liquid chromatographic analysis of the released leukotrienes revealed a mixture of leukotrienes C4 and D4, with a symmetrical reduction in product during anoxia. Although leukotriene release in response to hypoxia was not demonstrated, the findings do not preclude limited local release of leukotrienes, perhaps in association with increased smooth muscle responsiveness.     

Functional and biochemical characterization of rat bone marrow derived mast cells

The functional and biochemical characterization of rat bone marrow derived mast cells (RBMMC) confirms both species-related differences between rat and mouse bone marrow-derived mast cells (MBMMC) as well as mast cell heterogeneity in a single species. Such RBMMC have the staining characteristics of mucosal mast cells and contain the mucosal mast cell protease. The RBMMC release the preformed granule mediator beta-hexosaminidase both in response to immunologic stimulation with 200 ng Ag (net release 15.8 +/- 3.8%) and in response to 1 microM calcium ionophore A23187 (net release 21.8 +/- 6.8%). However, compound 48/80, substance P, and somatostatin did not induce mast cell degranulation. In experiments with optimal beta-hexosaminidase release, the RBMMC generated similar quantities of the newly formed arachidonic acid metabolites leukotriene C4 and PGD2 when stimulated with either Ag or calcium ionophore A23187. The RBMMC incorporate [35S]sulfate into proteoglycans consisting of 90% chondroitin sulfates and 10% heparin. The chondroitin sulfates were comprised of chondroitin 4 sulfate and chondroitin sulfate diB sulfated disaccharides in a ratio of 4/1. Although we show that RBMMC and MBMMC share a low histamine content, functional IgE receptors and unresponsiveness to cromolyn and selective secretagogues (compound 48/80, substance P, and somatostatin), we also provide evidence that RBMMC differ from MBMMC in their profile of newly generated mediators, preformed granule proteoglycan, and lack of proliferative response to mouse IL-3.     

Influence of estradiol and progesterone on the sensitivity of rat thoracic aorta to noradrenaline

The aim of this study was to investigate the effects of low and high doses of estradiol, and of progesterone on the response to noradrenaline in rat thoracic aorta. Two weeks after bilateral ovariectomy, female rats received a s.c. injection of vehicle (corn oil, 0.1 mL/day), estradiol (10 microg/kg/day or 4 mg/kg/day) and/or progesterone (20 mg/kg/day), for eight days. On the ninth day, the rats were sacrificed and aortic rings, with or without endothelium, were used to generate concentration-response curves to noradrenaline. Aortic rings with intact endothelium from the high-dose (4 mg/kg/day) estradiol group were supersensitive to noradrenaline compared to the vehicle or low-dose (10 microg/kg/day) estradiol groups (pD2 values = 7.86+/-0.09, 7.30+/-0.11 and 7.35+/-0.04, respectively). Endothelium-intact aortic rings from high-estradiol rats were supersensitive to noradrenaline when compared to vehicle-, progesterone- and progesterone + high-estradiol-treated rats (pD2 values = 7.77+/-0.12, 7.21+/-0.13, 6.93+/-0.04 and 7.22+/-0.18, respectively). There were no significant differences among the pD2 values for noradrenaline in aortic rings without endothelium. In conclusion, at high but not low doses, estradiol increased the sensitivity to noradrenaline and this was prevented by progesterone. Both of these effects were endothelium-dependent.     

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.     

Stimulation and inhibition of secretion by phorbol myristate acetate in different cell types

In washed human platelets and in HL60 granulocytes phorbol myristate acetate (PMA, 1-2000nM) synergised with threshold concentrations of secretogogues to induce a sustained maximum secretory response. Likewise, superoxide production from HL60 cells maintained a maximal response at PMA concentrations between 30-300nM. At concentrations up to 10nM PMA also augmented calcium ionophore, A23187, stimulated histamine release from rat peritoneal mast cells. However, in the mast cell PMA concentrations above 10nM reduced maximum histamine release in a dose-dependent manner.     

Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Characterization of the effects of phorbol esters on rat mast cell secretion

When applied to the skin, phorbol esters (PEs) elicit signs of acute inflammation, suggesting they may induce the release of mediators from mast cells. Therefore, we have studied the effects of PEs on purified rat peritoneal and thoracic mast cells both alone and in conjunction with the calcium ionophore, A23187, and various other secretagogues that interact with immunoglobulin E (e.g., anti-IgE and Con A) or other cell surface receptors, e.g., somatostatin and compd 48/80. PEs alone caused little or no release of histamine. However, the PE 12-O-tetradecanoylphorbol-13-acetate (TPA, 10 ng/ml) tremendously potentiated release induced by the calcium ionophore A23187, reducing the EC50 for A23187 from 832 ng/ml to 56 ng/ml. In the presence of suboptimal A23187 (50 ng/ml), only active tumor promoting PEs elicited histamine release. The EC50 values of the various active PEs were: TPA 5 ng/ml; 4 beta-PDD, 83 ng/ml; and 4-O-methyl-TPA, 807 ng/ml, with maximal histamine release ranging from 54 to 80%. TPA synergistically enhanced stimulation of histamine release by anti-IgE and Con A over the entire concentration-response range. In contrast, this synergism was absent when cells were stimulated with somatostatin and compd 48/80. Phorbol esters may act by increasing the activity of a calcium/phospholipid-dependent protein kinase (Ca/PL-PK). Mast cells do have Ca/PL-PK activity, and TPA in the presence of suboptimal A23187 induces protein phosphorylation comparable with other secretagogues. These results suggest that in the purified mast cell, PE-induced mediator release increases the sensitivity of release mechanisms for calcium, acts syngergistically with secretagogues interacting with IgE, and as suggested from structure-activity relationships, occurs via a specific mechanism of action perhaps involving the Ca/PL-PK.     

Effect of bile salts on the plasma concentration of immunoreactive vasoactive intestinal polypeptide in man

The effects of bile salts on the release of immunoreactive vasoactive intestinal polypeptide (IR-VIP) were investigated in men using a specific radioimmunoassay. Plasma IR-VIP was determined after extraction by the acid-acetone method (recovery 75 +/- 5%). Oral administration of 400 mg sodium taurocholate caused a rise in plasma IR-VIP from 18.5 +/- 1.3 pmol/l to 31.1 +/- 2.1 pmol/l after 30 min and 39.0 +/- 1.7 pmol/l after 60 min and return to the initial value after 120 min. Oral administration of chenodeoxycholic acid (CDCA) also increased plasma IR-VIP from a basal level of 14.5 +/- 1.5 pmol/l to 36.3 +/- 1.2 pmol/l after 60 min. Oral administration of ursodeoxycholic acid (UDCA) increased plasma IR-VIP from 11.9 +/- 1.1 pmol/l to 25.6 +/- 1.8 pmol/l after 30 min. Perifusion of 1 mM taurocholate stimulated release of IR-VIP from human duodenal mucosa into the perifusate. These results suggest that bile salts may participate, at least in part, in the release of IR-VIP from the gut.     

Differential calcium effects on prostaglandin D2 generation and histamine release from isolated rat peritoneal mast cells

We examined the role of Ca2+ mobilization in prostaglandin (PG) D2 generation and histamine release induced by A23187 from rat peritoneal mast cells. Both PGD2 generation and histamine release accompanied with 45Ca uptake were observed above 0.1 microM A23187. Although an increase of PGD2 generation was not exactly correlated with that of Ca2+ uptake, histamine release occurred in proportion to Ca2+ uptake. In contrast to PGD2 generation, below 0.1 microM A23187, about 20% of the total histamine was released without Ca2+ uptake and this response was inhibited by 10 microM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which is an intracellular Ca2+ antagonist. However, TMB-8 had no effect on PGD2 generation. These results suggest that Ca2+ dependency of histamine release is clearly different from that of PGD2 generation, and that histamine release is induced by not only Ca2+ uptake but also intracellular Ca2+ mobilization.     

Ionophore-stimulated lipoxygenase activity and histamine release in a cloned murine mast cell, MC9

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 microM ionophore, MC9 cells produce 270 +/- 43 pmoles 5-HETE, 74 +/- 40 pmoles 5,12 diHETEs and 65 +/- 31 pmoles LTC4/10(6) cells from 37 microM exogenously added [1-14C]arachidonic acid in two minutes. 5-HETE and 5,12-diHETES, including LTB4 were identified by GC/MS whereas LTC4 was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD2 and TxB2 (8.5 +/- 2.4 and 5.4 +/- 1.2 pmoles/10(6) cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca+2 and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 +/- 3% cellular histamine) and continued for 30 minutes (37 +/- 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.     

Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation

This study evaluated the effect of inhibitors of transmethylation on histamine release from rat mast cells and rat basophilic leukemia cells. IgE-mediated histamine release from rat basophilic leukemia cells (RBL-2H3 cells) was inhibited by 3-deazaadenosine (DZA) in the presence of L-homocysteine thiolactone (Hcy) or the combination of adenosine, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), and Hcy in a dose-dependent fashion. There were no significant changes in the cellular cAMP levels by these inhibitors. Histamine release induced by anti-IgE or dextran from normal rat mast cells was also blocked by DZA plus Hcy in a dose-dependent manner. DZA at 10(-3) M in the presence of 10(-4) M Hcy or the combination of 10(-3) M adenosine, 10(-4) M EHNA, and 10(-3) M Hcy inhibited lipid (perhaps phospholipid) methylation into RBL-2H3 cells without affecting choline incorporation. In the presence of 10(-3) M DZA plus 10(-4) M Hcy there was a 170-fold increase in [35S]AdoHcy with the concomitant appearance of 3-deaza-AdoHcy when the cells were incubated with [35S]methionine, thus indicating that these drugs inhibited methylation reaction(s) through the intracellular accumulation of AdoHcy and 3-deaza-AdoHcy. In contrast, histamine release from rat mast cells induced by the calcium ionophore A23187, compound 48/80, polymyxin B, or ATP was not inhibited by these compounds. These results suggest that IgE- or dextran-mediated histamine release involves methylation reactions(s), whereas the other secretagogues bypass this early step.     

Differential activation of membrane phospholipid turnover by compound 48/80 and ionophore A23187 in rat mast cells

Membrane phospholipid turnover was investigated during histamine release from rat mast cells. Addition of calcium ionophore A23187 (0.5 microgram/ml) to mast cells prelabeled with [3H]glycerol induced the rapid and progressive increase in phosphatidic acid (PA) and 1,2-diacylglycerol (DG), which was concomitant with the small rise in phosphatidylinositol (PI). Loss of the level in triacylglycerol (TG) was very marked. Polyamine compound 48/80 (5 micrograms/ml) was shown to cause rises in PA, 1,2-DG, and PI without any significant changes in TG. Both stimuli increased incorporation of exogenous [3H]glycerol into phospholipids, indicating the involvement of de novo synthesis in phospholipid metabolism. Studies with [3H]arachidonic acid-labeled mast cells showed an enhanced liberation of radioactive arachidonate and metabolites upon histamine release. There were associated decreases of radioactivity in phosphatidylcholine (PC) and TG when exposed to A23187, while phosphatidylethanolamine (PE) was degraded as a result of 48/80 activation. The transient increases of [3H]arachidonoyl-1,2-DG and PA were caused by 48/80, while A23187 showed a gradual rise in the radioactivity in these two lipid fractions. These findings reflect activation of phospholipase C. When mast cells were activated by low concentrations of A23187 (0.1 microgram/ml) and 48/80 (0.5 microgram/ml), different behaviors of PI metabolism were observed. An early degradation of PI and a subsequent formation of 1,2-DG and PA suggest that the lower concentrations of these agents stimulate the PI cycle initiated by PI breakdown rather than de novo synthesis. These results demonstrate that marked and selective changes in membrane phospholipid metabolism occur during histamine release from mast cells, and that these reactions seem to be controlled by the coordination of degradation and biosynthesis, depending on the type and the concentration of stimulants. A23187 stimulates arachidonate release perhaps via the cleavages of PC and TG, whereas 48/80 liberates arachidonate from PE.     

Regulation of adhesion of mouse bone marrow-derived mast cells to laminin

We have reported that mast cells adhere to laminin after activation with PMA. In this study, we demonstrate that the cross-linking of cell surface high-affinity IgE-R on mast cells derived from mouse bone marrow cultured for 3 wk in the presence of WEHI-3-conditioned media acts as a highly sensitive physiologic stimulus for this attachment and that receptor activation is also induced by calcium ionophore A23187. Adherence occurred at threefold log concentrations less of A23187 and Ag than required for histamine release in a selective subpopulation comprising 20 to 30% of the total cells. At higher concentrations of agonist that permitted histamine release, the time course for degranulation was shown to be more rapid than that of adherence. Adherence was inhibited by antibodies to laminin and laminin receptor and was calcium ion and temperature dependent. Treatment of cells with dibutyryl cAMP, which activates protein kinase A, inhibited both adherence and histamine release induced by Ag or calcium ionophore. Treatment of cells with staurosporin, which inhibits protein kinase C, also inhibited adherence and histamine release induced by calcium ionophore, but was not significantly active against either adherence or histamine release induced by Ag. It thus appears that agents which modulate intracellular signaling mechanisms are equally effective toward histamine release and adherence, suggesting these two events are intimately linked in stimulus secretion coupling. Specific cytokines stimulating mast cell adhesion to laminin could not be found; however, culture of mast cells with TGF-beta 1 was determined to enhance IgE-mediated adherence to laminin. Hence, the high-affinity IgE-R on the mast cell functions not only in exocytosis but also facilitates the process of mast cell adherence to laminin.     

Epigallocatechin gallate inhibits histamine release from rat basophilic leukemia (RBL-2H3) cells: role of tyrosine phosphorylation pathway

Some tea polyphenolic compounds including (-)-epigallocatechin gallate (EGCG) have been shown to inhibit histamine release from mast cells through poorly understood mechanisms. By using a mast cell model rat basophilic leukemia (RBL-2H3) cells we explored the mechanism of the inhibition. EGCG inhibited histamine release from RBL-2H3 cells in response to antigen or the calcium-ionophore A23187, while (-)-epicatechin (EC) had little effect. Increased tyrosine phosphorylation of several proteins including approximately 120 kDa proteins occurred in parallel with the secretion induced by either stimulation. EGCG also inhibited tyrosine phosphorylation of the approximately 120-kDa proteins induced by either stimulation, whereas EC did not. The tyrosine kinase-specific inhibitor piceatannol inhibited the secretion and tyrosine phosphorylation of these proteins induced by either stimulation also. Further analysis showed that the focal adhesion kinase pp125(FAK) was one of the approximately 120-kDa proteins. These findings suggest that EGCG prevents histamine release from mast cells mainly by inhibiting tyrosine phosphorylation of proteins including pp125(FAK).