Arachidonic acid release in rat peritoneal mast cells stimulated with antigen, ionophore A23187, and compound 48/80

Rat peritoneal mast cells respond to various types of secretagogues, such as antigen (receptor-mediated), A23187 (calcium mobilizing), and compound 48/80 (membrane perturbing), and release arachidonic acid from membrane phospholipids prelabeled with [3H]arachidonate. The rate of arachidonic acid liberation varied from one stimulant to the other. Ionophore A23187 (0.1 micrograms/ml) appeared to be most potent in releasing arachidonate among the three stimulants at which doses each secretagogue caused almost equivalent histamine secretion. However, upon stimulation with these three secretagogues, the radioactivity of phosphatidylcholine (PC) was markedly reduced with a concomitant increase of arachidonate radioactivity. Hydrolysis of PC by phospholipase A2 is likely to be the major route of arachidonic acid liberation in either IgE-mediated or non-IgE activation in mast cells.     

Arachidonic acid release in BW755C-pretreated rat peritoneal mast cells stimulated with A23187, concanavalin A and compound 48/80

Rat peritoneal mast cells respond to various secretagogues, such as ionophore A23187, concanavalin A (Ig E receptor cross-bridging) and compound 48/80 (membrane perturbing), to secrete histamine and to liberate arachidonic acid. Arachidonic acid release was made identifiable by pretreatment with BW755C, an inhibitor of both lipoxygenase and cyclo-oxygenase. The extent of arachidonic acid release varied among these three secretagogues. A23187 appeared to be most potent, whereas compound 48/80 was weakest. The sources of released arachidonic acids may be different depending on the types of stimulants. The stimulation with A23187 released arachidonic acid mainly from phosphatidylcholine and triacylglycerol. After treatment with concanavalin A and compound 48/80, in addition to phosphatidylcholine, phosphatidylinositol also appeared to serve as a donor of arachidonic acid.     

Calcium requirement in compound 48/80 induced histamine release from rat mast cells in vitro

The effect of magnesium and EDTA on compound $\text{48}\text{80}$-induced histamine release and adenosine triphosphate (ATP) content of mast cells has been studied. Inhibition of histamine release after preincubation of the cells with or without EDTA in the absence of calcium and the reversal by calcium indicate that calcium is required for compound $\text{48}\text{80}$-induced histamine release. The presence of magnesium potentiate the inhibition caused by the lack of calcium. The inhibition of histamine release is not related to changes in cellular ATP content. The observations with EDTA suggest that calcium may be provided for the release process from intracellular sources.     

Diabetes-induced rat hyposensitivity to compound 48/80

Rat mortality and contractile responses of isolated tracheas to compound 48/80 from rats made diabetic 4 days before by a single intravenous injection of alloxan and from diabetic rats that had been treated with insulin 6 h before were compared with control animals. Diabetic animals and tracheal segments from diabetic rats were significantly less responsive to compound 48/80 than control and insulin-treated diabetic animals. On the other hand, diabetic animals have a lower quantity of peritoneal mast cells than control rats, and insulin restored the normal quantity of cells in diabetic animals. These data indicate that diabetes elicits an hyposensitivity to compound 48/80, possibly related to a diabetes-induced decrease in the mast cell count.     

Extracellular magnesium decreases the secretory response of rat peritoneal mast cells to compound 48/80 in vitro.

Exposure of rat peritoneal mast cells to magnesium in the absence of extracellular calcium resulted in a time- and dose-dependent decrease in the secretory response induced by compound 48/80. The decrease was prevented by a low extracellular concentration of calcium. Furthermore, the decreased secretory responsiveness was dose-dependently restored by the addition of calcium to the cells simultaneously with compound 48/80. Preincubation with magnesium also inhibited antigen-induced histamine secretion in a dose-dependent manner. This was reversed by the simultaneous addition of calcium and the secretory stimulus. A dose-dependent decrease in antigen induced histamine secretion that was reversed by calcium was also observed. Exposure of the mast cells to magnesium for 15 min resulted in a parallel decrease in histamine secretion and in the cellular content of 45Ca2+. These observations suggest that magnesium may decrease the secretory response by displacing the cellular calcium which is utilized in stimulus-secretion coupling.     

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.     

Biphasic effect of phorbol myristate acetate on histamine secretion induced by compound 48/80 in rat peritoneal mast cells

Rat peritoneal mast cells which had been preincubated with phorbol myristate acetate (PMA, 100 ng/ml) for 30 sec elicited an enhanced histamine secretion induced by a potent secretagogue, compound 48/80. But a longer (5 min) preincubation with PMA followed by the agonist-stimulation induced a suppressed histamine secretion. A 5 min-PMA-pretreatment inhibited the compound 48/80-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in [32P]-labeled cells. PMA-treatment alone for 5 min induced an activation of Ca2+-efflux monitored by 45Ca2+. The inhibition of histamine secretion induced by a 5 min-PMA-pretreatment followed by the agonist-stimulation may partly be attributed to the decreased intracellular Ca2+ concentration, [Ca2+]i, probably due to the depressed PIP2 breakdown and enhanced Ca2+-efflux. On the other hand, a 30 sec-preincubation with PMA followed by compound 48/80-stimulation induced a slight but significant increase in histamine secretion. In this case, neither breakdown of PIP2 nor Ca2+-influx was enhanced to raise the [Ca2+]i. Although we are unable to explain the mechanism for the enhancement of histamine secretion by a short (30 sec) PMA-preincubation, these results suggest that the biphasic effects of PMA on histamine secretion are mediated by intracellular Ca2+ mobilization probably via protein kinase C activation.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using H NMR

¹H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events folowing exocytosis, activate glycolysis.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using 1H NMR.

1H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events following exocytosis, activate glycolysis.     

Staining properties of duodenal mast cells in 48/80-treated rats

Summary Mast cells in the tongue, mesentery and lamina propria of the duodenal mucosa in normal and 48/80-treated rats were observed at different time intervals. The tissues were studied comparatively after staining with toluidine blue, acridine orange or alcian bluesafranin. Under the experimental conditions used, the mast cells in the tongue and mesentery showed constant positive reactions to toluidine blue and acridine orange, both of which failed to demonstrate the presence of mast cells in the lamina propria of the duodenal mucosa. The combined alcian blue-safranin stain elicited a safranin-positive reaction in the mast cells of the tongue and mesentery and an alcian blue reaction in those of the lamina propria of the duodenal mucosa. This alcianophilia of the duodenal mast cells was not affected by compound 48/80. On the other hand, the safranin stain of the tongue and mesentery mast cells was altered to alcian blue by the drug. The results are discussed in the light of recent developments in mast cell research.This work was supported by grant MA-2236 of the Medical Research Council of Canada.     

Increased responsiveness of rat mast cells to compound 48/80 due to removal of extracellular magnesium. Effects of ouabain and EGTA.

A decreased secretory response of mast cells to compound 48/80 (12% of control value) after preincubation of the cells with magnesium but without calcium was partially restored by removal of magnesium. EGTA (10 microM) blocked the restoration and decreased the restored secretory activity again, while this was further increased by ouabain (1 mM). Furthermore, ouabain completely restored the decreased secretion (50% of control value) due to preincubation without the divalent cations. This may indicate that magnesium influences a pool of cellular calcium that is involved in the stimulus-secretion coupling and is available to EGTA, and ouabain did not counteract the inhibitory mechanism of magnesium.     

Strain differences in histamine release from mouse peritoneal mast cells induced by compound 48/80 or A23187

Strain differences among BALB/c, BDF1, CDF1, C3 H/He, C57 BL/6, DBA/2, ddy and ICR mice were investigated with respect to the ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80, a Ca2+ dependent histamine releaser, and the Ca2+ ionophore A23187. The ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80 were found to be high in BALB/c, ddY and ICR mice, but low in BDF1, CDF1, C3 H/He, C57 BL/6 and DBA/2 mice. Those induced by Ca2+ ionophore A23187 were high in BALB/c, BDF1, CDF1, C3 H/He, DBA2, ddy and ICR mice but low in C57 BL/6 mice. These results indicate that differences in histamine release from mouse peritoneal mast cells are strain dependent.     

Long term increase of mucosal mast cells in the rat induced by administration of Compound 48/80

Summary Administration of Compound 48/80 to rats for 5 days resulted in an increase of the specific type of mucosal mast cell, while connective tissue mast cells elsewhere were almost completely degranulated. The number of mucosal mast cells increased slowly for another 5 days and then returned to the control level, in an exponential manner. The half life of the newly formed mast cells was calculated to be about 40 days. This value may be taken as an estimate of the half life of mucosal mast cells. These cells, therefore, constitute a fraction of mast cells with rapid turnover. Available evidence indicates that the classical connective tissue mast cell has a very long life span, without significant turnover in terms of cell death and cell renewal. We suggest that the increase of mucosal mast cells is an indirect effect of Compound 48/80, related to its effect on other mast cells and mediated by material(s) released from these cells during the secretory process.Supported by grants from the Swedish Medical Research Council, Project no 2235     

The influence of Na+ ions on histamine secretion from mast cells induced by NGF or compound 48/80

It has been demonstrated that the release of histamine from mast cells by cytokines is strongly dependent on extracellular Ca2+ and Na+ ions. The results of our current research indicate that the removal of extracellular Na+ enhances NGF induced histamine release, but reduces induction by compound 48/80, suggesting that different mechanisms are involved in the secretory process induced by these agents.