DUAL MODES OF ACTION FOR DISODIUM CROMOGLYCATE IN INHIBITION OF ANTIGEN-INDUCED CONTRACTIONS OF GUINEA-PIG ISOLATED AIRWAYS SMOOTH MUSCLE

Disodium cromoglycate (DSCG; 10 mumol/l-1 mmol/l) relaxed the tone induced in the guinea-pig isolated trachea by histamine (5 mumol/l). Both isoprenaline and fenoterol were approximately 1000 times more potent than DSCG in relaxing tracheal smooth muscle. Ovalbumin (1 microgram/ml) contracted isolated tracheal and parenchymal strips from sensitized guinea-pigs. The sensitization procedure used was selective for eliciting IgG antibody production. In concentrations equieffective for relaxation of the isolated trachea, fenoterol (20 nmol/l), isoprenaline (60 nmol/l) and DSCG (100 mumol/l) inhibited ovalbumin-induced contractions of both tracheal and parenchymal strip preparations. DSCG produced a significantly greater (P less than 0.05) inhibition of ovalbumin-induced contractions of isolated trachea than did isoprenaline, suggesting that at 100 mumol/l DSCG produced a greater inhibition of mediator release than that produced by isoprenaline. Compound 48/80 (200 micrograms/ml) contracted the isolated trachea whereas this concentration had no contractile effect on the parenchymal strip. The failure of DSCG to inhibit compound 48/80 induced contractions of the isolated trachea together with the lack of contractile effect of compound 48/80 on the parenchymal strip suggest that compound 48/80 is inferior to antigenic stimulation as a reliable analogue of immunological activation of mast cells and consequent mediator release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compound 48/80 and substance P induced release of histamine and serotonin from rat peritoneal mast cells

The effect of substance P and compound 48/80 on histamine and serotonin release from not isolated and isolated mast cells have been compared in experiments in vitro. The response of not isolated and isolated mast cells were virtually identical. The release of both amines, in response to 48/80 and substance P, was dose-dependent. The percentage of histamine released by 48/80 was significantly higher than the percentage of serotonin, the difference being higher at lower concentrations of compound 48/80 after 15 min of incubation. Substance P also showed a tendency to higher efficiency for histamine than for serotonin release. In contrast to 48/80, the dose-response curves for histamine and serotonin release were parallel. These results support the view that the ratio between histamine and serotonin release depends on the liberator used. They also showed that this ratio can depend on the concentration of the agent inducing secretion. The results indicate that substance P as well as 48/80 act rather selectively as histamine liberators and that there is some difference in releasing properties of 48/80 and substance P.     

Inhibition of compound 48/80-mediated histamine release from isolated rat mast cells by oosponol-related compounds (4-acyl-isocoumarins).

Oosponol (4-hydroxymethylketone-8-hydroxyisocoumarin) is a metabolic product isolated from Oospora astringens which originated from house dust in a room of an asthmatic patient. The compound and the structurally related isocoumarins were studied to determine the inhibition of histamine release induced by compound 48/80 from isolated rat peritoneal mast cells. The released histamine was assayed by fluorometry. The compounds tested were not observed to release histamine. Some of 4-acyl-isocoumarins inhibited the histamine release at doses less than 10 micrometers, whereas the 3-acyl- and the 4-alkyl-compounds were not effective at doses over 100 microns. The pretreatment of mast cell with the compound for 15 min before the application of compound 48/80 was more effective than the simultaneous administration. The mode of inhibitory action of KIT-302, 4-(4'-carboxy-benzoyl)-isocoumarin, was non-competitive antagonism to compound 48/80 on the mast cells.     

Inhibitory action of 6-ethyl-3-(1H-tetrazol-5-YL)chromone (AA-344) on IgE, IgGa- or chemical agent-induced histamine release from isolated rat mast cells.

The antiallergic action of 6-ethyl-3-(1H-tetrazol-5-yl)chromone (AA-344) was studied on isolated rat peritoneal mast cells. AA-344 clearly inhibited the IgE-mediated release of histamine caused by various concentrations of antigen and the 50% inhibitory concentration was 0.1 microM. On the IgGa-mediated release of histamine, a peak inhibition of AA-344 was observed at 10 microM. The histamine release induced by chemical agents such as concanavalin A, dextran and compound 48/80 was depressed by AA-344 at the range of 0.1--1 mM. The results obtained in this study indicate that the antiallergic action of AA-344 is due to selective inhibition on the immunological release of the chemical mediator from mast cells.     

Increased Utilization of Endogenous ATP in Isolated Rat Mast Cells during Histamine Release Induced by Compound 48/80

Abstract: In order to investigate whether the energy dependence of histamine release induced by compound 48/80 from isolated rat mast cells reflects an increased utilization of ATP during the release process, the ATP content in samples of mast cells was determined 5 sec. before and 25 sec. after the addition of compound 48/80. No change in the ATP content was found when histamine release was induced from cells with an intact energy metabolism. However, when the cells had been preincubated for 1 min. with antimycin A to inhibit oxidative production of ATP, histamine release was accompanied by a greater reduction (P < 0.005) in the ATP content than that observed in the control samples. In contrast, when corresponding experiments were performed with chlorpromazine or n-decylamine as releasing agent, the ATP content was not lower in the samples in which histamine release had been induced than in the controls, regardless of whether antimycin A was present or not. The results indicate that histamine release from rat mast cells induced by compound 48/80, but not by chlorpromazine or n-decylamine, is accompanied by an increased utilization of ATP in the cells.     

Inhibitory Action of Exogenous Adenosine-5′-triphosphate on Glycolysis in Isolated Rat Mast Cells: Significance for Histamine Release

Abstract: Histamine release and lactate content were concomitantly determined in samples of isolated rat mast cells. Histamine release induced by exogenous ATP or compound 48/80 was inhibited by antimycin A (0.2 μM). Glucose (0.60 mM) restored the release induced by compound 48/80 but not that induced by ATP. ATP but not compound 48/80 inhibited the accumulation of lactate in suspensions of mast cells containing glucose (0.60 mM). ATP induced inhibition of lactate accumulation and release of histamine within the same concentration range. However, the time courses for the two processes were different. Antimycin A (0.2 μM) enhanced the accumulation of lactate, an effect which was counteracted by ATP. 0.05 mM ATP or more reduced the lactate accumulation to the same values as those found in the absence of antimycin A. The inhibitory action of ATP on glycolysis may explain the observed inability of glycolytic substrates to restore the ATP-induced histamine release blocked by inhibitors of oxidative metabolism.     

Studies on histamine-retaining granules obtained from isolated rat mast cells.

Histamine-retaining granules were isolated from rat mast cells after sonication in either sucrose of Ficoll-Hypaque media. The preparations obtained were compared in regard to recovery and spontaneous loss of histamine. The effect of agents known to release histamine from intact rat mast cells (antigen, compound 48/80, decylamine, the ionophores A23187 and X537A as well as ATP) was studied on the granules. Antigen and compound 48/80 did not release histamine. Decylamine and X537A induced a pronounced release independent of the presence of divalent cations. ATP caused a small, but significant release, which showed an absolute requirement for magnesium. A23187 released histamine only in the presence of either calcium or magnesium, and this release was unaffected by certain agents known to inhibit histamine release from intact rat mast cells. The results seem to exclude the possibility that agents known to induce release of histamine from intact rat mast cells by a calcium-and energy-dependent process would exert this action through a direct effect on intracellularly localized granules.     

On the mechanism of histamine release induced by thapsigargin from Thapsia garganica L.

Thapsigargin (Tg) is a pure chemical compound isolated from Thapsia garganica with a molecular weight of 650. It releases histamine from isolated rat mast cells but not from isolated histamine-retaining mast cell granules. The rate of release is markedly influenced by pretreatment of mast cells with Tg prior to the addition of calcium. In agreement with the effect of the ionophore A23187 but in contrast to many other calcium-dependent histamine-releasing agents, cells preincubated with Tg respond to the secretory action of calcium whenever the ion is introduced. However, after dilution of Tg-pretreated cells histamine release induced by the addition of calcium became dependent on the time of its addition. The secretory reaction induced by Tg and calcium can be divided into a two-step reaction at 37 degrees C. Pretreatment of mass cells with Tg renders the cells insensitive to the secretory action of compound 48/80 in the absence of calcium, and this effect could be partly counteracted if 1 mM of strontium was added together with compound 48/80. It is concluded that among various calcium- and energy-dependent histamine-releasing agents Tg most closely resembles the action of fluoride on isolated rat mast cells.     

Mechanism of histamine release from rat isolated peritoneal mast cells by dextran: the role of immunoglobulin E

In vivo passive sensitization of rat peritoneal mast cells with Nippostrongylus braziliensis antiserum or rat monoclonal myeloma IgE greatly enhanced histamine release in vitro by dextran or anti IgE, but did not alter release by compound 48/80 or A23187. Conversely, removal of IgE from the cells by acid pH abolished histamine release by dextran and anti IgE but did not impair release by compound 48/80. Whereas, histamine release from cells isolated from rats genetically resistant to dextran (NR rats) by anti IgE was potentiated by passive sensitization, dextran was unable to stimulate secretion from control or sensitized NR cells. The results suggest that dextran releases histamine by interaction with cell-fixed IgE and that the NR mast cell membrane lacks the ability to interpret this stimulus.     

Histamine H3 receptor activation inhibits neurogenic sympathetic vasoconstriction in porcine nasal mucosa

Histamine release from mast cells is a primary mediator of rhinorrhea, nasal mucosal swelling, increased secretion, sneezing, pruritis and congestion that occur in allergic rhinitis. It is well known that histamine H(1) receptor antagonists inhibit the itch and rhinorhea, but do not block the allergic nasal congestion. A growing body of evidence shows that in addition to histamine H(1) receptors, activation of H(3) receptors may contribute to the procongestant nasal actions of histamine. Activation of the prejunctional histamine H(3) receptor modulates sympathetic control of nasal vascular tone and resistance. The present study was conducted to further characterize the role of histamine H(3) receptors on neurogenic sympathetic vascular contractile responses in isolated porcine nasal turbinate mucosa. We presently found that the histamine H(3) receptor agonist, (R)-alpha-methylhistamine (10-1000 nM), inhibited electrical field stimulation-induced sympathetic vasomotor contractions in a concentration-dependent fashion. Pretreatment with either of the selective histamine H(3) receptor antagonists, thioperamide and clobenpropit, blocked the sympathoinhibitory effect of (R)-alpha-methylhistamine in porcine turbinate mucosa. The effect of compound 48/80, an agent that elicits the release of endogenous histamine from mast cells on nasal sympathetic contractile responses, was also tested. The action of compound 48/80 to release mast cell-derived histamine in the nose mimics many of the nasal responses associated with allergic rhinitis, extravascular leakage and decreased nasal patency. We presently found that compound 48/80 also inhibited the electrical field stimulation-induced sympathetic response. Pretreatment with the H(3) receptor antagonist clobenpropit blocked the sympathoinhibitory action of compound 48/80 on sympathetic contractile responses in nasal mucosa. Taken together, these studies indicate that histamine H(3) receptors modulate vascular contractile responses by inhibition of noradrenaline release from sympathetic nerve terminals in nasal mucosa. It is further suggested that histamine H(3) receptors may play a role in the regulation of vascular tone and nasal patency in allergic nasal congestive disease.     

Mechanisms of histamine release by compound 48/80

1. Rat and guinea-pig lung tissues were incubated for 20 min at 37 degrees C in Krebs-Ringer phosphate buffer at pH 7.4, or in Tyrode-Tris buffer at pH 8.2, and the release of histamine produced by adding different concentrations of compound 48/80 to the incubation medium was determined.2. At pH 7.4, increasing concentrations of 48/80 increased the release of histamine from the rat lung, with a tendency towards a maximum. No release of histamine from guinea-pig lung was observed at this pH. At pH 8.2, histamine release occurred both from rat and guinea-pig lung, and was proportional to the logarithm of the concentration of compound 48/80.3. Histamine release from rat lung by 20 mug/ml. of 48/80 decreased when the pH was raised from 7.4 to 8.2; but the release caused by 1 mg/ml. of 48/80 increased both in rat and guinea-pig lung as the pH was raised.4. 2-4-Dinitrophenol (DNP) inhibited the release of histamine from rat lung by a concentration of 20 mug/ml. of 48/80; the inhibition was prevented by glucose. DNP did not affect histamine release from rat or guinea-pig lung by a concentration of 1 mg/ml. of 48/80 and enhanced the release when the pH was raised from 7.4 to 8.2.5. 1 mg/ml. of 48/80 did not inhibit the enhanced oxygen consumption produced by DNP in the isolated rat diaphragm.6. Iodoacetic acid (IAA) or a Ca/Mg-free medium inhibited the release of histamine by 20 mug/ml. of 48/80 from rat lung but not the release produced by 1 mg/ml. in either rat or guinea-pig lung.7. The degranulation of rat mesentery mast cells caused by 20 mug/ml. of compound 48/80 was inhibited by DNP. The degranulation evoked by 1 mg/ml. of 48/80 was also sensitive to this inhibitor; in this instance, however, the metachromatic staining reaction of the mesentery mast cells was greatly diminished.8. It is concluded that two processes of histamine release by compound 48/80 occur in rat lung. One, dependent on cell metabolism, involves, mast cell granule secretion. The other, independent of cell metabolism, seems to consist of a simple exchange reaction between histamine and compound 48/80, and this is the only one occurring in guinea-pig lung.     

Inhibition by calcium antagonists of histamine release and calcium influx of rat mast cells: Difference between induction of histamine release by concanavalin A and compound 48/80

The relation between calcium influx and histamine release from rat mast cells was investigated. When purified mast cells pretreated with a calcium antagonist (MnCl₂ or methoxyverapamil (D-600)) were exposed to concanavalin A or compound 48/80 in Tyrode solution (pH 7.4) at 37°C, the calcium antagonists inhibited the extracellular calcium-dependent component of concanavalin A-induced histamine release. MnCl₂ also inhibited the extracellular calcium-dependent component of compound 48/80-induced histamine release, whereas D-600 did not inhibit the release. D-600 inhibited the ⁴⁵Ca uptake induced by concanavalin A, but did not inhibit the ⁴⁵Ca uptake induced by compound 48/80. It was found that the inhibitory action of calcium antagonists depended on the uptake of extracellular calcium. These observations suggest that concanavalin A and compound 48/80 stimulate different mechanisms of calcium influx. Studies on inactivation of the mechanisms of calcium influx showed that calcium influx into cells activated by concavalin A stopped when concanavalin A was washed out, whereas the influxactivated by compound 48/80 was still operative after compound 48/80 had been washed out.     

Changes in polyphosphoinositide metabolism during mediator release from stimulated rat mast cells

The metabolism of the polyphosphoinositides, diphosphoinositide (DPI) and triphosphoinositide (TPI), was studied during mediator release from rat mast cells. Serosal mast cells were purified by density gradient centrifugation and prelabeled with 32PO4. Incorporation of 32PO4 into DPI and TPI was determined by thin-layer chromatography on oxalic acid impregnated silica gel plates. 32PO4 incorporation into DPI and TPI was increased by Concanavalin A (ConA) or compound 48/80. The concentration of ConA causing a half-maximal increase in DPI labeling was less than that required for a comparable change in histamine release. The increases in DPI labeling and histamine release in response to ConA were enhanced by phosphatidylserine. The addition of alpha-methylmannoside to mast cells after challenge with ConA rapidly halted DPI and TPI labeling. The results of these studies indicate that changes in the metabolism of polyphosphoinositides may be an intrinsic part of the biochemical mechanisms that control mediator release from mast cells.     

The utilization of adenosine triphosphate in rat mast cells during histamine release induced by anaphylactic reaction and compound 48/80

Summary The ATP content of rat peritoneal mast cells has been studied in relation to histamine release induced by compound 48/80 and antigen-antibody (anaphylactic) reaction in vitro. When the ATP content of actively sensitized mast cells was reduced to different levels by oligomycin, a good correlation was obtained between the ATP levels and the amounts of histamine released by the anaphylactic reaction. A similar linear relation has previously been demonstrated between the ATP levels of mast cells and histamine release induced by compound 48/80. The ATP content of mast cells was also studied at different intervals after the exposure of the cells to antigen or compound 48/80. No significant change in the ATP content was observed in untreated mast cells during the short period when histamine release occurs. If, however, the mast cells were preincubated with oligomycin or 2-deoxyglucose to reduce the rate of ATP synthesis while a large part of the histamine release remained unaffected—a decrease in the ATP content could be demonstrated in close time relation to both anaphylactic and compound 48/80-induced histamine release. The observations indicate an increased utilization of ATP in mast cells during the release process.     

Sodium-potassium ATPase inhibition potentiates compound 48/80-induced histamine secretion from mast cells.

The effect of ouabain on the histamine secretion induced by compound 48/80 has been studied using rat peritoneal mast cells. Ouabain did not modify histamine release in the presence of millimolar concentrations of extracellular calcium. However, when mast cells were previously washed with a calcium-free buffer, ouabain strongly potentiated histamine release elicited by compound 48/80. The full potentiation of mast cell secretion by ouabain required 30 min preincubation before adding compound 48/80. It was inhibited by lanthanum and EGTA. Potassium deprivation mimicked the effect of ouabain. A 30 min preincubation time without potassium was also required. Potassium concentrations below 2.7 mM increased the effect of ouabain whereas higher potassium concentrations reversed this effect. The potentiation of compound 48/80-induced histamine release by ouabain or potassium deprivation was not immediately reversed by washing away ouabain or by adding potassium, respectively. The data confirm that sodium-potassium ATPase is involved, through a calcium-dependent process, in the regulation of histamine release from mast cells.     

Prostaglandin E2 prevents diclofenac-induced enhancement of histamine release and inflammation evoked by in vivo challenge with compound 48/80 in the hamster cheek pouch

Based on observations obtained by the use of intravital microscopy, we report that prostaglandins (PGs) can exert inhibitory effects on mast cell-dependent inflammation. Thus, the PG-synthesis inhibitors diclofenac and indomethacin potentiated extravasation of plasma evoked by challenge with the mast cell secretagogue compound 48/80. Although the plasma leakage induced by compound 48/80 was in large mediated by histamine, neither diclofenac nor indomethacin potentiated the plasma leakage caused by exogenous histamine. These findings indicated that endogenous PGs inhibited the mast cell-dependent reaction at the level of mediator release. This mode of action was confirmed, as diclofenac was found to enhance the in vivo release of histamine that ensued challenge with compound 48/80. Moreover, the enhancement of the response to compound 48/80 observed after diclofenac treatment was prevented by local administration of PGE2 (30 nM). This inhibition included both the histamine release and the plasma leakage. In addition, diclofenac enhanced the leukocyte emigration after compound 48/80 challenge, and PGE2 reversed also this effect, suggesting that endogenous PGs (e.g. PGE2) also inhibited the release of chemotactic mediators.     

Histamine Release Induced by Compound 48/80 from Isolated Rat Mast Cells: Dependence on Endogenous ATP

Abstract: Antimycin A (0.2 μM) reduced the ATP content in isolated rat mast cells to 25–30 % of the original value. Pyruvate (4.9 mM) did not restore the ATP content in antimycin A-treated cells, indicating a complete block of oxidative ATP production. 82–97 % of the glucose which disappeared from suspensions of mast cells in the presence of antimycin A was recovered as lactate. Therefore, the rate of lactate accumulation in suspensions of antimycin A-treated cells was used as a measure of the cellular ATP production rate. Maximal accumulation occurred with 1.1 mM glucose. Incubation with glucose (0.51 mM) for 2.5 min. completely restored the ability of mast cells pretreated with antimycin A to release histamine when exposed to compound 48/80. Concomitantly, the ATP content in the cells was restored to a steady-state level of about 75 % of the original value. 0.29 mM glucose partially restored the histamine release as well as the ATP level whereas 0.13 mM glucose did not affect either of these parameters. The turnover time of ATP at steady-state was about 30 sec. with all three concentrations of glucose, which indicates that the rate of ATP utilization by the cells is directly proportional to the ATP content. The present study supports the view that histamine release induced by compound 48/80 is dependent on the ATP content in the mast cells at the time of exposure to compound 48/80.     

Characterization of FPL-52694 [5-(2-hydroxypropoxyl)-8-propyl-4-oxo-4H-benzopyran-2-carboxylic acid Na] on histamine release from rat peritoneal mast cells induced by antigen, compound 48/80 and A 23187

We studied the in vitro effects of FPL-52694 [5-(2-hydroxypropoxyl)-8-propyl-4-oxo-4H-benzopyran-2-carboxylic acid Na] on histamine release from rat peritoneal mast cells. These cells exposed to ascaris antigen, compound 48/80 or the ionophore A 23187 concentration-dependently released histamine. About a 30-40% histamine release was obtained by 1 X 10(-4) g/ml of antigen, 1 X 10(-7) g/ml of compound 48/80 and A 23187. FPL-52694 (10(-9)-10(-4) g/ml) concentration-dependently inhibited the histamine release from mast cells in response to antigen (1 X 10(-4) g/ml) and compound 48/80 (1 X 10(-7) g/ml), but only slightly inhibited the histamine release induced by A 23187 (1 X 10(-7) g/ml). Similar results were obtained with disodium cromoglycate (DSCG), in the same dose ranges. However, the inhibitory activity of FPL-52694 on histamine release by antigen and compound 48/80 was approximately 10 times more potent than that of DSCG at certain concentrations. Tachyphylaxis was observed when these two agents were preincubated with mast cells for 10 min. These results show FPL-52694 to be a novel mast cell stabilizer.     

The influence of phosphatidyl serine on the release of histamine from isolated rat mast cells induced by different agents.

The influence of phosphatidyl serine (PS) on histamine release from isolated rat mast cells induced by antigen, compound 48/80, adenosine-5'-triphosphate (ATP), the ionophore A23187, and decylamine was studied. PS enhanced antigen-induced release but inhibited the release caused by compound 48/80, A23187, and decylamine. PS did not influence the release induced by ATP. The different effects of PS on the action of the various histamine releasing agents do not conform to a unifying model for the action of PS on the release process. Possible interactions between PS and the agents in the incubation medium as well as at specific reactive sites on the plasma membrane might explain some of the effects of PS. Consequently, the results cannot be used as evidence for the existence of basic differences in the release process induced by various calcium- and energy-dependent releasing agents.     

Investigation into the mast cell stabilizing activity of nature-identical and synthetic indanones

As part of an ongoing search for novel molecules with therapeutic potential we examined the mediator release inhibition activity of a number of indanones and their derivatives. The aldol condensation product 18 was approximately twice as potent as disodium cromoglycate as an inhibitor of compound 48/80-stimulated histamine release from rat peritoneal mast cells. The activity of this class of dimeric indanone compound is significantly higher than controls and may represent a new class of mast cell stabilizing agents. Compound 18 has been selected for further biological evaluation of its mast cell stabilization profile.