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.     

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.     

Cyclic AMP independent inhibition by papaverine of histamine release induced by compound 48/80.

Compound 48/80-induced histamine release from isolated rat peritoneal mast cells was inhibited in a dose-dependent manner by papaverine (ic₅₀ approx 20 μM). This effect of papaverine was not influenced by PGE₁ (14–140 μM), even though PGE₁ markedly increased must cell cAMP levels. Papaverine (0.5 mM) completely inhibited histamine release without causing any change in cAMP levels. Theophylline (0.1 and 0.5 mM) potentiated histamine release induced by submaximal concentrations of compound 48/80, while cAMP levels were increased. IBM X was as potent as papaverine in causing inhibition of mast cell phosphodiesterase. IBM X (0.14–0.7 mM) had no effect on histamine release but caused a 6–20 fold increase in mast cell cyclic AMP. Papaverine inhibition of histamine release was gradual at the onset and was parallelled by a depletion of mast cell ATP content. The inhibition of 48/80-induced histamine release and depletion of mast cell ATP levels was reversed by glucose. It is concluded that papaverine induced inhibition of 48/80-induced histamine release is independent of cAMP, is unrelated to phosphodiesterase inhibition but is dependent upon inhibition of energy production.     

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.     

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.     

Utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187.

The role of endogenous adenosine triphosphate (ATP) in histamine release from rat mast cells induced by the ionophore A23187 in vitro has been studied. 2 The amount of histamine released by calcium from rat mast cells primed with the ionophore A23187 was dependent on the ATP content of the mast cells. 3 In aerobic experiments a drastic reduction in mast cell ATP content was found during the time when histamine release induced by A23187 takes place. 4 Anaerobic experiments were performed with metabolic inhibitors (antimycin A, oligomycin, and carbonyl cyanide p-trifluorometroxyphenylnydrazone), which are known to block the energy-dependent calcium uptake by isolated mitochondria. The mast cell ATP content was reduced during A23187-induced histamine release under anaerobic conditions in the presence of glucose. This indicates an increased utilization of ATP during the release process. 5 The observations are consistent with the view that energy requiring processes are involved in ionophore-induced histamine release from rat mast cells although part of the ATP reduction in the aerobic experiments may be due to an uncoupling effect of calcium on the oxidative phosphorylation.     

THE EFFECT OF VARIOUS DRUGS ON ADENOSINE TRIPHOSPHATE CONTENT AND HISTAMINE RELEASE FROM RAT PERITONEAL CELL SUSPENSIONS RICH IN MAST CELLS

1. Iopanoic acid and iophenoxic acid were potent inhibitors of compound 48/80-induced histamine release from rat peritoneal cell suspensions rich in mast cells and caused a parallel dose-related reduction in ATP content of these cells. 2. Lignocaine was less potent and ouabain and probenecid were ineffective in inhibiting histamine release and reducing cellular ATP content. 3. Various drugs can inhibit 48/80-induced histamine release from rat peritoneal cells and this activity may result from depletion of cellular ATP. It is apparent that little structural specificity is required for activity in this cell system.     

Utilization of adenosine triphosphate in rat mast cells during and after secretion of histamine in response to compound 48/80

The adenosine triphosphate (ATP) content of rat mast cells and their lactate production were measured during and after secretion of histamine induced by compound 48/80. Antimycin A and oligomycin were used to block oxidative ATP synthesis, and 2-deoxyglucose (2-DG) was used to block glycolytic ATP synthesis. Histamine secretion was completed after 10 sec. exposure of the cells to compound 48/80. During that time period there was an increased ATP-utilization of 0.15 pmol/10(3) cells. After completion of the secretory process there seemed to be an enhanced utilization of ATP of 0.40 pmol/10(3) cells/min., which may be associated with recovery of the cells.     

Effects of adenosine-5'-triphosphate (ATP) on rat mast cells: influence on anaphylactic and compound 48/80-induced histamine release.

Anaphylactic histamine release from mast cells isolated from actively sensitized rats was inhibited by pre-incubation with micromolar concentrations of ATP. The inhibition was reversible under various experimental conditions and was counteracted by the presence of calcium in the incubation medium. Histamine release induced by compound 48/80 was similarly affected. Mast cells exposed to antigen under conditions when histamine release was inhibited by ATP became desensitized. The results indicate that ATP inhibits the release mechanism at a step which occurs after the binding of antigen to IgE.     

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.     

Inhibition of immediate allergic reactions by ethanol extract from Plumbago zeylanica stems

The antiallergic properties of the 70% ethanol extract from Plumbago zeylanica stems (EPZ) were investigated in the present study. The extract (500, 1000 mg/kg, p.o.) dose-dependently inhibited systemic anaphylactic shock induced by compound 48/80 in mice, reduced homologous passive cutaneous anaphylaxis and skin reactions induced by histamine or serotonin in rats, significant differences were observed at the dose of 1000 mg/kg. In vitro, EPZ (5, 20, 50 microg/ml) concentration-dependently reduced histamine release from rat peritoneal mast cells caused by compound 48/80 and antigen. EPZ (50 microg/ml) markedly increased intracellular cAMP content of rat mast cells. These findings demonstrate that EPZ inhibits mast cell-dependent immediate allergic reactions, which is probably mediated by reducing the release of mediators such as histamine from mast cells via elevating intracellular cAMP level and weakening the inflammatory action of mediators.     

Effect of TMB-8 on calcium transport in mast cells in relation to histamine secretion

We have previously reported an inhibition of histamine release by TMB-8 both in the presence and absence of calcium and with glucose in the medium. In the present investigation we have studied the effect of TMB-8 on calcium transport. The observations show that TMB-8 inhibits calcium uptake and enhances calcium efflux in mast cells. As antigen-induced histamine release from sensitized mast cells is primarily dependent on extracellular calcium, the inhibition of anaphylactic histamine release by TMB-8 is probably mainly due to an inhibition of calcium influx into the mast cells. We have shown an increased calcium efflux during histamine release from mast cells induced by compound 48/80 in the absence of calcium in the medium, suggesting the release of intracellular calcium stores. The increased calcium efflux was not inhibited by TMB-8. On the contrary, the enhanced calcium efflux caused by compound 48/80, was added to that by TMB-8. TMB-8 thus had no effect on the calcium release from intracellular stores by compound 48/80 but the enhanced calcium efflux by TMB-8 would tend to inhibit histamine release.     

Apomorphine-induced inhibition of histamine release in rat peritoneal mast cells.

The apomorphine-induced inhibition of histamine release in rat peritoneal mast cells was studied by means of secretagogues stimulating different pathways of mast cell activation. Apomorphine inhibited the mast cell response to all releasing agents (lysophosphatidylserine plus nerve growth factor, compound 48/80, substance P, ATP, tetradecanoylphorbolacetate, melittin). The IC50 ranged from 4 microM to 24 microM at concentrations of secretagogues releasing 30-50% of mast cell histamine. However, the potency of the drug decreased at higher secretagogue concentrations. Mast cells, pretreated with apomorphine and washed, released little histamine upon stimulation. The secretory response could be partially restored on increasing the concentration of secretagogues. The results suggest that apomorphine affects a regulatory step controlling the terminal sequence of mast cell secretory activity. As indicated by the reduced potency of the drug, the control by the apomorphine-sensitive reaction loses efficiency under conditions of massive histamine release.     

Dual regulation of the Na+/H(+)-exchange in rat peritoneal mast cells: role of protein kinase C and calcium on pHi regulation and histamine release.

1. The purpose of this study was to compare the actions of phorbol 12-myristate 13-acetate (PMA) and ionomycin on Na+/H+ exchange activation and histamine release to that of compound 48/80 in order to study the possible relationship between pHi and secretion of histamine in rat peritoneal mast cells. 2. Resting pHi in mast cells suspended in a bicarbonate-free physiological salt solution amounted to 6.73 +/- 0.05 (mean +/- s.d., n = 52). 3. PMA (20 nM) induced a substantial but rather slow increase in pHi. This response was very sensitive to inhibition by staurosporine, very sensitive to inhibition by 5-(N,N-hexamethylene)amiloride (HMA), insensitive to the absence of extracellular calcium (without EGTA), and sensitive to partial depletion of intracellular calcium with EGTA. 4. Ionomycin (1 microM) induced a biphasic change in pHi that was sensitive to inhibition by HMA, insensitive to staurosporine. In the absence of extracellular calcium using EGTA, the biphasic response disappeared, leaving only a slow, and diminished change in pHi. 5. The effects of ionomycin and PMA on pHi were additive. 6. Addition of the secretagogue compound 48/80 (1 microgram ml-1) increased pHi, substantially, delta pHi amounting to 0.29 +/- 0.05 pH-units (n = 4). The biphasic pHi-response was insensitive to the absence of extracellular calcium (without EGTA). The initial fast response in pHi was, however, inhibited by HMA, not staurosporine. 7. The finding that staurosporine and HMA each inhibited approximately half of the compound 48/80-induced pHi-response, whereas both inhibitors completely abolished the compound 48/80-induced pHi-response seems to indicate that two independent pathways for the activation of the Na+/H+ exchange were stimulated by compound 48/80. 8. The histamine release induced via both PKC activation (using PMA) and calcium (using ionomycin) were much larger than the sum of each activation pathway, whereas in the absence of extracellular calcium using EGTA, the histamine release in response to PMA and ionomycin was completely abolished. 9. The compound 48/80-induced histamine release was partially sensitive to inhibition by HMA (approximately 30% inhibition) and partially sensitive to inhibition by staurosporine (approximately 50% inhibition). Preincubation with staurosporine and HMA before stimulation with compound 48/80 showed the same degree of inhibition as observed after staurosporine alone, even though this combination of drugs completely inhibited the pHi-response. Furthermore, compound 48/80-induced histamine release was not dependent on the presence of extracellular calcium (with and without EGTA). 10. In spite of the similarities in second messenger pathways for pHi regulation and histamine release, it is, however, not very likely that these two processes are directly related. It is, however, possible, that an increase in pHi plays a permissive, rather than an essential role for histamine release in rat peritoneal mast cells. This hypothesis was supported by the finding that preincubation with the Na+/H+ exchange-inhibitor HMA inhibited 30% of the compound 48/80-induced histamine secretion.     

Effects of Clostridium botulinum C2 toxin-induced depolymerisation of actin on degranulation of suspended and attached mast cells

We studied the effects of C. botulinum C2 toxin, which ADP-ribosylates G-actin, on mast cell degranulation. C2 toxin inhibited degranulation of suspended rat peritoneal mast cells induced by compound 48/80 and dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) maximally by about 50 and 90%, respectively. Inhibition by C2 toxin occurred in a time- and concentration-dependent manner. Half-maximal inhibition of DNP-BSA-induced degranulation by C2 toxin occurred at about 0.015 ng/ml, whereas stimulation of mast cells induced by compound 48/80 was half-maximally inhibited at 0.15 ng/ml C2 toxin. C2 toxin also inhibited stimulated [³H]serotonin release from suspended mast cells. Phorbol 12-myristate 13-acetate (PMA)-induced histamine release of suspended mast cells was inhibited by C2 toxin by about 80–90%. C2 toxin had no effect on calcium ionophore A23187-induced histamine release. Toxin treatment of mast cells caused ADP-ribosylation of actin and depolymerisation of F-actin. Attachment of mast cells, which largely increased the diameter of the subcortical actin network, reduced degranulation stimulated by compound 48/80, antigen and calcium ionophore but not by PMA. Opposite to its effect on suspended cells, in adherent mast cells C2 toxin stimulated degranulation by compound 48/80, antigen, and calcium ionophore but not by PMA. The data indicate that mast cell degranulation and responsiveness towards the actin-depolymerising C2 toxin depend largely on mast cell attachment. Received: 7 October 1996 / Accepted: 22 November 1996     

The effect of a new antihistamine drug, Loderix (EGIS-2062), on stimulus-evoked histamine release from rat peritoneal mast cells

The ability of a new, non-sedative antihistamine drug, Loderix (EGIS-2062), to inhibit stimulus-evoked histamine release from rat peritoneal mast cells has been investigated and compared with that of ketotifen (Zaditen). At low concentrations Loderix preincubated with the cells for 10 min prior to the addition of various stimulants (immune aggregates, ionophore A23187 and compound 48/80) produced a concentration-dependent inhibition of histamine release, while at high concentrations it induced the release of histamine. The IC50 values were calculated as 0.2, 15 and 50 microM by using immune aggregate, (rat IgG2 alpha + anti rat IgG) calcium ionophore and 48/80 as stimulants, respectively. At the IC50 level Loderix was more effective than Zaditen (100 times more effective when immune aggregate and 4 times when 48/80 were used for stimulation). Both drugs had a dual effect on mast cells. The morphological observations confirmed the pharmacological action of the drugs, showing also that the histamine release at high concentrations is due to their cytotoxic effect.     

The action of local anaesthetics on histamine release.

The effect of the local anaesthetics lidocaine, procaine and tetracaine on compound 48/80-induced histamine release from isolated rat mast cells has been investigated. They inhibited histamine release in a dose-dependent manner; at a concentration of 20 mM there was almost total inhibition of histamine release by lidocaine and about 75% inhibition by procaine. Tetracaine exerted a biphasic effect: at concentrations below 1 mM it inhibited, but at concentrations above 1 mM it potentiated histamine release. The inhibitory effect of lidocaine on compound 48/80-evoked histamine release was dependent upon the time of preincubation of mast cells with this anaesthetic and it persisted after washing the cells and resuspension in a lidocaine-free medium. An increase of calcium ions antagonized the inhibitory action of lidocaine. These results can be explained by (1) blockade of membrane receptors for calcium binding which leads to a decrease in intracellular calcium concentration and (2) increase of cellular cyclic AMP content which subsequently inhibits the releasing process.     

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.     

Histamine release by compound 48/80: evidence for the depletion and repletion of calcium using chlortetracycline and 45calcium

Selective release of histamine from rat peritoneal mast cells in vitro was induced by compound 48/80 (1 microgram/ml). Most of the release (75-80%) occurred in a calcium(Ca)-free medium but optimum release was obtained in the presence of 0.9 mM Ca. The release in Ca-free medium still occurred after 180 min incubation. However, prolonged incubation (180 min) in a medium containing chelating agents (EDTA or EGTA) resulted in complete inhibition of histamine release, loss of fluorescence seen with chlortetracycline (CTC) and loss of previously loaded 45Ca from the mast cells. Addition of Ca to these cells resulted in rapid restoration of fluorescence with chlortetracycline. There was also a rapid uptake of 45Ca. Partial depletion of cellular Ca (60 min incubation with EDTA) reduced the rate as well as the amount of histamine release by compound 48/80. These data provide direct evidence for the depletion of cellular Ca which is utilized by compound 48/80 to induce histamine release.     

Adenosine triphosphatase in non-secreting and secreting mast cells

A Ca++-Mg++ ATPase has been demonstrated in the plasma membrane of rat peritoneal mast cells. The enzyme is localized by electron microscopy on the outer surface of the membrane. This agrees with the biochemical findings. A Ca++-Mg++ activated ATPase has also been shown to be present in the granule membrane. The optimal pH of the plasma membrane enzyme is close to the optimal pH for the histamine release. All the 14 inhibitors of plasma membrane ATPase tested - which caused varying degrees of inhibition of the enzyme - also inhibited histamine release induced by antigen, compound 48/80 and the divalent ionophore A23187. The conclusion from the study with the inhibitors is that a mild inhibition of the enzyme is compatible with histamine release, but a pronounced inhibition of the enzyme is always associated with inhibition of histamine release. ATP in low concentrations potentiates the release.