Early kinetics of Ca2+ fluxes and histamine release in rat mast cells stimulated with compound 48/80

The kinetics of Ca²⁺ uptake and efflux have been measured in rat peritoneal mast cells stimulated with compound 48/80 using rapid mixing and a silicone oil centrifugation technique. Responses at one-second time intervals were resolved beginning as early as three seconds after initial stimulation. The results clearly demonstrate that Ca²⁺ uptake occurs after the initiation of histamine release. Ca²⁺ efflux occurs simultaneously with histamine release. The implications of these findings are discussed and the technique is described.     

Effects of prolonged incubation of rat peritoneal mast cells with compound 48/80.

Rat peritoneal mast cells (MC) co-cultured on a monolayer of 3T3 fibroblasts (MC/3T3) were continuously exposed to compound 48/80 for 14 days. As early as 2 days following continuous exposure to compound 48/80, the MC/3T3 appeared as a heterogeneous population, with various MC appearing partially or fully degranulated or intact; this morphological pattern continued throughout the duration of the experiment. MC/3T3 remained functionally active as demonstrated by their ability to secrete histamine 15 min after each replacement with fresh medium containing compound 48/80, although this capacity diminished towards the end of the 14-day experiment. Concomitant with the histamine release, a significant increase in cellular histamine pools was observed. When MC/3T3 continuously exposed to compound 48/80 for 7 or 14 days were acutely challenged with anti-IgE antibodies, they were able to secrete histamine and prostaglandin D2 in amounts similar to those produced by control MC. In contrast, when these cells were challenged on day 7 or 14 with a higher dose of compound 48/80 or with substance P, the release of histamine was partially inhibited. Our results indicate that continuous in vitro exposure to compound 48/80, and the resulting MC degranulation product histamine, does not adversely affect the ability of MC/3T3 to synthesize histamine and to respond to activation stimuli of a related secretagogue for 7 days and a non-related one for at least 14 days.     

Differential effects of antioxidants and indomethacin on compound 48/80 induced histamine release and Ca2+ uptake in rat mast cells

The effect of nordihydroguaiaretic acid (NDGA), vitamin E, butylated hydroxytoluene (BHT) and indomethacin on histamine release and Ca2+ uptake in rat mast cells stimulated with compound 48/80 was studied. NDGA inhibited both the release of histamine and Ca2+ uptake in stimulated cells; however, there was no correlation between inhibition of Ca2+ uptake and the amount of histamine release. At a concentration of 5 microM, NDGA completely inhibited Ca2+ uptake, while histamine release was decreased by less than 50%. BHT (50 microM) inhibited both the Ca2+ uptake and histamine release. On the other hand, vitamin E (50 microM) inhibited histamine release by 70% without impairment in Ca2+ uptake. In the absence of the stimulus, vitamin E increased the cell-associated Ca2+; however, it had no effect on spontaneous release of histamine. Indomethacin (3 microM) inhibited Ca2+ uptake in stimulated cells by 50%, but did not affect the release of histamine. The results suggest that a part of Ca2+-influx may not be related to the coupled activation--secretion response and that lipid peroxidation through the lipoxygenase pathway may be involved in secretion of histamine from mast cells.     

Effect of anoxia, glucose and thioglycollate on anaphylactic and compound 48/80-induced histamine release in isolated rat mast cells

Histamine release by antigen from rat mast cells was strongly inhibited by prolonged anoxia. The inhibition was reversed by oxygen and by glucose. Thioglycollate in low concentration potentiated histamine release and in high concentration inhibited it. The effects of anoxia and thioglycollate are additive: inhibition was produced by a low concentration of thioglycollate combined with a short incubation in nitrogen. This inhibition was reversed by glucose and became a potentiation. The effects of anoxia, glucose and thioglycollate on histamine release by compound 48/80 were quantitatively similar to those on histamine release by antigen.

These results are consistent with the view that two opposing mechanisms are at work: a freeing of tissue SH groups which potentiates the anaphylactic mechanism and a disruption of tissue S—S bonds which inhibits it. Oxygen lack may inhibit by a reduction of S—S bonds as well as by an exhaustion of metabolic stores.

     

Thymus dependence of compound 48/80-induced mucosal mast cell proliferation

The mucosal and connective-tissue mast cells (MMC and CTMC, respectively) of the rat are histochemically and functionally distinct. MMC, unlike CTMC, are insensitive to the degranulating action of the mast cell secretagogue Compound 48/80 and instead increase in number after treatment with this drug. T cell-derived growth factors are necessary for the growth of MMC-like cells from hemopoietic tissues in vitro, as well as for nematode-induced MMC proliferation in vivo. We therefore examined the possible role of the thymus in the pharmacologically induced MMC expansion. Mast cell numbers and histamine content after treatment with Compound 48/80 were determined in the skin, tongue and gut of athymic male LEW/MOL-rnu/rnu rats and their normal littermates at the age of 6-7 weeks. The influence of the strain of rat and the mode of administration of the drug was assessed by studying age- and sex-matched Sprague-Dawley rats as well. Injections of Compound 48/80 for 5 days resulted in a statistically significant increase in the number of intestinal MMC and the histamine content of all thymus-bearing rats. The increase was more pronounced in the Sprague-Dawley rats, however, and unrelated to the mode of administration of the drug, indicating that the MMC-stimulating action of Compound 48/80 is at least partly controlled by genetic factors. Athymic rats showed no statistically significant MMC expansion. This suggests that the MMC response after Compound 48/80 treatment may be at least partly dependent on the thymus. The mast cell numbers and histamine content in the tissues of athymic control rats were higher than in their thymus-bearing littermates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in intracellular Ca2+ distribution of rat peritoneal mast cells before and after histamine release

Rat peritoneal mast cells were stained with quin 2, a fluorescent Ca²⁺ chelator. By means of a fluorescence microscope and real time image processer, it was revealed that the fluorescence derived from the Ca-quin 2 complex was weak in the area occupied by the nucleus and distributed unevenly in the cytoplasm of the resting cells so as to encompass the individual granules. When compound 48/80 or substance P was added in a Ca-free medium, the fluorescence intensity of quin 2 increased markedly all over the cell, suggesting that a large amount of Ca²⁺ was released from intracellular Ca stores. The increase in the fluorescence intensity produced by compound 48/80 or substance P in a Ca-free medium was inhibited by pretreatment with certain drugs eliciting an increase of c-AMP levels, such as dibutyryl c-AMP and theophylline, or by some anti-allergic drugs providing a membrane stabilizing action.     

Role of the cytoskeleton in Ca2+ release from the intracellular Ca store of rat peritoneal mast cells

In order to study the role of the cytoskeleton in histamine release from mast cells, the effects of cytochalasin D, cholchicine and vinblastine on Ca²⁺ release from the intracellular Ca store induced by compound 48/80 were investigated by means of a video-intensified microscopy system. When the quin 2-loaded mast cells were stimulated by 0.35 g/ml of compound 48/80, a rapid increase in intracellular Ca²⁺ was observed. At concentrations higher than 10^–6 M, both colchicine and vinblastine pretreatments significantly inhibited the increase in intracellular Ca²⁺ concentrations caused by compound 48/80, although cytochalasin D had no effect. When permeabilized mast cells were exposed to potassium-antimonate solution, microtubules became attached to the endoplasmic reticulum, where many dots of Ca-antimonate were observed; in some areas, the microtubules interconnected the endoplasmic reticulum and granules in the mast cells. From the results of the present study, it was assumed that microtubules play some important role in the processes leading to Ca²⁺ release from the intracellular Ca store.     

Effect of compound 48/80 on mast cells and histamine levels in rat brain and on corticosterone secretion

Inflammation Research -     

Freeze-fracture immunocytochemistry for intracellular localization of serotonin in mast cells stimulated with compound 48/80

Changes of intracellular localization of serotonin in rat mast cells were examined by freeze-fracture immunocytochemistry, to prevent the translocation of the serotonin antigen. Rat peritoneal cells including mast cells were stimulated in vitro with compound 48/80, at 17° C for 0, 30 or 60 s for exocytosis to occur. The mast cells were fixed, quickly frozen and freeze-fractured to expose the antigen on the fractured surface. They were immunostained with serotonin antibody, and the immunoreactions on the fractured surface were examined on ultrathin sections by electron microscopy. Unstimulated mast cells exhibited serotonin localization mostly in each intragranular matrix. In contrast, mast cells stimulated for 30 s exhibited increased serotonin in their intergranular cytoplasm. Mast cells showed more distinct immunoreactions in the cytoplasm where degranulation would be promoted after 60 s. It is suggested that intracellular release of serotonin occurred in the stimulated mast cells.     

Comparison of histamine and serotonin release from rat peritoneal mast cells induced by nerve growth factor and compound 48/80

Inflammation Research -     

In vitro uptake of 5-hydroxytryptamine and dopamine by rat mast cells after exocytosis induced by antigen or compound 48/80.

Mast cells from the peritoneal and pleural cavities of actively sensitized rats were isolated and incubated with biogenic amines (5-hydroxytryptamine and dopamine) with or without pretreatment with specific antigen. An anaphylactic reaction resulting in the release of 20-25% of the histamine in the cells led to a slightly reduced amine uptake. At concentrations which induced histamine release comparable to that during the anaphylactic reaction compound 48/80 had a similar effect on the uptake of the two amines. Histamine release induced by higher concentrations of compound 48/80 led to a more pronounced reduction in the uptake of the amines, the reduction being roughly proportional to the extent of the histamine release. It is concluded that the reduction in the in vitro amine uptake after anaphylactic and compound 48/80-induced histamine release is due to the fact that there are a fewer intact granules capable of storing histamine and not primarily due to a damage to the mechanisms by which mast cells take up biogenic amines in vitro. The observations further strengthen the view that anaphylactic and compound 48/80-induced histamine release are non-cytolytic processes.     

Tachykinin-1 receptor antagonism suppresses substance-P- and compound 48/80-induced mast cell activation from rat mast cells expressing functional mas-related GPCR B3

Inflammation Research - Mice and rats are important animal models for mast cell (MC) study. However, rat Mas-related-GPCR-B3 receptor (MRGPRB3) has been less studied than its mouse counterpart....     

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 preincubation 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.     

Compound 48/80-induced secretion of histamine from rat peritoneal mast cells depends on a tryptase controlled step also leading to chymase activity

Compound 48/80 caused activation of rat mast cell tryptase and chymase along with the release of histamine. Following low speed centrifugation, tryptase remained essentially in the supernatant while chymase sedimented. Enzyme activities as well as histamine release were inhibited by phenylmethyl-sulfonyl fluoride and tosyl-lysine chloromethyl ketone, which are respectively unspecific and specific inhibitors of trypsin-like enzymes. Chymostatin inhibited chymase, but not the release of histamine by 48/80. Tryptase activity thus seems to be essentially involved in the mechanism of histamine release, while chymase activity may be merely one of its accompanying events.     

Binding of compound 48/80 by isolated rat mast cells in connection with histamine release

The binding of compound 48/80 in connection with histamine release from isolated rat mast cells was investigated by a two-step incubation procedure. After incubation of mast cells with known concentrations of compound 48/80, the supernatants were collected and subsequently exposed to fresh mast cells. The response in the second incubation step provided a measure of the amount of compound 48/80 remaining in the supernatants. At noncytotoxic concentrations of the releasing agent, binding capacities for compound 48/80 of up to 4 micrograms/10(5) mast cells (4% w/v) were observed. The binding of compound 48/80 was of high affinity, giving mast cell concentrations of the compound exceeding that in the incubation medium by four orders of magnitude. The binding occurred rapidly with a substantial proportion bound within the first minute. These findings indicate that quantitation of exocytotic events by means of basic dyes may be compromised by competition for granular binding sites by basic releasing agents.     

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 experimentsin 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.     

X-ray microanalysis of rat mast cells stimulated with compound 48/80 in combination with quick-freezing method

X-ray microanalysis was performed on rat mast cells prepared by quick-freezing, cryosectioning and freeze-drying (QF-FD) method, or quick-freezing and freeze-substitution (QF-FS) method. Peritoneal cells including mast cells were stimulated with compound 48/80 for 0, 10 or 30 s at 17° C, and the mast cells stimulated for 30 s started exocytosis. In X-ray spectra of the QF-FD specimen, mast cells stimulated for 10 s increased their levels of phosphorus, sodium and chlorine in the intergranular cytoplasm prior to exocytosis, and kept this increase until 30 s after stimulation. In the QF-FS specimen, where soluble elements were removed, peaks of phosphorus, sulphur and potassium could be detected as elements in X-ray spectra. Phosphorus increased and potassium decreased in intergranular cytoplasm of mast cells stimulated for 10 s, and these changes became more obvious after 30 s. However, supplemental increase of other cations such as sodium could not be detected in the QF-FS specimens.