Nirmal Chakravarty

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

SummaryThe 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.

Dependence of histamine release from rat mast cells on adenosine triphosphate

Summaryusing pure populations of rat mast cells, the relation of the ATP content of the cells to histamine release induced by compound 48/80 has been studied. Variable ATP levels in the mast cells have been produced by incubation with appropriate concentrations of oligomycin.The dose-response curves of oligomycin for the inhibition of histamine release and for the reduction in the ATP content of the mast cells are similar. The concentration required for 50% inhibition of histamine release is, however, higher than that for 50% reduction of the ATP level.Comparative study of the reduction of the ATP content and the inhibition of histamine release in samples of the same suspension of mast cells shows a linear relation between 10 to 90% inhibition of histamine release and 40 to 95% inhibition of ATP synthesis.The observations support the hypothesis that ATP is involved in the process of histamine release from mast cells induced by compound 48/80.

Electron microscopic study of the regeneration in vitro of rat peritoneal mast cells after histamine secretion

SummaryRegeneration of rat mast cells was studied by TEM from 10 s to 48 h after secretion of histamine induced by compound 48/80. During the first 2 h, small intracellular cavities, formed during compound exocytosis and containing non-membrane-bound remnants of the granules, tended to coalesce, and after 2 h of incubation regeneration started. After 6 h, all the cavities had fused into one large central cavity which contained the remnants of the granules and remained open to the exterior during the entire period. The plasma membrane microfolds which disappeared just after secretion were reformed during regeneration. They were apparently involved in endocytotic-like activity and coated vesicles also appeared beneath the plasmalemma (membrane recycling?). The fate of the granule remnants in the cavity is unknown, as regeneration was not completed after 48 h which is the longest survival time obtained so far in ultrastructural studies of mast cell regeneration in vitro.

Plasma membrane adenosine triphosphatases in rat peritoneal mast cells and macrophages—the relation of the mast cell enzyme to histamine release

Abstract Adenosine triphosphatases activated by calcium or magnesium have been demonstrated on the outer surface of rat peritoneal mast cells and macrophages. The plasma membrane ATPases in the two types of cells have similar but not identical properties. Mg2+ is somewhat more effective than Ca2+ in stimulating both the enzymes. They are not influenced by sodium and potassium and not inhibited by ouabain and oligomycin. Ethacrynic acid inhibits both, but the mast cell enzyme is more sensitive to it. The enzyme on the macrophage has five to thirty-seven times higher activity (average seventeen times) than that on the mast cell. The apparent Km of the enzymes in intact cells, incubated with adenosine triphosphate for 5 min, is estimated to be 36 μM for mast cells and 30 μM for macrophages. The optimal pH for the mast cell and the macrophage enzymes is 6.7 and 7.1 respectively. The activities of the two enzymes rise similarly with temperature up to 37° but differ at 47°, the macrophage enzyme being less active at this temperature than at 37°. Phosphatidyl serine, which stimulates anaphylactic and dextran-induced histamine release, causes about 40 per cent stimulation of the plasma membrane ATPase of mast cells in the absence of Ca2+ and Mg2+ but has no appreciable effect in their presence. No change in the mast cell enzyme could, however, be observed in relation to histamine release induced by dextran, compound 48 80 and ATP. But ethacrynic acid, which in 1 mM concentration inhibits 50 per cent of the mast cell enzyme activity, also causes pronounced inhibition of histamine release induced by all the three agents in the same concentration. The inhibition is not influenced by the presence of glucose, suggesting that ethacrynic acid does not inhibit histamine release by blocking energy metabolism. Ethacrynic acid apparently acts at another site. The site of action could very well be plasma membrane ATPase. There is also a correlation between the inhibition of the mast cell enzyme by sodium fluoride and lack of calcium and their inhibitory effect on histamine release. The possible involvement of the plasma membrane ATPase of mast cells in the process of exocytosis leading to histamine release is discussed.

Role of intracellular calcium in histamine release from rat mast cells

Compound 48/80-induced histamine release may utilize both intracellular and extracellular calcium. The release is inhibited by La3+ and TMB-8 both in the presence and absence of calcium. The observations are consistent with a mobilization of calcium from the inner surface of the plasma membrane supporting the release. TMB-8 probably acts at more than one site. It has been shown to inhibit skeletal muscle contraction and this has been correlated to its inhibitory effect on the release of Ca2+ from the sarcoplasmic reticulum. TMB-8 also inhibits resting cellular influx and efflux of calcium in the ileum. Anaphylactic and dextran-induced histamine release is dependent on extracellular calcium; the inhibition of the release by TMB-8 seems to be primarily due to an interference with calcium transport across the plasma membrane. In the absence of extracellular calcium the inhibition of compound 48/80-induced histamine release by TMB-8 seems to be due to stabilization of Ca2+ binding to cellular stores.

Ca2+-Mg2+-activated adenosine triphosphatase in plasma and granule membranes in non-secreting and secreting mast cells

Abstract An adenosine triphosphatase (ATP) activated by Ca2+ or Mg2+ is shown morphologically on the outer surface of non-secreting and secreting rat peritoneal mast cells. ATPase having the same properties is also seen on the external surface of the other peritoneal cells, i.e. macrophages, mononuclear cells and lymphocytes. When histamine release from the mast cells was induced by exposing them to antigen (anaphylactic reaction) or compound 48/80, ATPase activated by Ca2+ or Mg2+ could in addition be demonstrated in the granule membranes. Granule membrane ATPase is also shown in non-secreting mast cells after freezing and thawing. ATPase on the outer surface of the plasma membrane is seen in the secreting mast cells as in the non-secreting cells except in the areas where the plasma membrane fuses with the granule membrane. The role of ATPase in granule secretion process has been discussed.

Histamine secretion from permeabilized mast cells by calcium

A transient increase in the permeability of the mast cell membrane was caused by the exposure of the cells to low concentrations of saponin, 5 or 10 micrograms/ml. These concentrations had very little effect in the absence of calcium but caused 35 to 50% histamine release, having the character of a secretory response, when 0.25 mM or more calcium was added to the medium. The dose-response curve was steep between 25 microM and 250 microM calcium and tended to flatten with higher concentrations. The release was associated with a pronounced increase in calcium uptake, which was faster than the histamine release. The membrane changes were slight as indicated by only 7 to 12% leakage of lactate dehydrogenase and by the absence of any detectable change in the electron micrographs. The transient nature of the membrane change is shown by the following experiment. When the cells were first exposed to saponin in the absence of calcium, the amount of histamine released by the subsequent incubation with calcium varied inversely with the time interval that elapsed before calcium was added. If calcium was added after 15 minutes no histamine release occurred. When calcium uptake was studied in the same manner, the stimulation of calcium uptake in saponin-treated cells also declined progressively with increasing intervals after the exposure to saponin when calcium was added. Stimulation of both histamine release and calcium uptake was inhibited by antimycin A, the inhibition curves with 10(-9)M to 10(-7)M antimycin A being similar. The effect on the calcium uptake by itself could explain the inhibition of histamine release. But the release was also inhibited by the calmodulin antagonists, W-7 and mepacrine, suggesting that the influx of calcium in the permeabilized cells acts primarily through calmodulin-mediated enzyme activation.

Glucose metabolism in rat mast cells during histamine release

Abstract The time course of the changes in the metabolism of exogenous glucose in rat peritoneal mast cells has been studied in relation to histamine release, induced by antigen-antibody (anaphylactic) reaction, dextran and compound 48 80 . Histamine is released from a suspension of mast cells at 37 °C in 10–40 sec in response to these selective releasing agents, and during the brief period when the release occurs, the metabolism of glucose is shown to be stimulated 82–183% above the basal level. Since histamine release from mast cells is preceded by exocytosis of the basophilic granules, the stimulation of glucose metabolism during the release appears to be coupled to an increased energy requirement for the exocytosis. The enhanced glucose metabolism, however, continues for varying periods—from 45 to 90 min or more with the different agents—after the release is completed. The persistent stimulation of energy metabolism seems to be related to the repair of the plasma membrane following exocytosis and to restorative processes within the cell.

The involvement of protein kinase C in exocytosis in mast cells

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.

Mechanisms of histamine release from isolated mast cell granules by calcium with phosphatidyl serine

Histamine is released from isolated mast cell granules with intact membranes by calcium (10 mM) in presence of phosphatidyl serine (25–50 μg/ml). The release occurs both in Krebs-Ringer solution and in sucrose solution without monovalent cations, but the release in Krebs-Ringer solution is somewhat higher. The histamine release is associated with increased calcium uptake. But calcium is taken up much faster, within 5 sec, while it takes several minutes before histamine release is completed. The observations suggest a rapid uptake of calcium to the granule membrane, from which it may be more slowly released to the matrix, displacing histamine from its binding sites. Phosphatidyl serine with calcium could also conceivably change the membrane permeability causing increased influx of sodium ions, thus accounting for the mild enhancement of the release in Krebs-Ringer solution.