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Arachidonic acid release in BW755C-pretreated rat peritoneal mast cells stimulated with A23187, concanavalin A and compound 4880
290
Biochimica et BwphVsica Acta 917 (1987) 296295 Elsevier
BBA 52332
Arachidonic acid release in BW755C-pretreated rat peritoneal mast cells stimulated with A23187, concanavalin A and compound 48/80 Kouji Yamada
a,*, Yukio Okano a, Kiyoshi Miura b and Yoshinori
Nozawa
a
a Depariment of Bwchemistry and h Department of 3rd Internal Medicine, Gifu lJniuersit.v School of Medicine, Tsukasamachi-40, (Received
Key words:
Arachidonic
Gifu 500 (Japan)
19 August
1986)
acid release; Phospholipid
metabolism;
(Rat mast cells)
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 cycle-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.
Introduction Mast cells respond to exposure to hyperosmolar solutions, anaphylatoxins, and several stimulants, such as anti-immunoglobulin E, concanavalin A, compound 48/80 and A23187. Such stimulants cause mobilization of calcium ions, enhanced phospholipid metabolism, release of arachidonic acid, and secretion of mediators, i.e., histamine, leukotrienes and prostaglandins [l]. Since arachidonic acid is a key substrate for generation
* On leave from the 3rd Department of Internal Medicine, Gifu University School of Medicine., Abbreviations: BW755C, 3-amino-l-[ m-(trif’luoromethyl)phenyl]-2-pyrazoline; compound 48/80, condensation product of N-methyl-p-methoxyphenetholamine with formaldehyde. Correspondence: Gifu University 500, Japan.
0005-2760/87/$03.50
K. Yamada, Department of Biochemistry, School of Medicine, Tsukasamachi-40, Gifu
0 1987 Elsevier Science Publishers
of prostanoids, the understanding of regulatory mechanism(s) for arachidonic acid release is of great importance from the pharmacological as well as the clinical viewpoints. However, because of the rapid conversion of arachidonic acid to peroxides, it has been difficult to elucidate fully the nature of arachidonic acid mobilization in stimulated mast cells. In the present study, we pretreated rat mast cells with BW755C [2], which is a known inhibitor of both lipoxygenase and cycle-oxygenase, to minimize further metabolic changes of arachidonic acid, and then stimulated cells with three functionally different secretagogues, A23187 (a bypassing stimulant), compound 48/80 (a membraneperturbing stimulant), and concanavalin A (a receptor-mediated stimulant). The three main pathways for arachidonic acid liberation in activated cells have been proposed to be: (1) phospholipase C, diacylglycerol lipase and monoacylglycerol lipase [3]; (2) phospholipase C followed by phos-
B.V. (Biomedical
Division)
291
phatidic acid-specific phospholipase A, [4]; and (3) phospholipase A, [5]. Current investigations suggest that the sources of liberated arachidonic acid in activated mast cells are phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) [6,7]. Materials and Methods Isolation of rat mast cells Mast cells were obtained from the peritoneal cavity of Wistar rats, and purified using a Percoll gradient method according to the procedure of Wells and Mann [8]. Briefly, rats were exsanguinated by decapitation and then injected with 20 ml of ice-cold medium A (137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH,PO,, 1.8 mM CaCl,, 1 mM MgCl,, 5.6 mM glucose, 10 mM Hepes, 1 mg/ml gelatin, 5 units/ml heparin, pH 7.4) into the cavity. The trunk of the rat was massaged more than 100 times. The medium was recovered by aspiration and centrifuged (150 x g, 5 min, 4“C). The pellet was resuspended in 1.6 ml of medium A. The cell suspension was mixed with 3.6 ml of Percoll and 0.4 ml of lo-fold concentrated phosphate-free medium A, and then overlaid with 1 ml medium A and centrifuged (200 x g, 15 min, 4OC). Mast cells were obtained from the pellet, washed twice, and resuspended in medium A. Mast cell preparations were about 95% pure, and more than 95% of cells were viable as inferred by trypan blue uptake. Lipid metabolism Purified mast cells suspended in medium A were prelabeled with [3H]arachidonic acid (2 pCi/106 cells) in the presece of 0.1% bovine serum albumin for 60 min at 37“C, and washed with medium A without bovine serum albumin twice. Mast cell suspension (2. 10’ cells/O.48 ml) was first incubated with 10 ~1 BW755C (30 PM) for 2 min and then stimulated by exposure to 10 ~1 A23187 (1 PM), compound 48/80 (5 pg/ml) or concanavalin A (30 pg/ml) for the indicated periods of time. A23187 was dissolved in dimethyl sulfoxide, compound 48/80 and concanavalin A were in water, and BW755C was in ethanol. Stimulation with concanavalin A was carried out in the presence of 30 pg/ml of P.S. The reactions
were terminated by the addition of 2 ml ice-cold chloroform/methanol (1 : 2, v/v), and lipid extraction was performed by the method of Bligh and Dyer [9]. The individual phospholipids were separated by two-dimensional thin-layer chromatography on Silica gel H 60 plates impregnated with magnesium acetate (2.5%) using chloroform/ methanol/ 13.5 N ammonia water (65 : 35 : 6, v/v) in the first-dimension system, and chloroform/ acetone/ methanol/ acetic acid/ water (30: 40: 10: 10: 5, v/v), in the second-dimension system [lo]. The neutral lipids were analysed on borate (0.4 M) -impregnated silica gel H 60 plates in chloroform/acetone (96 : 4, v/v) [lo]. Spots were identified by comigration with authentic standards. The areas corresponding to individual lipids were scraped into vials and the radioactivity was determined in a liquid scintillation counter (Beckmann LS-7500) with 2800 ml toluene/700 ml Triton X-100/175 ml water/l2 g 2,5-diphenyloxazol/ 0.84 g 2,2’-phenylenebis(5phenyloxazole) [lo]. Histamine assay Cell suspension ((l-2). 10’ cells/O.5 ml) was preincubated in the presence or absence of BW755C and stimulated with each secretagogue for 5 min. Histamine release was terminated by adding 1 ml of ice-cold 10 mM EDTA-containing medium A to the cell suspension. The histamine contents of the supernatant and the pellet were determined by the method of Shore et al. [ll]. The percent release was expressed as the ratio of histamine content in the supernatant to the sum of supernatant and pellet. Materials [5,6,8,9,11,12,14,15-3H]Arachidonate (56 Ci/ mmol) was obtained from Amersham. Compound 48/80, concanavalin A, and bovine serum albumin (fraction V, essentially fatty acid-free) were obtained from Sigma. A23187 and BW755C were obtained from Eli-Lilly and Teikoku Zoki Co., respectively. Silic age1 H 60 plates were products of Merck. Results and Discussion When rat [3H]arachidonic
mast cells were incubated with acid for 1 h, most of the radioac-
292
decrease drastically with the progressing time of incubation. Triacylglycerol also showed an appreciable decrease in 3H radioactivity level. Instead, diacylglycerol continued to increase, while phosphatidic acid reached a peak at 60 s and declined thereafter. PE and PI appeared to remain unchanged. These results suggested that two types of lipase are activated upon stimulation with A23187; phospholipase A, hydrolyzing PC to arachidonic acid and lysoPC, and triacylglycerol lipase hydrolyzing triacylglycerol to arachidonic acid and diacylglycerol. Therefore, the released arachidonic acid seems to originate from PC and triacylglycerol. The liberation of arachidonic acid upon stimulation with a receptor-mediated agent, concanavalin A, was less marked than that induced by A23187 (Fig. 2). Concanavalin A caused an increase in arachidonic acid radioactivity from 3.9 * lo3 dpm to 6.0. lo3 dpm. PC showed a gradual decrease immediately after stimulation. The reduction in triacylglycerol seen from A23187-stimulated cells did not occur within 300 s in concanavalin A-activated cells. Rather, a significant
tivity was incorporated into PC, amounting to 63.0% of total lipid radioactivity. The distribution of radioactivity among other lipids was 15.2% in PI, 10.3% in PE, 4.3% in triacylglycerol and 1.9% in PS. Arachidonic acid, diacylor monoacylglycerol accounted for less than 1%. When radiolabeled cells were pretreated with BW755C for 2 min and then stimulated with A23187 (1 PM), 3H radioactivity in the arachidonic acid fraction increased with the concentration of BW755C, indicating the prevention of arachidonic acid oxygenation by inhibition of both Iipoxygenase and cycle-oxygenase. But there was no further increase in radioactivity in the arachidonic acid fraction at concentrations more than 30 PM (data not shown). In the presence of 30 /.LM BW755C, remarkable increases were observed not only in arachidonic acid but also in diacylglycerol. Accordingly, we used 30 I_LM BW755C in the following experiments. When BW755C-pretreated mast cells were stimulated with 1 /.LM A23187, arachidonic acid increased 7-fold (3.4. lo3 dpm to 24.0. lo3 dpm) (Fig. 1). The radioactivity in PC was observed to
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Fig. 1. The time course of the changes in [3H]arachidonate in BW755C (30 PM) -pretreated mast cells stimulated with 1 gM A23187. Mast cells, prelabeled with [3H]arachidonic acid, were incubated with BW755C (30 PM) for 2 min at 37OC, and then exposed to 1 pM A23187 for the indicated periods of time. Open symbols (control) were without the stimulant. Each value is the mean of duplicate determinations from a representative of two similar experiments. The bars indicate the range. The radioactivity of total lipids is about 500000 dpm. The radioactivity for PC, triacylglycerol (TG), arachidonic acid (AA), diacylglycerol (DG), PI, phosphatidic acid (PA), PE and PL (total phospholipids) in unstimulated control was 3.8.105, 2.9.104, 3.4.103, 3.3.103, 5.5.104, 1.9.103, 5.9.104 and 4.9.105 dpm, respectively.
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Fig 2. The time course of the changes in [3H]arachidonate in BW755C (30 PM) -pretreated mast cells stimulated with 30 pg/ml concanavalin A. Mast cells, prelabeled with [ 3H]arachidonic acid, were incubated with BW755C (30 PM) for 2 min at 37’C. and then exposed to 30 pg/ml concanavahn A for the indicated periods of time. In the medium 30 pg/ml PS was added. Open symbols (control) were without the stimulant. Each value is the mean of duplicate determinations from a representative of two similar experiments. The bars indicate the range. The radioactivity of total lipids is about 3OOooO dpm. The radioactivity for PC, TG, AA, DG, PI, PA, PE and PL in unstimulated control was 1.6.105. 1.6.104, 2.0.103, 2.0.103, 5.2.104, 1.5.103, 2.8.104 and 2.6.105 dpm, respectively. The abbreviations for lipids are the same as in Fig. 1.
elevation was observed after 300 s. Diacylglycerol and phosphatidic acid tended to increase, with the latter being slightly decreased after a peak at 300 s. PI showed a transient increase at 30 s and then decreased to the minimum level at 120 s. It is thus conceivable that the decrease in PI may result from the action of phospholipase C, as observed with other secretory cells exposed to receptormediated stimulants. Major differences in lipid metabolism between A23187- and concanavalin A-stimulated cells were observed in triacylglycerol and PI. In concanavalin A-stimulated cells triacylglycerol increased and PI decreased, whereas A23187 caused a decrease in triacylglycerol without any significant change in PI. Therefore, these findings indicate that concanavalin A stimulation induces arachidonic acid liberation from PC by phospholipase A, and from PI by phospholipase C. By activation with compound 48/80, the level of 3H radioactivity in PC was considerably reduced (Fig. 3). However, triacylglycerol demonstrated an increase after 30 s which was also seen with concanavalin A stimulation. The level of
liberated [ 3Hlarachidonic acid showed a small but significant elevation. While a plateau was observed at 30-60 s, phosphatidic acid increased to a peak at 10 s, and decreased thereafter. The liberation rate of [ 3H]arachidonic acid in compound 48/80_stimulated cells was lowest among the three secretagogues with different sites of action. There was a small transient decrease in PI at 10 s. PE was not altered. The overall features of lipid metabolism were more or less similar in compound 48/80- and concanavalin A-stimulated cells. Although the amount of released arachidonic acid was small, the alterations in [ 3H]arachidonic acid radioactivity suggested that PC and PI serve as donors of arachidonic acid. On the other hand, the profiles of histamine secretion differed depending on the type of secretagogues (Fig. 4). Compound 48/80 induced an abrupt and full secretion of histamine within 10 s, whereas A23187 caused a rapid secretion within 10 s with a subsequent progressive increase. The situation was distinctly different with concanavalin A, which began to cause secretion of histamine after a 30-s lag period. These data,
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Fig. 3. The time course of the changes in [3H]arachidonate in BW755C (30 PM) -pretreated mast cells and arachidonic acid release induced by 5 pg/ml compound 48/80. Mast cells, prelabeled with [ ‘Hlarachidonic acid, were incubated with BW755C (30 PM) for 2 min at 37OC, and then exposed to 5 gg/ml compound 48/80 for the indicated periods of time. Open symbols (control) were without the stimulant. Each value is the mean of duplicate determinations from a representative of two similar experiments. The bars indicate the range. The radioactivity of total lipids is about 510000 dpm. The radioactivity for PC, TG, AA, DG, PI, PA, PE and PL in 1.8.103, 1.5.103, 8.2.104, 2.6.103, 5.2.104 and 4.9.10s dpm, respectively. The unstimulated control was 3.3. 105, 1.4.104, abbreviations for lipids are the same as in Fig. 1.
together with those for arachidonic acid release, lead us to consider that the potency of histamine secretion is not parallel with the rate of arachidonic acid liberation.
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Fig. 4. Time courses of secretagogue-induced Mast cells were incubated with BW755C (30 37OC, and then exposed to 1 PM A23187 and (0 -o), pg/ml compound 48/80 for the indicated canavalin A (0 -0) Stimulation with concanavalin A was carried ence of 30 pg/ml PS.
histamine release. PM) for 2 min at 0.5 (A -A), 30 pg/ml conperiods of time. out in the pres-
The sources of arachidonic acid released by activation have been investigated in several types of secretory cells, including platelets and neutrophils [6,12-141. Evidence has been presented to indicate that donors of arachidonic acid may be different depending on the type of cells and secretagogues. In addition to the source, the amounts and the time courses of arachidonic acid liberation are not uniform in secretory cells. In order to understand better the stimulussecretion coupling, the mechanisms of arachidonic acid mobilization need to be clarified; their elucidation is of great importance from the clinical point of view, i.e. with regard to allergy and inflammation. However, we have encountered some difficulties in calculating the liberated arachidonic acid content, because of its rapid oxygenation. It appears that the amount of free arachidonic acid has been underestimated in earlier works. In fact, Smith et al. [15] recently described measurement of arachidonic acid release in human platelets pretreated with BW755C. BW755C is an inhibitor of both lipoxygenase and cyclo-
295
oxygenase, and does not inhibit phospholipid metabolism but instead allows accumulation of released radioactive arachidonic acid [15]. By pretreatment of mast cells with BW755C, subtle changes of arachidonic acid release can be observed. Therefore, in the present study, we have examined changes in [ 3H]arachidonic acid distribution in rat peritoneal mast cells exposed to the three functionally different secretagogues, A23187 (receptor-bypassing), concanavalin A (receptormediated), and compound 48/80 (membrane perturbing). The accumulation of arachidonic acid after stimulation with all three secretagogues used here was found to be enhanced by pretreating mast cells with 30 PM BW755C. The level of diacylglycerol also increased. Three possibilities for diacylglycerol increase are considered. One is that the metabolism from diacylglycerol to arachidonic acid through monoacylglycerol is feedback-regulated by the accumulation of arachidonic acid. The second possibility is the hydrolysis of triacylglycerol by a lipase. Since the radioactivity in triacylglycerol of A23187-stimulated cells was decreased, this pathway may be operating in these cells. The release of arachidonic acid from triacylglycerol was suggested for rat platelets incubated with A23187 and thrombin [16]. The third is the enhanced PI turnover via phospholipase C generating diacylglycerol and inositol phosphates. In concanavalin A-stimulated cells, PI decreased and diacylglycerol and phosphatidic acid increased. It is conceivable that the receptor-mediated activation of phospholipase C caused a decrease in PI with increases in diacylglycerol and phosphatidic acid in concanavalin A-stimulated cells.
In summary, our results suggest that the quantity and time course of arachidonic acid liberation in mast cells differ depending on the type of stimulation: Ig E receptor (concanavalin A), cross-bridging membrane perturbation (compound 48/80), and Ca 2+ increase (A23187). References 1 Parker, C.W. (1986) Jpn. J. Allergol. 35, 305-312 2 Higgs, G.A., Flower, R.J. and Vane, J.R. (1979) Biochem. Pharmacol. 28, 1959-1961 3 Prescott, SM. and Majerus, P.W. (1983) J. Biol. Chem. 258, 764-769 4 Lapetina, E.G. (1982) Trends Pharmacol. Sci. 3, 115-118 5 Kannagi, R. and Koizumi, K. (1979) Arch. Biochem. Biophys. 196, 534-542 6 Okano, Y., Ishizuka, Y., Nakashima, S., Tohmatsu, T., Takagi, H. and Nozawa, Y. (1985) B&hem. Biophys. Res. Commun. 127, 126-732 7 Imai, A., Ishizuka, Y., Nakashima, S. and Nozawa, Y. (1984) Arch. B&hem. Biophys. 232, 259-268 8 Wells, E. and Mann, J. (1983) Biochem. Pharmacol. 32, 837-842 9 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917 10 Imai, A., Yano, K., Kameyama, Y. and Nozawa. Y. (1982) Jpn. J. Exp. Med. 52, 99-105 11 Shore, P.A., Burkhalter, A. and Cohn, V.H. (1959) J. Pharmacol. Exp. Ther. 127, 182-186 12 Neufeld, N.J. and Majerus, P.W. (1982) J. Biol. Chem. 258. 2461-2467 13 Takenawa, T., Homma, Y. and Nagai, Y. (1983) J. Immunol. 130, 2849-2855 14 Mahadevappa, V.G. and Holub, B.J. (1986) Biochem. Biophys. Res. Commun. 134, 1327-1333 15 Smith, J.B., Dangelmaier, C. and Mauco, G. (1985) Biochim. Biophys. Acta 835, 344-351 16 Colard, 0.. Breton, M. and Breziat. G. (1986) Biochem. J. 233. 691-695
Biochimica et BwphVsica Acta 917 (1987) 296295 Elsevier
BBA 52332
Arachidonic acid release in BW755C-pretreated rat peritoneal mast cells stimulated with A23187, concanavalin A and compound 48/80 Kouji Yamada
a,*, Yukio Okano a, Kiyoshi Miura b and Yoshinori
Nozawa
a
a Depariment of Bwchemistry and h Department of 3rd Internal Medicine, Gifu lJniuersit.v School of Medicine, Tsukasamachi-40, (Received
Key words:
Arachidonic
Gifu 500 (Japan)
19 August
1986)
acid release; Phospholipid
metabolism;
(Rat mast cells)
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 cycle-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.
Introduction Mast cells respond to exposure to hyperosmolar solutions, anaphylatoxins, and several stimulants, such as anti-immunoglobulin E, concanavalin A, compound 48/80 and A23187. Such stimulants cause mobilization of calcium ions, enhanced phospholipid metabolism, release of arachidonic acid, and secretion of mediators, i.e., histamine, leukotrienes and prostaglandins [l]. Since arachidonic acid is a key substrate for generation
* On leave from the 3rd Department of Internal Medicine, Gifu University School of Medicine., Abbreviations: BW755C, 3-amino-l-[ m-(trif’luoromethyl)phenyl]-2-pyrazoline; compound 48/80, condensation product of N-methyl-p-methoxyphenetholamine with formaldehyde. Correspondence: Gifu University 500, Japan.
0005-2760/87/$03.50
K. Yamada, Department of Biochemistry, School of Medicine, Tsukasamachi-40, Gifu
0 1987 Elsevier Science Publishers
of prostanoids, the understanding of regulatory mechanism(s) for arachidonic acid release is of great importance from the pharmacological as well as the clinical viewpoints. However, because of the rapid conversion of arachidonic acid to peroxides, it has been difficult to elucidate fully the nature of arachidonic acid mobilization in stimulated mast cells. In the present study, we pretreated rat mast cells with BW755C [2], which is a known inhibitor of both lipoxygenase and cycle-oxygenase, to minimize further metabolic changes of arachidonic acid, and then stimulated cells with three functionally different secretagogues, A23187 (a bypassing stimulant), compound 48/80 (a membraneperturbing stimulant), and concanavalin A (a receptor-mediated stimulant). The three main pathways for arachidonic acid liberation in activated cells have been proposed to be: (1) phospholipase C, diacylglycerol lipase and monoacylglycerol lipase [3]; (2) phospholipase C followed by phos-
B.V. (Biomedical
Division)
291
phatidic acid-specific phospholipase A, [4]; and (3) phospholipase A, [5]. Current investigations suggest that the sources of liberated arachidonic acid in activated mast cells are phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) [6,7]. Materials and Methods Isolation of rat mast cells Mast cells were obtained from the peritoneal cavity of Wistar rats, and purified using a Percoll gradient method according to the procedure of Wells and Mann [8]. Briefly, rats were exsanguinated by decapitation and then injected with 20 ml of ice-cold medium A (137 mM NaCl, 2.7 mM KCl, 0.4 mM NaH,PO,, 1.8 mM CaCl,, 1 mM MgCl,, 5.6 mM glucose, 10 mM Hepes, 1 mg/ml gelatin, 5 units/ml heparin, pH 7.4) into the cavity. The trunk of the rat was massaged more than 100 times. The medium was recovered by aspiration and centrifuged (150 x g, 5 min, 4“C). The pellet was resuspended in 1.6 ml of medium A. The cell suspension was mixed with 3.6 ml of Percoll and 0.4 ml of lo-fold concentrated phosphate-free medium A, and then overlaid with 1 ml medium A and centrifuged (200 x g, 15 min, 4OC). Mast cells were obtained from the pellet, washed twice, and resuspended in medium A. Mast cell preparations were about 95% pure, and more than 95% of cells were viable as inferred by trypan blue uptake. Lipid metabolism Purified mast cells suspended in medium A were prelabeled with [3H]arachidonic acid (2 pCi/106 cells) in the presece of 0.1% bovine serum albumin for 60 min at 37“C, and washed with medium A without bovine serum albumin twice. Mast cell suspension (2. 10’ cells/O.48 ml) was first incubated with 10 ~1 BW755C (30 PM) for 2 min and then stimulated by exposure to 10 ~1 A23187 (1 PM), compound 48/80 (5 pg/ml) or concanavalin A (30 pg/ml) for the indicated periods of time. A23187 was dissolved in dimethyl sulfoxide, compound 48/80 and concanavalin A were in water, and BW755C was in ethanol. Stimulation with concanavalin A was carried out in the presence of 30 pg/ml of P.S. The reactions
were terminated by the addition of 2 ml ice-cold chloroform/methanol (1 : 2, v/v), and lipid extraction was performed by the method of Bligh and Dyer [9]. The individual phospholipids were separated by two-dimensional thin-layer chromatography on Silica gel H 60 plates impregnated with magnesium acetate (2.5%) using chloroform/ methanol/ 13.5 N ammonia water (65 : 35 : 6, v/v) in the first-dimension system, and chloroform/ acetone/ methanol/ acetic acid/ water (30: 40: 10: 10: 5, v/v), in the second-dimension system [lo]. The neutral lipids were analysed on borate (0.4 M) -impregnated silica gel H 60 plates in chloroform/acetone (96 : 4, v/v) [lo]. Spots were identified by comigration with authentic standards. The areas corresponding to individual lipids were scraped into vials and the radioactivity was determined in a liquid scintillation counter (Beckmann LS-7500) with 2800 ml toluene/700 ml Triton X-100/175 ml water/l2 g 2,5-diphenyloxazol/ 0.84 g 2,2’-phenylenebis(5phenyloxazole) [lo]. Histamine assay Cell suspension ((l-2). 10’ cells/O.5 ml) was preincubated in the presence or absence of BW755C and stimulated with each secretagogue for 5 min. Histamine release was terminated by adding 1 ml of ice-cold 10 mM EDTA-containing medium A to the cell suspension. The histamine contents of the supernatant and the pellet were determined by the method of Shore et al. [ll]. The percent release was expressed as the ratio of histamine content in the supernatant to the sum of supernatant and pellet. Materials [5,6,8,9,11,12,14,15-3H]Arachidonate (56 Ci/ mmol) was obtained from Amersham. Compound 48/80, concanavalin A, and bovine serum albumin (fraction V, essentially fatty acid-free) were obtained from Sigma. A23187 and BW755C were obtained from Eli-Lilly and Teikoku Zoki Co., respectively. Silic age1 H 60 plates were products of Merck. Results and Discussion When rat [3H]arachidonic
mast cells were incubated with acid for 1 h, most of the radioac-
292
decrease drastically with the progressing time of incubation. Triacylglycerol also showed an appreciable decrease in 3H radioactivity level. Instead, diacylglycerol continued to increase, while phosphatidic acid reached a peak at 60 s and declined thereafter. PE and PI appeared to remain unchanged. These results suggested that two types of lipase are activated upon stimulation with A23187; phospholipase A, hydrolyzing PC to arachidonic acid and lysoPC, and triacylglycerol lipase hydrolyzing triacylglycerol to arachidonic acid and diacylglycerol. Therefore, the released arachidonic acid seems to originate from PC and triacylglycerol. The liberation of arachidonic acid upon stimulation with a receptor-mediated agent, concanavalin A, was less marked than that induced by A23187 (Fig. 2). Concanavalin A caused an increase in arachidonic acid radioactivity from 3.9 * lo3 dpm to 6.0. lo3 dpm. PC showed a gradual decrease immediately after stimulation. The reduction in triacylglycerol seen from A23187-stimulated cells did not occur within 300 s in concanavalin A-activated cells. Rather, a significant
tivity was incorporated into PC, amounting to 63.0% of total lipid radioactivity. The distribution of radioactivity among other lipids was 15.2% in PI, 10.3% in PE, 4.3% in triacylglycerol and 1.9% in PS. Arachidonic acid, diacylor monoacylglycerol accounted for less than 1%. When radiolabeled cells were pretreated with BW755C for 2 min and then stimulated with A23187 (1 PM), 3H radioactivity in the arachidonic acid fraction increased with the concentration of BW755C, indicating the prevention of arachidonic acid oxygenation by inhibition of both Iipoxygenase and cycle-oxygenase. But there was no further increase in radioactivity in the arachidonic acid fraction at concentrations more than 30 PM (data not shown). In the presence of 30 /.LM BW755C, remarkable increases were observed not only in arachidonic acid but also in diacylglycerol. Accordingly, we used 30 I_LM BW755C in the following experiments. When BW755C-pretreated mast cells were stimulated with 1 /.LM A23187, arachidonic acid increased 7-fold (3.4. lo3 dpm to 24.0. lo3 dpm) (Fig. 1). The radioactivity in PC was observed to
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Fig. 1. The time course of the changes in [3H]arachidonate in BW755C (30 PM) -pretreated mast cells stimulated with 1 gM A23187. Mast cells, prelabeled with [3H]arachidonic acid, were incubated with BW755C (30 PM) for 2 min at 37OC, and then exposed to 1 pM A23187 for the indicated periods of time. Open symbols (control) were without the stimulant. Each value is the mean of duplicate determinations from a representative of two similar experiments. The bars indicate the range. The radioactivity of total lipids is about 500000 dpm. The radioactivity for PC, triacylglycerol (TG), arachidonic acid (AA), diacylglycerol (DG), PI, phosphatidic acid (PA), PE and PL (total phospholipids) in unstimulated control was 3.8.105, 2.9.104, 3.4.103, 3.3.103, 5.5.104, 1.9.103, 5.9.104 and 4.9.105 dpm, respectively.
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Fig 2. The time course of the changes in [3H]arachidonate in BW755C (30 PM) -pretreated mast cells stimulated with 30 pg/ml concanavalin A. Mast cells, prelabeled with [ 3H]arachidonic acid, were incubated with BW755C (30 PM) for 2 min at 37’C. and then exposed to 30 pg/ml concanavahn A for the indicated periods of time. In the medium 30 pg/ml PS was added. Open symbols (control) were without the stimulant. Each value is the mean of duplicate determinations from a representative of two similar experiments. The bars indicate the range. The radioactivity of total lipids is about 3OOooO dpm. The radioactivity for PC, TG, AA, DG, PI, PA, PE and PL in unstimulated control was 1.6.105. 1.6.104, 2.0.103, 2.0.103, 5.2.104, 1.5.103, 2.8.104 and 2.6.105 dpm, respectively. The abbreviations for lipids are the same as in Fig. 1.
elevation was observed after 300 s. Diacylglycerol and phosphatidic acid tended to increase, with the latter being slightly decreased after a peak at 300 s. PI showed a transient increase at 30 s and then decreased to the minimum level at 120 s. It is thus conceivable that the decrease in PI may result from the action of phospholipase C, as observed with other secretory cells exposed to receptormediated stimulants. Major differences in lipid metabolism between A23187- and concanavalin A-stimulated cells were observed in triacylglycerol and PI. In concanavalin A-stimulated cells triacylglycerol increased and PI decreased, whereas A23187 caused a decrease in triacylglycerol without any significant change in PI. Therefore, these findings indicate that concanavalin A stimulation induces arachidonic acid liberation from PC by phospholipase A, and from PI by phospholipase C. By activation with compound 48/80, the level of 3H radioactivity in PC was considerably reduced (Fig. 3). However, triacylglycerol demonstrated an increase after 30 s which was also seen with concanavalin A stimulation. The level of
liberated [ 3Hlarachidonic acid showed a small but significant elevation. While a plateau was observed at 30-60 s, phosphatidic acid increased to a peak at 10 s, and decreased thereafter. The liberation rate of [ 3H]arachidonic acid in compound 48/80_stimulated cells was lowest among the three secretagogues with different sites of action. There was a small transient decrease in PI at 10 s. PE was not altered. The overall features of lipid metabolism were more or less similar in compound 48/80- and concanavalin A-stimulated cells. Although the amount of released arachidonic acid was small, the alterations in [ 3H]arachidonic acid radioactivity suggested that PC and PI serve as donors of arachidonic acid. On the other hand, the profiles of histamine secretion differed depending on the type of secretagogues (Fig. 4). Compound 48/80 induced an abrupt and full secretion of histamine within 10 s, whereas A23187 caused a rapid secretion within 10 s with a subsequent progressive increase. The situation was distinctly different with concanavalin A, which began to cause secretion of histamine after a 30-s lag period. These data,
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B
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Fig. 3. The time course of the changes in [3H]arachidonate in BW755C (30 PM) -pretreated mast cells and arachidonic acid release induced by 5 pg/ml compound 48/80. Mast cells, prelabeled with [ ‘Hlarachidonic acid, were incubated with BW755C (30 PM) for 2 min at 37OC, and then exposed to 5 gg/ml compound 48/80 for the indicated periods of time. Open symbols (control) were without the stimulant. Each value is the mean of duplicate determinations from a representative of two similar experiments. The bars indicate the range. The radioactivity of total lipids is about 510000 dpm. The radioactivity for PC, TG, AA, DG, PI, PA, PE and PL in 1.8.103, 1.5.103, 8.2.104, 2.6.103, 5.2.104 and 4.9.10s dpm, respectively. The unstimulated control was 3.3. 105, 1.4.104, abbreviations for lipids are the same as in Fig. 1.
together with those for arachidonic acid release, lead us to consider that the potency of histamine secretion is not parallel with the rate of arachidonic acid liberation.
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. 60
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Fig. 4. Time courses of secretagogue-induced Mast cells were incubated with BW755C (30 37OC, and then exposed to 1 PM A23187 and (0 -o), pg/ml compound 48/80 for the indicated canavalin A (0 -0) Stimulation with concanavalin A was carried ence of 30 pg/ml PS.
histamine release. PM) for 2 min at 0.5 (A -A), 30 pg/ml conperiods of time. out in the pres-
The sources of arachidonic acid released by activation have been investigated in several types of secretory cells, including platelets and neutrophils [6,12-141. Evidence has been presented to indicate that donors of arachidonic acid may be different depending on the type of cells and secretagogues. In addition to the source, the amounts and the time courses of arachidonic acid liberation are not uniform in secretory cells. In order to understand better the stimulussecretion coupling, the mechanisms of arachidonic acid mobilization need to be clarified; their elucidation is of great importance from the clinical point of view, i.e. with regard to allergy and inflammation. However, we have encountered some difficulties in calculating the liberated arachidonic acid content, because of its rapid oxygenation. It appears that the amount of free arachidonic acid has been underestimated in earlier works. In fact, Smith et al. [15] recently described measurement of arachidonic acid release in human platelets pretreated with BW755C. BW755C is an inhibitor of both lipoxygenase and cyclo-
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oxygenase, and does not inhibit phospholipid metabolism but instead allows accumulation of released radioactive arachidonic acid [15]. By pretreatment of mast cells with BW755C, subtle changes of arachidonic acid release can be observed. Therefore, in the present study, we have examined changes in [ 3H]arachidonic acid distribution in rat peritoneal mast cells exposed to the three functionally different secretagogues, A23187 (receptor-bypassing), concanavalin A (receptormediated), and compound 48/80 (membrane perturbing). The accumulation of arachidonic acid after stimulation with all three secretagogues used here was found to be enhanced by pretreating mast cells with 30 PM BW755C. The level of diacylglycerol also increased. Three possibilities for diacylglycerol increase are considered. One is that the metabolism from diacylglycerol to arachidonic acid through monoacylglycerol is feedback-regulated by the accumulation of arachidonic acid. The second possibility is the hydrolysis of triacylglycerol by a lipase. Since the radioactivity in triacylglycerol of A23187-stimulated cells was decreased, this pathway may be operating in these cells. The release of arachidonic acid from triacylglycerol was suggested for rat platelets incubated with A23187 and thrombin [16]. The third is the enhanced PI turnover via phospholipase C generating diacylglycerol and inositol phosphates. In concanavalin A-stimulated cells, PI decreased and diacylglycerol and phosphatidic acid increased. It is conceivable that the receptor-mediated activation of phospholipase C caused a decrease in PI with increases in diacylglycerol and phosphatidic acid in concanavalin A-stimulated cells.
In summary, our results suggest that the quantity and time course of arachidonic acid liberation in mast cells differ depending on the type of stimulation: Ig E receptor (concanavalin A), cross-bridging membrane perturbation (compound 48/80), and Ca 2+ increase (A23187). References 1 Parker, C.W. (1986) Jpn. J. Allergol. 35, 305-312 2 Higgs, G.A., Flower, R.J. and Vane, J.R. (1979) Biochem. Pharmacol. 28, 1959-1961 3 Prescott, SM. and Majerus, P.W. (1983) J. Biol. Chem. 258, 764-769 4 Lapetina, E.G. (1982) Trends Pharmacol. Sci. 3, 115-118 5 Kannagi, R. and Koizumi, K. (1979) Arch. Biochem. Biophys. 196, 534-542 6 Okano, Y., Ishizuka, Y., Nakashima, S., Tohmatsu, T., Takagi, H. and Nozawa, Y. (1985) B&hem. Biophys. Res. Commun. 127, 126-732 7 Imai, A., Ishizuka, Y., Nakashima, S. and Nozawa, Y. (1984) Arch. B&hem. Biophys. 232, 259-268 8 Wells, E. and Mann, J. (1983) Biochem. Pharmacol. 32, 837-842 9 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917 10 Imai, A., Yano, K., Kameyama, Y. and Nozawa. Y. (1982) Jpn. J. Exp. Med. 52, 99-105 11 Shore, P.A., Burkhalter, A. and Cohn, V.H. (1959) J. Pharmacol. Exp. Ther. 127, 182-186 12 Neufeld, N.J. and Majerus, P.W. (1982) J. Biol. Chem. 258. 2461-2467 13 Takenawa, T., Homma, Y. and Nagai, Y. (1983) J. Immunol. 130, 2849-2855 14 Mahadevappa, V.G. and Holub, B.J. (1986) Biochem. Biophys. Res. Commun. 134, 1327-1333 15 Smith, J.B., Dangelmaier, C. and Mauco, G. (1985) Biochim. Biophys. Acta 835, 344-351 16 Colard, 0.. Breton, M. and Breziat. G. (1986) Biochem. J. 233. 691-695