353
Biochimica et Biophysica Acta, 451 (1976) 353--362
© Elsevier/North-HollandBiomedicalPress
BBA 28113 PHOSPHOLIPID METABOLISM AND LYSOSOMAL ENZYME SECRETION BY LEUKOCYTES EFFECTS OF DIBUTYRYL CYCLIC ADENOSINE 3': 5'-MONOPHOSPHATE AND ATP
JEN-SIE TOU and CHRISTINE MAIER Department of Biochemistry, Tulane University School of Medicine, New Orleans, La. 70112 (U.S.A.)
(Received May 24th, 1976)
Summary The effects of dibutyryl cyclic adenosine 3' : 5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of ~-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3': 5'-monophosphate (0.1--1.0 mM) reduced fl-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 mM), but not other nucleotides, was found to potentiate /3glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition of the isotope incorporation into these acidic phospholipids by the two nucleotides has not been defined. Dibutyryl cyclic adenosine 3': 5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [7-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.
Introduction Phagocytosis augments the labeling of phosphatidic acid by 32Pi and the labeling of the phosphoinositides by 32Pi and myo-[2-3H]inositol [1--3]. It also enhances the formation of phosphatidylcholine and phosphatidylethanolamine from the respective 3~p-labeled 1-acyl phosphatides [4,5]. During phagocytosis of inert particles, the lysosomal enzymes are released from leukocytes
354 into the extracellular medium [6]. Protein secretion into the extracellular medium accompanying increased 32Pi incorporation into phosphatidic acid and phosphatidylinositol was also observed in pancreas slices stimulated with pancreozymin or acetylcholine [7], in parotid gland provoked by adrenaline [8,0] and in thyroid gland incubated with pituitary thyrotrophin [10]. Recently we have found an increased 32Pi incorporation into phosphatidic acid and the phosphoinositides accompanying t3-glucuronidase secretion by cytochalasin B-treated polymorphonuclear leukocytes in the presence and absence of inert particle's [11]. In order to understand the relationship between these two cellular events, enzyme secretion and phospholipid response in leukocytes, a study on the effects of agents that modulate enzyme secretion on phospholipid metabolism appears to be important. Cyclic adenosine 3' : 5'-monophosphate has been shown to inhibit lysosomal enzyme release into the extracellular medium from leukocytes in the presence of zymosan particles [12--14]. ATP has been found to potentiate 13-glucuronidase secretion by leukocidin-treated leukocytes in the presence of calcium [15] and to induce ~-glucuronidase release by glycerol-treated leukocytes [16]. In the present study, we examined the effects of dibutyryl cyclic adenosine 3': 5'-monophosphate and ATP on isotope incorporation into phospholipids and ~-glucuronidase secretion from leukocytes in the presence and absence of cytochalasin B. It was found that dibutyryl cyclic adenosine 3': 5'-monophosphate reduced both the phospholipid response and /3-glucuronidase secretion induced by cytochalasin B. ATP was found to potentiate 13.-glucuronidase secretion elicited by cytochalasin B, but it impaired the incorporation of myo-[2-3H]inositol into the phosphoinositides in leukocytes. Materials and Methods Ma terials Cytochalasin B, a product of ICI Ltd., Cheshire, U.K., was purchased from Aldrich Chemical Company, Milwaukee, Wisc. It was dissolved as a stock solution in dimethylsulfoxide at a concentration of 3 mg/ml and stored at --20°C until use. An aliquot of the stock solution was diluted with 0.9% NaCl to 4 pg/10 pl. The cytochalasin B concentration used contained 0.066% dimethylsulfoxide in the incubation medium. In control experiments it was shown that this concentration of dimethylsulfoxide had no apparent effect on the cells. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and phosphatidic acid were purchased from Supelco Co. The diphosphoinositide fraction of Folch containing diphosphoinositide and triphosphoinositide was prepared as described by Lees [17]. Carrier-free 32p-labeled sodium phosphate for injection was obtained from Mallinckrodt. myo-[2-3]Inositol (2.8 Ci/mmol, 98% pure) and [7-32P]ATP (sodium salt) were purchased from New England Nuclear Corp. Dibutyryl cyclic adenosine 3' : 5'-monophosphate (sodium salt) and ATP (disodium salt) were products of Sigma.
355
Me thods Preparation of leukocytes. Polymorphonuclear leukocytes from guinea pig peritoneal exudates were prepared as described previously [3]. For preparation of cell homogenates, the cells were suspended in ice-cold 0.34 M sucrose to give a cell concentration of 5 0 . 1 0 6 cells/ml and were homogenized in a glass homogenizer tube with a motor-driven Teflon pestle (A.H. Thomas Company, Pa.). After 10 up-and-down strokes of the pestle in 1 min, 1--2 min were allowed to elapse for dissipating any heat that had been produced. Homogenization was carried o u t for 3 1-min periods. The cell rupture was monitored by phase microscopy. Incubations. Separate aliquots of cell suspensions were used for measurements of isotope incorporation into phospholipids and the release of ~-glucuronidase. All incubations were performed in siliconized glass-stoppered tubes of 45 ml capacity at 37°C with shaking. For measurement of enzyme release, the cells ( 3 0 - - 4 5 . 1 0 6 in 1.9 ml of Krebs-Ringer phosphate buffer, pH 7.4) were preincubated with 0.1 ml of 20 mM dibutyryl cyclic adenosine 3' : 5'monophosphate or 0.1 ml of 20 mM ATP (the pH of ATP solution was adjusted to 7.0 with 0.1 M NaOH) in 0.9% NaCl for 10 min. Then 10 pl (4 pg) of cytochalasin B or 10 pl of 0.132% dimethylsulfoxide was added to each tube. The tubes were further incubated for 30 min. They were then placed in ice and centrifuged at 250 )< g for 5 min and the enzyme activity in the supernate was measured. Total enzyme activity was determined by diluting 1 volume of cell suspension with 9 volumes of 0.1% Triton X-100. Enzyme assays, fl-Glucuronidase was measured with phenolphthalein glucuronidate as substrate [18] b u t incubated at 56°C for 1 h as recommended by Sigma bulletin No. 325. Lactate dehydrogenase was determined according to Bermeyer et al. [ 19]. Measurement of isotope incorporation into phospholipids. The cells (30-4 5 . 1 0 6 in 1.8 ml of Krebs-Ringer phosphate b u f f e r ) w e r e preincubated with 0.1 ml of 20 mM dibutyryl cyclic adenosine 3' : 5'-monophosphate or 0.1 ml of 20 mM ATP in 0.9% NaC1 for 10 min, then 10 pl (4 pg) of cytochalasin B or 10 pl of 0.132% dimethylsulfoxide followed immediately by appropriate amount of isotope in 0.1 ml of 0.9% NaCl was added to each tube. After the tubes were further incubated for 30 min, the reaction was terminated by adding 10 ml of 0.1 M HC1 in methanol. The incorporation of 32P i and myo-[2-3H]inositol into phospholipids was measured as described previously [3].
Labeling of phospholipids by [7-32P]ATP in leukocyte homogenates. Leukocyte homogenates at protein concentration of 0.8 to 1.0 mg were incubated for 10 min at 37°C in a final volume of 1.0 ml containing sodium phosphate buffer (100 raM), pH 7.4, MgC12 (10 raM), and [ 7 ) 2 ] A T P (4 mM). The incubation was begun by the addition of MgC12. At the end of the incubation, the lipids were extracted as described before [ 3]. Results
Effect of dibutyryl cyclic adenosine 3': 5'-monophosphate on the release of fl-glucuronidase from leukocytes induced by cytochalasin B. The effect of
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Biochimica et Biophysica Acta, 451 (1976) 353--362
© Elsevier/North-HollandBiomedicalPress
BBA 28113 PHOSPHOLIPID METABOLISM AND LYSOSOMAL ENZYME SECRETION BY LEUKOCYTES EFFECTS OF DIBUTYRYL CYCLIC ADENOSINE 3': 5'-MONOPHOSPHATE AND ATP
JEN-SIE TOU and CHRISTINE MAIER Department of Biochemistry, Tulane University School of Medicine, New Orleans, La. 70112 (U.S.A.)
(Received May 24th, 1976)
Summary The effects of dibutyryl cyclic adenosine 3' : 5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of ~-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3': 5'-monophosphate (0.1--1.0 mM) reduced fl-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 mM), but not other nucleotides, was found to potentiate /3glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition of the isotope incorporation into these acidic phospholipids by the two nucleotides has not been defined. Dibutyryl cyclic adenosine 3': 5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [7-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.
Introduction Phagocytosis augments the labeling of phosphatidic acid by 32Pi and the labeling of the phosphoinositides by 32Pi and myo-[2-3H]inositol [1--3]. It also enhances the formation of phosphatidylcholine and phosphatidylethanolamine from the respective 3~p-labeled 1-acyl phosphatides [4,5]. During phagocytosis of inert particles, the lysosomal enzymes are released from leukocytes
354 into the extracellular medium [6]. Protein secretion into the extracellular medium accompanying increased 32Pi incorporation into phosphatidic acid and phosphatidylinositol was also observed in pancreas slices stimulated with pancreozymin or acetylcholine [7], in parotid gland provoked by adrenaline [8,0] and in thyroid gland incubated with pituitary thyrotrophin [10]. Recently we have found an increased 32Pi incorporation into phosphatidic acid and the phosphoinositides accompanying t3-glucuronidase secretion by cytochalasin B-treated polymorphonuclear leukocytes in the presence and absence of inert particle's [11]. In order to understand the relationship between these two cellular events, enzyme secretion and phospholipid response in leukocytes, a study on the effects of agents that modulate enzyme secretion on phospholipid metabolism appears to be important. Cyclic adenosine 3' : 5'-monophosphate has been shown to inhibit lysosomal enzyme release into the extracellular medium from leukocytes in the presence of zymosan particles [12--14]. ATP has been found to potentiate 13-glucuronidase secretion by leukocidin-treated leukocytes in the presence of calcium [15] and to induce ~-glucuronidase release by glycerol-treated leukocytes [16]. In the present study, we examined the effects of dibutyryl cyclic adenosine 3': 5'-monophosphate and ATP on isotope incorporation into phospholipids and ~-glucuronidase secretion from leukocytes in the presence and absence of cytochalasin B. It was found that dibutyryl cyclic adenosine 3': 5'-monophosphate reduced both the phospholipid response and /3-glucuronidase secretion induced by cytochalasin B. ATP was found to potentiate 13.-glucuronidase secretion elicited by cytochalasin B, but it impaired the incorporation of myo-[2-3H]inositol into the phosphoinositides in leukocytes. Materials and Methods Ma terials Cytochalasin B, a product of ICI Ltd., Cheshire, U.K., was purchased from Aldrich Chemical Company, Milwaukee, Wisc. It was dissolved as a stock solution in dimethylsulfoxide at a concentration of 3 mg/ml and stored at --20°C until use. An aliquot of the stock solution was diluted with 0.9% NaCl to 4 pg/10 pl. The cytochalasin B concentration used contained 0.066% dimethylsulfoxide in the incubation medium. In control experiments it was shown that this concentration of dimethylsulfoxide had no apparent effect on the cells. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and phosphatidic acid were purchased from Supelco Co. The diphosphoinositide fraction of Folch containing diphosphoinositide and triphosphoinositide was prepared as described by Lees [17]. Carrier-free 32p-labeled sodium phosphate for injection was obtained from Mallinckrodt. myo-[2-3]Inositol (2.8 Ci/mmol, 98% pure) and [7-32P]ATP (sodium salt) were purchased from New England Nuclear Corp. Dibutyryl cyclic adenosine 3' : 5'-monophosphate (sodium salt) and ATP (disodium salt) were products of Sigma.
355
Me thods Preparation of leukocytes. Polymorphonuclear leukocytes from guinea pig peritoneal exudates were prepared as described previously [3]. For preparation of cell homogenates, the cells were suspended in ice-cold 0.34 M sucrose to give a cell concentration of 5 0 . 1 0 6 cells/ml and were homogenized in a glass homogenizer tube with a motor-driven Teflon pestle (A.H. Thomas Company, Pa.). After 10 up-and-down strokes of the pestle in 1 min, 1--2 min were allowed to elapse for dissipating any heat that had been produced. Homogenization was carried o u t for 3 1-min periods. The cell rupture was monitored by phase microscopy. Incubations. Separate aliquots of cell suspensions were used for measurements of isotope incorporation into phospholipids and the release of ~-glucuronidase. All incubations were performed in siliconized glass-stoppered tubes of 45 ml capacity at 37°C with shaking. For measurement of enzyme release, the cells ( 3 0 - - 4 5 . 1 0 6 in 1.9 ml of Krebs-Ringer phosphate buffer, pH 7.4) were preincubated with 0.1 ml of 20 mM dibutyryl cyclic adenosine 3' : 5'monophosphate or 0.1 ml of 20 mM ATP (the pH of ATP solution was adjusted to 7.0 with 0.1 M NaOH) in 0.9% NaCl for 10 min. Then 10 pl (4 pg) of cytochalasin B or 10 pl of 0.132% dimethylsulfoxide was added to each tube. The tubes were further incubated for 30 min. They were then placed in ice and centrifuged at 250 )< g for 5 min and the enzyme activity in the supernate was measured. Total enzyme activity was determined by diluting 1 volume of cell suspension with 9 volumes of 0.1% Triton X-100. Enzyme assays, fl-Glucuronidase was measured with phenolphthalein glucuronidate as substrate [18] b u t incubated at 56°C for 1 h as recommended by Sigma bulletin No. 325. Lactate dehydrogenase was determined according to Bermeyer et al. [ 19]. Measurement of isotope incorporation into phospholipids. The cells (30-4 5 . 1 0 6 in 1.8 ml of Krebs-Ringer phosphate b u f f e r ) w e r e preincubated with 0.1 ml of 20 mM dibutyryl cyclic adenosine 3' : 5'-monophosphate or 0.1 ml of 20 mM ATP in 0.9% NaC1 for 10 min, then 10 pl (4 pg) of cytochalasin B or 10 pl of 0.132% dimethylsulfoxide followed immediately by appropriate amount of isotope in 0.1 ml of 0.9% NaCl was added to each tube. After the tubes were further incubated for 30 min, the reaction was terminated by adding 10 ml of 0.1 M HC1 in methanol. The incorporation of 32P i and myo-[2-3H]inositol into phospholipids was measured as described previously [3].
Labeling of phospholipids by [7-32P]ATP in leukocyte homogenates. Leukocyte homogenates at protein concentration of 0.8 to 1.0 mg were incubated for 10 min at 37°C in a final volume of 1.0 ml containing sodium phosphate buffer (100 raM), pH 7.4, MgC12 (10 raM), and [ 7 ) 2 ] A T P (4 mM). The incubation was begun by the addition of MgC12. At the end of the incubation, the lipids were extracted as described before [ 3]. Results
Effect of dibutyryl cyclic adenosine 3': 5'-monophosphate on the release of fl-glucuronidase from leukocytes induced by cytochalasin B. The effect of
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