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Arachidonic acid release induced by extracellular ATP in osteoblasts: role of phospholipase D
Prostaglandins, Leukotrienes and Essential FattyAcids (1997) 57(3), 335-339 © PearsonProfessionalLtd 1997
A r a c h i d o n i c acid r e l e a s e induced by e x t r a c e l l u l a r A T P in osteoblasts: role of phospholipase D Y. W a t a n a b e - T o m i t a , 1 A. Suzuki, 1 J. Shinoda, 1 Y. Oiso, 1 0 . K o z a w a 2 1First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466, Japan 2Department of Pharmacology, Gifu University School of Medicine, Gifu 500, Japan
Summary In a previous study, we have shown that extracellular ATP stimulates Ca2÷influx resulting in the release of arachidonic acid (AA) and prostaglandin E2 (PGE2) synthesis in osteoblast-like MC3T3-E1 cells. In addition, we have recently reported that extracellular ATP stimulates phosphatidylcholine hydrolysis by phospholipase D (PLD) independently from the activation of protein kinase C in these cells. It is well recognized that phosphatidylcholine is hydrolysed by PLD, generating phosphatidic acid, which can be further degraded by phosphatidic acid phosphohydrolase to diacylglycerol (DG). In the present study, we investigated the role of PLD activation in the extracellular ATP-induced AA release and PGE2 synthesis in osteoblast-like MC3T3-E1 cells. Extracellular ATP stimulated AA release dose-dependently in the range between 0.1 and 1 mM. Propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, significantly inhibited the AA release induced by extracellular ATP in a dosedependent manner in the range between 100 and 300 tIM. 1,6-Bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of DG lipase, significantly suppressed the AA release induced by extracellular ATP. Both the pretreatment of propranolol and RHC-80267 also inhibited the extracellular ATP-induced PGE2 synthesis. These results strongly suggest that the AA release induced by extracellular ATP is mediated at least in part by phosphatidylcholine hydrolysis by PLD in osteoblast-like cells.
INTRODUCTION It is well known that two functional cell types, osteoblasts and osteoclasts, regulate bone metabolismJ The former cells are responsible for bone formation and the latter for bone resorption. Accumulating evidence suggests that the receptors of bone resorbing agents such as parathyroid hormone and 1,25-dihydroxyvitamine D3 are found in osteoblasts.' Thus, osteoblasts are recognized to play important roles also in the regulation of bone resorption. A variety of hormonal factors and autacoids modify bone metabolismJ It has been reported that extraceUular ATP acts through its binding to specific receptor (P2purinoceptor) on the cell surface, 2'a and that the P2purinoceptor exists on osteoblasts. 4,5 It is generally recognized that stimulation of cellsurface receptors induced by many hormones and neuroReceived 11 December 1996 Accepted 4 January 1997 Correspondence to: Osamu Kozawa, Tel. 00 81 58 265 1241; Fax. 00 81 58 267 2959
transmitters initiates hydrolysis of phosphoinositides, which produces two second messengers, diacylglycerol (DG) and inositol 1,4,5-trisphosphate? '7 Protein kinase C is physiologically activated by DG. However, phosphatidylinositol hydrolysis by phospholipase C (PLC) is not the only pathway of DG formation. 8'9 Hydrolysis of phosphatidylcholine by phospholipase D (PLD) generates phosphatidic acid. a-l° Phosphatidic acid, which itself could be a potential intracellular mediator, can be further degraded by a phosphatidic acid phosphohydrolase to DG. Nowadays, it is recognized that PLD plays an important role in modulating cellular functions. We have recently reported H that extracellular ATP stimulates phosphatidylcholine hydrolysis by PLD independently from the activation of protein kinase C in osteoblast-like MC3T3-E1 ceils derived from newbom m o u s e c a l v a r i a . 12,1a In a previous study/4 we have shown that extraceilular ATP stimulates Ca2÷ influx resulting in the release of arachidonic acid (A_A)and the synthesis of prostaglandin E2 (PGE2) which has been shown to be a major eicosanoid product ~5,~6in osteoblast-like MC3T3-E1 cells. It is gener-
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ally accepted that the activation of phospholipase A2 is the major pathway of AA releaseY AA is also released via hydrolysis of phospholipids by other phospholipasesY In the present study, we investigated the role of phosphatidylcholine hydrolysis by PLD in the extracellular ATP-induced AA release in MC3T3-E1 cells. Herein, we show that the AA release induced by extracellular ATP is mediated at least in part by phosphatidylcholine hydrolysis by PLD in osteoblast-like cells. MATERIALS AND METHODS Materials
[5,6,8,9,11,12,14,15-aH]AA (212.1 Ci/mmol) and PGE2[~2sI] assay system were purchased from Amersham Japan (Tokyo, Japan). ATP and essentially fatty acid-free bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). d/-Propranolol hydrochloride (propranolol) was purchased from Wako Pure Chemical Industries (Osaka, Japan). 1,6-Bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267) was purchased from Funakoshi Pharmaceutical Co. (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. Propranolol and RHC-80267 were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide in the culture medium was 0.1%, which did not affect the measurement of AA release and PGE2 synthesis. Cell culture
Cloned osteoblast-like MC3T3-E1 cells were generously provided by Dr M. Kumegawa (Meikai University, Sakado, Japan) and maintained in a-minimum essential medium (a-MEM) containing 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% CO2/95% air. The cells (5 x 104) were seeded into 35-ram diameter dishes in 2 ml of c~-MEMcontaining 10% FCS. After 5 days, the medium was exchanged for 2 ml of a-MEM containing 0.3% FCS. The ceils were used for experiments after 48 h.
the medium was determined. When indicated, the cells were pretreated with various doses of propranolol or RHC-80267 for 20 min. Assay for PGE2
Procedures were done as described under 'Measurement of AA release' except for using unlabelled ceils. The cultured cells were pretreated with various doses of propranolol or RHC-80267 for 20 min, and then stimulated by 1 mM extracellular ATE After 120 min, the medium was collected and PGE2 in the medium was measured with a radioimmunoassay kit. Determinations
The radioactivity of 3H-samples was determined with a Beckman LS-6000IC liquid scintillation spectrometer. The radioactivity of 125I-samples was determined with an Aloka ARC-600 auto well gamma system. Statistical analysis
The data were analyzed by one-way analysis of variance, followed by the Bonferroni method for multiple comparison between pairs, and a P<0.05 was considered significant. All data are presented as the mean + SEM of triplicate determinations. RESULTS Effect of extracellular ATP on AA release in MC3T3-E1 cells
Extracellular ATP (1 mM) significantly stimulated the release of AA, compared to the control, and the effect was time-dependent up to 30 min in osteoblast-like MC3T3-E1 cells (Fig. 1). The effect of ATP was dose-dependent in the range between 0.1 and 1 mM (Fig. 2). The maximum effect of ATP on AA release was observed at 1 mM.
Measurement of AA release
Effect of propranolol on AA release induced by extracellular ATP in MC3T3-E1 cells
The cultured cells were labelled with [3H]AA (0.5 ~Ci/ dish) for 24 h. The medium was removed and the cells were then washed four times with 1 ml of the assay buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 135 mM NaC1, 5 mM KC1, 1 ~ MgSO4 and I mM CaCI2]. The cells were preincubated subsequently with 1 ml of the assay buffer containing 0.1% essentially fatty acid-free BSA at 37°C for 20 rain, and the ceils were then stimulated by extracellular ATE The reaction was terminated by collecting the medium, and the radioactivity of
We have demonstrated that extraceilular ATP stimulates phosphatidylcholine-hydrolysing PLD in osteoblast-like MC3T3-E1 cells.11It is well known that one of the products generated from the hydrolysis of phosphatidylcholine by PLD, phosphatidic acid, is a precursor of DG.s-l° Thus, we next examined the effect of propranolol, which is known to inhibit phosphatidic acid phosphohydrolase,18 on the extracellular ATP-induced AA release in these cells. The pretreatment with propranolol, which by itself had little effect on AA release, significantly inhibited the AA release
Prostaglandins, Leukotrienesand Essential Fatty Acids (1997) 57(3), 335-339
© Pearson Professional Ltd 1997
AA re~ease induced by extracellular A TP in osteoblasts
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Fig. 1 Time-dependent effect of extracellular ATP on AA release in MC3T3-E1 cells. The [aH]AA-labelled cells were stimulated by 1 mM ATP (e) or vehicle (©) for the indicated periods. Each value represents the mean + SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the control value.
induced by extracellular ATP (1 mM) in MC3T3-E1 cells (Fig. 3). The effect of propranolol was in a dose-dependent manner in the range between 100 and 300~M. The maximum inhibitory effect of propranolol (300 ~M) was about 59%. Effect of RHC-80267 on AA release induced by extracellular ATP in MC3T3-E1 cells
DG is recognized to be an important cellular source of arachidonate which may be released subsequently by DG lipase. 19 In addition, we examined the effect of RHC80267 on the extracellular ATP-induced AA release in MC3T3-E1 cells. RHC-80267 has been reported to selectively inhibit DG lipase activity. 2° The pretreatment with RHC-80267, which by itself had little effect on AA release, markedly suppressed the AA release induced by extracellular ATP in MC3T3-E1 cells (Fig. 4). The maximum inhibitory effect of RHC-80267 (30 ~ was about 35%. © Pearson Professional Ltd 1997
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Fig. 2 Dose-dependent effect of extracellular ATP on AA release in MC3T3-E1 cells. The [3H]AA-labelled cells were stimulated by various doses of ATP for 30 min. Values for unstimulated cells have been subtracted from each data point, Each value represents the mean + SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations, *P < 0.05 compared to the control value.
Effects of propranolol and RHC-80267 on PGE= synthesis induced by extracellular ATP in MC3T3-E1 cells
Among eicosanoid converted from AA, PGE2 is a major product in osteoblasts including MC3T3-E1 cells. 15'~6We previously reported that extracellular ATP stimulates the synthesis of PGE2 in MC3T3-E1 cells)* Next, we examined the effects of propranolol and RHC-80267 on the PGE2 synthesis induced by extracellular ATP in these cells. The pretreatment with propranolol, which by itself had no effect on PGE2 synthesis, significantly inhibited the PGE2 synthesis induced by extracellular ATP in MC3T3E1 cells (Table). The inhibitory effect of propranolol (300 ~VI) was about 60%. In addition, RHC-80267, which by itself had no effect on PGE2 synthesis, significantly suppressed the PGE2 synthesis induced by extracellular ATP (Table). The inhibitory effect of RHC-80267 (30 ~M) was about 47%.
Prostaglandins, Leukotrienes and Essential Fatty Acids (I 997) 57(3), 335-339
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Table Effects of propranolol and RHC-80267 on PGE2 synthesis induced by extracellular ATP in MC3T3-E1 cells. PGE= synthesis (pg/ml) 31 33 31 257 124 150
± ± ± ± ± +
RHC-80267 ATP
(pM)
Fig. 3 Effect of propranolol on the AA release induced by extracellular ATP in MC3T3-E1 cells. The [aH]AA-labelled cells were pretreated with various doses of propranolol for 20 min, and then stimulated by 1 mM ATP (0) or vehicle (©) for 30 min. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the value of ATP without propranolol pretreatment.
Control Propranolol RHC-80267 ATP Prepranolol + ATP RHC-80267 + A'I'P
0
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The cultured cells were pretreated with 300 I.tM propranolot, 30 I.tM RHC-80267 or vehicle for 20 min, and then stimulated by I mM ATP or vehicle for 120 min. Each value represents the mean + SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the value of ATP alone.
DISCUSSION
In the present study, we showed that propranolol, a phosphatidic acid phosphohydrolase inhibitor, TM significantly inhibited the AA release induced by extracellular ATP in osteoblast-like MC3T3-E 1 cells. We have recently reported
-
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Fig. 4 Effect of RHC-80267 on the AA release induced by extracellular ATP in MC3T3-E1 cells. The [3H]AA-labelled cells were pretreated with 30 ~tM RHC-80267 or vehicle for 20 min, and then stimulated by 1 mM ATP or vehicle for 30 min. Each value represents the mean + SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the value of ATP without RHC-80267 pretreatment.
that extracellular ATP stimulates phosphatidylcholine hydrolysis by PLD independently from protein kinase C activated by phosphoinositide hydrolysis in MC3T3-E1 cells, n Phosphatidylcholine can be hydrolysed by PLD to yield phosphatidic acid. Phosphatidic acid, which itself could be a potential intracellular mediator, can be further degrade d by a phosphatidic acid phosphohydrolase to DG.a-l° So, it seems that the conversion of phosphatidic acid to DG is involved in the extracellular ATP-induced AA release in MC3T3-E 1 cells. In addition, we showed that RHC-80267 markedly suppressed the AA release induced by extracellular ATP in MC3T3-E1 cells. RHC-80267 has been reported to inhibit DG lipase selectively.2° Thus, this finding suggests that the action of DG lipase is involved in the extracellular ATP-induced AA release in these cells. Therefore, from our results as a whole, it is most likely that the AA release from DG caused by phosphatidylcholine hydrolysis by PLD is an important pathway of extracellular ATP-induced AA release in osteoblast-like MC3T3-E 1 cells. Prostaglandins are recognized to be local modulators as autacoids. 17 Among them, PGE2 is a major
Prostaglandins, Leukotrienesand Essential Fatty Acids (1997) 57(3), 335-339
© Pearson Professional Ltd 1997
AA release induced by extracellular A TP in osteoblasts
eicosanoid product in osteoblasts including MC3T3-E1 cells. 15,16In a previous study, we have shown that extracellular ATP stimulates PGEz synthesis in MC3T3-E1 cells) 4 In the present study, we demonstrated that both propranolol and RHC-80267 suppressed the extracellular ATP-induced PGE2 synthesis as well as AA release in these cells. Our findings suggest that phosphatidylcholine hydrolysis by PLD, which results in the DG formation, is involved in the mechanism of extracellular ATP-induced arachidonic cascade in osteoblast-like MC3T3-E1 ceils. In conclusion, our results strongly suggest that the AA release induced by extracellular ATP is mediated at least in part by phosphatidylcholine hydrolysis by PLD in osteoblast-like ceils. REFERENCES 1. Nijweide P. J., Burger E. H., Feyen J. H. M. Cells of bone: proliferation, differentiation, and hormonal regulation. Phys Rev 1986; 66: 855-886. 2. Gordon J. L. Extracellular ATP: effects, sources and fate. Biochem J 1986; 233: 309-319. 3. Dubyak G. R., E1-Moatassim C. Signal transduction via Pz-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol 1993; 265 (Cell physiol. 34): C577-C606. 4. Sch6fl C., Cuthbertson K. S. IL, Walsh C. A. et al. Evidence for P2-purinoceptors in human osteoblast-like cells. J Bone Miner Res 1992, 7: 485-491. 5. Yu H., Ferrier J. Osteoblast-like cells have a variable mixed population of purino/nucleotide receptors. FEBSLett 1993; 328:209-214.
6. Nishizuka Y. Studies and perspectives of protein kinase C. Science 1986; 233:305-312. 7. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature 1989; 341: 197-205.
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8. Exton J. H. Signaling through phosphatidylcholine breakdown. JBiol Chem 1990; 265: 1--4. 9. Zeisel S. H. Choline phospholipids: signal transduction and carcinogenesis. FASEBJ 1993; 7: 551-557. 10. Bifiah M. M., Anthes J. C. The regulation and cellular functions of phosphatidylcholine hydrolysis. BiochemJ 1990; 269: 281-291. 11. Suzuki A., Shinoda J., Oiso Y., Kozawa O. Mechanism of phospholipase D activation induced by extracellular ATP in osteoblast-like cells. JEndocrino11995; 145: 81-86. 12. Kodama H., Amagai Y., Sudo H., Kasai S., Yamamoto S. Establishment of a clonal osteogenic cell line from newborn mouse calvalia. Jpn J Oral Bio11981; 23: 899-901. 13. Sudo H., Kodama H., Amagai Y., Yamamoto S., Kasai S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Bio11983; 96: 191-198. 14. Suzuki A., Kotoyori J., Oiso Y., Kozawa O. Prostaglandin E2 is a potential mediator of extracellular ATP action in osteoblast-like cells. CellAdh Commun 1993; 1:113-118. 15. Raisz L. G., Martin T. J. Prostaglandins in bone and mineral metabolism. In: Peck W. A., ed. Bone and Mineral Research, Annual 2. Amsterdam; Elsevier, 1984: 286-310. 16. Yokota K., Kusaka M., Ohshima T. et al. Stimulation of prostaglandin E2 synthesis in cloned osteoblastic ceils of mouse (MC3T3-E1) by epidermal growth factor. JBiol Chem 1986; 261: 15410-15415. 17. Smith W. L The eicosanoids and their biochemical mechanism of action. BiochemJ 1989; 2 5 9 : 3 1 5 - 3 2 4 . 18. Pappu A. S., Hauser G. Propranolol-induced inhibition of rat brain cytoplasmic phosphatidate phosphohydrolase. Neurochem Res 1983; 8: 1565-1575. 19. Balsinde J., Diez E., Mollinedo F. Arachidonic acid release from diacylglycerol in human neutrophils. J Biol Chem 1991; 266: 15638-15643. 20. Sutherland C. A., Amin D. Relative activities of rat and dog platelet phospholipase A2 and diglyceride lipase. J Biol Chem 1982; 257: 14006-14010.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 57(3), 335-339
A r a c h i d o n i c acid r e l e a s e induced by e x t r a c e l l u l a r A T P in osteoblasts: role of phospholipase D Y. W a t a n a b e - T o m i t a , 1 A. Suzuki, 1 J. Shinoda, 1 Y. Oiso, 1 0 . K o z a w a 2 1First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466, Japan 2Department of Pharmacology, Gifu University School of Medicine, Gifu 500, Japan
Summary In a previous study, we have shown that extracellular ATP stimulates Ca2÷influx resulting in the release of arachidonic acid (AA) and prostaglandin E2 (PGE2) synthesis in osteoblast-like MC3T3-E1 cells. In addition, we have recently reported that extracellular ATP stimulates phosphatidylcholine hydrolysis by phospholipase D (PLD) independently from the activation of protein kinase C in these cells. It is well recognized that phosphatidylcholine is hydrolysed by PLD, generating phosphatidic acid, which can be further degraded by phosphatidic acid phosphohydrolase to diacylglycerol (DG). In the present study, we investigated the role of PLD activation in the extracellular ATP-induced AA release and PGE2 synthesis in osteoblast-like MC3T3-E1 cells. Extracellular ATP stimulated AA release dose-dependently in the range between 0.1 and 1 mM. Propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, significantly inhibited the AA release induced by extracellular ATP in a dosedependent manner in the range between 100 and 300 tIM. 1,6-Bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of DG lipase, significantly suppressed the AA release induced by extracellular ATP. Both the pretreatment of propranolol and RHC-80267 also inhibited the extracellular ATP-induced PGE2 synthesis. These results strongly suggest that the AA release induced by extracellular ATP is mediated at least in part by phosphatidylcholine hydrolysis by PLD in osteoblast-like cells.
INTRODUCTION It is well known that two functional cell types, osteoblasts and osteoclasts, regulate bone metabolismJ The former cells are responsible for bone formation and the latter for bone resorption. Accumulating evidence suggests that the receptors of bone resorbing agents such as parathyroid hormone and 1,25-dihydroxyvitamine D3 are found in osteoblasts.' Thus, osteoblasts are recognized to play important roles also in the regulation of bone resorption. A variety of hormonal factors and autacoids modify bone metabolismJ It has been reported that extraceUular ATP acts through its binding to specific receptor (P2purinoceptor) on the cell surface, 2'a and that the P2purinoceptor exists on osteoblasts. 4,5 It is generally recognized that stimulation of cellsurface receptors induced by many hormones and neuroReceived 11 December 1996 Accepted 4 January 1997 Correspondence to: Osamu Kozawa, Tel. 00 81 58 265 1241; Fax. 00 81 58 267 2959
transmitters initiates hydrolysis of phosphoinositides, which produces two second messengers, diacylglycerol (DG) and inositol 1,4,5-trisphosphate? '7 Protein kinase C is physiologically activated by DG. However, phosphatidylinositol hydrolysis by phospholipase C (PLC) is not the only pathway of DG formation. 8'9 Hydrolysis of phosphatidylcholine by phospholipase D (PLD) generates phosphatidic acid. a-l° Phosphatidic acid, which itself could be a potential intracellular mediator, can be further degraded by a phosphatidic acid phosphohydrolase to DG. Nowadays, it is recognized that PLD plays an important role in modulating cellular functions. We have recently reported H that extracellular ATP stimulates phosphatidylcholine hydrolysis by PLD independently from the activation of protein kinase C in osteoblast-like MC3T3-E1 ceils derived from newbom m o u s e c a l v a r i a . 12,1a In a previous study/4 we have shown that extraceilular ATP stimulates Ca2÷ influx resulting in the release of arachidonic acid (A_A)and the synthesis of prostaglandin E2 (PGE2) which has been shown to be a major eicosanoid product ~5,~6in osteoblast-like MC3T3-E1 cells. It is gener-
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Watanabe-Tomita et al
ally accepted that the activation of phospholipase A2 is the major pathway of AA releaseY AA is also released via hydrolysis of phospholipids by other phospholipasesY In the present study, we investigated the role of phosphatidylcholine hydrolysis by PLD in the extracellular ATP-induced AA release in MC3T3-E1 cells. Herein, we show that the AA release induced by extracellular ATP is mediated at least in part by phosphatidylcholine hydrolysis by PLD in osteoblast-like cells. MATERIALS AND METHODS Materials
[5,6,8,9,11,12,14,15-aH]AA (212.1 Ci/mmol) and PGE2[~2sI] assay system were purchased from Amersham Japan (Tokyo, Japan). ATP and essentially fatty acid-free bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). d/-Propranolol hydrochloride (propranolol) was purchased from Wako Pure Chemical Industries (Osaka, Japan). 1,6-Bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267) was purchased from Funakoshi Pharmaceutical Co. (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. Propranolol and RHC-80267 were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide in the culture medium was 0.1%, which did not affect the measurement of AA release and PGE2 synthesis. Cell culture
Cloned osteoblast-like MC3T3-E1 cells were generously provided by Dr M. Kumegawa (Meikai University, Sakado, Japan) and maintained in a-minimum essential medium (a-MEM) containing 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% CO2/95% air. The cells (5 x 104) were seeded into 35-ram diameter dishes in 2 ml of c~-MEMcontaining 10% FCS. After 5 days, the medium was exchanged for 2 ml of a-MEM containing 0.3% FCS. The ceils were used for experiments after 48 h.
the medium was determined. When indicated, the cells were pretreated with various doses of propranolol or RHC-80267 for 20 min. Assay for PGE2
Procedures were done as described under 'Measurement of AA release' except for using unlabelled ceils. The cultured cells were pretreated with various doses of propranolol or RHC-80267 for 20 min, and then stimulated by 1 mM extracellular ATE After 120 min, the medium was collected and PGE2 in the medium was measured with a radioimmunoassay kit. Determinations
The radioactivity of 3H-samples was determined with a Beckman LS-6000IC liquid scintillation spectrometer. The radioactivity of 125I-samples was determined with an Aloka ARC-600 auto well gamma system. Statistical analysis
The data were analyzed by one-way analysis of variance, followed by the Bonferroni method for multiple comparison between pairs, and a P<0.05 was considered significant. All data are presented as the mean + SEM of triplicate determinations. RESULTS Effect of extracellular ATP on AA release in MC3T3-E1 cells
Extracellular ATP (1 mM) significantly stimulated the release of AA, compared to the control, and the effect was time-dependent up to 30 min in osteoblast-like MC3T3-E1 cells (Fig. 1). The effect of ATP was dose-dependent in the range between 0.1 and 1 mM (Fig. 2). The maximum effect of ATP on AA release was observed at 1 mM.
Measurement of AA release
Effect of propranolol on AA release induced by extracellular ATP in MC3T3-E1 cells
The cultured cells were labelled with [3H]AA (0.5 ~Ci/ dish) for 24 h. The medium was removed and the cells were then washed four times with 1 ml of the assay buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 135 mM NaC1, 5 mM KC1, 1 ~ MgSO4 and I mM CaCI2]. The cells were preincubated subsequently with 1 ml of the assay buffer containing 0.1% essentially fatty acid-free BSA at 37°C for 20 rain, and the ceils were then stimulated by extracellular ATE The reaction was terminated by collecting the medium, and the radioactivity of
We have demonstrated that extraceilular ATP stimulates phosphatidylcholine-hydrolysing PLD in osteoblast-like MC3T3-E1 cells.11It is well known that one of the products generated from the hydrolysis of phosphatidylcholine by PLD, phosphatidic acid, is a precursor of DG.s-l° Thus, we next examined the effect of propranolol, which is known to inhibit phosphatidic acid phosphohydrolase,18 on the extracellular ATP-induced AA release in these cells. The pretreatment with propranolol, which by itself had little effect on AA release, significantly inhibited the AA release
Prostaglandins, Leukotrienesand Essential Fatty Acids (1997) 57(3), 335-339
© Pearson Professional Ltd 1997
AA re~ease induced by extracellular A TP in osteoblasts
337
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Fig. 1 Time-dependent effect of extracellular ATP on AA release in MC3T3-E1 cells. The [aH]AA-labelled cells were stimulated by 1 mM ATP (e) or vehicle (©) for the indicated periods. Each value represents the mean + SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the control value.
induced by extracellular ATP (1 mM) in MC3T3-E1 cells (Fig. 3). The effect of propranolol was in a dose-dependent manner in the range between 100 and 300~M. The maximum inhibitory effect of propranolol (300 ~M) was about 59%. Effect of RHC-80267 on AA release induced by extracellular ATP in MC3T3-E1 cells
DG is recognized to be an important cellular source of arachidonate which may be released subsequently by DG lipase. 19 In addition, we examined the effect of RHC80267 on the extracellular ATP-induced AA release in MC3T3-E1 cells. RHC-80267 has been reported to selectively inhibit DG lipase activity. 2° The pretreatment with RHC-80267, which by itself had little effect on AA release, markedly suppressed the AA release induced by extracellular ATP in MC3T3-E1 cells (Fig. 4). The maximum inhibitory effect of RHC-80267 (30 ~ was about 35%. © Pearson Professional Ltd 1997
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Fig. 2 Dose-dependent effect of extracellular ATP on AA release in MC3T3-E1 cells. The [3H]AA-labelled cells were stimulated by various doses of ATP for 30 min. Values for unstimulated cells have been subtracted from each data point, Each value represents the mean + SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations, *P < 0.05 compared to the control value.
Effects of propranolol and RHC-80267 on PGE= synthesis induced by extracellular ATP in MC3T3-E1 cells
Among eicosanoid converted from AA, PGE2 is a major product in osteoblasts including MC3T3-E1 cells. 15'~6We previously reported that extracellular ATP stimulates the synthesis of PGE2 in MC3T3-E1 cells)* Next, we examined the effects of propranolol and RHC-80267 on the PGE2 synthesis induced by extracellular ATP in these cells. The pretreatment with propranolol, which by itself had no effect on PGE2 synthesis, significantly inhibited the PGE2 synthesis induced by extracellular ATP in MC3T3E1 cells (Table). The inhibitory effect of propranolol (300 ~VI) was about 60%. In addition, RHC-80267, which by itself had no effect on PGE2 synthesis, significantly suppressed the PGE2 synthesis induced by extracellular ATP (Table). The inhibitory effect of RHC-80267 (30 ~M) was about 47%.
Prostaglandins, Leukotrienes and Essential Fatty Acids (I 997) 57(3), 335-339
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< 0
0
I
I
I
100
200
300
propranolol
Table Effects of propranolol and RHC-80267 on PGE2 synthesis induced by extracellular ATP in MC3T3-E1 cells. PGE= synthesis (pg/ml) 31 33 31 257 124 150
± ± ± ± ± +
RHC-80267 ATP
(pM)
Fig. 3 Effect of propranolol on the AA release induced by extracellular ATP in MC3T3-E1 cells. The [aH]AA-labelled cells were pretreated with various doses of propranolol for 20 min, and then stimulated by 1 mM ATP (0) or vehicle (©) for 30 min. Each value represents the mean ± SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the value of ATP without propranolol pretreatment.
Control Propranolol RHC-80267 ATP Prepranolol + ATP RHC-80267 + A'I'P
0
3 4 3 18 11 * 12"
The cultured cells were pretreated with 300 I.tM propranolot, 30 I.tM RHC-80267 or vehicle for 20 min, and then stimulated by I mM ATP or vehicle for 120 min. Each value represents the mean + SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the value of ATP alone.
DISCUSSION
In the present study, we showed that propranolol, a phosphatidic acid phosphohydrolase inhibitor, TM significantly inhibited the AA release induced by extracellular ATP in osteoblast-like MC3T3-E 1 cells. We have recently reported
-
+
4"
4-
-
+
Fig. 4 Effect of RHC-80267 on the AA release induced by extracellular ATP in MC3T3-E1 cells. The [3H]AA-labelled cells were pretreated with 30 ~tM RHC-80267 or vehicle for 20 min, and then stimulated by 1 mM ATP or vehicle for 30 min. Each value represents the mean + SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the value of ATP without RHC-80267 pretreatment.
that extracellular ATP stimulates phosphatidylcholine hydrolysis by PLD independently from protein kinase C activated by phosphoinositide hydrolysis in MC3T3-E1 cells, n Phosphatidylcholine can be hydrolysed by PLD to yield phosphatidic acid. Phosphatidic acid, which itself could be a potential intracellular mediator, can be further degrade d by a phosphatidic acid phosphohydrolase to DG.a-l° So, it seems that the conversion of phosphatidic acid to DG is involved in the extracellular ATP-induced AA release in MC3T3-E 1 cells. In addition, we showed that RHC-80267 markedly suppressed the AA release induced by extracellular ATP in MC3T3-E1 cells. RHC-80267 has been reported to inhibit DG lipase selectively.2° Thus, this finding suggests that the action of DG lipase is involved in the extracellular ATP-induced AA release in these cells. Therefore, from our results as a whole, it is most likely that the AA release from DG caused by phosphatidylcholine hydrolysis by PLD is an important pathway of extracellular ATP-induced AA release in osteoblast-like MC3T3-E 1 cells. Prostaglandins are recognized to be local modulators as autacoids. 17 Among them, PGE2 is a major
Prostaglandins, Leukotrienesand Essential Fatty Acids (1997) 57(3), 335-339
© Pearson Professional Ltd 1997
AA release induced by extracellular A TP in osteoblasts
eicosanoid product in osteoblasts including MC3T3-E1 cells. 15,16In a previous study, we have shown that extracellular ATP stimulates PGEz synthesis in MC3T3-E1 cells) 4 In the present study, we demonstrated that both propranolol and RHC-80267 suppressed the extracellular ATP-induced PGE2 synthesis as well as AA release in these cells. Our findings suggest that phosphatidylcholine hydrolysis by PLD, which results in the DG formation, is involved in the mechanism of extracellular ATP-induced arachidonic cascade in osteoblast-like MC3T3-E1 ceils. In conclusion, our results strongly suggest that the AA release induced by extracellular ATP is mediated at least in part by phosphatidylcholine hydrolysis by PLD in osteoblast-like ceils. REFERENCES 1. Nijweide P. J., Burger E. H., Feyen J. H. M. Cells of bone: proliferation, differentiation, and hormonal regulation. Phys Rev 1986; 66: 855-886. 2. Gordon J. L. Extracellular ATP: effects, sources and fate. Biochem J 1986; 233: 309-319. 3. Dubyak G. R., E1-Moatassim C. Signal transduction via Pz-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol 1993; 265 (Cell physiol. 34): C577-C606. 4. Sch6fl C., Cuthbertson K. S. IL, Walsh C. A. et al. Evidence for P2-purinoceptors in human osteoblast-like cells. J Bone Miner Res 1992, 7: 485-491. 5. Yu H., Ferrier J. Osteoblast-like cells have a variable mixed population of purino/nucleotide receptors. FEBSLett 1993; 328:209-214.
6. Nishizuka Y. Studies and perspectives of protein kinase C. Science 1986; 233:305-312. 7. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature 1989; 341: 197-205.
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8. Exton J. H. Signaling through phosphatidylcholine breakdown. JBiol Chem 1990; 265: 1--4. 9. Zeisel S. H. Choline phospholipids: signal transduction and carcinogenesis. FASEBJ 1993; 7: 551-557. 10. Bifiah M. M., Anthes J. C. The regulation and cellular functions of phosphatidylcholine hydrolysis. BiochemJ 1990; 269: 281-291. 11. Suzuki A., Shinoda J., Oiso Y., Kozawa O. Mechanism of phospholipase D activation induced by extracellular ATP in osteoblast-like cells. JEndocrino11995; 145: 81-86. 12. Kodama H., Amagai Y., Sudo H., Kasai S., Yamamoto S. Establishment of a clonal osteogenic cell line from newborn mouse calvalia. Jpn J Oral Bio11981; 23: 899-901. 13. Sudo H., Kodama H., Amagai Y., Yamamoto S., Kasai S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Bio11983; 96: 191-198. 14. Suzuki A., Kotoyori J., Oiso Y., Kozawa O. Prostaglandin E2 is a potential mediator of extracellular ATP action in osteoblast-like cells. CellAdh Commun 1993; 1:113-118. 15. Raisz L. G., Martin T. J. Prostaglandins in bone and mineral metabolism. In: Peck W. A., ed. Bone and Mineral Research, Annual 2. Amsterdam; Elsevier, 1984: 286-310. 16. Yokota K., Kusaka M., Ohshima T. et al. Stimulation of prostaglandin E2 synthesis in cloned osteoblastic ceils of mouse (MC3T3-E1) by epidermal growth factor. JBiol Chem 1986; 261: 15410-15415. 17. Smith W. L The eicosanoids and their biochemical mechanism of action. BiochemJ 1989; 2 5 9 : 3 1 5 - 3 2 4 . 18. Pappu A. S., Hauser G. Propranolol-induced inhibition of rat brain cytoplasmic phosphatidate phosphohydrolase. Neurochem Res 1983; 8: 1565-1575. 19. Balsinde J., Diez E., Mollinedo F. Arachidonic acid release from diacylglycerol in human neutrophils. J Biol Chem 1991; 266: 15638-15643. 20. Sutherland C. A., Amin D. Relative activities of rat and dog platelet phospholipase A2 and diglyceride lipase. J Biol Chem 1982; 257: 14006-14010.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 57(3), 335-339