© 1997 American Heart Association, Inc.
Articles |
Involvement of Phosphatidylcholine Hydrolysis by Phospholipase D in Extracellular ATP-Induced Arachidonic Acid Release in Aortic Smooth Muscle Cells
the First Department of Internal Medicine, Nagoya University School of Medicine, (J.S., A.S., Y.O.); and the Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai (O.K.), Japan. Dr Kozawa is now with the Department of Pharmacology, Gifu University School of Medicine, Japan.
Correspondence to Osamu Kozawa, MD, PhD, Department of Pharmacology, Gifu University School of Medicine, Gifu 500, Japan.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
We investigated the effect of extracellular ATP on phosphatidylcholine-hydrolyzing phospholipase D activity and the role of phospholipase D activation in extracellular ATP-induced arachidonic acid release in cultured rat aortic smooth muscle cells. ATP significantly stimulated the formation of choline in a dose-dependent manner in the range between 0.01 and 0.5 mmol/L. However, ATP had no effect on the formation of phosphocholine. Staurosporine, an inhibitor of protein kinases, did not affect the ATP-induced formation of choline. ATP significantly stimulated arachidonic acid release in a dose-dependent manner in the range between 0.01 and 0.5 mmol/L. DL-Propranolol hydrochloride (propranolol), an inhibitor of phosphatidic acid phosphohydrolase, significantly inhibited the ATP-induced release of arachidonic acid. 1,6-Bis(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a potent and selective inhibitor of diacylglycerol lipase, reduced ATP-induced arachidonic acid release. Quinacrine, a phospholipase A2 inhibitor, suppressed ATP-induced arachidonic acid release. Both propranolol and RHC-80267 markedly inhibited the ATP-induced synthesis of 6-ketoprostaglandin F1
, a stable metabolite of prostacyclin. These results strongly suggest that extracellular ATP activates phosphatidylcholine-hydrolyzing phospholipase D independently of protein kinase C in aortic smooth muscle cells and that the arachidonic acid release induced by extracellular ATP is mediated, at least in part, through phosphatidylcholine hydrolysis by phospholipase D activation.
Key Words: extracellular ATP • phosphatidylcholine • phospholipase D • arachidonic acid • aortic smooth muscle cells
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
It is well recognized that vascular smooth muscle cells play a crucial role in the pathogenesis of atherosclerosis and hypertension.1 2 Extracellular ATP is generally accepted to influence many biological processes through binding to its specific cell-surface receptor.3 4 Extracellular ATP has been reported to induce contraction of vascular smooth muscle cells5 6 and stimulate proliferation of these cells.7 8 As for intracellular signaling, it has been reported that extracellular ATP induces phosphoinositide hydrolysis by phospholipase C and Ca2+ mobilization via P2-purinergic receptors in vascular smooth muscle cells.8 9 Phosphoinositide hydrolysis leads to the formation of diacylglycerol and inositol phosphates.10 Among these products, diacylglycerol and inositol 1,4,5-trisphosphate serve as messengers for the activation of PKC and the mobilization of intracellular Ca2+, respectively.10 11 It has been reported that inositol phosphate formation and PKC activation participate in mediating the extracellular ATP-induced mitogenesis in vascular smooth muscle cells.7 8
Diacylglycerol is formed not only from phosphoinositide hydrolysis by phospholipase C but also from phosphatidylcholine hydrolysis by phospholipase D.12 13 14 Phospholipase D is known to hydrolyze phosphatidylcholine, resulting in the formation of phosphatidic acid, which is further degraded into diacylglycerol by phosphatidic acid phosphohydrolase.13 It is currently recognized that phospholipase D plays an important role in modulating cellular functions that require long-term activation of PKC, since phosphatidylcholine is the principal phospholipid in cell membranes.12 13 14 Although it has been shown that phosphatidylcholine hydrolysis by phospholipase D is secondary to PKC activated by phosphoinositide hydrolysis, recent evidence suggests that phospholipase D is also activated independently of PKC.15 16 We previously reported that angiotensin II stimulates phosphatidylcholine-hydrolyzing phospholipase D activity independently of PKC in rat aortic smooth muscle cells.17
Extracellular ATP has been reported to stimulate the release of AA and PG synthesis in vascular smooth muscle cells.7 AA release is a rate-limiting step of PG synthesis.18 It is generally accepted that phospholipase A2 releases AA directly from esterified membrane phospholipid stores.18 AA is also released from phospholipids by other phospholipases, such as phospholipases C and D.18 19 PGI2, a most potent vascular relaxing agent,20 is the major PG synthesized in basal and vasoconstrictor-stimulated vascular smooth muscle cells.21 However, the exact mechanism of extracellular ATP-induced AA release has not yet been fully clarified in vascular smooth muscle cells.
In the present study, we investigated the effect of extracellular ATP on phosphatidylcholine-hydrolyzing phospholipase D activity and the role of phosphatidylcholine hydrolysis by phospholipase D in extracellular ATP-induced AA release in cultured rat aortic smooth muscle cells. Herein, we show that extracellular ATP activates phospholipase D independently of PKC in aortic smooth muscle cells and that the AA release induced by extracellular ATP is mediated, at least in part, through phosphatidylcholine hydrolysis by phospholipase D activation.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
[Methyl-3H]choline chloride (85 Ci/mmol), [methyl-14C]phosphocholine ammonium salt (50 mCi/mmol) and [5,6,8,9,11,12,14,15-3H]AA (208 Ci/mmol) were purchased from Amersham Japan. ATP, staurosporine, and quinacrine were purchased from Sigma Chemical Co. DL-Propranolol hydrochloride (propranolol) was purchased from Wako Pure Chemical Industries. 1,6-Bis(cyclohexyloximinocarbonylamino)-hexane (RHC-80267) was purchased from Funakoshi Pharmaceutical Co. The 6-keto-PGF1
enzyme immunoassay kit was purchased from Dainippon Pharmaceutical Co. Other materials and chemicals were obtained from commercial sources. Staurosporine, propranolol, RHC-80267, and quinacrine 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 the formation of choline and phosphocholine, the assay for AA release, or the measurement of PGI2 synthesis.
Cell Culture
Aortic smooth muscle cells were obtained from thoracic aorta of male Sprague-Dawley rats by the explantation method as previously described.22 The cells (1x105) were seeded into 35-mm-diameter dishes and maintained in 2 mL of Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum at 37°C in a humidified atmosphere of 5% CO2 / 95% air. The cells were used between the third and sixth passage. After 3 days, the medium was exchanged for fresh medium, and after 6 days from seeding, the medium was exchanged for 2 mL of serum-free DMEM. The cells were used for experiments after 48 hours.
Measurement of Choline and Phosphocholine Formation
To determine phosphatidylcholine-hydrolyzing phospholipase D activity, the cultured cells were labeled with [methyl-3H]choline chloride (2 µCi per dish) for 24 hours. The labeled cells were washed twice with 1 mL of an assay buffer (5 mmol/L HEPES, pH 7.4; 150 mmol/L NaCl; 5 mmol/L KCl; 5.5 mmol/L glucose; 0.8 mmol/L MgSO4; and 1 mmol/L CaCl2) and subsequently incubated in 1 mL of the assay buffer containing 0.01% bovine serum albumin at 37°C for 20 minutes. The cells were then stimulated by various doses of ATP. The reaction was terminated by adding 0.75 mL of ice-cold methanol. The dishes were placed on ice for 10 minutes. The contents were transferred to tubes to which chloroform was added and placed on ice for 60 minutes. Chloroform and water were then added at a final ratio of 1:1:0.9 (chloroform/methanol/water). The tubes were centrifuged at 14 000g for 5 minutes, and the upper aqueous methanolic phase was separated for analysis of the water-soluble [3H]choline-containing metabolites. Separation was conducted on a 1-mL Dowex 50-WH+ cation-exchange column (200 to 400 mesh) as previously described.23 In brief, the phase was diluted to 5 mL with water and applied to the column. Glycerophosphocholine was removed with 4 mL of water, and then radioactive phosphocholine and choline were eluted with 10 mL of water and 8 mL of 1 mol/L HCl, respectively. We have confirmed that standard [14C]phosphocholine and [3H]choline were eluted with the addition of 10 mL of water and 8 mL of 1 mol/L HCl, respectively. When indicated, the cells were pretreated with staurosporine for 20 minutes.
Assay for AA Release
Assay for AA release was performed as previously described.24 Briefly, the cultured cells were labeled with [3H]AA (0.5 µCi per dish) for 24 hours. The labeled cells were washed four times with 1 mL of the assay buffer. The cells were subsequently preincubated with 1 mL of the assay buffer containing 0.1% essentially fatty acid–free bovine serum albumin at 37°C for 20 minutes. The cells were then stimulated by various doses of ATP. After the indicated periods, the medium was collected and the radioactivity in the medium determined. When indicated, the cells were pretreated with propranolol, RHC-80267, or quinacrine for 20 minutes.
Measurement of PGI2 Synthesis
PGI2 synthesis was determined as described above except for using unlabeled cells. The cultured cells were pretreated with propranolol or RHC-80267 for 20 minutes and then stimulated by ATP. After 180 minutes, the medium was collected and 6-keto-PGF1, a stable metabolite of PGI2,25 in the medium determined with an enzyme immunoassay kit. The absorbance was determined at 450 nm with SLT-Labinstruments EAR 340 AT in the enzyme immunoassay.
Determination of Radioactivity
The radioactivity of 3H and 14C samples was determined with a Beckman LS-5000TD liquid scintillation spectrometer.
Statistical Analysis
The data were analyzed by one-way analysis of variance followed by the Bonferroni method for multiple comparison between pairs, and P<.05 was considered significant. All data are presented as the mean±SEM of triplicate determinations.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effects of Extracellular ATP on Choline and Phosphocholine Formation in Aortic Smooth Muscle Cells
Extracellular ATP significantly stimulated the formation of choline in aortic smooth muscle cells (Fig 1
). However, ATP did not affect the formation of phosphocholine in these cells (Fig 1). The formation of choline stimulated by ATP (0.5 mmol/L) increased time dependently up to 30 minutes. The stimulative effect of ATP was dose dependent in the range between 0.01 and 0.5 mmol/L (Fig 2). The maximum effect of ATP on choline formation was observed at 0.5 mmol/L.
|
, 
) or vehicle (
, 
) for the indicated periods, and the formation of choline (
|
Effect of Staurosporine on Extracellular ATP-Induced Choline Formation in Aortic Smooth Muscle Cells
The activation of phosphatidylcholine-hydrolyzing phospholipase D has been reported to be dependent on PKC or independent of PKC.15 16 In a previous study,26 we showed that TPA, a PKC-activating phorbol ester,11 stimulates phospholipase D activity in rat aortic smooth muscle cells and that staurosporine, an inhibitor of protein kinases,27 suppresses TPA-induced phospholipase D activation in a dose-dependent manner in the range between 0.1 and 3 µmol/L in these cells. In the present study, we examined the effect of staurosporine on the ATP-induced formation of choline in aortic smooth muscle cells. Staurosporine (3 µmol/L), which by itself had little effect on choline formation, did not affect the ATP-induced formation of choline in these cells (Table 1).
|
Effect of Extracellular ATP on AA Release in Aortic Smooth Muscle Cells
Extracellular ATP significantly stimulated AA release in aortic smooth muscle cells (Fig 3A). The AA release induced by ATP (0.5 mmol/L) increased time dependently up to 30 minutes. The stimulative effect of ATP was dose dependent in the range between 0.01 and 0.5 mmol/L (Fig 3B). The maximum effect of ATP on AA release was observed at 0.5 mmol/L.
|
Effect of Propranolol, RHC-80267, or Quinacrine on Extracellular ATP-Induced AA Release in Aortic Smooth Muscle Cells
To clarify whether the activation of phospholipase D is involved in extracellular ATP-induced AA release, we examined the effect of propranolol, an inhibitor of phosphatidic acid phosphohydrolase,28 on AA release in aortic smooth muscle cells. Propranolol (300 µmol/L), which by itself had little effect on AA release, significantly inhibited the ATP-induced AA release in these cells (Fig 4). We next examined the effect of RHC-80267, a potent and selective inhibitor of diacylglycerol lipase,29 on ATP-induced AA release in aortic smooth muscle cells. RHC-80267 (30 µmol/L), which by itself did not affect AA release, significantly suppressed the ATP-induced AA release in these cells (Fig 4). In addition, we examined the effect of quinacrine, a phospholipase A2 inhibitor,30 on the AA release in aortic smooth muscle cells. Quinacrine (50 µmol/L), which by itself had little effect on AA release, inhibited the ATP-induced AA release in these cells (Fig 4).
|
Effect of Propranolol or RHC-80267 on PGI2 Synthesis Induced by Extracellular ATP in Aortic Smooth Muscle Cells
It has been demonstrated that the major product of the released AA in vascular smooth muscle cells is PGI2.21 Therefore, we next examined the effect of propranolol and the effect of RHC-80267 on the ATP-induced synthesis of PGI2 as well as AA release in aortic smooth muscle cells. We confirmed that extracellular ATP increased the synthesis of 6-keto-PGF1, a stable metabolite of PGI2,25 in these cells. Propranolol (300 µmol/L) or RHC-80267 (30 µmol/L), which alone had no effect on 6-keto-PGF1 synthesis, significantly inhibited the ATP-induced synthesis of 6-keto-PGF1 (Table 2).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In the present study, we showed that ATP did not stimulate phosphocholine formation but did stimulate choline formation in cultured rat aortic smooth muscle cells. It is known that phosphatidylcholine is hydrolyzed by phosphatidylcholine-specific phospholipases C and D, resulting in the formation of phosphocholine and choline, respectively.12 13 14 15 Thus, it is most likely that extracellular ATP induces phosphatidylcholine hydrolysis mainly by phospholipase D in rat aortic smooth muscle cells. In a previous study,26 we reported that TPA, a PKC-activating phorbol ester,11 stimulates phospholipase D through the activation of PKC in rat aortic smooth muscle cells. However, in the present study we demonstrated that staurosporine, an inhibitor of protein kinases,27 did not affect the ATP-induced formation of choline in these cells. Therefore, it seems unlikely that PKC activation is involved in the ATP-induced formation of choline. These results suggest that extracellular ATP activates phospholipase D independently of PKC in rat aortic smooth muscle cells.
We next showed that propranolol, an inhibitor of phosphatidic acid phosphohydrolase,28 significantly inhibited ATP-induced AA release in rat aortic smooth muscle cells. It is well known that phosphatidylcholine hydrolysis by phospholipase D results in the formation of phosphatidic acid, which is further degraded into diacylglycerol by phosphatidic acid phosphohydrolase.13 So, it seems that the conversion of phosphatidic acid to diacylglycerol is involved in the ATP-induced AA release in rat aortic smooth muscle cells. Moreover, we demonstrated that RHC-80267, a potent and selective inhibitor of diacylglycerol lipase,29 significantly suppressed the ATP-induced AA release in these cells. This finding suggests that the action of diacylglycerol lipase is involved in ATP-induced AA release. Therefore, from our results as a whole, it is most likely that phosphatidylcholine hydrolysis by phospholipase D is an important pathway of the extracellular ATP-induced AA release in rat aortic smooth muscle cells. However, these inhibitory effects of propranolol and RHC-80267 on ATP-induced AA release were partial. We showed here that quinacrine, a phospholipase A2 inhibitor,30 also partially suppressed ATP-induced AA release in rat aortic smooth muscle cells. This finding suggests that phospholipase A2 participates in the extracellular ATP-induced AA release in these cells.
The released AA is metabolized to a variety of bioactive substances such as PGs. It has been reported that the major PG synthesized in vascular smooth muscle cells is PGI2.21 In the present study, we showed that extracellular ATP increased the synthesis of 6-keto-PGF1, a stable metabolite of PGI2,25 in rat aortic smooth muscle cells. In addition, both propranolol and RHC-80267 suppressed ATP-induced 6-keto-PGF1 synthesis as well as AA release in these cells. Thus, it is most likely that phosphatidylcholine hydrolysis by phospholipase D, which results in diacylglycerol formation, is involved in the extracellular ATP-induced synthesis of PGI2 in rat aortic smooth muscle cells.
Extracellular ATP has been shown to induce contraction of vascular smooth muscle cells5 6 and to stimulate the proliferation of these cells via activation of PKC.7 8 In the present study, as shown above, we demonstrated that extracellular ATP stimulates the synthesis of PGI2 in rat aortic smooth muscle cells. PGI2 is known to be a very potent vascular relaxing agent.20 We have earlier shown that carbacyclin, a stable analogue of PGI2,31 suppresses the proliferation of aortic smooth muscle cells at the late G1 phase.24 Thus, it is probable that extracellular ATP acts as a vasoconstrictor in aortic smooth muscle cells and that self-induced PGI2 synthesis may buffer the effect, resulting in the modulation of vasoactivity in these cells.
In conclusion, our results strongly suggest that extracellular ATP activates phospholipase D independently of PKC in aortic smooth muscle cells and that the AA release induced by extracellular ATP is mediated, at least in part, through phosphatidylcholine hydrolysis by phospholipase D activation.
|
Selected Abbreviations and Acronyms |
|---|
|
Received March 26, 1996;
revision received June 7, 1996;
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Jackson CL, Schwartz SM. Pharmacology of smooth muscle cell replication. Hypertension. 1992;20:713-736.
2. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809.[Medline] [Order article via Infotrieve]
3. Gordon JL. Extracellular ATP: effects, sources and fate. Biochem J. 1986;233:309-319.[Medline] [Order article via Infotrieve]
4.
Dubyak GR, El-Moatassim C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol. 1993;265:C577-C606.
5.
Burnstock G, Kennedy C. A dual function for adenosine 5'-triphosphate in the regulation of vascular tone. Circ Res. 1985;58:319-330.
6.
Olsson RA, Pearson JD. Cardiovascular purinoceptors. Physiol Rev. 1990;70:761-845.
7. Wang DJ, Huang NN, Heppel LA. Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells. J Cell Physiol. 1992;153:221-233.[Medline] [Order article via Infotrieve]
8. Erlinge D, You J, Wahlestedt C, Edvinsson L. Characterisation of an ATP receptor mediating mitogenesis in vascular smooth muscle cells. Eur J Pharmacol. 1995;289:135-149.[Medline] [Order article via Infotrieve]
9. Phaneuf S, Berta P, Casanova J, Cavadore JC. ATP stimulates inositol phosphates accumulation and calcium mobilization in a primary culture of rat aortic myocytes. Biochem Biophys Res Commun. 1987;143:454-460.[Medline] [Order article via Infotrieve]
10. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature. 1993;361:315-325.[Medline] [Order article via Infotrieve]
11.
Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986;233:305-312.
12. Billah MM, Anthes JC. The regulation and cellular functions of phosphatidylcholine hydrolysis. Biochem J. 1990;269:281-291.[Medline] [Order article via Infotrieve]
13.
Exton JH. Signaling through phosphatidylcholine breakdown. J Biol Chem. 1990;265:1-4.
14. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 1995;9:484-496.[Abstract]
15. Zeisel SH. Choline phospholipids: signal transduction and carcinogenesis. FASEB J. 1993;7:551-557.[Abstract]
16. Exton JH. Phosphatidylcholine breakdown and signal transduction. Biochim Biophys Acta. 1994;1212:26-42.[Medline] [Order article via Infotrieve]
17. Suzuki A, Shinoda J, Oiso Y, Kozawa O. Tyrosine kinase is involved in angiotensin II–stimulated phospholipase D activation in aortic smooth muscle cells: function of Ca2+ influx. Atherosclerosis. 1996;121:119-127.[Medline] [Order article via Infotrieve]
18. Smith WL. The eicosanoids and their biochemical mechanisms of action. Biochem J. 1989;259:315-324.[Medline] [Order article via Infotrieve]
19.
Balsinde J, Diez E, Mollinedo F. Arachidonic acid release from diacylglycerol in human neutrophils. J Biol Chem. 1991;266:15638-15643.
20. Lumley P, Humphrey PPA, Kennedy I, Coleman RA. Comparison of the potencies of some prostaglandins as vasodilators in three vascular beds of the anaesthetised dog. Eur J Pharmacol. 1982;81:421-430.[Medline] [Order article via Infotrieve]
21.
Hassid A, Williams C. Vasoconstrictor-evoked prostaglandin synthesis in cultured vascular smooth muscle. Am J Physiol. 1983;245:C278-C282.
22. Ito Y, Kozawa O, Tokuda H, Suzuki A, Watanabe Y, Kotoyori J, Oiso Y. Glucocorticoid inhibits cAMP production induced by vasoactive agents in aortic smooth muscle cells. Atherosclerosis. 1994;110:69-76.[Medline] [Order article via Infotrieve]
23.
Shinoda J, Suzuki A, Oiso Y, Kozawa O. Thromboxane A2–stimulated phospholipase D in osteoblast-like cells: possible involvement of PKC. Am J Physiol. 1995;269:E524-E529.
24. Murase T, Kozawa O, Miwa M, Tokuda H, Kotoyori J, Kondo K, Oiso Y. Regulation of proliferation by vasopressin in aortic smooth muscle cells: function of protein kinase C. J Hypertens. 1992;10:1505-1511.[Medline] [Order article via Infotrieve]
25. Siess W, Dray F, Seillan C, Ody C, Russo-Marie F. Prostanoid synthesis by vascular slices and cultured vascular cells of piglet aorta. Biochem Biophys Res Commun. 1981;99:608-616.[Medline] [Order article via Infotrieve]
26. Miwa M, Kozawa O, Suzuki A, Watanabe Y, Shinoda J, Oiso Y. Vasopressin activates phospholipase D through pertussis toxin–insensitive GTP-binding protein in aortic smooth muscle cells: function of Ca2+/calmodulin. Biochem Cell Biol. 1995;73:191-199.[Medline] [Order article via Infotrieve]
27. Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase. Biochem Biophys Res Commun. 1986;135:397-402.[Medline] [Order article via Infotrieve]
28. Pappu AS, Hauser G. Propranolol-induced inhibition of rat brain cytoplasmic phosphatidate phosphohydrolase. Neurochem Res. 1983;8:1565-1575.[Medline] [Order article via Infotrieve]
29.
Sutherland CA, Amin D. Relative activities of rat and dog platelet phospholipase A2 and diglyceride lipase. J Biol Chem. 1982;257:14006-14010.
30. Lapetina EG, Billah MM, Cuatrecasas P. The phosphatidylinositol cycle and the regulation of arachidonic acid production. Nature. 1981;292:367-369.[Medline] [Order article via Infotrieve]
31. Whittle BJR, Moncada S, Whiting F, Vane JR. Carbacyclin: a potent stable prostacyclin analogue for the inhibition of platelet aggregation. Prostaglandins. 1980;19:605-627.[Medline] [Order article via Infotrieve]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||