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(Hypertension. 2001;37:623.)
© 2001 American Heart Association, Inc.

Scientific Contributions

20-Hydroxyeicosatetraenoic Acid Mediates Angiotensin II–Induced Phospholipase D Activation in Vascular Smooth Muscle Cells

Jean-Hugues Parmentier; Mubarack M. Muthalif; Andrew T. Nishimoto; Kafait U. Malik

From the Department of Pharmacology, College of Medicine, The University of Tennessee Health Science Center, Memphis, Tenn.

Correspondence to Kafait U. Malik, PhD, DSc, Professor of Pharmacology, College of Medicine, The University of Tennessee at Memphis, 874 Union Avenue, Memphis, TN 38163. E-mail kmalik{at}utmem.edu

	Abstract

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Angiotensin II (Ang II) activates cytosolic phospholipase A₂ (cPLA₂) and phospholipase D (PLD) in rabbit vascular smooth muscle cells (VSMCs). Ang II also activates ras/mitogen-activated protein (MAP) kinase in VSMCs; this activation is mediated by 20-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, which are metabolites of arachidonic acid generated by cytochrome P450 4A and lipoxygenase, respectively, produced on activation of cPLA₂. The purpose of this study was to determine if Ang II–induced PLD activation in VSMCs is mediated through the ras/extracellular signal-regulating kinase (ERK) pathway by arachidonic acid metabolites that are generated consequent to cPLA₂ stimulation. Inhibitors of PLD (C₂ ceramide), phosphatidate phosphohydrolase (propranolol), and diacylglycerol lipase (RHC 80267) attenuated Ang II–induced arachidonic acid release. Ang II–induced PLD activation, as measured by [³H]phosphatidylethanol production, was inhibited by C₂ ceramide but not by propranolol or RHC 80267. Ang II–induced PLD activation was decreased by the inhibitor methyl arachidonylfluorophosphate (MAFP) and the antisense oligonucleotide of cPLA₂. Inhibitors of lipoxygenases (baicalein) and cytochrome P450 4A (ODYA) attenuated Ang II–induced PLD activation. 20-HETE and 12(S)-HETE increased PLD activity. Inhibitors of ras farnesyltransferase (FPT III and BMS-191563) and MAP kinase kinase (UO126) attenuated the increase in PLD activity elicited by 20-HETE and Ang II. PLD2 was the main isoform activated by Ang II in VSMCs. These data suggest that the CYP4A metabolite 20-HETE, which is generated from arachidonic acid after cPLA₂ activation by Ang II, stimulates the ras/MAP kinase pathway, which in turn activates PLD2 and releases further arachidonic acid for prostaglandin synthesis through the phosphatidate phosphohydrolase/diacylglycerol lipase pathway.

Key Words: phospholipases • angiotensin • cytochrome • muscle, smooth, vascular • arachidonic acid • 20-HETE

	Introduction

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Angiotensin II (Ang II), an octapeptide, stimulates the migration, hypertrophy, and hyperplasia of vascular smooth muscle cells (VSMCs).^{1 2 3} Ang II produces these biological actions by generating lipid mediators, including arachidonic acid and its metabolites, consequent to the activation of phospholipase A₂ (PLA₂), phospholipase C, and phospholipase D (PLD) and by producing superoxide and hydrogen peroxide.^{4 5} The activation of PLA₂ releases arachidonic acid directly from phospholipids, whereas PLD catalyzes the hydrolysis of phosphatidylcholine into choline and phosphatidic acid.⁶ The latter can be converted into arachidonic acid for prostaglandin synthesis by PLA₂ or sequentially by phosphatidate phosphohydrolase and diacylglycerol lipase.⁷ Products generated by lipoxygenase, 12(S)-hydroxyeicosatetraenoic acid (HETE), and/or cytochrome P 450 4A (CYP4A), 20-HETE, from arachidonic acid liberated by cytostolic PLA₂ (cPLA₂) in response to Ang II, cause smooth muscle hyperplasia and contribute to the hypertension caused by this peptide.^{8 9 10}

Recently, we reported that 20-HETE activates ras/mitogen-activated protein (MAP) kinase, which stimulates VSMC proliferation.² Moreover, we demonstrated that norepinephrine stimulates PLD in VSMC via the ras/MAP kinase pathway.¹¹ Ang II also activates ras/MAP kinase via arachidonic acid metabolites formed through CYP4A (20-HETE) and lipoxygenase (12-HETE).¹² These observations, together with recent reports that the PLD activation in cardiac sarcolemma,¹³ leukocytes,¹⁴ and HEK 293 cells¹⁵ is mediated through the stimulation of cPLA₂, raised the possibility that the activation of PLD in VSMCs by Ang II might be mediated by the ras/MAP kinase pathway metabolites of arachidonic acid that are generated by the initial activation of cPLA₂. To test this hypothesis, we examined the effect of various inhibitors of this pathway on Ang II–induced PLD activation in rabbit VSMCs. We show that 20-HETE is the major eicosanoid involved in mediating Ang II–induced PLD activation in these cells.

	Methods

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Materials
[³H]arachidonic acid was purchased from Dupont-NEN, and [³H]oleic acid was obtained from American Radiolabeled Chemicals. Ang II was from Bachem, and methyl arachidonylfluorophosphonate (MAFP), 12(S)-HETE, and 20-HETE were from Cayman Chemicals. Phosphatidylethanol, C₂ ceramide, C₂ dihydroceramide, RHC-80267, propranolol, baicalein, indomethacin, and ODYA were obtained from Biomol. FPTIII was purchased from Calbiochem, and BMS-191563 was a gift from Bristol-Myers Squibb (Princeton, NJ). UO126 was from Promega.

Culture of VSMCs
Male New Zealand white rabbits (1 to 2 kg) were anesthetized with pentobarbital (Abbot Laboratories), and the thorax and abdomen were opened by a midline incision. The aorta was rapidly removed, and VSMCs were isolated as previously described.¹⁶ Cells between 4 and 8 passages were plated in 24-well clusters or 100-mm plates. Cells were maintained under 5% CO₂ in M-199 medium containing penicillin, streptomycin, Fungizone, and 10% fetal bovine serum.

Transient Transfections
VSMCs were transfected with antisense or sense oligonucleotides designed from cPLA₂ or secretory PLA₂ cDNA sequences.¹⁷ Phosphorothioate oligonucleotides (Molecular Resource Center) were complexed with lipofectamine PLUS reagents (Life Technologies) according to the manufacturer’s instructions. The oligonucleotide mix was added to the cells for 24 hours, and the medium was replaced with fresh M-199 for another 24 hours and exposed to Ang II (100 nmol/L) or its vehicle for 10 minutes and assayed for PLD as described below. Efficiency of transfection with oligonucleotides was measured by Western blot analysis.

For experiments with hemagglutinin (HA)-tagged pCGN-PLD plasmids (a gift from Dr Michael Frohman from SUNY at Stony Brook, NY),^{18 19} cells in 100-mm dishes were transfected with wild-type and catalytically inactive variants of PLD1 and PLD2 (K898R-PLD1 and K758R-PLD2) using a calcium phosphate method. Briefly, 10 µg of DNA was combined with CaCl₂ and mixed with Hepes buffer salt (HeBS) buffer. After 20 minutes of incubation, the solution mix was slowly added to the cells in the presence of serum-free M-199 and incubated for 6 hours. Cells were then washed twice with HBSS and allowed to recover in M-199 containing 10% fetal bovine serum for 24 hours. Cells were arrested overnight in 0.5% fetal bovine serum and then treated with Ang II (100 nmol/L) or its vehicle for 10 minutes and assayed for PLD activity as described below. Transfection efficiencies were determined by Western blot analysis.

Arachidonic Acid Release
VSMCs cultured in 24-well clusters were incubated with [³H]arachidonic acid (1 µCi/mL) in M-199 for 18 hours, as previously described.¹⁷ Cells were incubated with inhibitors for 1 to 4 hours, washed 3 times with HBSS, and exposed to Ang II or its vehicle for 10 minutes. The amount of radioactivity released into the medium (mainly prostaglandins) was determined by liquid scintillation spectroscopy. Total radioactivity in the cells was determined after overnight treatment with 1 mol/L NaOH. Radioactivity released into the medium was calculated as a percent of the total cellular radioactivity and referred to as fractional release of [³H]arachidonic acid. Data were expressed as percent increase over basal fractional [³H]arachidonic acid release.

PLD Assay
PLD activity in VSMCs was assayed as described previosly²⁰ with slight modifications.¹¹ Briefly, near-confluent rabbit VSMCs were incubated with [³H]oleic acid (1 µCi/µL) for 16 hours. Cells were then incubated with inhibitors and exposed to Ang II (100 nmol/L), 20-HETE, 12(R)-HETE, or 12(S)-HETE (0.5 µmol/L for all HETE) for an additional 10 minutes in the presence of ethanol. VSMCs were scraped into 2 mL of ice-cold methanol and 2 mol/L HCl, and lipids were separated by chloroform extraction. The aqueous and insoluble fractions were saved to determine transfection efficiencies, and a 40-µL aliquot was removed from the chloroform phase to determine the content of radioactivity in the total lipid fraction. The chloroform phase (0.8 mL) was evaporated under nitrogen and redissolved in chloroform/methanol (9:1) containing phosphatidylethanol standard. Samples were spotted onto a silica gel thin-layer chromatography plate, and lipids were separated with the solvent system of chloroform/acetone/methanol/acetic acid/water (50:20:12.5:10:7.5). Phosphatidylethanol was identified by the mobility of authentic standard visualized with iodine vapor. Lanes containing phosphatidylethanol were scraped, and radioactivity was measured by scintillation spectroscopy. Data were expressed as the ratio of [³H]phosphatidylethanol to [³H]total lipids. Figure 1 summarizes the tools used to inhibit the pathways described in this study.

View larger version (11K): [in this window] [in a new window]   Figure 1. Pathways for PLD activation and the site of action of various inhibitors. AA indicates arachidonic acid; PPH, phosphatidate phosphohydrolase; and DAG, diacylglycerol.

Western Blotting
The efficiency of transient transfection with both oligonucleotides and plasmids was determined by Western blotting. Briefly, VSMCs treated with oligonucleotides were washed twice and scraped in ice-cold PBS. Cells were pelleted by quick centrifugation and resuspended in boiling Laemmli sample buffer. Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Blocking was performed with TBS buffer (20 mmol/L Tris-HCl [pH 7.6] and 200 mmol/L NaCl) containing 3% nonfat dry milk powder. The membrane was then incubated with anti-cPLA₂ antibody (1:200; Santa-Cruz Biotechnology) for 2 hours at room temperature. The immunoblots were subsequently washed, incubated with horseradish peroxidase–linked secondary antibodies, and rinsed and developed with enhanced chemiluminescence reagents (Amersham). For transfection efficiencies with HA-tagged pCGN-PLD plasmids, the top aqueous layer and insoluble fraction from the PLD assay were precipitated with 4 volumes of ice-cold acetone, incubated for 1 hour at -80°C, and pelleted and dried under nitrogen. The pellet was resuspended in boiling Laemmli buffer and treated as described above. HA-PLD expression was detected with an HA probe (Santa-Cruz Biotechnology).

Statistical Analysis
Results are expressed as the mean±SD from different batches of cells for PLD activity or arachidonic acid release. Data were analyzed by 1-way ANOVA. Student’s t test was applied to determine differences between treatments and their respective control values. The Newman-Keuls multiple range test was applied to compare treatments among multiple groups. The null hypothesis was rejected at P<0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of Inhibitors of PLD, Phosphatidate Phosphohydrolase, and Diacylglycerol Lipase on Ang II–Induced Arachidonic Acid Release
To assess the contribution of PLD to arachidonic acid release for prostaglandin synthesis in rabbit VSMCs, we examined the effects of inhibitors of PLD (C₂ ceramide), phosphatidate phosphohydrolase (propranolol), and diacylglycerol lipase (RHC-80267)¹⁵ on Ang II–induced arachidonic acid release. PLD activity was reduced by C₂ ceramide (10 µmol/L for 4 hours) but not by C₂ dihydroceramide, an inactive analog of C₂ ceramide (Figure 2A). In addition, treatment with propranolol (10 µmol/L for 30 minutes) or RHC-80267 (10 µmol/L for 30 minutes) also inhibited Ang II–induced arachidonic acid release. These inhibitors did not alter basal arachidonic acid release (data not shown).

View larger version (36K): [in this window] [in a new window]   Figure 2. Effect of inhibitors of PLD/phosphatidate phosphohydrolase/diacylglycerol pathway on Ang II–induced arachidonic acid (AA) release and PLD activity in VSMCs. A, [3H]arachidonic acid–labeled VSMCs pretreated with inhibitors of PLD (C2 ceramide, 10 µmol/L for 4 hours, or C2 dihydroceramide [di-C2], inactive analog), phosphatidate phosphohydrolase (propranolol, 10 µmol/L for 30 minutes), diacylglycerol lipase (RHC-80267, 10 µmol/L for 30 minutes), or vehicle were stimulated with 100 nmol/L Ang II for 10 minutes. Arachidonic acid release was then measured as described in Methods. Data are expressed as percent increase in arachidonic acid release over basal fractional release in unstimulated cells. Values are mean±SD of 3 independent experiments performed in sextuplicate. *Value significantly different from Ang II, P<0.05. B, Effect of PLD/diacylglycerol/arachidonic acid pathway inhibitors on Ang II–induced PLD activity. [3H]oleic acid–labeled VSMCs were treated as described above and PLD activity was measured as described in Methods. Data are expressed as -fold increase in PLD activity over basal activity in unstimulated cells. Values are mean±SD of 3 independent experiments performed in duplicate. *Value significantly different from Ang II, P<0.05.

To confirm the selectivity of these inhibitors, we examined the effects of C₂ ceramide, C₂ dihydroceramide, propranolol, and RHC-80267 on basal and Ang II–induced PLD activity. Ang II–induced PLD activation was inhibited by C₂ ceramide but not by C₂ dihydroceramide, propranolol, or RHC-80267 (Figure 2B). These results suggest that Ang II activates PLD and releases arachidonic acid/prostaglandins via the phosphatidate phosphohydrolase and diacylglycerol lipase pathway (PLD phosphatidate phosphohydrolase diacylglycerol lipase arachidonic acid) in addition to the conventional cPLA₂ pathway in rabbit VSMCs.

cPLA₂, and Cytochrome P450 Product 20-HETE, Mediate Ang II–Induced PLD Activity
Recently, it was reported that cPLA₂ is involved in PLD activation in rat heart sarcolemma,¹³ leukocyte cell lines,¹⁴ and HEK293 cells.¹⁵ To assess the contribution of cPLA₂ to Ang II–induced PLD activation, we used MAFP (50 µmol/L),²¹ a PLA₂ inhibitor, and HELSS (haloenol lactone suicide substrate), a selective calcium-independent PLA₂ (iPLA₂) inhibitor with a 1000-fold selectivity for iPLA₂ over cPLA₂ at 10 µmol/L.²² As shown in Figure 3, Ang II–induced PLD activity was inhibited by MAFP but not by HELSS in VSMCs. Moreover, Ang II–induced PLD activity was inhibited in cells treated for 48 hours with cPLA₂ antisense but not sense oligonucleotides (Figure 4). These data indicate that the products of cPLA₂ activation, arachidonic acid, and/or the metabolites of arachidonic acid mediate the PLD activation elicited by Ang II in VSMCs.

View larger version (32K): [in this window] [in a new window]   Figure 3. Effect of inhibitors of PLA2 and arachidonic acid metabolism on Ang II–induced PLD activity. Cells were incubated with inhibitors of PLA2 (HELSS, 10 µmol/L; MAFP, 50 µmol/L), cyclooxygenase (indomethacin, 10 µmol/L), lipoxygenase (baicalein, 5 µmol/L), or CYP4A (ODYA, 5 µmol/L) or their vehicle for 30 minutes. They were then exposed to 100 nmol/L Ang II, and PLD activity was measured. Data are expressed as -fold increase in PLD activity over basal activity. Values are mean±SD of 3 independent experiments performed in duplicate. *Value significantly different from Ang II, P<0.05.

View larger version (31K): [in this window] [in a new window]   Figure 4. Effect of cPLA2 antisense oligonucleotides on AngII–induced PLD activity. A, [3H]oleic acid–labeled VSMCs were incubated with cPLA2 antisense (AS) or sense (SE) oligonucleotides or vehicle (lipofectamine) for 48 hours, and PLD activity was measured as described in Methods. Data are expressed as -fold increase in PLD activity over basal activity in unstimulated cells. Values are mean±SD of 3 independent experiments performed in duplicate. *Value significantly different from Ang II, P<0.05. B, Representative Western blot showing effect of treatment with cPLA2 antisense and sense oligonucleotides on cPLA2 protein level. Lane 1 shows vehicle; lane 2, 0.5 µmol/L cPLA2 antisense; lane 3, 1 µmol/L cPLA2 antisense; and lane 4, 1 µmol/L cPLA2 sense.

Arachidonic acid (10 µmol/L) increased PLD activity to the same extent as Ang II, and the activation was blocked by the inhibitor of arachidonic acid metabolism 5,8,11,14-eicosatetraynoic acid (data not shown). To determine the contribution of arachidonic acid metabolites to cPLA₂-dependent PLD activation, VSMCs were treated with inhibitors of the 3 enzymatic pathways metabolizing arachidonic acid in VSMCs (Figure 3). Ang II–induced PLD activation was reduced by the lipoxygenase inhibitor baicalein (5 µmol/L) but not by the cyclooxygenase inhibitor indomethacin (10 µmol/L), suggesting a significant contribution of arachidonic acid metabolite(s) from the lipoxygenase pathway to PLD activation. However, Ang II–induced PLD activation was blocked by treatment with ODYA (5 µmol/L), a specific CYP4A inhibitor.²³ CYP4A metabolizes arachidonic acid to 20-HETE in VSMCs.¹² As shown in Figure 5, 20-HETE (0.5 µmol/L) stimulated PLD activity to approximately the same extent as Ang II and arachidonic acid. 12(S)-HETE, a 12-lipoxygenase-derived metabolite of ara-chidonic acid, also increased PLD activity, whereas the stereoisomer 12(R)-HETE was inactive. 15(S)-HETE also stimulated PLD to the same extent as 12- or 20-HETE (data not shown). On the basis of these observations, it seems that Ang II–induced cPLA₂-dependent PLD activation is mediated mainly by the CYP4A metabolite 20-HETE and to a lesser degree by lipoxygenase metabolites.

View larger version (14K): [in this window] [in a new window]   Figure 5. Effect of HETEs on PLD activity in VSMCs. [3H]oleic acid–labeled VSMCs were treated with 20-HETE, 12(R)-HETE, or 12(S)-HETE (0.5 µmol/L for 10 minutes for each), and PLD activity was measured as described in Methods. Data are expressed as -fold increase in PLD activity over basal activity in unstimulated cells. Values are mean±SD of 3 independent experiments performed in duplicate. *Value significantly different from basal level, P<0.05.

20-HETE Stimulates PLD Through ras/MAP Kinase Pathway
We previously reported that 20-HETE mediates the Ang II- and norepinephrine-induced activation of ras and MAP kinase in rabbit VSMCs.¹² We also showed that ras/MAP kinase mediates norepinephrine-induced PLD activation in VSMCs.¹¹ These findings raised the possibility that 20-HETE mediates Ang II–induced PLD stimulation through the ras/MAP kinase pathway. To test this hypothesis, we examined the effect of inhibitors of the ras/MAP kinase pathway on the PLD activity stimulated by Ang II and 20-HETE. Ras is post-translationally modified by farnesylation. Farnesyltransferase inhibitors, such as FPT III and BMS-191563, decrease ras farnesylation and its translocation to plasma membrane, resulting in the loss of its function.^{8 12} Ang II–induced PLD activity was inhibited by FPT III (25 µmol/L) and BMS-191563 (10 µmol/L; Figure 6A). Consistent with the inhibitory effect of FPT III and BMS-191563 on Ang II-stimulated PLD activity, the 20-HETE-induced increase in PLD activity was also blocked by these agents (Figure 6B). The ras-farnesyltransferase inhibitors FPT III and BMS-191563, when preincubated with VSMCs for 18 hours, did not cause cytotoxicity or a change in basal PLD activity (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 6. Effect of inhibitors of ras/MEK pathway on Ang II– (A) and 20-HETE– (B) induced PLD activity. Cells were incubated with inhibitors of ras farnesyltransferase (FPT III, 25 µmol/L; BMS-191563, 10 µmol/L; each for 18 hours) or MEK (UO126, 10 µmol/L for 1 hour) and then exposed to 100 nmol/L Ang II or 0.5 µmol/L 20-HETE for 10 minutes. PLD activity was measured as described in Methods. Data are expressed as -fold increase in PLD activity over basal activity in unstimulated cells. Values are mean±SD of 3 independent experiments performed in duplicate in each group. *Value significantly different from Ang II, P<0.05.

To further establish the importance of the ras/MAP kinase pathway in 20-HETE-mediated Ang II–induced PLD activity, we treated the cells with an inhibitor of MEK (MAP kinase kinase), UO126. Ang II- and 20-HETE–induced PLD activity were reduced in the presence of UO126 (10 µmol/L; Figure 6). UO126 required a shorter incubation and lower concentration than PD-98059, another widely used MEK inhibitor that also reduced Ang II–induced PLD activation (data not shown). These findings support the conclusion that 20-HETE–mediated PLD activity is regulated by the ras and MAP kinase pathway in VSMCs.

Ang II Selectively Activates PLD2
Agonist-induced PLD1 and constitutively-expressed PLD2 isoforms have been cloned and extensively characterized.⁶ The finding that PLD2 activity may also be upregulated by several agonists, primarily through ADP-ribosylation factor (ARF),²⁴ has led to a renewed interest in the identification of agonist-induced PLD isoforms. To assess the contribution of each PLD isoform to overall Ang II–induced PLD activity, we overexpressed wild-type and catalytically inactive PLD1 and PLD2^{18 19} in rabbit VSMCs. Basal PLD activity was increased by 20% in cells overexpressing wild-type PLD2, which is consistent with a constitutive basal expression of PLD2, whereas the overexpression of wild-type PLD1 or inactive variants of PLD1 and PLD2 had no significant effect on basal PLD activity (data not shown). Overexpression of catalytically inactive K758R-PLD2 but not K898R-PLD1 markedly reduced Ang II–induced PLD activation (Figure 7). In VSMCs expressing wild-type PLD2 but not wild-type PLD1, Ang II–stimulated PLD activity was enhanced (Figure 7). These results suggest that PLD2 is the major isoform activated by Ang II in VSMCs.

View larger version (33K): [in this window] [in a new window]   Figure 7. Effect of PLD isoform mutants on Ang II–induced PLD activity. A, Cells were transiently transfected with wild-type or catalytically inactive mutants of PLD1 or PLD2 for 48 hours, and PLD activity was determined after stimulation with Ang II as described in Methods. Data are expressed as -fold increase in PLD activity over basal activity in unstimulated cells. Values are mean±SD of 3 independent experiments performed in duplicate. *Value significantly different from Ang II alone, P<0.05. B, Representative Western blot showing efficiency of transfection of HA-tagged pCGN-PLD constructs using HA antibody. Lane 1 shows HA-PLD2; lane 2, HA-K758R-PLD2; lane 3, calcium phosphate alone; lane 4, HA-PLD1; and lane 5, HA-K898R-PLD1.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The sustained activation of PLD by Ang II plays an important role in VSMC signaling and growth.^{4 25 26} However, the regulatory mechanisms involved in Ang II–induced PLD activation have not been clearly defined. The present study demonstrates that in rabbit VSMCs, the Ang II–induced increase in PLD activity is mediated by cPLA₂ stimulation. Moreover, the arachidonic acid metabolites generated primarily by CYP4A and, to a lesser extent, by lipoxygenase (20-HETE and 12(S)-HETE, respectively) activate PLD through the ras/MAP kinase pathway.

In a previous study, we reported that Ang II stimulates arachidonic acid release for prostaglandin synthesis through selective cPLA₂ activation and its translocation to the nuclear envelope.²⁷ In the present study, the Ang II–induced release of arachidonic acid was reduced by the PLD inhibitor C₂ ceramide but not by its inactive analogue C₂ dihydroceramide. Moreover, propranolol and RHC-80267, inhibitors of phosphatidate phosphohydrolase and diacylglycerol lipase, reduced arachidonic acid release without altering PLD activity. The latter 2 agents also reduce Ang II–induced 6-keto-PGF₁ production in rat VSMCs.²⁸ Therefore, it seems that Ang II also promotes arachidonic acid release and prostaglandin synthesis by PLD activation through the phosphatidate phosphohydrolase/diacylglycerol lipase pathway in rabbit VSMCs. The activation of -adrenergic receptors in rat-1 fibroblasts also releases arachidonic acid via PLD activation through the phosphatidate phosphohydrolase/diacylglycerol lipase pathway.²⁰

In rabbit VSMCs, cPLA₂ mediates norepinephrine and Ang II–stimulated arachidonic acid release.²⁷ Ang II–induced PLD activation was also abolished in the absence of extracellular calcium, and cPLA₂ activation was an early event compared with PLD stimulation in rabbit VSMCs (data not shown). It has been reported that the activation of PLD depends on cPLA₂ in rat heart sarcolemma,¹³ leukocytes,¹⁴ and HEK293 cells.¹⁵ Supporting this hypothesis was our demonstration that the PLA₂ inhibitor MAFP²¹ and cPLA₂ antisense blocked Ang II–induced PLD activation in rabbit VSMCs. Although MAFP inhibits both cPLA₂ and iPLA₂, our data with cPLA₂ antisense oligonucleotides and the lack of inhibition of Ang II–induced PLD activation by iPLA₂ inhibitor HELSS²² suggest that cPLA₂ mediates Ang II–induced PLD activation.

The mechanism by which cPLA₂ stimulation promotes Ang II–induced PLD activation could involve arachidonic acid and/or its metabolites. Our findings that arachidonic acid–stimulated PLD activity was blocked by an inhibitor of arachidonic acid metabolism (our unpublished observations) suggest that metabolites of arachidonic acid mediate the Ang II–induced increase in PLD activity. Supporting this view is our demonstration that an inhibitor of CYP4A (ODYA) and, to a lesser degree, lipoxygenase (baicalein) but not cyclooxygenase (indomethacin) blocked Ang II–induced PLD. Arachidonic acid is metabolized by CYP4A and lipoxygenase into HETEs, including 20-HETE and 12(S)-HETE, respectively, in rabbit VSMCs.¹² The fact that 20-HETE and 12(S)-HETE but not 12(R)-HETE increased PLD activity supports our proposition that arachidonic acid metabolites generated via CYP4A and lipoxygenase mediate the cPLA₂-induced activation of PLD in response to Ang II. 15-HETE could also be involved in Ang II–induced PLD activation because it increased PLD activity in VSMCs. Recently, 12(S)-HETE was also reported to cause activation of PLD in lymphocytes.²⁹

Although in our study 12(S)-HETE increased PLD activity to the same extent as 20-HETE, the partial reduction by the lipoxygenase inhibitor baicalein suggests that products of lipoxygenases contribute to a smaller degree than CYPA4 (20-HETE) to Ang II–induced PLD activation. However, we cannot rule out the contribution of other arachidonic acid metabolites formed through lipoxygenase/CYP pathway, such as trihydroxyeicosatrienoic acids.³⁰ These findings indicate a novel role for 20-HETE in the signaling mechanism of Ang II in PLD activation. The mechanism by which 12(S)- and 20-HETE cause PLD activation could involve the ras/ERK pathway for the following reasons. First, we and others have shown that 20-HETE stimulates the ras/raf/MEK/ERK pathway in VSMCs.^{12 31} Second, PLD activation by norepinephrine is mediated through the ras/MAP kinase pathway.¹¹ Therefore, our finding that Ang II- and 20-HETE–induced PLD activation was blocked by inhibitors of ras farnesyltransferase (FPTIII and BMS-191563) and MEK (U0126) supports the view that ras/MAP kinase mediates the PLD activation caused by the 20-HETE generated by cPLA₂ activation in response to Ang II in rabbit VSMCs.

The signaling mechanism leading to ras/MAP kinase activation by 20-HETE is not known. Palmitoylation of ras has been shown to cause its activation.³² Recently, it was reported that 12(S)-HETE activated p21-activated kinase through a cdc42/rac-dependent pathway.³³ Because the localization/activation of small G proteins, including ras or cdc42/rac, is regulated by post-translational lipidation and p21-activated kinase is upstream of the MEK/MAP kinase pathway,³³ it is possible that 20-HETE promotes the activation of ras by its lipidation.

PLD exists in at least 2 isoforms, PLD1 and PLD2.⁶ In a recent study with A10 cells, a dedifferentiated cell line bearing a resemblance to neointimal cells, Ang II primarily activated ARF-mediated PLD2 rather than PLD1.²⁴ In the present study in VSMCs transfected with HA-tagged PLD1 and PLD2, Ang II–induced PLD activation was enhanced in VSMCs expressing PLD2 but not PLD1. Moreover, in cells expressing dominant-negative mutant K758 PLD2 but not K898R PLD1, Ang II–induced PLD activation was diminished. Therefore, it seems that PLD2 is the main isoform activated by Ang II in rabbit VSMCs.

In conclusion, this study provides evidence that Ang II–induced PLD activation is mediated by the initial stimulation of cPLA₂ and the generation of arachidonic acid metabolites, mainly 20-HETE, through the CYP4A pathway in rabbit VSMCs. 20-HETE activates PLD via ras/MAP kinase, which releases further arachidonic acid for prostanoid through the phosphatidate phosphohydrolase/diacylglycerol lipase pathway. Furthermore, arachidonic acid metabolites generated through this pathway do not participate in PLD regulation, because the inhibitors of the phosphatidate phosphohydrolase/diacylglycerol lipase pathway do not alter Ang II–induced PLD activation. Our study also demonstrates that Ang II selectively activates the PLD2 but not PLD1 isoform in rabbit VSMCs. Overall, this study provides evidence for a novel signaling pathway for PLD activation by Ang II in VSMCs (Ang II cPLA₂ arachidonic acid 20-HETE ras/ERK PLD).

	Acknowledgments

 
This work was supported by NIH-NHLBI grant 19134–26. Dr Jean-Hugues Parmentier is the recipient of a Postdoctoral Fellowship from the Center for Neuroscience and an American Heart Association Southeast Affiliate. We thank Anne Estes for excellent technical assistance, Jin Emerson-Cobb for editorial assistance, and Dr Lauren Cagen for editorial comments. We gratefully acknowledge Dr Michael Frohman for the PLD constructs.

Received October 24, 2000; first decision December 11, 2000; accepted December 18, 2000.

	References

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