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Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca²⁺-dependent phospholipase A₂

Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
Angiotensin II (ANG II) promotes vascular smooth muscle cell (VSMC) growth, stimulates Ca²⁺-calmodulin (CaM)-dependent kinase II (CaMKII), and activates cytosolic Ca²⁺-dependent phospholipase A₂ (cPLA₂), which releases arachidonic acid (AA). ANG II also generates H₂O₂ and activates Akt, which have been implicated in ANG II actions in VSMC. This study was conducted to investigate the relationship of these signaling molecules to Akt activation in rat aortic VSMC. ANG II increased Akt activity, as measured by its phosphorylation at serine-473. ANG II (200 nM)-induced Akt phosphorylation was decreased by extracellular Ca²⁺ depletion and calcium chelator EGTA and inhibitors of CaM [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and CaMKII {(2-[N-(2-hydroxyethyl)]-N-(4-me-thoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine)}. cPLA₂ inhibitor pyrrolidine-1, antisense oligonucleotide, and retroviral small interfering RNA also attenuated ANG II-induced Akt phosphorylation. AA increased Akt phosphorylation, and AA metabolism inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) blocked ANG II- and AA-induced Akt phosphorylation (199.03 ± 27.91% with ANG II and 110.18 ± 22.40% with ETYA + ANG II; 405.00 ± 86.22% with AA and 153.97 ± 63.26% with ETYA + AA). Inhibitors of lipoxygenase (cinnamyl-3,4-dihydroxy--cyanocinnamate) and cytochrome P-450 (ketoconazole and 17-octadecynoic acid), but not cyclooxygenase (indomethacin), attenuated ANG II- and AA-induced Akt phosphorylation. Furthermore, 5(S)-, 12(S)-, 15(S)-, and 20-hydroxyeicosatetraenoic acids and 5,6-, 11,12-, and 14,15-epoxyeicosatrienoic acids increased Akt phosphorylation. Catalase inhibited ANG II-increased H₂O₂ production but not Akt phosphorylation. Oleic acid, which also increased H₂O₂ production, did not cause Akt phosphorylation. These data suggest that ANG II-induced Akt activation in VSMC is mediated by AA metabolites, most likely generated via lipoxygenase and cytochrome P-450 consequent to AA released by CaMKII-activated cPLA₂ and independent of H₂O₂ production.

angiotensin II; Akt; hydroxyeicosatetraenoic acids; lipoxygenase; Ca²⁺-dependent phospholipase A₂; epoxyeicosatrienoic acid; reactive oxygen species

ANG II also activates cPLA₂, which releases arachidonic acid (AA) from tissue phospholipids (4, 29–30, 35). AA is metabolized by cyclooxygenase into prostaglandins and thromboxane A₂ by lipoxygenase into leukotrienes and 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids (HETEs) and by cytochrome P-450 into 12(R)-, 19-, and 20-HETE and 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) (15, 28, 33, 37–38). In mesangial cells, PLA₂-activated AA release and H₂O₂ production mediate ANG II-induced Akt activation (16). Akt activation has also been shown to be dependent on Ca²⁺-calmodulin (CaM) in endothelial cells (32). In VSMC, reactive oxygen species (ROS) play an important role in Akt activation caused by ANG II (52). However, the relationship between these signaling molecules involved in the activation of Akt by ANG II has not yet been established. We have previously shown that ANG II, by stimulating Ca²⁺-CaM-dependent kinase II (CaMKII), activates cPLA₂ and releases AA; by activating the Ras-ERK1/2 pathway, AA metabolites of lipoxygenase [12(S)- and 15(S)-HETE] and cytochrome P-450 (20-HETE) amplify cPLA₂ activity (28). It is therefore possible that ANG II-induced Akt activation is mediated by CaMKII-stimulated cPLA₂, release of AA, and generation of AA metabolites and ROS. To test this hypothesis, we have investigated the effect of Ca²⁺ depletion and inhibitors of CaM, CaMKII, cPLA₂, and AA metabolism on ANG II-induced Akt activation in rat VSMC. The effect of ANG II and AA on ROS production and the effect of oleic acid that also generates ROS on Akt phosphorylation were determined. The results of our study show that in rat aortic VSMC, ANG II-induced Akt activation is mediated by metabolites of AA-derived via lipoxygenase and cytochrome P-450 consequent to CaMKII-activated cPLA₂ and is independent of ROS production.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
Materials

Chemicals were purchased from the following sources: aprotinin, dithiothreitol, phenylmethylsulfonyl fluoride, sodium orthovanadate, leupeptin, HEPES, and antipain from Sigma-Aldrich (St. Louis, MO); haloenol lactone suicide substrate (HELSS), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), ketoconazole, 5,8,11,14-eicosatetraynoicacid (ETYA), cinnamyl-3,4-dihydroxy--cyanocinnamate (CDC), and indomethacin were from BioMol Research Laboratories (Plymouth Meeting, PA); 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN-93) and 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-92) were from Calbiochem (San Diego, CA); 17-octadecynoic acid (17-ODYA), (all Z)-(20-HETE), 15(S)-HETE, 12(S)-HETE, 5(S)-HETE, 5,6-, 11,12-, and 14,15 EETs, and AA were from Cayman Chemical (Ann Arbor, MI); human ANG II (H-Asp-Arg-Val-tyr-Ile-His-Pro-Phe-OH) was from Bachem Bioscience (King of Prussia, PA); anti-Akt1 (c-20) was from Santa Cruz Biotechnology (Santa Cruz, CA); phospho-Akt (Ser473) antibody was from Cell Signaling Technology (Beverly, MA); anti-rabbit horseradish peroxidase (HRP)-conjugated Ig serum was from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK); and anti-goat HRP conjugated-IgG (H + L) was from Vector Laboratories (Burlingame, CA). Pyrrolidine-1 was kindly provided by Dr. M. H. Gelb (University of Washington, Seattle, WA). Anti-PLD₂ antibody was kindly supplied by Dr. Sylvain Bourgoin (Universite Laval, QC, Canada). N-hydroxy-N'-(4-butyl-2 methylphenyl)formamidine (HET0016) was kindly supplied by Dr. Richard J. Roman (Medical College of Wisconsin, Milwaukee, WI).

Methods

VSMC isolation and culture. Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 250–350 g were anesthetized with pentobarbital sodium (Abbott, North Chicago, IL), and the thorax was opened. The thoracic aorta was excised and rapidly removed. VSMC were isolated and cultured as described (51). The cultured cells were maintained under 5% CO₂ in medium 199 (M199) with penicillin-streptomycin and 10% fetal bovine serum. VSMC between the fourth and tenth passages were made quiescent for 48 h in M199 containing 0.1% fetal bovine serum before exposure to various agents. VSMC were characterized by using smooth muscle-specific anti-actin antibody.

Preparation of thiooligonucleotides and transient transfection of VSMC. Phosphorothioate oligonucleotides against the translation initiation site of rat cPLA₂ were synthesized by Invitrogen (Carlsbad, CA). The sequences of oligonucleotides used in this study were the following: cPLA₂ sense, 5'-AZF GAT CCT TAT CAG CAC FZA-3'; and cPLA₂ antisense, 5'-TFZ GTG CTG ATA AGG ATC ZFT-3' (where F = A-phosphorothioate and Z = T-phosphorothioate). VSMC, about 80% confluent without serum and antibiotics, were transiently transfected by incubating for 6 h in M199 with either sense or antisense oligonucleotide complexed with oligofectamine reagent according to the manufacturer's protocol (Invitrogen). The cells were then made quiescent in the presence of oligonucleotides for 48 h before exposure to ANG II (200 nM) or its vehicle for 5 min.

Preparation of small interfering RNA and transient infection of VSMC with retroviral genesuppressor system. cPLA₂ small interfering RNA (siRNA) forward (5'-TCGAG ACAGT AGTGG TTCTA CGTGCC gagtactg GGCAC GTAGA ACCAC TACTG TTTTTT-3') and reverse (5'-CTAGA AAAAA CAGTA GTGGT TCTAC GTGCC cagtactc GGCAC GTAGA ACCAC TACTGTC-3') primers were synthesized by Integrated DNA Technologies (Coralville, IA). Forward and reverse primer were mixed and annealed at 95°C for 10 min and then gradually cooled to room temperature. The annealed oligonucleotide was inserted into linearized pSuppressorRetro viral vector using ready-to-go T4 DNA ligase kit (Amersham, Buckinghamshire, UK). The ligated plasmid DNA was transformed into competent DH5 cells (GIBCO-BRL, Carlsbad, CA). After transformation, the colonies were picked up, amplified, purified using miniprep purification kit (Qiagen, The Netherlands), and sequenced using the primer against the pSupressorRetro viral vector. The sequenced plasmid was then purified with Qiagen Maxi plasmid DNA kit and transfected into HEK-293 cells using the following method.

HEK-293 cells were grown to 30% confluence in Dulbecco's modified Eagle's medium (DMEM) supplement with 10% fetal bovine serum and penicillin-streptomycin. These cells were transfected with the plasmid DNAs (pECO packaging vector and pSuppressorRetro Vector containing the cPLA₂ siRNA insert) using the calcium phosphate precipitation method. The transfected cells were incubated for 3–4 h. Fresh medium was then added and replaced on the second day. The virus was harvested by filtering the virus-containing supernatant. VSMC were made quiescent and infected with the viral supernatant in M199 containing 8 µg/ml polybrene and 0.1% fetal bovine serum for 48 h before stimulation. The LacZ retrovirus was used to check infection efficiency and to exclude any possible nonspecific effect of the virus on ANG II-induced Akt phosphorylation in VSMC. The infection efficiency in VSMC was confirmed by -galactosidase staining (Invitrogen, Carlsbad, CA).

Western blot analysis. VSMC were dispersed into lysis buffer [1% IGEPAL CA-630, 25 mM HEPES (pH 7.5), 50 mM NaCl, 50 mM NaF, 10 nM okadaic acid, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml antipain, 10 µg/ml aprotinin, and 10 µg/ml leupeptin]. The lysates were sonicated and centrifuged, and protein concentration was determined. Equal amounts of protein (2080 µg) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. The blots were blocked and incubated with primary antibody (1:1,000) overnight at 4°C. The blots were then exposed to their respective secondary antibodies conjugated with horseradish peroxidase and developed by using the ECL Plus Western Blot Detection kit (Amersham). The density of bands was measured using the NIH Image 1.6 program.

cPLA₂ assay. Quiescent VSMC were stimulated with ANG II or its vehicle after pretreatment with inhibitors, and cPLA₂ activity was determined as described (29). The cells were lysed and sonicated in lysis buffer (350 mM sucrose, 1 mM EGTA, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 20 µg/ml soybean trypsin inhibitor). The protein concentration was determined by the Bradford method (Bio-Rad). L--1-Palmitoyl-2-arachidonyl-[¹⁴C]phosphatidylcholine (American Radiolabeled Chemicals) was concentrated under N₂ and cosonicated in substrate buffer [9 µM dioleoglycerol, 25 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM CaCl₂, 1 mM dithiothreitol, and 1 mg/ml bovine serum albumin]. The lysate (containing 30 µg protein) was incubated in substrate buffer (25 µl) at 37°C for 1 h. The reaction was stopped by the addition of Dole's reagent (2-propanol-heptane-0.5 M H₂SO₄, 20:5:1 vol/vol/vol), heptane, and 20 µg/ml AA. The upper heptane phase was drawn out and passed through a Sep-Pak silica column (Waters Chromatography, Milford, MA). The elutate was collected in glass vials and air dried. The radioactivity of free AA was determined by liquid scintillation spectrometry.

ROS determination. ROS production was measured according to the method described (24). Briefly, quiescent VSMC were incubated with the H₂O₂-sensitive fluorescent probe 2',7'-dichloroflurescein diacetate (DCF-DA) (10 µM; Molecular Probes, Eugene, OR) for 10 min and treated with ANG II (200 nM), oleic acid (100 µM), AA (20 µM), or their vehicle for 5 or 10 min. Nonfluorescent DCF-DA is deacetylated by cellular esterases to the polar compound 2',7'-dichloroflurescein, which remains trapped in the cell and oxidized by oxygen radicals (H₂O₂, lipid hydroperoxides, HOO·, and ONOO^–) into the highly fluorescent 2',7'-dichlorodihydrofluorescein. Relative fluorescence intensity and fluorescent images were obtained with an Eclipse TE200 inverted microscope (Nikon, Melville, NY) using a SPOT RT slider digital camera (Diagnostic Instruments, Sterling Heights, MI) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. To examine the inhibitory effect of catalase or ETYA on the production of H₂O₂, quiescent VSMC were pretreated with catalase (2,000 U/ml) for 48 h or ETYA (10 µM) for 30 min before incubation with DCF-DA and stimulated with the various agonists as described above. Treatment of VSMC with catalase for this period has been reported to increase cellular expression of catalase and reduce H₂O₂ level in rat VSMC (46).

Cytosolic Ca²⁺ measurement. Cytosolic Ca²⁺ concentration was measured as described (11, 18). Briefly, coverslips were washed with nitric acid and water and sterilized under UV light. Trypsinized VSMC were cultured to confluence on sterilized coverslips and then arrested overnight in serum-free and antibiotic-free M-199. Quiescent VSMC were loaded for 30 min with 5 µM fura 2-AM mixed with 0.05% bovine serum albumin and 0.025% pluronic acid in Krebs-Ringer (KB) solution (in mM: 120 NaCl, 5 KCl, 0.62 MgSO₄, 10 HEPES, 6 glucose, and 1.8 CaCl₂; adjusted to pH 7.4). Coverslips were placed in cuvettes and superfused with KB solution with or without Ca²⁺ (0.89 mM MgCl₂ and 1 mM EGTA replaced CaCl₂). After a stable basal level of cytosolic Ca²⁺ was achieved, the cells were exposed to ANG II (200 nM) in KB solution with or without Ca²⁺. Cytosolic levels of Ca²⁺ were measured using luminescence spectrometer (Perkin-Elmer LS 50B, Norwalk, CT) with an excitation wavelength alternately at 340 and 380 nm and emission wavelength 510 nm. Calibration of the Ca²⁺-dependent fluorescence (F) for each measurement was obtained by the addition of 5 µM ionomycin [maximal F (F_max)] in the presence of 4 mM Ca²⁺, followed by the addition of 5 µM ionomycin in the presence of 4 mM EGTA in Ca²⁺-free KB solution [minimal F (F_min)]. Intracellular Ca²⁺ concentration ([Ca²⁺l_i) was calculated using a previously described formula (11, 18): [Ca²⁺]_i=K_d (F–F_min)/(F_max–F), with the dissociation constant (K_d) = 224 nM, F_max = 10.7125 (n = 4), and F_min = 1.175 (n = 4).

Analysis of Data

Data were analyzed by ANOVA; the unpaired Student's t-test was used to determine the difference between two groups. A value of P < 0.05 was considered to be statistically significant.

	RESULTS

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ANG II-Induced Akt Activation Is Dependent on Extracellular Ca²⁺

In rat aortic VSMC, ANG II stimulated phosphorylation of Akt, as measured by phosphorylation of serine-473, in a concentration-dependent manner (Fig. 1A). The peak of Akt phosphorylation was observed at 200 nM and 5 min (Fig. 1, A and B). Therefore, in all of our experiments, VSMC were exposed to 200 nM ANG II for 5 min. ANG II-induced Akt phosphorylation was abolished by pretreating the cells with the extracellular Ca²⁺ chelator EGTA (10 mM) for 30 min (Fig. 2A). Because high concentrations of EGTA may affect [Ca²⁺]_i, the effect of lower concentrations of EGTA (1 mM) in the presence and absence of BAPTA-AM (intracellular Ca²⁺ chelator) and the effect of depletion of extracellular Ca²⁺ on Akt phosphorylation were also examined. Quiescent VSMC were depleted of extracellular Ca²⁺ by replacing M199 (1.8 mM) with Ca²⁺-free Hanks’ buffered saline solution (HBSS). The cells were then stimulated with 200 nM ANG II for 5 min in Ca²⁺-free HBSS or M199 (1.8 mM Ca²⁺). Depletion of the extracellular Ca²⁺ (5 min) blocked the Akt phosphorylation induced by ANG II (Fig. 2B). However, the Akt phosphorylation was restored by the addition of Ca²⁺ (1.8 mM CaCl₂) (Fig. 2C). The addition of Ca²⁺ to the Ca²⁺-free HBSS increased both basal and ANG II-induced Akt phosphorylation (Fig. 2C). BAPTA-AM (20 µM) or EGTA (1 mM) in the presence of Ca²⁺ (M199) produced a smaller but significant decrease in ANG II-induced Akt phosphorylation (Fig. 2B). The effect of EGTA (1 mM) was not further reduced by BAPTA-AM (Fig. 2B). BAPTA-AM (20 µM) did not alter Akt phosphorylation elicited by ANG II in the presence of 1.8 mM CaCl₂ (Fig. 2C). It is well known that ANG II induces a biphasic [Ca²⁺]_i response (a rapid and a sustained phase). The initial [Ca²⁺]_i transient is mediated primarily by Ca²⁺ mobilization from intracellular stores; the sustained phase is caused by extracellular Ca²⁺ influx (9, 50). Although ANG II failed to cause phosphorylation of Akt in the absence of extracellular Ca²⁺, it increased cytosolic [Ca²⁺]_i by releasing Ca²⁺ from intracellular stores. The sustained phase of cytosolic [Ca²⁺]_i increase caused by ANG II was abolished in the absence of extracellular Ca²⁺ (Fig. 2D).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Angiotensin II (ANG II) stimulates Akt activation in rat aortic vascular smooth muscle cells (VSMC). A: quiescent VSMC were stimulated with 200 nM ANG II for indicated time. Bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 5). B: quiescent VSMC were stimulated with the indicated concentration of ANG II for 5 min. Bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 4). Equal amounts of protein from each cell lysate sample were resolved by SDS-PAGE and transferred to nitrocellulose membranes, and total Akt and phospho-Akt were measured by Western blot analysis as described in Methods. Intensity of phospho(P)-Akt bands are normalized to the abundance of Akt and shown relative to the values obtained with vehicle alone. Values are means ± SE. *Value significantly different from the corresponding value obtained in the absence of ANG II (vehicle alone) (P < 0.05).

View larger version (34K): [in this window] [in a new window]   Fig. 2. ANG II-induced Akt phosphorylation is dependent on extracellular Ca2+. A: quiescent VSMC were treated with EGTA (10 mM) for 30 min before addition of ANG II (200 nM) as indicated. Bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 4). B: quiescent VSMC were depleted of Ca2+ by replacing medium 199 (M199) with Ca2+-free Hanks' buffered saline solution (HBSS), or pretreated with EGTA (1 mM) for 30 min or BAPTA-AM (20 µM) alone, or combined with Ca2+ depletion-EGTA (1 mM) for 30 min before stimulation with ANG II (200 nM) in the presence of Ca2+ or in the absence of Ca2+ as indicated. Bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 4). C: quiescent VSMC were stimulated with ANG II (200 nM) in Ca2+-free HBSS with or without exogenous Ca2+ (1.8 mM) as indicated, or VSMC were stimulated with ANG II (200 nM) in Ca2+-free HBSS containing BAPTA-AM (20 µM) with or without exogenous Ca2+ (1.8 mM). The blots are representative of three different experiments. D: cytosolic levels of Ca2+ [intracellular Ca2+ concentration ([Ca2+]i)] in VSMC stimulated with ANG II (200 nM) in the presence or absence of Ca2+. The results shown in traces are means ± SE (n = 4 for each group). Phospho-Akt and Akt were detected, and the intensity of phospho-Akt bands was calculated as described in Fig 1. Values are means ± SE. *Value significantly different from the corresponding value obtained with vehicle alone or inhibitors alone (P < 0.05). Value significantly different from the corresponding value obtained with ANG II in the absence of inhibitors (P < 0.05).

Increased [Ca²⁺]_i is known to activate CaM, a small and highly conserved Ca²⁺-binding protein, which in turn activates CaMKII (28–30, 55). To assess the contribution of CaM and CaMKII to ANG II-induced Akt phosphorylation, we examined the effect of their respective inhibitors. The CaM inhibitor W-7 at 30 µM (1) reduced Akt phosphorylation, whereas its inactive analog W-5 at the same concentration had no effect (Fig. 3A). The isoquinoline sulfonamide derivative KN-93 (30 µM), an inhibitor of CaMKII (1), but not its inactive structural analog KN-92, also inhibited Akt phosphorylation elicited by ANG II (Fig. 3B).

View larger version (18K): [in this window] [in a new window]   Fig. 3. ANG II-induced Akt phosphorylation in rat aortic VSMC is regulated by Ca2+-calmodulin (CaM) and CaM kinase II (CaMKII). A: ANG II-induced Akt phosphorylation in rat aortic VSMC is inhibited by CaM inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) but not by its inactive analog N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5). Quiescent VSMC were pretreated with W-7 or W-5 (30 µM) for 30 min before stimulation with ANG II (200 nM) for 5 min as indicated. Top, Western blot analysis; bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 6). B: ANG II-induced Akt phosphorylation in rat aortic VSMC is inhibited by CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN-93) but not by its inactive analog 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-92). Quiescent VSMC were pretreated with KN-92 or KN-93 (30 µM) for 30 min before stimulation with ANG II (200 nM) for 5 min as indicated. Top, Western blot analysis; bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 5). Phospho-Akt and Akt were detected, and the intensity of phospho-Akt bands was calculated as described in Fig 1. Values are means ± SE. *Value significantly different from the corresponding value obtained with vehicle alone or inhibitors alone (P < 0.05). Value significantly different from the corresponding value obtained with ANG II in the absence of inhibitors (P < 0.05).

Activation of CaMKII as a result of increased influx of extracellular Ca²⁺ has been shown to stimulate cPLA₂ activity in rabbit VSMC (28–30). Therefore, we examined the possible contribution of cPLA₂ in ANG II-induced Akt phosphorylation. cPLA₂ inhibitor pyrrolidine-1 (100 nM) (14, 40) and Ca²⁺-independent PLA₂ inhibitor HELSS (10 µM) (4) attenuated ANG II-induced Akt phosphorylation (Fig. 4A). Because ANG II-induced Akt phosphorylation was dependent on extracellular Ca²⁺, and pyrrolidine-1 inhibited the activity of cPLA₂ as measured from the hydrolysis of its substrate arachidonyl phosphatidylcholine (Fig. 4D), the effect of ANG II on Akt phosphorylation in rat VSMC is most likely mediated by Ca²⁺ and cPLA₂. Pyrrolidine-1, at the concentration that inhibited ANG II-induced Akt phosphoryltion, did not alter exogenous AA-induced Akt phosphorylation (http://ajpheart.physiology.org/cgi/content/full/0571.2004/DC1), which is consistent with its cPLA₂ inhibitory effect in reducing ANG II-stimulated AA release. To further assess the role of cPLA₂ in ANG II-induced Akt activation, we examined the effect of cPLA₂ sense and antisense oligoneucleotides on Akt phosphorylation in VSMC. In VSMC transfected with cPLA₂ antisense but not sense oligonucleotide, ANG II-induced Akt phosphorylation was inhibited (Fig. 4B). In three separate experiments, the mean protein levels of cPLA₂, as determined by Western blot analysis, for vehicle, ANG II alone, cPLA₂ sense alone, cPLA₂ sense + ANG II, cPLA₂ antisense alone, and cPLA₂ antisense + ANG II were 1.00, 1.07, 1.02, 1.07, 0.71, and 0.57, respectively (Fig. 4C). We also designed retroviral cPLA₂ siRNA. As cPLA₂ antisense, cPLA₂ siRNA, but not LacZ virus alone, markedly reduced cPLA₂ protein levels and ANG II-induced phosphorylation of Akt (Fig. 4C). Furthermore, cPLA₂ siRNA inhibited PLA₂ activity in VSMC (Fig. 4E). cPLA₂ siRNA did not alter Akt protein levels and produced only a slight decrease in PLD₂ protein levels, indicating the specificity of its effect (Fig. 4C). These data indicate that ANG II-induced Akt phosphorylation is mediated by cPLA₂, which is activated by CaMKII (28–30).

View larger version (40K): [in this window] [in a new window]   Fig. 4. ANG II-induced Akt phosphorylation is mediated through cytosolic Ca2+-dependent phospholipase A2 (cPLA2) activation. A: quiescent VSMC were pretreated with cPLA2 inhibitor pyrrolidine-1 (100 nM) and Ca2+-independent PLA2 inhibitor haloenol lactone suicide substrate (HELSS, 10 µM) for 30 min before addition of ANG II (200 nM) for 10 min as indicated. Bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 4). B: VSMC were transfected with cPLA2 sense or antisense oligonucleotides complexed with oligofectamine for 48 h in the absence of serum and then stimulated with ANG II (200 nM) for 5 min. Bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 5). C: quiescent VSMC were infected with retrovirus containing full-length LacZ or cPLA2 small interfering RNA (siRNA) insert for 48 h and then stimulated with ANG II (200 nM) for 5 min. Equal amounts of protein from each cell lysate sample were used for the detection of phospho-Akt, Akt, phospholipase D2 (PLD2), and cPLA2 as described in Fig. 1. Bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 6). Right, infection efficiency of VSMC with retrovirus containing LacZ by using -galactosidase staining. D: cPLA2 activation induced by ANG II in rat aortic VSMC was inhibited by pretreatment of quiescent VSMC with pyrrolidine-1 (100 nM). Equal amounts of protein from each cell lysate sample were used for measuring cPLA2 activity as described in Methods (n = 4). E: cPLA2 activity induced by ANG II in rat aortic VSMC was inhibited by infecting quiescent cells with retrovirus containing cPLA2 siRNA insert. Quiescent VSMC were infected with retrovirus containing full-length LacZ or cPLA2 siRNA insert for 48 h and then stimulated with ANG II (200 nM) for 5 min (n = 5). For determination of cPLA2 activity, equal amounts of protein from each sample were used. The cPLA2 activity was determined from the radioactivity of [14C] arachidonic acid (AA) released from [14C]phosphatidylcholine. Activity of cPLA2 is shown relative to that obtained with vehicle in the absence of ANG II. Phospho-Akt and Akt were detected, and the intensity of phospho-Akt bands was calculated as described in Fig. 1. Values are means ± SE. *Value significantly different from the corresponding value obtained with vehicle alone or inhibitors alone (P < 0.05). Value significantly different from the corresponding value obtained with ANG II in the absence of inhibitors (P < 0.05).

Activation of cPLA₂ promotes the release of AA, which is metabolized via cyclooxygenase (lipoxygenase) and cytochrome P-450 into various biologically active products (15). Exogenous AA has been reported to increase Akt phosphorylation in mesangial cells and VSMC (16, 31). To assess the contribution of endogenous AA released by activation of cPLA₂ by ANG II, we examined the effect of the inhibitor of AA metabolism ETYA on the action of ANG II and exogenous AA on Akt phosphorylation. Exogenous AA (20 µM) caused phosphorylation of Akt in VSMC (Fig. 5A). ETYA (10 µM) blocked this effect of exogenous AA as well as ANG II-induced Akt phosphorylation (Fig. 5B). It has been reported that ANG II-induced Akt activation in mesangial cells is mediated by ROS production consequent to Rac-1-regulated NAD(P)H oxidase (Nox4) stimulated by AA released via cPLA₂ activation (17). Moreover, inhibition of H₂O₂ production reduces ANG II-induced Akt phosphorylation (17, 52). ROS can stimulate or inhibit AA metabolism under different conditions (2, 45). To assess the relative contribution of ROS and of AA metabolites to Akt activation, we measured the production of H₂O₂ in response to ANG II and to exogenous AA in rat VSMC treated with the inhibitor of AA metabolism ETYA and the ROS production inhibitor catalase. In VSMC exposed to ANG II (200 nM) for 5 min, there was a significant increase in H₂O₂ production. Exposure of the cells to exogenous AA (20 µM) for 10 min also markedly increased the production of H₂O₂. However, treatment of cells with oleic acid (100 µM) for 10 min, which also increased catalase-reducible H₂O₂ production (Fig. 6A), failed to increase the phosphorylation of Akt (Fig. 6B). ETYA, which inhibited ANG II- and AA-induced Akt phosphorylation, did not reduce but rather increased ROS production that is reducible by catalase, although it has been reported that ETYA inhibits oxidase activity (58). Moreover, ETYA produced an additive effect on stimuli-induced ROS production (Fig. 6A). ETYA by itself did not exhibit any autofluorescence (data not shown). The effect of ANG II to increase ROS production was not altered in VSMC infected with cPLA₂ siRNA (http://ajpheart.physiology.org/cgi/content/full/0571.2004/DC1), suggesting that endogenous AA does not contribute to ANG II-induced ROS production. Pretreatment of cells with exogenous catalase for 48 h, which is known to increase expression of catalase (46), inhibited both ANG II- and AA-induced ROS production (Fig. 6A). Akt phosphorylation elicited by AA, but not that produced by ANG II, was reduced by catalase (Fig. 6C). These observations suggest that H₂O₂ does not mediate ANG II-induced phosphorylation of Akt in rat VSMC. However, in VSMC cultured in DMEM medium containing high glucose, both the basal and ANG II-induced ROS production was increased (Fig. 6E). Moreover, in this high-glucose medium, both ANG II and AA-induced Akt phosphorylation were inhibited by catalase (Fig. 6D).

View larger version (24K): [in this window] [in a new window]   Fig. 5. AA metabolites mediate Akt phosphorylation induced by ANG II. A: exogenous AA stimulates Akt phosphorylation in rat VSMC. Quiescent VSMC were stimulated with AA (20 µM) for indicated time. Top, Western blot analysis; bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 4). *Value significantly different from that obtained in the absence of AA (vehicle alone). B: Akt phosphorylation induced by ANG II and AA is inhibited by inhibitor of AA metabolism 5,8,11,14-eicosatetraynoicacid (ETYA). Quiescent VSMC were pretreated with cPLA2 metabolism inhibitor ETYA (10 µM) for 30 min before stimulation with ANG II (200 nM) for 5 min or AA (20 µM) for 10 min as indicated. Top, Western blot analysis; bottom, densitometric analysis of ANG II or AA-induced Akt phosphorylation (n = 4). Phospho-Akt and Akt were detected, and the intensity of phospho-Akt bands was calculated as described in Fig. 1. Values are means ± SE. *Value significantly different from the corresponding value obtained with vehicle alone or ETYA alone. Value significantly different from the corresponding value obtained with ANG II or AA in the absence of ETYA (P < 0.05).

View larger version (48K): [in this window] [in a new window]   Fig. 6. Akt phosphorylation stimulated by ANG II is independent of H2O2 production in rat aortic VSMC. A: ANG II, AA, oleic acid, and ETYA increased reactiv oxygen species (ROS) production, which was reducible by catalase. Quiescent VSMC were pretreated with ETYA (10 µM) for 30 min or catalase (2,000 IU/ml) for 48 h and then incubated with the H2O2-sensitive fluorescent probe 2',7'-dichloroflurescein diacetate (DCF-DA, 10 µM) for 10 min. ANG II (200 nM), AA (20 µM), oleic acid (100 µM), or their vehicles were added to the cells for 5 or 10 min. Top, intensity of the fluorescent images (n = 5). Intensity of fluorescent is shown relative to control (vehicle alone). Bottom, representative fluorescent images of cells treated with different agents. B: oleic acid did not increase Akt phosphorylation. Quiescent VSMC were treated with oleic acid (100 µM) for 5 and 10 min or AA (20 µM) for 10 min. Blots are representative of three different experiments. C: catalase inhibits AA-, but not ANG II-induced, Akt phosphorylation in normal glucose medium (5.5 mM). Quiescent cells were pretreated with catalase (2,000 IU/ml) for 48 h before stimulation with ANG II (200 nM) for 5 min or AA (20 µM) for 10 min in M199. Bottom, densitometric analysis of ANG II-or AA-induced Akt phosphorylation (n = 4). D: catalase inhibited both ANG II and AA-induced Akt phosphorylation in DMEM medium with high glucose (27.5 mM). Quiescent cells were pretreated with catalase (2,000 IU/ml) for 48 h before stimulation with ANG II (200 nM) for 5 min or AA (20 µM) for 10 min in DMEM with high glucose. E: high glucose increases basal ROS production as well as ANG II-induced ROS production. VSMC were cultured with M199 and DMEM (high glucose) containing 10% fetal bovine serum and penicillin-streptomycin for 2 passages and then arrested with M199 and DMEM (high glucose) containing 0.1% fetal bovine serum. Quiescent VSMC were then incubated with the H2O2-sensitive fluorescent probe DCF-DA (10 µM) for 10 min before stimulation with ANG II (200 nM) in consequent phenol-free M199 and DMEM (high glucose). Fluorescent images were acquired and quantified. Phospho-Akt and Akt were detected, and the intensity of phospho-Akt bands was calculated as described in Fig. 1. Values are means ± SE. *Value significantly different from the corresponding value obtained with vehicle alone or inhibitors alone (P < 0.05). Value significantly different from the corresponding value obtained with ANG II, AA, or oleic acid in the absence of inhibitors (P < 0.05).

ETYA inhibited both ANG II- and AA-stimulated Akt phosphorylation (Fig. 5B). This suggests that AA metabolites released by CaMKII-activated cPLA₂ promote Akt phosphorylation. To determine whether AA metabolites generated via cyclooxygenase, lipoxygenase, and/or cytochrome P-450 contribute to the phosphorylation of Akt elicited by ANG II, we examined the effect of inhibitors of these enzymes on ANG II-induced Akt phosphorylation. The inhibitors of lipoxygenase CDC (10 µM) (54), but not cyclooxygenase (indomethacin) (35), decreased Akt phosphorylation caused by ANG II and AA (Fig. 7, A and B). Cytochrome P-450 inhibitors 17-ODYA (27, 37, 60) and ketoconazole (6, 37), which inhibit both cytochrome P-450 -hydroxylase and epoxygenase (6, 27, 37, 60), also diminished ANG II- and AA-induced Akt phosphorylation (Fig. 7, A–C). However, HET0016, a selective inhibitor of -hydroxylase (22, 26), did not alter ANG II- and AA-induced Akt phosphorylation in VSMC (Fig. 7C). The metabolism of AA via lipoxygenase results in generation of 5(S)-, 12(S)-, and 15(S)-HETE and via cytochrome P-450 -hydroxylase 20-HETE and epoxygenase EETs (37). All of these metabolites (500 nM) enhanced the phosphorylation of Akt (Fig. 7, D and E). These data indicate that one or more AA metabolites, generated via lipoxygenase (HETEs) and cytochrome P-450 (EETs), mediate Akt phosphorylation elicited by ANG II in rat VSMC.

View larger version (47K): [in this window] [in a new window]   Fig. 7. ANG II-induced Akt phosphorylation is mediated by one or more AA metabolites. A: ketoconazole and cinnamyl-3,4-dihydroxy-cyanocnnamate (CDC), but not indomethacin, decreased ANG II-induced Akt phosphorylation. Quiescent VSMC were pretreated with inhibitors of lipoxygenase (CDC, 10 µM), cytochrome P-450 (ketoconazole, 20 µM), or cyclooxygenase (indomethacin, 50 µM) for 30 min before addition of ANG II (200 nM) as indicated. Bottom, densitometric analysis of ANG II-induced Akt phosphorylation (n = 4). B: ketoconazole and CDC, but not indomethacin, decreased AA-induced Akt phosphorylation. Quiescent VSMC were pretreated with CDC (10 µM), ketoconazole (20 µM), or indomethacin (50 µM) for 30 min before addition of AA (20 µM) as indicated. Blots are representative of three different experiments. C: 17-octadecynoic acid (17-ODYA), but not HET0016, attenuated both ANG II- and AA-stimulated Akt phosphorylation. Quiescent VSMC were pretreated with 17-ODYA (10 µM) and HET0016 for 60 min before addition of ANG II (200 nM) or AA (20 µM) as indicated. Blots are representative of three different experiments. D: exogenous 5(S)-, 12(S)-, 15(S)-, and 20-HETE promote Akt phosphorylation in rat aortic VSMC. Quiescent VSMC were stimulated with 500 nM 5(S)-, 15(S)-, 12(S), and 20-HETE for 5 min. Bottom, densitometric analysis of HETEs-induced Akt phosphorylation (n = 5). E: exogenous 5,6-, 11,12-, and 14,15-EET stimulate Akt phosphorylation in rat aortic VSMC. Quiescent VSMC were stimulated with 500 nM 5,6-, 11,12-, and 14,15-EET for different time periods. Blots represent three different experiments. Phospho-Akt and Akt were detected, and the intensity of phospho-Akt bands was calculated as described in Fig. 1. Values are means ± SE. *Value significantly different from the corresponding value obtained with vehicle alone or inhibitors alone (P < 0.05). Value significantly different from the corresponding value obtained with ANG II in the absence of inhibitors (P < 0.05).

	DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

Our demonstration that inhibitors of CaM, W-7, CaMKII, and KN-93, but not their inactive structural analogs, W-5 and KN-92, attenuated ANG II-induced Akt phosphorylation suggests that increased cytosolic [Ca²⁺]_i promotes Akt phosphorylation by activating CaMKII in rat VSMC. CaMK kinase, which activates CaMK I and IV (39), has also been implicated in Akt activation in neuroblastoma cells (56). Moreover, mepacrine and aristolochic acid, nonselective inhibitors of PLA₂, block Akt activation elicited by ANG II (16) and exogenous AA mimics this effect of ANG II in mesengial cells (16). In our study, selective cPLA₂ inhibitor pyrrolidine-1 and Ca²⁺-independent PLA₂ inhibitor HELSS decreased the phosphorylation of Akt in rat VSMC. Because EGTA (10 mM), an extracellular Ca²⁺ chelator, or depletion of extracellular Ca²⁺ abolished ANG II-induced phosphorylation of Akt, it appears that Ca²⁺-dependent cPLA₂ is involved in Akt phosphorylation elicited by ANG II. Therefore, HELSS appears to inhibit ANG II-induced Akt phosphorylation by a nonselective effect, because in renal proximal tubule cells, it also inhibits dopamine-stimulated PI3K activity (8), which regulates intracellular Ca²⁺ mobilization through extracellular calcium-sensing receptor (53). Because Ca²⁺-independent PLA₂ (iPLA₂) has been implicated in store-operated Ca²⁺ entry (44), it is possible that HELSS by inhibiting iPLA₂ reduces Ca²⁺ entry and subsequently reduces Akt phosphorylation. The involvement of cPLA₂ in ANG II-induced Akt phosphorylation was further supported by our demonstration that cPLA₂ antisense, but not sense oligonucleotide, and cPLA₂ siRNA inhibited Akt phosphorylation elicited by ANG II. These observations strongly suggest that ANG II-induced Akt activation is mediated via cPLA₂, which is stimulated by CaMKII (28–30).

Activation of cPLA₂ in response to ANG II releases AA (35); ANG II and exogenous AA promote Akt activation via generation of H₂O₂, independent of its metabolism in mesangial cells (16). ANG II stimulated the production of ROS, which have been implicated in Akt phosphorylation in rat VSMC (52). However, it is not known whether AA released by ANG II in VSMC phosphorylates Akt directly or through ROS production and/or through the action of its metabolites generated by cyclooxygenase, lipoxygenase, and/or cytochrome P-450. In the present study, the concentration of ANG II (200 nM) that caused the maximal increase in Akt phosphorylation also produced ROS. However, treatment with catalase, which increased intracellular catalase expression (46) and reduced H₂O₂ production, inhibited Akt phosphorylation stimulated by exogenous AA but not that by ANG II. Moreover, our demonstration that the inhibitor of AA metabolism ETYA, which stimulated ROS production by itself and had an additive effect on ANG II-stimulated ROS production, inhibited both the ANG II- and AA-induced Akt phosphorylation strongly suggests that AA generated by activation of cPLA₂ in response to ANG II activates Akt through one or more of its metabolites rather than through ROS production. Furthermore, oleic acid, which increased ROS production, failed to cause Akt phosphorylation. Because exogenous AA produced a marked increase in H₂O₂ production and both ETYA and catalase reduced its effects on Akt phosphorylation, it would appear that Akt activation by exogenous AA is mediated through generation of ROS as well as through one or more of its metabolites. In view of the ability of ANG II to stimulate ROS production, but inability of catalase to inhibit ANG II-induced Akt phosphorylation in rat VSMC cultured in M199 that contain normal glucose (5.5 mM), it is possible that the higher level of ROS produced by exogenous AA potentiates the effect of one or more of its metabolites on Akt activation but is unable to activate Akt alone (Fig. 8). Supporting this view was our finding that the ETYA, which also stimulated and further increased ROS production caused by ANG II, did not activate but rather attenuated ANG II-induced Akt activation by inhibiting the metabolism of endogenous AA metabolism stimulated by ANG II. The lack of contribution of ROS to ANG II-induced Akt phosphorylation in our study seems controversial to previous reports (36, 52). This could be related to the difference in the medium used to culture the rat VSMC. For an example, ANG II causes Akt phosphorylation in DMEM containing high glucose (27.5 mM) but not in DMEM containing lower glucose (5.5 mM) (23), which is equivalent to M199 used in our study. Rat VSMC cultured in high glucose (25 mM) show significant increases in basal and ANG II-induced ROS production and proliferative activities compared with VSMC cultured in normal glucose (5.5 mM) (41). Moreover, high glucose increases Akt activity as well as ROS production in mesangial cells (42). Therefore, it is possible that ROS-mediated Akt phosphorylation by ANG II is dependent on the experimental conditions, especially the glucose level. Supporting this view, we found that in VSMC cultured in DMEM with high glucose concentration (27.5 mM), there was an increase in the production of ROS, and under this condition catalase inhibited ANG II-induced Akt phosphorylation. Therefore, it appears that high levels of ROS act as a permissive factor in potentiating Akt activation caused by endogenous AA released by ANG II (Fig. 8).

View larger version (14K): [in this window] [in a new window]   Fig. 8. Proposed mechanism of ANG II on Akt phosphorylation in VSMC cultured in M199 containing normal glucose (5.5 mM). Release of endogenous AA is required for ANG II-induced Akt phosphorylation, and high concentration of ROS generated such as by high glucose (27.5 mM) can act as a permissive factor to potentiate Akt phosphorylation.

In conclusion, this study demonstrates that in rat VSMC, ANG II stimulates Akt phosphorylation by a mechanism that is dependent on extracellular Ca²⁺. ANG II-induced increase in cytosolic [Ca²⁺]_i by stimulating CaMKII activates cPLA₂ and releases AA; AA metabolites generated via lipoxygenase and cytochrome P-450, possibly HETEs and EETs, respectively, by activating p38MAPK, phosphorylates Akt by a mechanism independent of H₂O₂ in rat VSMC.

	GRANTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-19134-29 and American Heart Association Predoctoral Fellowship (to F. Li).

	ACKNOWLEDGMENTS

 
We thank Anne Estes for excellent technical assistance and Dr. Lauren Cagen for editorial comments. We gratefully acknowledge Dr. M. H. Gelb for generously supplying the pyrrolidine-1, Dr. Sylvain Bourgoin for providing anti-PLD₂ antisera, and Dr. Richard J. Roman for HET0016.

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. U. Malik, Dept. of Pharmacology, College of Medicine, Univ. of Tennessee Health Science Center, Rm. 115, Crowe Bldg., 874 Union Ave., Memphis, TN 38163 (E-mail: kmalik{at}utmem.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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