Introduction

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Angiotensin (1-7) is a bioactive component of the renin angiotensin system that may play an important role in the regulation of blood pressure (Benter et al., 1993). Ang-(1-7) is generated endogenously from both Ang I and Ang II (Chappell et al., 1989, 1990) and stimulates PG production in neural, endothelial and VSMC (Jaiswal et al., 1991, 1992, 1993). Studies in human subjects and animal models of hypertension have shown that inhibition of angiotensin-converting enzyme results in significant increases in circulating levels of Ang-(1-7) (Ferrario et al., 1991; Kohara et al., 1993). Dellipizzi et al. (1994) showed that Ang-(1-7) at low doses is more potent than Ang II in causing diuresis and natriuresis in isolated perfused kidney. Similarly, Ang-(1-7) causes vasodilation by stimulating production of PGs and nitric oxide when injected directly into feline isolated mesenteric or hindquarter vascular beds (Osei et al., 1993). Long-term intravenous infusion of Ang-(1-7) in spontaneously hypertensive rats produces a decrease in arterial pressure accompanied by a significant diuresis and natriuresis and an increase in urinary prostaglandins (Benter et al., 1993, 1995). However, the mechanism by which Ang-(1-7) stimulates AA release and PG synthesis has not yet been elucidated. Ang-(1-7) stimulates release of PG in porcine endothelial and smooth muscle cells and human astrocytes via AT₂ receptors (Jaiswal et al., 1991, 1992, 1993). The release of AA for PG production in response to various stimuli has been reported to be the result of activation of cytosolic (c) and secretory (s) PLA₂. Although Ang-(1-7) stimulates PLA₂ activity in renal proximal tubular cells (Andreatta-Van Leyen et al., 1993), it is not known whether it stimulates AA release for PG synthesis by activating cPLA₂. Previous studies have shown that MAP kinase can activate cPLA₂ by phosphorylation (Lin et al., 1993). In view of the demonstration that Ang II activates MAP kinase (Duff et al., 1992; Tsuda et al., 1992; Lucchesi et al., 1996; Liao et al., 1996) and cPLA₂ phosphorylation (Rao et al., 1994), it is possible that Ang II and Ang-(1-7) stimulate AA release by activating cPLA₂ via MAP kinase. Because the CaM inhibitors W-7 and calmidazolium have been reported to attenuate the Ang II-induced increase in MAP kinase activity (Eguchi et al., 1996), it is possible that Ang II and Ang-(1-7) stimulate cPLA₂ via MAP kinase by activating CaM kinase II. To test these hypotheses and compare the signaling mechanisms mediating the actions of Ang-(1-7) and Ang II, we have investigated the effects of these peptides on AA release and PGI₂ synthesis in VSMC, which is the major site of action of prostanoids in modulating the effect of Ang peptides in vascular tone (McGiff, 1981; Nasjletti and Malik, 1982; Benter et al., 1993).

	Methods

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Preparation of VSMC. Male New Zealand rabbits (1-2 kg) and male Sprague-Dawley rats (300 g, Charles Rivers, Wilmington, MA) were anesthetized with 30 mg/kg pentobarbital (Abbott Laboratories, North Chicago, IL), and the abdomen was opened with a midline incision. The thoracic aorta was rapidly removed, and VSMC were isolated (Nebigil and Malik, 1990). Cells that were between 4 and 8 passages were plated in 12- or 24-well or 100-mm plates. Cells were maintained under 5% CO₂ in M-199 medium with penicillin, streptomycin and 10% FBS.

Preparation of thiooligonucleotides and transient transfection of VSMC. Phosphorothioate oligonucleotides directed against the translation initiation sites of cPLA₂, CaM kinase II and MAP kinase were synthesized at the Molecular Resource Center, University of Tennessee, Memphis. The sequences of oligonucleotides used in this study were: cPLA₂ antisense, 5-TAC AGT AAA TAT CTA GGA ATG-3; cPLA₂ sense, 5-ATG TCA TTT ATA GAT CCT TAC-3 (Roshak et al., 1994); MAP kinase (ERK1) antisense, 5-AGC CGC CGC CGC CGC CGC CAT-3; MAP kinase (ERK1) sense, 5-ATG GCG GCG GCG GCG GCG GCT-3 (Marquardt and Stabel, 1992); CAM kinase II antisense, 5-GCA GGT GGC GGT GGT CTC CAT-3; and CaM kinase II sense, 5-ATG GAG ACC ACC GCC ACC TGC-3 (Zhou and Ikebe, 1994). VSMC were transfected with either sense or antisense oligonucleotides complexed with 2 µg/ml of lipofectamine (Gibco-BRL, Bethesda, MD) and incubated in serum-free M-199 for 6 hr. Thereafter, fresh M-199 containing 5% FBS and oligonucleotides were added, and the cells were incubated for another 30 hr. In ERK1, VSMC were transiently transfected with two pulses of sense and antisense oligonucleotides at 48-hr intervals. Cells were incubated with [³H]AA (0.3 µCi/ml) during transfection. The cells were washed three times with HBSS and then exposed to Ang peptides for 15 min. In preliminary experiments several concentrations of antisense oligonucleotides (0.5-10 µM) were tested; the concentration that produced maximal effect on its respective effector system without exerting nonselective effect, as determined by changes in its protein levels and that of another effector system by Western blots, was used in our experiments.

[³H]Arachidonic acid release. Release of radiolabeled AA and metabolites (referred to as [³H]AA) from wild-type VSMC and transfectants stimulated by Ang peptides was measured. Cells labeled with [³H]AA for 18 hr were washed with HBSS and then treated with Ang peptides in BSS containing BSA for 15 min at 37°C. The [³H]AA released into the medium and that remaining in the VSMC were measured by liquid scintillation spectroscopy. Total radioactivity in the cells was determined after treating the cells with 1 M NaOH overnight. [³H]AA released into the medium was expressed as percent of the total cellular radioactivity and referred to as fractional release.

Radioimmunoassay of 6-keto-PGF₁. Cultured cells were washed twice with HBSS and incubated with different peptides at 37°C for the times indicated. The content of 6-keto-PGF₁ (the stable hydrolysis product of PGI₂) in the incubation buffer was measured by radioimmunoassay as described previously (Jaiswal and Malik, 1988). Samples (100 µl) were mixed with 3000 to 4000 cpm [³H]-6-keto-PGF₁ (150 Ci/mM) tracer plus an appropriate concentration of antibody (kindly provided by Dr. Leffler, Department of Physiology, University of Tennessee, Memphis) in polystyrene tubes. Tracer and antibody were prepared in buffer containing (g/l): 1.0, NaN₃; 9.0, NaCl; 6.8, KH₂PO₄; 26.1, K₂HPO₄; and 2.0, gelatin. Tubes were then vortexed and incubated overnight at 4°C. Dextran-coated charcoal (1 ml) was added to each tube to separate bound from free tracer, and radioactivity was determined by liquid scintillation spectroscopy. Cross-reactivity of the 6-keto-PGF₁ antibody was <0.1% with thromboxane B₂, 13,14-dihydro-15-keto-PGE₂ and PGI₂ and <0.5% with PGE₂ and PGF₁. None of the drugs used in this study interfered with the radioimmunoassay of 6-keto-PGF₁.

Lipoxygenase assay. Lipoxygenase activity was determined in VSMC cell lysates with the method described by Waslidge and Hayes (1995). Lysates from VSMC were diluted on ice with 50 mM Tris-HCl buffer, pH 7.4, and 40 µg of protein samples were transferred to an ice-cold 96-well plate. The assay was initiated by the addition of 50 µl AA (final concentration, 70 µM) in 50 mM Tris-HCl buffer, pH 7.4, and incubated at 37°C for 10 min. The assay was terminated by the addition of 100 µl FOX reagent: sulfuric acid (25 mM), xylenol orange (100 µM) and methanol/water (9:1). Blanks contained enzyme during the incubation, but substrate was added after the FOX reagent. The yellow color of the acidified xylenol orange was converted to a blue color by the lipid hydroperoxide-mediated oxidation of Fe⁺⁺ ions and interaction of the resulting Fe⁺⁺⁺ ions with the dye (Jiang et al., 1991). Absorbance of the Fe⁺⁺⁺ complex at 650 nm was measured by spectrophotometry on a micro plate reader (Molecular Devices, Sunnyvale, CA).

CaM kinase II assay. CaM kinase II activity was assayed in VSMC cell lysates with CaM kinase II assay kits (Upstate Biotechnology Incorporated, Lake Placid, NY) with a peptide substrate (KKALRRQETVDAL) with relative selectivity for CaM kinase II, according to the manufacturer's recommendations. The reaction mixture contained 10 µl of substrate cocktail (500 µM auto camtide II and 40 µg/l CaM), 10 µl of an inhibitor cocktail (2 µM PKA inhibitor peptide and 2 µM PKC inhibitor peptide [Upstate Biotechnology, Inc.]) and 10 µl of Mg⁺⁺-ATP cocktail (1 µCi [-³²P]ATP); it was incubated at 37°C for 10 min. The phosphorylated substrate was separated from the residual [-³²P]ATP with p81 phosphocellulose paper. The papers were washed twice in 0.75% H₃PO₄ and then in acetone for 2 min, and the bound radioactivity was quantified with a scintillation counter. Blanks to correct for nonspecific binding of [-³²P]ATP and its breakdown products to the phosphocellulose paper and controls for phosphorylation of endogenous proteins in the sample were performed, and CaM kinase II activity was expressed as picomoles per minute per milligram protein. This assay measures the phosphotransferase activity of CaM kinase II in crude cell lysates. This enzyme assay is rapid, convenient and fairly specific for CaM kinase II. This assay is limited, however, because phosphorylation of substrate by certain unknown kinases in the crude lysate cannot be ruled out.

Phospholipase A₂ assay. Cells grown in 100-mm plates were arrested for 24 hr and stimulated with Ang peptides or vehicle and lysed in HEPES buffer containing protease and phosphatase inhibitors (350 mM sucrose, 1 mM EGTA, 100 µg/ml PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 20 µg/ml soybean trypsin inhibitor). The concentration of protein was determined by Bradford assay (Bio-Rad Laboratories, Richmond, CA). PLA₂ activity in lysates of VSMC fractions (20-30 µg protein/assay) was measured with [¹⁴C]arachidonyl phosphatidylcholine as substrate as described previously (Leslie, 1990). Radiolabeled phospholipid stock (11 µl) was dried under N₂, and added to 0.5 ml of reaction mixture (9 µM dioleoylglycerol, 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl₂, 1 mM dithiothreitol and 1 mg/ml BSA) and sonicated for 15 min on ice. The reaction mixture (50 µl) containing 25 µg of protein from cell lysate was incubated at 37°C for 1 hr. The reaction was stopped by adding 2.5 ml of Dole's reagent (2-propanol/heptane/0.5 M H₂SO₄, 20:5:1); 1.5 ml heptane and 1 ml water containing 20 µg of unlabeled AA were added and mixed. The heptane phase containing radiolabeled fatty acid was passed through a silicic acid chromatography column (Sep-pak silica cartridges; Waters Chromatography, Milford, MA). The eluates were collected in a scintillation vial and air dried, and radioactivity was determined by liquid scintillation spectrometry with use of high flash-point LSC cocktail (Packard Instrument Company, Meriden, CT).

MAP kinase assay. The activity of MAP kinase was determined in cell lysates of the VSMC with a BIOTRAK kit (Amersham, Arlington Heights, IL) with a peptide substrate relatively selective for MAP kinase (KRELVEPLTPAGEAPNQALLR) per the manufacturer's instructions. Transfer of [-³²P]ATP to the Thr on the substrate was measured. Cells were homogenized in buffer (10 mM Tris, 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM orthovanadate, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, pH 7.4) and centrifuged at 25,000 × g for 20 min to remove cellular debris. For each assay, 5 µl of Mg[³²P]-ATP buffer (1 µCi ³²P), 15 µl of sample (10 µg protein) and 10 µl of substrate buffer were added and incubated at 30°C for 10 min. The reactions were terminated by adding a stop reagent, and 30 µl of this mixture was spotted onto phosphocellulose discs. The papers were gently washed with 75 mM orthophosphoric acid or 1% acetic acid for 2 min and with distilled water and radioactivity was determined. Enzyme activity was expressed as picomoles per minute per milligram protein.

Western blot analysis. VSMC lysates (30 µg protein) obtained from parental cells and transfectants were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The blots were blocked with 3% BSA in TBS at room temperature for 1 hr and then incubated for 2 hr with primary monoclonal antibodies (1:1000 dilution). The blots were developed with use of biotinylated secondary antibodies and horseradish peroxidase, and signals were detected using ECL Western blotting detection reagents (Amersham, Arlington Heights, IL).

Confocal microscopy. Cells were grown to approximately 70% confluency on chamber slides (Nunc Inc., Naperville, IL) and arrested for 24 hr. Then the cells were washed with 1 ml of BSS containing CaCl₂ and treated with Ang II and Ang-(1-7) (10 nM) for 15 min. Cells were fixed in cold methanol/acetone solution (1:1) for 3 min at room temperature. The cells were then washed in TBS and blocked in TBS containing 3% BSA for 30 min. Monoclonal antibody (cPLA₂ diluted 200-fold with 3% BSA in TBST) was applied to each well. After 1 hr, the cells were washed three times (10 min each) and exposed to TRITC-conjugated goat-antimouse IgG (1:200 dilution). After 45 min incubation in the dark, the cells were washed three times (10 min each) with TBST and rinsed quickly with water. Galvetol (10 µl; Sigma) was applied to the cell surface, and cover slips were mounted. Controls were carried out with nonimmune IgG. Nuclei were visualized with propidium iodide (Sigma). Slides were viewed by confocal fluorescence microscopy (BioRad MRC-1000 laser scanning confocal imaging system using an argon/krypton lamp located in the NeuroScience Center, University of Tennessee, Memphis) with a 100 × objective lens.

Drugs. [³H]6-keto-PGF₁ (150 Ci/mmol) and [³H]AA (100 Ci/mmol) were purchased from Du Pont-New England Nuclear (Boston, MA), and L-[1-¹⁴C]phosphatidylcholine (57 mCi/mmol) and [-³²P]ATP (3000 Ci/mmol) were purchased from ARC Inc., St. Louis, MO). Ang II, Ang-(1-7) and the AT₂ receptor antagonist PD-123319, and the nonselective antagonist [Sar¹,Thr⁸]-Ang II were purchased from Bachem Bioscience Inc. (King of Prussia, PA). The AT₁ receptor antagonist losartan (DUP-753) was obtained from Du Pont (Wilmington, DE). The Ang-(1-7) receptor antagonist D-Ala⁷-Ang-(1-7) (Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-D-Ala⁷) (Santos et al., 1994) was synthesized at the Molecular Resource Center, University of Tennessee, Memphis. HBSS, M-199, BSA, EGTA, PMSF, soybean trypsin inhibitor, xylenol orange, lipoxidase and the cyclooxygenase inhibitor indomethacin (Salari et al., 1984) were purchased from Sigma (St. Louis, MO); the MEK inhibitor PD-98059 (Dudley et al., 1995) from New England Biolabs (Beverly, MA); cPLA₂ inhibitor MAFP (Balsinde and Dennis, 1996) from Cayman Chemicals (Ann Arbor, MI); the CaM kinase II inhibitor KN-93 (Sumi et al., 1991) from Calbiochem (San Diego, CA); and lipoxygenase inhibitor baicalein (Sekiya and Ohuda, 1982) from Biomol (Plymouth Meeting, PA). cPLA₂ monoclonal antibody was kindly provided by Dr. Knopf of Genetics Institute (Cambridge, MA); CaM kinase II and MAP kinase (ERK1) monoclonal antibodies were from Life Technologies Inc. (Gaithersburg, MD); and 1,2-dioleoyl-sn-glycerol was from Avanti Polar Lipids (Alabaster, AL). Stock solutions of PD-98059, KN-93, MAFP, baicalein and indomethacin were prepared in dimethyl sulfoxide. Ang peptides were dissolved in double-distilled water. In preliminary experiments, various concentrations of these inhibitors were tested; the concentration that produced maximal inhibitory effect on its respective effector system without exerting nonselective action was used in this study. The selectivity of inhibitors was tested by examining their effect on the effector system not expected to be inhibited and also by performing in vitro enzyme assays.

Analysis of data. The results are expressed as mean ± S.E. Data were analyzed by analysis of variance; the unpaired Student's t test was applied to determine the difference between two groups and the Neuman-Keuls test was used for multiple comparisons. A value of P  .05 was considered significant.

	Results

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Effects of Ang peptides on [³H]AA release and 6-keto-PGF₁ synthesis in VSMC of rabbit. In VSMC prelabeled with [³H]AA, both Ang-(1-7) and Ang II produced dose-dependent stimulation of [³H]AA release and 6-keto-PGF₁ production (fig. 1, A and B). Ang-(1-7) was more potent than Ang II at 100 nM in releasing [³H]AA from tissue lipids and in stimulating 6-keto-PGF₁ production (fig. 1B). Ang-(1-7) produced a maximal increase in [³H]AA release of 70 ± 10% above basal and in 6-keto-PGF₁ synthesis of 190 ± 10% above basal. Ang II produced a maximal increase in [³H]AA release of 40 ± 6% above basal and in 6-keto-PGF₁ synthesis of only 35 ± 4% above basal. The effect of Ang II and Ang-(1-7) at higher concentrations to increase 6-keto-PGF₁ was diminished. Even though Ang II and Ang-(1-7) released significant amounts of [³H]AA, the synthesis of 6-keto-PGF₁ was stimulated by Ang-(1-7) to a much greater extent than by Ang II.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effects of Ang peptides Ang-(1-7) and Ang II on [3H]AA release (A) and 6-keto-PGF1 production (B) in rabbit VSMC. VSMC were treated with various concentrations of peptides for 15 min. Fractional release is the percent of tritium released into the medium from the total cellular radioactivity. Data are expressed as mean ± S.E. of six experiments. * Value significantly different from the basal value (P < .05).

Effects of cyclooxygenase and LO inhibitors on Ang II- and Ang-(1-7)-stimulated 6-keto-PGF₁ production in VSMC of rabbit. Chronic infusion of Ang II in rats has been reported to increase the conversion of AA to LO products and decrease the conversion of AA to 6-keto-PGF₁ in the aorta (Lin et al., 1994). Hence, we examined the effects of Ang II, Ang-(1-7) and the LO inhibitor baicalein on LO activity, to determine whether the decreased effect of Ang II as compared with Ang-(1-7) on 6-keto-PGF₁ synthesis in VSMC is the result of increased LO activity. In rabbit VSMC, Ang II and Ang-(1-7) increased LO activity; the effect of Ang II was much greater than that of Ang-(1-7) (132 ± 14% above vehicle with Ang II vs. 56 ± 18% above vehicle for Ang-(1-7), P < .05) (fig. 2). Baicalein inhibited both Ang II- and Ang-(1-7)-induced increase in LO activity (fig. 2). Ang II (1 nM) which failed to stimulate 6-keto-PGF₁ synthesis caused a significant increase in its synthesis in the presence of baicalein (60%) (P < .05), whereas Ang-(1-7) at 1 nM did not alter 6-keto-PGF₁ synthesis in the presence or absence of baicalein. The effect of a higher concentration of Ang II (100 nM) was also markedly enhanced (80 ± 30% over vehicle) in the presence of baicalein (fig. 3A) (P < .05). However, Ang-(1-7) (100 nM)-induced 6-keto-PGF₁ production was increased only 30 ± 10% above basal in the presence of baicalein (fig. 3B). Indomethacin, a cyclooxygenase inhibitor, attenuated both Ang II- and Ang-(1-7)-induced 6-keto-PGF₁ production in VSMC (fig. 3, A and B) (P < .05).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Effects of Ang-(1-7) and Ang II on lipoxygenase activity, measured as absorbance by lipid hydroperoxide-mediated oxidation, in the presence and absence of baicalein (BACL) in rabbit VSMC. Cells were preincubated with baicalein (10 µM) or vehicle (VEH) for 15 min and treated with Ang II and Ang-(1-7) for 15 min at 37°C. VSMC protein lysates (40 µg) were prepared with Triton X-100 (0.1% v/v final concentration) and were diluted as required with 50 mM Tris-HCl buffer, pH 7.4. The samples were incubated at 37°C for 10 min with AA. The results shown are the mean ± S.E. (n = 3). * Value significantly different from that obtained with vehicle of Ang peptides;  value different from that obtained in the absence of BACL (P < .05).

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Effects of indomethacin (IND) and baicalein (BACL) on (A) Ang II- and (B) Ang-(1-7)-stimulated 6-keto-PGF1 production in VSMC. Cells were preincubated with indomethacin (10 µM) and baicalein (10 µM) for 15 min and treated with Ang II and Ang-(1-7) for 15 min at 37°C. Data are expressed as mean ± S.E. (n = 3). * Value significantly different from basal values;  significant difference in 6-keto-PGF1 production compared with vehicle (VEH) (P < .05).

Effects of Ang receptor antagonists on the increase in [³H]AA release induced by Ang II and Ang-(1-7) in VSMC. Cells were pretreated with the AT₁ receptor antagonist DUP-753 (100 nM), AT₂ receptor antagonist PD-123319 (100 nM), nonselective receptor antagonist [Sar¹,Thr⁸]-AngII, Ang-(1-7) receptor antagonist D-Ala⁷-Ang-(1-7) (100 nM) and a combination of receptor antagonists; the cells were stimulated with Ang II and Ang-(1-7). Ang II-, but not Ang-(1-7)-induced [³H]AA release was inhibited by DUP-753, whereas PD-123319 inhibited both Ang II- and Ang-(1-7)-induced [³H]AA release (fig. 4). D-Ala⁷-Ang-(1-7) inhibited the effect of Ang-(1-7) but not that of Ang II. Higher concentrations of DUP-753 (1 µM) and D-Ala⁷-Ang-(1-7) (1 µM) also failed to alter Ang-(1-7)- and Ang II-induced AA release, respectively (data not shown). The combination of D-Ala⁷-Ang-(1-7) (100 nM) and PD-123319 (100 nM) inhibited Ang-(1-7)-stimulated AA release, and the combination of DUP-753 (100 nM) and PD-123319 (100 nM) reduced Ang II-stimulated AA release to a greater degree than did each of the antagonists alone. None of the antagonists altered the basal [³H]AA release in VSMC (data not shown).

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Effects of angiotensin receptor antagonists on (A) Ang-(1-7)- and (B) Ang II-induced [3H]AA release in rabbit aortic VSMC. Cells prelabeled with 0.3 µCi/well of [3H]AA were preincubated with DUP-753 (100 nM), PD-123177 (100 nM), Sar1-Thr8-Ang II (100 nM), D-Ala7-Ang-(1-7) antagonist (100 nM) and a combination of DUP-753 (100 nM) and PD-123319 (100 nM), and D-Ala7-Ang-(1-7) and PD-123319 (100 nM) for 10 min and stimulated with 100 nM of Ang-(1-7) or Ang II for 15 min. Data are expressed as mean ± S.E. (n = 3). * Value significantly different from basal;  represents value significantly different from vehicle (VEH)-treated cells;  value significantly different from that obtained in the presence of the corresponding antagonist alone (P < .05).

Effects of cPLA₂ sense and antisense oligonucleotides and cPLA₂ inhibitor MAFP on Ang-(1-7)- and Ang II-stimulated [³H]AA release and PG synthesis in VSMC. Antisense oligonucleotides directed against the translation initiation sites of cPLA₂ and its sense complement and the cPLA₂ inhibitor MAFP were used to study the contribution of cPLA₂ to the release of [³H]AA in response to Ang peptides. The PLA₂ antisense oligonucleotide has been used to block lipopolysaccharide and platelet-activating factor-induced PG production in macrophage-like P388D1 cells (Barbour and Dennis, 1993), and lipopolysaccharide-induced PG production in monocytes (Roshak et al., 1994). MAFP is an irreversible inhibitor of the cPLA₂ and has no effect on the sPLA₂ (Balsinde and Dennis, 1996). Treatment of VSMC with cPLA₂ antisense oligonucleotides and cPLA₂ inhibitor MAFP decreased the release of [³H]AA and 6-keto-PGF₁ elicited by Ang-(1-7) (fig. 5). The inhibitory effect of cPLA₂ antisense on [³H]AA and 6-keto-PGF₁ synthesis elicited by Ang-(1-7) was prevented in VSMC cotransfected with cPLA₂ sense oligonucleotides. Moreover, Ang II-stimulated [³H]AA was also attenuated by cPLA₂ antisense oligonucleotides and by MAFP (fig. 6). MAFP did not alter the basal [³H]AA release (data not shown). Treatment of VSMC with cPLA₂ antisense oligonucleotides decreased the level of cPLA₂ protein, whereas the sense complement did not alter the level of cPLA₂ immunoreactive protein (fig. 6).

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Effects of cPLA2 antisense oligonucleotides and the cPLA2 inhibitor MAFP on Ang-(1-7)-stimulated [3H]AA release (A) and 6-keto-PGF1 production (B) in rabbit VSMC. Cells were transiently transfected with antisense (AS) and sense (S) oligonucleotides with lipofectamine or preincubated with MAFP (50 µM for 30 min) and exposed to Ang-(1-7) as described under "Methods." Data represent the mean ± S.E. of four experiments from two batches of cells. * Value significantly different from basal;  value significantly different from that obtained with Ang-(1-7) alone (VEH) (P < .05).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   (A) Effects of cPLA2 antisense oligonucleotides and the cPLA2 inhibitor MAFP on Ang II-stimulated [3H]AA release. Cells were transiently transfected with antisense (AS) and sense (S) oligonucleotides with lipofectamine or preincubated with MAFP (50 µM for 30 min) and exposed to Ang II. Data represent the mean ± S.E. of four experiments from two batches of cells. * Value significantly different from basal;  value significantly different from that obtained with Ang II alone (VEH). (B) Inhibition of cPLA2 protein levels by cPLA2 antisense oligonucleotides in rabbit VSMC. Cells were transiently transfected with 1 µM oligonucleotides or vehicle (VEH) for 6 hr in medium containing lipofectamine. Cells were allowed to recover in 0.5% FBS/M-199 for 30 hr. Total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by Western blot analysis with mouse monoclonal cPLA2 antibody.

Effects of CaM kinase II and MAP kinase sense and antisense oligonucleotides, the CaM kinase II inhibitor KN-93 and the MEK inhibitor PD-98059 on Ang-(1-7)- and Ang II-stimulated [³H]AA release and 6-keto-PGF₁ synthesis. Treatment of VSMC with CaM kinase II antisense, but not sense, oligonucleotides attenuated the effect of Ang-(1-7) to increase the release of [³H]AA and 6-keto-PGF₁ (fig. 7, A and B). The inhibitory effect of CaM kinase II antisense oligonucleotide on Ang-(1-7)-stimulated [³H]AA release and 6-keto-PGF₁ production was abolished when cells were cotransfected with CaM kinase II sense oligonucleotide. The CaM kinase II inhibitor KN-93 also attenuated the Ang-(1-7)-stimulated [³H]AA release and 6-keto-PGF₁ synthesis. Moreover, treatment of VSMC with MAP kinase antisense oligonucleotide and PD-98059, which inhibits activation of MEK, also reduced Ang-(1-7)-induced [³H]AA release (fig. 7C). The Ang II-induced [³H]AA release was reduced by the CaM kinase II inhibitor KN-93, by the CaM kinase II antisense oligonucleotide and by PD-98059 (fig. 8). The basal [³H]AA release was not altered by either KN-93, PD-98059 or antisense oligonucleotides (data not shown). Ang-(1-7) and Ang II increased cPLA₂ activity, measured as release of radioactive fatty acid from the hydrolysis of L-[1-¹⁴C]phosphatidylcholine. Ang-(1-7) increased cPLA₂ activity by 154 ± 13% above vehicle (2592 ± 653 cpm) and KN-93 and PD-98059 reduced it to 44 ± 8% and 22 ± 9%, respectively (n = 3-5, P < .05). Ang II also increased cPLA₂ activity by 113 ± 9% above vehicle and KN-93 and PD-98059 reduced it to 28 ± 8% and 30 ± 3%, respectively (n = 3-5, P < .05). KN-93 or PD-98059 did not alter the basal cPLA₂ activity (data not shown). Because Ang II (100 nM) produced only a 35% increase in 6-keto-PGF₁ above basal, the effect of later agents on Ang II-induced 6-keto-PGF₁ was not examined. CaM kinase II antisense but not sense oligonucleotides reduced CaM kinase II and not MAP kinase protein levels. Conversely, MAP kinase antisense but not sense oligonucleotides decreased MAP kinase protein and not CaM kinase II protein levels (fig. 8).

View larger version (48K): [in this window] [in a new window]   Fig. 7.   Effects of CaM kinase II antisense oligonucleotides, the CaM kinase II inhibitor KN-93, MAP kinase antisense oligonucleotides and the MEK inhibitor PD-98059 on Ang-(1-7)-stimulated [3H]AA release (A, C) and 6-keto-PGF1 production (B, D). Cells were transiently transfected with antisense (AS) and sense (S) oligonucleotides with lipofectamine for 36 hr (CaM kinase II) and for 96 hr (MAP kinase, two pulses of oligonucleotides at 48-hr intervals) or preincubated with KN-93 (20 µM for 4 hr) or PD-98059 (20 µM for 4 hr) and exposed to Ang-(1-7). Data represent the mean ± S.E. of four experiments from two batches of cells. * Value significantly different from basal;  value significantly different from that obtained with Ang-(1-7) alone (VEH) (P < .05).

View larger version (38K): [in this window] [in a new window]   Fig. 8.   (A) Effect of CaM kinase II antisense oligonucleotides, the CaM kinase II inhibitor KN-93, MAP kinase antisense oligonucleotides and the MEK inhibitor, PD-98059 on Ang II-stimulated [3H]AA release. Cells were transiently transfected with antisense (AS) and sense (S) oligonucleotides using lipofectamine for 36 hr (CaM kinase II) and for 96 hr (MAP kinase, two pulses at 48-hr intervals) or preincubated with KN-93 (20 µM for 4 hr) or PD-98059 (20 µM for 4 hr) and exposed to Ang II. Data represent the mean ± S.E. of four experiments from two batches of cells. * Value significantly different from basal;  value significantly different from that obtained with Ang II alone (VEH) (P < .05). (B) Inhibition of MAP kinase and CaM kinase II protein levels by their respective antisense oligonucleotides in rabbit VSMC.

Effects of the CaM kinase II inhibitor KN-93, MEK inhibitor PD-98059, and CaM kinase II and MAP kinase antisense and sense oligonucleotides on Ang-(1-7)- and Ang II-stimulated CaM kinase II and MAP kinase activation. To determine the sequence of events involved in Ang-(1-7)- and Ang II-stimulated AA release, experiments were designed to study the effect of these peptides on MAP kinase activity and the CaM kinase II activity in the presence of CaM kinase II inhibitor KN-93, the MEK inhibitor PD-98059 and CaM kinase II and MAP kinase antisense and sense oligonucleotides. Both Ang-(1-7) and Ang II stimulated MAP kinase and CaM kinase II activity (fig. 9). KN-93 and CaM kinase II antisense oligonucleotides attenuated CaM kinase II activity and MAP kinase activity elicited by Ang-(1-7) and Ang II. On the other hand, PD-98059, which significantly reduced the MAP kinase activity, did not reduce Ang peptides-stimulated CaM kinase II activity (fig. 9).

View larger version (66K): [in this window] [in a new window]   Fig. 9.   Effects of the CaM kinase II inhibitor KN-93 (20 µM), the MEK inhibitor PD-98059 (PD, 20 µM) and CaM kinase II and MAP kinase antisense (AS) and sense (S) oligonucleotides on the Ang-(1-7)- and Ang II-induced increase in MAP kinase (A) and CaM kinase II (B) activity. Cells were transiently transfected with AS and S oligonucleotides with lipofectamine for 36 hr (CaM kinase II) and for 96 hr (MAP kinase, two pulses at 48-hr intervals) or preincubated with KN-93 (20 µM for 4 hr) or PD-98059 (20 µM for 4 hr) and exposed to Ang peptides. Proteins (10 µg) were used to measure phosphotransferase activity of MAP and CaM kinases with use of their specific synthetic substrates. * Value significantly different from control (CON);  value significantly different from that obtained with Ang peptides alone (VEH), P < .05.

Translocation of cPLA₂ induced by Ang-(1-7) and Ang II. Translocation of cPLA₂ to the nuclear envelope, which is Ca⁺⁺ mediated, is required for the release of AA in response to various stimuli (Glover et al., 1995; Schievella et al., 1995). To determine whether Ang peptides also promote the translocation of cPLA₂ to the nuclear envelope or plasma membrane, we performed immunofluorescence experiments; confocal images were obtained with anti-cPLA₂ antibody in Ang II and Ang-(1-7)-stimulated VSMC and in unstimulated VSMC. Figure 10 shows that these enzymes are initially dispersed throughout the cytoplasm and that upon stimulation with Ang peptides, cPLA₂ translocate around the nucleus. In control experiments in which cells were treated with nonimmune IgG, only faint background fluorescence was observed. The nuclei were detected by staining with propidium iodide.

View larger version (68K): [in this window] [in a new window]   Fig. 10.   Translocation of cPLA2 in response to Ang-(1-7) and Ang II as visualized by confocal microscopy. Arrested VSMC were exposed to nonimmune IgG (A). VSMC that were exposed to vehicle (veh, B), Ang-(1-7) (C) and Ang II (D) were visualized by anti-cPLA2 and TRITC-conjugated goat-anti-mouse IgG. VSMC was exposed to propidium iodide; a fluorescent marker for nuclei is used as a stain to identify nuclei (E).

	Discussion

Top Abstract Introduction Methods Results Discussion References

This study demonstrates that Ang-(1-7) and Ang II stimulate cPLA₂ and release AA, which appears to be mediated by MAP kinase via activation of CaM kinase II in VSMC of the rabbit aorta (fig. 11). Ang-(1-7) and Ang II stimulate AA release in VSMC for the production of prostacyclin, measured as immunoreactive 6-keto-PGF₁. The effect of higher concentrations of Ang-(1-7) and Ang II in increasing 6-keto-PGF₁ was diminished. Whether this is because of desensitization of Ang peptide receptors is not known. Although Ang-(1-7) and Ang II produced similar increases in AA release, Ang-(1-7) produced a much greater increase in 6-keto-PGF₁ than Ang II. The smaller increase in 6-keto-PGF₁ synthesis elicited by Ang II as compared with Ang-(1-7) could be caused by activation by Ang II of LO enzymes that convert AA into hydroperoxyeicosatetraenoic acid, which is known to decrease the activity of prostacyclin synthase and decrease 6-keto-PGF₁ synthesis (Lin et al., 1994). AA is metabolized by LO into hydroxyeicosatetraenoic acids in VSMC (Natarajan et al., 1994). Although Ang II increases LO product levels (Lin et al., 1994), it seems that Ang-(1-7) produces a smaller increase in LO products of AA and that this may have contributed to the high levels of 6-keto-PGF₁ synthesis observed in response to Ang-(1-7) in VSMC. Supporting this conclusion was our finding that Ang II produced a significantly greater increase than Ang-(1-7) in LO activity in rabbit VSMC. Moreover, the LO inhibitor baicalein enhanced the effect of Ang II but failed to alter that of Ang-(1-7) at 1 nM on 6-keto-PGF₁ synthesis. However, baicalein increased the effect of the higher concentration of Ang-(1-7) (100 nM) on 6-keto-PGF₁ synthesis; but the degree of increase was less than that produced by Ang II at the same concentration. These observations together with the demonstration that Ang-(1-7) but not Ang II stimulates PG production in the endothelial cells (Leung et al., 1992; Jaiswal et al., 1992) raises the possibility that higher PG production by Ang-(1-7) (VSMC and endothelial cells) than Ang II might contribute to the vasodepressor action of Ang-(1-7) (Benter et al., 1995). Supporting this view is the report that inhibition of PG synthesis with indomethacin minimized the vasodepressor effect of Ang-(1-7) (Benter et al., 1993).

View larger version (33K): [in this window] [in a new window]   Fig. 11.   Model of Ang-(1-7)- and Ang II-stimulated AA release in rabbit VSMC. AT = Angiotensin type receptor. In this model, activation of AT receptor leads to an influx of Ca++ ions through Ca++ channels. Ca++ binds to CaM and activates CaM kinase II. CaM kinase II activates MAP kinase cascade through MEK. Upon activation, cPLA2 translocates to the nuclear membrane to release AA from phospholipids.

Ang II-stimulated AA release was inhibited by both AT₁ and AT₂ receptor antagonists. However, inhibition by the AT₂ receptor antagonist was less than that by DUP-753, which indicates that Ang II-stimulated AA release is predominantly mediated via the AT₁ receptor. On the other hand, the magnitude of inhibition of the Ang-(1-7)-stimulated AA release by the Ang-(1-7)-selective receptor antagonist D-Ala⁷-Ang-(1-7) and the AT₂ receptor antagonist PD-123319 was about the same. The combination of D-Ala⁷-Ang-(1-7) and PD-123319, however, inhibited the effect of Ang-(1-7) and the combination of DUP-753 and PD-123319 inhibited Ang II's ability to stimulate AA release to a greater degree than did either of the antagonists alone. These results agree with previous observations that Ang-(1-7) has been shown to produce various biological actions in vivo and in vitro by stimulating an Ang-(1-7)-specific receptor and an AT₂ receptor (Benter et al., 1993; Jaiswal et al., 1991; Santos et al., 1994; Fontes et al., 1994). It is well established that pressor and proliferative effects of Ang II are mediated via the AT₁ receptor (Timmermans et al., 1993; Murphy et al., 1991; Sasaki et al., 1991). Recently, disruption of AT₂ receptors was shown to increase blood pressure (Ichiki et al., 1995), and transfection of the AT₂ receptor resulted in reduced proliferation of VSMC (Nakajima et al., 1995). Munzenmaier and Greene (1996) showed that the AT₂ receptor antagonist PD-123319 selectively inhibited angiogenesis and elevated blood pressure. Our observation that the effects of Ang-(1-7) on AA release and PG synthesis are inhibited by the AT₂ receptor antagonist PD-123319 and by the Ang-(1-7)-selective receptor antagonist suggest that Ang-(1-7), like Ang II, may also act on AT₂ receptors as well as on Ang-(1-7)-specific receptor (Jaiswal et al., 1993).

Several types of PLA₂ have been characterized, and two Ca⁺⁺-dependent phospholipases, cPLA₂ and sPLA₂, have been implicated in the release of AA in response to various stimuli (Lin et al., 1992; Leslie, 1990; Murakami et al., 1993; Xing and Insel, 1996; Jacobs and Douglas, 1996). Our demonstration that both Ang II- and Ang-(1-7)-induced AA release was inhibited by cPLA₂ antisense oligonucleotides and by the cPLA₂ inhibitor MAFP suggests that cPLA₂ is involved in the release of AA. cPLA₂ antisense oligonucleotides depleted the cPLA₂ protein level but did not completely inhibit the AA release elicited by Ang II and Ang-(1-7). In addition, the cPLA₂ inhibitor MAFP did not totally block AA release elicited by Ang peptides. Therefore, the involvement of sPLA₂ and other lipases in the actions of Ang peptides in AA release cannot be ruled out.

The activity of cPLA₂ is regulated by MAP kinase (Lin et al., 1993; Xing and Insel, 1996). Ang II has been reported to cause phosphorylation of cPLA₂ in VSMC of rat aortae (Rao et al., 1994). Moreover, in these cells, Ang II has been shown to increase MAP kinase activity (Liao et al., 1996; Rao et al., 1994; Eguchi et al., 1996), which is attenuated by CaM inhibitors (Eguchi et al., 1996). Our finding in VSMC of rabbit aortae that Ang-(1-7)- and Ang II-induced AA release was attenuated by a CaM kinase II inhibitor, KN-93, by an inhibitor that inactivates MEK, PD-98059 and their respective antisense oligonucleotides suggests that both CaM kinase II and MAP kinase are involved in cPLA₂ activation by these peptides. The inhibitors of CaM kinase II and MEK and the antisense oligonucleotides of CaM kinase II and MAP kinase did not totally abolish either the Ang peptide-induced increase in CaM kinase II and MAP kinase activity or the AA release. Hence, we cannot exclude the involvement of other kinases in the activation of cPLA₂ or the involvement of CaM kinase II and MAP kinase in the activation of other lipases that might have a role in AA release in response to Ang peptides in rabbit VSMC.

An important finding of the present study was that the CaM kinase II inhibitor KN-93 attenuated MAP kinase activity, but the MEK inhibitor PD-98059 did not alter CaM kinase II activity elicited by Ang peptides. These observations suggest that CaM kinase II acts upstream of MAP kinase in response to Ang peptides. Because the MEK inhibitor PD-98059 reduced the increase in MAP kinase, but not CaM kinase II, activity produced by Ang-(1-7) and Ang II, it appears that MAP kinase activation by these peptides is mediated by MEK via activation of CaM kinase II. Ang II activates p21ras activity in VSMC of the rat aorta (Eguchi et al., 1996), and ras is known to activate MEK via stimulation of Raf-1. Therefore, Ang-(1-7) and Ang II, by increasing CaM kinase II activity, could lead to activation of ras, which in turn would promote activation of MEK via Raf-1. However, recently it has been reported that Ang II stimulates MAP kinase via a pathway involving PKC independent of c-Raf-1 and phorbol ester-sensitive isozymes in the VSMC of rat aortae (Liao et al., 1996, 1997). Therefore, it is possible that CaM kinase II activates MAP kinase by stimulating MEK directly or via some other signaling molecules.

The site of AA release by cPLA₂ in response to Ang peptides is not known. It has been reported that cPLA₂ translocates to the nuclear envelope, a site enriched in its substrate and in cyclooxygenase (Glover et al., 1995; Schievella et al., 1995). Nuclear membrane has high specific activity for [³H]AA labeling (Capriotti et al., 1988). Our finding that cPLA₂ translocates to the nuclear envelope in response to Ang-(1-7) and Ang II suggests that these peptides may stimulate AA release from cytoskeletal structures around the nucleus such as the endoplasmic reticulum. Translocation of cPLA₂ to the nuclear envelope appears to be independent of its phosphorylation because mutation at the MAP kinase phosphorylation site of cPLA₂ has been reported not to alter ionophore-induced translocation of the enzyme to the nuclear envelope (Schievella et al., 1995).

In conclusion, this study demonstrates that Ang-(1-7) and Ang II stimulate prostacyclin synthesis by promoting the release of AA from tissue lipids via activation of distinct types of AT receptors. Both peptides appear to stimulate CaM kinase II, which in turn via MAP kinase activation enhances cPLA₂ activity and releases AA for prostacyclin synthesis. Previously observed vasodepressor actions of Ang-(1-7) could be explained by the observation that it does not stimulate AT₁ receptors and it is more potent than Ang II in stimulating synthesis of vasodilatory prostaglandins probably because of its low potency in stimulating LO activity.