Calhoun Research Laboratory, Department of Internal Medicine, Akron General Medical Center, Akron 44307; and Department of Biological Sciences, Kent State University, Kent, Ohio 44242
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ABSTRACT |
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The accumulation of radiolabeled arachidonic acid (AA), immunoblot analysis of subcellular fractions, and immunofluorescence tagging of proteins in intact cells were used to examine the coupling of ANG II receptors with the activity and location of a cytosolic phospholipase A2 (cPLA2) in vascular smooth muscle cells (VSMC). ANG II induced the accumulation of AA, which peaked by 10 min and was downregulated by 20 min. A large proportion of the AA released in response to ANG II was due to the activation of a Ca2+-dependent lipase coupled to an AT1 receptor. However, regulation of Ca2+ availability failed to completely block AA release, and a small but significant reduction in ANG II-mediated AA release was observed in the presence of an AT2 antagonist. These findings, coupled with a 25% reduction in the ANG II-induced AA release by an inhibitor specific for a Ca2+-independent PLA2, are consistent with the presence and activation of a Ca2+-independent PLA2. In contrast, immunoblot analysis and immunofluorescence detection showed that the ANG II-mediated translocation of cPLA2 to a membrane fraction was exclusively AT1 dependent and regulated by Ca2+ availability. Furthermore, the nucleus was the membrane target. We conclude that ANG II regulates the Ca2+-dependent activation and translocation of cPLA2 through an AT1 receptor and that this event is targeted at the nucleus in VSMC.
angiotensin II; arachidonic acid; cytosolic phospholipase A2; angiotensin receptors
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INTRODUCTION |
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ANGIOTENSIN (ANG) II is a potent vasoconstrictor that stimulates the growth of vascular smooth muscle cells (VSMC) (12, 38). There is interest in the proliferative effects of this hormone in VSMC, since growth of these cells within the arterial intima plays a role in vascular occlusive disorders, such as atherosclerosis and restenosis. Involvement of ANG II in these processes has been documented using ANG-converting enzyme inhibitors and selective antagonists of the AT1 receptor subtype (11, 31). Although the signal transduction system that couples ANG II receptors with proliferative responses is uncertain, a variety of biochemical pathways respond to ANG II in these cells. For example, ANG II stimulates phospholipase C (PLC), which results in the formation of diacylgycerols (DAG) and inositol trisphosphates (16). The production of these lipid metabolites appears to be coupled to the ANG II-dependent activation of protein kinase C (PKC) (17) and the mobilization of Ca2+ from intracellular stores (13, 36). Consistent with its actions as a growth factor, ANG II also increases mitogen-activated protein (MAP) kinase activity and stimulates transcriptional processes (34, 37).
ANG II also stimulates the release of the second messenger arachidonic acid (AA) from phospholipid substrates in many cell types, including pituitary cells (1), renal epithelial cells (19), and VSMC (10). Although some of this AA may be derived from the deacylation of DAG (28) or from the activation of a Ca2+-independent phospholipase A2 (PLA2) (19, 23), there is substantial evidence that the receptor-dependent accumulation of AA depends on the activation of a cytosolic form of PLA2 (cPLA2) (3), which has been identified in a variety of cell types, including VSMC (32). In general, cPLA2 is characterized by a high molecular mass (80-160 kDa), activation by micromolar concentrations of Ca2+, selectivity for AA at the sn-2 position of the glycerolipid (2, 6), and potent inhibition by arachidonyl trifluoromethyl ketone (AACOCF3) (4, 15). Phosphorylation of cPLA2 by PKC (32, 33) or MAP kinase (25, 26) may be required for full activation of this enzyme (35), but effective activation involves translocation of the enzyme from the cytosol to its substrates located in a target membrane (8).
A variety of agonists have been shown to stimulate cPLA2, including platelet-derived growth factor and other mitogens in Chinese hamster ovary (CHO) cells (25, 26), thrombin in platelets (21), acetylcholine in astrocytoma cells (5), and ANG II in vascular endothelial cells (30). To date, this has not been shown for ANG II in VSMC. In addition, ANG II-dependent translocation of cPLA2 to a membrane target has not been shown in VSMC, but the target has been demonstrated to be the nucleus in vascular endothelial cells (30).
The purpose of the work described here was to link the activity and location of cPLA2 with the activation of ANG II receptors in VSMC. The activity of the enzyme was measured as the release of [3H]AA from prelabeled cells, and localization of the enzyme was determined using both immunoblot analysis of isolated proteins and immunofluorescence detection of cPLA2 in intact cells. ANG II stimulated the activity of the enzyme and its translocation to the nucleus. Both of these effects depended on the activation of the AT1 receptor and the presence of Ca2+, while [3H]AA release was blocked by AACOCF3. These results are consistent with a role for cPLA2 in the transduction of the ANG II signal in VSMC.
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METHODS |
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VSMC culture. VSMC were isolated from rat thoracic aorta by explant culture, as previously described (14). Cells were identified as VSMC by their characteristic morphology, as well as with antibodies to VSMC-specific actin. Cells were routinely used between passages 3 and 10.
[3H]AA release. VSMC were grown in six-well culture dishes in Dulbecco's modified Eagle's medium-Ham's F-12 (DMEM-F12) containing 10% fetal bovine serum (FBS). Subconfluent monolayers were growth arrested for 24 h in DMEM-F12 without FBS. Cells were labeled using 0.25 µCi/ml [3H]AA (100 Ci/mmol; DuPont NEN) for 24 h in serum-free medium. The cells were washed three times in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) to remove unincorporated label. Cells were stimulated in serum-free medium also containing 0.5% BSA to trap released fatty acids. The cells were placed on ice, and the medium was removed and extracted using chloroform-methanol-water (2:2:1 vol/vol/vol) after exposure to various treatments. The organic phase was evaporated under a stream of N2, reconstituted in 30 µl of chloroform-methanol (1:1 vol/vol), and spotted on Whatman LK6D silica gel thin-layer plates. The plates were developed in ethyl acetate-hexane-acetic acid-water (17:7:3:18 vol/vol/vol/vol). The fatty acids were visualized using iodine vapor, and the bands corresponding to authentic AA standard were scraped into scintillation vials and counted in 5 ml of Ecolite (ICN Biomedicals). Data are expressed as means ± SE in counts per minute per well.
Immunoblot analysis of cPLA2.
Subconfluent monolayers of VSMC grown in 100-mm dishes
were washed twice with PBS before exposure to various treatments. Cells were incubated at 37°C in Krebs-Ringer buffer [in mM: 128 NaCl, 5 KCl, 1.2 MgSO4, 1 Na2HPO4,
10 glucose, 1 CaCl2 and 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES); pH 7.4] in the presence of various treatments. The treatment buffer was removed, and the cells were scraped in ice-cold PBS [containing 1 mM ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA)] and pelleted by centrifugation. The pellet was
resuspended in PBS with protease inhibitors (50 µg/ml
phenylmethylsulfonyl fluoride, 0.1 mg/ml aprotinin, 0.1 mg/ml
leupeptin, and 0.1 mg/ml ovalbumin, as well as 1 mM EGTA) and
resedimented. The supernatant was removed, and the pellet was
immediately frozen in liquid N2.
Immunofluorescence staining of cPLA2. VSMC were passed into 25-mm2 glass-bottom culture dishes and allowed to adhere overnight. The cells were washed twice with PBS before application of treatments. Cells were fixed with ice-cold methanol for 1 min, washed twice with PBS, and incubated in blocking buffer (3% BSA in PBS) at 37°C for 40 min. The cells were exposed to a polyclonal cPLA2 antibody (Santa Cruz Biotechnology) diluted in blocking buffer (1 µg/ml). The cells were incubated for 2 h at 37°C before four washes with blocking buffer and incubation for 45 min at 37°C with Texas red-conjugated anti-rabbit immunoglobulin G (Jackson Immuno Research Laboratories) diluted in blocking buffer (7.5 µg/ml). Finally, the cells were washed four times with blocking buffer and twice with PBS and viewed using a Nikon inverted fluorescent microscope (×100, oil immersion). Cells were observed under white light or with a Texas red filter (XF43; excitation 580 nm, emission 630 nm). Images were acquired from experiments done on 3 separate days using the same camera settings (brightness = 0, contrast = +4, gamma = 0.45, shutter speed = 1), and pictures were saved using Image-Pro software. The relative intensity of cPLA2 immunofluorescence was estimated for 10 × 30-pixel blocks for the cytoplasm, whereas 7 × 10-pixel blocks were used for the nuclear envelope, to narrow the field of view at the surface of that organelle. The relative fluorescence intensities for cytoplasmic and nuclear sites from each cell were summed, and percent distribution at each site was determined. The data are expressed as means ± SE from six different cells for each treatment.
Statistical analyses. Statistical differences between experimental groups were determined using analysis of variance and the Newman-Keuls pairwise comparison test. Differences were considered significant at P < 0.05.
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RESULTS |
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Effects of ANG II on PLA2 activity in VSMC. The effects of ANG II on PLA2 activities in VSMC were determined by measuring the release of [3H]AA from prelabeled cells. Exposure of the 3H-labeled cells to 100 nM ANG II stimulated the time-dependent release of [3H]AA, which was marked by 30 s and peaked at 10 min as a 218% increase in unesterified AA compared with untreated cells (Fig. 1). The rate of [3H]AA release in ANG II-treated cells was reduced to control levels by 20 or 30 min of exposure. This decline in the rate of AA release is shown in Fig. 1, inset, in which accumulated [3H]AA is reported as the difference between treated and untreated cells.
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ANG II-induced translocation of cPLA2. Membrane and cytosolic fractions were prepared from control and ANG II-treated VSMC, to examine cPLA2 translocation. Proteins from both fractions were separated by SDS-PAGE and immunoblotted with anti-cPLA2. In both fractions, the monoclonal antibody against the lipase detected a protein with an apparent molecular mass of 160 kDa. That this band was authentic cPLA2 was verified using other cell types, in which both the polyclonal and monoclonal antibodies detected a single band at 85 kDa in CHO or at ~120 kDa in rat liver, brain, or kidney cells. In VSMC, the relative distributions of cPLA2 between the two fractions in untreated cells were 80% cytosolic to 20% particulate, when equal amounts of protein were separated from each fraction. The immunoblot data for the membrane fraction are shown in Fig. 3. Exposure of the cells to 1 µM ANG II stimulated a time-dependent increase of cPLA2 in the particulate fraction, since at 2.5 or 7 min the membrane-associated cPLA2 increased to 117 or 125% of the time 0 control value, respectively. However, this protein declined to 77% of control at 15 min of treatment. The receptor dependence of the ANG II-induced translocation is also shown in Fig. 3. The cPLA2 content in the membrane fraction was reduced to 93% of the control value when the cells were incubated with the AT1 antagonist losartan for 2 min before exposure to ANG II for 7 min.
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Immunofluorescence detection of cPLA2 in intact VSMC. Immunofluorescence was used to localize cPLA2 in intact VSMC. In nonstimulated cells, the fluorescence appeared as a diffuse signal throughout the cell. However, treatment with ANG II caused the movement of the cPLA2 from the cytoplasm to the nuclear membrane, where antibody staining revealed a bright halo around the nucleus. Through pixel analysis, increases in fluorescence intensity were detected at the surface of the nuclei, which corresponded to a 24% increase over control by 7 min of exposure. This ANG II-induced fluorescent labeling of the nuclei returned to near baseline at 15 min of stimulation. These data and the effects of removing extracellular Ca2+ are expressed as percent of total immunofluorescence, to show the changes in the relative distribution of the cPLA2 between the cytoplasm and the nucleus (Fig. 5). The baseline distributions of cPLA2 in cells maintained in the presence of 1 mM Ca2+ were 60% associated with the nucleus and 40% in the cytosol. ANG II increased the distributions of cPLA2 at the nucleus to 67 or 72% at 2.5 or 7 min of treatment, respectively. In contrast, the cPLA2 associated with the nucleus declined to 58% by 15 min of exposure to the peptide. The amount of cPLA2 in the cytoplasm reflected this translocation, since the distributions were reduced to 33 or 28% at 2.5 or 7 min of treatment, respectively. This value increased to 42% when the cells were incubated with ANG II for 15 min in the presence of Ca2+.
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DISCUSSION |
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AA acts as a second messenger and is released from membrane phospholipids in response to the activation of receptors by a variety of agonists. Such receptor-coupled accumulation of unesterified AA may involve the activation of cPLA2. This enzyme requires micromolar concentrations of Ca2+, has a high molecular mass, translocates from the cytosol to a membrane target on exposure to Ca2+, and specifically removes AA from its substrates (6, 7). The activation and/or translocation of this cPLA2 is stimulated by numerous agonists, including ATP in CHO cells (9), phenylephrine in VSMC (29), and ANG II in vascular endothelial cells (30). However, the translocation and/or activation of cPLA2 has not been demonstrated in VSMC exposed to ANG II. Here, we show that the peptide hormone acts through the AT1 receptor to induce the Ca2+-dependent accumulation of AA and the movement of cPLA2 from the cytosol to the nuclear membrane.
ANG II stimulates the proliferation of VSMC, and this effect is mediated through the AT1 receptor (12). AA may be involved in this response, since it regulates DNA synthesis and gene transcription in other cell types (18, 20, 24). Consistent with this suggestion, the ANG II-induced accumulation of [3H]AA was attenuated by the AT1 receptor antagonist. However, the AT2 antagonist PD-123319 also mediated a minor reduction in AA release. In contrast, the translocation of cPLA2 to a membrane target appeared to be coupled exclusively to an AT1 receptor, since the ANG II-dependent translocation of cPLA2 to the nucleus was blocked by losartan. This effect was shown using both immunoblot analysis of separated proteins and immunofluorescence in intact cells. In addition, AACOCF3, which is an inhibitor of cPLA2, eliminated the ANG II-dependent [3H]AA release. Thus ANG II induced the activation and translocation of cPLA2 via the AT1 receptor subtype.
ANG II induces an increase in Ca2+ in VSMC through a combination of influx and mobilization from intracellular stores (13), and this response may be involved in the regulation of cPLA2. The ANG II-induced accumulation of [3H]AA was reduced by two-thirds when EGTA was used to remove extracellular Ca2+. In addition, attenuation of ANG II-dependent Ca2+ mobilization via inhibition of PLC with U-73122 (13) resulted in a 52% reduction in AA accumulation, whereas ionomycin-induced Ca2+ influx stimulated a 47% increase in unesterified [3H]AA. This apparent dependence of ANG II-induced cPLA2 activation on both the influx and mobilization of Ca2+ was consistent with observations for ANG II stimulation of phospholipase D in VSMC (13). However, the persistent release of AA in the presence of treatments that attenuate the accumulation of Ca2+ raises the possibility that some fraction of the ANG II-mediated AA release involved a Ca2+-independent PLA2. Consistent with this suggestion, inhibition of Ca2+-independent PLA2 with HELSS reduced ANG II-mediated AA release by 25%.
The translocation of cPLA2 from cytosol to the nucleus was also affected by available Ca2+, since ionomycin induced this movement, whereas removal of extracellular Ca2+ reduced the ANG II-dependent movement of cPLA2 to the nucleus by about one-half. Furthermore, incubation of unstimulated, intact cells in the absence of Ca2+ for 2 min was sufficient to reduce the amount of cPLA2 associated with the nucleus (Fig. 5). It appeared, therefore, that resting levels of intracellular Ca2+ are sufficient to maintain a constitutive localization of cPLA2 at the nucleus and that the ANG II-dependent increase in available Ca2+ is needed for the activation and translocation of the enzyme.
The time courses for ANG II-induced activation and translocation of cPLA2 were similar. Both showed rapid initiations following exposure of the VSMC to the agonist, and both appeared to be downregulated within 15-20 min of stimulation. Immunoblot analysis of cPLA2 associated with the nuclear fraction showed the translocation of a protein doublet with apparent molecular masses near 160 kDa. This value is likely to be higher than the actual mass, since cPLA2 generally migrates slowly when separated by SDS-PAGE (6, 7). That the translocated lipase separates as a doublet is consistent with the suggestion that kinase-dependent phosphorylation is required for full activation of the enzyme (35). Although this has not yet been shown in ANG II-stimulated VSMC, it may explain the observation that ANG II was a more potent effector of [3H]AA release than was ionomycin, since ANG II stimulates the phosphorylation of cPLA2 (32) and Ca2+ accumulation (13), whereas ionomycin directly affects the availability of Ca2+.
In sum, a cPLA2 was identified in VSMC. It had a high molecular mass and separated on gels as a protein doublet, which may represent its phosphorylation state. This protein doublet rapidly migrated from the cytosolic compartment to the nucleus on exposure of the cells to ANG II. The time course for this movement was chronologically consistent with the activation of the enzyme and the consequent release of [3H]AA from prelabeled glycerolipids, which was eliminated by the cPLA2 inhibitor AACOCF3. Both the stimulation of the lipase and its translocation to the nuclear target required activation of the AT1 receptor subtype, and both depended on available Ca2+. It is possible that this AT1-coupled, Ca2+-dependent production of unesterified AA plays a central role in the proliferation of VSMC induced by ANG II. However, further work is needed to localize the production of unesterifed AA to specific subcellular compartments and to show the coupling of this second messenger with VSMC growth.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant-in-aid from the American Heart Association, Ohio-West Virginia Affiliate (to E. J. Freeman).
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FOOTNOTES |
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Address for reprint requests: E. J. Freeman, Calhoun Research Laboratory, Dept. of Internal Medicine, Akron General Medical Center, 400 Wabash Ave., Akron, OH 44307.
Received 23 July 1997; accepted in final form 2 October 1997.
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