Calcium/calmodulin-dependent protein kinase IIalpha mediates activation of mitogen-activated protein kinase and cytosolic phospholipase A2 in norepinephrine-induced arachidonic acid release in rabbit aortic smooth muscle cells.

We have investigated the contribution of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and mitogen-activated protein kinase (MAP kinase) in norepinephrine (NE)-induced arachidonic acid (AA) release in rabbit aortic vascular smooth muscle cells (VSMC). NE enhanced release of AA via activation of cytosolic phospholipase A2 (cPLA2) but not secretory PLA2 in VSMC prelabeled with [3H]AA. NE (10 microM) enhanced CaM kinase II and MAP kinase activity. In cells transiently transfected with antisense oligonucleotides complementary to the translation initiation sites of CaM kinase II and MAP kinase, NE-induced AA release was inhibited by 100 and 35% respectively. Treatment of cells with PD-098059, a MAP kinase kinase inhibitor, or with MAP kinase antisense oligonucleotide reduced NE-induced activation of MAP kinase and cPLA2. NE-induced MAP kinase and cPLA2 activation was also inhibited in cells treated with a CaM kinase II inhibitor, KN-93, or with CaM kinase II antisense oligonucleotide. On the other hand, inhibition of MAP kinase kinase with PD-098059 or of MAP kinase with antisense oligonucleotides did not alter the NE-induced increase in CaM kinase II activity. Phosphorylation of MAP kinase and CaM kinase II by NE, studied by 32P incorporation and immune complex kinase assays, was inhibited by KN-93. Collectively, these data suggest that CaM kinase II can activate MAP kinase, which in turn activates cPLA2 to release AA for prostacyclin synthesis in the rabbit VSMC. This novel pathway for activation of MAP kinase by CaM kinase II appears to be mediated through stimulation of MAP kinase kinase. Activation of adrenergic receptors with NE in VSMC caused translocation of CaM kinase II, MAP kinase, and cPLA2 to the nuclear envelope only in the presence of extracellular Ca2+. Okadaic acid, which increased phosphorylation and activity, did not translocate these enzymes. Therefore, it appears that in rabbit VSMC, NE, by promoting extracellular Ca2+ influx, increases CaM kinase II activity, leading to activation of MAP kinase and cPLA2 and translocation to the nuclear envelope, resulting in release of AA from the nuclear envelope for prostacyclin synthesis.

Norepinephrine (NE) 1 stimulates prostaglandin synthesis in the cardiovascular system via activation of distinct types of adrenergic receptor (AR) at the postjunctional effector cells, e.g. ␤ 1 AR in the heart, ␣ 1 AR in the kidney and spleen, and ␣ 1 AR and/or ␣ 2 AR in blood vessels (1,2). PGI 2 synthesis elicited by NE in VSMC is primarily due to activation of ␣ 2 and to a lesser extent ␣ 1 AR (2). Activation of both ␣ 1 and ␣ 2 AR in VSMC promotes PGI 2 synthesis by increasing Ca 2ϩ influx, primarily through voltage-dependent Ca 2ϩ channels, via a pertussis toxin-sensitive G i␣ -like protein (2). The increased Ca 2ϩ influx, by interacting with calmodulin, activates PLA 2 , which releases AA from tissue lipids for PGI 2 synthesis (2). The release of AA for prostaglandin synthesis in response to various stimuli has been reported to be due to the activation of cPLA 2 or sPLA 2 species. sPLA 2 can hydrolyze phospholipids containing different fatty acids at the sn-2-position, requires millimolar Ca 2ϩ concentrations for activation, and is sensitive to disulfide reducing agents (3). On the other hand, cPLA 2 selectively hydrolyzes phospholipids containing AA at the sn-2-position, is activated by micromolar Ca 2ϩ concentrations, and is not sensitive to disulfide reducing agents (4,5). Whether NE stimulates AA release for PGI 2 synthesis in the VSMC by activation of cPLA 2 and/or sPLA 2 is not known. cPLA 2 activity has been reported to be regulated by phosphorylation by MAP kinase (6 -8) and by Ca 2ϩ -dependent translocation to the nuclear envelope (9,10), allowing its access to arachidonyl-containing phospholipid substrate. However, recent studies have provided evidence for cPLA 2 activation independent of MAP kinase in human platelets in response to the thrombin agonist SFLLRN (11) and in human neutrophils in response to TNF-␣ (12). Our previous findings in the VSMC of rabbit aorta that PGI 2 synthesis elicited by activation of ␣ 1 and ␣ 2 AR was attenuated by the calmodulin (CaM) inhibitor W-7 (2) raises the possibility that Ca 2ϩ /CaM might stimulate cPLA 2 directly or indirectly via activation of CaM kinase II or MAP kinase.
CaM kinase II is abundant in the brain and has been implicated in neurotransmitter release (13). In this study, we report that CaM kinase II␣ is also expressed in VSMC and report for the first time that CaM kinase II promotes MAP kinase-induced activation of cPLA 2 . We further show that, upon NE treatment, cPLA 2 translocates to the nucleus along with CaM kinase II and MAP kinase.
Preparation of VSMC-Male New Zealand rabbits (1-2 kg) were anesthetized with 30 mg/kg pentobarbital (Abbott Laboratories, North Chicago, IL), and the thorax and abdomen were opened by a midline incision. The aorta was rapidly removed, and VSMC were isolated as described previously (15). Cells between four and eight passages were plated in 12 or 24 wells or 100-mm plates. Cells were maintained under 5% CO 2 in M-199 medium (Sigma) with penicillin, streptomycin, and 10% FBS.
Preparation of Thiooligonucleotides and Transient Transfection of VSMC-Antisense oligonucleotides directed against the translation initiation sites of cPLA 2 , sPLA 2 , CaM kinase II␣, and MAP kinase were designed (Table I). VSMC were transfected with sense and/or antisense oligonucleotides complexed with 4 g/ml of lipofectamine and incubated in serum-free M-199 for 6 h. Thereafter, fresh M-199 containing 10% FBS and oligonucleotides was added, and the cells were incubated with [ 3 H]AA for another 18 h to label tissue lipids.
[ 3 H]AA Release-After transient transfection, cells were washed with Hanks' balanced salt solution and exposed to NE in balanced salt solution containing BSA for 15 min at 37°C. 3 H released into extracellular medium and that remaining in the VSMC was measured by liquid scintillation spectroscopy. Total radioactivity in the cells was determined after treating the cells with 1 M NaOH overnight. 3 H released into the medium was expressed as percentage of the total cellular radioactivity and referred to as fractional release.
Phospholipase A 2 Assay-Cells grown in 100-mm plates were arrested for 24 h and stimulated with or without NE and lysed in HEPES buffer containing protease and phosphatase inhibitors (350 mM sucrose, 1 mM EGTA, 100 g/ml phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, and 20 g/ml soybean trypsin inhibitor). The concentration of protein was determined by Bradford assay (Bio-Rad). PLA 2 activity in lysates of VSMC fractions (20 -30 g of protein/ assay) was measured using [ 14 C]arachidonyl phosphatidylcholine as substrate as described previously (16). 11 l of radiolabeled phospholipid stock was dried under N 2 and added to 0.5 ml of reaction mixture (9 M dioleoylglycerol, 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl 2 , 1 mM DTT, 1 mg/ml BSA) and sonicated for 15 min on ice. The reaction mixture (50 l) containing 25 g of protein from cell lysate was incubated at 37°C for 1 h. The reaction was stopped by adding 2.5 ml of Dole's reagent (2-propanol, heptane, 0.5 M H 2 SO 4 , 20:5:1), and then 1.5 ml of heptane and 1 ml of water containing 20 g 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 using a high flash point LSC mixture (Packard Instrument Company, Meriden, CT).
CaM Kinase II Assay-CaM kinase II activity was assayed in cell lysates using CaM kinase II assay kits (Upstate Biotechnology Inc., Lake Placid, NY) utilizing a peptide substrate (KKALRRQETVDAL) with relative selectivity for CaM kinase II. The reaction mixture containing 10 l of substrate mixture, 10 l of a mixture containing inhibitors of other Ser/Thr kinases such as protein kinase A and protein kinase C, and 10 l of Mg 2ϩ /ATP mixture containing ␥-32 P was incubated at 30°C for 10 min. The phosphorylated substrate was separated from the residual [␥-32 P]ATP using p81 phosphocellulose paper. The papers were washed twice in 0.75% H 3 PO 4 and then in acetone for 2 min, and the bound radioactivity was quantified with a scintillation counter. Blanks to correct for nonspecific binding of [␥-32 P]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 pmol/min/mg protein.
MAP Kinase Assay-The activity of MAP kinase was determined in cell lysates of the VSMC with a BIOTRAK kit (Amersham), using a peptide substrate relatively selective for CaM kinase II (KRELVEPLT-PAGEAPNQALLR). Transfer of ␥-32 P of 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 DTT, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, pH 7.4) and centrifuged at 25,000 ϫ g for 20 min to remove cellular debris. For each assay, 5 l of Mg[ 32 P]ATP buffer, 15 l of sample (10 g of protein), 10 l of substrate buffer were added and incubated at 30°C for 10 min. The reactions were terminated by adding a stop reagent, and 30 l 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 radioactively was determined. Enzyme activity was expressed as pmol/min/mg protein.
CaM Kinase Phosphorylation-VSMC were washed three times with phosphate-free DMEM and then prelabeled for 4 h with [ 32 P]orthophosphate (300 Ci/ml) along with inhibitors and treated with NE (10 M) for 10 min. The cells were quickly washed three times with ice-cold phosphate-buffered saline and immersed in a slurry of ice and ethanol. The cells were scraped and sonicated in buffer containing 10 mM HEPES, 250 mM sucrose, 5 mM EDTA, protease inhibitors (100 g/ml phenylmethylsulfonyl fluoride, 100 g/ml leupeptin, 10 g/ml aprotinin, and 20 g/ml soybean trypsin inhibitor), and phosphatase inhibitors (5 M phosphoserine, phosphothreonine, phosphotyrosine, ␤-glycerophosphate, p-nitrophenyl phosphate, and sodium vanadate). The amount of protein was adjusted to 1 mg/ml and split into two halves. One half was incubated with rat monoclonal CaM kinase II␣ antibody, and the other half was incubated with mouse IgG for 4 h at 4°C and then with protein A-agarose beads for 1 h. The immunoprecipitate was centrifuged at 12,000 rpm for 2 min, and the pellets were washed with ice-cold phosphate-buffered saline containing phosphatase inhibitor. The pellets were resuspended in Laemmli buffer, and the supernatants were subjected to SDS-PAGE (10% gel) and autoradiography.
MAP Kinase Phosphorylation-Phosphospecific MAP kinase antibody (New England Biolabs) that detects phosphorylated Tyr residues of p44 and p42 MAP kinases but does not appreciably cross-react with the unphosphorylated forms was used. Lysates from cells that were stimulated by NE in the presence or absence of inhibitors were resolved on an SDS-PAGE and transferred to polyvinylidene difluoride membrane. The blots were processed as per the manufacturer's instructions.
Immune Complex Kinase Assays-Cell lysates containing equal amounts of proteins (500 g) from control and NE-treated samples in the presence and absence of inhibitors were incubated with 5 l of ERK-1 and CaM kinase II antibody or anti-mouse IgG for 1 h at 4°C. The immune complexes were captured by protein A-agarose beads and washed three times with 1 ml of radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% Nonidet P-40, 0.25% sodium deoxycholate containing 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , 2 mM EGTA, 50 g/ml leupeptin, and 0.5% aprotinin). The pellets were suspended in 50 l of protein kinase assay buffer containing 10 Ci of [␥-32 P]ATP and assayed for MAP kinase and CaM kinase II as described above.
Western Blot Analysis-Lysates from transfectants and parental cells were prepared in buffer. Samples containing 30 g of protein were resolved by SDS-polyacrylamide gel electrophoresis before transfer to nitrocellulose. The blots were blocked with 3% BSA in TBS at room temperature for 2 h and then incubated for 2 h with primary monoclonal antibodies (1:1000 dilution). The blots were developed using biotinylated secondary antibodies and horseradish peroxidase, and signals were detected using ECL Western blotting detection reagents (Amersham). Western blotting experiments were carried out at least three times on the transfectants.
Confocal Microscopy-Cells were grown to approximately 70% confluency on chamber slides (Nunc, Inc., Naperville, IL) and arrested for 24 h. Then the cells were washed with 1 ml of balanced salt solution containing CaCl 2 and treated with NE (10 M) for 10 min in the presence or absence of extracellular Ca 2ϩ or okadaic acid, a phosphatase inhibitor. 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 antibodies (cPLA 2 or CaM kinase II or MAP kinase, diluted 200-fold with 3% BSA in TBS containing 0.1% Tween 20 (TBST)) were applied to each well. After 1 h, the cells were washed three times (10 min each) and exposed to tetramethyl rhodamine (TRITC)-conjugated goat anti-mouse IgG (1:200 dilution). After a 45-min incubation in the dark, the cells were washed three times (10 min each) with TBST and rinsed quickly with water. 10 l of Galvetol (Sigma) was applied to the cell surface, and coverslips were mounted. Controls were carried out, replacing the primary antibody with IgG. Nuclei were visualized with 4Ј,6-diamidino-2phenylindole (Sigma). Slides were viewed by confocal fluorescence microscopy (Bio-Rad MRC-1000 Laser Scanning Confocal Imaging system using an argon/krypton lamp) with a ϫ 100 objective lens.

Effect of NE on AA Release in the VSMC of Rabbit Aorta-
Rabbit aortic VSMC contain ␣ 1 and ␣ 2 AR, and both are coupled to prostacyclin synthesis (2). To determine the effect of AR agonist NE on release of AA from tissue lipids, the [ 3 H]AAlabeled VSMC were incubated for different time periods with NE, which acts on both ␣ 1 and ␣ 2 AR. The release of [ 3 H]AA and its metabolites into the medium and the cell content of tritium were measured. NE enhanced release of [ 3 H]AA in a time-dependent manner, with the maximal release at 15 min (Fig. 1). NE was more efficacious than methoxamine, an ␣ 1 AR †, value significantly different from that obtained with NE alone; † †, value significantly different from that obtained with cPLA 2 antisense alone (p Ͻ 0.05). agonist, and UK-14304, an ␣ 2 AR agonist (data not shown).
Type of Lipases Involved in the NE-stimulated AA Release-To delineate the type of PLA 2 involved in the release of AA in response to NE, antisense oligonucleotides directed against the translation initiation sites of cPLA 2 and sPLA 2 were used. Phosphorothioate oligonucleotides have been successfully targeted to inhibit c-Myb, c-Fos, c-Myc, and many other signal transduction and effector molecules including PLA 2 (17). The phosphorothioate oligonucleotides penetrate the cell membrane easily and allow sequence-specific inhibition of the processes of translation of mRNA into protein. Moreover, they are more stable to endo-and exonucleases that degrade naked DNA. However, antisense oligonucleotides may also act by a nonantisense mechanism, particularly when continuous four-G-nucleotide sequences occur (18). After careful consideration of the above criteria, antisense oligonucleotides directed against translation initiation sites of cPLA 2 , sPLA 2 , CaM kinase II, and MAP kinase were designed (Table I). The PLA 2 antisense oligonucleotide has been used to block lipopolysaccharide-and platelet-activating factor induced prostaglandin production in macrophage-like P388D1 cells (19) and lipopolysaccharide-induced prostaglandin production in monocytes (20).
VSMC were transfected with either cPLA 2 or sPLA 2 sense and antisense oligonucleotides complexed with lipofectamine (4 g/ml) and incubated with medium (Opti-MEM from Life Technologies, Inc.) for 6 h. Thereafter, fresh M-199 medium containing 10% FBS was added, and the cells were incubated for another 18 h. Treatment of VSMC with cPLA 2 , but not with sPLA 2 , antisense oligonucleotides decreased the release of [ 3 H]AA elicited by NE. The inhibitory effect of cPLA 2 antisense on [ 3 H]AA release elicited by NE did not occur when VSMC cotransfected with cPLA 2 sense oligonucleotides ( Fig. 2A). The effect of cPLA 2 antisense oligonucleotides on PLA 2 activity was also examined. VSMC transfected with sense or antisense oligonucleotides were exposed to NE for 15 min, and cell lysates were prepared to study AA release from [ 14 C]arachidonyl phosphatidylcholine (Fig. 2B) antisense, but not sense, oligonucleotides reduced PLA 2 activity in the lysate of VSMC exposed to NE. These data suggest that NE stimulates AA release for prostanoid synthesis in the VSMC via activation of cPLA 2 . Fig. 3 shows an immunoblot of VSMC total lysate proteins probed with monoclonal anti-cPLA 2 . The cPLA 2 migrates at ϳ100 kDa on SDS-PAGE. Western analysis of cells transfected with cPLA 2 antisense oligonucleotides for a longer duration (24 h) showed a significant reduction in the immunoreactive protein. These data positively correlate with the observed PLA 2 activity (Fig. 2B) and also indicate that the action of cPLA 2 antisense was specific for the cPLA 2 . The sense oligonucleotide had little or no effect on the 85-kDa PLA 2 immunoreactive protein.
CaM Kinase II and MAP Kinase Mediate NE-stimulated AA Release-It has been shown that MAP kinase can activate cPLA 2 in vitro by phosphorylating Ser-505 in response to platelet-derived growth factor (7). MAP kinase activation can occur through both protein kinase C-dependent and protein kinase C-independent mechanisms (21,22). MAP kinase-independent regulation of cPLA 2 has also been reported (23). To study the type of kinases involved in NE-induced AA release, VSMC were transfected for 6 h with CaM kinase II or MAP kinase sense and/or antisense oligonucleotides, and the cells were incubated in media containing 10% FBS for another 18 h at 37°C. The specificity of CaM kinase II antisense oligonucleotides to reduce CaM kinase II protein is shown in Fig. 3.
Treatment of VSMC with CaM kinase II antisense, but not sense, oligonucleotides completely abolished the ability of NE to increase [ 3 H]AA release (Fig. 4A). The inhibitory effect of CaM kinase II antisense oligonucleotides on NE-induced AA release was abolished in VSMC that were co-transfected with sense oligonucleotides. The CaM kinase inhibitor KN-93 abolished the NE-induced AA release. KN-93 blocks activation of the kinase by binding to an unidentified site on CaM kinase II and interferes with CaM binding (24). On the other hand, treatment of VSMC with MAP kinase antisense reduced the NE effect by only 30% (Fig. 5A). Moreover, the MEK inhibitor, PD-098059, also partially reduced NE-induced AA release. These results indicate that both CaM kinase II and MAP kinase are involved in NE-stimulated AA release and that CaM kinase II plays a predominant role. NE stimulated CaM kinase and MAP kinase activities in a time-dependent manner (data not shown). Treatment of VSMC with CaM kinase II antisense oligonucleotides and the CaM kinase II inhibitor, KN-93, significantly reduced NE-stimulated CaM kinase II activity (Fig. 4B). Treatment of VSMC with MAP kinase antisense or the MEK inhibitor PD-098059 significantly reduced the NE-stimulated MAP kinase activity (Fig. 5B). These results support a role for CaM kinase II and MAP kinase in NE-stimulated AA release in VSMC of rabbit aorta.
CAM Kinase II and MAP Kinase Mediates NE-induced PLA 2 Activity-To establish the role of CaM kinase and MAP kinase in mediating NE-induced PLA 2 activity, experiments were designed to study PLA 2 activity in the presence of CaM kinase II antisense oligonucleotide, its inhibitor KN-93, and a MEK inhibitor, PD-098059. Treatment of VSMC with CaM kinase II antisense and KN-93 significantly reduced NE-stimulated PLA 2 activity (Fig. 6A). Treatment of VSMC with MAP kinase antisense and MEK inhibitor also significantly decreased NEstimulated PLA 2 activity (Fig. 6B). These results further sup- port our finding that cPLA 2 is activated by both CaM kinase II and MAP kinase. The activity of purified cPLA 2 is insensitive to reduction by DTT (7); likewise, NE-stimulated PLA 2 activity in VSMC was found to be insensitive to DTT included in the buffer. This provides further confirmation that cPLA 2 and not sPLA 2 mediates the ability of NE to release AA in VSMC (data not shown).
CaM Kinase II Acts Upstream of MAP Kinase in the NEinduced PLA 2 Activation-MAP kinase has been reported to activate cPLA 2 and AA release in response to various stimuli in different cell systems (6 -8, 22). In most of the experiments with growth factors, MAP kinase has been implicated in the cPLA 2 activation and AA release. There are no reports on the involvement of any other kinase in cPLA 2 activation. Our results suggest that both CaM kinase II and MAP kinase are involved in NE-induced cPLA 2 activation in VSMC. To determine the sequence of events in cPLA 2 activation, experiments were designed to study the effect of NE on 1) MAP kinase activity in the presence of CaM kinase II antisense oligonucleotides or the CaM kinase II inhibitor, KN-93, and 2) CaM kinase activity in the presence of MAP kinase antisense oligonucleotides and MEK inhibitor, PD-098059. CaM kinase II antisense and KN-93 inhibited MAP kinase activity elicited by NE (Fig.  7A). The MEK inhibitor, PD-098059, also significantly reduced the NE-stimulated MAP kinase activity. On the other hand, MAP kinase antisense oligonucleotides and PD-098059 did not reduce NE-stimulated CaM kinase II activity (Fig. 7B). This suggests that MAP kinase does not activate CaM kinase II and that CaM kinase II acts upstream of MAP kinase in NEstimulated AA release.
It has been reported that phosphorylation is required for the full activation of many enzymes. To determine phosphorylation and activation of MAP kinase and CaM kinase II in response to NE, two approaches were employed. The first approach was to measure 32 P incorporation into these kinases, and the second approach was to measure kinase activity in MAP kinase and CaM kinase II immunoprecipitates using synthetic substrates. Fig. 8 shows that NE increased MAP kinase and CaM kinase II phosphorylation and that MAP kinase phosphorylation was inhibited by CaM kinase II inhibitor, KN-93, and MEK inhib- itor, PD-098059 (Fig. 8A). On the other hand, MEK inhibitor, PD-098059, did not have any effect on CaM kinase II phosphorylation (Fig. 8B). These results suggest that CaM kinase II may be involved in MAP kinase phosphorylation. Table II shows the transfer of phosphate by MAP kinase and CaM kinase II to their respective substrates with NE treatment, and this kinase activity was inhibited by KN-93. In contrast, PD-098059 did not affect the transfer of phosphate to CaM kinase II substrate in VSMC exposed to NE. Collectively, enzyme assays, 32 P incorporation, and immune complex kinase assay confirm the activation of MAP kinase by CaM kinase II. Okadaic acid, a phosphatase inhibitor, which increased phosphorylation of MAP kinase, CaM kinase II (Fig. 8, A and B), and cPLA 2 did not increase [ 3 H]AA release in VSMC (data not shown).
Translocation of cPLA 2 , CaM Kinase II, and MAP Kinase to the Nucleus by NE-cPLA 2 , CaM kinase II, and MAP kinase translocate in response to various agents. MAP kinase and CaM kinase II exhibit isoform-specific targeting to the nucleus (26,27). On the other hand, cPLA 2 is targeted to nuclear membrane (9), and this translocation is Ca 2ϩ -mediated (10). The contribution of phosphorylation and translocation of these enzymes, particularly cPLA 2 , in response to endogenous ligands including NE has not yet been characterized. Therefore, we performed immunofluorescence experiments, and the confocal images were obtained using anti-cPLA 2 , anti-CaM kinase II, and anti-MAP kinase antibodies in NE-stimulated and unstimulated cells in the presence or absence of Ca 2ϩ . Fig. 9 shows the confocal images of VSMC exposed to NE in the presence and absence of extracellular Ca 2ϩ . We also examined the effect of okadaic acid on the translocation of these enzymes in VSMC. It can be clearly seen that these enzymes are initially dispersed throughout the cytoplasm and that, upon stimulation with NE, they translocate to the nucleus. However, in cells that were stimulated with NE in the absence of extracellular Ca 2ϩ , cPLA 2 , CaM kinase II, and MAP kinase did not translocate to the nuclear envelope. The confocal images of VSMC exposed to vehicle of NE (not shown in Fig. 9) were similar to those that were exposed to NE in the absence of extracellular Ca 2ϩ . Okadaic acid, which increased phosphorylation and activities of these enzymes, also did not translocate cPLA 2 , MAP kinase, or CaM kinase II to the nuclear envelope (data not shown). In control experiments, in which the cells were treated with secondary antibody (TRITC-conjugated goat anti-mouse IgG) in the absence of primary antibody but in the presence of IgG, or in cells that were treated with secondary antibody alone, only faint background fluorescence was observed (data not shown). DISCUSSION The present study has led to the following conclusions. 1) NE-induced AA release is mediated via activation of cPLA 2 . 2) This requires concomitant activation of CaM kinase II and   9. Translocation of cPLA 2 (without Ca 2؉ (A) and with Ca 2؉ (B)), CaM kinase II (without Ca 2؉ (C) and with Ca 2؉ (D)), and MAP kinase (without Ca 2؉ (E) and with Ca 2؉ (F)) in response to NE as visualized by confocal microscopy. Arrested VSMC that were exposed to NE (10 M) were visualized using anti-cPLA 2 , CaM kinase II, MAP kinase, and TRITC-conjugated goat anti-mouse IgG.
MAP kinase. CaM kinase II stimulates cPLA 2 by activation of MAP kinase, most probably via MEK. 3) Upon exposure of VSMC to NE, cPLA 2 , CaM kinase II, and MAP kinase translocate to the nuclear membrane in a Ca 2ϩ -dependent manner. We have proposed a novel signaling pathway of MAP kinase activation by CaM kinase II (Fig. 10).
Our results provide evidence that in rabbit VSMC, NE stimulates AA release by activating the 85-kDa cPLA 2 . NE-stimulated PLA 2 activity in VSMC was not altered by the reducing agent DTT, which inactivates sPLA 2 but not cPLA 2 . cPLA 2 but not sPLA 2 antisense oligonucleotides attenuated NE-induced AA release in VSMC. Moreover, cotransfection of VSMC with cPLA 2 but not sPLA 2 sense oligonucleotides prevented the inhibitory effect of cPLA 2 AS on NE-induced AA release.
We also investigated the contribution of CaM kinase II and MAP kinase to the activation of cPLA 2 . MAP kinase has been shown to phosphorylate and activate cPLA 2 in vitro (7,22,28). Several studies have shown that AA release in response to various stimuli is mediated via the activation of cPLA 2 by MAP kinase (7,8,28). However, it has recently been suggested that other uncharacterized kinases are involved in the activation of cPLA 2 (11,12). The demonstration that prostacyclin synthesis elicited by NE in VSMC was inhibited by a calmodulin inhibitor, W-7 (2), suggests that Ca 2ϩ /calmodulin can activate cPLA 2 by stimulating CaM kinase II. This kinase is known to be activated by Ca 2ϩ influx as well as by Ca 2ϩ released from intracellular stores (13). Our results indicate that activation of ␣ 1 /␣ 2 AR with NE in VSMC leads to activation of CaM kinase II and MAP kinase, which in turn stimulate cPLA 2 to release AA by promoting the influx of extracellular Ca 2ϩ . Inhibition of NE-induced release of AA in VSMC transiently transfected with CaM kinase II, MAP kinase antisense oligonucleotides, CaM kinase II inhibitor, KN-93, and MEK inhibitor, PD-098059, strongly support this conclusion. This observation is further supported by our findings that CaM kinase II and MAP kinase antisense oligonucleotides and their respective inhibitors reduced the activity of CaM kinase II and MAP kinase, respectively. In addition, CaM kinase II inhibitor and antisense oligonucleotide reduced the MAP kinase activity. However, MAP kinase antisense and the MEK inhibitor, PD-098059 failed to decrease CaM kinase II activity. This suggests the sequential activation of MAP kinase and cPLA 2 by CaM kinase II. Our findings that NE increased 32 P incorporation into both MAP kinase and CaM kinase II and transfer of phosphates into their respective substrates and that CaM kinase II inhibitor, KN-93, reduced these effects suggest that CaM kinase II mediates MAP kinase phosphorylation. Since MAP kinase antisense oligonucleotides abolished NE-induced MAP kinase activity but reduced only partially the release of AA, we cannot exclude the possibility that CaM kinase II may also directly activate cPLA 2 . Supporting this view was our finding that CaM kinase II antisense or its inhibitor, KN-93, abolished NE-induced PLA 2 activity, whereas a MEK inhibitor, PD-098059, reduced PLA 2 activity by ϳ50%.
For AA to be released from phospholipids, cPLA 2 has to translocate to nuclear membrane, and a kinase must phosphorylate cPLA 2 . However, the sequence of events is not well defined. Translocation of cPLA 2 from cytosol to nuclear membrane has been reported in response to ionophore or IgE/antigen (9,10). This translocation of cPLA 2 appears to be very crucial for its function because of the localization of its substrate and of AA-metabolizing enzymes. The nuclear membrane is an important compartment for uptake and release of arachidonate. EM autoradiography studies have shown that the nuclear membrane exhibits the highest specific activity of [ 3 H]arachidonate labeling (29). Arachidonate compartmentalization within the nuclear membrane, and possibly within certain phospholipids in this membrane, is important for AA release and conversion to eicosanoids (30). Our finding that cPLA 2 and the enzymes that increase its activity, CaM kinase II and MAP kinase, translocate to the nuclear envelope strongly suggests that NE promotes AA release from the nuclear membrane. It has been demonstrated that the Ca 2ϩ -dependent lipid binding domain exists in cPLA 2 with homology to protein kinase C, p65, GTPase-activating protein, and phospholipase C (5). This domain facilitates agonist-stimulated translocation to membranes. Our results showed that cPLA 2 , CaM kinase II, and MAP kinase translocated to the nuclear envelope in a Ca 2ϩ -dependent manner. It has also been reported that mutation at the MAP kinase phosphorylation site of cPLA 2 did not affect ionophore-induced translocation of the enzyme to the nuclear envelope (10), suggesting that translocation is phosphorylation-independent. Okadaic acid, a protein phosphatase inhibitor, which increased MAP kinase, CaM kinase II, and cPLA 2 phosphorylation did not cause release of [ 3 H]AA or translocation of cPLA 2 to the nuclear envelope. Thus phosphorylation of these enzymes does not appear to require translocation to the nuclear envelope. On the other hand, upon NE stimulation, cPLA 2 , CaM kinase II, and MAP kinase translocate to the nuclear envelope in a Ca 2ϩ -dependent manner. Thus Ca 2ϩ may play a significant role in the translocation of these enzymes and in interaction of cPLA 2 with the phospholipid substrate, which is mediated by a Ca 2ϩ -dependent phospholipid binding region.
Ca 2ϩ -dependent MAP kinase activation has been documented in rat cardiac myocytes upon angiotensin II stimulation (31). Recently, Ca 2ϩ -dependent MAP kinase activation by angiotensin II was demonstrated to be mediated by intracellular Ca 2ϩ and CaM (32). Our results provide evidence that CaM kinase II activates MAP kinase in rabbit VSMC. It has been shown that a protein-tyrosine kinase (Pyk2) is involved in relaying messages from protein kinase C and Ca 2ϩ , thus activating Ras-mediated regulation of MAP kinase (33). In Gprotein-coupled muscarinic acetylcholine receptors, tyrosine kinases Lyn and Syk are involved in the MAP kinase signaling cascade (25). Our results indicate that Ca 2ϩ -dependent MAP kinase activation by NE is mediated by CaM kinase II, most probably via MEK. CaM kinase is a Ser/Thr kinase that may activate MEK. The protein-tyrosine kinase that has been implicated in the MAP kinase activation does not have a Ca 2ϩsensing region and CaM-binding motif (32), suggesting the possible involvement of CaM kinase II, which fulfills both criteria. In conclusion, we have demonstrated that NE-induced MAP kinase activation in VSMC is mediated by CaM kinase II. CaM kinase II may be a key player orchestrating several mitogenic signaling pathways via activation of MAP kinase and cPLA 2 , leading to uncontrolled cell growth in VSMC.