Several growth factors, hormones and neurotransmitters, including norepinephrine, increase cellular calcium levels, promoting the translocation of cytosolic phospholipase A2 to the nuclear envelope. This study was conducted to investigate the contributions of the calcium-binding protein calmodulin and of calcium—calmodulin-dependent protein kinase II to cytosolic phospholipase A2 translocation to the nuclear envelope elicited by norepinephrine in rabbit aortic smooth-muscle cells. Norepinephrine caused cytosolic phospholipase A2 accumulation around the nuclear envelope as determined from its immunofluorescence; cytosolic phospholipase A2 translocation was blocked by inhibitors of calmodulin and calcium—calmodulin-dependent protein kinase II or calcium—calmodulin-dependent protein kinase IIα antisense oligonucleotide. Calmodulin and calcium—calmodulin-dependent protein kinase II inhibitors did not prevent cytosolic calcium increase but attenuated cytosolic phospholipase A2 phosphorylation caused by norepinephrine or ionomycin. In vascular smooth-muscle cells reversibly permeabilized with β-escin and treated with alkaline phosphatase, norepinephrine failed to cause cytosolic phospholipase A2 phosphorylation and translocation to the nuclear envelope; these effects of norepinephrine were minimized by the phosphatase inhibitor okadaic acid. Recombinant cytosolic phospholipase A2 phosphorylated by purified calcium—calmodulin-dependent protein kinase II, but not unphosphorylated or dephosphorylated cytosolic phospholipase A2, introduced into permeabilized vascular smooth-muscle cells in the absence of calcium accumulated around the nuclear envelope. These data suggest that norepinephrine-induced translocation of cytosolic phospholipase A2 to the nuclear envelope is mediated by its phosphorylation by calcium—calmodulin-dependent protein kinase II and that calcium alone is insufficient for cytosolic phospholipase A2 translocation to the nuclear envelope in rabbit vascular smooth-muscle cells.

Introduction

Mammalian phospholipase A2 (PLA2) is a large superfamily of enzymes with a common function of catalysing the deacylation of fatty acid from the sn-2 position of membrane phospholipids but with distinct structural and biochemical characteristics (Dennis, 1997; Kramer and Sharp, 1997; Leslie, 1997; Murakami, 1997). Cytosolic PLA2 (cPLA2) is an 85-kDa protein with no sequence homology to other PLA2 species (Kramer and Sharp, 1997; Leslie, 1997; Harwalkar et al., 1998; Kramer et al., 1991; Clark et al., 1991). In a variety of cells, it acts as a neurohumoral receptor-regulated enzyme that mediates release of arachidonic acid (AA) for the biosynthesis of eicosanoids with potent biological actions (Leslie, 1997; Clark et al., 1995). Studies conducted with cPLA2 inhibitors or knockout mice have implicated this enzyme in several pathophysiological processes, including reproduction, allergic responses, post-ischaemic brain injury, cell proliferation and cancer (Uozumi et al., 1997; Bonventre et al., 1997; Anderson et al., 1997; Heasley et al., 1997).

cPLA2 is activated by low levels of calcium (Ca2+) (Kramer et al., 1991; Gronich et al., 1990; Clark et al., 1990; Wijkander and Sundler, 1991), which is not directly involved in its catalytic activity but is believed to be required for its binding to membranes (Wijkander and Sundler, 1992; Nalefski et al., 1994). cPLA2 translocates from cytosol to membranes in the presence of submicromolar Ca2+ concentrations (Clark et al., 1991; Channon and Leslie, 1990; Yoshihara and Watanabe, 1990). The increase in intracellular Ca2+ triggered by Ca2+ ionophores or agonists such as norepinephrine (NE), angiotensin II, bradykinin, epidermal growth factor, IgE-antigen, histamine and thrombin translocates the enzyme from the cytosol to the nuclear envelope or promotes its association with cell membrane fraction (Muthalif et al., 1996; Muthalif et al., 1998; Freeman et al., 1998; Schievella et al., 1995; Kast et al., 1993; Schalkwijk et al., 1995; Glover et al., 1995; Sierra-Honigmann et al., 1996; McNicol and Shibou, 1998). Recently, it has been shown that short duration intracellular Ca2+ [Ca2+]i transients translocate cPLA2 to the Golgi, whereas long [Ca2+]i transients promote its translocation to Golgi, endoplasmic reticulum and perinuclear membrane (Evans et al., 2001). These observations, and the demonstration that AA-metabolizing enzymes also localize to the nuclear envelope, suggest that cPLA2 releases AA for prostanoid production from the membrane phospholipids of the nuclear envelope and adjacent endoplasmic reticulum (Woods et al., 1993; Regier et al., 1995; Coffey et al., 1997; Serhan et al., 1996).

The mechanism by which Ca2+ influx promotes translocation of cPLA2 is not known. Ca2+ produces several of its cellular actions, including activation of Ca2+-calmodulin (CaM)-dependent protein kinase II (CaMKII) and cPLA2, by binding to CaM (Dupont and Goldbeter, 1998). These observations raise the possibility that CaMKII might mediate cPLA2 translocation to the nuclear envelope. To test this hypothesis, we have investigated the effect of inhibitors of CaM and CaMKII on cPLA2 translocation and its phosphorylation in response to NE and ionomycin. The present study demonstrates that phosphorylation of cPLA2 by CaMKII in response to NE mediates its translocation by a mechanism independent of its catalytic activity and that Ca2+ alone is not sufficient for cPLA2 translocation to the nuclear envelope in rabbit vascular smooth-muscle cells (VSMCs).

Materials and Methods

General experimental protocol

VSMC from rabbit aortae were isolated and cultured as described (Nebigil and Malik, 1992). Cells from 3-4 passages were grown to ∼70% or complete confluence on chamber slides (Nalge Nunc, Naperville, IL). The density of cells on slides (∼100-125) for studies on cPLA2 translocation and levels of Ca2+ measurements were similar. The cells were growth arrested overnight in M-199 medium (Sigma, St Louis, MO) containing 0.1% foetal bovine serum. After rinsing three times with 1 ml balanced salt solution with or without Ca2+, they were incubated in the same solution with 10 μM NE (Sigma, St Louis, MO), 1 μM ionomycin (Calbiochem, La Jolla, CA), or their vehicle for 10 minutes at 37°C. For experiments involving treatment with inhibitors, cells were growth arrested in M-199 medium supplemented with 0.1% serum for 16-18 hours. `Serum-starved' cells were then treated with inhibitors of CaM (1 μM calmidazolium (CLMD) and 10 μM W-7, Calbiochem, La Jolla, CA; 10 μM E6-Berbamine (E6-B), Biomol, Plymouth Meeting, PA), CaMKII (10 μM KN-93, Calbiochem, La Jolla, CA) or cPLA2 (10 μM arachidonyl trifluoromethyl ketone (AACOCF3) and 10 μM methyl arachidonyl fluorophosphonate (MAFP) Calbiochem, La Jolla, CA) for 15 minutes followed by treatment with NE (10 μM) or its vehicle for 10 minutes at 37°C. The concentrations of these inhibitors used are similar to those used by other investigators (Asano, 1989; Nebigil and Malik, 1993; Hu et al., 1992; Muthalif et al., 1996; LaBelle and Polyak, 1998). W-5 (10 μM; Biomol, Plymouth Meeting, PA) and KN-92 (10 μM; Calbiochem, La Jolla, CA) are structural analogues of W-7 and KN-93, respectively, and do not inhibit CaM and CaMKII activity; their effects were also examined. In another series of experiments, cells were transiently transfected with CaMKII antisense (GCA GGT GGC GGT GGT CTC CAT, 5 μM), sense (ATG GAG ACC ACC GCC ACC TGC, 5 μM) or scrambled (CCA TGC GTG GTC GTG CGA TGG, 5 μM) oligonucleotides or their vehicle mixed with 2 μg ml-1 lipofectamine and incubated in serum-free M-199 medium for 6 hours and then exposed to NE (10 μM) or its vehicle for 10 minutes as described (Muthalif et al., 1996). The cells treated with various agents were washed with phosphate-buffered saline and cPLA2 distribution was determined by confocal microscopy.

Confocal microscopy and quantitation of confocal images

Cells were viewed by confocal fluorescence microscopy (BioRad MRC-1000, Laser Scanning Confocal Imaging system using an argon-krypton lamp with a 100× objective lens) with anti-cPLA2 monoclonal antibody as described (Muthalif et al., 1996). The original Texas Red confocal images, obtained in red, were converted to black and white images in Adobe® Photoshop® 7.0. This made it easier to quantify the density of cPLA2 accumulation around the nuclear envelope. A fixed number of pixels were quantified in several cells using NIH image 1.62 and the values were averaged for each experiment and grouped for different batches of cells for statistical analysis. Then, the images were converted into pseudocolour in the RGB mode of Adobe Photoshop 7.0 to enhance the visual appearance of cPLA2 distribution around the nuclear envelope.

Measurement of cytosolic Ca2+ levels

VSMCs were loaded with fura-2 (5 μM for 30 minutes at 37°C) and the level of cytosolic Ca2+ was determined as described (Cornwell and Lincoln, 1989). The effects of NE (10 μM) and ionomycin (1 μM) were determined in the presence of inhibitors of CaM (10 μM W-7, 1 μM CLMD and 10 μM E6-B), CaMKII (10 μM KN-93) or their vehicle.

Treatment of cells with phosphatases and phosphatase inhibitors

Cells grown to sub-confluence on slides and arrested for growth were permeabilized with β-escin (Sigma, St Louis, MO) as described (Kobayashi et al., 1989). They were washed to remove β-escin, incubated with 0.5 Unit ml-1 phosphatase (alkaline phosphatase, pH 8.0, or potato acid phosphatase, pH 4.8; Calbiochem, La Jolla, CA) for 15 minutes at 37°C in the presence and absence of serine/threonine phosphatase inhibitor (1 μM okadaic acid; Biomol, Plymouth Meeting, PA), allowed to reseal for 1 hour at 37°C, and then exposed to NE (10 μM), ionomycin (1 μM) or their vehicle for 10 minutes. Cells were washed and processed for confocal microscopy. Permeabilization and resealing were verified by uptake of Texas-Red-conjugated bovine serum albumin (Molecular Probes, Eugene, OR). Earlier studies from our laboratory have shown that VSMCs transiently permeabilized with β-escin and resealed maintain the same responsiveness to NE as nonpermeabilized cells (Nebigil and Malik, 1993). Cell viability was also determined with trypan blue exclusion. More than 95% of the cells treated with phosphatases were viable.

cPLA2 assay

cPLA2 activity was determined from the hydrolysis of substrate L-[1-14C]arachidonyl phosphatidylcholine (40-100 μM, 200 Ci mmol-1; American Radiolabeled Chemicals, St Louis, MO) using 25 μg protein from cell lysates as described (Muthalif et al., 1996).

Phosphorylation and immunoprecipitation of cPLA2

Cells were grown on 100 mm tissue culture dishes to sub-confluence and arrested for growth. Phosphorylation and immunoprecipitation was performed as described (Akiba et al., 1995). Briefly, cells were labelled with 300 μCi ml-132P-orthophosphoric acid (Amersham Pharmacia Biotech, Piscataway, NJ) for 4 hours in phosphate-free Dulbecco's modified Eagle's medium (DMEM) along with inhibitors and treated with NE (10 μM) or ionomycin (1 μM) for 10 minutes. The cells were lysed in HEPES buffer containing protease and phosphatase inhibitors (350 mM sucrose, 1 mM EGTA, 100 μg ml-1 aprotinin and 20 μg ml-1 soybean trypsin inhibitor) and cPLA2 was immunoprecipitated using anti-cPLA2 monoclonal antibodies (Genetics Institute, Cambridge, MA). 32P-labelled cPLA2 immunoprecipitate was subjected to 10% SDS-PAGE. The gel was dried and the radioactivity was detected by autoradiography.

Immunoblotting

To analyse CaMKIIα activity, samples (20 μg of protein) were resolved on 10% SDS-polyacrylamide gels and then transferred to a nitrocellulose membrane. After blocking with 2% milk and 2% BSA in TTBS for 1-2 hours, the membrane was incubated overnight with anti-phospho-CaMKIIα polyclonal antibody (Santa Cruz, San Diego, CA) at 1:1000 dilution in 20 mM Tris, pH 7.6, 137 mM NaCl and 0.05% Tween (TTBS buffer) containing 5% BSA, followed by incubation with anti-goat-IgG-horseradish-peroxidase antibody (1:20,000 dilution in TTBS) for 1 hour at 25°C. The immunoreactive protein was detected using the Amersham ECL Plus system. CaMKII protein levels were detected using anti-CaMKII goat polyclonal antibody (Santa Cruz, San Diego, CA).

In vitro phosphorylation of cPLA2 by CaMKII and its loading in VSMCs

A recent study from our laboratory has shown that CaMKII phosphorylates recombinant cPLA2 or that immunoprecipitated from VSMC in the presence but not absence of Ca2+ and CaM (Muthalif et al., 2001). To prepare phosphorylated cPLA2 for introduction into VSMCs grown on slides, 3 μg of recombinant cPLA2 (Genetics Institute, Cambridge, MA) was incubated for 4 hours with 60 ng of purified rat brain CaMKII (Calbiochem, La Jolla, CA) and 0.4 μg of CaM (Calbiochem, La Jolla, CA) at 30°C in kinase buffer containing 20 mM MOPS (pH 7.2), 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM CaCl2, 75 mM MgCl2, and 500 μM ATP in a total volume of 30 μl. To prepare dephosphorylated cPLA2, 3 μg of phosphorylated cPLA2 was incubated with 0.5 Unit ml-1 alkaline phosphatase at 37°C for 15 minutes in a total volume of 30 μl with 20 mM MOPS (pH 8.0). For unphosphorylated cPLA2, 3 μg of recombinant cPLA2 in 30 μl of 20 mM MOPS (pH 7.2) was used. Aliquots (0.5 μl) of cPLA2 solutions were used to load VSMC reversibly permeabilized with β-escin as described (Nebigil and Malik, 1993). The cells were washed to remove β-escin, incubated with 0.5 μl of cPLA2 solution (unphosphorylated, phosphorylated or dephosphorylated) in the presence of 10 mM ethyleneglycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and 1 μM 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA, both from Calbiochem, La Jolla, CA) for 10 minutes, and then allowed to reseal for 1 hour at 37°C. Permeabilization and resealing were verified, and the cells were processed for confocal microscopy to determine the localization of cPLA2 as described above.

The phosphorylation of cPLA2 by CaMKII and its dephosphorylation by alkaline phosphatase were confirmed by gel-shift analysis of cPLA2 protein. A sample (1 μg) of cPLA2 protein was resolved on 20-cm 10% SDS-polyacrylamide gels (1% bisacrylamide, pH 8.3) and then transferred to a nitrocellulose membrane. Immunoblot analysis with anti-cPLA2 polyclonal antibody at 1:2000 dilution was carried out as described above.

In vitro labelling of phosphorylated, unphosphorylated, and dephosphorylated cPLA2 with fluorescent dye Alexa 488 and its loading in VSMCs

Phosphorylated, unphosphorylated and dephosphorylated recombinant cPLA2 were tagged with the fluorescent dye Alexa 488 according to the manufacturer's protocol (Molecular Probes, Eugene, OR). Briefly, the recombinant cPLA2 was labelled with the reactive dye and the unincorporated dye was separated from that incorporated into the protein by passing through a column packed with the purification resin provided by Molecular Probes. The labelled cPLA2 protein was quantified, and 0.5 μg was loaded into VSMCs reversibly permeabilized with β-escin (20 μM) in the presence of 10 mM EGTA and 1 μM BAPTA; its localization in the cells was viewed by confocal microscopy as described above.

Analysis of data

The density of immunostaining of cPLA2 and its catalytic activity are expressed as mean ± s.e.m. Data were analysed by one-way analysis of variance (ANOVA); the Newman-Keuls multiple-range test was used to determine the difference between multiple groups. The unpaired Student's t-test was used to determine the difference between two groups. A value of P<0.05 was considered to be statistically significant.

Results

Effect of NE and ionomycin on cPLA2 translocation to the nuclear envelope in sub-confluent and confluent VSMCs

Previous studies from our laboratory have shown that extracellular Ca2+ is required for cPLA2 activation and translocation from the cytoplasm to the nuclear envelope in response to NE (10 μM) (Muthalif et al., 1996). In subconfluent VSMCs, cPLA2 was localized in the nucleus as well as in the cytoplasm; in confluent cells; however, cPLA2 was localized mainly in the cytoplasm. NE and ionomycin caused translocation of cPLA2 from both the nucleus and cytoplasm to the nuclear envelope in sub-confluent cells and from the cytoplasm to the nuclear envelope in confluent cells (Fig. 1). In the absence of extracellular Ca2+, NE and ionomycin failed to cause cPLA2 translocation in both sub-confluent and confluent cells (data not shown). When the primary antibody was omitted, no fluorescence was detected, indicating that the fluorescence pattern resulted from primary antibody recognition alone. A similar result was obtained when the secondary antibody was omitted or mouse IgG was used instead of cPLA2 antibody. Propidium iodide was used to visualize nuclei.

Fig. 1.

cPLA2 is localized to the nuclear envelope in sub-confluent and confluent cells in response to norepinephrine (NE) and ionomycin (ION) in VSMCs. Sub-confluent (70% confluent) or confluent (100% confluent) VSMCs from rabbit aorta were stimulated with NE (10 μM), ION (1 μM) or their vehicles (V) for 10 minutes in the presence of 1.8 mM Ca2+. (A) cPLA2 was detected using a monoclonal antibody specific for cPLA2 and a biotinylated second antibody recognized by Texas-Red—streptavidin. Immunofluorescent staining was visualized by confocal microscopy (100× magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope was quantified using the NIH Image 1.62 program as described in Materials and Methods (n=5). *Value significantly different from the corresponding value obtained in the presence of V (P<0.05).

Fig. 1.

cPLA2 is localized to the nuclear envelope in sub-confluent and confluent cells in response to norepinephrine (NE) and ionomycin (ION) in VSMCs. Sub-confluent (70% confluent) or confluent (100% confluent) VSMCs from rabbit aorta were stimulated with NE (10 μM), ION (1 μM) or their vehicles (V) for 10 minutes in the presence of 1.8 mM Ca2+. (A) cPLA2 was detected using a monoclonal antibody specific for cPLA2 and a biotinylated second antibody recognized by Texas-Red—streptavidin. Immunofluorescent staining was visualized by confocal microscopy (100× magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope was quantified using the NIH Image 1.62 program as described in Materials and Methods (n=5). *Value significantly different from the corresponding value obtained in the presence of V (P<0.05).

Effect of inhibitors of CaM and CaMKII on NE-induced cPLA2 translocation to the nuclear envelope in subconfluent cells

Ca2+ is known to produce several of its biological actions by interacting with CaM, including AA release for prostacyclin synthesis in VSMCs (Nebigil and Malik, 1993; Muthalif et al., 1996). To investigate the possible contribution of CaM to NE-induced translocation of cPLA2, the effects of three structurally distinct CaM inhibitors were examined: CLMD (Gietzen et al., 1982), E6-B (Hu et al., 1992) and W-7 (Tanaka et al., 1983). All three inhibitors blocked NE-induced cPLA2 translocation to the nuclear envelope from the cytoplasm and nucleus (Fig. 2). W-5, a structural analogue of W-7 that does not inhibit CaM activity (Hidaka et al., 1981) in concentrations similar to that of W-7, failed to alter NE-induced cPLA2 translocation (data not shown).

Fig. 2.

CaM inhibitors block NE-induced cPLA2 translocation to the nuclear envelope. VSMCs were treated with CaM inhibitors calmidazolium (CLMD, 1 μM), E6-berbamine (E6-B, 10 μM), W-7 (10 μM) or their vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells treated with 10 μM NE or its vehicle (V) and visualized by confocal microscopy (100× magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with V of NE (P<0.05).

Fig. 2.

CaM inhibitors block NE-induced cPLA2 translocation to the nuclear envelope. VSMCs were treated with CaM inhibitors calmidazolium (CLMD, 1 μM), E6-berbamine (E6-B, 10 μM), W-7 (10 μM) or their vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells treated with 10 μM NE or its vehicle (V) and visualized by confocal microscopy (100× magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with V of NE (P<0.05).

Because CaM activates CaMKII in VSMCs (Muthalif et al., 2001), we studied the effect of the CaMKII inhibitor KN-93 and that of an inactive structural analogue, KN-92 (Fan et al., 1997), on NE-induced cPLA2 translocation and CaMKIIα activity. KN-93 (Fig. 3) but not KN-92 (data not shown) inhibited NE-induced cPLA2 translocation from the cytoplasm and nucleus to the nuclear envelope. The CaMKIIα subtype has been shown to be involved in the activation of cPLA2 in VSMCs (Muthalif et al., 1996). To determine the possible involvement of CaMKIIα in the NE-induced cPLA2 translocation, the effect of CaMKIIα antisense, sense and scrambled oligonucleotides were tested. Treatment of cells with CaMKIIα antisense (Fig. 3), but not sense or scrambled, oligonucleotides (data not shown) inhibited NE-induced cPLA2 translocation from the nucleus and cytoplasm to the nuclear envelope. The concentration of CaMKIIα antisense used in these experiments has been previously shown in our laboratory selectively to deplete CaMKII protein levels in rabbit VSMCs (Muthalif et al., 1996). In sub-confluent VSMCs, ionomycin also caused cPLA2 translocation from the nucleus and cytoplasm to the nuclear envelope, which was blocked by inhibitors of CaM (W-7) and CaMKII (KN-93; Fig. 4) but not by their corresponding inactive structural analogues (W-5 and KN-92) at similar concentrations (data not shown). The effect of other CaM inhibitors (CLMD, E6-B) on ionomycin-induced cPLA2 translocation was not tested. In confluent VSMCs, the inhibitors of CaM and CaMKII (data not shown) also blocked VSMC translocation of cPLA2 from the cytoplasm to the nuclear envelope elicited by NE or ionomycin.

Fig. 3.

NE-induced translocation of cPLA2 to the nuclear envelope depends on CaMKIIα. VSMCs were treated with the CaMKII inhibitor KN-93 (10 μM) or its vehicle (VEH), or CaMKIIα antisense oligonucleotide (5 μM) or its vehicle (lipofectamine; LIPO, 2 mg ml-1). (A) cPLA2 translocation to the nuclear envelope in cells exposed to 10 μM NE or its vehicle (V) was visualized by confocal microscopy (100× magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with V of NE (P<0.05).

Fig. 3.

NE-induced translocation of cPLA2 to the nuclear envelope depends on CaMKIIα. VSMCs were treated with the CaMKII inhibitor KN-93 (10 μM) or its vehicle (VEH), or CaMKIIα antisense oligonucleotide (5 μM) or its vehicle (lipofectamine; LIPO, 2 mg ml-1). (A) cPLA2 translocation to the nuclear envelope in cells exposed to 10 μM NE or its vehicle (V) was visualized by confocal microscopy (100× magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with V of NE (P<0.05).

Fig. 4.

Ionomycin (ION)-induced translocation of cPLA2 to the nuclear envelope depends on CaM and CaMKII. VSMCs were treated with CaM inhibitor W-7 (10 μM) CaMKII inhibitor KN-93 (10 μM) or their vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells exposed to 1 μM ION or its vehicle (V) was visualized by confocal microscopy (100× magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with V of ION (P<0.05).

Fig. 4.

Ionomycin (ION)-induced translocation of cPLA2 to the nuclear envelope depends on CaM and CaMKII. VSMCs were treated with CaM inhibitor W-7 (10 μM) CaMKII inhibitor KN-93 (10 μM) or their vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells exposed to 1 μM ION or its vehicle (V) was visualized by confocal microscopy (100× magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with V of ION (P<0.05).

Effect of inhibitors of CaM, CaMKII and their structural analogues on CaMKIIα activity in VSMCs

To determine the selectivity of inhibitors of CaM and CaMKII, the effects of W-5 and KN-92 (the inactive structural analogues of W-7 and KN-93) on CaMKII activity was determined. The inhibitors of CaM (W-7) and CaMKII (KN-93) but not their corresponding structural analogues (W-5 and KN-92) attenuated an NE- or ionomycin-induced increase in CaMKII activity, as detected from immunoblot analysis using phospho-CaMKIIα monoclonal antibodies (Fig. 5). KN-93 at the 10 μM concentration used in this study did not alter the activity of NE-induced increase in phosphorylation of protein kinase Cα, another serine/threonine kinase. Moreover, KN-93 was also effective at concentration as low as 1 μM at inhibiting NE-induced increase in CaMKII activity and cPLA2 translocation to the nuclear envelope (data not shown). These studies and those presented below were performed only on sub-confluent cells.

Fig. 5.

NE- and ionomycin (ION)-induced CaMKIIα activation is attenuated by inhibitors of CaM and CaMKII, and not their structural analogues. VSMCs were treated with NE (10 μM) (A,C), ionomycin (1 μM) (B,D) or vehicle (VEH) in the presence of inhibitors of CaM (10 μM W-7) (A,B), CaMKII (10 μM KN-93) (C,D), their corresponding structural analogues (10 μM W-5, 10 μM KN-92) or their vehicles (V) as described in Materials and Methods. CaMKIIα activity was determined by immunoblot analysis using a polyclonal antibody against active CaMKIIα (phospho-CaMKIIα). The figure shows a representative blot and the densitometric analysis of three experiments performed with each inhibitor and their vehicles on different batches of cells grown in 100 mm tissue culture dishes. The density of CaMKII phosphorylation was quantified using NIH Image 1.62 (n=3). *Value significantly different from the corresponding value obtained with V of NE (A,C) or ION (B,D) (P<0.05).

Fig. 5.

NE- and ionomycin (ION)-induced CaMKIIα activation is attenuated by inhibitors of CaM and CaMKII, and not their structural analogues. VSMCs were treated with NE (10 μM) (A,C), ionomycin (1 μM) (B,D) or vehicle (VEH) in the presence of inhibitors of CaM (10 μM W-7) (A,B), CaMKII (10 μM KN-93) (C,D), their corresponding structural analogues (10 μM W-5, 10 μM KN-92) or their vehicles (V) as described in Materials and Methods. CaMKIIα activity was determined by immunoblot analysis using a polyclonal antibody against active CaMKIIα (phospho-CaMKIIα). The figure shows a representative blot and the densitometric analysis of three experiments performed with each inhibitor and their vehicles on different batches of cells grown in 100 mm tissue culture dishes. The density of CaMKII phosphorylation was quantified using NIH Image 1.62 (n=3). *Value significantly different from the corresponding value obtained with V of NE (A,C) or ION (B,D) (P<0.05).

Effect of CaM and CaMKII inhibitors on NE- and ionomycin-induced increase in cytosolic Ca2+ and cPLA2 phosphorylation in VSMCs

To determine whether Ca2+ is required to activate CaMKII to phosphorylate cPLA2 or directly for cPLA2 translocation to the nuclear envelope, the effects of NE and ionomycin on cytosolic Ca2+ and cPLA2 phosphorylation were investigated in the presence of inhibitors of CaM (W-7) and CaMKII (KN-93). Both NE and ionomycin increased the level of cytosolic Ca2+, which was not altered by W-7 and KN-93 (Fig. 6A). The CaM inhibitors E6-B and CLMD also failed to interfere with the NE-or ionomycin-induced rise in cytosolic Ca2+ (data not shown). NE and ionomycin increased cPLA2 phosphorylation, which was inhibited in the absence of extracellular Ca2+ and by W-7 and KN-93, whereas W-5 and KN-92 had no effect on cPLA2 phosphorylation (Fig. 6B-F).

Fig. 6.

Inhibitors of CaM and CaMKII do not change intracellular Ca2+ levels but block NE and ionomycin (ION)-induced cPLA2 phosphorylation. VSMCs were treated with inhibitors of CaM (10 μM W-7) and CaMKII (10 μM KN-93) or their vehicle (VEH) as described in Materials and Methods. (A) Effect of inhibitors of CaM (10 μM W-7) and CaMKII (10 μM KN-93) or vehicle (VEH) on cytosolic Ca2+ ([Ca]i) was measured using fura-2 in the presence of NE (10 μM), ION (1 μM) or vehicle (V). The figure shows a representative of three experiments performed with each agonist and vehicle, and in the presence of different inhibitors on different batches of cells grown on coverslips. (B) Phosphorylation of cPLA2 in response to NE (10 μM), ION (1 μM) or vehicle (V) in the presence (1.8 mM) or absence of extracellular Ca2+. (C,D) Phosphorylation of cPLA2 in response to NE (10 μM) and ION (1 μM) in the presence or absence of inhibitors of CaM (10 μM W-7), CaMKII (10 μM KN-93). (E,F) Phosphorylation of cPLA2 in response to NE (10 μM) and ION (1 μM) in the presence or absence of the corresponding structural analogues of W-7 and KN-93 (10 μM W-5; 10 μM KN-92). Phosphorylation of cPLA2 was determined by incorporation of 32P and detected by autoradiography as described in Materials and Methods. The figure shows a representative autoradiogram and the densitometric analysis from three experiments repeated with each agonist and the vehicle in the presence and absence of each inhibitor performed in different batches of cells grown in 100 mm tissue culture dishes. The density of cPLA2 phosphorylation was quantified using NIH Image 1.62 (n=3). *Value significantly different from the corresponding value obtained in the absence of Ca2+ (B), in the presence of V of NE (C,E) or ION (D,F) (P<0.05).

Fig. 6.

Inhibitors of CaM and CaMKII do not change intracellular Ca2+ levels but block NE and ionomycin (ION)-induced cPLA2 phosphorylation. VSMCs were treated with inhibitors of CaM (10 μM W-7) and CaMKII (10 μM KN-93) or their vehicle (VEH) as described in Materials and Methods. (A) Effect of inhibitors of CaM (10 μM W-7) and CaMKII (10 μM KN-93) or vehicle (VEH) on cytosolic Ca2+ ([Ca]i) was measured using fura-2 in the presence of NE (10 μM), ION (1 μM) or vehicle (V). The figure shows a representative of three experiments performed with each agonist and vehicle, and in the presence of different inhibitors on different batches of cells grown on coverslips. (B) Phosphorylation of cPLA2 in response to NE (10 μM), ION (1 μM) or vehicle (V) in the presence (1.8 mM) or absence of extracellular Ca2+. (C,D) Phosphorylation of cPLA2 in response to NE (10 μM) and ION (1 μM) in the presence or absence of inhibitors of CaM (10 μM W-7), CaMKII (10 μM KN-93). (E,F) Phosphorylation of cPLA2 in response to NE (10 μM) and ION (1 μM) in the presence or absence of the corresponding structural analogues of W-7 and KN-93 (10 μM W-5; 10 μM KN-92). Phosphorylation of cPLA2 was determined by incorporation of 32P and detected by autoradiography as described in Materials and Methods. The figure shows a representative autoradiogram and the densitometric analysis from three experiments repeated with each agonist and the vehicle in the presence and absence of each inhibitor performed in different batches of cells grown in 100 mm tissue culture dishes. The density of cPLA2 phosphorylation was quantified using NIH Image 1.62 (n=3). *Value significantly different from the corresponding value obtained in the absence of Ca2+ (B), in the presence of V of NE (C,E) or ION (D,F) (P<0.05).

Effect of phosphatases and phosphatase inhibitors on NE-induced cPLA2 translocation in VSMCs

To assess the contribution of phosphorylation to NE-induced cPLA2 translocation to the nuclear envelope, alkaline phosphatase (0.5 Unit ml-1) was introduced to VSMCs by reversibly permeabilizing them with β-escin in the presence or absence of the phosphatase inhibitor okadaic acid (1 μM). Earlier studies from our laboratory have shown that VSMCs transiently permeabilized and resealed maintained the same responsiveness to NE as nonpermeabilized cells (Nebigil and Malik, 1993). Okadaic acid alone did not cause cPLA2 translocation to the nuclear envelope. Treatment with alkaline phosphatase in the absence, but not the presence, of okadaic acid blocked NE-induced cPLA2 translocation (Fig. 7A,B), suggesting that Ca2+-induced phosphorylation of cPLA2 requires its translocation to the nuclear envelope. Similar results were obtained with potato acid phosphatase (data not shown).

Fig. 7.

NE-induced translocation of cPLA2 to the nuclear envelope depends on phosphorylation. VSMCs were treated with okadaic acid (OA; 10 μM), alkaline phosphatase (AP; 0.5 Unit ml-1; loaded by reversible permeabilization with β-escin), OA in combination with AP (OA+AP) or vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells treated with 10 μM NE or its vehicle (V) was visualized by confocal microscopy (100× magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from that obtained with V of NE (P<0.05). (C) cPLA2 activity in VSMCs treated with OA, AP, OA+AP or VEH as described in Materials and Methods (n=5). (D) Phosphorylation of cPLA2 as determined by 32P incorporation in cells treated with OA, AP, OA+AP or VEH. A representative of three experiments performed with each agent or its vehicle on different batches of cells grown in 100 mm tissue culture dishes. (E) Density of cPLA2 phosphorylation was quantified using NIH Image 1.62 (densitometric analysis of D).

Fig. 7.

NE-induced translocation of cPLA2 to the nuclear envelope depends on phosphorylation. VSMCs were treated with okadaic acid (OA; 10 μM), alkaline phosphatase (AP; 0.5 Unit ml-1; loaded by reversible permeabilization with β-escin), OA in combination with AP (OA+AP) or vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells treated with 10 μM NE or its vehicle (V) was visualized by confocal microscopy (100× magnification). (B) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from that obtained with V of NE (P<0.05). (C) cPLA2 activity in VSMCs treated with OA, AP, OA+AP or VEH as described in Materials and Methods (n=5). (D) Phosphorylation of cPLA2 as determined by 32P incorporation in cells treated with OA, AP, OA+AP or VEH. A representative of three experiments performed with each agent or its vehicle on different batches of cells grown in 100 mm tissue culture dishes. (E) Density of cPLA2 phosphorylation was quantified using NIH Image 1.62 (densitometric analysis of D).

NE and okadaic acid increased cPLA2 activity about fourfold over their vehicle controls. The activity of cPLA2 in response to NE was further enhanced in the presence of okadaic acid. Alkaline phosphatase reduced cPLA2 activity in both the absence and presence of NE; the reduction was minimized in the presence of okadaic acid. However, NE in the presence of okadaic acid and alkaline phosphatase caused the same order of increase in cPLA2 activity as NE alone (Fig. 7C).

NE-induced cPLA2 phosphorylation was also increased in the presence of okadaic acid. Alkaline phosphatase inhibited NE-induced cPLA2 phosphorylation, which was restored in the presence of okadaic acid (Fig. 7D,E). Alkaline phosphatase also inhibited ionomycin-induced phosphorylation of cPLA2, which was restored in the presence of okadaic acid (data not shown). Treatment of cells with alkaline phosphatase also caused dephosphorylation of CaMKII, which was prevented by okadaic acid (data not shown). Therefore, it appears that phosphorylation caused by an increase in cytosolic Ca2+ in response to NE and ionomycin is required both for cPLA2 activation and for its translocation to the nuclear envelope in VSMCs.

Effect of inhibitors of cPLA2 activity on NE-induced cPLA2 translocation in VSMCs

To determine whether both phosphorylation and catalytic activity of cPLA2 are required for NE-induced translocation to the nuclear envelope, VSMCs were treated with two different inhibitors of cPLA2 catalytic activity — AACOCF3 (10 μM) and MAFP (10 μM) — and the effects of NE on cPLA2 translocation, activity and phosphorylation were examined. Neither inhibitor prevented NE from causing cPLA2 translocation to the nuclear envelope (Fig. 8A,B); the inhibitors attenuated cPLA2 activity (Fig. 8C) but failed to alter NE-induced cPLA2 phosphorylation (Fig. 8D,E). These observations indicate that the phosphorylation, but not the catalytic activity of cPLA2, is essential for its translocation to the nuclear envelope from the cytosol and nucleus in VSMCs.

Fig. 8.

NE-induced translocation of cPLA2 to the nuclear envelope depends on its phosphorylation and not its activity. VSMCs were treated with inhibitors of cPLA2 activity arachidonyltrifluoromethyl ketone (AACOCF3; 10 μM) and methyl arachidonyl fluorophosphonate (MAFP; 10 μM) or their vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells treated with 10 μM NE or its vehicle (V) was visualized by confocal microscopy (100× magnification). (B) Density of fluorescence around the nuclear envelope was quantified (n=5). *Value significantly different from that obtained with V of NE (P<0.05). (C) cPLA2 activity in VSMCs treated with AACOCF3, MAFP or VEH in the presence of NE (10 μM) or its vehicle (V) as described in Materials and Methods (n=5). (D) Phosphorylation of cPLA2 as determined by 32P incorporation in VSMCs treated with AACOCF3, MAFP or their vehicle (VEH) and treated with NE (10 μM) or its vehicle (V). The figures shows a representative of three experiments performed with each agent and its vehicle on different batches of cells grown in 100 mm tissue culture dishes. (E) Density of cPLA2 phosphorylation was quantified using NIH Image 1.62 (densitometric analysis of D).

Fig. 8.

NE-induced translocation of cPLA2 to the nuclear envelope depends on its phosphorylation and not its activity. VSMCs were treated with inhibitors of cPLA2 activity arachidonyltrifluoromethyl ketone (AACOCF3; 10 μM) and methyl arachidonyl fluorophosphonate (MAFP; 10 μM) or their vehicle (VEH). (A) cPLA2 translocation to the nuclear envelope in cells treated with 10 μM NE or its vehicle (V) was visualized by confocal microscopy (100× magnification). (B) Density of fluorescence around the nuclear envelope was quantified (n=5). *Value significantly different from that obtained with V of NE (P<0.05). (C) cPLA2 activity in VSMCs treated with AACOCF3, MAFP or VEH in the presence of NE (10 μM) or its vehicle (V) as described in Materials and Methods (n=5). (D) Phosphorylation of cPLA2 as determined by 32P incorporation in VSMCs treated with AACOCF3, MAFP or their vehicle (VEH) and treated with NE (10 μM) or its vehicle (V). The figures shows a representative of three experiments performed with each agent and its vehicle on different batches of cells grown in 100 mm tissue culture dishes. (E) Density of cPLA2 phosphorylation was quantified using NIH Image 1.62 (densitometric analysis of D).

Distribution of exogenous phosphorylated, unphosphorylated, and dephosphorylated cPLA2 in VSMCs

Phosphorylation of recombinant cPLA2 by CaMKIIα and its dephosphorylation by alkaline phosphatase was confirmed by its gel shift (Fig. 9A). In VSMCs loaded with cPLA2 after reversible permeabilization with β-escin in the presence of EGTA and BAPTA, the phosphorylated, but not unphosphorylated or dephosphorylated, form of cPLA2, as detected by immunofluorescence (Fig. 9B) or by conjugation to Alexa 488 (Fig. 9C), was found to accumulate around the nucleus.

Fig. 9.

Phosphorylation-dependent gel shift of cPLA2 (A) and localization of phosphorylated recombinant cPLA2 to the nuclear envelope in cells reversibly permeabilized with β-escin (B,C). (A) Recombinant cPLA2 unphosphorylated (UP), phosphorylated (P) by purified CaMKII or after dephosphorylation (DP) by alkaline phosphatase (0.5 U ml-1) was separated on 10% SDS-PAGE and analysed for gel shift as described in Materials and Methods. (B) VSMCs were reversibly permeabilized with β-escin to introduce cPLA2 (0.5 μg) that was unphosphorylated, phosphorylated by CaMKII or dephosphorylated by AP (0.5 Unit ml-1) and detected by immunofluorescence staining. Phosphorylated, but not unphosphorylated or dephosphorylated, cPLA2 translocated to the nuclear envelope (representative of three experiments). In nonpermeabilized cells, unphosphorylated, phosphorylated and dephosphorylated cPLA2 did not translocate to the nuclear envelope. (C) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with unphosphorylated (UP) cPLA2 in permeabilized cells (P<0.05). (D) Unphosphorylated, phosphorylated and dephosphorylated cPLA2 (0.5 μg) conjugated with fluorescence-tagged Alexa 488 was introduced in VSMCs reversibly permeabilized with β-escin. The figure shows a representative of three experiments. Phosphorylated, but not unphosphorylated or dephosphorylated, cPLA2 accumulated around the nuclear envelope.

Fig. 9.

Phosphorylation-dependent gel shift of cPLA2 (A) and localization of phosphorylated recombinant cPLA2 to the nuclear envelope in cells reversibly permeabilized with β-escin (B,C). (A) Recombinant cPLA2 unphosphorylated (UP), phosphorylated (P) by purified CaMKII or after dephosphorylation (DP) by alkaline phosphatase (0.5 U ml-1) was separated on 10% SDS-PAGE and analysed for gel shift as described in Materials and Methods. (B) VSMCs were reversibly permeabilized with β-escin to introduce cPLA2 (0.5 μg) that was unphosphorylated, phosphorylated by CaMKII or dephosphorylated by AP (0.5 Unit ml-1) and detected by immunofluorescence staining. Phosphorylated, but not unphosphorylated or dephosphorylated, cPLA2 translocated to the nuclear envelope (representative of three experiments). In nonpermeabilized cells, unphosphorylated, phosphorylated and dephosphorylated cPLA2 did not translocate to the nuclear envelope. (C) Density of cPLA2 fluorescence around the nuclear envelope (n=5). *Value significantly different from the corresponding value obtained with unphosphorylated (UP) cPLA2 in permeabilized cells (P<0.05). (D) Unphosphorylated, phosphorylated and dephosphorylated cPLA2 (0.5 μg) conjugated with fluorescence-tagged Alexa 488 was introduced in VSMCs reversibly permeabilized with β-escin. The figure shows a representative of three experiments. Phosphorylated, but not unphosphorylated or dephosphorylated, cPLA2 accumulated around the nuclear envelope.

Discussion

Several neurohumoral agents, including NE, angiotensin II, bradykinin and histamine, increase cellular levels of Ca2+, which promotes the translocation of cPLA2 from the cytosol to the nuclear envelope and AA release (Muthalif et al., 1996; Muthalif et al., 1998; Freeman et al., 1998; Kast et al., 1993; Sierra-Honigmann et al., 1996). The present study demonstrates that, in rabbit VSMCs, NE-induced cPLA2 translocation to the nuclear envelope is mediated by its phosphorylation by CaMKII and that increased cytosolic Ca2+ alone is not sufficient for its translocation to the nuclear envelope in response to NE.

In sub-confluent cells, cPLA2 (as detected by immunostaining) was distributed in both the cytosol and the nucleus. However, in confluent VSMCs, cPLA2 was localized primarily to the cytosol. Exposure to NE or ionomycin of both sub-confluent and confluent cells caused accumulation of cPLA2 in the perinuclear region and the nuclear envelope. Similar distribution of cPLA2 in sub-confluent and confluent endothelial cells, and its translocation to the perinuclear region and the nuclear envelope in response to histamine have been reported (Sierra-Honigmann et al., 1996). Ca2+ has been shown to be essential for the binding of cPLA2 to phospholipid vesicles or membranes (Clark et al., 1991; Wijkander and Sundler, 1992; Nalefski et al., 1994; Channon and Leslie, 1990; Yoshihara and Watanabe, 1990). The enzyme contains an N-terminal Ca2+-dependent phospholipid-binding domain (CaLB) that is believed to bind Ca2+ and to promote the attachment of cPLA2 to membranes (Nalefski and Falke, 1998; Zhang et al., 1996; Xu et al., 1998); deletion of this but not the C-terminal domain prevents its membrane binding (Nalefski et al., 1994). In our study, in the absence of extracellular Ca2+, NE and ionomycin failed to cause translocation of cPLA2 to the nuclear envelope in both sub-confluent and confluent VSMCs. Although NE causes release of intracellular Ca2+ in the VSMCs via activation of α-1 adrenergic receptors (Nebigil and Malik, 1993), this appears to be insufficient to cause redistribution and activation of cPLA2 (Muthalif et al., 1996). Our findings that, first, three structurally distinct inhibitors of the Ca2+-binding protein CaM (CLMD, E6-B and W-7) blocked NE- or ionomycin-induced cPLA2 accumulation around the nuclear envelope in both sub-confluent and confluent cells and, second, that W-5 (a structural analogue of W-7 that does not inhibit CaM activity at similar concentrations) failed to alter NE- or ionomycin-induced cPLA2 translocation suggest an involvement of CaM in cPLA2 translocation to the nuclear envelope in VSMCs.

CaM is known to produce several of its cellular actions by activating CaMKII (Soderling et al., 1990). CaMKII has been reported to cause activation of cPLA2 and to be translocated to the nucleus in response to NE in VSMCs (Muthalif et al., 1996). Our demonstration that CaMKII inhibitor KN-93 and CaMKII antisense, which decrease CaMKII phosphorylation and activity (Muthalif et al., 1998), but not KN-92 (an inactive structural analogue of KN-93), blocked translocation of cPLA2 to the nuclear envelope in both sub-confluent and confluent cells suggests that CaMKII mediates cPLA2 translocation. Inhibitors of CaM and CaMKII did not alter the NE-induced rise in cytosolic Ca2+ levels. Moreover, the effect of the Ca2+ ionophore ionomycin to cause cPLA2 translocation, but not to increase cytosolic Ca2+ levels, was also blocked by inhibitors of CaM (W-7) and CaMKII (KN-93). These observations strongly suggest that CaM-activated CaMKII mediates the effect of increased cytosolic Ca2+ to promote cPLA2 translocation to the nuclear envelope in VSMCs. Because NE and ionomycin caused cPLA2 phosphorylation and inhibitors of CaM (W-7) and CaMKII (KN-93) but not their respective structural analogues (W-5 and KN-92) blocked cPLA2 phosphorylation and increase its catalytic activity in response to NE and ionomycin, it would appear that phosphorylation and activation of cPLA2 is required for its translocation to the nuclear envelope. However, our demonstration that AACOCF3 and MAFP inhibited cPLA2 activity but not its phosphorylation in response NE in VSMCs suggests that cPLA2 phosphorylation, but not its activity, is an essential component of the mechanism for its translocation to the nuclear envelope.

That cPLA2 phosphorylation by CaMKII mediates its translocation induced by NE and ionomycin is supported by our observation that this was prevented by treatment of VSMCs with alkaline or potato acid phosphatase, which inhibited the phosphorylation of cPLA2 and CaMKII. Moreover, the inhibitory effect of phosphatases on NE- and ionomycin-induced cPLA2 phosphorylation and translocation was attenuated in the presence of the phosphatase inhibitor okadaic acid. Although okadaic acid alone increased the extent of cPLA2 phosphorylation and its catalytic activity, this was less than that caused by NE or ionomycin and was insufficient to cause cPLA2 translocation to the nuclear envelope. Therefore, it appears that phosphorylation of cPLA2 by Ca2+—CaM-dependent CaMKII in response to NE and ionomycin is responsible for its translocation to the nuclear envelope. The effect of phosphatases to prevent cPLA2 translocation and phosphorylation in response to NE or ionomycin was not due to their cytotoxic effects, because they did not cause any morphological change or entry of Texas-Red-conjugated BSA or trypan blue into VSMCs.

Direct evidence that phosphorylation of cPLA2 by CaMKII promotes its accumulation around the nuclear envelope was provided by our demonstration that recombinant cPLA2 phosphorylated by purified CaMKII in vitro (but not cPLA2 that was unphosphorylated or dephosphorylated by treatment with alkaline phosphatase) was localized around the nuclear envelope in VSMCs that had been reversibly permeabilized by β-escin. Similarly, cPLA2 cross-linked with Alexa 488 and phosphorylated by CaMKII, but not unphosphorylated or dephosphorylated cPLA2, was found to be localized around the nuclear envelope in reversibly permeabilized cells in the absence of extracellular Ca2+ and the presence of EGTA and the intracellular Ca2+ chelator BAPTA. These findings also support the view that cPLA2 phosphorylation by CaMKII mediates its translocation from the nucleus and cytosol to the nuclear envelope in the rabbit VSMCs. The accumulation of phosphorylated but not dephosphorylated cPLA2 to the nuclear envelope in the absence of Ca2+ suggests that dephosphorylation and not lack of Ca2+ promotes the dissociation of cPLA2 from the nuclear envelope in the rabbit VSMCs.

The site of phosphorylation of cPLA2 by CaMKII that is involved in its translocation and association with components of the nuclear envelope and/or endoplasmic reticulum is not known. Recently, we have reported that CaMKII phosphorylates cPLA2 at Ser515 in response to NE in VSMCs (Muthalif et al., 2001). Therefore, it is possible that phosphorylation of cPLA2 at this site might be responsible for its translocation to the nuclear envelope by NE. However, further studies on the translocation of cPLA2 mutated at this site (S515A) are required to address this issue. cPLA2 is also known to be phosphorylated on Ser505 and/or on Ser727 by mitogen-activated-protein kinases (MAPK) ERK1/2 and p38 MAPK and by MAPK-activated protein kinase 1 (MNK-1) or a related kinase (Lin et al., 1993; Borsch-Haubold et al., 1998; Gijon and Leslie, 1999; Gijon et al., 2000; Hefner et al., 2000). In embryonic chick heart cells, β-2-adrenergic-receptor-stimulated cPLA2 translocation to the perinuclear region and AA release have been reported to be mediated by ERK1/2 and p38 MAPK activation (Magne et al., 2001). However, in CHO cells the Ca2+ ionophore A23187 caused translocation of cPLA2 that was mutated at the site (S505A) phosphorylated by these kinases (Schievella et al., 1995). Because, in VMSCs, ERK1/2 is phosphorylated by AA metabolites generated by CaMKII-stimulated cPLA2, and the inhibitor of cPLA2 activity MAFP [which attenuates ERK1/2 phosphorylation (Muthalif et al., 1998)] did not alter cPLA2 translocation to the nuclear envelope, it is unlikely that ERK1/2 phosphorylation is required for cPLA2 translocation to the nuclear envelope in response to NE in VSMCs. p38 MAPKs, which also cause phosphorylation and/or activation of cPLA2 in some cell systems (Kramer et al., 1995; Waterman et al., 1996; Kramer et al., 1996; Borsch-Haubold et al., 1998; Hiller and Sundler, 1999), do not mediate NE-induced activation or translocation of cPLA2 to the nuclear envelope in VSMCs (Fatima et al., 2001).

The mechanism by which cPLA2 binds to the perinuclear membranes is not known. In MDCK cells, depletion of ATP has been shown to result in Ca2+-induced translocation of cPLA2 to the perinculear region (Scheridan et al., 2001). It is not known whether the translocation of cPLA2 produced by neurohumoral agents is due to decreased ATP levels in VSMCs. In HEK 293 cells stably transfected with cPLA2, the Ca2+ ionophore A23187 promotes co-localization of cPLA2 with the intermediate filament protein vimentin around the perinuclear region by an interaction between C2 domain of cPLA2 and the head domain of vimentin (Nakatani et al., 2000). Therefore, it is possible that phosphorylated cPLA2 introduced into permeabilized VSMCs, which was translocated to the nuclear envelope in the presence of EGTA and BAPTA, might bind with vimentin upon translocation to the nuclear envelope.

Although phosphorylation of cPLA2 results in its activation, it has been reported to be insufficient for its maximal activation and AA release (Shalkwijk et al., 1995; Qiu et al., 1998; Gijon and Leslie, 1999). Moreover, it has been shown that, for continuous membrane localization and full activation of cPLA2, a critical time period of increased cytosolic Ca2+ level is required (Hirabayashi et al., 1999). It has been reported that mutation of the Ca2+-binding residues in the C2 domain leads to a higher Ca2+ requirement for cPLA2 binding to its substrates and its activity (Bittova et al., 1999). However, our demonstration that the inhibitors of cPLA2 AACOCF3 and MAFP, which attenuated NE-induced cPLA2 activation and AA release in VSMCs (Muthalif et al., 1996; LaBelle and Polyak, 1998), failed to block the phosphorylation and translocation of cPLA2 to the nuclear envelope suggests that the release of AA in response to NE is independent of cPLA2 translocation to the nuclear envelope. These observations also raise the possibility that NE might release AA from sites other than the nuclear envelope in VSMCs. The significance of cPLA2 translocation to the nuclear envelope during inhibition of its activity in response to NE is not known. One might speculate that the translocation of cPLA2 to the nuclear envelope in response to NE in VSMCs could be a part of an inactivating rather than an activating mechanism of cPLA2 (i.e. transient removal of cPLA2 away from its site(s) of action).

In conclusion, the present study provides an evidence that cPLA2 translocation from the cytosol to the nuclear envelope in response to the adrenergic transmitter NE is mediated via phosphorylation of cPLA2 by Ca2+—CaM-dependent CaMKII, and that Ca2+ does not directly cause cPLA2 translocation to the nuclear envelope. Moreover, cPLA2 translocation to the nuclear envelope is independent of its catalytic activity.

Acknowledgements

We thank Anne Estes for her excellent technical assistance, Genetics Institute (Cambridge, MA; a division of Wyeth) for providing us with a generous supply of cPLA2 and its antibody, Lauren M. Cagen for scientific discussions and Jin Emerson-Cobb for editing the manuscript. This work was supported by NHLBI, National Institutes of Health, Grant 19134-28 (to K.U.M.) and Centers for Connective Tissue Diseases and Vascular Biology, and by postdoctoral fellowships from the Neuroscience Center and American Heart Association, Southeast Affiliate (to S.F.), NIH Training Grants HL-07641-11 (to A.A. and F.A.Y.) and NIAMS 07317-22 (Z.K.).

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