Introduction

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Signal transduction from the cell surface to the nucleus largely relies on a family of cytoplasmic protein kinases, the mitogen-activated protein kinases (MAPKs), which upon activation, translocate into the nucleus where they phosphorylate and regulate transcription factors. MAPKs consist of at least three groups: the extracellular signal-regulated kinases (ERK1 and ERK2), the N-terminal c-Jun kinases, and p38 kinase (Whitmarsh and Davis, 1996; Robinson and Cobb, 1997). A widely used strategy to explore the role of ERK and two members of the p38 group (p38 and p38) in various cell functions is the use of specific inhibitors. U0126 inhibits ERK activation by MEK (MAPK kinase; Favata et al., 1998), whereas SB203580 directly inhibits p38 and p38 but not other p38 kinases (Lee et al., 1994; Cuenda et al., 1997; Kumar et al., 1997). A prerequisite for unambiguous interpretation of experimental data using these inhibitors is the knowledge of possible additional effects. We used SB203580 to explore the possible role of p38 in norepinephrine (NE)-induced cPLA₂ translocation in rabbit aortic vascular smooth muscle cells (VSMCs). However, we made an unexpected observation that SB203580 activates and promotes translocation of cPLA₂ to the nuclear envelope in VSMCs. In quiescent cells, cPLA₂ is located in the cytoplasm; but in response to growth factors and neurohumoral agents, cPLA₂ is phosphorylated, activated, and translocated to the nuclear envelope (Yoshihara and Watanabe, 1990; Glover et al., 1995; Schalkwijk et al., 1995; Muthalif et al., 1996; Freeman et al., 1998). Calcium (Ca²⁺) has been shown to play an important role in cPLA₂ translocation (Glover et al., 1995; Schievella et al., 1995; Muthalif et al., 1996; Gijon et al., 1999; Perisic et al., 1999). Studies from our laboratory have provided evidence for the role of calmodulin (Nebigil and Malik, 1993) and Ca²⁺/calmodulin-dependent kinase II (CaMKII; Muthalif et al., 1996) in the increase in cPLA₂ activity in response to NE in VSMCs. In this study, we present evidence that SB203580 causes phosphorylation and activation of cPLA₂ in a Ca²⁺-, calmodulin-, and CaMKII-dependent fashion, promotes cPLA₂ translocation to the nuclear envelope, and releases AA.

	Experimental Procedures

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Materials. Monoclonal antibodies against phospho-CaMKII and SB203580, SB202190, PD169316, SB202474, W-7, W-5, KN-92, nifedipine, FPT-III, and baicalein were purchased from Calbiochem (San Diego, CA), U0126 from Promega (Madison, WI) and 17-octadecynoic acid (17-ODA) from Cayman Chemical (Ann Arbor, MI). Monoclonal cPLA₂ antibody was provided as a gift from Genetics Institute, Inc. (Cambridge, MA). Phospho-p38- and phospho-ERK1/2-specific antibodies were obtained from New England Biolabs, Inc. (Beverly, MA), tissue culture media from Meditech, Inc. (Herndon, VA), and p38 antibody, anisomycin, and fetal bovine serum from Sigma (St. Louis, MO). Protein A agarose was purchased from Roche Molecular Biochemicals (Indianapolis, IN). [³H]Arachidonic acid (200 Ci/mmol) and L-[1-¹⁴C]arachidonyl phosphatidylcholine (200 Ci/mmol) were obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). [³²P]Orthophosphoric acid and the ECL kit were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). KN-93 was purchased from Seikagaku America (Falmouth, MA).

VSMC Cultures and Experimental Protocol. Rabbit aortic VSMCs were prepared as described previously (Nebigil and Malik, 1992). Cells were grown in M-199 medium supplemented with 10% fetal bovine serum, 200 U/ml penicillin, and 0.2 mg/ml streptomycin. VSMCs were starved in serum-free medium for 12 to 16 h. After being rinsed three times with M-199 medium, they were incubated in the same medium with 0.8, 10, 20, 30, or 50 µM SB203580 or vehicle for 15 min at 37°C. For experiments involving treatment with inhibitors, cells were growth-arrested in serum-free M-199 medium for 16 to 18 h. "Serum-starved" cells were then treated with an inhibitor of Ca²⁺ channels (nifedipine, 1 µM); calmodulin (W-7, 10 µM; or its inactive analog W-5, 10 µM); CaMKII (KN-93, 10 µM; or its inactive analog KN-92, 10 µM) for 15 min; with MEK inhibitor (U0126, 10 µM) for 1 h; and with Ras inhibitor (FPT-III, 25 µM) for 12 h; followed by treatment with SB203580 (20 µM) and with 17-ODA (5 µM) and baicalein (5 µM). VSMCs were preincubated with anisomycin (1 µM) or its vehicle for 15 min at 37°C followed by treatment with SB203580 (20 µM), SB202474 (20 µM), SB202190 (20 µM), PD169316 (20 µM), or their vehicles for an additional 15 min at the same temperature. The concentrations of all the drugs used in this study are similar to those employed by other investigators (Börsch-Haubold et al., 1998; Hedges et al., 2000; Wang et al., 2000). The cells treated with various agents were washed with phosphate-buffered saline and cPLA₂ distribution was determined by confocal microscopy.

Measurement of cPLA₂, CaMKII, ERK1/2, and p38 Kinase Activities. After stimulation as indicated, cells were washed three times with phosphate-buffered saline and lysed in kinase buffer (1 ml/10-cm plate). The activities of cPLA₂, CaMKII, ERK1/2, and p38 kinase were measured by the following methods.

cPLA₂ Assay For cPLA₂ assay, cells grown in 10-cm culture plates were growth-arrested for 24 h and treated with or without SB203580 and lysed in Tris buffer containing protease and phosphatase inhibitors [10 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin]. The concentration of protein was determined by Bradford assay (Bio-Rad, Hercules, CA). cPLA₂ activity in VSMC lysate fractions (20-30 µg of protein/assay) was measured using [¹⁴C]arachidonyl phosphatidylcholine as substrate as described previously (Muthalif et al., 1996).

CaMKII, ERK1/2, and p38 Activities. CaMKII, ERK1/2, and p38 activities were measured by determining the phosphorylation status of CaMKII, ERK1/2, and p38, respectively. Cell lysates (80 µg) were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting. The blots were incubated with phospho-specific antibodies (1:1000), and the bands were visualized by ECL using a protocol supplied by the manufacturer.

Phosphorylation and Immunoprecipitation of cPLA₂. Cells were grown on 10-cm culture plates to subconfluency and arrested for growth. Phosphorylation and immunoprecipitation were performed as described (Akiba et al., 1995). Briefly, cells were labeled with 300 µCi/ml [³²P]orthophosphoric acid for 4 h in phosphate-free Dulbecco's modified Eagle's medium and treated with SB203580 (10, 20, 30, or 50 µM) or vehicle for 15 min. When inhibitors were used, the cells were treated with the inhibitor after 4 h of labeling with [³²P]orthophosphoric acid and then stimulated with SB203580 (20 µM) or anisomycin (1 µM). The cells were lysed in HEPES buffer containing protease and phosphatase inhibitors (10 mM Tris [pH 7.4], 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and cPLA₂ was immunoprecipitated using anti-cPLA₂ monoclonal antibodies. ³²P-Labeled cPLA₂ immunoprecipitate was subjected to 10% SDS-polyacrylamide gel electrophoresis. The gel was dried and the radioactivity was detected by autoradiography.

Measurement [³H]AA Release from VSMCs. The release of AA and its tritiated products from VSMCs was measured as described (Muthalif et al., 1996). Briefly, cells were washed with Hanks' balanced salt solution and exposed to SB203580 in balanced salt solution containing bovine serum albumin for 15 min at 37°C. [³H]AA released into extracellular medium and that remaining in the VSMCs was measured by liquid scintillation spectroscopy. Total radioactivity in the cells was determined after treating the cells with 1 M NaOH overnight. [³H] released into the medium was expressed as a percentage of the total cellular radioactivity and referred to as fractional release.

Confocal Microscopy. Cells were viewed by confocal fluorescence microscopy (Bio-Rad MRC-1000 Laser Scanning Confocal Imaging system using an argon/krypton lamp with a 100 objective lens) with anti-cPLA₂ monoclonal antibody as a primary antibody and Texas Red-conjugated mouse IgG as a secondary antibody as described earlier (Muthalif et al., 1998a).

Analysis of Data. Data were analyzed by one-way analysis of variance; the Newman-Keuls multiple range test was used to determine the difference among 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 statistically significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

SB203580 Stimulates Phosphorylation and Activation of cPLA₂ and [³H]AA Release. Treatment of quiescent VSMCs with p38 kinase inhibitor SB203580 caused a concentration-dependent increase in cPLA₂ phosphorylation, as indicated by an increase in ³²P incorporation, and in cPLA₂ activity measured as release of [¹⁴C]AA from the hydrolysis of phosphatidylcholine labeled with [¹⁴C]AA at sn-2 position (Fig. 1, A and B). This effect of SB203580 was selective because its inactive derivative, SB202474 (25 µM), had no effect on cPLA₂ activity (data not shown). Lower concentrations of SB203580 also increased cPLA₂ activity from 178 ± 18 cpm with vehicle versus 419 ± 18 cpm with 0.8 µM SB203580 (p < 0.05; n = 3, average values ± S.E. from experiments performed in cells from three different rabbits). SB203580, but not SB202474, inhibited the increase in the activity of p38 kinase caused by anisomycin, an activator of p38 kinase (Kochi and Collier, 1993) in rabbit VSMCs (Fig. 2). Time course experiments demonstrated that a 15-min incubation with SB203580 was sufficient to elicit an increase in cPLA₂ activity that reached a maximum at 60 min; after that time point, cPLA₂ activity declined (Fig. 1C). SB203580 at concentrations that increased cPLA₂ activity also increased release of [³H]AA and/or its products measured as fractional tritium output (Fig. 1D).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   cPLA2 is phosphorylated and activated by p38 inhibitor SB203580. A, concentration-dependent activation of cPLA2 by SB203580. Quiescent VSMCs were stimulated for 15 min with 10, 20, 30, and 50 µM SB203580 or its vehicle (VEH). Amount of 32P incorporated into cPLA2 activity was determined by immunoprecipitating cPLA2 as described under Experimental Procedures. The gel shown in A is representative of experiments performed in cells (10-cm plates) from three different rabbits. B, concentration-dependent activation of cPLA2. Average values ± S.E. from experiments performed in cells (10-cm plates) from three different rabbits. *, denotes value significantly different from that obtained in the presence of vehicle (p < 0.05). C, time course of cPLA2 activity induced by SB203580. Quiescent VSMCs were stimulated with 20 µM of SB203580 for 0, 15, 30, 60, 90, and 120 min. cPLA2 activity was measured as described under Experimental Procedures. The maximum cPLA2 activity that was obtained at 15 min was considered to be 100%. Average values ± S.E. from experiments performed in cells (10-cm plates) from three different rabbits. D, arachidonic acid released with 0.8 and 20 µM of SB203580. Average values ± S.E. from experiments performed in cells (10-cm plates) from three different rabbits.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Anisomycin-induced increase in p38 activity is inhibited by SB203580 but not by its inactive analog SB202474. Quiescent VSMCs were stimulated for 15 min with 1 µM anisomycin (ANIS) or its vehicle (V) in the presence and absence of 20 µM SB203580, 20 µM SB202474, or their vehicle (VEH). The activity of p38 was determined using anti-phospho-p38 antibodies; protein levels were determined by using anti-p38 antibodies as described under Experimental Procedures. A representative blot of experiments was performed in cells (10-cm plates) from three different rabbits.

SB203580 Stimulates Phosphorylation of ERK1/2 and CaMKII. The activity of cPLA₂ in different cell systems has been shown to be regulated by ERK1/2 (Lin et al., 1993), p38 kinases (Kramer et al., 1996; Waterman et al., 1996; Hiller and Sundler, 1999; Hazan-Halevy and Levy, 2000), and/or CaMKII (Muthalif et al., 1996). To determine the contribution of MAPKs and CaMKII to cPLA₂ activation by SB203580 in rabbit VSMCs, we examined the phosphorylation state of these MAPKs (ERK1/2 and p38) and of CaMKII, using antibodies specific for the phosphorylated form of these enzymes. SB203580 increased phosphorylation of CaMKII and ERK1/2, but not p38 kinase (Fig. 3). Although the protein level of p38 kinase, as detected by polyclonal antibody, was quite high, the level of phosphorylated p38 was much lower in quiescent VSMCs. However, anisomycin markedly increased phosphorylation of p38 (Fig. 2).

View larger version (69K): [in this window] [in a new window]   Fig. 3.   SB203580 stimulates ERK1/2 and CaMKII activities. Quiescent VSMCs were stimulated for 15 min with 10, 20, 30, and 50 µM SB203580 or its vehicle (VEH) for 15 min. The activities of ERK1/2, CaMKII, and p38 were determined using phospho-specific antibodies; protein levels were determined by anti-ERK1/2, anti-CaMKII, and anti-p38 antibodies, respectively, as described under Experimental Procedures. A representative blot from experiments was performed in cells (10-cm plates) from three different rabbits.

SB203580-Induced cPLA₂ Phosphorylation is Attenuated by Inhibitors of Ca²⁺ Channel, Calmodulin, and CaMKII. The effect of SB203580 in causing phosphorylation of CaMKII raised the possibility that it might increase cPLA₂ activity by increasing the influx of Ca²⁺, which, by binding with calmodulin, causes activation of CaMKII that in turn increases cPLA₂ activity. To test this hypothesis, we investigated the effect of Ca²⁺ depletion from the medium and of Ca²⁺ channel blocker (nifedipine) and inhibitors of calmodulin (W-7) and CaMKII (KN-93).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   SB203580-induced cPLA2 phosphorylation is inhibited by Ca2+ depletion from the medium, by Ca2+ channel blocker (nifedipine), and inhibitors of calmodulin (W-7) and CaMKII (KN-93). Top panel, quiescent VSMCs were stimulated for 15 min with 20 µM SB203580 (SB) in the presence of vehicle with Ca2+ (VEH + Ca2+) or without Ca2+ (VEH  Ca2+) or in the presence of Ca2+ and Ca2+ channel blocker nifedipine (NIF+Ca2+; NIF, 1 µM). 32P incorporation into cPLA2 was determined as described under Experimental Procedures. This is representative of experiments performed in cells (10-cm plates) from three different rabbits. Bottom panel, quiescent VSMCs were stimulated for 15 min with 20 µM SB203580 (SB) or its vehicle (V) in the presence of calmodulin inhibitor (W-7, 10 µM) or its inactive analog (W-5, 10 µM) and CaMKII inhibitor (KN-93, 10 µM) or its inactive analog (KN-92, 10 µM) or their vehicle (VEH). This is representative of experiments performed in cells (10-cm plates) from three different rabbits.

U0126 and FPT-III Inhibit ERK1/2 and cPLA₂ but Not CaMKII Phosphorylation Induced by SB203580. Earlier studies from our laboratory have shown stimulation of the Ras/MAPK pathway by the products of AA released by the initial activation of cPLA₂ by CaMKII (Muthalif et al., 1998a). To determine the contribution of the Ras/MAPK pathway to SB203580-induced cPLA₂ activation, the effects of MEK inhibitor U0126 and Ras inhibitor FPT-III were examined. Both these antagonists attenuated ERK1/2 activity and cPLA₂ phosphorylation, but not the CaMKII activity (Fig. 5).

View larger version (49K): [in this window] [in a new window]   Fig. 5.   Inhibitors of MEK (U0126) and Ras (FPT-III) attenuate SB203580-induced increase in phosphorylation of ERK1/2 and cPLA2 but not CaMKII. Quiescent VSMCs were stimulated for 15 min with SB203580 (20 µM) or its vehicle (VEH) in the presence of inhibitors of MEK (U0126, 10 µM), Ras (FPT-III, 25 µM), or their vehicle (V). The activities of ERK1/2 and CaMKII were determined using phospho-specific antibodies; protein levels were determined by anti-ERK1/2 and anti-CaMKII antibodies, respectively, as described under Experimental Procedures. 32P incorporation into cPLA2 was determined as described under Experimental Procedures. This is representative of three experiments performed in cells from three different rabbits.

17-ODA and Baicalein Inhibit SB203580-Induced Increase in ERK1/2 Phosphorylation. Products of AA generated via cytochrome P450 and lipoxygenase have been shown to cause activation of ERK1/2 via the Ras/Raf/MEK pathway (Muthalif et al., 1998a). To determine whether SB203580 increases ERK1/2 phosphorylation by generating AA metabolites via these pathways, the effect of inhibitors of cytochrome P450 (17-ODA) and lipoxygenase (baicalein) on SB203580-induced increase in ERK1/2 phosphorylation was examined. Incubation of VSMCs with 17-ODA and baicalein inhibited the increase in ERK1/2 activity caused by SB203580 (Fig. 6).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Inhibitors of cytochrome P450 (17-ODA) and 12-lipoxygenase (baicalein) attenuate SB203580-induced increase in ERK1/2 phosphorylation. Quiescent VSMCs were stimulated for 15 min with SB203580 (20 µM) or its vehicle (V) in the presence of inhibitors of cytochrome P450 (17-ODA, 5 µM) and 12-lipoxygenase (baicalein, 5 µM) or its vehicle (VEH). The activity of ERK1/2 was determined using phospho-specific antibodies and protein level was determined by anti-ERK1/2 antibodies, as described under Experimental Procedures. This is representative of experiments performed in cells (10-cm plates) from three different rabbits.

SB203580 Causes Translocation of cPLA₂. The increase in intracellular levels of Ca²⁺ in response to various agents that cause cPLA₂ activation has been shown to be required for cPLA₂ translocation from the cytoplasm to the nuclear envelope (Glover et al., 1995; Schievella et al., 1995; Kan et al., 1996; Muthalif et al., 1996; Gijon et al., 1999; Perisic et al., 1999). We found that SB203580 also caused cPLA₂ translocation from the cytoplasm to the nuclear envelope in quiescent VSMCs treated with 20 µM for 15 min. This translocation was not observed in the absence of extracellular Ca²⁺ (Fig. 7A) or in the presence of CaMKII inhibitor KN-93 (Fig. 7B). SB203580-induced ERK1/2 and CaMKII activity were attenuated by KN-93 but not its inactive analog KN-92 (Fig. 7C).

View larger version (55K): [in this window] [in a new window]   Fig. 7.   cPLA2 translocation to the nuclear envelope is blocked by extracellular Ca2+ depletion and CaMKII inhibitor KN-93 (A and B), and KN-93 inhibits SB203580-induced increase in phosphorylation of ERK1/2 and CaMKII. A, quiescent VSMCs were stimulated for 15 min with 20 µM SB203580 or vehicle (VEH) in the presence (+Ca2+) or absence (Ca2+) of Ca2+. cPLA2 translocation was determined by confocal microscopy as described under Experimental Procedures. This is representative of three experiments performed in cells from three different rabbits. Bar indicates 10 µm. B, quiescent VSMCs were stimulated for 15 min with 20 µM SB203580 or vehicle (VEH) in the presence of 10 µM of KN-93 or its vehicle (V). cPLA2 translocation was determined by confocal microscopy as described under Experimental Procedures. This is representative of three experiments performed in cells from three different rabbits. Bar indicates 10 µm. C, quiescent VSMCs were stimulated for 15 min with 20 µM SB203580 (SB) or its vehicle (V) in the presence of 10 µM KN-93, 10 µM KN-92, or their vehicle (VEH). The activities of ERK1/2 and CaMKII were determined using phospho-specific antibodies; protein levels were determined by anti-ERK1/2 and anti-CaMKII antibodies, respectively, as described under Experimental Procedures. This is representative of experiments performed in cells (10-cm plates) from three different rabbits.

p38 Kinase Inhibitors SB202190 and PD169316 Did Not Cause CaMKII Activation or cPLA₂ Phosphorylation and Translocation. To determine whether CaMKII and cPLA₂ are also activated by other p38 MAPK inhibitors, we examined the effects of SB202190 (20 µM; Lee et al., 1994) and PD169316 (20 µM; Kummer et al., 1997) on p38 kinase and CaMKII activation as well as cPLA₂ phosphorylation in VSMCs. Both SB202190 and PD169316 inhibited anisomycin-induced p38 activation, but failed to stimulate CaMKII and cPLA₂ phosphorylation (Fig. 8, A, B, and C). While SB202190 and PD169316 had no effect on cPLA₂ translocation, concentrations of SB203580 as low as 0.8 µM were effective in promoting cPLA₂ translocation from cytoplasm to the nuclear envelope (Fig. 9). NE has been shown to increase cPLA₂ translocation and its activity in rabbit VSMCs (Muthalif et al., 1996). To investigate if p38 is involved in cPLA₂ activation in VSMCs, we examined the effect of SB202190 and PD169316 on NE-induced increase in cPLA₂ activity and its translocation to the nuclear envelope. Both these agents failed to alter either of the effects NE had on cPLA₂, i.e. translocating it to the nuclear envelope (Fig. 10A) and increasing its activity (Fig. 10B).

View larger version (37K): [in this window] [in a new window]   Fig. 8.   SB202190 and PD169316 inhibited anisomycin-induced phosphorylation of p38 but not CaMKII or cPLA2. Quiescent VSMCs were treated for 15 min with the p38 inhibitors SB203580 (20 µM), SB202190 (20 µM), PD169316 (20 µM), or their vehicle (VEH) in the presence of anisomycin (ANIS) or its vehicle (V). The activities of p38 (A) and CaMII (B) were determined by Western blot analysis using anti-phospho-specific p38 and CaMKII antibodies; protein levels were determined by anti-p38 and anti-CaMKII antibodies as described under Experimental Procedures. C, cPLA2 phosphorylation was determined by 32P incorporation, immunoprecipitation and autoradiography as described under Experimental Procedures. This is representative of experiments performed in cells (10-cm plates) from three different rabbits.

View larger version (52K): [in this window] [in a new window]   Fig. 9.   SB202190 and PD169316 do not cause cPLA2 translocation to the nuclear envelope. Quiescent VSMCs were treated for 15 min with SB203580 (0.8 and 20 µM), SB202190 (20 µM) and PD169316 (20 µM) or vehicle (VEH). cPLA2 translocation was determined by confocal microscopy as described under Experimental Procedures. This is representative of experiments performed in cells from three different rabbits. Bar indicates 10 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 10.   SB202190 and PD169316 had no effect on norepinephrine-induced cPLA2 translocation (A) or its activity (B). Quiescent VSMCs were treated for 15 min with the p38 inhibitors SB202190 (20 µM), PD169316 (20 µM) or their vehicle (VEH) in the presence of NE (10 µM) or its vehicle (V). A, cPLA2 translocation was determined by confocal microscopy as described under Experimental Procedures. A representative of three experiments performed in cells from three different rabbits. Bar indicates 10 µm. B, cPLA2 activity presented as an increase in counts per minute was determined from the hydrolysis of phosphatidylcholine labeled with [14C]AA at the sn-2 position as described under Experimental Procedures. This is representative of experiments performed in cells (10-cm plates) from three different rabbits. *, denotes value significantly different from that obtained in the presence of vehicle (V) (p < 0.05).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The present study demonstrates that SB203580, used as an inhibitor of p38 kinase activity, caused CaMKII and cPLA₂ activation and translocation of cPLA₂ from the cytoplasm to the nuclear envelope in rabbit VSMCs and increased [³H]AA release. This effect of SB203580 was not shared by other p38 kinase inhibitors, SB202190 and PD169316. p38 has been shown to activate cPLA₂ in human neutrophils (Waterman et al., 1996; Hazan-Halevy and Levy, 2000), platelets (Kramer et al., 1996), and macrophages (Hiller and Sundler, 1999). It has been reported that the IC₅₀ values for the SB203580-sensitive p38 MAPK isoforms p38 and p38 are 0.8 µM (Kumar et al., 1997; Cuenda et al., 1997). However, in our study, SB203580 at concentrations (of 0.8 µM or higher) that block anisomycin-induced increase in p38 activity caused cPLA₂ translocation to the nuclear envelope, increased cPLA₂ activity, and AA release. Moreover, SB202190 and PD169316 in concentrations that inhibited anisomycin-induced increase in p38 activity failed to alter NE-induced increase in cPLA₂ activity or its translocation to the nuclear envelope. Therefore, it appears that SB203580 increases cPLA₂ activity by a mechanism unrelated to its inhibitory effect on p38 activity and that p38 is not involved in cPLA₂ activation elicited by NE in rabbit VSMCs. Moreover, these observations raise the possibility that activation of cPLA₂ by p38 might be cell-specific. Whether it is due to lack of a specific isoform of p38 and/or a mediator involved in cPLA₂ activation in rabbit VSMCs remains to be determined.

NE and angiotensin II have been reported to increase the release of AA by stimulating cPLA₂ activity via CaMKII activation in rabbit VSMCs (Muthalif et al., 1996; Muthalif et al., 1998b). Our findings that SB203580 increased CaMKII activity and that an inhibitor of CaMKII, KN-93, blocked CaMKII activity and cPLA₂ phosphorylation, suggest that the effect of SB203580 on cPLA₂ activation is mediated by CaMKII. In the present study, SB203580 also increased ERK1/2 activity in rabbit VSMCs. ERK1/2 has been reported to phosphorylate and increase cPLA₂ activity in various cell types (Lin et al., 1993; Nemenoff et al., 1993). However, in rabbit VSMCs it has been shown that activation of cPLA₂ by CAMKII causes AA release and the metabolites of AA generated via cytochrome P450 and lipoxygenase activate ERK1/2 via the Ras/Raf/MEK pathway and amplify cPLA₂ activity (Muthalif et al., 1998a). Our demonstration that the inhibitor of CaMKII KN-93 blocked the effect of SB203580 had in increasing ERK1/2 activity and phosphorylation of cPLA₂ supports the view that activation of ERK1/2 by this compound is mediated by CaMKII, most likely through generation of AA metabolites and activation of the Ras/MEK pathway. Supporting this conclusion was our finding that FPT-III, which inhibits farnesylation of Ras (Manne et al., 1995), and U0126, an inhibitor of MEK (Favata et al., 1998), abolished ERK1/2 activation and cPLA₂ phosphorylation in VSMCs. SB203580 has been shown to increase Raf-1 activity in quiescent baboon VSMCs and prolongs the activation of MEK1 in these cells when they are stimulated with pervanadate (Kalmes et al., 1999). Although these investigators failed to observe an increase in Ras or ERK1/2 activity in baboon VSMCs in response to SB203580, they found a slight increase in MEK1 activity in intact cells. Moreover, SB203580-activated Raf-1 from these cells was able to phosphorylate and activate MEK1 immunoprecipitated from the same cells in vitro. Whether MEK1 from SB203580-treated cells activates ERK1/2 in these cells was not determined. Previously, we have shown that the AA metabolites generated via cytochrome P450 and lipoxygenase pathway, mainly 12(S)-, 15(S)-, and 20-hydroxyeicosatetraenoic acids, mediate its effect on ERK1/2 activation (Muthalif et al., 1998a). Our finding that the inhibitors of cytochrome P450 (17-ODA) and lipoxygenase (baicalein) attenuated SB203580-induced increase in ERK1/2 phosphorylation suggest that the increase in ERK1/2 activation by this compound is mediated by the metabolites of AA generated via these enzymatic pathways in rabbit VSMCs.

It is possible that the effect of SB203580 to increase CaMKII activity, and consequently cPLA₂ phosphorylation, results from increasing the influx of extracellular Ca²⁺. The evidence supporting this hypothesis was our finding that the depletion of Ca²⁺ from the medium and an inhibitor of calmodulin, W-7, attenuated the cPLA₂ phosphorylation elicited by SB203580 in VSMCs. Since the effect of SB203580 to phosphorylate cPLA₂ was inhibited by nifedipine, a voltage-sensitive Ca²⁺ channel blocker, but not by SKF96365, a receptor-operated Ca²⁺ channel blocker (Blayney et al., 1991; data not shown), it appears that SB203580 acts by increasing the activity of voltage-sensitive Ca²⁺ channels. Although SKF96365 is not highly selective and can also block voltage-sensitive Ca²⁺ channels, its inability to inhibit SB203580-induced phosphorylation of cPLA₂ indicates that the voltage-sensitive Ca²⁺ channels activated by SB203580 in rabbit VSMCs are insensitive to SKF96365. We were unable to determine the effect of SB203580 on intracellular levels of Ca²⁺ because this compound is light-sensitive and exhibited very high basal levels of fluorescence in the absence of VSMCs.

It is well established that many agonists that increase cPLA₂ activity produce their effect by increasing the levels of cytosolic Ca²⁺ required for translocating cPLA₂ from cytoplasm to the nuclear envelope (Glover et al., 1995; Schievella et al., 1995; Gijon et al., 1999; Perisic et al., 1999). In the present study, SB203580 also caused cPLA₂ translocation from the cytoplasm to the nuclear envelope that was blocked by depletion of Ca²⁺ from the medium, consistent with its effect to increase cPLA₂ activity by increasing entry of Ca²⁺ via voltage-sensitive Ca²⁺ channels. The other p38 kinase inhibitors, SB202190 and PD169316, which did not increase phosphorylation of CaMKII and cPLA₂, also failed to increase cPLA₂ translocation from the cytoplasm to the nuclear envelope.

In conclusion, the present study demonstrates that SB203580, used as a selective inhibitor of p38 kinase, exerts effects unrelated to its inhibitory effect on p38 kinase in rabbit VSMCs. It increases CaMKII, ERK1/2, and cPLA₂ activity, translocation of cPLA₂ from the cytoplasm to the nuclear envelope, and AA release, most likely by increasing the influx of extracellular Ca²⁺ via voltage-sensitive Ca²⁺ channels. It is interesting that SB202190, which has a very slight structural difference from SB203580 (i.e., the radical bound to pyridinyl imidazole is hydroxyphenyl for SB202190 and methylsulfinylphenyl for SB203580) did not increase ERK1/2, CaMKII, or cPLA₂ activity and its translocation to the nuclear envelope. Therefore, the data obtained with SB203580 and derived from using it as an inhibitor of p38 kinase must be interpreted with great caution. SB202190 and PD169316, which lack the above nonselective effects of SB203580, may be preferable as inhibitors of p38 kinase.