Involvement of the mitogen-activated protein kinase cascade in peroxynitrite-mediated arachidonic acid release in vascular smooth muscle cells

Rita K. Upmacis, Ruba S. Deeb, Matthew J. Resnick, Rochelle Lindenbaum, Caryn Gamss, Dev Mittar, and David P. Hajjar

Departments of Biochemistry and Pathology, Center of Vascular Biology, Weill Medical College of Cornell University, New York, New York 10021

Submitted 10 April 2003 ; accepted in final form 22 January 2004


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Eicosanoid production is reduced when the nitric oxide (NO·) pathway is inhibited or when the inducible NO synthase gene is deleted, indicating that the NO· and arachidonic acid pathways are linked. We hypothesized that peroxynitrite, formed by the reaction of NO· and superoxide anion, may cause signaling events leading to arachidonic acid release and subsequent eicosanoid generation. Western blot analysis of rat arterial smooth muscle cells demonstrated that peroxynitrite (100–500 µM) and 3-morpholinosydnonimine (SIN-1; 200 µM) stimulate phosphorylation of extracellular signal-regulated kinase (ERK), p38, and cytosolic phospholipase A2 (cPLA2). We found that peroxynitrite-induced arachidonic acid release was completely abrogated by the mitogen-activated protein/ERK kinase (MEK) inhibitor U0126 and by calcium chelators. With the p38 inhibitor SB-20219, we demonstrated that peroxynitrite-induced p38 phosphorylation led to minor arachidonic acid release, whereas U0126 completely blocked p38 phosphorylation. Addition of arachidonic acid caused p38 phosphorylation, suggesting that arachidonic acid or its metabolites are responsible for p38 activation. KN-93, a specific inhibitor of Ca2+/calmodulin-dependent kinase II (CaMKII), revealed no role for this kinase in peroxynitrite-induced arachidonic acid release in our cell system. Together, these results show that in response to peroxynitrite the cell initiates the MEK/ERK cascade leading to cPLA2 activation and arachidonic acid release. Thus studies investigating the role of the NO· pathway on eicosanoid production must consider the contribution of signaling pathways initiated by reactive nitrogen species. These findings may provide evidence for a new role of peroxynitrite as an important reactive nitrogen species in vascular disease.

reactive nitrogen species; prostaglandin H2 synthase; extracellular signal-regulated kinase; p38; cytosolic phospholipase A2


REACTIVE NITROGEN SPECIES (RNS), such as nitric oxide (NO·) and peroxynitrite (ONOO), play an important role in host defense mechanisms. However, when released in an unrestrained manner, they may become involved in the pathogenesis of several inflammatory diseases that include rheumatoid arthritis (31) and atherosclerosis (5, 13, 64). These diseases involve arachidonic acid metabolites. One route of arachidonic acid metabolism, which results in eicosanoid generation, involves the constitutive and inducible forms of prostaglandin H2 synthase (PGHS-1 and PGHS-2, respectively). A disruption in the balance of essential eicosanoids is thought to contribute to the pathophysiology of inflammatory vascular disease (11). Because RNS and eicosanoids are often found at sites of inflammation, where their synthesis is often elicited by the same stimuli, an understanding of the interactions between RNS and the arachidonic acid pathway is of great interest. Thus the determination of those processes involving RNS that contribute to changes in eicosanoid levels during inflammatory disease is of clinical importance.

There is now strong evidence linking the NO· and arachidonic acid pathways. For instance, mice bearing a targeted deletion in the inducible NO synthase (iNOS) gene demonstrate reduced ability to synthesize prostaglandin E2 (PGE2) (42). Similarly, administration of NOS inhibitors to rats leads to a reduction in prostaglandin production in inflammatory lesions (56, 57). Furthermore, inflamed brain tissue from iNOS-knockout mice reveals decreased levels of PGE2 compared with wild-type mice (46, 54). In addition, arginine depletion can decrease prostaglandin synthesis in mouse macrophages stimulated with lipopolysaccharide (LPS) (54, 55). These observations indicate that NO· and/or NO-derived species can alter PGHS activity and eicosanoid production in vivo.

The chemistry of NO· under physiological conditions is complex, leading to the formation of various RNS that include NO·, nitrosonium ion (NO+), nitroxyl anion (NO), nitrite (NO2), nitrate (NO3), peroxynitrite (ONOO) (62) and nitrosoperoxocarbonate (ONOOCO2) (50). For this reason, the type of interaction between RNS and PGHS enzymes is highly dependent on the nature of the species under consideration. For example, ONOO, which arises from the reaction between NO· and superoxide radical anion (27), activates PGHS (34, 63), whereas NO· inhibits activation (63). The mechanism of ONOO-induced PGHS activation is likely similar to that of peroxide activation of PGHS, which involves oxidation of the heme moiety (15). ONOO has also been shown to cause Tyr nitration in PGHS in vascular smooth muscle cells (13) and in platelets (7). These results indicate that it is possible for a specific RNS such as ONOO to have both an inhibitory and activating effect.

Although RNS directly affect the activity of PGHS enzymes, the effect of RNS on the signaling pathways that lead to arachidonic acid release and increased PGHS activity has not been fully investigated. Arachidonic acid release from arachidonyl phospholipids is catalyzed by cytosolic phospholipase A2 (cPLA2) (9, 37). cPLA2 activation, which requires both phosphorylation and an increase in intracellular Ca2+ (25, 37), is achieved by several agonists, including growth factors, neurotransmitters, angiotensin II, vasopressin, LPS, colony-stimulating factor-1, and thrombin (32, 37, 38). cPLA2 is phosphorylated on Ser505 in vitro by mitogen-activated protein (MAP) kinases (MAPKs), including the extracellular signal-regulated kinases (ERKs) and p38 MAPK (32, 39, 45, 66), although the specific signaling mechanism varies in response to different agonists in different cell types. In addition, cPLA2 is phosphorylated on Ser727 by MAPK-interacting kinase I (MNK-I), or a closely related isoform (24), and on Ser515 by calcium/calmodulin-dependent protein kinase II (CaMKII) (44). When expressed in a baculovirus/Sf9 cell system, cPLA2 is phosphorylated on four serine residues (Ser437, Ser454, Ser505, and Ser727) (12). However, the signaling mechanisms by which RNS might activate cPLA2 are not yet fully elucidated.

The actions of RNS, such as ONOO, which is known to initiate lipid peroxidation (49), react with sulfhydryl groups (48), and cause oxidative damage via Tyr nitration (13, 28), are not limited to immediate chemical damage. ONOO has been found to activate calcium-dependent PKC, the ERKs, c-Jun NH2-terminal kinase (JNK), and p38 MAPK (3, 21, 29, 58). ONOO also affects cellular signaling by causing dimerization of the epidermal growth factor receptor (EGFR) in PC-12 cells, possibly through intermolecular dityrosine cross-linking (65), but may also occur independently of the EGFR receptor (71). It has been reported that ONOO causes the release of arachidonic acid from PC-12 cells (23) due to stimulation of a low-molecular-mass form of cPLA2 in mitochondria isolated from these cells (22). However, the specific role of ONOO in the signaling cascade leading to cPLA2 activation and arachidonic acid release in vascular smooth muscle cells has not yet been examined.

In this study, we found that arachidonic acid is released in vascular smooth muscle cells after challenge with ONOO. To investigate the hypothesis that ONOO stimulates the phosphorylation of signaling molecules leading to the activation of cPLA2, we examined the effect of ONOO on the p38 MAPK, CaMKII, and ERK signaling pathways. We believe our new findings define the contributions of specific signaling pathways linking ONOO to changes in eicosanoid metabolism in vascular cells.


    EXPERIMENTAL PROCEDURES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from GIBCO-BRL. Sodium peroxynitrite (110–200 mM in 4.7% sodium hydroxide) and 3-morpholinosydnonimine (SIN-1) were obtained from Calbiochem and Alexis Biochemicals, respectively. Phosphospecific polyclonal antibodies to the activated forms of MEK1/2 (Ser217/221), ERK1/2 (Thr202/Tyr204), p38 MAPK (Thr180/Tyr182), cPLA2 (Ser505), Raf-1 (Ser259), and MKK3/6 (Ser189/207) were obtained from Cell Signaling Technologies (Beverly, MA). Antibodies specific to the nonphosphorylated forms of these signaling molecules were also obtained from Cell Signaling Technologies. The polyclonal antibody specific for phosphorylated CaMKII (Thr286) was obtained from Promega. Horseradish peroxidase-conjugated antirabbit antibodies were purchased from Amersham International (Arlington Heights, IL). 4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imadazole (SB-202190), 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), KN-93, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM) were obtained from Calbiochem. Ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was obtained from J. T. Baker. 3H-labeled arachidonic acid (100 Ci/mmol) was purchased from New England Nuclear (Boston, MA), whereas nonlabeled arachidonic acid was obtained from Aldrich. All the materials for SDS-PAGE were obtained from Bio-Rad Laboratories (Hercules, CA).

UV/Vis spectroscopy and handling of ONOO. The concentration of ONOO was determined with a Perkin Elmer Lambda 20 spectrophotometer by measuring its absorbance at 302 nm ({epsilon} = 1,670 M–1·cm–1). Matching quartz precision cells (Hellma, 1-cm path length; 1-ml or 100-µl volume) were used in our measurements. On receiving ONOO, the sample was thawed, its concentration was determined by UV/Vis spectroscopy, and it was divided into smaller aliquots, snap frozen, and stored at –80°C until required. During an experiment, an aliquot of ONOO was kept on dry ice until its addition was required, at which point it was thawed and added directly to the well. The remnants of the sample were immediately snap frozen on dry ice, and the concentration of the sample was determined after completion of the experiment. A comparison of the ONOO concentration before and after the experiment allowed us to determine the quality and consistency of ONOO used in the experiment. These protocols ensured reproducibility. Aliquots used in an experiment were not reused.

Smooth muscle cell culture. Primary passage rat smooth muscle cells were isolated and characterized as described previously (47). Cells were plated either in six-well dishes (for arachidonic acid release or PGE2/6-keto-PGF1{alpha} measurements in the supernatant) or in 100-mm dishes (for the measurement of phosphorylated signaling molecules in the cell lysate) in DMEM supplemented with 10% (vol/vol) fetal bovine serum and 1% (vol/vol) glutamine. All cells were incubated at 37°C in 5% CO2 in air. Cells were rendered quiescent for 3–4 days with serum-free medium (quiescent medium) containing insulin, transferrin, and selenium (ITS; Life Technologies) to suppress PGHS-2 expression (52). Nitrate/nitrite levels were measured with a colorimetric assay (Cayman Chemical) and were found to be nondetectable in the supernatant medium of quiescent cells. Cells between passages 6 and 15 were used in experiments. We did not see significant variation in our results related to passage number.

Treatment of smooth muscle cells. Quiescent rat smooth muscle cells were exposed to either quiescent medium or quiescent medium with added ONOO or arachidonic acid for 1 h at 37°C. In experiments requiring ONOO, ONOO from a stock solution (100–200 mM) was added directly to the medium in the form of a single bolus while the dish was swirled (63). This immediate addition strategy was performed because ONOO has an extremely short half-life. In control experiments, ONOO was allowed to decompose (as determined spectrophotometrically) at room temperature for 2–3 days. In other control experiments, cells were treated with NaOH as vehicle control (data not shown). In experiments using the inhibitors (including SB-202190, U0126, KN-93, or BAPTA-AM and EGTA) cells were first pretreated with the inhibitor (1 h for SB-202190, KN-93, BAPTA-AM, and EGTA; 40 min for U0126) and then stimulated with ONOO (200 or 500 µM), or in some cases arachidonic acid (10 µM), for time points that ranged between 0 and 60 min, depending on the type of experiment being performed.

PGE2 and 6-keto-PGF1{alpha} measurements. Quiescent smooth muscle cells in six-well dishes were exposed to ONOO (100, 200, and 500 µM) for 1 h (or for time points between 0 and 60 min), and the supernatant medium was assayed for either PGE2 or 6-keto-PGF1{alpha} formation with enzyme immunoassay assay kits (Amersham Pharmacia). Quiescent smooth muscle cells in six-well dishes were also exposed to 100–500 µM nitrite, S-nitroso-N-acetyl-penicillamine (SNAP), 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7), and SIN-1. Total protein in the cell lysate was determined by using either the Lowry or the modified Lowry method (41). After exposure of the cells to 100, 200, and 500 µM ONOO, nitrate/nitrite levels in the supernatant medium were found to be 114 ± 7, 222 ± 4, and 473 ± 10 µM nitrate/nitrite, respectively, which closely reflects the expected concentration of the decomposition products of ONOO.

Western blotting. After treatments, cells were washed with PBS and lysed in buffers, the composition of which varied depending on which phosphorylated signaling molecule was under investigation. The modified lysis buffer used for detecting the phospho-specific forms of p38, MEK1/2, ERK1/2, cPLA2, Raf-1, and MKK3/6 comprised (in mM) 20 Tris·HCl pH 7.5, 1 EDTA, 1 EGTA, 150 NaCl, 2.5 sodium pyrophosphate, 1 {beta}-glycerolphosphate, 1 sodium vanadate, and 1 phenylmethylsulfonyl fluoride (PMSF) with 1% Triton X-100 and 1 µg/ml leupeptin. The modified lysis buffer used for detecting the phospho-specific form of CaMKII comprised (in mM) 20 Tris·HCl pH 8, 2 EDTA, 2 EGTA, 10 sodium phosphate, 2 dithiothreitol, 25 benzamidine, and 1 PMSF with 25 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin, and 10 µg/ml aprotinin. The cells were scraped, transferred to an Eppendorf tube, vortexed, and put on ice for 30 min. The lysates were clarified by centrifugation (10,000 g at 4°C) for 10 min. The protein sample (25–50 µg) was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blocked for 90 min at 25°C with 5% nonfat milk in a PBS-1% Tween buffer. The membrane was then incubated overnight at 4°C with an appropriate dilution of the phospho-specific antibody, followed by incubation for 75 min with a 1:2,000 dilution of the appropriate horseradish peroxidase-conjugated secondary antibody. The immunoblot signal was visualized through enhanced chemiluminescence.

Determination of arachidonic acid release. Confluent rat smooth muscle cells in six-well dishes were labeled for 24 h with [3H]arachidonic acid (0.5–1 µCi/ml) in quiescent DMEM. The cells were washed three times with quiescent DMEM containing 0.2% fatty acid-free bovine serum albumin (BSA) and once in PBS to remove all unincorporated [3H]arachidonic acid. Approximately 80–90% of the added [3H]arachidonic acid was incorporated into the cells. The cells were treated with and without inhibitors and stimulated with ONOO as described above. Before ONOO addition, 100 µl of the medium was removed and counted for the presence of 3H label. After ONOO treatment, another 100 µl of the medium was removed and counted. The difference obtained served as the measure of arachidonic acid release.

Miscellaneous. Cell viability was assessed by in situ staining with Trypan blue, and cell number was determined by hemocytometer. At 500 µM ONOO, cell viability experiments demonstrated no significant cell death (data not shown). ONOO becomes toxic under our experimental conditions at levels of 1 mM and higher. Unless otherwise stated, data are reported as averages ± SE, with significant differences determined by a single-factor ANOVA or by Student's t-test.


    RESULTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ONOO increases PGE2 synthesis and arachidonic acid release in a time- and concentration-dependent manner. Figure 1A shows that, in the absence of exogenously added arachidonic acid, treatment of smooth muscle cells with ONOO caused a dose-dependent increase in PGE2 formation. This effect was also observed with SIN-1, which generates ONOO on decomposition (Ref. 53 and data not shown). In contrast, the following had no effect on PGE2 formation: nitrite (a decomposition product of ONOO), SNAP, which contains a nitrosonium group (NO+), and NOC-7, which releases NO·, and sodium hydroxide vehicle (data not shown). ONOO was found to induce PGE2 synthesis in a time-dependent manner, as shown in Fig. 1B, with maximal PGE2 formation occurring in ~15 min. To examine further whether ONOO induces cPLA2 activation, we measured 3H-labeled arachidonic acid release in smooth muscle cells after ONOO treatment. Figure 1C shows that arachidonic acid release in the medium increased proportionally with ONOO concentration. In addition, arachidonic acid release occurred maximally after ~15 min following the addition of ONOO, as shown in Fig. 1D. Formation of 6-keto-PGF1{alpha} [which is the stable end product of prostacyclin (PGI2)] also increased in response to ONOO, as shown in Fig. 1E.



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Fig. 1. Concentration- and time-dependent effect of ONOO on prostaglandin (PG)E2 synthesis, arachidonic acid (AA) release, and 6-keto-PGF1{alpha} synthesis in vascular smooth muscle cells. A: quiescent smooth muscle cells were treated with ONOO for 1 h at 37°C, and the supernatant medium was assayed for PGE2 formation. Statistical analysis (ANOVA) revealed a P value < 0.001 between groups. Note that the measurement obtained without any added ONOO is not statistically different from Dulbecco's modified Eagle's medium (DMEM) alone. B: quiescent smooth muscle cells were treated with ONOO (500 µM), and the supernatant medium was assayed at different time intervals for PGE2 formation. C: quiescent smooth muscle cells were labeled with [3H]arachidonic acid and treated with ONOO for 1 h. The supernatant medium was assayed for radioactivity. [3H]arachidonic acid release was expressed as disintegrations per minute (dpm) per milliliter of supernatant. Statistical analysis (ANOVA) revealed a P value of 2.6 x 10–5 between groups. D: quiescent smooth muscle cells were labeled with [3H]arachidonic acid and treated with ONOO (500 µM), and the supernatant medium was assayed at different time intervals for [3H]arachidonic acid release (dpm/ml supernatant). E: quiescent smooth muscle cells were treated with ONOO for 1 h at 37°C, and the supernatant medium was assayed for 6-keto-PGF1{alpha} formation. Note that the measurement obtained without any added ONOO is not statistically different from DMEM alone. Statistical analysis (ANOVA) revealed a P value of 4.3 x 10–9 between groups. The results are expressed as averages ± SE of 3 replicates per treatment and are representative of 1 experiment repeated 3 times.

 
ONOO and SIN-1 activate signaling molecules in a time-dependent manner. Figure 2 demonstrates the temporal effect of ONOO and SIN-1 on the signaling molecules p38, MEK1/2, and ERK1/2 (also known as p44/42) and on cPLA2. The effect of ONOO and SIN-1 on the activation of these molecules was studied by Western blot analysis using antibodies specific for the phosphorylated and nonphosphorylated forms of these molecules. Figure 2, A–D, shows that ONOO and SIN-1 induced p38, MEK1/2, ERK1/2, and cPLA2 phosphorylation in a time-dependent manner. Phosphorylation in each case was observed by ~15 min. Thus ONOO and SIN-1 activate several different signaling molecules, although these results do not yet indicate whether their activation is instrumental in causing arachidonic acid release.



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Fig. 2. ONOO and 3-morpholinosydnonimine (SIN-1) activate signaling molecules in a time-dependent manner. Quiescent smooth muscle cells were exposed to ONOO (500 µM) and SIN-1 (200 µM) for different time periods, and cell lysates were subjected to Western blotting with antibodies specific for the phosphorylated and nonphosphorylated forms of p38 (A), MEK1/2 (B), extracellular signal-related kinase (ERK)1/2 (C), and cytosolic phospholipase A2 (cPLA2, D). Results shown are representative of 3 independent experiments.

 
ONOO activates signaling molecules in a concentration-dependent manner. Figure 3 demonstrates the dose-dependent ability of ONOO (100–500 µM) to induce the phosphorylation of p38 (Fig. 3A), MEK1/2 (Fig. 3B), ERK1/2 (Fig. 3C) and cPLA2 (Fig. 3D). The effect of ONOO on the activation of these signaling molecules was again studied by Western blot analysis with antibodies specific for their phosphorylated and nonphosphorylated forms. In all cases, ONOO induced phosphorylation in a concentration-dependent manner. There was little or no response below 100 µM ONOO.



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Fig. 3. ONOO activates signaling molecules in a concentration-dependent manner. Quiescent smooth muscle cells were exposed to ONOO (0, 100, 200, and 500 µM) at 37°C for 1 h (A and D) or 20 min (B and C), and cell lysates were subjected to Western blotting with antibodies specific for the phosphorylated and nonphosphorylated forms of p38 (A), MEK1/2 (B), ERK1/2 (C), and cPLA2 (D). Results shown are representative of 3 independent experiments.

 
p38 MAPK inhibitor SB-202190 partially inhibits ONOO-dependent arachidonic acid release. To evaluate the role of p38 MAPK in the signaling pathway of ONOO-dependent arachidonic acid release and PGE2 synthesis, cells were pretreated with SB-202190 (25 µM), a specific inhibitor of p38, and then exposed to ONOO (500 µM). Figure 4A shows that the p38 MAPK-specific inhibitor SB-202190 did not fully inhibit ONOO-dependent arachidonic acid release (~37% inhibition) from smooth muscle cells prelabeled with [3H]arachidonic acid. This inhibition was significant (P < 0.004). These results indicate that although ONOO induces p38 phosphorylation, the p38 MAPK pathway is not fully responsible for ONOO-dependent arachidonic acid release. Because these results implicate p38 MAPK in ONOO-induced arachidonic acid release, we decided to investigate the involvement of MKK3/6, which is known to activate p38 MAPK, in this process (20). Figure 4B shows that MKK3/6 is phosphorylated in the presence of ONOO at 5 min. Thus p38 MAPK activation is likely induced by MKK3/6.



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Fig. 4. The p38 inhibitor SB-202190 partially reduces ONOO-induced arachidonic acid release. A: quiescent [3H]arachidonic acid-labeled smooth muscle cells were pretreated with SB-202190 (25 µM) for 1 h, followed by incubation with ONOO (500 µM) for 1 h. The supernatant medium was assayed for radioactivity. [3H]arachidonic acid release is expressed as dpm/ml supernatant. The results are given as an average of 6 measurements and are representative of >3 experiments. Statistical analysis (Student's t-test) revealed that SB-202190 caused a significant reduction in ONOO-induced arachidonic acid release (P < 0.004). B: quiescent smooth muscle cells were exposed to ONOO (200 µM) for different time periods, and cell lysates were subjected to Western blotting with an antibody specific for the phosphorylated form of MKK3/6.

 
CaMKII activation by ONOO does not lead to arachidonic acid release. Figure 5A demonstrates the temporal effect of ONOO on the signaling molecule CaMKII, which has been shown to be involved in cPLA2 activation (43, 44). ONOO-dependent CaMKII phosphorylation occurred maximally at 5 min but thereafter dissipated. ONOO-dependent CaMKII phosphorylation was blocked by KN-93 (40 µM), which is a CaMKII inhibitor. Figure 5B, however, shows that KN-93 did not inhibit ONOO-induced arachidonic acid release. Thus ONOO-induced CaMKII signaling does not lead to arachidonic acid release in smooth muscle cells.



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Fig. 5. The Ca2+/calmodulin-dependent kinase (CaMKII) inhibitor KN-93 does not prevent ONOO-induced arachidonic acid release. A: quiescent smooth muscle cells were treated with ONOO (500 µM) for 1 h with and without preincubation with KN-93 (40 µM) for 1 h. Cell lysates were subjected to Western blotting with an antibody specific for phospho-CaMKII. B: [3H]arachidonic acid-labeled quiescent smooth muscle cells were pretreated with KN-93 (40 µM) for 1 h, followed by incubation with ONOO (500 µM) for 1 h. The supernatant medium was then assayed for radioactivity. [3H]arachidonic acid release is expressed as dpm/ml supernatant. The results represent an average of averages obtained from 4 separate experiments in which 3–6 measurements were made per experiment. Statistical analysis (Student's t-test) revealed that KN-93 did not significantly reduce ONOO-induced arachidonic acid release.

 
MEK1/2 inhibitor U0126 completely abolishes ONOO-induced arachidonic acid release. To determine the role of MAPK in ONOO-dependent PGE2 synthesis, we used the MEK1/2 inhibitor U0126. Figure 6A shows that preincubation of smooth muscle cells with U0126 (40 µM) followed by stimulation with ONOO abolished ERK1/2 phosphorylation. These results clearly show that phosphorylation of ERK1/2 by ONOO requires MEK1/2. To determine whether cPLA2 activation is dependent on the MEK/ERK pathway, the effect of U0126 on arachidonic acid release and cPLA2 phosphorylation was studied. Arachidonic acid release was completely blocked with U0126, as shown in Fig. 6B, because there was no significant difference between cells exposed to both U0126 and ONOO vs. control cells. In addition, Fig. 6C shows that ONOO-induced cPLA2 phosphorylation is completely abolished by U0126. Because Raf-1 is a known upstream activator of MEK1/2 (33), we investigated the involvement of Raf-1 in this process. Figure 6D shows that Raf-1 is phosphorylated in the presence of ONOO within 5 min. Together, these data demonstrate that ONOO-induced arachidonic acid release requires MEK1/2, ERK1/2, and cPLA2 phosphorylation and that the upstream activator of these signaling molecules is likely Raf-1.



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Fig. 6. The MEK1/2 inhibitor U0126 prevents ONOO-induced arachidonic acid release and blocks cPLA2 phosphorylation. A: quiescent smooth muscle cells were preincubated with and without U0126 (40 µM) for 40 min and then exposed to ONOO (200 µM) for 1 h. Cell lysates were subjected to Western blotting with an antibody specific for phospho-ERK1/2. B: [3H]arachidonic acid-labeled quiescent smooth muscle cells were pretreated with U0126 (40 µM) for 40 min, followed by incubation with ONOO (500 µM) for 1 h. The supernatant medium was then assayed for radioactivity. [3H]arachidonic acid release is expressed as dpm/ml supernatant. The results represent an average of averages obtained from 3 separate experiments with 3–6 measurements per experiment. Statistical analysis (Student's t-test) revealed that U0126 significantly reduced ONOO-induced arachidonic acid release (P < 0.001). There was no significant difference in cells exposed to U0126 + ONOO vs. control cells. C: quiescent smooth muscle cells were preincubated with and without U0126 (40 µM) for 40 min and exposed to ONOO (200 µM) for 1 h. Cell lysates were subjected to Western blotting with antibodies specific to phospho-cPLA2 and cPLA2. D: quiescent smooth muscle cells were exposed to ONOO (500 µM) for 1 h. Cell lysates were subjected to Western blotting with an antibody specific for phospho-Raf-1. Results shown are representative of 3 independent experiments.

 
p38 Phosphorylation is inhibited by MEK1/2 inhibitor U0126 and is induced by arachidonic acid. In addition to inhibiting ONOO-induced ERK1/2 phosphorylation as shown in Fig. 6A, the MEK1/2 inhibitor U0126 also prevented ONOO-induced p38 phosphorylation as shown in Fig. 7A. This result indicates that a product of the MEK/ERK pathway leads to p38 activation. Conversely, the p38 inhibitor SB-202190 did not inhibit ONOO-induced ERK1/2 phosphorylation, indicating that ERK1/2 activation is not dependent on the p38 MAPK pathway (data not shown). To test whether this product is related to arachidonic acid or its metabolites, quiescent smooth muscle cells were incubated with arachidonic acid (10 µM) for 1 h and cell lysates were examined for p38 phosphorylation. The results in Fig. 7B, left, clearly demonstrate that arachidonic acid (or its metabolites) induces p38 phosphorylation, as previously demonstrated (30). In parallel experiments, we also tested for the involvement of the MEK/ERK pathway in arachidonic acid-induced p38 phosphorylation by again using the MEK1/2 inhibitor U0126. The results in Fig. 7B, right, show that p38 phosphorylation was not prevented by U0126. Thus the MEK/ERK pathway is not involved in arachidonic acid-induced p38 phosphorylation.



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Fig. 7. p38 Phosphorylation is inhibited by the MEK1/2 inhibitor U0126 and is induced by arachidonic acid. A: quiescent smooth muscle cells were preincubated with and without U0126 (40 µM) for 40 min and then exposed to ONOO (200 µM) for 15 or 30 min. Cell lysates were subjected to Western blotting with an antibody specific for phospho-p38. B: quiescent smooth muscle cells were incubated with arachidonic acid (10 µM) for 5, 15, 30, and 60 min, with and without U0126 (40 µM) for 40 min. Cell lysates were subjected to Western blotting with antibodies specific for phospho-p38 and p38.

 
Calcium chelators BAPTA-AM and EGTA inhibit ONOO-induced arachidonic acid release. To examine whether ONOO-dependent arachidonic acid release is mediated by cPLA2, which is known to require Ca2+ for its activity, we incubated smooth muscle cells with the membrane-permeant calcium chelator BAPTA-AM along with EGTA for 1 h followed by stimulation with ONOO. Figure 8 shows that the calcium chelators decreased ONOO-induced arachidonic acid release to a level similar to that observed in control cells. These results indicate that a calcium-dependent form of PLA2 is involved in ONOO-dependent arachidonic acid release.



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Fig. 8. The effect of calcium chelators on ONOO-induced arachidonic acid release. [3H]arachidonic acid-labeled quiescent rat smooth muscle cells were pretreated with BAPTA-AM (30 µM) and EGTA (500 µM) for 1 h, followed by incubation with ONOO (500 µM) for 1 h, and the supernatant medium was assayed for radioactivity. [3H]arachidonic acid release is expressed as dpm/ml supernatant (average ± SE for n = 6 separate cell preparations). Statistical analysis (Student's t-test) revealed that BAPTA-AM and EGTA significantly reduced ONOO-induced arachidonic acid release (P < 0.003). Results shown are representative of 3 independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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It was previously demonstrated that in the presence of arachidonic acid, ONOO enhances purified PGHS activity and also stimulates PGE2 production in arterial smooth muscle cells (7, 34, 63). Under these conditions, ONOO most likely activates the PGHS isozymes in a manner similar to peroxide activation of PGHS, involving heme oxidation (15). We now demonstrate for the first time that in vascular smooth muscle cells a separate mechanism exists whereby the presence of ONOO leads to arachidonic acid release and subsequent eicosanoid production (Fig. 1). An essential clue to the mechanism of its action is the observation that maximal PGE2 production occurs after ONOO addition after ~15 min. In contrast, eicosanoid synthesis after the addition of exogenous arachidonic acid is known to be complete within 1 min of PGHS-1 activation (35). The sole addition of ONOO appears to induce a signaling cascade, thus accounting for the delayed release of arachidonic acid and subsequent synthesis of PGE2 and 6-keto-PGF1{alpha}. The observation that PGE2 and 6-keto-PGF1{alpha} production is increased after ONOO exposure is important because several studies have observed a link between the NO· and arachidonic acid pathways (42, 46, 5457). Increased eicosanoid production in the presence of functional NOS enzymes may be occurring, in part, by an ONOO-induced signaling mechanism that results in arachidonic release. The fact that we observed an enhancement of 6-keto-PGF1{alpha} production in the presence of ONOO indicates that we are not observing significant ONOO-induced PGI2 synthase (PGI2S) inhibition, as previously reported (59, 73, 75). Although ONOO inhibits purified PGI2S with an IC50 of 50 nM (75), substantially higher levels of ONOO are required to inhibit PGI2S in cells (59) because ONOO can deactivate by many different routes. Thus the levels of ONOO that we are adding are not leading to PGI2S deactivation.

In this study, we have examined the specific role of ONOO in initiating cellular signaling cascades leading to the activation of cPLA2 and the eventual release of arachidonic acid in smooth muscle cells, which has not yet been characterized. We investigated pathways that have shown precedence in cPLA2 activation: p38, CaMKII, and the ERKs (32, 39, 44, 45, 66). We observed phosphorylation of MEK1/2, ERK1/2, p38, and cPLA2 in smooth muscle cells after incubation with ONOO in both a time- and concentration-dependent manner (Figs. 2 and 3). Using SIN-1, a molecule that generates both NO· and superoxide anion on decomposition, we demonstrated that in situ generation of ONOO activates the same signaling molecules as added ONOO (Fig. 2). The ONOO concentrations used are consistent with those previously reported by others to activate MAPKs (29) and the EGFR (65). Although the ONOO concentrations are high, it is necessary to consider that ONOO decomposition occurs with a half-life of ~1 s at pH 7.4. This suggests that 500 µM ONOO decays to 2 µM in <10 s, which is within the range of physiological concentrations of ONOO (16). Thus only a small fraction of the total ONOO concentration added comes into contact with the cell (72). In addition, given the reactivity of ONOO with multiple targets in the cell (13, 28, 48, 49), it is not possible to define the local concentration of ONOO required to initiate the signaling molecules that we have studied.

Although it has been established that ONOO stimulates both ERK and p38 MAPK in PC-12 cells (29), until this study it was not clear which MAPK is responsible for the ONOO-mediated activation of cPLA2 in vascular smooth muscle cells. To determine which MAPK is responsible for the ONOO-mediated activation of cPLA2, we used specific inhibitors aimed at blocking either MEK1/2 or p38 MAPK. The highly specific MEK1/2 inhibitor U0126 served to prevent ERK1/2 phosphorylation (14), whereas SB-202190 specifically inhibited p38 MAPK (36). On incubation with the MEK-1/2 inhibitor U0126, ONOO-induced arachidonic acid release and cPLA2 phosphorylation were significantly reduced to control levels, indicating full inhibition (Fig. 6). Upstream activation of ERK1/2 and MEK1/2 likely involves Raf-1, which is the main effector recruited by GTP-bound Ras (2). In these studies, we have demonstrated that Raf-1 is also phosphorylated after ONOO incubation. These findings suggest its involvement in the activation of the ERK-MAPK pathway. The initial signal leading to the activation of the Raf-1/MEK/ERK pathway, however, remains unknown. ONOO is known to permeate the membrane (1), where it could cause chemical modification of specific molecules that, in turn, initiate phosphorylation of signaling molecules. Use of the p38 inhibitor SB-202190 also led to a significant reduction in ONOO-induced arachidonic acid release, although only partial reduction was attained, indicating incomplete involvement of the p38 MAPK pathway (Fig. 4). p38 MAPK phosphorylation is mediated by the upstream signaling kinases MKK6/MKK3, which were also phosphorylated after ONOO incubation. p38 MAPK is known to participate in signal transduction initiated by cellular stress (10, 68), and thus it is reasonable to assume that ONOO, a molecule that is capable of inducing oxidative reactions, mediates the activation of p38 MAPK. The fact that complete inhibition of arachidonic acid release was achieved with the MEK1/2 inhibitor U0126 suggests that the p38 MAPK pathway is initiated by metabolites downstream of MEK1/2. This finding is supported by the fact that ONOO-induced p38 phosphorylation was inhibited by U0126 (Fig. 7A). Here, it was also demonstrated that the addition of arachidonic acid activates p38 in smooth muscle cells, a process that is independent of the MEK/ERK pathway (Fig. 7B). Recently, it was shown that cPLA2-dependent arachidonic acid metabolites contribute to norepinephrine-induced activation of p38 MAPK in vascular smooth muscle cells (30). In particular, the arachidonic acid metabolites of lipoxygenase, 5(S)-hydroxyeicosatetraenoic acid (HETE), 12(S)-HETE, and 15(S)-HETE, are known to activate p38 MAPK (30, 51, 67). The PGHS metabolite, PGE2, is also known to cause p38 MAPK activation (18). Thus formation of arachidonic acid metabolites may cause a positive feedback mechanism in the cPLA2-catalyzed arachidonic acid process. Taken in total, our findings indicate that the major ONOO-driven pathway in smooth muscle cells leading to arachidonic acid release and subsequent eicosanoid production is the MEK/ERK pathway and that initiation of the p38 MAPK pathway is dependent on metabolites generated from the MEK/ERK pathway.

Involvement of CaMKII in ONOO-mediated activation of ERK1/2 was previously reported in PC-12 cells (29). Here, we demonstrate that CaMKII is also activated by ONOO in rat smooth muscle cells (Fig. 5). CaMKII requires Ca2+ and calmodulin (CaM) for activation, and, once activated, CaMKII autophosphorylates at Thr286, which allows phosphorylation of target substrates (17, 61). Phosphorylation of Thr286 results in partial activation of the kinase, even in the absence of Ca2+/CaM (61). The CaMKII-specific inhibitor KN-93 (40) can prevent CaMKII autophosphorylation. Although norepinephrine-induced activation of CaMKII leads to cPLA2 phosphorylation on Ser515 (but not on Ser505 or Ser727) with concomitant arachidonic acid release (44), this was not observed in our studies using ONOO as an agonist. Our studies indicate that full-length cPLA2 is activated with phosphorylation occurring at Ser505.

In addition to phosphorylation, cPLA2 activation also requires a source of Ca2+ (25, 37). In this regard, ONOO exposure is known to cause increased cytosolic Ca2+, which depends on the presence of extracellular Ca2+ (3). Coupled with the role of ONOO in cellular signaling, we thus examined the role of Ca2+ as a necessary requirement for the activation of cPLA2. Ca2+ chelation was found to prevent the release of arachidonic acid after ONOO addition (Fig. 8). These results implicate a role for the calcium-dependent form of PLA2, which requires Ca2+ for the translocation of cPLA2 from the cytosol to membranes where phospholipids are present (70). It could be argued that the increased permeability of Ca2+ is due to membrane damage by ONOO. However, the fact that maximal cPLA2 activation and prostaglandin production occurred 20–30 min after ONOOaddition and the fact that this process was prevented by specific inhibitors of cell signaling molecules indicate that cell damage is unlikely to be the cause.

Finally, the role of ONOO in cellular signaling has implications in the context of inflammation and, in particular, vascular disease. Atherosclerotic lesions have been shown to contain iNOS, which is capable of generating both NO· and superoxide (69) and may promote the activity of ONOO (8). The rate of ONOO production is three times faster than the rate by which superoxide is scavenged by superoxide dismutase (4), indicating that ONOO formation is favorable in a pathophysiological setting. Prolonged exposure to ONOO leads to Tyr nitration that will inhibit PGI2 synthase (59, 7375) and PGHS (13), thus limiting the production of PGI2 (64). Indeed, nitrated PGHS-1 has been observed in human atherosclerotic lesions, although the mechanism of its formation remains unknown (13). Although the direct effects of ONOO in the pathology of vascular disease remain a question, it is apparent that ONOO mediates the production of eicosanoids via the activation of ERK in vascular smooth muscle cells. In this regard, it is interesting to note that elevated levels of activated ERK1/2 have been found in rabbit atherosclerotic lesions compared with control tissue (26). It is possible that the localization of iNOS in the vicinity of an atherosclerotic lesion elevates ONOO levels, thereby activating the ERK family of MAPKs. Thus, although the actions of ONOO are considered deleterious on an acute chemical basis, ONOO may provide the cell with a means to combat some of the clinical manifestations of inflammation through specific eicosanoids before Tyr nitration becomes prominent. Increased PGHS-2 expression in atherosclerosis (60) can contribute to the increased eicosanoid production that is observed in atherosclerotic patients (6, 19). However, the fact that iNOS gene removal causes a reduction in PGE2 synthesis in mice (42) argues for a role of RNS in this process. It is intriguing to consider that ONOO-induced signaling pathways (in addition to ONOO activation of PGHS) may be contributing to increased eicosanoid output in atherosclerotic patients. Our results indicate that studies investigating the role of the NO· pathway on eicosanoid production need to consider the contribution of signaling pathways initiated by RNS. These findings may provide evidence for a new role of ONOO during vascular disease.


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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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This study was made possible by National Heart, Lung, and Blood Institute Grants HL-46403, HL-07423, and HL-49666 awarded to D. P. Hajjar. R. S. Deeb was supported by a National Institutes of Health T32 training grant in Cardiovascular Biology awarded to D. P. Hajjar. R. K. Upmacis was supported, in part, by an American Heart Association Scientist Development Award (9630223N) and an Atorvastatin Research Award (Pfizer Incorporated).


    ACKNOWLEDGMENTS
 
We are grateful to Dr. Domenick Falcone for helpful discussions.

M. J. Resnick and R. Lindenbaum are currently medical students at the University of Pennsylvania School of Medicine and the Sackler Institute of Graduate Biomedical Sciences at New York University School of Medicine, respectively.

Present address of D. Mittar: Dept. of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar 143 005, India.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. K. Upmacis, Dept. of Pathology and Center of Vascular Biology, Rm. A626, Weill Medical College of Cornell Univ., 1300 York Ave., New York, NY 10021 (E-mail rupmacis{at}med.cornell.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Augusto O, Bonini MG, Amanso AM, Linares E, Santos CC, and De Menezes SL. Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology. Free Radic Biol Med 32: 841–859, 2002.[CrossRef][ISI][Medline]

2. Avruch J, Zhang XF, and Kyriakis JM. Raf meets Ras: completing the framework of a signal transduction pathway. Trends Biochem Sci 19: 279–283, 1994.[CrossRef][ISI][Medline]

3. Bapat S, Verkleij A, and Post JA. Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C. FEBS Lett 499: 21–26, 2001.[CrossRef][ISI][Medline]

4. Beckman JS, Chen J, Ischiropoulos H, and Crow JP. Oxidative chemistry of peroxynitrite. Methods Enzymol 233: 229–240, 1994.[ISI][Medline]

5. Beckmann JS, Ye YZ, Anderson PG, Chen J, Accavitti MA, Tarpey MM, and White CR. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe Seyler 375: 81–88, 1994.[ISI][Medline]

6. Belton O, Byrne D, Kearney D, Leahy A, and Fitzgerald DJ. Cyclooxygenase-1 and -2-dependent prostacyclin formation in patients with atherosclerosis. Circulation 102: 840–845, 2000.[Abstract/Free Full Text]

7. Boulos C, Jiang H, and Balazy M. Diffusion of peroxynitrite into the human platelet inhibits cyclooxygenase via nitration of tyrosine residues. J Pharmacol Exp Ther 293: 222–229, 2000.[Abstract/Free Full Text]

8. Buttery LD, Springall DR, Chester AH, Evans TJ, Standfield EN, Parums DV, Yacoub MH, and Polak JM. Inducible nitric oxide synthase is present within human atherosclerotic lesions and promotes the formation and activity of peroxynitrite. Lab Invest 75: 77–85, 1996.[ISI][Medline]

9. Clark JD, Lin LL, Kriz RW, Ramesha CS, Sultzman LA, Lin AY, Milona N, and Knopf JL. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell 65: 1043–1051, 1991.[ISI][Medline]

10. Cohen P. The search for physiological substrates of MAP and SAP kinases in mammalian cells. Trends Cell Biol 7: 353–361, 1997.[CrossRef][ISI]

11. Davidge ST. Prostaglandin H synthase and vascular function. Circ Res 89: 650–660, 2001.[Abstract/Free Full Text]

12. De Carvalho MG, McCormack AL, Olson E, Ghomashchi F, Gelb MH, Yates JR 3rd, and Leslie CC. Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A2 expressed in insect cells and present in human monocytes. J Biol Chem 271: 6987–6997, 1996.[Abstract/Free Full Text]

13. Deeb RS, Resnick MJ, Mittar D, McCaffrey T, Hajjar DP, and Upmacis RK. Tyrosine nitration in prostaglandin H2 synthase. J Lipid Res 43: 1718–1726, 2002.[Abstract/Free Full Text]

14. De Silva DR, Jones EA, Favata MF, Jaffee BD, Magolda RL, Trzaskos JM, and Scherle PA. Inhibition of mitogen-activated protein kinase kinase blocks T cell proliferation but does not induce or prevent anergy. J Immunol 160: 4175–4181, 1998.[Abstract/Free Full Text]

15. Dietz R, Nastainczyk W, and Ruf HH. Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2. Eur J Biochem 171: 321–328, 1988.[Abstract]

16. Estevez AG, Radi R, Barbeito L, Shin JT, Thompson JA, and Beckman JS. Peroxynitrite-induced cytotoxicity in PC12 cells: evidence for an apoptotic mechanism differentially modulated by neurotrophic factors. J Neurochem 65: 1543–1550, 1995.[ISI][Medline]

17. Feinmesser RL, Wicks SJ, Taverner CJ, and Chantry A. Ca2+/calmodulin-dependent kinase II phosphorylates the epidermal growth factor receptor on multiple sites in the cytoplasmic tail and serine 744 within the kinase domain to regulate signal generation. J Biol Chem 274: 16168–16173, 1999.[Abstract/Free Full Text]

18. Fiebich BL, Schleicher S, Spleiss O, Czygan M, and Hull M. Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C. J Neurochem 79: 950–958, 2001.[CrossRef][ISI][Medline]

19. Fitz Gerald GA, Smith B, Pedersen AK, and Brash AR. Increased prostacyclin biosynthesis in patients with severe atherosclerosis and platelet activation. N Engl J Med 310: 1065–1068, 1984.[Abstract]

20. Garrington TP and Johnson GL. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol 11: 211–218, 1999.[CrossRef][ISI][Medline]

21. Go YM, Patel RP, Maland MC, Park H, Beckman JS, Darley-Usmar VM, and Jo H. Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-Jun NH2-terminal kinase. Am J Physiol Heart Circ Physiol 277: H1647–H1653, 1999.[Abstract/Free Full Text]

22. Guidarelli A and Cantoni O. Pivotal role of superoxides generated in the mitochondrial respiratory chain in peroxynitrite-dependent activation of phospholipase A2. Biochem J 366: 307–314, 2002.[ISI][Medline]

23. Guidarelli A, Palomba L, and Cantoni O. Peroxynitrite-mediated release of arachidonic acid from PC12 cells. Br J Pharmacol 129: 1539–1541, 2000.[Abstract/Free Full Text]

24. Hefner Y, Borsch-Haubold AG, Murakami M, Wilde JI, Pasquet S, Schieltz D, Ghomashchi F, Yates JR, Armstrong CG, Paterson A, Cohen P, Fukunaga R, Hunter T, Kudo I, Watson SP, and Gelb MH. Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases. J Biol Chem 275: 37542–37551, 2000.[Abstract/Free Full Text]

25. Hirabayashi T, Kume K, Hirose K, Yokomizo T, Iino M, Itoh H, and Shimizu T. Critical duration of intracellular Ca2+ response required for continuous translocation and activation of cytosolic phospholipase A2. J Biol Chem 274: 5163–5169, 1999.[Abstract/Free Full Text]

26. Hu Y, Dietrich H, Metzler B, Wick G, and Xu Q. Hyperexpression and activation of extracellular signal-regulated kinases (ERK1/2) in atherosclerotic lesions of cholesterol-fed rabbits. Arterioscler Thromb Vasc Biol 20: 18–26, 2000.[Abstract/Free Full Text]

27. Huie RE and Padmaja S. The reaction of NO with superoxide. Free Radic Res Commun 18: 195–199, 1993.[ISI][Medline]

28. Ischiropoulos H and al-Mehdi AB. Peroxynitrite-mediated oxidative protein modifications. FEBS Lett 364: 279–282, 1995.[CrossRef][ISI][Medline]

29. Jope RS, Zhang L, and Song L. Peroxynitrite modulates the activation of p38 and extracellular regulated kinases in PC12 cells. Arch Biochem Biophys 376: 365–370, 2000.[CrossRef][ISI][Medline]

30. Kalyankrishna S and Malik KU. Norepinephrine-induced stimulation of p38 mitogen-activated protein kinase is mediated by arachidonic acid metabolites generated by activation of cytosolic phospholipase A2 in vascular smooth muscle cells. J Pharmacol Exp Ther 304: 761–772, 2003.[Abstract/Free Full Text]

31. Kaur H and Halliwell B. Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid patients. FEBS Lett 350: 9–12, 1994.[CrossRef][ISI][Medline]

32. Kramer RM and Sharp JD. Structure, function and regulation of Ca2+-sensitive cytosolic phospholipase A2 (cPLA2). FEBS Lett 410: 49–53, 1997.[CrossRef][ISI][Medline]

33. Kyriakis JM and Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81: 807–869, 2001.[Abstract/Free Full Text]

34. Landino LM, Crews BC, Timmons MD, Morrow JD, and Marnett LJ. Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis. Proc Natl Acad Sci USA 93: 15069–15074, 1996.[Abstract/Free Full Text]

35. Lassmann G, Odenwaller R, Curtis JF, De Gray JA, Mason RP, Marnett LJ, and Eling TE. Electron spin resonance investigation of tyrosyl radicals of prostaglandin H synthase. Relation to enzyme catalysis. J Biol Chem 266: 20045–20055, 1991.[Abstract/Free Full Text]

36. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, Strikler JE, McLaughlin MM, Siemens IR, Fisher SM, Livi GP, White JR, Adams JL, and Young PR. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372: 739–746, 1994.[CrossRef][ISI][Medline]

37. Leslie CC. Properties and regulation of cytosolic phospholipase A2. J Biol Chem 272: 16709–16712, 1997.[Free Full Text]

38. Lin LL, Lin AY, and Knopf JL. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc Natl Acad Sci USA 89: 6147–6151, 1992.[Abstract]

39. Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, and Davis RJ. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72: 269–278, 1993.[ISI][Medline]

40. Mamiya N, Goldenring JR, Tsunoda Y, Modlin IM, Yasui K, Usuda N, Ishikawa T, Natsume A, and Hidaka H. Inhibition of acid secretion in gastric parietal cells by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-93. Biochem Biophys Res Commun 195: 608–615, 1993.[CrossRef][ISI][Medline]

41. Markwell MA, Haas SM, Bieber LL, and Tolbert NE. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87: 206–210, 1978.[ISI][Medline]

42. Marnett LJ, Wright TL, Crews BC, Tannenbaum SR, and Morrow JD. Regulation of prostaglandin biosynthesis by nitric oxide is revealed by targeted deletion of inducible nitric-oxide synthase. J Biol Chem 275: 13427–13430, 2000.[Abstract/Free Full Text]

43. Muthalif MM, Benter IF, Uddin MR, and Malik KU. Calcium/calmodulin-dependent protein kinase II{alpha} mediates activation of mitogen-activated protein kinase and cytosolic phospholipase A2 in norepinephrine-induced arachidonic acid release in rabbit aortic smooth muscle cells. J Biol Chem 271: 30149–30157, 1996.[Abstract/Free Full Text]

44. Muthalif MM, Hefner Y, Canaan S, Harper J, Zhou H, Parmentier JH, Aebersold R, Gelb MH, and Malik KU. Functional interaction of calcium-/calmodulin-dependent protein kinase II and cytosolic phospholipase A2. J Biol Chem 276: 39653–39660, 2001.[Abstract/Free Full Text]

45. Nemenoff RA, Winitz S, Qian NX, Van Putten V, Johnson GL, and Heasley LE. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J Biol Chem 268: 1960–1964, 1993.[Abstract/Free Full Text]

46. Nogawa S, Forster C, Zhang F, Nagayama M, Ross ME, and Iadecola C. Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia. Proc Natl Acad Sci USA 95: 10966–10971, 1998.[Abstract/Free Full Text]

47. Pomerantz KB, Tall AR, Feinmark SJ, and Cannon PJ. Stimulation of vascular smooth muscle cell prostacyclin and prostaglandin E2 synthesis by plasma high and low density lipoproteins. Circ Res 54: 554–565, 1984.[Abstract]

48. Radi R, Beckman JS, Bush KM, and Freeman BA. Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J Biol Chem 266: 4244–4250, 1991.[Abstract/Free Full Text]

49. Radi R, Beckman JS, Bush KM, and Freeman BA. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 288: 481–487, 1991.[ISI][Medline]

50. Radi R, Denicola A, and Freeman BA. Peroxynitrite reactions with carbon dioxide-bicarbonate. Methods Enzymol 301: 353–367, 1999.[CrossRef][ISI][Medline]

51. Reddy MA, Thimmalapura PR, Lanting L, Nadler JL, Fatima S, and Natarajan R. The oxidized lipid and lipoxygenase product 12(S)-hydroxyeicosatetraenoic acid induces hypertrophy and fibronectin transcription in vascular smooth muscle cells via p38 MAPK and cAMP response element-binding protein activation. Mediation of angiotensin II effects. J Biol Chem 277: 9920–9928, 2002.[Abstract/Free Full Text]

52. Rimarachin JA, Jacobson JA, Szabo P, Maclouf J, Creminon C, and Weksler BB. Regulation of cyclooxygenase-2 expression in aortic smooth muscle cells. Arterioscler Thromb 14: 1021–1031, 1994.[Abstract]

53. Rosenkranz B, Winkelmann BR, and Parnham MJ. Clinical pharmacokinetics of molsidomine. Clin Pharmacokinet 30: 372–384, 1996.[ISI][Medline]

54. Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie MG, and Needleman P. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci USA 90: 7240–7244, 1993.[Abstract]

55. Salvemini D, Seibert K, Masferrer JL, Misko TP, Currie MG, and Needleman P. Endogenous nitric oxide enhances prostaglandin production in a model of renal inflammation. J Clin Invest 93: 1940–1947, 1994.[ISI][Medline]

56. Salvemini D, Settle SL, Masferrer JL, Seibert K, Currie MG, and Needleman P. Regulation of prostaglandin production by nitric oxide; an in vivo analysis. Br J Pharmacol 114: 1171–1178, 1995.[Abstract]

57. Sautebin L, Ialenti A, Ianaro A, and Di Rosa M. Modulation by nitric oxide of prostaglandin biosynthesis in the rat. Br J Pharmacol 114: 323–328, 1995.[Abstract]

58. Schieke SM, Briviba K, Klotz LO, and Sies H. Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: attenuation by selenite supplementation. FEBS Lett 448: 301–303, 1999.[CrossRef][ISI][Medline]

59. Schmidt P, Youhnovski N, Daiber A, Balan A, Arsic M, Bachschmid M, Przybylski M, and Ullrich V. Specific nitration at tyrosine 430 revealed by high resolution mass spectrometry as basis for redox regulation of bovine prostacyclin synthase. J Biol Chem 278: 12813–12819, 2003.[Abstract/Free Full Text]

60. Schonbeck U, Sukhova GK, Graber P, Coulter S, and Libby P. Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions. Am J Pathol 155: 1281–1291, 1999.[Abstract/Free Full Text]

61. Soderling TR. Protein kinases. Regulation by autoinhibitory domains. J Biol Chem 265: 1823–1826, 1990.[Free Full Text]

62. Stamler JS, Singel DJ, and Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Science 258: 1898–1902, 1992.[ISI][Medline]

63. Upmacis RK, Deeb RS, and Hajjar DP. Regulation of prostaglandin H2 synthase activity by nitrogen oxides. Biochemistry 38: 12505–12513, 1999.[CrossRef][ISI][Medline]

64. Upmacis RK, Deeb RS, and Hajjar DP. Role of nitrogen oxides on eicosanoid production during atherosclerosis: understanding the controversies. Curr Atheroscler Rep 3: 181–182, 2001.[Medline]

65. Van der Vliet A, Hristova M, Cross CE, Eiserich JP, and Goldkorn T. Peroxynitrite induces covalent dimerization of epidermal growth factor receptors in A431 epidermoid carcinoma cells. J Biol Chem 273: 31860–31866, 1998.[Abstract/Free Full Text]

66. Waterman WH, Molski TF, Huang CK, Adams JL, and Sha'afi RI. Tumour necrosis factor-alpha-induced phosphorylation and activation of cytosolic phospholipase A2 are abrogated by an inhibitor of the p38 mitogen-activated protein kinase cascade in human neutrophils. Biochem J 319: 17–20, 1996.[ISI][Medline]

67. Wen Y, Gu J, Knaus UG, Thomas L, Gonzales N, and Nadler JL. Evidence that 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid activates p21-activated kinase. Biochem J 349: 481–487, 2000.[CrossRef][ISI][Medline]

68. Widmann C, Gibson S, Jarpe MB, and Johnson GL. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev 79: 143–180, 1999.[Abstract/Free Full Text]

69. Xia Y and Zweier JL. Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci USA 94: 6954–6958, 1997.[Abstract/Free Full Text]

70. Yoshihara Y and Watanabe Y. Translocation of phospholipase A2 from cytosol to membranes in rat brain induced by calcium ions. Biochem Biophys Res Commun 170: 484–490, 1990.[ISI][Medline]

71. Zhang P, Wang YZ, Kagan E, and Bonner JC. Peroxynitrite targets the epidermal growth factor receptor, Raf-1, and MEK independently to activate MAPK. J Biol Chem 275: 22479–22486, 2000.[Abstract/Free Full Text]

72. Zhu L, Gunn C, and Beckman JS. Bactericidal activity of peroxynitrite. Arch Biochem Biophys 298: 452–457, 1992.[ISI][Medline]

73. Zou M, Martin C, and Ullrich V. Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite. Biol Chem 378: 707–713, 1997.[ISI][Medline]

74. Zou MH, Leist M, and Ullrich V. Selective nitration of prostacyclin synthase and defective vasorelaxation in atherosclerotic bovine coronary arteries. Am J Pathol 154: 1359–1365, 1999.[Abstract/Free Full Text]

75. Zou MH and Ullrich V. Peroxynitrite formed by simultaneous generation of nitric oxide and superoxide selectively inhibits bovine aortic prostacyclin synthase. FEBS Lett 382: 101–104, 1996.[CrossRef][ISI][Medline]