J Biol Chem, Vol. 273, Issue 44, 28766-28772, October 30, 1998

Direct Inhibition of Cyclooxygenase-1 and -2 by the Kinase Inhibitors SB 203580 and PD 98059
SB 203580 ALSO INHIBITS THROMBOXANE SYNTHASE^*

Angelika G. Börsch-Haubold^§, Sophie Pasquet, and Steve P. Watson

From the Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom

	ABSTRACT

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The kinase inhibitors SB 203580 and PD 98059 have been reported to be specific inhibitors of the 38- and 42/44-kDa mitogen-activated protein kinase (MAPK) pathways, respectively. In this study, the two inhibitors were found to decrease platelet aggregation induced by low concentrations of arachidonic acid, suggesting that they also interfere with the metabolism of arachidonic acid to thromboxane A₂. In support of this, SB 203580 and PD 98059 inhibited the conversion of exogenous [³H]arachidonic acid to [³H]thromboxane in intact platelets. Measurement of platelet cyclooxygenase-1 activity following immunoprecipitation revealed that SB 203580 and PD 98059 are direct inhibitors of this enzyme. Both compounds were shown to inhibit purified cyclooxygenase-1 and -2 by a reversible mechanism. In addition, SB 203580 (but not PD 98059) inhibited platelet aggregation induced by prostaglandin H₂ and the conversion of prostaglandin H₂ to thromboxane A₂ in intact platelets. SB 203580 also inhibited this pathway in platelet microsome preparations, suggesting a direct inhibitory effect on thromboxane synthase. These results demonstrate that direct effects of the two kinase inhibitors on active arachidonic acid metabolites have to be excluded before using these compounds for the investigation of MAPKs in signal transduction pathways. This is of particular relevance to studies on the regulation of cytosolic phospholipase A₂ as these two MAPKs are capable of phosphorylating cytosolic phospholipase A₂, thereby increasing its intrinsic activity.

	INTRODUCTION

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Since specific inhibitors of the mitogen-activated protein kinase (MAPK)¹ and stress-activated protein kinase (SAPK) cascades were first described, they have been widely exploited to investigate the involvement of p38^mapk (also called SAPK2a) and p42/p44^mapk in intracellular signal transduction pathways. The pyridinylimidazole compound SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole) was developed from a series of bicyclic pyridin-4-ylimidazoles that exhibited potent anti-inflammatory actions mediated via inhibition of cyclooxygenase, 5-lipoxygenase, and inflammatory cytokine biosynthesis (for review, see Ref. 1). The lipopolysaccharide-stimulated production of interleukin-1 and tumor necrosis factor- was decreased in human monocytes in the presence of these compounds, and they were therefore termed cytokine-suppressive anti-inflammatory drugs. Further studies revealed that the molecular target of the pyridinylimidazoles was the stress-activated p38^mapk (2, 3). SB 203580 inhibits p38^mapk and its isoform p38 (SAPK2b) with in vitro IC₅₀ values of 0.3-0.6 µM, but has no inhibitory action on SAPK3 and SAPK4 (4-6).

The flavone compound PD 98059 (2-(2-amino-3-methoxyphenyl)oxanaphthalen-4-one) is a specific inhibitor of the mammalian MAPK kinase (MEK) (7). It acts by binding to the inactivated form of MEK, thereby preventing its phosphorylation by c-Raf or MEK kinase (8). Depending on cell type and stimulation, the IC₅₀ determined in intact cells ranges from 2 to 10 µM, values that are similar to those determined for inhibition of activation of MEK1 by Raf in vitro (7, 9). Activation of MEK2 is inhibited with an IC₅₀ of 50 µM (8). Kinetic properties suggest an allosteric mechanism for inhibition, which might be the reason for its high selectivity toward MEK (7, 8).

We have previously used SB 203580 and PD 98059 to investigate the involvement of p38^mapk and p42/p44^mapk, respectively, in the regulation of cytosolic phospholipase A₂ and the activation of human platelets (10, 11). During the course of these studies, we have not observed an action of either drug consistent with inhibition of kinases other than their known targets. However, we have obtained evidence that SB 203580 and PD 98059 interfere with the conversion of arachidonic acid to the pro-aggregatory metabolite thromboxane (Tx) A₂ (11). In platelets, the metabolism of arachidonic acid by the cyclooxygenase pathway results in the production of several active eicosanoids. The prostaglandin (PG) endoperoxide PGH₂ activates platelets (12), whereas its stable product, PGE₂, can have pro-aggregatory or inhibitory effects depending on concentration (13). PGH₂ is the substrate for thromboxane synthase and is converted by this enzyme to TxA₂ in platelets. In this study, we have investigated whether the reduction of TxA₂ formation by SB 203580 and PD 98059 is due to inhibition of p38^mapk and p42/p44^mapk, respectively, or to a direct effect on arachidonic acid metabolism.

	MATERIALS AND METHODS

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Reagents and Antibodies-- SB 203580 and PD 98059 were kindly provided by Dr. J. C. Lee (SmithKline Beecham, King of Prussia, PA) and Dr. A. R. Saltiel (Parke-Davis), respectively. SB 203580 was also obtained from Alexis Corp. (Nottingham, United Kingdom). Purity of these compounds was >98%. Polyclonal anti-cyclooxygenase-1 antibody, purified cyclooxygenase-1 and -2, and enzyme immunoassay (EIA) kits for measuring PGE₂ were purchased from Cayman Chemical Co., Inc.(Ann Arbor, MI). Polyclonal rabbit anti-p42^mapk antibody was from Santa Cruz Biotechnology, and rabbit anti-p38^mapk antiserum was a gift from Dr. R. J. Ulevitch (Scripps Research Institute, La Jolla, CA). 5-[1,2-³H]Hydroxytryptamine (specific radioactivity of 25 Ci/mmol), [5,6,8,9,11,12,14,15-³H]arachidonic acid (specific radioactivity of 212 Ci/mmol), and radioimmunoassay and EIA kits for measuring TxB₂ were supplied by Amersham Pharmacia Biotech. [-³²P]ATP (specific activity of 3000 Ci/mmol) was obtained from NEN Life Science Products. Arachidonic acid (Sigma) was dissolved in ethanol and stored at 20 °C. PGH₂ (Calbiochem), dissolved in hexane/isopropyl alcohol (9:1), was stored at 70 °C; before experimentation, the solvent was evaporated under a stream of N₂, and PGH₂ was dissolved in acetone (100 µg/ml) (14). Furegrelate (Sigma) was dissolved in Tyrode's buffer. Prostacyclin was kindly donated by Wellcome Laboratories (Beckenham, Kent, UK). Bovine thrombin and protein A-Sepharose CL-4B were obtained from Sigma. Collagen was purchased from Nycomed Arzneimittel (Munich, Germany). All other reagents were of analytical grade.

Preparation of Washed Platelets-- 50 ml of blood was drawn on the day of experimentation from healthy volunteers by venipuncture using 7.5 ml of acidic citrate dextrose (120 mM sodium citrate, 110 mM glucose, and 80 mM citric acid) as anticoagulant. All solutions were prewarmed to 30 °C. Platelet-rich plasma was obtained by centrifugation at 200 × g for 20 min, and platelets were collected by centrifugation at 1000 × g for 10 min in the presence of 0.1 µg/ml prostacyclin. The platelet pellet was gently resuspended in Tyrode's buffer (20 mM HEPES, 135 mM NaCl, 3 mM KCl, 0.25 mM Na₂HPO₄, 12 mM NaHCO₃, 1 mM MgCl₂, and 5 mM glucose, pH 7.3) and 150 µl of acidic citrate dextrose solution. Platelets were washed with a mixture of 25 ml of Tyrode's buffer and 3 ml of acidic citrate dextrose and centrifuged at 1000 × g for 10 min in the presence of prostacyclin. The pellet was resuspended in 1 ml of Tyrode's buffer, and the volume was adjusted to give 4 × 10⁸ cells/ml. Platelets were allowed to rest for 30 min at 30 °C prior to experimentation.

Platelet Aggregation, TxB₂ Release, and Kinase Activity-- Platelets (500 µl) were incubated with Me₂SO (0.4%), SB 203580 (20 µM), or PD 98059 (20 µM) for 10 min at 37 °C and stimulated with collagen, thrombin, arachidonic acid, or PGH₂ under stirred conditions (1200 rpm). Aggregation was monitored in a Born lumi-aggregometer for 5 min. The reaction was stopped by the addition of 2 volumes of EIA buffer (100 mM phosphate, pH 7.5, containing 150 mM NaCl, 0.1% bovine serum albumin, and 0.01% NaN₃). The amount of TxB₂ released was determined by radioimmunoassay or EIA as described (15).

Activation of p42^mapk and p38^mapk was measured after immunoprecipitation from platelet lysates using 5 µl of anti-p42^mapk antibody or 2 µl of anti-p38^mapk antiserum. Proteins were separated on 10% SDS-polyacrylamide gels that contained 0.5 mg/ml myelin basic protein co-polymerized with the acrylamide. Kinases were denatured in 6 M guanidine HCl and renatured for 16 h at 4 °C as described previously (16). Gels were incubated in kinase buffer (50 mM Tris, pH 8.0, 5 mM MgCl₂, 1 mM EGTA, 5 mM dithiothreitol, 50 µM ATP, and 20 µCi/ml [-³²P]ATP) for 1 h at 37 °C, extensively washed in 5% trichloroacetic acid and 1% Na₄O₂P₇, and dried. After autoradiography, the region of MAPK was cut from the gel and Cerenkov-counted for radioactivity.

Release of 5-[³H]Hydroxytryptamine-- Platelets were incubated with 10 µCi of 5-[³H]hydroxytryptamine for 1 h at 30 °C, washed, and resuspended in Tyrode's buffer. Stimulation of platelets was stopped with an equal volume of 6% (v/v) glutaraldehyde solution. Platelets were pelleted by centrifugation at 13,000 × g for 15 min, and the radioactivity of the supernatant was determined by liquid scintillation spectrometry in a Beckman scintillation counter to a 5% level of significance.

Preparation of Platelet Microsomes-- Washed platelets were suspended at 2 × 10⁹ cells/ml in 25 mM HEPES, pH 7.0, containing 25 mM NaCl, 100 mM KCl, 2 mM MgSO₄·7H₂O, 12 mM trisodium citrate, 10 mM glucose, 5 mM dithiothreitol, 1 mM benzamidine HCl, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM EGTA, and microsomal fractions were prepared as described (17). The suspension was sonicated on ice four times for 5 s and then centrifuged at 1500 × g. This process was repeated on the pellet, and both sonicates were centrifuged at 13,000 × g for 30 min at 4 °C to remove insoluble material. The supernatant was centrifuged at 150,000 × g for 60 min at 4 °C, and the pellet, representing the microsomal fraction, was suspended in 20 mM Tris, pH 8.0, containing 1 mM EDTA, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1 mM benzamidine HCl. Protein concentrations were determined by the Bradford reaction (Bio-Rad) using bovine serum albumin as a standard. Microsomes were stored at 70 °C. Thromboxane synthase activity was verified by the capacity of the platelet microsomes to convert PGH₂ into TxB₂. The amount of TxB₂ produced was determined by EIA as described by Maclouf (15). In some experiments, microsomes were preincubated for 15 min at 37 °C with Me₂SO (0.4%), SB 203580 (20 µM), or PD 98059 (20 µM).

Cyclooxygenase and Thromboxane Synthase Activities in Intact Platelets-- Platelets were preincubated with Me₂SO (0.4%), SB 203580 (20 µM), or PD 98059 (20 µM) for 10 min and then incubated for 5 min with 1 µM arachidonic acid containing 1 µCi of [³H]arachidonic acid at 37 °C. The reaction was stopped by acidification (addition of 1 N HCl to reach pH 4), and lipids were extracted by incubation overnight at 4 °C with 3 volumes of ethyl acetate. Samples were concentrated by evaporation and applied to silica thin-layer chromatography plates (Merck). Lipids were separated by ascending thin-layer chromatography in ethyl acetate/isooctane/acetic acid/H₂O (110:50:20:100, v/v/v/v) as described (18). 1-cm fractions of the silica plate were scraped off, and the radioactive products were quantitated by liquid scintillation spectrometry in a Beckman scintillation counter. Radioactive lipids were identified compared with the migration of standards. The 12-lipoxygenase product 12-[³H]hydroxyeicosatetraenoic acid migrated close to [³H]arachidonic acid, and radioactivity from this lipid was added to untransformed [³H]arachidonic acid for the calculation of metabolite formation.

Cyclooxygenase Assays-- For immunoprecipitation of cyclooxygenase-1, platelets were lysed in ice-cold buffer (50 mM Tris-HCl, pH 8.0, 30 mM n-octyl glucoside, 1 mM EDTA, and 1 mM benzamidine HCl) (19). Lysates were precleared with protein A-Sepharose CL-4B, and cyclooxygenase-1 was immunoprecipitated using 5 µl of polyclonal anti-cyclooxygenase-1 antibody and 25 µl of protein A-Sepharose CL-4B slurry overnight at 4 °C. Immunoprecipitates were recovered by microcentrifugation and washed twice in the ice-cold lysis buffer. Immunoprecipitates were resuspended in 20 µl of 50 mM Tris, pH 8, containing 0.1% bovine serum albumin and 1 mM phenol at 37 °C and were incubated with buffer, Me₂SO (2%; control), SB 203580 (20 µM), or PD 98059 (20 µM) for 10 min followed by hematin (1 µM) for 1 min. Cyclooxygenase-1 was stimulated with 25 µM arachidonic acid containing 1 µCi of [³H]arachidonic acid at 37 °C. After 10 min, the reaction was terminated by the addition of 3 volumes of ice-cold Tris-buffered saline and centrifugation for 1 min at 5000 × g. Lipids were extracted from the supernatant in 3 volumes of ethyl acetate and separated by thin-layer chromatography as described above. Cyclooxygenase-1 bound to protein A-Sepharose was solubilized in Laemmli sample buffer and subjected to SDS-polyacrylamide electrophoresis. Cyclooxygenase-1 immunoprecipitation was verified by immunoblotting.

For measuring transformation of arachidonic acid using purified cyclooxygenase, cyclooxygenase-1 and -2 were diluted to give PGE₂ production within the linear range of the EIA kit. Cyclooxygenase was incubated with buffer, Me₂SO, SB 203580, PD 98059, or indomethacin for 15 min, and the reaction was initiated by the addition of arachidonic acid (25 µM) as described above. Formation of PGE₂ was measured using EIA according to the instructions of the manufacturer.

	RESULTS

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Inhibitory Effects of SB 203580 and PD 98059 on Platelet Responses-- Platelet aggregation induced by collagen is dependent on the formation of thromboxane A₂ from arachidonic acid by the action of cyclooxygenase and thromboxane synthase. We have reported earlier that SB 203580 and PD 98059 inhibit platelet aggregation induced by low concentrations of collagen. PD 98059 is more powerful than SB 203580 in inhibiting this response (11). Its inhibitory action was only overcome by collagen at 20 µg/ml (Fig. 1A), whereas the effect of SB 203580 was overcome at 5 µg/ml collagen (11). In contrast to collagen, aggregation stimulated by thrombin (0.1 unit/ml) was not dependent on the release of TxA₂, as demonstrated by preincubation with the cyclooxygenase inhibitor indomethacin (Fig. 1B). The presence of neither PD 98059 (20 µM) nor SB 203580 (20 µM) altered the aggregation traced to thrombin (Fig. 1B). These concentrations of PD 98059 and SB 203580 are sufficient to inhibit thrombin-induced activation of p42^mapk and p38^mapk, respectively (10, 20). The inclusion of Me₂SO did not have any significant effect on platelet aggregation as tested with thrombin (0.1 units/ml) and collagen (10 µg/ml) (data not shown). SB 203580 and PD 98059 partially inhibited aggregation stimulated by the Ca²⁺ ionophore A23187 (2 µM) (data not shown), a platelet stimulus that induces profound liberation of arachidonic acid from phospholipids (21). In contrast, aggregation induced by A23187 in the presence of the cyclooxygenase blocker indomethacin was not altered in the presence of PD 98059 or SB 203580 (data not shown). Thus, SB 203580 and PD 98059 impaired platelet aggregation only under conditions where formation of TxA₂ contributed to the response.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effect of SB 203580 and PD 98059 on the aggregation of human platelets. Washed platelets were preincubated with Me2SO (0.4-1%), PD 98059 or SB 203580 (both 20 µM if not indicated otherwise on the figure), or indomethacin (10 µM) for 10 min at 37 °C and stimulated with collagen (A), thrombin (B), arachidonic acid (C), or PGH2 (D) in a stirred solution. Aggregation was recorded in a Born lumi-aggregometer as increase in light transmission. Traces shown are representative of several independent experiments with blood obtained from different donors. IC50 values (C) were assessed by measuring the distances of maximum aggregation against inhibitor concentration.

Thromboxane formation stimulated by collagen (2 and 5 µg/ml), as measured by EIA, was partially inhibited by SB 203580 and completely blocked by PD 98059 (Fig. 2), which is in agreement with the effects of the compounds on aggregation. In addition, release of 5-hydroxytryptamine stimulated by collagen in the absence of indomethacin was reduced by both compounds, but could be overcome at higher concentrations of collagen (Fig. 3).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effect of SB 203580 and PD 98059 on TxB2 release from platelets. Washed platelets (4 × 108 cells/ml) were treated with Me2SO, SB 203580 (20 µM), or PD 98059 (20 µM) and stimulated with collagen for 5 min at 37 °C. Release of TxB2 into the buffer was measured by EIA. Data shown are the means ± S.D. from triplicate determinations and are representative of two independent measurements.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Release of 5-[3H]hydroxytryptamine from platelets. 5-[3H]Hydroxytryptamine-radiolabeled platelets (4 × 108 cells/ml) were incubated with Me2SO (1%; ), SB 203580 (20 µM; ), or PD 98059 (20 µM; ) and stimulated with collagen for 2 min at 37 °C under stirred conditions. Platelets were fixed in glutaraldehyde solution, and the release of 3H into the buffer was determined. Typical values for basal and stimulated samples (20 µg/ml collagen) were 4000 and 11,000 dpm, respectively. Data are expressed as the means ± S.E. from quadruplicate determinations.

Platelet Activation by Arachidonic Acid-- To investigate the effect of the compounds on the metabolism of arachidonic acid, we incubated platelets with low concentrations of arachidonic acid, which induce aggregation as a consequence of conversion to TxA₂. SB 203580 and PD 98059 inhibited platelet aggregation induced by 0.2 µM arachidonic acid with approximate IC₅₀ values of 3 and 0.8 µM, respectively (Fig. 1C). Aggregation stimulated by 1 µM arachidonic acid was reduced by 50% in the presence of SB 203508 (20 µM) and was blocked by PD 98059 (20 µM) (Fig. 1C). Moreover, both compounds inhibited formation of TxB₂ from arachidonic acid (1 µM) as measured by EIA (Table I). These observations demonstrate that both inhibitors interfere with the metabolism of arachidonic acid to TxA₂.

Arachidonic acid (0.2 and 1 µM) did not stimulate platelet p42^mapk and caused weak activation of p38^mapk relative to activation by thrombin (Fig. 4). However, activation of MAPK-activated protein kinase-2, the in vivo substrate of p38^mapk, was not significantly enhanced above basal levels by arachidonic acid (data not shown). The TxA₂ receptor agonist U46619 has been reported to cause weak phosphorylation of p42^mapk and p38^mapk (20). Thus, arachidonic acid and its metabolites seem to be only weak stimulators of MAPKs.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Activation of p42mapk and p38mapk. Platelets were incubated with buffer (basal), arachidonic acid (aa; 0.2 and 1 µM), or thrombin (1 unit/ml) for 2 min at 37 °C under stirred conditions. p42mapk and p38mapk were immunoprecipitated from platelet lysates and run on SDS-polyacrylamide gels containing myelin basic protein (MBP). Gels were denatured, renatured, and incubated with kinase buffer. After extensive washing, gels were dried. Phosphorylated proteins were visualized by autoradiography, cut from the gels, and Cerenkov-counted for incorporation of 32P into myelin basic protein.

Because the kinase inhibitors could interfere with the release of endogenous arachidonic acid through an effect on the activity of cytosolic phospholipase A₂, it was important to determine the transformation of arachidonic acid to TxB₂ independent of the release of endogenous thromboxane. For this reason, we incubated platelets with [³H]arachidonic acid (1 µM) and measured the formation of ³H-labeled metabolites. 29.0 ± 4.6% of [³H]arachidonic acid (mean ± S.E., n = 4 (n = number of independent experiments)) added to platelets was metabolized to [³H]TxB₂ in 10 min with little formation of the endoperoxide [³H]PGH₂ (1.8 ± 0.3%) or its stable product, [³H]PGE₂ (2.8 ± 0.7%) (Fig. 5). Untransformed [³H]arachidonic acid and the 12-lipoxygenase product 12-[³H]hydroxyeicosatetraenoic acid constituted 42.9 ± 3.7% of total radioactivity. When platelets were incubated in the presence of the cyclooxygenase blocker indomethacin, formation of [³H]TxB₂, [³H]PGH₂, and [³H]PGE₂ was completely inhibited (Fig. 5A). This was in contrast to incubation with the thromboxane synthase inhibitor furegrelate (10 µM) (19, 22), which completely blocked the formation of [³H]TxB₂, but increased the formation of its precursor, [³H]PGH₂, to 13.5 ± 1.3% (n = 4) and of [³H]PGE₂ to 14.4 ± 3.0% (n = 4) (Fig. 5A). In the presence of SB 203580 or SB 203580 plus furegrelate, the metabolism of [³H]arachidonic acid was substantially inhibited with [³H]TxB₂, [³H]PGH₂, and [³H]PGE₂, accounting for 2-5% of total radioactivity (Fig. 5B). Similarly, in the presence of furegrelate, PD 98059 fully inhibited the formation of all three products (data not shown). These results confirm that SB 203580 and PD 98059 inhibit platelet cyclooxygenase.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Conversion of [3H]arachidonic acid to metabolites. Washed platelets were incubated with Me2SO (), furegrelate (10 µM; ), indomethacin (10 µM; ), SB 203580 (20 µM; ) or SB 203580 and furegrelate () for 10 min at 37 °C and stimulated with arachidonic acid (1 µM) containing [3H]arachidonic acid (1 µCi). The reaction was stopped after 10 min by acidification, and lipids were extracted by ethyl acetate and separated on silica thin-layer chromatography plates. Fractions of the silica plate were scraped off, and radioactivity was measured by liquid scintillation spectrometry. The positions of TxB2 and PGH2 were determined in comparison with standards ([3H]TxB2 and unlabeled PGH2, visualized by iodine vapor); PGE2 was identified by its RF value. The background represented 1-2% of total radioactivity. Data shown are the means ± S.D. from triplicate determinations and are representative of four independent experiments. 12-HETE, 12-[3H]hydroxyeicosatetraenoic acid.

To test the possibility that both compounds cause direct inhibition of cyclooxygenase, we immunoprecipitated cyclooxygenase-1 from unstimulated platelets and measured its activity in vitro. Under resting conditions, neither p42^mapk nor p38^mapk is activated (11, 16). Cyclooxygenase-1 converted [³H]arachidonic acid to [³H]PGH₂, which was measured as the chemically stable [³H]PGE₂ (Fig. 6). There was no significant formation of [³H]PGE₂ in the presence of either SB 203580 (20 µM) or PD 98059 (20 µM) (Fig. 6). The presence of Me₂SO had no significant effect on enzyme activity (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   In vitro activity of platelet cyclooxygenase-1. Cyclooxygenase-1 was immunoprecipitated from human platelets, and its activity was determined in vitro in the presence of Me2SO (), SB 203580 (20 µM; ), or PD 98059 (20 µM; ) using [3H]arachidonic acid (25 µM; 1 µCi) as substrate. Lipids were extracted in ethyl acetate and separated by thin-layer chromatography as described in the legend of Fig. 5. Cyclooxygenase activity was estimated by conversion to [3H]PGE2, the stable metabolite of [3H]PGH2. Inclusion of Me2SO did not have any effect on cyclooxygenase activity.

Human platelets contain the constitutively expressed cyclooxygenase-1, but not the inducible cyclooxygenase-2 (23). To further characterize the inhibition of cyclooxygenase by the two kinase inhibitors, we measured the effect of increasing inhibitor concentrations on purified cyclooxygenase-1 and -2. SB 203580 and PD 98059 inhibited cyclooxygenase-1 with approximate IC₅₀ values of 2 and 1 µM, respectively (Fig. 7A). Inhibition was completely reversible for both compounds, as tested by preincubation of cyclooxygenase with 3 µM inhibitor before a 10-fold dilution and incubation with substrate (data not shown). Cyclooxygenase-2 was also inhibited by both compounds in a reversible manner. IC₅₀ values were ~2 and 4 µM for SB 203580 and PD 98059, respectively (Fig. 7B). Inclusion of the solvent Me₂SO did not reduce enzyme activity. The degree of inhibition of cyclooxygenase-1 and -2 by SB 203580 and PD 98059 (20 µM) was similar to that by a maximally effective concentration of the cyclooxygenase inhibitor indomethacin (data not shown). Inhibition by SB 203580 could be overcome by increasing arachidonic acid concentrations; in the presence of SB 203580 (3 µM), the K_m for arachidonic acid was increased without a change in the V_max, suggesting a competitive mechanism of inhibition (data not shown). In contrast, PD 98059 (3 µM) decreased the V_max even at the highest concentration of arachidonic acid used (50 µM) (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Inhibition of purified cyclooxygenase-1 and -2. Cyclooxygenase-1 (A) and -2 (B) were incubated with SB 203580 (A, ; and B, ) or PD 98059 (A, ; and B, ) for 15 min. Cyclooxygenase activity was measured using arachidonic acid (25 µM) transformation. Formation of PGE2 was quantified by EIA. The presence of Me2SO did not significantly decrease cyclooxygenase activity. Similar data were obtained in several independent experiments.

Platelet Activation by PGH₂-- To distinguish between the effects of the kinase inhibitors on cyclooxygenase and thromboxane synthase, we stimulated platelets with the cyclooxygenase product PGH₂. SB 203580 inhibited PGH₂-induced platelet aggregation, whereas PD 98059 did not alter the response (Fig. 1D). In addition, the formation of TxB₂ from 1 µg/ml PGH₂ was inhibited by SB 203580, but not by PD 98059, as determined by EIA (Table I).

It was important to determine the activity of thromboxane synthase independent of the stimulation of receptor-activated pathways and of activation of cytosolic phospholipase A₂. We therefore measured the transformation of PGH₂ to TxB₂ on platelet microsomes, a membrane fraction that contains thromboxane synthase (17). Microsomes were obtained from unstimulated platelets, which means that p42^mapk and p38^mapk were not activated. PGH₂ added to microsomes was transformed to TxB₂ as determined by radioimmunoassay (Table I). The presence of PD 98059 in this assay did not significantly alter the response, whereas the addition of SB 203580 blocked TxB₂ formation (Table I). These results demonstrate that SB 20350 (but not PD 98059) acts as a direct inhibitor of thromboxane synthase.

	DISCUSSION

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Several studies have confirmed the selectivity of the kinase inhibitors SB 203580 and PD 98059 against a variety of kinases. SB 203580 was tested against 16 protein kinases and two phosphatases and was found to be selective against p38 and p38 (SAPK2a and SAPK2b), but to have no inhibitory effect on SAPK3, SAPK4, p42/p44^mapk, c-Jun N-terminal kinase (SAPK1), or any of the upstream kinases (3, 6). Similarly, the selectivity of PD 98059 has been described in several studies (7-9). We (Refs. 11 and 20 and this study) and others (24) have observed that platelet responses dependent on the formation of TxA₂ are inhibited by these compounds. However, since SB 203580 has been developed from drugs that are inhibitors of cyclooxygenase and lipoxygenase, it is uncertain whether the inhibitory actions of SB 203580 on platelet responses can be interpreted as effects of p38^mapk or whether they are due to inhibition of arachidonic acid metabolism. Furthermore, we have also observed inhibition of platelet activation by PD 98059 under conditions that have little effect on p42^mapk activation. For these reasons, we set out to investigate the effects of these compounds on the arachidonic acid cascade.

We obtained evidence for direct inhibition of platelet cyclooxygenase by SB 203580 from a variety of approaches: first, inhibition of platelet aggregation induced by stimuli that are dependent on TxA₂ to elicit aggregation; second, inhibition of TxB₂ release; third, inhibition of the conversion of [³H]arachidonic acid to ³H-labeled metabolites after the addition of [³H]arachidonic acid to intact platelets; and fourth, inhibition of the activity of immunoprecipitated cyclooxygenase-1 as measured in vitro. In addition, we found direct effects of SB 203580 on thromboxane synthase using PGH₂ as agonist, as the following responses were inhibited: platelet aggregation, TxB₂ formation from intact platelets, and conversion of PGH₂ to TxB₂ in platelet microsomes.

PD 98059 was a more powerful inhibitor of thromboxane-dependent platelet aggregation than SB 203580. In intact platelets, PD 98059 was a stronger inhibitor of cyclooxygenase (approximate IC₅₀ for inhibition of arachidonic acid-induced platelet aggregation = 0.8 µM) than of MEK activation by Raf (IC₅₀ = 2-10 µM in vivo) (7, 10). Alessi et al. (8) have previously pointed out that relatively high concentrations of PD 98059 (20-50 µM) have to be used on intact cells to completely block the activation of p42/p44^mapk, and our studies on the activation of p42/p44^mapk in human platelets have confirmed these results (10). However, a 10-fold lower concentration of PD 98059 was sufficient to inhibit platelet aggregation, suggesting that this inhibition is independent of p42^mapk. In fact, p42^mapk was not activated by arachidonic acid under the conditions of the aggregation experiment. Moreover, PD 98059 does not alter phosphorylation of cytosolic phospholipase A₂ in collagen-activated platelets (11). The interference with collagen-induced aggregation would therefore seem to reflect its inhibitory effect on cyclooxygenase. In contrast to SB 203580, PD 98059 had no direct effect on thromboxane synthase.

Inhibition of purified cyclooxygenase-1 and -2 was reversible for both compounds. The IC₅₀ values for purified cyclooxygenase-1 agree with the values determined in platelet aggregation experiments. Cyclooxygenase-2 was slightly less susceptible to inhibition by SB 203580 and PD 98059 compared with cyclooxygenase-1. Marshall et al. (25) have previously analyzed the effects of an analogue of SB 203580 on cyclooxygenase activity; inhibition by 2-(4-methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole was reversible and competitive. They speculated that this compound and other nonsteroidal anti-inflammatory drugs such as acetoamidophenol and phenylbutazone act as radical scavengers and thereby decrease the availability of hydrogen peroxide intermediates during the enzymatic reaction (25). It is possible that a similar mechanism applies to SB 203580.

The rate-limiting step for the liberation of TxA₂ in human platelets is the formation of arachidonic acid from membrane phospholipids by the activity of cytosolic phospholipase A₂ (26). Cytosolic phospholipase A₂ is regulated by changes in the intracellular Ca²⁺ concentration (27) and by phosphorylation of serine 505 (28) and possibly on other serine residues (29, 30). SB 203580 and PD 98059 are important tools to investigate MAPK-mediated phosphorylation of cytosolic phospholipase A₂ and the regulation of arachidonic acid release. However, as we show in this report, both compounds interfere directly with arachidonic acid metabolism through the cyclooxygenase pathway. Moreover, in other cases where arachidonic acid metabolites regulate the cell's response, a clear distinction between the effects of kinase inhibition and cyclooxygenase inhibition by SB 203580 and PD 98059 has to be made. For routine application of the inhibitors, we suggest that the compounds be used in conjunction with a cyclooxygenase blocker, such as indomethacin or aspirin, in order to be able to interpret results unambiguously.

	ACKNOWLEDGEMENTS

We thank Prof. J. Maclouf (INSERM U 346, Paris, France) for advice on the cyclooxygenase assay. We are grateful to Dr. A. R. Saltiel for PD 98059 and to Dr. J. C. Lee for SB 203580.

	FOOTNOTES

^* This work was supported by the British Heart Foundation.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.

These two authors contributed equally to this work.

^§ To whom correspondence should be addressed. Fax: 44-1865-271853; E-mail: angelika.borsch{at}pharmacology.oxford.ac.uk.

The abbreviations used are: MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; Tx, thromboxane; PG, prostaglandin; EIA, enzyme immunoassay.

	REFERENCES

Top Abstract Introduction Materials & Methods Results Discussion References

1. Lee, J. C., and Young, P. R. (1996) J. Leukocyte Biol. 59, 152-157[Abstract]
2. Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., Strickler, J. E., McLaughlin, M. M., Siemens, I. R., Fisher, S. M., Livi, G. P., White, J. R., Adams, J. L., and Young, P. R. (1994) Nature 372, 739-746[CrossRef][Medline] [Order article via Infotrieve]
3. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) FEBS Lett. 364, 229-233[CrossRef][Medline] [Order article via Infotrieve]
4. Jiang, Y., Chen, C., Li, Z., Guo, W., Gegner, J. A., Lin, S., and Han, J. (1996) J. Biol. Chem. 271, 17920-17926[Abstract/Free Full Text]
5. Cuenda, A., Cohen, P., Buée-Scherrer, V., and Goedert, M. (1997) EMBO J. 16, 295-305[CrossRef][Medline] [Order article via Infotrieve]
6. Goedert, M., Cuenda, A., Craxton, M., Jakes, R., and Cohen, P. (1997) EMBO J. 16, 3563-3571[CrossRef][Medline] [Order article via Infotrieve]
7. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689[Abstract/Free Full Text]
8. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489-27494[Abstract/Free Full Text]
9. Pang, L., Sawada, T., Decker, S. J., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 13585-13588[Abstract/Free Full Text]
10. Börsch-Haubold, A. G., Kramer, R. M., and Watson, S. P. (1996) Biochem. J. 318, 207-212
11. Börsch-Haubold, A. G., Kramer, R. M., and Watson, S. P. (1997) Eur. J. Biochem. 245, 751-759[Medline] [Order article via Infotrieve]
12. Gresele, P., Deckmyn, H., Nenci, G. G., and Vermylen, J. (1991) Trends Pharmacol. Sci. 12, 158-163[CrossRef][Medline] [Order article via Infotrieve]
13. Vezza, R., Roberti, R., Nenci, G. G., and Gresele, P. (1993) Blood 82, 2704-2713[Abstract/Free Full Text]
14. Hamberg, M., Svensson, J., Wakabayashi, T., and Samuelsson, B. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 345-349[Abstract/Free Full Text]
15. Maclouf, J. (1981) Methods Enzymol. 86, 273-286
16. Börsch-Haubold, A. G., Kramer, R. M., and Watson, S. P. (1995) J. Biol. Chem. 270, 25885-25892[Abstract/Free Full Text]
17. Habib, A., and Maclouf, J. (1992) Arch. Biochem. Biophys. 298, 544-552[CrossRef][Medline] [Order article via Infotrieve]
18. Daret, D., Blin, P., Dorian, B., Rigaud, M., and Larrue, J. (1993) J. Lipid Res. 34, 1473-1482[Abstract]
19. Karim, S., Habib, A., Lévy-Toledano, S., and Maclouf, J. (1996) J. Biol. Chem. 271, 12042-12048[Abstract/Free Full Text]
20. Kramer, R. M., Roberts, E. F., Ulm, S. L., Börsch-Haubold, A. G., Watson, S. P., Fisher, M. J., and Jakubowski, J. A. (1996) J. Biol. Chem. 271, 27723-27729[Abstract/Free Full Text]
21. Halenda, S. P., Zavoico, G. B., and Feinstein, M. B. (1985) J. Biol. Chem. 260, 12484-12491[Abstract/Free Full Text]
22. Gorman, R. R., Johnson, R. A., Spilman, C. H., and Aiken, J. W. (1983) Prostaglandins 26, 325-342[CrossRef][Medline] [Order article via Infotrieve]
23. Habib, A., Créminon, C., Frobert, Y., Grassi, J., Pradelles, P., and Maclouf, J. (1993) J. Biol. Chem. 268, 23448-23454[Abstract/Free Full Text]
24. Saklatvala, J., Rawlinson, L., Waller, R. J., Sarsfield, S., Lee, J. C., Morton, L. F., Barnes, M. J., and Farndale, R. W. (1996) J. Biol. Chem. 271, 6586-6589[Abstract/Free Full Text]
25. Marshall, P. J., Griswold, D. E., Breton, J., Webb, E. F., Hillegass, L. M., Sarau, H. M., Newton, J. J., Lee, J. C., Bender, P. E., and Hanna, N. (1991) Biochem. Pharmacol. 42, 813-824[CrossRef][Medline] [Order article via Infotrieve]
26. Kramer, R. M. (1994) in Signal-activated Phospholipases (Liscovitch, M., ed), pp. 13-30, R. G. Landes Co., Austin, TX
27. Clark, J. D., Lin, L.-L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., and Knopf, J. L. (1991) Cell 65, 1043-1051[CrossRef][Medline] [Order article via Infotrieve]
28. Lin, L.-L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J. (1993) Cell 72, 269-278[CrossRef][Medline] [Order article via Infotrieve]
29. de Carvalho, M. G. S., McCormack, A. L., Olson, E., Ghomashchi, F., Gelb, M. H., Yates, J. R., III, and Leslie, C. C. (1996) J. Biol. Chem. 271, 6987-6997[Abstract/Free Full Text]
30. Börsch-Haubold, A. G., Bartoli, F., Asselin, J., Dudler, T., Kramer, R. M., Apitz-Castro, R., Watson, S. P., and Gelb, M. H. (1998) J. Biol. Chem. 273, 4449-4458[Abstract/Free Full Text]