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W1985888026 abstract "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. 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. mitogen-activated protein kinase stress-activated protein kinase MAPK/extracellular signal-regulated kinase kinase thromboxane prostaglandin enzyme immunoassay. 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 p38mapk(also called SAPK2a) and p42/p44mapk 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. 1Lee J.C. Young P.R. J. Leukocyte Biol. 1996; 59: 152-157Crossref PubMed Scopus (374) Google Scholar). The lipopolysaccharide-stimulated production of interleukin-1 and tumor necrosis factor-α 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 p38mapk (2Lee 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. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3143) Google Scholar, 3Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1981) Google Scholar). SB 203580 inhibits p38mapk and its isoform p38β (SAPK2b) with in vitro IC50values of 0.3–0.6 μm, but has no inhibitory action on SAPK3 and SAPK4 (4Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1996; 271: 17920-17926Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, 5Cuenda A. Cohen P. Buée-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (316) Google Scholar, 6Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (358) Google Scholar). The flavone compound PD 98059 (2-(2-amino-3-methoxyphenyl)oxanaphthalen-4-one) is a specific inhibitor of the mammalian MAPK kinase (MEK) (7Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar). It acts by binding to the inactivated form of MEK, thereby preventing its phosphorylation by c-Raf or MEK kinase (8Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar). 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 (7Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar, 9Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar). Activation of MEK2 is inhibited with an IC50 of 50 μm (8Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar). Kinetic properties suggest an allosteric mechanism for inhibition, which might be the reason for its high selectivity toward MEK (7Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar, 8Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar). We have previously used SB 203580 and PD 98059 to investigate the involvement of p38mapk and p42/p44mapk, respectively, in the regulation of cytosolic phospholipase A2 and the activation of human platelets (10Börsch-Haubold A.G. Kramer R.M. Watson S.P. Biochem. J. 1996; 318: 207-212Crossref PubMed Scopus (71) Google Scholar, 11Börsch-Haubold A.G. Kramer R.M. Watson S.P. Eur. J. Biochem. 1997; 245: 751-759Crossref PubMed Scopus (141) Google Scholar). 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 (11Börsch-Haubold A.G. Kramer R.M. Watson S.P. Eur. J. Biochem. 1997; 245: 751-759Crossref PubMed Scopus (141) Google Scholar). 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 (12Gresele P. Deckmyn H. Nenci G.G. Vermylen J. Trends Pharmacol. Sci. 1991; 12: 158-163Abstract Full Text PDF PubMed Scopus (126) Google Scholar), whereas its stable product, PGE2, can have pro-aggregatory or inhibitory effects depending on concentration (13Vezza R. Roberti R. Nenci G.G. Gresele P. Blood. 1993; 82: 2704-2713Crossref PubMed Google Scholar). 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 p38mapk and p42/p44mapk, respectively, or to a direct effect on arachidonic acid metabolism. 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-p42mapk antibody was from Santa Cruz Biotechnology, and rabbit anti-p38mapk 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. [γ-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) (14Hamberg M. Svensson J. Wakabayashi T. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 345-349Crossref PubMed Scopus (872) Google Scholar). 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. 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 mmNa2HPO4, 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. 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 (15Maclouf J. Methods Enzymol. 1981; 86: 273-286Crossref Scopus (39) Google Scholar). Activation of p42mapk and p38mapk was measured after immunoprecipitation from platelet lysates using 5 μl of anti-p42mapk antibody or 2 μl of anti-p38mapkantiserum. 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 (16Börsch-Haubold A.G. Kramer R.M. Watson S.P. J. Biol. Chem. 1995; 270: 25885-25892Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). 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 [γ-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. 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. Washed platelets were suspended at 2 × 109 cells/ml in 25 mmHEPES, pH 7.0, containing 25 mm NaCl, 100 mmKCl, 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 mmEGTA, and microsomal fractions were prepared as described (17Habib A. Maclouf J. Arch. Biochem. Biophys. 1992; 298: 544-552Crossref PubMed Scopus (19) Google Scholar). 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 mmphenylmethylsulfonyl 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 (15Maclouf J. Methods Enzymol. 1981; 86: 273-286Crossref Scopus (39) Google Scholar). 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). 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 (18Daret D. Blin P. Dorian B. Rigaud M. Larrue J. J. Lipid Res. 1993; 34: 1473-1482Abstract Full Text PDF PubMed Google Scholar). 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. 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) (19Karim S. Habib A. Lévy-Toledano S. Maclouf J. J. Biol. Chem. 1996; 271: 12042-12048Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). 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 mmTris, pH 8, containing 0.1% bovine serum albumin and 1 mmphenol 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 μmarachidonic 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. 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 (11Börsch-Haubold A.G. Kramer R.M. Watson S.P. Eur. J. Biochem. 1997; 245: 751-759Crossref PubMed Scopus (141) Google Scholar). Its inhibitory action was only overcome by collagen at 20 μg/ml (Fig. 1 A), whereas the effect of SB 203580 was overcome at 5 μg/ml collagen (11Börsch-Haubold A.G. Kramer R.M. Watson S.P. Eur. J. Biochem. 1997; 245: 751-759Crossref PubMed Scopus (141) Google Scholar). 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. 1 B). The presence of neither PD 98059 (20 μm) nor SB 203580 (20 μm) altered the aggregation traced to thrombin (Fig. 1 B). These concentrations of PD 98059 and SB 203580 are sufficient to inhibit thrombin-induced activation of p42mapk and p38mapk, respectively (10Börsch-Haubold A.G. Kramer R.M. Watson S.P. Biochem. J. 1996; 318: 207-212Crossref PubMed Scopus (71) Google Scholar, 20Kramer R.M. Roberts E.F. Ulm S.L. Börsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). 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 (21Halenda S.P. Zavoico G.B. Feinstein M.B. J. Biol. Chem. 1985; 260: 12484-12491Abstract Full Text PDF PubMed Google Scholar). 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. 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).Figure 3Release 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 IC50values of 3 and 0.8 μm, respectively (Fig. 1 C). Aggregation stimulated by 1 μmarachidonic acid was reduced by 50% in the presence of SB 203508 (20 μm) and was blocked by PD 98059 (20 μm)(Fig. 1 C). 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.Table IT×B2 formation from intact platelets and platelet microsomesIntact plateletsng T×B2/4 × 108 plateletsPart A Me2SO/buffer0.15 ± 0.01 Me2SO/collagen4.25 ± 0.30 Me2SO/arachidonic acid5.08 ± 0.70 SB 203580/arachidonic acid0.24 ± 0.04 (p < 0.05) PD 98059/arachidonic acid0.30 ± 0.02 (p < 0.05) Me2SO/PGH23.67 ± 0.25 SB 203580/PGH20.26 ± 0.01 (p < 0.05) PD 98059/PGH23.84 ± 0.22Platelet microsomesng T×B2/4 × 108plateletsPart B Me2SO/PGH21.38 ± 0.16 SB 203580/PGH20.31 ± 0.04 (p < 0.05) PD 98059/PGH21.18 ± 0.10Platelets (4 × 108 platelets/ml) (Part A) or platelet microsome preparations (Part B) were preincubated with Me2SO (0.4%), SB 203580 (20 μm), or PD 98059 (20 μm) for 10 min and stimulated with collagen (10 μg/ml), arachidonic acid (1 μm), or PGH2 (1 μg/ml) for 5 min at 37 °C. T×B2 release was determined using enzyme immunoassay (Part A) or radioimmunoassay (Part B). Data shown are the means ± S.D. from one experiment performed in triplicate and are representative of two similar experiments. Statistical significance compared with the Me2SO control was assessed by Student'st test. Open table in a new tab Platelets (4 × 108 platelets/ml) (Part A) or platelet microsome preparations (Part B) were preincubated with Me2SO (0.4%), SB 203580 (20 μm), or PD 98059 (20 μm) for 10 min and stimulated with collagen (10 μg/ml), arachidonic acid (1 μm), or PGH2 (1 μg/ml) for 5 min at 37 °C. T×B2 release was determined using enzyme immunoassay (Part A) or radioimmunoassay (Part B). Data shown are the means ± S.D. from one experiment performed in triplicate and are representative of two similar experiments. Statistical significance compared with the Me2SO control was assessed by Student'st test. Arachidonic acid (0.2 and 1 μm) did not stimulate platelet p42mapk and caused weak activation of p38mapkrelative to activation by thrombin (Fig. 4). However, activation of MAPK-activated protein kinase-2, the in vivo substrate of p38mapk, 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 p42mapk and p38mapk (20Kramer R.M. Roberts E.F. Ulm S.L. Börsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Thus, arachidonic acid and its metabolites seem to be only weak stimulators of MAPKs. 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. 5 A). This was in contrast to incubation with the thromboxane synthase inhibitor furegrelate (10 μm) (19Karim S. Habib A. Lévy-Toledano S. Maclouf J. J. Biol. Chem. 1996; 271: 12042-12048Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 22Gorman R.R. Johnson R.A. Spilman C.H. Aiken J.W. Prostaglandins. 1983; 26: 325-342Crossref PubMed Scopus (60) Google Scholar), 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. 5 A). 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. 5 B). 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. 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 p42mapk nor p38mapkis activated (11Börsch-Haubold A.G. Kramer R.M. Watson S.P. Eur. J. Biochem. 1997; 245: 751-759Crossref PubMed Scopus (141) Google Scholar, 16Börsch-Haubold A.G. Kramer R.M. Watson S.P. J. Biol. Chem. 1995; 270: 25885-25892Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). 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). Human platelets contain the constitutively expressed cyclooxygenase-1, but not the inducible cyclooxygenase-2 (23Habib A. Créminon C. Frobert Y. Grassi J. Pradelles P. Maclouf J. J. Biol. Chem. 1993; 268: 23448-23454Abstract Full Text PDF PubMed Google Scholar). 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. 7 A). 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. 7 B). 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 theV max even at the highest concentration of arachidonic acid used (50 μm) (data not shown). 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. 1 D). 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 (17Habib A. Maclouf J. Arch. Biochem. Biophys. 1992; 298: 544-552Crossref PubMed Scopus (19) Google Scholar). Microsomes were obtained from unstimulated platelets, which means that p42mapk and p38mapk 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 TxB2formation (Table I). These results demonstrate that SB 20350 (but not PD 98059) acts as a direct inhibitor of thromboxane synthase. 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 p38β (SAPK2a and SAPK2b), but to have no inhibitory effect on SAPK3, SAPK4, p42/p44mapk, c-Jun N-terminal kinase (SAPK1), or any of the upstream kinases (3Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1981) Google Scholar, 6Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (358) Google Scholar). Similarly, the selectivity of PD 98059 has been described in several studies (7Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar, 8Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar, 9Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar). We (Refs. 11Börsch-Haubold A.G. Kramer R.M. Watson S.P. Eur. J. Biochem. 1997; 245: 751-759Crossref PubMed Scopus (141) Google Scholar and 20Kramer R.M. Roberts E.F. Ulm S.L. Börsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar and this study) and others (24Saklatvala J. Rawlinson L. Waller R.J. Sarsfield S. Lee J.C. Morton L.F. Barnes M.J. Farndale R.W. J. Biol. Chem. 1996; 271: 6586-6589Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar) 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 p38mapk 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 p42mapk 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) (7Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar, 10Börsch-Haubold A.G. Kramer R.M. Watson S.P. Biochem. J. 1996; 318: 207-212Crossref PubMed Scopus (71) Google Scholar). Alessi et al. (8Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar) 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/p44mapk, and our studies on the activation of p42/p44mapk in human platelets have confirmed these results (10Börsch-Haubold A.G. Kramer R.M. Watson S.P. Biochem. J. 1996; 318: 207-212Crossref PubMed Scopus (71) Google Scholar). However, a 10-fold lower concentration of PD 98059 was sufficient to inhibit platelet aggregation, suggesting that this inhibition is independent of p42mapk. In fact, p42mapk 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 (11Börsch-Haubold A.G. Kramer R.M. Watson S.P. Eur. J. Biochem. 1997; 245: 751-759Crossref PubMed Scopus (141) Google Scholar). 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. (25Marshall 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. Hanna N. Biochem. Pharmacol. 1991; 42: 813-824Crossref PubMed Scopus (34) Google Scholar) 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 (25Marshall 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. Hanna N. Biochem. Pharmacol. 1991; 42: 813-824Crossref PubMed Scopus (34) Google Scholar). 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(26Kramer R.M. Liscovitch M. Signal-activated Phospholipases. R. G. Landes Co., Austin, TX1994: 13-30Google Scholar). Cytosolic phospholipase A2 is regulated by changes in the intracellular Ca2+ concentration (27Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar) and by phosphorylation of serine 505 (28Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1659) Google Scholar) and possibly on other serine residues (29de Carvalho M.G.S. McCormack A.L. Olson E. Ghomashchi F. Gelb M.H. Yates J.R., III Leslie C.C. J. Biol. Chem. 1996; 271: 6987-6997Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 30Börsch-Haubold A.G. Bartoli F. Asselin J. Dudler T. Kramer R.M. Apitz-Castro R. Watson S.P. Gelb M.H. J. Biol. Chem. 1998; 273: 4449-4458Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). 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. 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." @default.
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W1985888026 title "Direct Inhibition of Cyclooxygenase-1 and -2 by the Kinase Inhibitors SB 203580 and PD 98059" @default.
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