Role of prostaglandin H synthase isoforms in murine ear edema induced by phorbol ester application...

Role of prostaglandin H synthase isoforms in murine earedema induced by phorbol ester application on skin

Teresa Sa´nchez, Juan J. Moreno*

Department of Physiological Sciences, School of Pharmacy, Barcelona University, Barcelona, Spain

Received 8 August 1998; received in revised form 31 August 1998; accepted 4 November 1998

Abstract

Topical application of TPA to a murine ear induced an edema that was accompanied byeicosanoid biosynthesis and an early enhancement of prostaglandin H synthase 2 (PGHS-2)expression. PGHS-2 induction may be correlated with the time-course of TPA-induced edemaformation. Treatment with drugs that inhibit AA mobilization such as dexamethasone or manoalideor inhibitors of leukotriene formation such as zileuton or baicalein, reduced TPA-induced edemadevelopment and PGHS-2 levels. On the other hand, arachidonic acid (AA) application on themurine ear induced rapid expression of PGHS-2. This effect was not reproduced by other fattyacids such as oleic, linoleic, eicosatetraynoic or eicosapentaenoic acids. PGHS-2 expressioninduced by AA application was independent of PGHS and lipoxygenase metabolite synthesis.However, topical application of PGE2 on skin induced PGHS-2 overexpression. This studysuggests that AA release and/or subsequent metabolism by PGHS may be involved in theinduction of PGHS-2 expression in murine TPA- and AA-induced ear oedema. © 1999 ElsevierScience Inc. All rights reserved.

Keywords: Arachidonic acid; Cyclooxygenase; Arachidonic acid; Acute inflammation; Leukotrienes; Anti-inflammatory drugs

Prostaglandin H synthase (PGHS; EC 1.14.99.1), also called cyclooxygenase, catalyzesthe key step in the conversion of arachidonic acid to prostaglandin (PG) H2 (PGH2), theimmediate precursor of prostaglandins and thromboxanes. It also plays a crucial role in

* Corresponding author. Tel.:134-93-402-4505; fax:134-93-402-1896.E-mail address:[email protected] (J.J. Moreno)

Prostaglandins & other Lipid Mediators 57 (1999) 119–131

0090-6980/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.PII: S0090-6980(98)00078-1

responses to inflammation. Two isoforms of PGHS have been characterized. PGHS-1, ahomodimer of 72 KDa protein, was originally purified from bovine and ovine seminalvesicles [1,2] and later human [3] and murine [4] PGHS-1 proteins were deduced fromcDNAs. More recently, a second form of PGHS encoded by a 4.6 Kb mRNA was discoveredin certain cell cultures and designated as PGHS-2 [5,6]. These isoforms appear to havedifferent biological roles. However, their relative contributions to the maintenance of normalphysiological functions and in diseased states are not entirely clear.

The expression of PGHS-2 is induced by stimuli, including mitogens, cytokines, andbacterial lipopolysaccharides, and it is inhibited by anti-inflammatory steroids [7]. Thissuggests that PGHS-2 may participate in inflammation by producing prostanoids.

Recently, PGHS-2 has been detected in the inflammatory site of models such as carrageenin-induced pleurisy [8,9], air-pouch [10] and paw edema [11] in rats. Moreover, the expres-sion of PGHS-2 is increased in synovia from patients with rheumatoid arthritis [12]. Theseobservations, which show that this cyclooxygenase isoform is detectable in inflammatorysites in vivo, indicate a role for this enzyme in the development of the inflammatoryresponse. However, its exact contribution is not clear because of the concomitant expressionof the PGHS-1 isoform [8,12]. Thus, there is considerable interest in understanding thephysiological roles of PGHS-1 and PGHS-2 and in developing drugs that inhibit themdifferentially.

Using phorbol ester (TPA)-induced ear edema and arachidonic acid (AA)-induced earedema, we have previously shown [13] that prostanoid levels increase at an early stage ofedema, and we suggested that these prostanoids may play an important role in cell migrationand plasma exudation. The purpose of this paper is to study the role of PGHS-2 in the earedema inflammatory process, and to clarify the contribution of the PGHS-2 isoform in theformation of prostanoids and the development of acute inflammation.

1. Materials and methods

1.1. Materials

Arachidonic acid (AA), eicosatetraynoic acid (ETYA), eicosapentaenoic acid, linoleicacid, oleic acid, 12-O-tetra-decanoylphorbol 13-acetate (TPA), phenylmethylsulfonylfluo-ride (PMSF), aprotinin, leupeptin, diethyldithiocarbamic acid, dexamethasone, ketoprofen,indomethacin, manoalide, zileuton, baicalein, and staurosporine were obtained from SigmaChemical Co. (St. Louis, MO, USA). PGE2, a peptido-leukotriene mixture that containsLTC4, LTD4, LTE4, and LTF4 (5 mg/each), and leukotriene B4 were purchased from CaymanChemicals Co. (Ann Arbor, MI, USA). All other reagents were of analytical grade.

1.2. Animals

Ear edema was induced in male Swiss Webster mice (Interfauna Iberica SA, Barcelona,Spain). Animals were housed with a 12-h lighting schedule (06:00–18:00 h on) with access

120 T. Sanchez, J.J. Moreno / Prostaglandins & other Lipid Mediators 57 (1999) 119–131

to food and water ad libitum. Housing and experimental procedures were conducted accord-ing to standard conditions.

1.3. AA and TPA ear edema inflammation models

TPA or AA was dissolved in acetone, and 20ml of solution was applied to the inner andouter surface of the right ear of the mice. The left ear received acetone, delivered in the samemanner. Treatments were given topically in a volume of 20ml per ear 15 min before TPAor AA application, except for dexamethasone, which was administered topically severalhours before inflammation was induced. All drugs were dissolved in acetone, a solvent thatdid not have any effect on the inflammatory processes. Finally, mice were killed by CO2

inhalation and a 7-mm diameter section of the right and left auditory pinna, measured fromthe apex, was cut, and the samples were weighed and used for PGHS determinations. Earedema was measured as the differences in weight between the challenged and the unchal-lenged ear. Percent inhibition was calculated by using (C-T)/C3 100 (%), where C and Tindicate non-treated (vehicle) edema and drug-treated edema, respectively. Measurementswere taken from 0 to 24 h after TPA-induced ear edema and from 0 to 4 h after arachidonicacid application.

1.4. Measurements of eicosanoid metabolites

Mouse ears were homogenized with 1 ml of methanol containing 1% 1 M HCl. Then, 2ml of distilled water was added to each tube, and the tubes were kept on ice for 30 min. Theresulting insoluble proteins were removed by centrifugation (30 0003 g, 20 min) andarachidonic acid metabolites were measured as previously described [13,14]. The eico-sanoids were extracted with ethyl acetate (6 ml) and the aqueous phase was then discarded;the organic phase was evaporated in a stream of nitrogen. PGE2, 6-keto-PGF1a, and LTB4

in the extracts were measured using the specific protocols described for EIA kits fromCayman Chemicals Co.

1.5. Protein determination and western blot analysis

Mouse ears were homogenized with 0.5 ml of phosphate-buffered saline (PBS) containing2 mM EDTA, 250mg/ml PMSF, 250mg/ml aprotinin, 250mg/ml leupeptin, and 200mg/mldimethyldithiocarbamic acid and lysates were normalized using the BioRad protein assay kit(BioRad, Richmond, CA, USA) and stored at280°C until use. Bromophenol blue (0.05%w/v) and 2-mercaptoethanol (6% v/v) were added to equal amounts of protein (20mg) andthe mixture was boiled for 10 min. SDS-PAGE electrophoresis was performed using 4.5%stacking and 10% resolving gels [15] with a Mini-PROTEAN II electrophoresis cell (Bio-Rad) in 25 mM Tris, 190 mM glycine, and 2 mM SDS buffer. Proteins were transferred ontoTrans-Blot nitrocellulose membrane (Bio-Rad) using Bio-Rad’s Mini Trans-Blot electro-phoretic transfer cell in 25 mM Tris and 192 mM glycine buffer with 20% methanol for 1 hat 100 V. For immunodetection, the nitrocellulose membranes were blocked with 5% skimmilk in Tris-buffered saline (150 mM NaCl, 10 mM Tris, pH 7.6, 0.1% Tween 20) for 1 h,

121T. Sanchez, J.J. Moreno / Prostaglandins & other Lipid Mediators 57 (1999) 119–131

washed, and then incubated with antiPGHS-1 or antiPGHS-2 (Cayman Chemicals Co.)diluted to 1:2000 in blocked buffer for 1 h. These rabbit polyclonal antisera against PGHS-1(sheep seminal vesicles) or PGHS-2 (synthetic peptide from murine PGHS-2) did notcross-react with PGHS-2 or PGHS-1, respectively [16]. Antigen-antibody complexes weredetected by a second incubation of the membrane with Amersham’s anti-rabbit IgG horse-radish peroxidase-linked secondary antibody at 1:2000 in blocked buffer. After washing,chemiluminescence was performed according to the manufacturer’s protocol (Amersham,Buckinghamshire, UK) and bands were detected by autoradiography after exposure to KodakX-OMAST-LS film (Eastman Kodak, Rochester, NY, USA). Quantitation was by videodensitometry. Ovine PGHS-1 and PGHS-2 (Cayman Chemicals Co.) were used as a stan-dard.

1.6. Statistical analysis

Data are expressed as the mean6 SEM and experiments were performed on tissue pairs(control ear and ear administered with pro-inflammatory agent), the determinations were fitsimultaneously to pairs of ears in order to obtain parameter estimates free of animal-to-animal variation.

Statistical significance was assessed by the one-tailed Student’st-test for unpaired sam-ples, withP , 0.05 regarded as significant.

2. Results

2.1. Edema and PGHS-2 expression were induced by TPA in murine ear skin

Consistent with earlier reports, TPA produced a long-lasting response with a delayedonset. The maximum edema (1386 3%) was observed 6 h after phorbol application (Fig.1B). Further, TPA edema showed measurable changes in the levels of 6-keto-PGF1a andLTB4. Thus, we detected 4366 12 pg/ear of 6-keto-PGF1a and 10286 60 pg/ear of LTB4at 6 h (Table 1).

To examine the expression of PGHS-1 and PGHS-2 in TPA-induced ear inflammation, weperformed immunoblot analyses using specific antibodies against both PGHS isoforms. Asshown in Figure 1A, PGHS-1 and PGHS-2 were barely detectable in the homogenate of thecontrol murine ear. However, TPA application increased PGHS-2 levels by 69-fold reachinga maximum at 6 h, and remaining significantly high at 24 h (13-fold). Interestingly, thetime-course of the PGHS-2 levels was parallel to that of the TPA-induced edema (Fig. 1). Incontrast, the magnitude of induction of PGHS-1 was less than that of PGHS-2.

Topical treatment with dexamethasone (5mg/ear) or manoalide (1mg/ear), both of whichaffect AA mobilization [17,18], significantly attenuated eicosanoid biosynthesis (Table 2)and TPA-induced edema response (78% and 41%, respectively), and inhibited the increaseof PGHS-2 levels (51% and 60%, respectively) (Fig. 2). Drugs that inhibit leukotrieneformation such as zileuton (100mg/ear), a selective 5-lipoxygenase inhibitor [19] or baica-lein (100 mg/ear), a more selective 12-lipoxygenase inhibitor [20] also decreased edema


(23% and 35%, respectively) and PGHS-2 levels (51% and 50%, respectively). In contrast,inhibitors of prostanoid biosynthesis, such as ketoprofen (1 mg/ear) and indomethacin (0.5mg/ear) did not affect the expression of PGHS-2 in the inflammatory tissues (Fig. 2). Takentogether, these results strongly support the hypothesis that AA mobilization and/or leuko-triene formation following TPA application could be related to PGHS-2 expression.

2.2. PGHS-2 overexpression induced by AA was not reproduced by other fatty acids

PGHS-2 levels increased 40-fold 30 min after AA application and remained high for atleast 4 h (45-fold) (Fig. 3A), whereas the extent of induction of PGHS-1 was less than thatof PGHS-2. These results indicate that PGHS-2 is present at low levels in the normal tissue,but was induced early in the inflammatory tissues when AA was applied. The impairment ofPGHS-2 expression after AA application was also dose-dependent, an increment was

Fig. 1. Time-course of PGHS-1 and PGHS-2 expression in TPA-induced ear edema model. Mice received atopical application of TPA (6mg/ear) on ear skin. A, Tissue was homogenized and analyzed by Western blot.Purified PGHS-1 (from sheep seminal vesicular, 100 ng) or PGHS-2 (from ovine placenta, 100 ng) were used asstandard. Results are from a representative experiment. B, Ear edema (M) is given as a percentage increase overthe control value (mean6 SEM from 4 to 5 animals). Optical density of the PGHS-2 Western blot representedin A (f) is expressed as arbitrary units.


observed at 100mg/ear and maximal at 500mg/ear (Fig. 4A). To determine whether PGHS-2expression is unique to AA, we compared the effect of topical application of various fattyacids; AA was the most potent stimulator of PGHS-2 expression. Oleic acid and linoleic acid,a precursor of AA, had no significant effect. ETYA, a non-metabolized analogue of AA alsohad no effect; whereas eicosapentaenoic acid, another fatty acid metabolized by cyclooxy-genase or lipoxygenase pathways, also induced PGHS-2 expression, although it was lesspotent than AA (Fig. 4B).

Table 1Eicosanoid levels in TPA- or AA-induced ear edema

Treatments PGE2(ng/ear)

LTB4

(pg/ear)6-keto-PGF1a

(pg/ear)

Control 96 1 66 1 676 13TPA-edema

(2 h) ND 4066 21* 3986 11*(6 h) ND 10286 60* 4366 12*(24 h) ND 8676 24* ND

AA-odema(0.5 h) 766 4* 16 6 2 ND(1 h) 386 3* 17 6 1* ND(2 h) 216 1* 23 6 2* ND(4 h) 186 1* 21 6 1* ND

TPA- or AA-induced ear edema was induced and eicosanoid tissue levels were measured. Values are mean6SEM of 4-5 animals.

* P , 0.05 vs. control value. (ND, not determined).

Table 2Effect of dexamethasone, manoalide, ketoprofen, indomethacin, zileuton and baicalein on prostaglandin andleukotriene levels

Treatments PGE2(ng/ear)

LTB4

(pg/ear)6-keto-PGF1a

(pg/ear)

TPA ND 12586 49 3966 12TPA 1 dexamethasone ND 3456 12* 2016 9*TPA 1 manoalide ND 5676 13* 2266 11*TPA 1 ketoprofen ND 14566 32 1986 8*TPA 1 zileuton ND 5136 10* 3986 11TPA 1 baicalein ND 5556 11* 4016 12AA 42 6 3 176 2 NDAA 1 indomethacin 186 1* 16 6 1 NDAA 1 ketoprofen 226 2* 18 6 2 NDAA 1 zileuton 436 3 86 1* NDAA 1 baicalein 416 2 96 1* ND

Eicosanoid tissue levels were tested in TPA- and AA-induced ear edema, 6 h and 1 h after phlogogens wereapplied, respectively. Manoalide (1mg/ear), ketoprofen (1 mg/ear), indomethacin (0.5 mg/ear), zileuton (100mg/ear), and baicalein (100mg/ear) were administered topically 15 min before TPA or AA application.Dexamethasone (5mg/ear) was applied 2 h before inflammation was elicited. Values are mean6 SEM of 4–5animals.

* P , 0.05 vs. non treated sample. (ND, not determined).


2.3. AA metabolism is not necessary for PGHS-2 overexpression

AA was rapidly metabolized through the cyclooxygenase and lipoxygenase pathways,leading to the formation of several biologically active metabolites, including prostaglandinsand leukotrienes (Table 1). In order to determine whether the induction of PGHS-2 expres-sion could be a direct effect of AA or one of its metabolites, we assessed the effect ofcyclooxygenase inhibitors (indomethacin, ketoprofen) or lipoxygenase inhibitors (zileuton,baicalein) on AA-induced PGHS-2 expression. None of the inhibitors significantly influ-enced the PGHS-2 levels increased after AA application (Fig. 4C), at doses that abrogateeicosanoid biosynthesis (Table 2).

Pretreatment with dexamethasone for 6 h followed by AA application revealed a concen-tration-dependent decrease in PGHS-2 tissue levels (Fig. 5). Thus, AA-stimulated PGHS-2expression was reduced most markedly at a corticosteroid dosage of 50mg/ear. Pretreatmentwith dexamethasone for 2 h before AA-stimulated PGHS-2 overexpression significantlyreduced PGHS-2 levels (Fig. 5).

2.4. Prostaglandin application stimulates an enhancement of PGHS-2 expression inmouse ear skin

Although we had observed that AA-induced PGHS-2 overexpression could not requirearachidonate metabolism, we assessed the effect of AA metabolites on PGHS-2 levels in theskin. Thus, the application of LTB4 (1 and 5mg/ear) or a mixture of leukotrienes (LTC4,LTD4, LTE4, LTF4, 1 mg/each/ear) did not induce significant changes in tissue PGHS-2levels. However, PGE2 (50 mg/ear) administration led to a significant increase in tissuePGHS-2 levels (Fig. 6). None of these arachidonate metabolites produced edema, althoughPGE2 produced marked vasodilatation and erythema (data not shown). Application of allleukotrienes together with PGE2 led to a significant edema (436 2%), whereas the PGHS-2overexpression induced in this experimental condition was similar to that obtained withPGE2 (Fig. 6).

Fig. 2. Effect of dexamethasone (5mg/ear, Dxt), manoalide (1mg/ear, Ml), ketoprofen (1 mg/ear, Kp),indomethacin (500mg/ear, I), zileuton (200mg/ear, Z) or baicalein (100mg/ear, B) on PGHS-2 expressioninduced by TPA. Treatments were applied topically 15 min before TPA (6mg/ear), and samples were homog-enized 6 h after TPA application. Western blot is representative from 4. Edema inhibition expressed as mean6SEM of 4–5 animals are indicated below each lane.


3. Discussion

Until recently, only one form of PGHS had been characterized, PGHS-1 [21] but, morerecently, a second form (PGHS-2) was discovered [5]. These PGHS isoforms are encoded bygenes located on different chromosomes [22], and these genes differ in their regulation at thetranscriptional level. Recent data reinforce the likelihood that the isoforms mediate differentbiological functions [23,24]. PGHS-2 is often referred to as the inducible form of PGHSbecause it is encoded by an immediate early gene [25]. Thus, PGHS-2 expression in certaincell types can be rapidly induced by proinflammatory or mitogenic agents. Moreover, theinduction of PGHS-2 during inflammation is consistent with the results of Masferrer et al.[26], and the magnitude of PGHS-2 induction could be correlated with the production of alarge amount of prostanoids and with the time-course of edema formation as an index ofinflammation. Thus, several reports have suggested a role for PGHS-2 in several animalmodels of inflammation induced by carrageenan or phorbol esters [11,27,28]

Fig. 3. Time-course of PGHS-1 and PGHS-2 expression after AA application on ear skin. Mice received topicallyAA (1 mg/ear) on ear skin. A, Tissue was homogenized and analyzed by Western blot. Purified PGHS-1 (fromsheep seminal vesicular, 100 ng) or PGHS-2 (from ovine placenta, 100 ng) were used as standard. Results arefrom a representative experiment. B, Ear edema (M) is given as a percentage increase over the control value(mean6 SEM from 4–5 animals). Optical density of the PGHS-2 Western blot represented in A (f) is expressedas arbitrary units.


Data presented here show that TPA application on murine ear increased PGHS-2 tissueprotein levels. Thus, PGHS-2 expression had significantly increased 2 h after TPA applica-tion, reached a maximum at 4–6 h, and declined at 24 h. These findings are consistent withresults obtained in vitro in cultured fibroblasts [16,29], and endothelial cells [30,31]. Incontrast, PGHS-1 expression was much less induced than PGHS-2. Nevertheless, TPAincreased basal levels of PGHS-1 and these results are consistent with the notion that TPAincreases the transcriptional rate of PGHS-1 gene in human endothelial cells [31].

Herschman et al. [32] reported that the induction of PGHS-2 by TPA, epidermal growthfactor or platelet-derived growth factor (PDGF) does not require the expression of other

Fig. 4. PGHS-2 overexpression was specifically induced by AA and it was not reduced by lipoxygenase pathwayinhibitors. A, We applied various doses of AA (10–1000mg/ear) and 1 h later, animals were killed and PGHS-2levels were assayed. B, Murine ear were exposed for 1 h to 500mg of oleic (O), linoleic (L), arachidonic (AA),ETYA (ET) and eicosapentaenoic acid (EPA), and PGHS-2 analyzed. C, Zileuton (200mg/ear, Z), baicalein (100mg/ear, B), zileuton plus baicalein (200 and 100mg/ear, respectively, Z1 B), and indomethacin (500mg/ear, I)were applied 15 min before AA (500mg/ear). Tissues were homogenized 1 h later and PGHS-2 levels wereassayed. Western blot is representative from 4. The percentage of ear edema or ear edema inhibition valuesexpressed as mean6 SEM of 4–5 animals are indicated below each lane.


proteins because transcription is stimulated by the activation of latent pre-existing transcrip-tion factors, which permits immediate expression of proteins. Besides, TPA, a tumor-promoting phorbol ester that can directly activate protein kinase C [33], elicited AAmobilization and eicosanoid formation, which are limiting-steps in edema induction. Ourresults show that drugs that inhibit AA release, such as dexamethasone and manoalide, orinhibitors of leukotriene formation such as zileuton and baicalein, decreased TPA-inducededema. These data were correlated with a reduction in PGHS-2 levels. From these findings,we could speculate that arachidonic acid mobilization and/or the subsequent leukotrieneformation could be involved in PGHS-2 expression in this model of inflammation. If weconsider that TPA induces an increase in LTB4 levels and a subsequent infiltration ofneutrophils and mononuclear cells, we can propose that TPA-induced PGHS-2 expression indermal tissue could be the result of AA mobilization and/or a consequence of cell influx.

Fig. 5. Dexamethasone inhibits in a time- and dose-dependent way PGHS-2 expression induced by AA.Dexamethasone (5mg/ear) were applied 2, 4, and 6 h before induction of PGHS-2 overexpression by AAapplication (1 mg/ear) on ear skin. Moreover, dexamethasone (5 and 50mg/ear) was applied 6 h before PGHS-2overexpression-stimulated by AA. Western blot is representative from 4. The percentage of ear edema inhibitionvalues expressed as the mean6 SEM of 4–5 animals are indicated below each lane.

Fig. 6. Topical application of PGE2 induced PGHS-2 expression in murine ear skin. Mice received a topicalapplication of ethanol (C), LTB4 (1–5mg/ear), a mixture of leukotrienes (LTC4, LTD4, LTE4, LTF4; 1 mg/ear ofeach), PGE2 (50mg/ear), and PGE2 (50mg/ear) plus all leukotrienes (1mg/ear of each). Mice were killed 1 h aftereicosanoid application. Western blot is representative from 4. The percentage of ear edema values expressed asthe mean6 SEM of 4–5 animals are indicated below each lane.


Since the application of arachidonic acid to murine ear can induce a very rapid increasein PGHS-2 protein levels, it is possible that free-arachidonic acid or an arachidonatemetabolite can up-regulate PGHS-2 expression in murine skin. It has been reported that fattyacids, in addition to AA, activate signalling including calcium mobilization, protein kinaseC and ERK [34]. Interestingly, we observed that AA was the most potent stimulator ofPGHS-2 expression. These results are consistent with those of Danesch et al. [35], whoreported that AA induced other immediate early genes such as Egr-1 and c-fos, whereaseicosapentaenoic and docosahexaenoic acids only slightly increased their mRNA levels. Themechanisms involved in the regulation of protein expression by fatty acids are not wellknown. Recently, arachidonic acid has been shown to increase the expression of thegrowth-related early genes c-fos and Egr-1 in 3T3 fibroblasts, through its conversion to PGE2

[36]. In addition, polyunsaturated fatty acids, including arachidonic acid, may transcription-ally modify the expression of genes encoding for enzymes of fatty acid metabolism [37].

There are many lines of evidence showing that the gene encoding PGHS-2 is inducible byseveral varieties of hormones. Thus, iloprost, a stable analogue of prostacyclin, increases thesteady-state levels of PGHS-2 mRNA in the mouse osteoblastic MC3T3-E1 cell line [38].Recently, Minghetti et al. [39] using cultured microglia found that LPS-induced PGHS-2expression was up-regulated by elevation of cAMP, and that exogenous PGE2 increasedPGHS-2 expression by a mechanism involving, cAMP. To clarify the effect of eicosanoidson PGHS-2 expression in our experimental model, we tested whether the application of themain eicosanoids produced in the TPA-induced ear edema reproduced the overexpression ofPGHS-2. Our results show that PGE2 could be involved in the control of PGHS-2 expression,which is in apparent contrast with the lack of effect of cyclooxygenase inhibitors onTPA-induced PGHS-2 overexpression. This PGHS-2 overexpression induced by PGE2 maybe considered a positive feedback mechanism to amplify PGE2 synthesis. However, we mustconsider that AA itself may also increase PGHS-2 expression.

Thus, our results indicate that arachidonic acid release and/or the subsequent metabolismby the cyclooxygenase pathway may be involved in the induction of PGHS-2 expressionduring TPA-induced ear edema. Although, we must consider the role of leukotrienes incellular infiltration, other important step that could be involved in PGHS-2 overexpressionduring TPA-induced inflammation.

Acknowledgments

The authors are very grateful to Mr Robin Rycroft for his valuable assistance in thepreparation of the English manuscript. Teresa Sa´nchez had a personal grant from theGeneralitat of Catalunya. This study was supported by a grant from the Spanish Ministry ofEducation and Science (DGICYT PB94–0934) and (DGES PM97–0110).

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Role of prostaglandin H synthase isoforms in murine ear edema induced by phorbol ester application...

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