QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.102806v1 318/2/611    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Claudino, R. F. Articles by Calixto, J. B. Search for Related Content PubMed PubMed Citation Articles by Claudino, R. F. Articles by Calixto, J. B.

INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Pharmacological and Molecular Characterization of the Mechanisms Involved in Prostaglandin E₂-Induced Mouse Paw Edema

Department of Pharmacology, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil (R.F.C., C.A.L.K., J.B.C.); and Department of Chemistry, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil (J.F.)

Received February 10, 2006; accepted April 25, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The present study evaluated some of the mechanisms underlying prostaglandin E₂ (PGE₂)-induced paw edema formation in mice. Intraplantar (i.pl.) injection of PGE₂ (0.10-10.0 nmol/paw) into the hindpaw elicited a dose-related edema formation, with a mean ED₅₀ value of 0.42 nmol/paw. The coinjection of selective E-prostanoid (EP)₃ [(2E)-N-[(5-bromo-2-methoxyphenyl)-sulfonyl]-3-[5-chloro-2-(2-naphthylmethyl)phenyl]acrylamide; L826266), but not EP₂ or EP₄ (all 10 nmol/paw), receptor antagonists significantly inhibited PGE₂-induced paw edema. Like L826266, the PGE₂-induced paw edema was markedly reduced by treatment with pertussis toxin and phospholipase C (PLC) inhibitor 1-[6-[[17-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122). Likewise, the selective neurokinin (NK)₁ receptor antagonist N-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-prolyl]-N-methyl-N-phenyl-methyl-3-(2-aphthyl)-L-alaninamide (FK888) and the antagonist of vanilloid receptor (TRPV1) receptors 4'-chloro-3-methoxycinnamanilide (SB366791) (both 1 nmol/paw) also significantly inhibited PGE₂-mediated paw edema. Conversely, the selective NK₂, NK₃, and calcitonin gene-related peptide (CGRP) CGRP_8-37 receptor antagonists all failed to interfere with PGE₂-induced paw edema. The neonatal treatment of mice with capsaicin was also able to reduce PGE₂-induced paw edema. The inhibitors of protein kinase C (PKC) 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X) and mitogen protein-activated kinases (MAPKs; 30 nmol/paw) c-Jun NH₂-terminal kinase (JNK) (anthra[1,9-cd]pyrazol-6(2H)-one; SP600125), extracellular signal-regulated kinase (PD98059), and p38 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SB203580], but not protein kinase A, markedly decreased the PGE₂-mediated edema formation. The i.pl. injection of PGE₂ (3 nmol/paw) induced a significant activation of MAPKs, namely, JNK and p38, an effect that was largely prevented by the selective EP₃ receptor antagonist L826266 (10 nmol/paw). Collectively, these findings indicate that edematogenic responses elicited by PGE₂ are mediated by EP₃ receptor activation, also involving the stimulation of PLC, PKC, and MAPKs pathways and the participation of TRPV1 and NK₁ receptors. These results make a considerable contribution to our comprehension of the mechanisms involved in PGE₂-mediated inflammatory responses in mice.

PGE₂, a major cyclooxygenase product, exhibits a broad range of biological actions in diverse tissues through its binding to specific receptors on plasma membrane. These receptors belong to the family of G protein-coupled receptors, and they can be divided into four subtypes (EP_1-4), each of which is encoded by distinct genes (Negishi et al., 1995). Whereas the EP₁ receptor induces calcium mobilization by phospholipase C activation via G_q protein, EP₂ and EP₄ receptors are known to activate adenylyl cyclase via stimulatory G protein. On the other hand, the EP₃ receptor reduces cAMP levels as it is coupled to inhibitory G proteins. In addition, the EP₃ receptors are the only receptors to display multiple splice variants identified in several species (Namba et al., 1993; for review, see Kobayashi and Narumiya, 2002; Bos et al., 2004; Hata and Breyer, 2004).

PGE₂ is generally considered as a key proinflammatory mediator, and its role has been extensively studied in several inflammatory events (Ikeda et al., 1975; Flower et al., 1976; Raud, 1990). Thus, high levels of PGE₂ have been found in inflammatory exudates, and the injection of PGE₂ directly into tissue has been shown to induce a number of classical sign of inflammation. However, despite the importance of PGE₂ in this process being recognized, the receptor subtypes and the signaling pathways involved in the PGE₂-mediated inflammatory actions remain to be further elucidated. In this regard, using pharmacological and molecular approaches, the present study aimed at investigating the receptor subtypes as well the possible signal transduction pathways involved in PGE₂-induced paw edema formation in mice.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Animals. The experiments were conducted using male Swiss mice (25-35 g) kept in chambers under a 12-h light/dark cycle, with controlled humidity (60-80%) and temperature (22 ± 1°C). Food and water were freely available. Experiments were performed during the light phase of the cycle. The animals were acclimatized to the experimental laboratory for at least 1 h before testing and were used once throughout the experiments. All experimental protocols used in this study were approved by the Ethics Committee of the Federal University of Santa Catarina (263/CEUA and 23080035336-16/UFSC).

Measurement of Mouse Paw Edema. Experiments were conducted according to the procedures described by Campos and Calixto (1995). In brief, the animals received a 20-µl intraplantar (i.pl.) injection, into the right hindpaw, of phosphate-buffered saline (PBS; composition 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate buffer) containing PGE₂ (0.1-10 nmol/paw). The left paw received the same volume of PBS and was used as the control. The paw edema formation was measured by means of a plethysmometer (Ugo Basile, Comerio, Italy) at several time points (15, 30, 45, and 60 min) after the i.pl. injection of PGE₂ and was expressed in microliters as the difference between the right and left paws.

Characterization of the Mechanisms Involved in PGE₂-Induced Mouse Paw Edema. To evaluate the EP receptor subtypes involved in the edematogenic responses induced by PGE₂, animals received an i.pl. injection of AH6809 (an EP₂ receptor antagonist; 10 nmol/paw), L826266 (a selective EP₃ receptor antagonist; 10 nmol/paw), or L161982 (an EP₄ receptor antagonist; 10 nmol/paw) coinjected with PGE₂ (3 nmol/paw). In a separate set of experiments, animals received L826266 (0.1-10 nmol/paw) 30 min before PGE₂ (3 nmol/paw) injection.

In another experiment to assess the participation of pertussis toxin-sensitive G proteins in PGE₂ paw edema formation, animals were pretreated with pertussis toxin (10 ng/paw) 20 min before PGE₂ (3 nmol/paw) injection. The control group received the same volume (20 µl) of vehicle before PGE₂ administration. In another experimental group, to test whether the phospholipase C (PLC) was involved in PGE₂-induced paw edema, animals received an i.pl. injection of PGE₂ (3 nmol/paw) in association with U-73122 (1 pmol/paw), a selective PLC antagonist.

In another experimental group, to evaluate the participation of sensorial neuropeptides and vanilloid receptors in PGE₂-mediated mouse paw edema, animals received an i.pl. injection of the selective NK₁ FK888 (1 nmol/paw), NK₂ SR48968 (0.5 nmol/paw), NK₃ SR142801 (1 nmol/paw), or calcitonin gene-related peptide (CGRP) CGRP_8-37 (0.5 nmol/paw) receptor antagonists or the TRPV1 SB366791 (1 nmol/paw) receptor antagonist coinjected with PGE₂ (3 nmol/paw).

To investigate to what extent some groups of kinases are involved in PGE₂-induced edema formation in the mouse paw, we assessed the effects of coadministration of the following selective enzyme inhibitors with PGE₂ (3 nmol/paw): H89 (PKA; 3 nmol/paw), GF109203X (PKC; 3 nmol/paw), SP600125 (JNK; 30 nmol/paw), PD98059 (mitogen-activated protein kinase kinase; 30 nmol/paw), and SB203580 (p38; 30 nmol/paw).

The doses of all inhibitors used in this study were chosen on the basis of literature data or preliminary experiments (Santos and Calixto, 1997; Beirith et al., 2003; Inoue et al., 2003; da Cunha et al., 2004; Ferreira et al., 2004, 2005).

Neonatal Capsaicin Treatment. To further explore the role of capsaicin-sensitive C fibers in PGE₂-induced paw edema formation, mice were treated during the neonatal period (on the second day of life) with capsaicin (50 mg/kg, subcutaneously) or vehicle alone (10% ethanol, 10% Tween 80, and 80% PBS), as described previously (Gamse, 1982). Animals were used at 6 to 7 weeks after the neonatal administration of capsaicin or its vehicle (used as control). To discover whether the degeneration of capsaicin-sensitive primary afferent C fibers had occurred, we applied the wiping eye test described by Ikeda et al. (2001). In brief, 50 µl of 0.01% (w/v) capsaicin was instilled into one eye, and the number of wiping motions that occurred in the subsequent 1-min period was counted. The animals that wiped their eyes 5 times or less were used as the neonatal capsaicin-treated group. The edema was induced by i.pl. injection of PGE₂ (3 nmol/paw) and measured as described above.

Preparation of Tissue for Western Blot Studies. The right paws of the mice were isolated at different times (5-60 min) after PGE₂ treatment (3 nmol/paw). In another experimental group, 30 min before i.pl. injection of PGE₂ (3 nmol/paw), the animals received an i.pl injection of L826266 (10 nmol/paw). Fifteen minutes after PGE₂ treatment, the animals were killed, and the subcutaneous tissue of the paws was removed.

The subcutaneous tissue of the paws was rapidly removed and homogenized in an ice-cold buffer containing protease and phosphatase inhibitors (100 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 µg/ml aprotinin, 0.1 mM phenylmethanesulfonyl fluoride, 200 mM NaF, and 2 mM sodium orthovanadate). The homogenate was first centrifuged at 1000g for 10 min at 4°C. The pellet was discarded, and the supernatant was further centrifuged at 35,000g for 30 min at 4°C. The resulting supernatant was collected as a cytoplasm-rich fraction. Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA). The samples were aliquoted and stored at -80°C until Western blot analysis.

Western Blot Analysis. To assess the possible activation of MAPKs following PGE₂ injection into the mouse paw, Western blot analysis was carried out as described previously (Ferreira et al., 2005) with minor modifications. Equivalent amounts of protein (40 µg for JNK and 70 µg for p38 of cytoplasm-rich fraction) were mixed with sample buffer (200 mM Tris, 10% glycerol, 2% SDS, 2.75 mM -mercaptoethanol, and 0.04% bromphenol blue) and boiled for 5 min. Proteins were separated in a 10% SDS-polyacrylamide gel by electrophoresis and transferred onto a polyvinylidene difluoride membrane, according to the manufacturer's instructions (Millipore Corporation, Billerica, MA). The membrane was blocked by incubation overnight with a 10% nonfat dry milk solution and then incubated with antiphosphorylated forms of JNK (phospho-JNK) or p38 (phospho-p38) antibodies. After washing, the membrane was incubated with adjusted peroxidase-coupled secondary antibodies. The immunocomplexes were visualized using the ECL chemiluminescence detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The membrane was then incubated for 10 min in a stripping buffer at room temperature and reincubated with an antibody against actin, which served as a loading control.

Drugs and Reagents. The following drugs and reagents were used: AH6809 (Cayman Chemical, Ann Arbor, MI); capsaicin, CGRP_8-37 (CGRP fragment 8-37), H89, pertussis toxin, PGE₂, and U-73122 (Sigma-Aldrich, St. Louis, MO); GF109203X, PD98059, SB203580, SB366791, and SP600125 (Tocris Cookson Inc., Ellisville, MO); and polyclonal antibodies antiactin and antiphosphorylated forms of JNK and p38 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). FK888 was kindly provided by Fujisawa Pharmaceutical (Osaka, Japan). SR48968 and SR142801 were donated by Sanofi-Aventis (Bridgewater, NJ). L826266 and L161982 were kindly provided by Merck Frosst (Kirkland, QC, Canada).

Stock solutions for most drugs (0.1-1 M) were prepared in ethanol, except for AH6809 and CGRP_8-37 that were made in a 1% NaHCO₃ solution and distilled water, respectively. All drugs were stocked in siliconized plastic tubes and maintained at -18°C until use. Solutions of these drugs were prepared in PBS (137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate buffer) to the desired concentration just before use. The final concentration of ethanol did not exceed 0.5%, which alone had no effect on PGE₂-induced edema response. In addition, NaHCO₃ alone also had no effect on PGE₂-induced edema response.

Statistical Analysis. The results are presented as the mean ± S.E.M. of four to seven animals per group, except the mean ED₅₀ or ID₅₀ values (i.e., the dose of agonist necessary to produce 50% of the maximal edematogenic response or the dose of antagonists necessary to reduce the agonist response by 50% relative to the control value, respectively), which are reported as geometric means accompanied by their respective 95% confidence limits. The ED₅₀ or ID₅₀ values were calculated by use of the GraphPad Prism software (GraphPad Software Inc., San Diego, CA). The percentages of inhibition are reported as mean ± S.E.M. of inhibitions obtained for each individual experiment in relation to the control values. The ED₅₀, ID₅₀, and percentages of inhibition were calculated at the peak of PGE₂-induced paw edema (30 min). Statistical comparison of the data were performed by analysis of variance (ANOVA) followed by Dunnett's or Student-Newman-Keuls tests or by Student's unpaired t test, as appropriate. P values less than 0.05 (P < 0.05) were considered significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
PGE₂-Induced Paw Edema Formation. The intraplantar injection of PGE₂ (0.1-10 nmol/paw) into the mouse hindpaw elicited a dose- and time-dependent edema formation (Fig. 1). The edematogenic response induced by i.pl. injection of PGE₂ was evident as early as 15 min after reaching the maximum at 30 min, and remained significant for up to 60 min. The calculated mean ED₅₀ value and the 95% confidence limits for this effect were 0.42 (0.29-0.54) nmol/paw, and the maximum effect was 80.0 ± 3.0 µl (Fig. 1).

View larger version (33K): [in this window] [in a new window]   Fig. 1. Dose-response and time-course effect of PGE2-induced paw edema formation in mice. Values represent the differences of volume (in microliters) between vehicle-injected (0.02 ml of PBS solution) and drug-injected paws. Each point represents the mean ± S.E.M. of four to seven animals. In some cases, the error bars are hidden within the symbols. Significantly different from control values: **, P < 0.01 (one-way ANOVA followed by post hoc Dunnett's test).

Characterization of EP Receptor Subtypes in PGE₂-Induced Mouse Paw Edema. PGE₂-induced paw edema formation was significantly inhibited by coinjection of the selective EP₃ receptor antagonist L826266 (10 nmol/paw), with a percentage of inhibition of 52.9 ± 8.3. On the other hand, the edematogenic response evoked by PGE₂ was not significantly affected by the coadministration of either EP₂ (AH6809; 10 nmol/paw) or EP₄ (L161982; 10 nmol/paw) receptor antagonists (Fig. 2A). However, L826266 was not able to inhibit the edematogenic response induced by PGE₂ at the 15-min time point. To elucidate whether the lack of inhibition was due to antagonist pharmacokinetics, we administered it locally 30 min before the edema induction. This pretreatment caused an accentuated inhibition (80.8 ± 5.5%) (Fig. 2A) of PGE₂-mediated paw edema, an effect that was dependent on the used dose (0.1-10 nmol/paw) (Fig. 2B). The mean ID₅₀ value calculated using the values obtained with the pretreatment was 0.36 (0.05-1.82) nmol/paw. Thus, these results indicate that edematogenic responses caused by PGE₂ are probably mediated mainly through the activation of the EP₃ receptor subtype.

View larger version (17K): [in this window] [in a new window]   Fig. 2. A, effect of EP2, EP3, and EP4 receptor antagonists on PGE2-induced paw edema formation in mice. AH6809 (EP2 receptor antagonist; 10 nmol/paw), L826266 (EP3 receptor antagonist; 10 nmol/paw), and L161982 (EP4 receptor antagonist; 10 nmol/paw) were coadministered with PGE2 (3 nmol/paw). EP3 receptor antagonist L826266 (10 nmol/paw) was also administered 30 min before PGE2 injection. B, dose-response effect for the selective EP3 receptor antagonist on PGE2-induced paw edema in mice. L826266 (0.1-10 nmol/paw) was administered locally 30 min before PGE2 (3 nmol/paw). Values represent the differences of volume (in microliters) between vehicle-injected (0.02 ml of PBS solution) and drug-injected paws. Each point represents the mean ± S.E.M. of four to seven animals. Significantly different from control values: **, P < 0.01 (one-way ANOVA followed by post hoc Dunnett's test).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effect of pertussis toxin-sensitive G protein and PLC inhibitor U-73122 on PGE2-induced paw edema formation in mice. A, pertussis toxin-sensitive G protein (10 ng/paw) was administered 20 min before PGE2 (3 nmol/paw) injection. B, U-73122 (a selective PLC inhibitor; 1 pmol/paw) was coadministered with PGE2 (3 nmol/paw). Values represent the differences of volume (in microliters) between vehicle-injected (0.02 ml of PBS solution) and drug-injected paws. Each point represents the mean ± S.E.M. of four to seven animals. Significantly different from control values: **, P < 0.01 (one-way ANOVA followed by post hoc Dunnett's test).

View larger version (15K): [in this window] [in a new window]   Fig. 4. A, effect of neonatal treatment with vehicle (1 ml/kg s.c.) or with capsaicin (50 mg/kg s.c.) on PGE2-induced paw edema formation in mice. B, effect of selective NK1, NK2, NK3, and CGRP receptor antagonists and selective TRPV1 receptor antagonists on PGE2-induced paw edema formation in mice. FK888 (a selective NK1 receptor antagonist; 1 nmol/paw), SR48968 (a selective NK2 receptor antagonist; 0.5 nmol/paw), SR142801 (a selective NK3 receptor antagonist; 1 nmol/paw), CGRP8-37 (a selective CGRP receptor antagonist), and SB366791 (a selective TRPV1 receptor antagonist) were coadministered with PGE2 (3 nmol/paw). Values represent the differences of volume (in microliters) between vehicle-injected (0.02 ml of PBS solution) and drug-injected paws. Each point represents the mean ± S.E.M. of four to seven animals. Significantly different from control values: **, P < 0.01 (Student's unpaired t test or one-way ANOVA followed by post hoc Dunnett's test).

Involvement of PKC, PKA, and Mitogen-Activated Protein Kinases in PGE₂-Induced Paw Edema Formation. Next, we investigated how the activation of some groups of kinases might be implicated in paw edema induced by PGE₂ in mice. Figure 5, A and B, demonstrates that the i.pl. coinjection of the selective inhibitors of p38 (SB203580; 30 nmol/paw), JNK (SP600125; 30 nmol/paw), ERK (PD98059; 30 nmol/paw), and PKC (GF109203X; 3 nmol/paw), all significantly reduced PGE₂-induced paw edema formation. The percentages of inhibition observed for these drugs were 48.6 ± 2.2, 51.5 ± 4.9, 64.0 ± 3.6, and 54.5 ± 5.8, respectively. In contrast, the coinjection of the selective inhibitor of PKA (H89; 3 nmol/paw) had no significant effect on PGE₂ response.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Effect of PKC, PKA (A), and MAPK (B) inhibitors on PGE2-induced paw edema formation in mice. H89 (a selective PKA inhibitor; 3 nmol/paw), GF109203X (a selective PKC inhibitor; 3 nmol/paw), SP600125 (a selective JNK inhibitor; 30 nmol/paw), PD98059 (a selective mitogen-activated protein kinase kinase inhibitor; 30 nmol/paw), and SB203589 (a selective p38 inhibitor; 30 nmol/paw) were coadministered with PGE2 (3 nmol/paw). Values represent the differences of volume (in microliters) between vehicle-injected (0.02 ml of PBS solution) and drug-injected paws. Each point represents the mean ± S.E.M. of four to seven animals. Significantly different from control values: *, P < 0.05; **, P < 0.01 (one-way ANOVA followed by post hoc Dunnett's test).

The i.pl. injection of PGE₂ (3 nmol/paw) resulted in a marked and time-dependent activation of MAPKs. A statistically significant increase in phosphorylation levels of JNK was observed from 5 to 30 min following PGE₂ administration (Fig. 6A). Moreover, p38 MAPK phosphorylation was significant from 15 to 60 min after PGE₂ injection (Fig. 6B). We did not detect any alteration in actin expression after PGE₂ injection.

View larger version (34K): [in this window] [in a new window]   Fig. 6. Western blots showing the time course of p38 (A), JNK (B), and actin (C) activation in response to i.pl. injection of PGE2 (3 nmol/paw) into the mouse paw. Mouse paw tissues were obtained from PBS (basal) or PGE2-injected mice at the indicated time points. Cytosolic levels of phosphorylated p38 (P-p38), phosphorylated JNK (P-JNK), and actin were determined using specific antibodies. Results were normalized by arbitrarily setting the densitometry of the basal group and are expressed as the mean ± S.E.M. (n = 3). *, P < 0.05, compared with basal values (one-way ANOVA followed by Dunnett's test).

View larger version (32K): [in this window] [in a new window]   Fig. 7. Representative images of Western immunoblotting and densitometry analyses showing the effect of EP3 receptor antagonists on PGE2-induced phosphorylation of p38 (A) or JNK (B) and the internal control actin (C). Thirty minutes before PGE2 administration, animals received i.pl. injection of L826266 (10 nmol/paw). Paw tissues were obtained from PBS (basal) mice or from mice 15 min after PGE2 injection. Cytosolic levels of phosphorylated p38 (P-p38), phosphorylated JNK (P-JNK) and actin were determined using specific antibodies. Results were normalized by arbitrarily setting the densitometry of the basal group, and they are expressed as the mean ± S.E.M. (n = 3). #, P < 0.05, compared with basal values (B); *, P < 0.05, compared with vehicle group (one-way ANOVA followed by Student-Newman-Keuls test).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we sought to investigate, by the use of pharmacological tools and molecular procedures, the EP receptor subtypes as well as some of the transducing mechanisms involved in the edematogenic responses mediated by PGE₂ in mice. Our results clearly demonstrate that PGE₂ injection into the mouse paw produces a dose- and time-dependent edema formation at a nanomolar range. This evidence extends previous studies showing that PGE₂ injection evokes an increase of vascular permeability in the skin of rats, rabbits, and humans (Williams and Morley, 1973; Ikeda et al., 1975; Flower et al., 1976; Raud, 1990). Interestingly, the present data allow us to suggest that PGE₂-elicited paw edema in mice is mainly, if not solely, mediated by the activation of EP₃ receptors. This conclusion derives from the results indicating that edema formation induced by PGE₂ injection is markedly prevented by the selective EP₃ L826266, but not the EP₂ AH6809 or EP₄ L161982 receptor antagonists. In fact, it has been recently demonstrated that edema formation and plasmatic extravasation induced by the topical application of arachidonic acid is found significantly decreased in mice with genic deletion of EP₃ receptors, whereas these responses remain unaffected in knockout mice for EP₁, EP₂, or EP₄ receptors (Goulet et al., 2004) In addition, Yuhki et al. (2004) showed that IP, EP₂, and EP₃, but not EP₁ or EP₄, receptor subtypes are largely implicated in exudate formation in the model of pleurisy induced by carrageenan in the mouse. Notably, we have also observed that the EP₃ receptor antagonist L826266 consistently inhibits carrageenan-induced edema in mice, to the same extent that it is able to reduce PGE₂-mediated edema formation (data not shown). Although our data visibly point to the EP₃ receptor as being mainly responsible for the edematogenic effect of PGE₂ in the mouse paw, we cannot completely discard the participation of the EP₁ receptor in this response, because no selective EP₁ receptor antagonist is currently available for mice. This possibility remains to be further evaluated in future studies.

The EP₃ receptor is distributed throughout most mouse tissues (Narumiya et al., 1999). Its alternative splicing forms are capable of inducing a broad range of effects (Bos et al., 2004). Murine EP₃, EP₃, and EP₃ receptors usually activate inhibitory G proteins, but they may also interact with G_s and G₁₃ proteins (for reviews, see Hatae et al., 2002; Hata and Breyer, 2004). It was first affirmed that EP₃ receptor would be able to decrease the adenylyl cyclase activity (reducing the cAMP production) through an interaction with the G_i subunit. Furthermore, the stimulation of PLC by EP₃ receptor was reported to induce the accumulation of inositol triphosphate and diacylglycerol that, in turn, promoted intracellular Ca²⁺ mobilization and PKC activation (Irie et al., 1994). Moreover, it has been hypothesized that subunits of G_i might stimulate PLC and activate p21^ras (Irie et al., 1994; Burkey and Regan, 1995). The results of the present study confirm and also extend these previous findings by demonstrating that the pretreatment with pertussis toxin significantly inhibited PGE₂-induced paw edema. Furthermore, PGE₂-induced paw edema formation was significantly inhibited by coinjection of the selective PLC inhibitor U-73122. We might assume that one or more of the above-mentioned signaling pathways are involved in EP₃ receptor-mediated PGE₂-induced responses in the mouse paw.

Another interesting aspect investigated in the present study was the possible participation of certain groups of kinases in the mouse paw edema induced by PGE₂. Our results demonstrate that the selective PKC blocker GF109203X, but not the PKA inhibitor H89, was able to consistently reduce PGE₂-induced mouse paw edema. Additional sets of experiments also indicated the relevance of MAPKs in our model, because the selective JNK SP600125, ERK PD98059, or p38 SB203580 inhibitors were all markedly effective in reducing PGE₂-induced edema formation. The functional results were confirmed by Western blot analysis, which indicated that i.pl. injection of PGE₂ resulted in a marked activation of PKC (our unpublished data), JNK and p38 MAPKs in the mouse paw. Of interest, the increased expression of MAPKs, but not PKC, induced by PGE₂, seems to be driven by the activation of EP₃ receptor subtype, because this response was almost completely prevented by the selective EP3 receptor antagonist. However, we cannot discard the hypothesis that EP₃ receptor may activate other forms of PKC. Therefore, we might suggest that EP₃-mediated PGE₂ edematogenic responses in the mouse paw involve the coordinated activation of PKC, but not PKC isoform, and MAPKs. In this regard, there are reports in the literature demonstrating that stimulation of PKC is able to activate the ERK signaling pathway (Qiu and Leslie, 1994; Vlahos et al., 2003).

The activation of peripheral terminals of sensory fibers, especially C fibers, causes the release of neuropeptides (such as tachykinins) both peripherally and in the dorsal spinal cord (Holzer, 1998). These inflammatory mediators, particularly substance P, act on target cells in the periphery, producing the classic signals of inflammation in a phenomenon known as "neurogenic inflammation" (Holzer, 1998; Richardson and Vasko, 2002). The present study also analyzed the possible role played by sensory fibers in PGE₂-induced paw edema. Capsaicin is a pharmacological tool that produces the selective degeneration of C and some A fibers, associated with the irreversible loss of more than 80% of small-diameter sensory neuron cell bodies (for review, see Holzer, 1991). Our results demonstrated that neonatal treatment with capsaicin was able to inhibit PGE₂-induced mouse paw edema, suggesting that stimulation of primary sensory fibers is an important event for PGE₂ inflammatory responses. In addition, we showed that coinjection of the selective NK₁ FK888, but not NK₂ SR48969, NK₃ SR142801, or CGRP CGRP_8-37 receptor antagonists, significantly reduced PGE₂-evoked edema formation. Consequently, it is possible to conclude that proedematogenic effects of PGE₂ in the mouse paw are mediated, at least partially, by the release of tachykinins, probably substance P, acting on NK₁ receptors. In fact, White (1996) has shown that PGE₂ is capable of releasing substance P from cultured rat sensory neurons. The colocalization of tachykinin and TRPV1 receptors (nonselective cation channels expressed predominantly in small-diameter sensory fibers) strongly suggests the involvement of the latter in the PGE₂-induced edematogenic response. The TRPV1 receptor is exogenously activated by the pungent plant-derived compound capsaicin, but it is also endogenously stimulated by protons, heat, and some lipid-derived mediators (for review, see Calixto et al., 2005). It is now well known that TRPV1 activation augments the release of neuropeptides in a calcium-dependent manner (Bevan and Geppetti, 1994; Kessler et al., 1999). We found that, at least in part, PGE₂-induced edematogenic response in the mouse paw is associated with the activation of TRPV1. This conclusion derives from the results indicating that the coinjection of the selective TRPV1 receptor antagonist SB366791 significantly inhibited the PGE₂-induced paw edema. Recent evidence has suggested that regulation and activation of TRPV1 are defined by complex mechanisms such as phospholipid-mediated inhibition and phosphorylation (Premkumar and Ahern, 2000; Chuang et al., 2001). It is noteworthy that TRPV1 can be modulated by the PKA and PKC phosphorylation (Lopshire and Nicol 1998; Cesare et al., 1999). Interestingly, it has recently been shown that functional interaction of TRPV1 with PGE₂ occurs through a PKC-dependent mechanism by coupling of EP₁ receptor (Moriyama et al., 2005). Thus, our results allow us to suggest that PGE₂ might modulate TRPV1 through the PKC-dependent mechanisms. Whether MAPKs are associated with the activation of sensory fibers following EP₃ receptor activation by PGE₂ remains to be further assessed.

Together, the present results provide convincing experimental evidence indicating that PGE₂ induces paw edema in mice via the stimulation of EP₃ receptors, in a process dependent on the activation of TRPV1 and NK1 receptors as well as PKC and MAPKs. These findings help to elucidate the possible mechanisms underlying PGE₂-elicited inflammatory responses in mice and point to EP₃ receptor antagonists as possible therapeutic options for treating inflammatory disorders.

	Acknowledgements

 
We thank the pharmaceutical companies for the kind donations of FK888, L826266, L161982, SR48968, and SR142801 used in this study.

	Footnotes

 
This study was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação de Apoio a Ciência e Tecnologia do Estado de Santa Catarina, and Programa de Apoio aos Núcleos de Excelência, Brazil. R.F.C. is a postgraduate student in pharmacology.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.102806.

ABBREVIATIONS: PGE₂, prostaglandin E₂; EP, E-prostanoid; i.pl., intraplantar; PBS, phosphate-buffered saline; AH6809, 6-isopropoxy-9-oxoxanthene-2-carboxylic acid; L826266, (2E)-N-[(5-bromo-2-methoxyphenyl)sulfonyl]-3-[5-chloro-2-(2-naphthylmethyl)phenyl]acrylamide; L161982, N-{[4'-({3-butyl-5-oxo-1-[2-(trifluoromethyl) phenyl]-1,5-dihydro-4H-1,2,4-triazol-4-yl}methyl) biphenyl-2-yl]sulfonyl}-3-methylthiophene-2-carboxamide; PLC, phospholipase C; U-73122, 1-[6-[[17-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; NK, neurokinin; CGRP, calcitonin gene-related peptide; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; SB203580, 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine); SB366791, 4'-chloro-3-methoxycinnamanilide; SP600125, anthra[1,9-cd]pyrazol-6-(2H)-one; FK888, N-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-prolyl]-N-methyl-N-phenyl-methyl-3-(2-aphthyl)-L-alaninamide; SR48968, (S)-N-methyl-(N[4-acetylamino-4-phenylpiperidine)-2-(3,4-dichlorophenyl)butyl]benzamide; SR142801, (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)-piperiden-3-yl)propyl)-4-phenilpipedidin-4-yl)-N-methyl-acetamide; TRPV1, vanilloid receptor; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; PKA, protein kinase A; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; PKC, protein kinase C; JNK, c-Jun NH₂-terminal kinase; ANOVA, analysis of variance; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase.

Address correspondence to: Dr. João Batista Calixto, Departamento de Farmacologia, Universidade Federal de Santa Catarina, Campus Universitário-Trindade, Bloco D-CCB-Cx, Postal 476-CEP, 88049-900-Florianópolis, Santa Catarina, Brazil. E-mail: calixto{at}farmaco.ufsc.br or calixto3{at}terra.com.br

	References

Top Abstract Materials and Methods Results Discussion References

 

Beirith A, Santos AR, and Calixto JB (2003) The role of neuropeptides and capsaicin-sensitive fibres in glutamate-induced nociception and paw oedema in mice. Brain Res 969: 110-116.[CrossRef][Medline]

Bevan S and Geppetti P (1994) Protons: small stimulants of capsaicin-sensitive sensory nerves. Trends Neurosci 17: 509-512.[CrossRef][Medline]

Bos CL, Richel DJ, Ritsema T, Peppelenbosch MP, and Versteeg HH (2004) Prostanoids and prostanoid receptors in signal transduction. Int J Biochem Cell Biol 36: 1187-1205.[CrossRef][Medline]

Burkey TH and Regan JW (1995) Activation of mitogen-activated protein kinase by the human prostaglandin EP3A receptor. Biochem Biophys Res Commun 211: 152-158.[CrossRef][Medline]

Campos MM and Calixto JB (1995) Involvement of B₁ and B₂ receptors in bradykinin-induced rat paw oedema. Br J Pharmacol 114: 1005-1013.[Medline]

Calixto JB, Kassuya CA, Andre E, and Ferreira J (2005) Contribution of natural products to the discovery of the transient receptor potential (TRP) channels family and their functions. Pharmacol Ther 106: 179-208.[CrossRef][Medline]

Cesare P, Dekker LV, Sardini A, Parker PJ, and McNaughton PA (1999) Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heat. Neuron 23: 617-624.[CrossRef][Medline]

Chuang HH, Prescott ED, Kong H, Shields S, Jordt SE, Basbaum AI, Chao MV, and Julius D (2001) Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature (Lond) 411: 957-962.[CrossRef][Medline]

da Cunha JM, Rae GA, Ferreira SH, and Cunha F de Q (2004) Endothelins induce ETB receptor-mediated mechanical hypernociception in rat hindpaw: roles of cAMP and protein kinase C. Eur J Pharmacol 501: 87-94.[CrossRef][Medline]

Ferreira J, da Silva GL, and Calixto JB (2004) Contribution of vanilloid receptors to the overt nociception induced by B₂ kinin receptor activation in mice. Br J Pharmacol 141: 787-794.[CrossRef][Medline]

Ferreira J, Triches KM, Medeiros R, and Calixto JB (2005) Mechanisms involved in the nociception produced by peripheral protein kinase C activation in mice. Pain 117: 171-181.[CrossRef][Medline]

Flower RJ, Harvey EA, and Kingston WP (1976) Inflammatory effects of prostaglandin D₂ in rat and human skin. Br J Pharmacol 56: 229-233.[Medline]

Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science (Wash DC) 294: 1871-1875.[Abstract/Free Full Text]

Gamse R (1982) Capsaicin and nociception in the rat and mouse. Possible role of substance P. Naunyn-Schmiedeberg's Arch Pharmacol 320: 205-216.[CrossRef][Medline]

Goulet JL, Pace AJ, Key ML, Byrum RS, Nguyen M, Tilley SL, Morham SG, Langenbach R, Stock JL, McNeish JD, et al. (2004) E-prostanoid-3 receptors mediate the proinflammatory actions of prostaglandin E₂ in acute cutaneous inflammation. J Immunol 173: 1321-1326.[Abstract/Free Full Text]

Hata AN and Breyer RM (2004) Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation. Pharmacol Ther 103: 147-166.[CrossRef][Medline]

Hatae N, Sugimoto Y, and Ichikawa A (2002) Prostaglandin receptors: advances in the study of EP₃ receptor signaling. J Biochem (Tokyo) 131: 781-784.[Abstract/Free Full Text]

Holzer P (1991) Capsaicin: cellular targets, mechanisms of action and selectivity for thin sensory neurons. Pharmacol Rev 43: 143-201.[Medline]

Holzer P (1998) Neurogenic vasodilatation and plasma leakage in the skin. Gen Pharmacol 30: 5-11.[Medline]

Ikeda K, Tanaka K, and Katori M (1975) Potentiation of bradykinin-induced vascular permeability increase by prostaglandin E₂ and arachidonic acid in rabbit skin. Prostaglandins 10: 747-758.[CrossRef][Medline]

Ikeda Y, Ueno A, Naraba H, and Oh-ishi S (2001) Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice. Life Sci 69: 2911-2919.[CrossRef][Medline]

Inoue M, Kawashima T, Allen RG, and Ueda H (2003) Nocistatin and prepronociceptin/orphanin FQ 160-187 cause nociception through activation of G_i/_o in capsaicin-sensitive and of G_s in capsaicin-insensitive nociceptors, respectively. J Pharmacol Exp Ther 306: 141-146.[Abstract/Free Full Text]

Irie A, Segi E, Sugimoto Y, Ichikawa A, and Negishi M (1994) Mouse prostaglandin E receptor EP₃ subtype mediates calcium signals via G_i in cDNA-transfected Chinese hamster ovary cells. Biochem Biophys Res Commun 204: 303-309.[CrossRef][Medline]

Kessler F, Habelt C, Averbeck B, Reeh PW, and Kress M (1999) Heat-induced release of CGRP from isolated rat skin and effects of bradykinin and the protein kinase C activator PMA. Pain 83: 289-295.[CrossRef][Medline]

Kobayashi T and Narumiya S (2002) Function of prostanoid receptors: studies on knockout mice. Prostaglandins Other Lipid Mediat 68-69: 557-573.[Medline]

Lopshire JC and Nicol GD (1998) The cAMP transduction cascade mediates the prostaglandin E₂ enhancement of the capsaicin-elicited current in rat sensory neurons: whole-cell and single-channel studies. J Neurosci 18: 6081-6092.[Abstract/Free Full Text]

Moriyama T, Higashi T, Togashi K, Iida T, Segi E, Sugimoto Y, Tominaga T, Narumiya S, and Tominaga M (2005) Sensitization of TRPV1 by EP₁ and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol Pain 1: 1-13.[CrossRef][Medline]

Namba T, Sugimoto Y, Negishi M, Irie A, Ushikubi F, Kakizuka A, Ito S, Ichikawa A, and Narumiya S (1993) Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP₃ determines G-protein specificity. Nature (Lond) 365: 166-170.[CrossRef][Medline]

Narumiya S, Sugimoto Y, and Ushikubi F (1999) Prostanoid receptors: structures, properties and functions. Physiol Rev 79: 1193-1226.[Abstract/Free Full Text]

Negishi M, Sugimoto Y, and Ichikawa (1995) Prostaglandin E receptors. J Lipid Mediat Cell Signal 12: 379-391.[CrossRef][Medline]

Premkumar LS and Ahern GP (2000) Induction of vanilloid receptor channel activity by protein kinase C. Nature (Lond) 408: 985-990.[CrossRef][Medline]

Qiu ZH and Leslie CC (1994) Protein kinase C-dependent and -independent pathways of mitogen-activated protein kinase activation in macrophages by stimuli that activate phospholipase A₂. J Biol Chem 269: 19480-19487.[Abstract/Free Full Text]

Raud J (1990) Vasodilatation and inhibition of mediator release represent two distinct mechanisms for prostaglandin modulation of acute mast cell-dependent inflammation. Br J Pharmacol 99: 449-454.[Medline]

Richardson JD and Vasko MR (2002) Cellular mechanisms of neurogenic inflammation. J Pharmacol Exp Ther 302: 839-845.[Abstract/Free Full Text]

Santos AR and Calixto JB (1997) Further evidence for the involvement of tachykinin receptor subtypes in formalin and capsaicin models of pain in mice. Neuropeptides 31: 381-389.[CrossRef][Medline]

Vlahos CJ, McDowell SA, and Clerk A (2003) Kinases as therapeutic targets for heart failure. Nat Rev Drug Discov 2: 99-113.[CrossRef][Medline]

White DM (1996) Mechanism of prostaglandin E₂-induced substance P release from cultured sensory neurons. Neuroscience 70: 561-565.[CrossRef][Medline]

Williams TJ and Morley J (1973) Prostaglandins as potentiators of increased vascular permeability in inflammation. Nature (Lond) 246: 215-217.[CrossRef][Medline]

Yuhki K, Ueno A, Naraba H, Kojima F, Ushikubi F, Narumiya S, and Oh-ishi S (2004) Prostaglandin receptors EP₂, EP₃ and IP mediate exudate formation in carrageenin-induced mouse pleurisy. J Pharmacol Exp Ther 311: 1218-1224.[Abstract/Free Full Text]

This article has been cited by other articles:

				A.M. Patwardhan, J. Vela, J. Farugia, K. Vela, and K.M. Hargreaves Trigeminal Nociceptors Express Prostaglandin Receptors Journal of Dental Research, March 1, 2008; 87(3): 262 - 266. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.102806v1 318/2/611    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Claudino, R. F. Articles by Calixto, J. B. Search for Related Content PubMed PubMed Citation Articles by Claudino, R. F. Articles by Calixto, J. B.