Intraplantar PGE2 causes nociceptive behaviour and mechanical allodynia: the role of prostanoid E receptors and protein kinases

Abstract

Background and purpose: Receptor subtypes involved in PGE(2)-induced nociception are still controversial. The present study investigated the prostanoid E receptor (EP) subtypes and the protein kinase (PK) pathways involved in the nociception induced by PGE(2) injection in the mouse paw.

Experimental approach: Paw-licking and mechanical allodynia were measured in vivo and protein kinase activation ex vivo by Western blots of extracts of paw skin.

Key results: Intraplantar (i.pl.) injection of PGE(2) into the mouse paw caused nociceptive behaviour of short duration with mean ED(50) of 1.43 nmol. PGE(2) produced a longer-lasting mechanical allodynia, with an ED(50) of 0.05 nmol. Intraplantar injection of antagonists at EP(3) or EP(4), but not at EP(1) or EP(2) receptors inhibited PGE(2)-induced paw-licking. Paw-licking caused by PGE(2) was blocked by an inhibitor of PKA but only partially decreased by inhibition of the extracellular-regulated kinase (ERK). Selective inhibitors of PKC, c-Jun N-terminal kinase (JNK) or p38, all failed to affect PGE(2)-induced paw-licking. An EP(3) antagonist inhibited PGE(2)-induced mechanical allodynia. However, inhibitors of PKA, PKC or ERK, but not p38 or JNK, also partially inhibited PGE(2)-induced mechanical allodynia. Western blot analyses confirmed that i.pl. injection of PGE(2) activated PKA, PKCalpha, and mitogen activated kinases (MAPKs) in the paw. Co-treatment with EP(3) or EP(4) receptor antagonists reduced PGE(2)-induced PKA and ERK, but not PKCalpha activation.

Conclusions and implications: The present results indicate that the nociceptive behaviour and mechanical allodynia caused by i.pl. PGE(2) are mediated through activation of distinct EP receptors and PK-dependent mechanisms.

Figure 1

Dose and time dependence for PGE₂-induced paw licking (0.3–10 nmol per paw) (a) or mechanical allodynia (0.01–0.3 nmol per paw) (b) in mice. The paw licking is expressed as licking time (s) and the mechanical allodynia is expressed as threshold (mg). Each point on the curve represents the mean of 6–10 animals and vertical lines show the s.e.m. Asterisks denote the significance levels in comparison with the vehicle-treated group (one-way ANOVA followed by Dunnett's test). ^*P<0.05, ^**P<0.01 and ^***P<0.001.

Figure 2

Effect of i.pl. treatment with selective receptor antagonists for EP₃ (L-826266, 3–30 nmol per paw (a)) EP₄ (L-161982, 1–30 nmol per paw (b)) EP_1/2 receptors (AH-6809, 10 and 100 nmol per paw (c)) or a combination of EP₃ and EP₄ receptor antagonists (L-826266 plus L-161982, each 10 nmol per paw) (d) on PGE₂-induced paw licking in mice (PGE₂, 3 nmol per paw). The drugs were co-injected with PGE₂ and the effects of the drugs are expressed as licking time (s). Each column represents the mean of 7–8 animals and vertical lines show the s.e.m. Asterisks denote significant difference levels, ^*P<0.05, ^**P<0.01 and ^***P<0.001, compared with the PGE₂ plus vehicle-injected mice (black bar, one-way ANOVA followed by Dunnett's (a and b) or Student–Newman–Keuls' (c and d) test).

Figure 3

Effect of i.pl. treatment with selective receptor antagonists for EP₃ (L-826266, 0.1–10 nmol per paw (a)) EP_1/2 (AH-6809, 100 nmol per paw) or EP₄ receptors (L-161982, 10 nmol per paw (b)) on PGE₂-induced mechanical allodynia (0.1 nmol per paw) in mice. The drugs were co-injected with PGE₂ and the effects of the drugs are expressed as threshold (mg). Each column represents the mean of 6–12 animals and vertical lines show the s.e.m. ^#Denotes significant difference from basal group (P<0.01) (Student-Newman-Keuls' test). Asterisks denote the significance levels, ^**P<0.01, compared with the PGE₂ plus vehicle-injected mice (black bar, one-way ANOVA followed by Dunnett's (a) or Student–Newman–Keuls' (b) test).

Figure 4

Effect of i.pl. treatment with the selective EP₃ receptor antagonist (L-826266, 10 nmol per paw) on 17-phenyl-trinor-PGE₂-induced paw licking (3 nmol per paw (a)) or mechanical allodynia (0.1 nmol per paw (b)). In c and d, the effects of i.pl. treatment with the EP₃ receptor antagonist (L-826266, 10 nmol per paw) or the EP₄ receptor antagonist (L-161982, 10 nmol per paw) on paw-licking or mechanical allodynia induced by carrageenan (300 μg per paw) are shown. The effects of the drugs are expressed as licking time (s) or threshold (mg). Each column represents the mean of six animals and vertical lines show the s.e.m. ^#denotes significant difference from basal group (P<0.01) (Student–Newman–Keuls' test). Asterisks denote the significance levels, ^**P<0.01 and ^***P<0.001, compared with the PGE₂ plus vehicle-injected mice (black bar, one-way ANOVA followed by Student–Newman–Keuls' test).

Figure 5

Effect of i.pl. treatment with an inhibitor of PKA (KT-5720, 0.1–3 nmol per paw) (a), inhibitors of PKC (GF109203X, 10 nmol per paw), ERK (PD98059, 30 nmol per paw), p38 MAPK (SB203580, 30 nmol per paw) or JNK (SP600125, 30 nmol per paw) on PGE₂-induced paw licking (3 nmol per paw) (b), inhibitors of PKC (GF109203X, 10 nmol per paw), PKA (KT-5720, 3 nmol per paw), ERK (PD98059, 30 nmol per paw), p38 MAPK (SB203580, 30 nmol per paw) or JNK (SP 600125, 30 nmol per paw) on mechanical allodynia induced by PGE₂ (0.1 nmol per paw) (c). The effects of the drugs are expressed as licking time (s) or threshold (mg). Each column represents the mean of six animals and vertical lines show the s.e.m. ^#denotes significant difference from basal group, P<0.01 (Student–Newman–Keuls' test). Asterisks denote the significance levels, ^*P<0.05, ^**P<0.01 and ^***P<0.001, compared with the PGE₂ plus vehicle-injected mice (black bar, one-way ANOVA followed by Dunnett's (a) or Student–Newman–Keuls' (b and c) test).

Figure 6

Effect of post-treatment with GF109203X (10 nmol per paw), KT5720 (3 nmol per paw) or PD98059 (30 nmol per paw) on PGE₂-induced paw-licking (a) or mechanical allodynia (b) in mice. The effects of the drugs are expressed as licking time (s) or threshold (mg). Each column represents the mean of six animals and vertical lines show the s.e.m. ^#denotes significant difference from basal group, P<0.01 (Student–Newman–Keuls' test). Asterisks denote the significance levels, ^***P<0.001, compared with the PGE₂ plus vehicle-injected mice (black bar, one-way ANOVA followed by Student–Newman–Keuls' test).

Figure 7

Representative images of western immunoblotting and densitometry analyses showing the time course of phosphorylation of PKA RII (phospho-PKA RII; (a)) ERK (phospho-ERK1, (b)) PKCα translocation from cytosol (c) to membrane (d) in response to i.pl. injection of PGE₂ (3 nmol per paw) into mouse paw. Densities for actin are shown in (e). Mouse paw tissues were obtained from naive (basal, B) or PGE₂-injected mice at the indicated times. Membrane levels of PKC-α and cytosolic levels of phospho-PKA RII, PKCα, phospho-ERK1 and actin were determined using specific antibody and indicated PK activation. Results were normalized by arbitrarily setting the densitometry of the basal group and are expressed as mean±s.e.m. (n=3). ^*P<0.05, as compared with basal values (one-way ANOVA followed by Dunnett's test).

Figure 8

Representative images of western immunoblotting and densitometry analyses showing the effect of EP₃ or EP₄ receptor antagonists in PGE₂-induced phosphorylation of PKA RII (phospho-PKA RII, (a)) of ERK1 (phospho-ERK1, (b)) or PKCα activation (PKCα membrane (c)) and PKCα cytosol (d)). Mouse paw tissues were obtained from naive (basal, B) or 15 min after PGE₂ injection. Cytosolic levels of phospho-PKA RII were determined using a specific antibody. Results were normalized by arbitrarily setting the densitometry of the basal group and are expressed as mean±s.e.m. (n=3). ^#P<0.05, as compared with basal values (B), ^*P<0.05, as compared with vehicle group (one-way ANOVA followed by Student–Newman–Keuls' test).