Figure 1.
Intraplantar injection effect of crotalphine (CRP) or opioid receptor agonists in the rat nociceptive threshold.
Pain threshold was obtained in the rat paw pressure test, before (dotted line) and 1 h after intraplantar injection of CRP (0.6 ng/paw), DAMGO (μ opioid receptor agonist, 5 µg/paw), DPDPE (δ opioid receptor agonist, 20 µg/paw), U-50488 (κ opioid receptor agonist, 10 µg/paw). CRP or opioid agonists were injected in naïve rats (Panel A) or in rats injected with PGE2 (100 ng/paw) 2 h before opioids or peptide administration (Panel B). CTOP (20 µg/paw), ICI 174,864 (ICI, 10 µg/paw) or nor-Binaltorphimine (NOR, 50 µg/paw), μ, δ and κ opioid receptor antagonists, respectively, or vehicle (50 µl/paw) were injected by the intraplantar route immediately before the opioid agonists or CRP administration. Panel C represents PGE2-induced hyperalgesia in the contralateral paw. Data represent mean values ± S.E.M. for six rats per group. * significantly different from baseline (dotted line), # significantly different from control (saline = SAL). Data were analyzed by two-way analysis of variance (ANOVA) with post-hoc testing by Tukey.
Figure 2.
Intraplantar injection effect of prostaglandin E2 (PGE2) on gene (A, C, E) expression of μ (A), κ (B) and δ (C) opioid receptors.
The changes in mRNA levels of opioid receptors were determined by real time PCR, in dorsal root ganglia (DRG) obtained 3 h after PGE2 injection (100 ng/paw). Dotted line represents the values obtained in naïve animals. Data are presented as mean of 7 animals ± SEM and expressed as % of control (naïve) animals. * Significantly different from mean values of naïve animals (p<0.05). Data were analyzed by one-way analysis of variance (ANOVA) with post-hoc testing by Tukey.
Figure 3.
Intraplantar injection effect of prostaglandin E2 (PGE2) on protein expression of μ (A), κ (B) and δ (C) opioid receptors.
The changes in protein levels of opioid receptors were determined by immunobloting, in dorsal root ganglia (DRG) and plantar tissue obtained 3 h after PGE2 injection (100 ng/paw). Data are presented as mean ± SEM and expressed as % of control (naïve) animals. *Significantly different from mean values of naïve animals, n = 6 per group (p<0.05). Data were analyzed by one-way analysis of variance (ANOVA) with post-hoc testing by Tukey.
Figure 4.
PGE2, per se, does not activate opioid receptors.
The plantar tissue (A) and dorsal root ganglia (ipsi and contralateral) (B) were obtained 3 h after PGE2 injection (100 ng/paw). Tissues obtained from naïve animals were used as controls. The tissues were sectioned and probed with anti-μ, κ or δ antibodies. (A) The black bar represents the results obtained with the conformation state-sensitive antibodies whereas the white bar represents the results obtained using the control antibodies (western blotting assay antibodies). (B) The first couple of bars represents the results obtained using the control antibodies (western blotting assay antibodies). The second one represents the conformation state-sensitive antibodies. The dotted line represents the values obtained for naïve animals. * Significantly different from mean values of naïve animals (p<0.05). Data were analyzed by one-way analysis of variance (ANOVA) with post-hoc testing by Tukey.
Figure 5.
PGE2 improves agonist-mediated opioid receptor activation.
Naïve or prostaglandin E2 (PGE2, 100 ng/paw) injected rats were treated with DAMGO (5 µg/paw), U50,488 (10 µg/paw), DPDPE (20 µg/paw), (Panel A) or crotalphine (CRP, 0.6 ng/paw) (Panel B). The agonists, CRP, or vehicle (control) were administered 2 h after PGE2 injection. ELISA assay was performed in slices of plantar tissue, using conformation state-sensitive antibodies (μ, κ, δ activated). Tissues were collected 3 h after PGE2 injection. *Significantly different from mean values of control animals (dotted line), n = 6 animals per group (p<0.05). #Significantly different from mean values of CRP or agonist alone (p<0.05). Data were analyzed by one-way analysis of variance (ANOVA) with post-hoc testing by Tukey.
Figure 6.
PGE2 improves agonist-mediated opioid receptor activation in primary neuronal cell culture.
Cells from dorsal root ganglia were obtained from naïve rats and incubated at 37°C, in a 5% of CO2, for two days. On the third day, the cells were incubated with PGE2 (1 µM) or vehicle for 15 min and further incubated with 1 µM of crotalphine (CRP, A) or DAMGO, U50,488, DPDPE (B) for 15 min. ELISA assay was performed directly on fixed cells, using conformation state-sensitive antibodies (activated). Three separate experiments were carried out on different occasions.* Significantly different from mean values of naïve animals (dotted line) (p<0.05), # Significantly different from mean values of CRP or agonist alone, average of 3 independent experiments (p<0.05). Data were analyzed by one-way analysis of variance (ANOVA) with post-hoc testing by Tukey.
Figure 7.
Effect of crotalphine on cyclic GMP levels in primary culture of DRG cells.
Cells from DRG were obtained from naïve rats and incubated at 37°C in a 5% of CO2, for two days. On the third day, the cells were incubated with Nor-BNI (1 µM) for 30 min and PGE2 (1 µM) or vehicle (saline - Sal) for 15 min, and further incubated with 1 µM of crotalphine (CRP), U50,488, or saline, for 15 min. Cells were lysed and subjected to enzyme immunoassay.*Significantly different from mean values of control cells (p<0.05), # Significantly different from mean values of CRP alone (p<0.05), Significantly different from mean values of U 50,488 alone (p<0.05). cGMP levels were analysed by one-way analysis of variance with a post-hoc Tukey test. Data were analyzed by one-way analysis of variance (ANOVA) with post-hoc testing by Tukey.
Figure 8.
Effect of crotalphine (CRP) on AKT phosphorylation in primary culture of DRG cells.
Cells from DRG were obtained from naïve rats and incubated at 37°C in a 5% of CO2, for two days. On the third day, the cells were incubated with PGE2 (1 µM) or vehicle (saline - Sal) for 15 min. and further incubated with 1 µM of CRP, U50,488, or saline, for 15 min. Cells were scrapped and homogenized, and the total homogenate was subjected to western blotting. (A) The ratio of phospho-protein to total protein is expressed as percentage from control (Sal+Sal). Three separate experiments were carried out on different occasions.*Significantly different from mean values of control cells (p<0.05). AKT levels were analysed by one-way analysis of variance with a post-hoc Tukey test. (B) Representative blots showing the levels AKT in the total lisate of DRGs.
Figure 9.
Effect of crotalphine (CRP) on phosphorylated form of ERK (A) and JNK (B) in primary culture of DRG cells.
Cells from DRG were obtained from naïve rats and incubated at 37°C in a 5% of CO2, for two days. On the third day, cells were incubated with Nor-BNI (1 µM) for 30 min and PGE2 (1 µM) or vehicle (saline - Sal) for 15 min and further incubated with 1 µM of CRP, U50,488, or saline, for 15 min. Cells were scrapped and homogenized, and the total homogenate was subjected to western blotting. The ratio of phospho-protein to total protein is expressed as % of control (sal+sal). GAPDH was used as the internal loading control. Three separate experiments were carried out on different occasions.*Significantly different from mean values of control cells (p<0.05). (A) p-ERK1/2 and (B) p-JNK levels were analysed by one-way analysis of variance with a post-hoc Tukey test. Representative blots showing the levels p-ERK1/2 and p-JNK in the total lysate of DRGs. # Significantly different from mean values of CRP alone (p<0.05).
Figure 10.
Involvement of PKCζ on crotalphine-mediated ERK1/2 and JNK phosphorylation in DRG primary culture.
Cells from DRG were obtained from naïve rats and incubated at 37°C in a 5% of CO2, for two days. On the third day, cells were incubated for 30 minutes with TAT carrier (control) or ζ pseudosubstrate (1 µM) followed by incubation with PGE2 or vehicle (15 minutes). The neurons were then incubated with 1 µM of crotalphine (CRP) and TAT or ζ pseudosubstrate for 15 minutes. Cells were scrapped and homogenized, and the total homogenate was subjected to western blotting for (A) pERK1/2 and (B) pJNK. The ratio of phospho-protein to total protein isexpressed as % of control (Sal+Sal). GAPDH was used as the internal loading control. Three separate experiments were carried out on different occasions.*Significantly different from mean values of control cells (p<0.05). # Significantly different from mean values of CRP alone (p<0.05). p-ERK1/2 and p-JNK levels were analysed by one-way analysis of variance with a post-hoc Tukey test. Representative blots showing the levels pERK1/2 or pJNK in the total lysate of DRGs.