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. 2018 May;159(5):864-875.
doi: 10.1097/j.pain.0000000000001155.

Role of GPCR (mu-opioid)-receptor tyrosine kinase (epidermal growth factor) crosstalk in opioid-induced hyperalgesic priming (type II)

Dionéia Araldi  1 , Luiz F Ferrari, Jon D Levine
Affiliations

Role of GPCR (mu-opioid)-receptor tyrosine kinase (epidermal growth factor) crosstalk in opioid-induced hyperalgesic priming (type II)

Dionéia Araldi et al. Pain. 2018 May.

Abstract

Repeated stimulation of mu-opioid receptors (MORs), by an MOR-selective agonist DAMGO induces type II priming, a form of nociceptor neuroplasticity, which has 2 components: opioid-induced hyperalgesia (OIH) and prolongation of prostaglandin-E2 (PGE2)-induced hyperalgesia. We report that intrathecal antisense knockdown of the MOR in nociceptors, prevented the induction of both components of type II priming. Type II priming was also eliminated by SSP-saporin, which destroys the peptidergic class of nociceptors. Because the epidermal growth factor receptor (EGFR) participates in MOR signaling, we tested its role in type II priming. The EGFR inhibitor, tyrphostin AG 1478, prevented the induction of prolonged PGE2-induced hyperalgesia, but not OIH, when tested out to 30 days after DAMGO. However, even when repeatedly injected, an EGFR agonist did not induce hyperalgesia or priming. A phosphopeptide, which blocks the interaction of Src, focal adhesion kinase (FAK), and EGFR, also prevented DAMGO-induced prolongation of PGE2 hyperalgesia, but only partially attenuated the induction of OIH. Inhibitors of Src and mitogen-activated protein kinase (MAPK) also only attenuated OIH. Inhibitors of matrix metalloproteinase, which cleaves EGF from membrane protein, markedly attenuated the expression, but did not prevent the induction, of prolongation of PGE2 hyperalgesia. Thus, although the induction of prolongation of PGE2-induced hyperalgesia at the peripheral terminal of peptidergic nociceptor is dependent on Src, FAK, EGFR, and MAPK signaling, Src, FAK, and MAPK signaling is only partially involved in the induction of OIH.

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Figures

Figure 1
Figure 1
Schematic depicting the protocol used to evaluate the expression and induction of Type II hyperalgesic priming (OIH and prolongation of PGE2-induced hyperalgesia) induced by repeated exposure to DAMGO.
Figure 2
Figure 2. Induction of Type II hyperalgesic priming (OIH and prolongation of PGE2-induced hyperalgesia) is mu-opioid receptor (MOR) dependent
Rats were treated daily with an intrathecal injection of MM-ODN (120 μg/20 μL/day; black bars) or AS-ODN (120 μg/20 μL/day; dotted bars) for mu-opioid receptor (MOR) mRNA for 3 consecutive days. The average baseline mechanical nociceptive threshold, before ODNs, was 131.3 ± 1.9 g for the MM-ODN group and 132.1 ± 1.8 g for the AS-ODN group. Upper panel (A – D). On the 4th day, ∼17 hours after the last injection of MM- or AS-ODN, repeated (hourly × 4) intradermal injections of DAMGO (1 μg) were administered and, the mechanical nociceptive threshold evaluated 30 min after the 4th injection of DAMGO. Average baseline mechanical nociceptive threshold, before repeated injections of DAMGO, was 133.3 ± 1.1 g for the MM-ODN group and 131.2 ± 1.5 g for the AS-ODN group (A). In the AS-ODN-treated group, hyperalgesia induced by the 4th injection of DAMGO (OIH) was prevented (t(10) = 17.51, *** p < 0.0001, when MOR MM-ODN- and MOR AS-ODN-treated groups was compared at 30 min after the 4th injection of DAMGO; unpaired Student's t test). Treatment with intrathecal injections of MOR MM- or AS-ODN was then continued for 2 additional days and, on the 6th day (∼17 hours after the last injection of MM- or AS-ODN), DAMGO (1 μg) was injected intradermally on the dorsum of the hind paw and the mechanical nociceptive threshold evaluated 30 min after injection. The average baseline mechanical nociceptive threshold, before a single injection of DAMGO, was 130.7 ± 1.9 g for the MM-ODN group and 129.8 ± 2.2 for the AS-ODN group. When compared to MOR MM-ODN-treated group, DAMGO-induced hyperalgesia was completely blocked in the MOR AS-ODN-treated group (F(1,20) = 53.57, *** p < 0.0001, when MOR MM-ODN-treated and AS-ODN-treated group was compared 30 min after the injection of DAMGO; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). Five (B), 15 (C) and 30 (D) days after the last injection of MOR MM- or AS-ODN, when DAMGO (1 μg) was intradermally again injected, DAMGO-induced hyperalgesia at 30 min was still blocked in the MOR AS-ODN-treated group (F(1,10) = 34.16, *** p = 0.0002, for 5 days (B), F(1,10) = 46.10, *** p < 0.0001 for 15 days (C), and F(1,10) = 56.50, *** p < 0.0001 for 30 days (D) when the MOR MM-ODN-treated and MOR AS-ODN-treated groups were compared, 30 min after injection of DAMGO; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). Of note, the average baseline mechanical nociceptive threshold, before DAMGO, was 131.9 ± 1.6 g (15 days) and 130.7 ± 2.2 g (30 days) for the MM-ODN group and 130.8 ± 1.5 g (15 days) and 129.9 ± 1.9 g (30 days) for the AS-ODN group. Lower panel (E – H). On the 6th day (∼17 hours after the last injection of MOR MM- or AS-ODN, and 2 days after having received repeated injections of DAMGO), groups of rats were treated with an intradermal injection of PGE2 (100 ng). The average baseline mechanical nociceptive threshold, before repeated injections of DAMGO, was 129.8 ± 1.9 g for the MM-ODN group and 131.8 ± 1.3 g for the AS-ODN group; before the injection of PGE2, was 128.5 ± 2.4 g for the MM-ODN group and 129.9 ± 1.9 g for the AS-ODN group. (E). Treatment with MOR AS-ODN was able to prevent the prolongation of PGE2 hyperalgesia at the 4th hour, compared to the MOR MM-ODN-treated group (F(2,20) = 159.45, *** p < 0.0001, when MOR MM-ODN-treated group is compared to MOR AS-ODN-treated group at the 4th hour after the injection of PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). Five (F) and 15 (G) days after the last injection of MOR MM- or AS-ODN, PGE2 (100 ng) was again injected. The prolongation of PGE2 hyperalgesia at the 4th hour was still blocked in the group that had been treated with MOR AS-ODN (F(2,20) = 259.52, *** p < 0.0001, for 5 days (F) and, F(2,20) = 202.30, *** p < 0.0001 for 15 days (G), when MOR MM-ODN-treated group is compared to MOR AS-ODN-treated group at the 4th hour after the injection of PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). Of note, the average baseline mechanical nociceptive threshold, before PGE2, was 130.7 ± 1.2 g (15 days) and 129.7 ± 1.9 g (30 days) for the MM-ODN group and 129.9 ± 2.1 g (15 days) and 131.2 ± 1.6 g (30 days) for the AS-ODN group. H. Thirty days after the last injection of ODN, when the average baseline mechanical nociceptive threshold was 130.1 ± 1.3 g for the MM-ODN group and 132.1 ± 1.1 g for the AS-ODN group, PGE2 was again injected intradermally. The prolongation of PGE2-induced hyperalgesia was again markedly attenuated at the 4th hour in the MOR AS-ODN-treated group (F(2,20) = 108.36, *** p < 0.0001, when MOR MM-ODN-treated group is compared to MOR AS-ODN-treated group at the 4th hour after the injection of PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). These findings indicate that MOR is necessary for the induction of Type II priming (OIH and prolongation of PGE2 hyperalgesia). n = 6 rats/6 paws per treated-group.
Figure 3
Figure 3. Lesion of IB4-negative nociceptors prevents Type II priming
Rats were treated with vehicle (20 μL; black bars) or SSP-saporin (100 ng/20 μL; gray bars) by intrathecal injection. The average baseline mechanical nociceptive threshold, before treatments, was 128.3 ± 1.8 g for the vehicle group and 130.3 ± 1.5 g for the SSP-saporin group. Two weeks later, when the average baseline mechanical nociceptive threshold was 130.5 ± 1.5 g for the vehicle group and 131.1 ± 1.8 g for the SSP-saporin group, DAMGO (1 μg) was repeatedly (hourly × 4) injected on the dorsum of the hind paw. In the SSP-saporin-treated group, the 4th injection of DAMGO did not induce hyperalgesia (t(10) = 7.481, *** p < 0.0001, when vehicle- and SSP-saporin-treated groups are compared 30 min after the 4th injection of DAMGO; unpaired Student's t test). Five days later, when mechanical nociceptive threshold was not different from the pre-DAMGO baseline (129.1 ± 2.2 g, t(5) = 1.581; p = 0.1747, for the vehicle-treated group; 130.8 ± 1.0 g, t(5) = 1.536; p = 0.1852, for the SSP-saporin-treated group, when the mechanical nociceptive threshold is compared before and 5 days after DAMGO; paired Student's t test), PGE2 (100 ng) was injected and the mechanical nociceptive threshold evaluated 30 min and 4 hours later. Two-way repeated-measures ANOVA followed by Bonferroni post hoc test showed PGE2 hyperalgesia at 30 min in both groups, with no significant (ns) difference between the groups. However, the prolongation of PGE2 hyperalgesia was markedly attenuated in the SSP-saporin-treated group at the 4th hour (F(2,14) = 45.53, *** p < 0.0001, when vehicle-treated group is compared to SSP-saporin-treated group at the 4th hour after the injection of PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that IB4-negative nociceptors are necessary for OIH and the prolongation of PGE2 hyperalgesia induced by repeated exposure to DAMGO. n = 6 paws per group.
Figure 4
Figure 4. MOR-EGFR crosstalk in the induction of prolongation of PGE2 hyperalgesia
Rats received an intradermal injection of vehicle (5 μL; black bars) or EGFR inhibitor (Tyrphostin AG 1478, 1 μg; dotted bars). The average baseline mechanical nociceptive threshold, before treatments, was 130.1 ± 1.4 g for the vehicle group and 132.0 ± 1.3 g for the EGFR inhibitor group. Ten minutes later, repeated (hourly × 4) intradermal injections of DAMGO (1 μg) were performed on the dorsum of the hind paw. Five days later, when mechanical threshold was not different from the pre-DAMGO baseline (128.8 ± 2.1 g, t(5) = 0.3162; p = 0.7646, for vehicle-treated group; 130.7 ± 2.0 g, t(5) = 2.169; p = 0.0822, for EGFR inhibitor-treated group, when the mechanical nociceptive threshold is compared before and 5 days after DAMGO; paired Student's t test), PGE2 (100 ng) was injected at the same site and mechanical nociceptive threshold evaluated 30 min and 4 hours later. In the group previously treated with EGFR inhibitor, the prolongation of PGE2 hyperalgesia was almost completely eliminated at the 4th hour (F(2,16) = 175.68, *** p < 0.0001, when vehicle-treated group is compared to EGFR inhibitor treated group at the 4th hour after the injection of PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). B – C. When PGE2 (100 ng) was again injected on the dorsum of the hind paw, 15 (B) or 30 (C) days after repeated exposure to DAMGO, the prolongation of PGE2-induced hyperalgesia was not present at the 4th hour in the EGFR inhibitor-treated group (F(2,16) = 98.89, *** p < 0.0001 for 15 days (B) and, F(2,16) = 120.35, *** p < 0.0001 for 30 days (C), when the vehicle-treated group is compared to EGFR inhibitor-treated group at the 4th hour after the injection of PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). These data support a role of crosstalk between mu-opioid and EGF receptors in the induction of the prolongation of PGE2 hyperalgesia by repeated exposure to DAMGO. Of note, the average baseline mechanical nociceptive threshold, before PGE2, was 129.7 ± 1.7 g (15 days) and 131.4 ± 1.8 g (30 days) for the vehicle group and 131.1 ± 1.1 g (15 days) and 130.6 ± 1.2 g (30 days) for the EGFR inhibitor group. D. A different group of rats, with average baseline mechanical nociceptive threshold of 130.9 ± 2.0 g, was treated with repeated (hourly × 4) intradermal injections of the EGFR agonist (EGF ligand; 1 μg/5 μL; gray bars) and the mechanical nociceptive threshold evaluated 30 min after each injection. No change in nociceptive threshold was observed after the 1st (not significant, ns, 130.3 ± 2.2 g, t(5) = 1.267, p = 0.2610), 2nd (ns, 130.0 ± 1.6 g, t(5) = 1.066, p = 0.3352), 3rd (ns, 128.0 ± 2.1 g, t(5) = 2.236, p = 0.0756) or 4th (ns, 128.3 ± 1.9 g, t(5) = 2.214, p = 0.0778) injection of EGFR agonist (when the mechanical nociceptive threshold before the 1st injection and 30 min after each injection of EGFR agonist, is compared; paired Student's t test). Five days (5 d) later, when the mechanical threshold was not different from the pre-EGFR agonist baseline (132.3 ± 1.7 g, t(5) = 2.485; p = 0.0555, when the mechanical nociceptive threshold is compared before and 5 days after repeated injections of EGFR agonist; paired Student's t test), PGE2 (100 ng) was injected at the same site, on the dorsum of the hind paw, and the mechanical nociceptive threshold evaluated 30 min and 4 hours after injection. PGE2 induced hyperalgesia 30 min after injection (t(5) = 10.03, *** p = 0.0002, when mechanical thresholds before and 30 min after PGE2, were compared; paired Student's t test), but not at the 4th hour (t(5) = 0.7895, p = 0.4656, when mechanical thresholds before and 4 hours after PGE2, were compared; paired Student's t test), indicating that repeated activation of EGF receptor does not induce hyperalgesic priming. n = 6 paws per group.
Figure 5
Figure 5. EGFR signaling in the induction of prolongation of PGE2 hyperalgesia
Rats were treated intradermally with vehicle (5 μL; black bars) or phosphopeptide SH2 (SH2, which blocks the Src interaction's with FAK and EGFR; 1 μg, withe bars). The average baseline mechanical nociceptive threshold, before treatments, was 131.3 ± 1.8 g for the vehicle group and 129.7 ± 1.7 g for the SH2 group. Starting ten minutes later, repeated (hourly × 4) intradermal injections of DAMGO (1 μg) were performed on the dorsum of the hind paw. Five days later, when the mechanical nociceptive threshold was not different from the pre-DAMGO baseline (129.3 ± 1.2 g, t(5) = 0.2758; p = 0.738, for the vehicle-treated group; 128.7 ± 2.1 g, t(5) = 0.1644; p = 0.8759, for the SH2-treated group; when the mechanical nociceptive threshold is compared before and 5 days after DAMGO; paired Student's t test), PGE2 (100 ng) was injected at the same site on the dorsum of the hind paw and the mechanical nociceptive threshold evaluated 30 min and 4 hours later. In the SH2-treated group, the prolongation of PGE2 hyperalgesia was inhibited at the 4th hour (F(2,30) = 144.07, *** p < 0.0001, when vehicle-treated group was compared to SH2-treated group at the 4th hour after the injection of PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). B. Fifteen days later, when the average baseline mechanical nociceptive threshold was 131.5 ± 1.0 g for the vehicle group and 128.9 ± 1.9 g for the SH2 group, PGE2 (100 ng) was again injected intradermally. We observed that the 4th hour of PGE2-induced hyperalgesia was still attenuated in the SH2-treated group (F(2,30) = 309.92, *** p < 0.0001, when SH2-treated group was compared to vehicle-treated group at the 4th hour after PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). C. Thirty days after repeated (hourly × 4) injections of DAMGO, when the average baseline mechanical nociceptive threshold was 131.7 ± 1.7 g for the vehicle group and 129.3 ± 1.4 g for the SH2 group, PGE2 (100 ng) was again injected intradermally. In the SH2-treated group the prolongation of PGE2 hyperalgesia was completely inhibited (F(2,30) = 168.26, *** p < 0.0001, when SH2-treated group is compared to vehicle-treated group at the 4th hour after PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), demonstrating that the interaction of Src with FAK and EGFR plays a role in the induction of prolongation of PGE2 hyperalgesia by repeated exposure to DAMGO. n = 6 paws per group.
Figure 6
Figure 6. Second messengers in induction of OIH
Rats were treated intradermally with vehicle (5 μL), MAPK inhibitor (U0126, 1 μg; dark gray bars), Src inhibitor (SU 6656, 1 μg; light gray bars), EGFR inhibitor (tyrphostin AG 1478; 1 μg; white bars) or phosphopeptide SH2 (SH2, which blocks the interaction of Src with FAK and EGFR; 1 μg; dotted bars). The average baseline mechanical nociceptive threshold, before treatments, was 132.3 ± 1.8 g for the vehicle group, 129.9 ± 1.8 g for the MAPK inhibitor, 133.3 ± 1.3 g for the Src inhibitor group, 130.7 ± 1.1 g for the EGFR inhibitor group, and 130.3 ± 2.0 g for the SH2 group. Starting ten minutes later, repeated (hourly × 4) intradermal injections of DAMGO (1 μg) were performed on the dorsum of the hind paw and the mechanical nociceptive threshold evaluated 30 min after the 4th injection of DAMGO. A. The hyperalgesia induced by the 4th injection of DAMGO was prevented by all inhibitors (F(4,40) = 65.72, *** p < 0.001, when vehicle-treated group is compared to all inhibitor-treated groups; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). Five days later, when the mechanical thresholds were not different from the pre-DAMGO baseline (130.3 ± 1.4 g, t(5) = 2.215; p = 0.0776, for the vehicle-; 130.7 ± 1.2 g, t(5) = 0.5407; p = 0.6119, for MAPK inhibitor; 129.9 ± 1.0 g, t(5) = 1.064; p = 0.3358, for Src inhibitor-; 128.5 ± 2.2 g, t(5) = 1.766; p = 0.1377, for EGFR inhibitor-; and 129.9 ± 1.8 g, t(5) = 0.8596; p = 0.4293, for the SH2-treated group, when the mechanical nociceptive threshold is compared before and 5 days after DAMGO; paired Student's t test), DAMGO (1 μg) was injected at the same site on the dorsum of the hind paw and the mechanical nociceptive threshold evaluated 30 min later. DAMGO-induced hyperalgesia was partially attenuated in the groups previously treated with inhibitors for MAPK (* p < 0.05) and EGFR (** p < 0.01) and completely inhibited in the Src inhibitor- and SH2-treated groups (F(2,40) = 38.15, *** p < 0.001, when vehicle-treated group is compared to inhibitors-treated groups; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). B. Fifteen days later, when the average baseline mechanical nociceptive threshold was 127.3 ± 2.6 g for the vehicle group, 132.3 ± 1.7 g for the MAPK inhibitor group, 134.3 ± 2.4 g for the Src inhibitor group, 134.3 ± 2.5 g for the EGFR inhibitor group, and 133.3 ± 1.9 g for the SH2 group, DAMGO (1 μg) was injected again on the dorsum of the hind paw. An attenuation on DAMGO-induced hyperalgesia was observed in the groups previously treated with MAPK and EGFR inhibitors (** p < 0.01, when vehicle-treated group is compared to MAPK and EGFR inhibitor-treated groups; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). However, a marked inhibition of DAMGO-induced hyperalgesia was observed in the groups pretreated with the Src inhibitor and SH2 (F(4,20) = 27.13, *** p < 0.001, when vehicle-treated group is compared to Src inhibitor- and SH2-treated groups; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). C. To verify that pretreatment with these inhibitors is able to prevent the induction of OIH, DAMGO (1 μg) was injected again, 30 days after the repeated (hourly × 4) injections of DAMGO. Of note, the average baseline mechanical nociceptive threshold, 30 days after treatments, was 132.0 ± 1.7 g for the vehicle group, 134.0 ± 2.0 g for the MAPK inhibitor, 132.8 ± 1.1 g for the Src inhibitor group, 129.7 ± 2.1 g for the EGFR inhibitor group, and 132.7 ± 1.7 g for the SH2 group. At this time, only a partial attenuation of DAMGO-induced hyperalgesia was observed in the groups previously treated with the MAPK and Src inhibitors, and SH2, when compared to the vehicle-treated group (F(4,40) = 3.47, ** p < 0.01, when vehicle-treated group is compared to MAPK or Src inhibitors- or SH2-treated groups; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). However, no significant (ns) difference was observed between vehicle- and EGFR inhibitor-treated groups (ns, two-way repeated-measures ANOVA followed by Bonferroni post hoc test). These findings indicate a partial contribution of a Src, FAK and MAPK signaling in induction of OIH by repeated exposure to DAMGO. n = 6 paws per group.
Figure 7
Figure 7. Role of matrix metalloproteinases in DAMGO-induced prolongation of PGE2 hyperalgesia
Rats received an intradermal injection of vehicle (5 μL; black bars), ilomastat (a general MMP inhibitor, 1 μg; gray bars) or MMP-9 inhibitor (1 μg; dotted bars) followed, 10 min later, by repeated (hourly × 4) injections of DAMGO (1 μg) at the same site. The average baseline mechanical nociceptive threshold, before treatments, was 129.4 ± 2.2 g for the vehicle group, 131.3 ± 1.9 g for the MMP inhibitor group, and 130.7 ± 1.1 g for the MMP-9 inhibitor group. Five days later, when the mechanical nociceptive threshold was not different from the pre-DAMGO baseline (127.0 ± 2.5 g, t(5) = 1.395; p = 0.2215, for the vehicle-treated group; 132.3 ± 1.7 g, t(5) = 0.4416; p = 0.6772, for the general MMP inhibitor-treated group, 127.7 ± 2.2 g, t(5) = 0.2548; p = 0.8090, for the MMP-9 inhibitor-treated group, when the mechanical nociceptive threshold is compared before and 5 days after DAMGO; paired Student's t test), PGE2 (100 ng) was injected intradermally on the dorsum of the hind paw and, a significant inhibition of PGE2 hyperalgesia at the 4th hour was observed in both MMP and MMP-9 inhibitor-treated groups (F(2,30) = 223.71, *** p < 0.0001, when inhibitor-treated groups are compared to vehicle-treated group at the 4th hour after PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). B. However, at day 15, when the average baseline mechanical nociceptive threshold was 128.9 ± 1.5 g for the vehicle group, 130.3 ± 2.3 g for the MMP inhibitor, and 129.9 ± 2.1 g for the MMP-9 inhibitor group, PGE2 (100 ng) was again injected intradermally. We observed that prolongation of PGE2 hyperalgesia at the 4th hour was only weakly attenuated in both the general MMP and the MMP-9 inhibitor-treated groups (F(2,30) = 7.89, * p = 0.0088, when inhibitors-treated groups are compared to vehicle-treated group at the 4th hour after PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test), indicating that MMPs play a role in the expression, but not induction, of DAMGO-induced prolongation of PGE2 hyperalgesia. n = 6 paws per group.
Figure 8
Figure 8. Second messengers involved in MOR-EGFR crosstalk
Rats were treated intradermally with vehicle (5 μL; black bars), salirasib (RAS inhibitor, 1 μg; gray bars) or GW5074 (cRaf1 inhibitor, 1 μg; white bars) on the dorsum of the hind paw. The average baseline mechanical nociceptive threshold, before treatments, was 127.7 ± 2.4 g for the vehicle group, 132.7 ± 2.2 g for the RAS inhibitor group, and 129.3 ± 2.8 g for the cRaf1 inhibitor group. Ten minutes later, repeated (hourly × 4) injections of DAMGO (1 μg) was performed at the same site on the dorsum of the hind paw. Five days later, PGE2 (100 ng) was injected at the same site and the mechanical nociceptive threshold evaluated 30 min and 4 hours later. Compared to vehicle-treated group, in all inhibitors treated-groups PGE2 was able to induce hyperalgesia 30 min after its injection (data not shown). A small attenuation in the prolongation of PGE2 hyperalgesia at the 4th hour was observed only in the group previously treated with cRaf1 inhibitor (F(5,30) = 3.10, * p = 0.0226, when inhibitor-treated groups were compared to vehicle-treated group at the 4th hour after PGE2; two-way repeated-measures ANOVA followed by Bonferroni post hoc test). The RAS inhibitor and vehicle did not inhibit the prolongation of PGE2 hyperalgesia at the 4th hour. These findings indicate that RAS and cRaf1 do not play a role in the induction of prolongation of PGE2 hyperalgesia.
Figure 9
Figure 9. Schematic of mechanisms involved in induction of Type II hyperalgesic priming (OIH and prolongation of PGE2-induced hyperalgesia)
Chronic opioid use changes MOR signaling, that may reflect intracellular signal switching. Our findings show that repeated (hourly × 4) intradermal injections of a MOR selective agonist DAMGO induces Type II hyperalgesic priming (OIH [A] and prolongation of PGE2-induced hyperalgesia [B]), in peptidergic IB4-negative nociceptors. Induction of both components present in Type II priming are MOR dependent, which activates diverse downstream second messengers. A. Activation of MOR (a Gαi-protein-coupled receptor) by repeated (hourly × 4) injections of DAMGO, stimulates Src and FAK, leading to activation of MAPK, which ultimately produces sensitization in the peripheral terminal of IB4-negative nociceptor. B. After the peripheral terminal of IB4-negative nociceptors have received repeated injections of DAMGO, PGE2 was injected, which activates a signaling cascade, involving EGFR, Src and FAK, leading to stimulation of MAPK, which ultimately prolongs PGE2-induced hyperalgesia. Thus, while the induction of OIH is partially attenuated by the inhibition of Src, FAK and MAPK signaling (A), the prolongation of PGE2 hyperalgesia is completely dependent on Src, FAK, EGFR and MAPK signaling (B). Schematics summarize the signaling pathways involved in the induction of OIH (A) and prolongation of PGE2 hyperalgesia (B) induced by repeated exposure to DAMGO. Abbreviations: βγ, G-protein βγ subunit; DAMGO, [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin acetate salt (a mu-opioid receptor agonist); EGFR, epidermal growth factor receptor; EP, prostaglandin receptor; FAK, focal adhesion kinase; Gαi, G-protein αi subunit; IB4, isolectin B4; MAPK, mitogen-activated protein kinase; MOR, mu-opioid receptor; PGE2, prostaglandin-E2; Src, proto-oncogene tyrosine-protein kinase.
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