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

Araldi, Dionéia; Ferrari, Luiz, F.; Levine, Jon, D.*

doi: 10.1097/j.pain.0000000000001155
Research Paper

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.

We investigate the mechanisms for the induction of type II hyperalgesic priming (opioid-induced hyperalgesia and prolongation of PGE2 hyperalgesia), a form of nociceptor neuroplasticity.

Division of Neuroscience, Departments of Medicine and Oral Surgery, University of California, San Francisco, San Francisco, CA, USA

Corresponding author. Address: Division of Neuroscience, Departments of Medicine and Oral Surgery, University of California, San Francisco, 513 Parnassus Ave, San Francisco, CA 94143-0440, USA. Tel.: 476-5108; fax: 476-6305. E-mail address: Jon.Levine@ucsf.edu (J. D. Levine).

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Received August 22, 2017

Received in revised form December 29, 2017

Accepted January 04, 2018

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1. Introduction

Hyperalgesic priming refers to a neuroplastic change in nociceptors that have been shown to contribute to the transition from acute to chronic pain.3,41,47 We have recently described a second form of hyperalgesic priming (type II), induced by repeated exposure to DAMGO, a mu-opioid receptor (MOR) agonist.5,8 Type II priming is characterized not only by prolongation of prostaglandin-E2 (PGE2)-induced hyperalgesia, as observed in type I priming, but also by the onset of opioid-induced hyperalgesia (OIH).5,31 A second important difference between these 2 types of priming is that type I is induced by the activation of protein kinase epsilon (PKCε)3,23,41 and maintained by protein translation23 in the terminals of nonpeptidergic, IB4-positive nociceptors,30 whereas the maintenance of type II priming is dependent on the simultaneous activation of Src tyrosine kinase (Src) and mitogen-activated protein kinase (MAPK).8

Mechanisms mediating hyperalgesic priming can be divided into those mediating induction, expression,5 and maintenance.8 In this study, we investigated the mechanisms mediating the induction of OIH and the prolongation of PGE2-induced hyperalgesia in type II hyperalgesic priming. In particular, we examined the role of the interaction of MOR, a Gαi-protein coupled receptor (GPCR) with epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) that has been demonstrated to have crosstalk with opioid receptors.10,16,42 We also investigated second messenger, downstream of MOR, involved in the induction of OIH and prolongation of PGE2 hyperalgesia in type II hyperalgesic priming.

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2. Materials and methods

2.1. Animals

Male Sprague-Dawley rats (240-400 g; Charles River Laboratories, Hollister, CA), housed 3 per cage, under a 12-hour light/dark cycle, in a humidity and temperature-controlled animal care facility at the University of California, San Francisco, were used in the presented experiments. Water and food were available ad libitum. Nociceptive evaluations were performed between 10:00 AM and 5:00 PM. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of California at San Francisco and adhered to the National Institutes of Health Guide for the care and use of laboratory animals.

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2.2. Testing mechanical nociceptive threshold

An Ugo Basile Analgesymeter (Randall-Selitto paw-withdrawal test; Stoelting, Chicago, IL) was used to quantify mechanical nociceptive threshold, by the application of a linearly increasing mechanical force to the dorsum of the rat's hind paw, as previously described.5,8,27,54,55 Rats were placed in acrylic restrainers for 30 minutes before experiments. The restrainers provided proper ventilation and allowed for the extension of the hind legs from lateral ports in the cylinder during assessment of nociceptive threshold. The nociceptive threshold was defined as the force (expressed in grams) at which the rat withdrew its paw. Baseline paw-pressure nociceptive threshold was defined as the mean of the 3 readings taken before the test agents were injected. Only 1 paw was used in an experiment, and each experiment was performed on a separate group of rats. To minimize experimenter bias, individuals conducting the behavioral experiments (D.A. and L.F.F.) were blinded to experimental interventions.

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2.3. Drug administration

The following drugs were used in this study: Prostaglandin-E2 (PGE2, a direct-acting hyperalgesic agent that sensitizes nociceptors), DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt, an MOR agonist), salirasib (a RAS inhibitor), GW5074 (a cRaf1 kinase inhibitor), SU 6656 (a Src family kinase inhibitor), and Ilomastat (an inhibitor of a wide variety of matrix metalloproteases [MMPs]), all from Sigma-Aldrich (St. Louis, MO); recombinant rat EGF protein (an EGF ligand [EGFR agonist]) from Abcam (Cambridge, MA); MMP-9 inhibitor from Calbiochem (Billerica, MA); Tyrphostin AG 1478 hydrochloride (an EGFR inhibitor), U0126 (MAPK/ERK inhibitor), and N-acetyl-O-phosphono-Tyr-Glu-Glu-Ile-Glu (peptide SH2; a phosphopeptide ligand for the Src SH2 domain that blocks Src interactions with EGFR and focal adhesion kinase [FAK]) from Tocris (Avonmouth, Bristol, United Kingdom).

The PGE2 stock solution (1 μg/μL), initially prepared in ethanol 100%, was diluted in saline (0.9% NaCl), yielding a final ethanol concentration <1%. DAMGO, recombinant rat EGF protein and peptide SH2 were dissolved in saline. All other drugs were dissolved in dimethyl sulfoxide 100% (Sigma-Aldrich) and further diluted in saline containing 2% of Tween 80 (Sigma-Aldrich). The final concentration of Tween 80 and dimethyl sulfoxide was ∼2%. Intradermal administration of drugs was performed on the dorsum of the hind paw, using a 30-gauge hypodermic needle adapted to a 50μL Hamilton syringe by a segment of PE/10 polyethylene tubing (Becton Dickinson, Franklin Lakes, NJ). The administration of all drugs, except PGE2, recombinant rat EGF and DAMGO, was preceded by a hypotonic shock (injection of 1 μL of distilled water, separated from the drug in the same syringe by a bubble, avoiding their mixing) to facilitate their entry into the nerve terminal through the cell membrane.12,13

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2.4. Mu-opioid receptor antisense

To investigate the role of MOR in induction of DAMGO-induced type II priming, oligodeoxynucleotides (ODNs) antisense (AS) for MOR mRNA was used.8,33,49 The AS-ODN sequence for MOR, 5′-CGC-CCC-AGC-CTC-TTC-CTC-T-3′, (Invitrogen Life Technologies, Carlsbad, CA) was directed against a unique region of rat MOR (UniProtKB database entry P33535 [OPRM_RAT] AS sequence to block translation and downregulate the gene expression of all 8 known isoforms [MOR]). The ODN mismatch (MM) sequence, 5′-CGC-CCC-GAC-CTC-TTC-CCT-T-3′ for MOR, was a scrambled version of the AS sequence that has the same base pairs and GC ratio, but scrambled nucleotide order, with little or no homology to any mRNA sequences posted at GeneBank.

Mismatch- or AS-ODNs were reconstituted in nuclease-free 0.9% NaCl, before use, and then injected intrathecally at a dose of 6 μg/μL in a volume of 20 μL (120 μg/20 μL). Oligodeoxy-nucleotides were injected for 3 consecutive days, once a day and, at the fourth day, repeated (hourly × 4) intradermal injections of DAMGO (1 μg) were performed and the mechanical nociceptive threshold evaluated 30 minutes after the fourth injection. Injections of MOR MM- or AS-ODN were performed for 2 more days (until day 5) and, on the sixth day (∼17 hours after the last injection of ODN), DAMGO (1 μg) or PGE2 (100 ng) was injected intradermally. DAMGO or PGE2 was injected again 5, 15, and 30 days after the last injection of ODN. Rats were anesthetized with isoflurane (2.5% in O2) and, the ODNs, administered using a microsyringe adapted to a 30-gauge needle, placed into the subarachnoid space, between the L4 and L5 vertebrae, as previously described.1 During the injections, the sudden flick of the rat's tail confirmed the access to the subarachnoid space.39 Rats recovered consciousness approximately 2 minutes after the injection. Of note, the attenuation of the expression of proteins involved in nociceptor sensitization by using intrathecal AS-ODNs has been previously demonstrated by several studies.5,6,8,11,25,40,46,51–53

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2.5. Intrathecal administration of SSP-saporin

[Sar9, Met(O2)11]-substance P-saporin, a SP-positive nociceptor neurotoxin (SSP-Saporin; Advanced Targeting Systems, San Diego, CA) was prepared in saline (5 ng/μL), and 20 μL was injected intrathecally, 14 days before the nociceptive tests.4,7 The addition of [Sar9, Met(O2)11] to substance P conjugated to saporin makes the agent more stable and a more potent toxin than when substance P alone is bound to saporin. The pretreatment interval and dose were established by Wiley et al59 and Choi et al,18 who observed prominent loss of neurons expressing the neurokinin 1 (NK1) receptor in the laminae 1 of the lumbar dorsal horn after using this protocol.32,34,56,58

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2.6. Induction of opioid-induced hyperalgesia and prolongation of PGE2-induced hyperalgesia

The repeated (hourly × 4) intradermal injections of the MOR agonist DAMGO (1 μg) produces OIH and a latent state of hyperresponsiveness to subsequent injection of proalgesic mediators,5,8,31 referred to as type II hyperalgesic priming.5,8 This neuroplasticity is expressed as prolongation of PGE2-induced mechanical hyperalgesia, lasting more than 4 hours, as opposed to the injection of PGE2 in naive paws, in which hyperalgesia is no longer present by 2 hours.2 To investigate the signaling pathways involved in the induction of type II hyperalgesic priming (OIH and prolongation of PGE2-induced hyperalgesia) by repeated exposure to an MOR agonist, all inhibitors were injected 10 minutes before the first injection of DAMGO. The OIH produced by repeated injections of DAMGO, was evaluated 30 minutes after the fourth injection of DAMGO and 5, 15, and 30 days after the repeated injections of DAMGO, by a single injection of DAMGO at each time point. The presence of hyperalgesia, 30 minutes after the injection of DAMGO, is a characteristic of OIH. To evaluate the signaling pathways involved in the prolongation of PGE2-induced hyperalgesia, all inhibitors were injected 10 minutes before the first injection of DAMGO. PGE2 was injected intradermally 5, 15, and 30 days after repeated injections of DAMGO. Mechanical hyperalgesia was evaluated for after 30 minutes and again at 4 hours after the injection of PGE2; the presence of hyperalgesia at the fourth hour is characteristic of hyperalgesic priming.3,5–8,24,41 If inhibitors were able to attenuate the expression (Fig. 1) of type II hyperalgesic priming (OIH and prolongation of PGE2-induced hyperalgesia), we also evaluated their role 30 days after injection, when DAMGO or PGE2 was again injected. If at this time, the DAMGO-induced hyperalgesia or prolongation of PGE2-induced hyperalgesia was still not present at 30 minutes (for DAMGO) or the fourth hour (for PGE2), then the inhibitor was considered able to prevent the induction (Fig. 1) of DAMGO-induced type II hyperalgesic priming (OIH and/or prolongation of PGE2-induced hyperalgesia).

Figure 1

Figure 1

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2.7. Data analysis

Data from all experiments are presented as mean ± SEM of n independent observations; the dependent variable was change in mechanical paw-withdrawal threshold, expressed as percentage change from baseline. Statistical evaluations were made using GraphPad Prism 5.0 statistical software (GraphPad Software). A P value <0.05 was considered statistically significant. To evaluate the role of second messengers in the induction of type II priming, inhibitors were injected in only 1 paw. In the experiments in which the MM- or AS-ODN was injected intrathecally, only the left paws were used (6 rats per group). No significant difference in mechanical nociceptive threshold was observed before the repeated injections of DAMGO and 5, 15, or 30 days later, when PGE2 or DAMGO was administered (average mechanical nociceptive threshold before repeated [hourly × 4] injections of DAMGO: 132.7 ± 1.11 g; average mechanical nociceptive threshold before PGE2 or DAMGO injection: 129.3 ± 1.23 g; n = 132 paws [132 rats]; paired Student t test, t (131) = 1.017, P = 0.4219). As indicated in the figure legends, Student t test or 2-way repeated-measures analysis of variance, followed by Bonferroni post hoc test, was performed to compare the magnitude of the hyperalgesia induced by the fourth injection of DAMGO or by the subsequent injection of PGE2 or DAMGO, in the different groups, or to compare the effect produced by different treatments on the DAMGO-induced OIH (evaluated 30 minutes after the fourth injection or 5, 15, and 30 days after injection) or the prolongation of PGE2-induced hyperalgesia (evaluated 4 hours after its injection at days 5, 15, and 30 after repeated injections of DAMGO) with the control (vehicle) groups.

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3. Results

3.1. Mu-opioid receptor dependence

Oligodeoxynucleotides MM or AS to MOR mRNA were used to study the role of MOR, in the peripheral terminal of the nociceptor, in the induction of type II hyperalgesic priming (OIH and prolongation of PGE2 hyperalgesia). Rats were treated daily with MM- or AS-ODN, by intrathecal administration, for 3 consecutive days and on the fourth day, ∼17 hours after an injection of ODN, rats received repeated (hourly × 4) intradermal injections of DAMGO. In the group treated with AS, DAMGO-induced hyperalgesia was prevented when the mechanical nociceptive threshold was evaluated 30 minutes after the injection of fourth dose of DAMGO (Fig. 2A). Mismatch- or AS-ODN was injected for 2 more days (days 4 and 5) and on the sixth day, ∼17 hours after the last injection of ODN, a single injection of DAMGO was administered. DAMGO did not induce hyperalgesia in the AS-ODN–treated group (Fig. 2A). When DAMGO was injected again 5 (Fig. 2B), 15 (Fig. 2C), and 30 (Fig. 2D) days after the last injection of MOR ODN, the mechanical hyperalgesia evaluated 30 minutes after its injection was not present in AS-ODN–treated rats. Another group of rats was pretreated with either MM- or AS-ODN for 5 consecutive days and on the fourth day, received repeated (hourly × 4) intradermal injections of DAMGO. On the sixth day, ∼17 hours after the injection of ODN, an intradermal injection of PGE2 was performed. In the AS-ODN–treated group, prolongation of PGE2 hyperalgesia was not present (Fig. 2E). The prolongation of PGE2 hyperalgesia was also not present, 5 (Fig. 2F), 15 (Fig. 2G), and 30 (Fig. 2H) days after the last injection of MOR AS-ODN. Taken together, these findings indicate that DAMGO acts at MOR to induce both components of type II priming (OIH and prolongation of PGE2 hyperalgesia).

Figure 2

Figure 2

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3.2. Lesion of IB4-negative nociceptors prevents priming

We previously demonstrated that IB4-saporin, which destroys nonpeptidergic IB4-positive nociceptors and mediates type I priming,7,30 did not attenuate DAMGO-induced type II priming (OIH or prolongation of PGE2 hyperalgesia).5 In rats pretreated with SSP-saporin, which destroys IB4-negative peptidergic nociceptors, repeated (hourly × 4) injections of DAMGO did not induce hyperalgesia, evaluated after its fourth injection, or produce prolongation of PGE2 hyperalgesia, when tested 5 days later (Fig. 3). These data support the suggestion that type II hyperalgesic priming occurs in peptidergic, IB4-negative nociceptors.

Figure 3

Figure 3

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3.3. Epidermal growth factor receptor in DAMGO-induced prolongation of PGE2 hyperalgesia

We next determined if inhibition of EGFR is able to prevent DAMGO-induced prolongation of PGE2 hyperalgesia. Preadministration of the EGFR inhibitor (tyrphostin AG 1478) prevented the prolongation of PGE2 hyperalgesia at the fourth hour when tested 5 (Fig. 4A), 15 (Fig. 4B), or 30 (Fig. 4C) days after repeated injections of DAMGO. These findings indicate that induction of DAMGO-induced prolongation of PGE2 hyperalgesia is EGFR dependent. An EGFR agonist (EGF ligand) alone was, however, unable to induce either hyperalgesia or prolongation of PGE2 hyperalgesia (Fig. 4D).

Figure 4

Figure 4

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3.4. Src, focal adhesion kinase, and epidermal growth factor receptor signaling in the induction of prolongation of PGE2 hyperalgesia

We next examined signaling pathways downstream of EGFR that mediates the EGFR-dependent induction of prolonged PGE2 hyperalgesia by repeated exposure to DAMGO. SH2, a peptide that blocks the interaction of Src, FAK, and EGFR, was able to prevent the development of the prolongation of PGE2-induced hyperalgesia when tested 5 (Fig. 5A), 15 (Fig. 5B), and 30 (Fig. 5C) days after repeated exposure to DAMGO. These findings indicate that signaling through Src, FAK, and EGFR is required for the induction of the prolongation of PGE2 hyperalgesia by an MOR agonist.

Figure 5

Figure 5

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3.5. Induction of opioid-induced hyperalgesia

Since our previous5,8 and current findings demonstrate the participation of Src, MAPK, FAK, and EGFR in the induction of prolongation of PGE2-induced hyperalgesia by repeated exposure to DAMGO, we tested inhibitors for these second messengers in OIH. Mitogen-activated protein kinase, Src and EGFR inhibitors, and SH2 were able to block the hyperalgesia induced by the fourth injection of DAMGO (Fig. 6A). Five (Fig. 6A) and 15 (Fig. 6B) days after the treatment with these inhibitors, we observed that in the Src inhibitor– and SH2-treated groups DAMGO was unable to induce hyperalgesia, whereas in MAPK- and EGFR inhibitors–treated groups, DAMGO-induced hyperalgesia was only partially developed. When DAMGO was again injected 30 days (Fig. 6C) after treatment with these inhibitors, a partial attenuation of DAMGO-induced hyperalgesia was still observed in the MAPK and Src inhibitors and SH2-treated groups (Fig. 6C), whereas no significant attenuation was observed between vehicle- and EGFR inhibitor–treated groups. These findings suggest a partial contribution of Src, FAK, and MAPK to the induction of OIH.

Figure 6

Figure 6

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3.6. Matrix metalloprotease in the expression of DAMGO-induced prolongation of PGE2 hyperalgesia

Since it has previously been shown that MOR signals to EGFR by activating MMP, which cleaves EGF from a membrane surface precursor molecule,9,42 we next tested if MMPs play a role in MOR-EGFR crosstalk associated with the induction of prolongation of PGE2-induced hyperalgesia, induced by repeated exposure to DAMGO. Ilomastat, which inhibits a wide variety of MMPs, and an MMP-9 selective inhibitor, were both able to block the prolongation of PGE2-induced hyperalgesia when PGE2 was administered 5 days after the repeated exposure to DAMGO (Fig. 7A). However, when PGE2 was again injected, 15 days after the repeated exposure to DAMGO, the prolongation of PGE2-induced hyperalgesia at the fourth hour was present (Fig. 7B). These findings indicate that MMPs play a role in the expression, but not the induction, of DAMGO-induced prolongation of PGE2 hyperalgesia.

Figure 7

Figure 7

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3.7. Mu-opioid receptor–epidermal growth factor receptor crosstalk

We also evaluated the role of 2 other signaling molecules downstream of MOR, RAS, and cRaf160 in the induction of the prolongation of PGE2 hyperalgesia. Inhibitors of RAS (salirasib) and cRaf1 (GW5074) did not prevent the prolongation of PGE2 hyperalgesia induced by repeated exposure to DAMGO (Fig. 8).

Figure 8

Figure 8

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4. Discussion

DAMGO-induced type II hyperalgesic priming consists of 2 phenomena: OIH and prolongation of PGE2-induced hyperalgesia. Induction of both phenomena is dependent on the action of DAMGO at the MOR, because AS-ODN against MOR, prevented the induction of both components of type II priming. These findings agree with a recent report, which found that activation of MOR expressed in primary afferent nociceptors initiates OIH.19 We also found that intrathecal administration of SSP-saporin, which destroys peptidergic nociceptors (which contain MOR50), prevented DAMGO-induced OIH and the prolongation of PGE2-induced hyperalgesia. Thus, DAMGO induces type II priming by action at the MOR in peptidergic IB4-negative nociceptors.

Chronic use of opioids modifies multiple MOR signaling mechanisms, including receptor phosphorylation, change in downstream signaling pathways, and receptor multimerization and trafficking, which may contribute to OIH.48 Previous studies have shown crosstalk between RTK and opioid receptors, which mediate changes in opioid receptor signaling.10,16,42 We found that an EGFR inhibitor (tyrphostin AG 1478) was able to prevent the prolongation of PGE2 hyperalgesia, when tested out to 30 days after repeated exposure to DAMGO, indicating that the induction of the prolongation of PGE2-induced hyperalgesia is EGFR dependent. The EGFR inhibitor was, however, not able to prevent the induction of OIH. On the other hand, although EGFR was necessary for the induction of prolongation of PGE2 hyperalgesia, an EGFR agonist (EGF ligand) alone was not sufficient to induce hyperalgesia and/or hyperalgesic priming. Our result is similar to those reported in a recent study,38 in which the late phase of the formalin test was not enhanced by intrathecal treatment with an EGF ligand, indicating that EGF ligand is not able to induce hypersensitivity.

It has been demonstrated that stimulation of several GPCRs, including opioid receptors, can transactivate EGFR signaling.10,16,29,42 Receptor tyrosine kinase transactivation by GPCRs occurs through activation of membrane-bound MMPs, which play a role in the processing of EGF-like precursor molecules expressed in the plasma membrane.9,14,38,45 Src may also activate metalloproteinases,43 which in turn cleaves EGF from a precursor molecule in the cell membrane. However, we found that MMPs are only involved in the expression, not the induction, of prolongation of PGE2 hyperalgesia. Therefore, we sought out a role of ligand-independent intracellular signaling pathways (ie, Src family proteins).15,21,28,36,44 Importantly, a phosphopeptide ligand for the Src SH2 domain, which blocks the interaction of Src with FAK and EGFR, was able to prevent the induction of prolonged PGE2 hyperalgesia by repeated exposure to DAMGO. Thus, chronic exposure to opioids is able to increase Src activity, which phosphorylates EGFR in its cytosolic domain.57 Still, how MOR, EGFR, Src, and FAK interact in the induction of prolongation of PGE2-induced hyperalgesia by DAMGO, remains to be established.

Because Src, MAPK, and FAK participate in the prolongation of PGE2 hyperalgesia produced by repeated exposure to DAMGO,8 we tested the effect of inhibitors for these second messengers on DAMGO-induced OIH. Thirty days after DAMGO (hourly × 4) was injected, all inhibitors, injected before DAMGO (induction protocol), only partially attenuated DAMGO-induced hyperalgesia, indicating that the mechanism involved in the induction of OIH differs substantially from that involved in the prolongation of PGE2 hyperalgesia. Thus, although both are produced by action of DAMGO at the MOR, the mechanisms involved in the induction of OIH and prolongation of PGE2 hyperalgesia are, at least in part, different. Changes in MOR signaling produced by chronic use of opioids cannot be entirely explained by the typical GPCR signaling pathway and it is possibly a result from shifts in intracellular signaling. In this direction, a previous study suggested a crucial role of Src mediating MOR phosphorylation, producing long-term changes in the signals downstream the receptor.60 Similarly, a switch in intracellular signaling produced by phosphorylated GPCR activation involving the participation of β-arrestins has also been observed. In a 2005 study, Lefkowitz and Shenoy35 suggested that the binding of β-arrestins to phosphorylated GPCR receptors was not just a mechanism that resulted in signaling termination, but could also produce changes in the receptor, allowing it to recruit messengers that were not previously activated directly by the GPCR, such as Src, and to interact with downstream effectors such as MAP kinases.37 This could explain, for example, events such as biased agonism35 or, in our case, Type II hyperalgesic priming.22 In fact, a previous study has suggested a novel, noncanonical signaling pathway for MOR observed after chronic exposure to opioid agonists, in which phosphorylation of MOR led to the recruitment and activation of cRaf1 and RAS in a Src kinase-dependent manner.60 Interaction between GPCRs and MAPK cascades has been shown to either depend on RAS (possibly involving Src) or to be RAS independent, in which case it would require the GPCR to transactivate RTKs such as EGFR.20 However, although Src, MAPK, and EGFR do play a role in our model, our results are not totally compatible with this mechanism, since we found that inhibitors for RAS and cRaf1 did not affect the induction of prolongation of PGE2 hyperalgesia.

The prolongation of PGE2 hyperalgesia in type I priming is mediated by an autocrine mechanism involving the release of cAMP from the peripheral terminal of the primary afferent nociceptor, its metabolism to adenosine by 2 ectonucleotidases (ie, ecto-5′-phosphodiesterase and ecto-5′-nucleotidase),17,26 and the action of the end product of this metabolic pathway, adenosine, at A1-adenosine receptors on the peripheral terminal of the nociceptor, to produce PKCε-dependent prolonged mechanical hyperalgesia.26 Thus, it would seem that the coupling of receptors to second messengers that mediate hyperalgesia differs between type I and type II priming, coupling to PKCε in type I hyperalgesic priming26 and to PKA in type II.5 Type I and type II priming occur in different populations of nociceptors, type I in IB4-positive (nonpeptidergic)7,30 and type II in IB4-negative (peptidergic) nociceptors. In addition, the induction of type I priming can also be prevented by an injection of a protein translation inhibitor (ie, cordycepin) in the peripheral terminal of the nociceptor,7,23 which failed to prevent the induction of DAMGO-induced type II priming.5

In conclusion, our results demonstrate a crucial role of MOR, in IB4-negative peptidergic nociceptors, in the induction of type II priming (OIH and prolongation of PGE2 hyperalgesia), which is partially dependent of Src, FAK, and MAPK signaling to develop OIH and completely dependent on Src, FAK, EGFR, and MAPK to induce prolongation of PGE2 hyperalgesia. The current findings are summarized in Figure 9. Understanding the mechanisms responsible for the induction of type II hyperalgesic priming, a form of neuroplasticity in the peripheral terminal of the primary afferent nociceptor, may provide useful information for the design of drugs with improved therapeutic profiles to treat neuroplasticity induced by chronic use of opioids.

Figure 9

Figure 9

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Conflict of interest statement

The authors declare no competing financial interests.

This study was funded by a grant from the National Institutes of Health (NIH), NS084545.

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Acknowledgments

The authors thank Marie Kern for technical assistance.

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Keywords:

Hyperalgesic priming; Hyperalgesia; EGFR; MOR; Chronic pain

© 2018 International Association for the Study of Pain