Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2018 Apr 6;175(10):1760–1769. doi: 10.1111/bph.14191

Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia

Wenying Wang 1,†, Xiaqing Ma 1,3,†, Limin Luo 1, Min Huang 1, Jing Dong 1, Xiaoli Zhang 1, Wei Jiang 1,✉, Tao Xu 1,2,✉
PMCID: PMC5913404  PMID: 29500928

Abstract

Background and Purpose

The P2X3 receptor is a major receptor in the processing of nociceptive information in dorsal root ganglia. We investigated the role of the P2X3 receptor and the detailed mechanisms underlying chronic morphine‐induced analgesic tolerance in rats.

Experimental Approach

Repeated i.t. morphine treatment was used to induce anti‐nociceptive tolerance. The expression of spinal P2X3 receptor, phosphorylated PKCε and exchange factor directly activated by cAMP (Epac) were evaluated. Effects of A‐317491 (P2X3 antagonist), ε‐V1‐2 (PKCε inhibitor) and ESI‐09 (Epac inhibitor) on mechanical pain thresholds and tail‐flick latency after chronic morphine treatment were determined. Co‐localization of P2X3 receptor with NeuNs (marker of neuron), IB4 (marker of small DRG neurons), peripherin, PKCε and Epac were performed by double immunofluorescence staining.

Key Results

Chronic morphine time‐dependently increased the expression of P2X3 receptor, phosphorylated PKCε and Epac in DRGs. ε‐V1‐2 prevented chronic morphine‐induced expression of P2X3 receptor. ESI‐09 decreased the phosphorylation of PKCε and up‐regulated expression of Epac after chronic morphine exposure. Mechanical pain thresholds and tail‐flick latency showed that A317491, ε‐V1‐2 and ESI‐09 significantly attenuated the loss of morphine's analgesic potency. Morphine‐induced P2X3 receptor expression mainly occurred in neurons staining for IB4 and peripherin. Co‐localization of P2X3 receptor with PKCε and Epac was demonstrated in the same neurons.

Conclusions and Implications

Chronic morphine exposure increased the expression of P2X3 receptor, and i.t. P2X3 receptor antagonists attenuated the loss of morphine's analgesic effect. Inhibiting Epac/PKCε signalling was shown to play a significant inhibitory role in chronic morphine‐induced P2X3 receptor expression and attenuate morphine‐induced tolerance.

Abbreviations

DRGs

dorsal root ganglia

Epac

exchange factor directly activated by cAMP

IB4

isolectin B4

NF200

neurofilament 200

P2X3 receptor

purine P2X3 receptor

Introduction

http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1627 is a good choice for managing moderate‐to‐severe pain, providing effective pain relief. However, its side effects, especially the development of tolerance, cannot be underestimated. Although many mechanisms have been implicated in the development of the analgesic tolerance elicited by prolonged morphine administration (Bekhit, 2010), the neurotransmitters/receptors involved are still unclear.

The purinergic http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=480, one of the seven members of the ionotropic P2X receptor family, is highly expressed in small‐diameter sensory neurons of the dorsal root ganglia (DRGs) and have been reported to be involved in both acute (Souslova et al., 2000) and chronic pain (Jarvis et al., 2002). Antagonists of P2X3 receptor have been reported to attenuate inflammatory and neuropathic pain when used systemically (Jarvis et al., 2002) and locally (McGaraughty et al., 2003). Since the processes by which both morphine tolerance and chronic pain may share common pathways (Mayer et al., 1999), in a previous study, we investigated the involvement of P2X3 receptor in the development of morphine anti‐nociceptive tolerance and showed that http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4115, a P2X3 antagonist, not only partially prevents the development of morphine anti‐nociceptive tolerance but also potently reverses the tolerance established by bolus administration in mice (Ma et al., 2015). Here, we explored the detailed mechanisms involving P2X3 receptor in morphine‐induced anti‐nociceptive tolerance.

Methods

Animals

Adult male Sprague–Dawley rats (Fudan University Medical Animal Center, Shanghai, China) weighing 200–250 g were housed in groups of five in cages (64 × 40 × 22 cm) before cannulation and housed individually after cannulation with a chew stick and nesting material for environmental enrichment. All the animals were housed on corn cob in a room maintained at a constant temperature of 22–23°C with an alternating 12 h light/dark cycle (lights on at 07:00 h) with water and food available ad libitum. In all behavioural experiments, six rats were included in each group. The protocol followed the NIH Guide for the Care and Use of Laboratory Animals (1996) and was approved by the Animal Care and Use Committee of the Sixth People's Hospital Affiliated to Shanghai Jiao Tong University [SYXK (Shanghai, China) 2011‐0128, 1 January 2011]. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). All efforts were made to minimize suffering and reduce the number of animals used. The overall experimental design is illustrated in Figure 2A.

Figure 2.

Figure 2

Open in a new tab

Daily i.t. injection of the selective P2X3 receptor antagonist A317491 attenuates morphine‐induced anti‐nociceptive tolerance. (A) Schematic of the experimental timeline. (B) Thermal and (C) mechanical thresholds measured daily in all groups (n = 6; *P < 0.05 vs. saline group; # P < 0.05 vs. morphine group).

Intrathecal cannulation

For repeated delivery of drugs, i.t. cannulation was performed using the method described by Storkson et al. (1996). Briefly, animals were anaesthetized with isoflurane inhalation in 100% oxygen (induced at 5% and maintained at 2%), then an 8 cm PE‐10 catheter (Becton Dickinson, Sparks, MD, USA) was inserted into the subarachnoid space at the L4–L5 level. During surgery, the concentration of isoflurane was increased when necessary. Involuntary movements of the tail or hindlimb were regarded as signs of dura penetration. The catheter was then advanced 1 cm into the i.t. space to reach the level of the lumbar enlargement. The external end of the catheter was sealed by heat. The correct location of the catheter was verified by i.t. injection of lidocaine (2%, 10 μL) on the next day, which caused reversible bilateral hindlimb paresis for 10–15 min. The rats were allowed to recover for 3 days before drug administration; penicillin was administered to prevent infection.

Drugs and groups

Rats were briefly anaesthetized with isoflurane to isolate the i.t. catheter; following recovery, they were placed in a transparent Plexiglas box, and drugs were slowly (1 μL·min−1) injected through the exteriorized portion of the catheter with a micro‐syringe (Hamilton, Reno, NV, USA) in a 10 μL volume followed by a flush with 10 μL of 0.9% saline (Baxter Healthcare, New York, NY, USA). Successfully catheterized rats were treated randomly with i.t. morphine (15 μg in 10 μL saline; Shenyang First Pharmaceutical Factory, Shenyang, China) for 3, 5 and 7 days; morphine plus A317491 (15 μg in 10 μL saline; Sigma, St. Louis, MO, USA) or A‐317491 alone for 7 days; morphine plus the http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1486 inhibitor ε‐V1‐2 (10 μg in 10 μL saline; AnaSpec Inc., Fremont, CA, USA) or ε‐V1‐2 alone for 7 days; morphine plus the exchange factor directly activated by cAMP (http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=259) inhibitor http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7917 (5 μg in 10 μL 20% DMSO; Sigma) or ESI‐09 alone for 7 days; or saline control for 7 days. The inhibitors were given 30 min before morphine treatment.

Behavioural tests

Paw‐withdrawal test

Rats were habituated to the experimental conditions for 4 days before experiments. Paw pressure thresholds were assessed using a paw pressure analgesia meter (Ugo Basile, Varese, Italy) before and 30 min after i.t. injection. The cut‐off threshold was set at 250 g. The paw pressure thresholds were determined by averaging three consecutive trials separated by 5 min.

Tail‐flick latency

The pain response to thermal stimuli was assessed using a Tail‐Flick Analgesia Meter (Columbus Instruments, Columbus, OH, USA). In brief, radiant heat was applied to the animal's tail ~4 cm from the tip. The intensity of the thermal stimulus was adjusted to provide an average baseline tail‐flick latency of 3–4 s. A cut‐off time of 10 s was set to avoid injury. Only rats with baseline reaction times between 3 and 4 s were used in experiments. The control reaction time and the latency of the response were recorded twice, with an interval of 15 min between readings.

Western blotting

Under deep anaesthesia with pentobarbital (50 mg·kg−1, i.p.) and hypoxia with carbon dioxide, the L4–L5 DRGs were removed and immediately homogenized in ice‐chilled tissue protein extraction reagent. Samples were prepared as reported previously (Xu et al., 2015). Membranes were incubated with the following primary antibodies: rabbit anti‐rat P2X3 receptor (1:1000; Alomone Labs, Jerusalem, Israel), mouse anti‐GAPDH (1:1000; CST, Danvers, MA, USA), rabbit anti‐rat pPKCε (1:1000; Abcam, Cambridge, MA, USA), mouse anti‐rat PKCε (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti‐rat Epac (1:200; Santa Cruz Biotechnology). The blots were washed in Tris‐buffered saline/Tween 20 and then incubated with HRP‐conjugated goat anti‐rabbit or anti‐mouse IgG secondary antibody (1:5000; HuaBio Inc., Cambridge, MA, USA). Protein bands were detected by ImageQuant Ai600 (General Electric Co., Boston, MA, USA) with an enhanced ECL substrate (Thermo Fisher, Scientific, Waltham, MA, USA). The results were analysed and quantified using ImageJ software (version 2.0.0, National Institutes of Health, Rockville, MA, USA). Each subject gave an independent value to be analysed.

Immunohistochemistry

For fluorescence immunohistochemistry, rats were anaesthetized with pentobarbital (50 mg·kg−1, i.p.) and made hypoxic with carbon dioxide and transcardially perfused with 4% cold paraformaldehyde. DRGs were harvested, post‐fixed for 4 h at 4°C in 4% paraformaldehyde and then dehydrated sequentially in 10, 20 and 30% sucrose overnight for 3 days. Frozen sections (10 μm) were cut on a cryostat and air dried on microscope slides for 30 min at room temperature before use. In all groups, double labelling was used to assess the co‐localization of P2X3 receptor with ionized Ca2+‐binding adapter molecule 1, isolectin B4 (IB4), neurofilament 200 (NF200), peripherin, PKCε or Epac in the DRGs. Frozen sections were prepared as previously reported (Xu et al., 2015), and the slides were incubated with the primary antibody (rabbit anti‐P2X3 receptor, 1:500; mouse anti‐PKCε, 1:50, Santa Cruz; mouse anti‐rat Epac, 1:50, Santa Cruz; mouse anti‐peripherin, 1:100, Millipore, Billerica, MA, USA; mouse anti‐NF200, 1:100, Sigma; or FITC‐conjugated IB4, 2 μg·mL−1, Sigma) overnight at 4°C. Subsequently, the slides were rinsed in PBS and incubated with the secondary antibody for 2 h at room temperature. The secondary antibodies goat anti‐mouse IgG H&L (Alexa Fluor® 594; 1:500) and goat anti‐rabbit IgG H&L (Alexa Fluor 488; 1:500) were from Abcam. Images were acquired using an inverted microscope (Leica DM IL LED, Leica Microsystems, Buffalo Grove, IL, USA).

Statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). All data are presented as the mean ± SEM and were analysed with Graph‐Pad Prism 5 (Graph Pad Software, San Diego, CA, USA). Behavioural data were converted to a percentage maximum possible effect (MPE) as follows: %MPE = 100 × (post‐drug latency/threshold − pre‐drug latency/threshold)∕(cut‐off latency/threshold − pre‐drug latency/threshold). Paw‐withdrawal thresholds and tail‐flick latencies were analysed using two‐way (time and treatment) ANOVA followed by one‐way ANOVA (at each time point) and Tukey's test for multiple comparisons. To control for unwanted sources of variation, the relative expression of target proteins in different groups was normalized to GAPDH, and the phosphorylation level of target proteins was compared with their total level. We set the average value of the saline group to 1 and standardized each set of data. The value of each group including the saline control group was normalized to the mean value of the saline group (Park et al., 2013; Chu et al., 2014). The Kolmogorov–Smirnov test showed the data followed Gaussian distribution. If data follow Gaussian distribution, one‐way ANOVA (protein levels) or Student's t‐test (number of positive cells) was carried out. Post hoc tests were run only when F achieved P < 0.05. If data were not normally distributed, non‐parametric tests were used. Differences were considered significant at P < 0.05. Behavioural tests, cell counting, protein quantitation and all data analyses were performed by observers who were blinded to the experimental groups.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a,b).

Results

Chronic morphine treatment induces P2X3 receptor expression

Western blot analyses were used to determine the effect of repeated morphine treatment on P2X3 receptor expression in the DRG (Figure 1A). Analysis of the time course revealed that such treatment for 5 or 7 days increased the P2X3 receptor expression by 1.91 ± 0.33‐ and 2.16 ± 0.24‐fold compared with the saline group (Figure 1A, P < 0.05). The number of P2X3 receptor‐positive cells in DRGs after morphine treatment increased by 2.15 ± 0.59‐fold compared with control (Figure 1B, P < 0.05).

Figure 1.

Figure 1

Open in a new tab

(A) Expression of P2X3 receptor and (B) number of P2X3 receptor‐positive cells per section per animal in DRGs increase after repeated morphine treatment. The DRGs were removed 2 h after morphine injection on day 7 (n = 6,*P < 0.05 vs. saline (S) group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.

A‐317491 diminishes the degree of morphine‐induced anti‐nociceptive tolerance

The overall experimental design was shown in Figure 2A. Repeated i.t. administration of morphine‐induced acute increases in both tail‐flick latency to thermal stimulation (Figure 2B) and paw‐withdrawal threshold to mechanical stimulation (Figure 2C) on days 1 to 3 (P < 0.05 vs. saline group). In thermal nociceptive testing (Figure 2B), morphine‐induced anti‐nociceptive efficacy was attenuated through days 4 to 7 in the morphine‐alone group (%MPE 17.1 ± 3.1 on day 7, P > 0.05 vs. saline group), while in the A317491 plus morphine group, the analgesic potency of morphine was still powerful on day 7 (%MPE 69.8 ± 6.8, P < 0.05 vs. saline group). Daily A317491 injection partially prevented the morphine‐induced anti‐nociceptive tolerance throughout the 7 days.

In mechanical threshold testing (Figure 2C), the efficacy of morphine declined to the baseline level (%MPE 11.8 ± 2.4) in the morphine‐alone group on day 7 (P > 0.05). On days 4 to 7, the morphine‐induced analgesia in the morphine‐alone group showed a markedly decreasing mechanical threshold compared with the saline group. However, the degree of tolerance was diminished by daily injection of A317491 and morphine through day 7 (%MPE 82.2 ± 5.4, P < 0.05 vs. saline and morphine groups).

Assessment of morphine potency 24 h after the chronic tolerance protocol (only morphine was given in the dose–response test on day 8) revealed a rightward shift in the dose–response curve in rats that had been repeatedly given morphine compared with those given saline. A317491 prevented the rightward shift in tail‐flick assays (Supporting Information Figure S1A). Calculation of the morphine ED50 from the dose–response curves revealed that rats receiving morphine alone had higher values [17.84 to 23.51, 95% confidence interval (CI)] in the tail‐flick test than animals that had received saline (0.87 to 1.16, 95% CI), morphine with A‐317491 (2.25 to 2.69, 95% CI) or A‐317491 alone (0.90 to 1.34, 95% CI) (P < 0.05 for all), suggesting that A‐317491 prevents the decline in morphine potency (Supporting Information Figure S1B). There was no difference in the morphine ED50 values for morphine plus A‐317491, A‐317491 alone and saline (Supporting Information Figure S1B).

Co‐localization of P2X3 receptor with IB4 and peripherin

P2X3 receptor were strongly co‐localized with IB4, a marker of non‐peptidergic C‐fibre neurons (Figure 3B). Higher magnification images of DRG sections from morphine‐tolerant rats showed that P2X3 receptor were localized in the entire cytoplasm (Figure 3D). We also confirmed that P2X3 receptor did not co‐localize with microglia (Figure 3A) or astrocytes (data not shown). The majority of P2X3 receptor were co‐localized with peripherin, a marker of neurons with unmyelinated or small‐calibre fibres (Figure 3D), and a minority was co‐localized with NF200, a marker of neurons with myelinated fibres (Figure 3C).

Figure 3.

Figure 3

Open in a new tab

Morphine‐induced P2X3 receptor up‐regulation is co‐localized with IB4 and peripherin in the DRG. (A) Confocal images of P2X3 receptor (green) and ionized Ca2+‐binding adapter molecule 1 (Iba1) (red) immunofluorescence with no co‐localization. (B) Immunofluorescence images of P2X3 receptor (red) and IB4 (green) co‐localization in DRGs. (C) There was less co‐localization of P2X3 receptor (green) with NF200 (red) in myelinated neurons. (D) Co‐localization of P2X3 receptor (green) with peripherin (red) in unmyelinated/small‐calibre neurons. DRG samples from the 7 day morphine group (scale bars: A/B, 80 μm; C/D, 20 μm).

Pharmacological inhibition of phosphorylation of PKCε prevents morphine anti‐nociceptive tolerance and suppresses chronic morphine‐induced P2X3 receptor expression

To identify the contributors to the increased P2X3 receptor expression in the DRGs after chronic morphine treatment, we assessed the phosphorylation of the possible upstream regulator, PKCε, and determined whether its inhibition reduces the chronic morphine‐induced P2X3 receptor expression and relieves morphine tolerance. We found that the phosphorylation of PKCε had the same pattern as the P2X3 receptor expression after chronic morphine exposure: morphine treatment for 3, 5 or 7 days increased PKCε phosphorylation by 1.94 ± 0.42‐fold, 2.99 ± 0.56‐fold and 3.19 ± 0.39‐fold compared with the saline group (Figure 4A, P < 0.05). Although morphine markedly increased the threshold on day 1, the anti‐nociception effect was reduced within 3 days of treatment, and by day 7, morphine had no effect (Figures 4D and 5D), demonstrating the development of antinociceptive tolerance. Co‐administration of the PKCε‐selective inhibitor ε‐V1‐2 with morphine for 7 consecutive days greatly suppressed the morphine‐induced expression of P2X3 receptor (Figure 4B, P < 0.05 vs. morphine group), while the anti‐nociceptive effect of morphine remained throughout the treatment (Figure 4D, P < 0.05). ε‐V1‐2 alone had no effect on the mechanical threshold and P2X3 receptor expression. Double‐labelling immunofluorescence showed clear co‐localization of P2X3 receptor and PKCε in the same DRG neurons (Figure 4C). Bolus ε‐V1‐2 injection (10 μg, i.t.) tended to potentiate the morphine‐induced anti‐nociception in morphine‐tolerant rats on day 8, although the difference did not reach statistical significance (Supporting Information Figure S2, P > 0.05 vs. day 7 morphine‐induced anti‐nociception).

Figure 4.

Figure 4

Open in a new tab

Involvement of PKCε in morphine (Mor)‐induced antinociceptive tolerance and P2X3 receptor expression. (A) The phosphorylation of PKCε in the DRG increased time dependently with chronic Mor treatment. (B) The inhibitor of PKCε, ε‐V1‐2, reversed the increased P2X3 receptor expression induced by repeated Mor treatment. (C) PKCε co‐localized with P2X3 receptor in DRGs (scale bar: 50 μm for the up; 20 μm for the down). (D) Mor tolerance was attenuated when Mor was administered with ε‐V1‐2 for 7 days (n = 6,*P < 0.05 vs. saline (S) group; # P < 0.05 vs. morphine group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.

Figure 5.

Figure 5

Open in a new tab

Epac is responsible for the chronic morphine (Mor)‐induced phosphorylation of PKCε and increase in P2X3 receptor expression. (A) The expression of Epac in the DRG increased time dependently after chronic Mor exposure. (B) The inhibitor of Epac, ESI‐09, reversed the increased P2X3 receptor expression and phosphorylation of PKCε induced by repeated morphine treatment. (C) Epac co‐localized with P2X3 receptor in DRGs (scale bar: 50 μm for the left; 20 μm for the right). (D) Morphine tolerance was attenuated by co‐administration of morphine and ESI‐09 for 7 days (n = 6,*P < 0.05 vs. saline (S) group; # P < 0.05 vs. morphine group). M3, 3 days of morphine injection; M5, 5 days of morphine injection; M7, 7 days of morphine injection.

Inhibition of Epac prevents morphine anti‐nociceptive tolerance and suppresses both P2X3 receptor expression and PKCε phosphorylation after chronic morphine treatment

To further explore the mechanism of increased P2X3 receptor expression after prolonged morphine exposure, we evaluated the effect of ESI‐09 (an Epac inhibitor) on the expression of P2X3 receptor and PKCε phosphorylation. Chronic morphine treatment time dependently increased the expression of Epac (Figure 5A, P < 0.05 vs. saline group), indicating its involvement in morphine‐induced tolerance. Co‐treatment with the Epac inhibitor ESI‐09 and morphine reversed the increase in P2X3 receptor and PKCε phosphorylation after chronic morphine injection (Figure 5B, P < 0.05 vs. morphine group). Furthermore, ESI‐09 markedly attenuated the development of morphine‐induced anti‐nociceptive tolerance, while the anti‐nociceptive effect of morphine was retained (Figure 5D, P < 0.05). The immunofluorescence results showed typical co‐localization of P2X3 receptor with Epac (Figure 5C).

Discussion

Consistent with our previous work in mice (Ma et al., 2015), we found that administration of the selective P2X3 receptor antagonist A317491 attenuated the morphine‐induced anti‐nociceptive tolerance in rats. Co‐localization of P2X3 receptor with IB4 and peripherin showed that the P2X3 receptor is mainly expressed in non‐peptidergic C‐fibre neurons and neurons with unmyelinated or small‐calibre fibres. Co‐localization of P2X3 receptor with Epac and PKCε supports the idea that chronic morphine increases P2X3 receptor expression through the Epac‐PKCε pathway.

The P2X3 receptor is mainly expressed in nociceptive sensory neurons in the DRG and plays vital roles in sensory processing and transmission (Wirkner et al., 2007). ATP is released from different cell types around primary sensory neurons, especially under conditions of pathological pain (Burnstock, 2000). Evidence has emerged that the P2X3 receptor participates in models of chronic pain. Inhibition of P2X3 receptor expression has been reported to alleviate neuropathic pain (Xu et al., 2012) and reverse mechanical hyperalgesia (Brown and Yule, 2007). Prado et al. (2013) suggested that neuronal P2X3 receptor activation and the consequent PKCε translocation increase the susceptibility of nociceptors to inflammatory mediators, allowing the development of inflammatory hyperalgesia. Our previous behavioural experiments in mice showed that an antagonist of P2X3 receptor delays the development of morphine‐induced analgesic tolerance (Ma et al., 2010). But the details of the involvement of P2X3 receptor in such tolerance remained unclear. Here, we demonstrated that expression of P2X3 receptor increases during the development of morphine‐induced anti‐nociceptive tolerance in rats.

Among the three types of P2X current induced by ATP in adult rat DRG neurons, the fast currents mainly occur in small IB4‐positive DRG neurons (Grubb and Evans, 1999), and the intracellular signalling pathway for the activation of PKCε (Epac/PLC/PKCε) is also closely correlated with the subtype of small DRG neurons expressing IB4 (Hucho et al., 2005). Our results here showing the co‐localization of P2X3 receptor with IB4 in DRG neurons. And the co‐localization of P2X3 receptor with peripherin showed that the P2X3 receptor is mainly expressed in neurons with unmyelinated or small‐calibre fibres. Epac‐PKCε signalling may be responsible for chronic morphine‐induced P2X3 receptor expression. Epac has been reported to play a key role in the activation of PKCε in DRG neurons (Hucho et al., 2005; Wang et al., 2007). Epac‐PKCα signalling in P2X3 receptor‐mediated hyperalgesia after inflammation was proposed by Gu et al. (2016a), and it has been reported that an Epac‐PKC‐dependent increase in F‐actin in DRG neurons enhances the membrane expression of P2X3 receptor to bring about their sensitization after inflammation (Gu et al., 2016b).

The participation of PKCε in receptor desensitization and morphine‐induced tolerance has been extensively reported (Chu et al., 2010). PKCε activation has been reported to modulate the heterologous regulation of μ‐opioid receptor phosphorylation (Illing et al., 2014) and play an important role in the development of morphine‐induced tolerance (Qiu et al., 2014). Consistent with these studies, our results showed a robust increase in PKCε phosphorylation after long‐term morphine exposure. Co‐administration of ε‐V1‐2, a PKCε‐specific inhibitor, along with morphine clearly delayed the development of morphine‐induced anti‐nociceptive tolerance. Meanwhile, this inhibitor effectively inhibited the chronic morphine‐induced increase of P2X3 receptor expression in the rat DRG. Co‐localization of P2X3 receptor with PKCε provides strong evidence that the morphine‐induced increased expression of P2X3 receptor can be suppressed by reducing the PKCε phosphorylation in the same neuron. Phosphorylation of PKCε in DRGs could be regarded as an upstream target of P2X3 receptor expression during the development of chronic morphine‐induced analgesic tolerance.

Epac is known to be up‐regulated in inflammation and to play a critical role in P2X3 sensitization by activation of PKCε‐dependent signalling via PGE2 (Wang et al., 2007). Gu et al. (2016b) showed that F‐actin links Epac‐PKCε signalling to P2X3 receptor sensitization in the DRG in inflammation. Another work by this group also showed that Epac plays an essential role in the P2X3 receptor‐mediated hyperalgesia in inflammation (Gu et al., 2016a). However, there is no previous work exploring the role of Epac in the development of morphine‐induced anti‐nociceptive tolerance. Here, we showed that chronic morphine exposure increased the expression of Epac in DRGs. Pretreatment with ESI‐09, a specific Epac inhibitor, not only decreased chronic morphine‐induced P2X3 receptor expression but also reduced repeated morphine‐induced PKCε phosphorylation. In behavioural tests, rats pretreated with the Epac inhibitor showed resistance to morphine anti‐nociceptive tolerance after repeated treatment. The co‐localization of P2X3 receptor with Epac provides a basis for this chronic morphine‐induced signalling process to occur in the same neuron during the establishment of anti‐nociceptive tolerance.

Activation of P2X3 receptor within the CASK/P2X3 complex has important consequences for neuronal plasticity (Fabbretti, 2013) and possibly for the release of neuromodulators and neurotransmitters that may accelerate the formation of morphine‐induced analgesic tolerance. P2X3‐positive neurons belong almost exclusively to the GDNF‐sensitive population (Bradbury et al., 1998), and 75% of P2X3‐positive neurons contain TRPV1 (Guo et al., 1999). Involvement of TRPV1 in morphine‐induced effects has been reported (Chen et al., 2007; Vardanyan et al., 2009). Besides co‐localization, the P2X3 receptor and TRPV1 may functionally interact, which suggests that the two pain‐relevant receptors P2X3 and TRPV1 probably interact in an inhibitory manner by a close physical association that is established by means of a structural motif located at the C‐terminal end of the P2X3 receptor distal to Glu362 (Stanchev et al., 2009). This evidence also indicates a possible role of P2X3 receptor in the development of morphine‐induced analgesic tolerance.

As morphine‐induced analgesic tolerance is considered to lead to neuroimmune activation and neuroinflammation (Raghavendra et al., 2002; DeLeo et al., 2004), the up‐regulation of P2X3 receptor during morphine‐induced analgesic tolerance maybe a consequence of the activation of Epac‐PKCε signalling, morphine‐induced sensitization and neuronal inflammation. We did not evaluate neuroinflammation during the chronic morphine exposure and its relationship with Epac‐PKCε signalling and P2X3 receptor sensitization. Such data would be an important supplement to this work. The mediation of P2X3 receptor via Epac‐PKCε signalling could be an effective target to alleviate chronic morphine‐induced anti‐nociceptive tolerance.

Author contributions

W.J. and T.X. were responsible for conceiving and designing the study. W.W. and X.M. participated in establishing the animal model and behavioural test. L.L. and M.H performed the molecular biological experiments. X.Z., J.D. and T.X. performed the data analysis and interpretation. W.J. and T.X. obtained funding and provided administrative support. W.W. and T.X. were responsible for drafting the manuscript. W.J. supervised the study.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This http://onlinelibrary.wiley.com/doi/10.1111/bph.13405/abstract acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Figure S1 Effect of A‐317491 on the cumulative dose–response curve of morphine. Morphine‐induced antinociceptive dose–responses were assessed on day 8. (A) A rightward shift of the dose–response curve in the 7‐day morphine group was revealed on day 8, and co‐administration of A‐317491 with morphine prevented this shift. (B) The ED50 of morphine in the morphine‐alone group was higher than that in other groups. The development of morphine tolerance was attenuated when morphine was administered along with A‐317491 for 7 days (n = 6/group; ***P < 0.001 vs morphine group).

Figure S2 Bolus injection of epsilon‐V1–2 partly restored the analgesic efficacy of morphine on day 8. Mechanical thresholds were measured daily (n = 6).

Click here for additional data file. (217.1KB, pdf)

Acknowledgements

We thank Dr IC Bruce for reading the manuscript. This work was supported by the Young Scholarship Program of the National Natural Science Foundation of China to T.X. (81200855) and the Shanghai Natural Science Foundation to T.X. (17ZR1421100).

Wang, W. , Ma, X. , Luo, L. , Huang, M. , Dong, J. , Zhang, X. , Jiang, W. , and Xu, T. (2018) Exchange factor directly activated by cAMP–PKCε signalling mediates chronic morphine‐induced expression of purine P2X3 receptor in rat dorsal root ganglia. British Journal of Pharmacology, 175: 1760–1769. doi: 10.1111/bph.14191.

Contributor Information

Wei Jiang, Email: jiangw@sjtu.edu.cn.

Tao Xu, Email: balor@sjtu.edu.cn.

References

  1. Alexander SPH, Fabbro D, Kelly E, Marrion NV, Peters JA, Faccenda E et al (2017a). The Concise Guide to PHARMACOLOGY 2017/18: Enzymes. Br J Pharmacol 174: S272–S359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander SPH, Peters JA, Kelly E, Marrion NV, Faccenda E, Harding SD et al (2017b). The Concise Guide to PHARMACOLOGY 2017/18: ligand‐gated ion channels. Br J Pharmacol 174: S130–S159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bekhit MH (2010). Opioid‐induced hyperalgesia and tolerance. Am J Ther 17: 498–510. [DOI] [PubMed] [Google Scholar]
  4. Bradbury EJ, Burnstock G, McMahon SB (1998). The expression of P2X3 purinoreceptors in sensory neurons: effects of axotomy and glial‐derived neurotrophic factor. Mol Cell Neurosci 12: 256–268. [DOI] [PubMed] [Google Scholar]
  5. Brown DA, Yule DI (2007). Protein kinase C regulation of P2X3 receptors is unlikely to involve direct receptor phosphorylation. Biochim Biophys Acta 1773: 166–175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burnstock G (2000). P2X receptors in sensory neurones. Br J Anaesth 84: 476–488. [DOI] [PubMed] [Google Scholar]
  7. Chen SR, Prunean A, Pan HM, Welker KL, Pan HL (2007). Resistance to morphine analgesic tolerance in rats with deleted transient receptor potential vanilloid type 1‐expressing sensory neurons. Neuroscience 145: 676–685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chu J, Zheng H, Zhang Y, Loh HH, Law PY (2010). Agonist‐dependent μ‐opioid receptor signaling can lead to heterologous desensitization. Cell Signal 22: 684–696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chu SC, Chen PN, Hsieh YS, Yu CH, Lin MH, Lin YH et al (2014). Involvement of hypothalamic PI3K‐STAT3 signalling in regulating appetite suppression mediated by amphetamine. Br J Pharmacol 171: 3223–3233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SP, Giembycz MA et al (2015). Experimental design and analysis and their reporting: new guidance for publication in BJP. Br J Pharmacol 172: 3461–3471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeLeo JA, Tanga FY, Tawfik VL (2004). Neuroimmune activation and neuroinflammation in chronic pain and opioid tolerance/hyperalgesia. Neuroscientist 10: 40–52. [DOI] [PubMed] [Google Scholar]
  12. Fabbretti E (2013). ATP P2X3 receptors and neuronal sensitization. Front Cell Neurosci 7: 236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grubb BD, Evans RJ (1999). Characterization of cultured dorsal root ganglion neuron P2X receptors. Eur J Neurosci 11: 149–154. [DOI] [PubMed] [Google Scholar]
  14. Gu Y, Li G, Chen Y, Huang LY (2016a). Epac‐protein kinase C alpha signaling in purinergic P2X3R‐mediated hyperalgesia after inflammation. Pain 157: 1541–1550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gu Y, Wang C, Li G, Huang LY (2016b). EXPRESS: F‐actin links Epac‐PKC signaling to purinergic P2X3 receptors sensitization in dorsal root ganglia following inflammation. Mol Pain 12, 174480691666055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guo A, Vulchanova L, Wang J, Li X, Elde R (1999). Immunocytochemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites. Eur J Neurosci 11: 946–958. [DOI] [PubMed] [Google Scholar]
  17. Harding SD, Sharman JL, Faccenda E, Southan C, Pawson AJ, Ireland S et al (2018). The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY. Nucl Acids Res 46: D1091–D1106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hucho TB, Dina OA, Levine JD (2005). Epac mediates a cAMP‐to‐PKC signaling in inflammatory pain: an isolectin B4(+) neuron‐specific mechanism. J Neurosci 25: 6119–6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Illing S, Mann A, Schulz S (2014). Heterologous regulation of agonist‐independent μ‐opioid receptor phosphorylation by protein kinase C. Br J Pharmacol 171: 1330–1340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jarvis MF, Burgard EC, McGaraughty S, Honore P, Lynch K, Brennan TJ et al (2002). A‐317491, a novel potent and selective non‐nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat. Proc Natl Acad Sci U S A 99: 17179–17184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010). Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160: 1577–1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ma X, Xu T, Xu H, Jiang W (2015). Blockade and reversal of spinal morphine tolerance by P2X3 receptor antagonist. Behav Pharmacol 26: 260–267. [DOI] [PubMed] [Google Scholar]
  23. Mayer DJ, Mao J, Holt J, Price DD (1999). Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions. Proc Natl Acad Sci U S A 96: 7731–7736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McGaraughty S, Wismer CT, Zhu CZ, Mikusa J, Honore P, Chu KL et al (2003). Effects of A‐317491, a novel and selective P2X3/P2X2/3 receptor antagonist, on neuropathic, inflammatory and chemogenic nociception following intrathecal and intraplantar administration. Br J Pharmacol 140: 1381–1388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McGrath JC, Lilley E (2015). Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol 172: 3189–3193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Park JM, Hu JH, Milshteyn A, Zhang PW, Moore CG, Park S et al (2013). A prolyl‐isomerase mediates dopamine‐dependent plasticity and cocaine motor sensitization. Cell 154: 637–650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Prado FC, Araldi D, Vieira AS, Oliveira‐Fusaro MC, Tambeli CH, Parada CA (2013). Neuronal P2X3 receptor activation is essential to the hyperalgesia induced by prostaglandins and sympathomimetic amines released during inflammation. Neuropharmacology 67: 252–258. [DOI] [PubMed] [Google Scholar]
  28. Qiu Y, Zhao W, Wang Y, Xu JR, Huie E, Jiang S et al (2014). FK506‐binding protein 12 modulates μ‐opioid receptor phosphorylation and protein kinase Cε‐dependent signaling by its direct interaction with the receptor. Mol Pharmacol 85: 37–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Raghavendra V, Rutkowski MD, DeLeo JA (2002). The role of spinal neuroimmune activation in morphine tolerance/hyperalgesia in neuropathic and sham‐operated rats. J Neurosci 22: 9980–9989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Souslova V, Cesare P, Ding Y, Akopian AN, Stanfa L, Suzuki R et al (2000). Warm‐coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors. Nature 407: 1015–1017. [DOI] [PubMed] [Google Scholar]
  31. Stanchev D, Blosa M, Milius D, Gerevich Z, Rubini P, Schmalzing G et al (2009). Cross‐inhibition between native and recombinant TRPV1 and P2X3 receptors. Pain 143: 26–36. [DOI] [PubMed] [Google Scholar]
  32. Storkson RV, Kjorsvik A, Tjolsen A, Hole K (1996). Lumbar catheterization of the spinal subarachnoid space in the rat. J Neurosci Methods 65: 167–172. [DOI] [PubMed] [Google Scholar]
  33. Vardanyan A, Wang R, Vanderah TW, Ossipov MH, Lai J, Porreca F et al (2009). TRPV1 receptor in expression of opioid‐induced hyperalgesia. J Pain 10: 243–252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wang C, Gu Y, Li GW, Huang LY (2007). A critical role of the cAMP sensor Epac in switching protein kinase signalling in prostaglandin E2‐induced potentiation of P2X3 receptor currents in inflamed rats. J Physiol 584 (Pt 1): 191–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wirkner K, Sperlagh B, Illes P (2007). P2X3 receptor involvement in pain states. Mol Neurobiol 36: 165–183. [DOI] [PubMed] [Google Scholar]
  36. Xu C, Xu W, Xu H, Xiong W, Gao Y, Li G et al (2012). Role of puerarin in the signalling of neuropathic pain mediated by P2X3 receptor of dorsal root ganglion neurons. Brain Res Bull 87: 37–43. [DOI] [PubMed] [Google Scholar]
  37. Xu H, Xu T, Ma X, Jiang W (2015). Involvement of neuronal TGF‐β activated kinase 1 in the development of tolerance to morphine‐induced antinociception in rat spinal cord. Br J Pharmacol 172: 2892–2904. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Effect of A‐317491 on the cumulative dose–response curve of morphine. Morphine‐induced antinociceptive dose–responses were assessed on day 8. (A) A rightward shift of the dose–response curve in the 7‐day morphine group was revealed on day 8, and co‐administration of A‐317491 with morphine prevented this shift. (B) The ED50 of morphine in the morphine‐alone group was higher than that in other groups. The development of morphine tolerance was attenuated when morphine was administered along with A‐317491 for 7 days (n = 6/group; ***P < 0.001 vs morphine group).

Figure S2 Bolus injection of epsilon‐V1–2 partly restored the analgesic efficacy of morphine on day 8. Mechanical thresholds were measured daily (n = 6).

Click here for additional data file. (217.1KB, pdf)

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES