Morphine-induced hyperalgesia involves mu opioid receptors and the metabolite morphine-3-glucuronide

Abstract

Opiates are potent analgesics but their clinical use is limited by side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). The Opiates produce analgesia and other adverse effects through activation of the mu opioid receptor (MOR) encoded by the Oprm1 gene. However, MOR and morphine metabolism involvement in OIH have been little explored. Hence, we examined MOR contribution to OIH by comparing morphine-induced hyperalgesia in wild type (WT) and MOR knockout (KO) mice. We found that repeated morphine administration led to analgesic tolerance and hyperalgesia in WT mice but not in MOR KO mice. The absence of OIH in MOR KO mice was found in both sexes, in two KO global mutant lines, and for mechanical, heat and cold pain modalities. In addition, the morphine metabolite morphine-3beta-D-glucuronide (M3G) elicited hyperalgesia in WT but not in MOR KO animals, as well as in both MOR flox and MOR-Nav1.8 sensory neuron conditional KO mice. M3G displayed significant binding to MOR and G-protein activation when using membranes from MOR-transfected cells or WT mice but not from MOR KO mice. Collectively our results show that MOR is involved in hyperalgesia induced by chronic morphine and its metabolite M3G.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

WT but not MOR KO mice show hyperalgesia under repeated morphine analgesic tolerance conditions. (A) The experimental design shows the schedule for nociceptive measures (arrows). Following baseline (BL), mice received 3 mg/kg morphine (ip) on day-1 (d1) to evaluate morphine-induced analgesia. Mice received thereafter 20 mg/kg morphine or saline control each day until day-7 (d7). On d8, nociceptive levels were measured before morphine administration to evaluate hyperalgesia, and following 3 mg/kg morphine to measure analgesic tolerance. Maintenance of hyperalgesia (OIH) was scored on the indicated days and latent sensitization on day 27. (B) Tail pressure (n = 18–19/group) and (C) tail immersion (48 °C, n = 19–25/group) results show analgesic tolerance (upper panels) in WT mice with repeated morphine. Pressure and heat hyperalgesia in WT but not KO mice are shown with the same mouse groups in bottom panels. **p < 0.01; ***p < 0.001 compared to baseline or d1. (D) Analgesic tolerance and hyperalgesia occur in MOR-flox but not MOR-CMV mice. **p < 0.01; ***p < 0.001 compared to baseline or to d1 (tail immersion 48 °C, n = 8–9/group). Two-way repeated ANOVA, Newman-Keuls test. (E) WT but not MOR KO mice show cold allodynia under morphine analgesic tolerance conditions. Following baseline (BL) cold response scoring on the 5 °C cold plate, mice received 20 mg/kg morphine or saline control once a day until day 7 as described in (A) and paw responses to cold were measured to evaluate cold allodynia. n = 13–16/group. ***p < 0.001 compared to BL. ANOVA repeated measures, Newman-Keuls test. Detailed statistical analyses are presented in Supplementary Table S2.

Figure 2

MOR KO mice do not show OIH persistence. Persistence of hyperalgesia after cessation from seven-day 20 mg/kg morphine in WT but not MOR-KO mice. (B, C) On day-27 when WT mice had recovered from OIH (Fig. 1A), the opioid antagonist naltrexone induced hyperalgesia i.e. latent sensitization in WT (B) but no MOR KO (C) mice. Tail immersion (48 °C, n = 11–14/group for OIH persistence and n = 6–8/group for latent sensitization following 20 mg/kg morphine; n = 8–10/group for persistence of OIH following 60 mg/kg morphine). **p < 0.01; ***p < 0.001; repeated measures ANOVA followed by Newman-Keuls test. Detailed statistical analyses are presented in Supplementary Table S3.

Figure 3

Morphine induced hyperalgesia in female and male WT mice but not MOR KO mice. Analgesia, analgesic tolerance and OIH were measured following the protocol described in Fig. 1A (see sex-grouped analysis in Fig. 1) on female (left panels) and male (right panels) mice with the tail immersion (A–D), pressure analgesimeter (E–H) and cold plate (I,J) tests for heat, pressure and cold hypersensitivities, respectively. Data are expressed as mean ± SEM. n = 7–11 mice/group. *, **, ***p < 0.05, 0.01 and 0.001 compared to the corresponding group (ANOVA repeated measures, Newman-Keuls. BL, Baseline. Detailed statistical analyses are presented in Supplementary Table S4.

Figure 4

MOR KO mice show no morphine-induced hyperalgesia following partial sciatic nerve ligation (pSNL). (A) The experimental design shows the schedule for nociceptive measures (arrows). Following determination of baseline (BL) and neuropathic hypersensitivity (14 days post-pSNL, partial sciatic nerve ligation) on a 5 °C cold plate, WT and MOR KO mice were treated for 7 days with either morphine or saline control solution. On d8, allodynia was measured with the cold plate, von Frey filaments and heat plantar tests. (B) Cold pSNL allodynia and OIH (n = 10–12/group), two-way ANOVA, Newman-Keuls test. Morphine induced OIH in WT mice,*p < 0.05 compared to post-pSNL. (C) Mechanical pSNL allodynia (n = 10–12/group), two-way ANOVA, Newman-Keuls test. pSNL induced mechanical allodynia; WT saline group and WT morphine group, p < 0.001 pSNL vs BL; KO saline group and KO morphine group, p < 0.01 pSNL vs BL. Morphine induced OIH in WT mice, *p < 0.05 compared to post-pSNL. (D) Heat pSNL allodynia (n = 10–12/group), two-way ANOVA. Morphine induced no OIH. pSNL induced cold, mechanical and heat allodynia. Detailed statistical analyses are presented in Supplementary Table S5.

Figure 5

MOR KO mice show no morphine-3-glucuronide-induced hyperalgesia. M3G concentration in mouse plasma (A), brain (B) and spinal cord (C) 2 hr following 10 mg/kg morphine administration. M3G was quantified using LC-MS/MS. Data are expressed as mean ± SEM. n = 9–11 mice/group. ***p < 0.001 in KO mice as compared to WT mice. (D,E) Acute M3G induces heat and touch hyperalgesia in WT but not KO mice. (n = 9–10/group) one-way ANOVA, Newman-Keuls test *p < 0.05; **p < 0.01 compared to baseline (BL). (F,G) Acute M3G induces heat and touch hypersensitivity in MOR Flox and cKO mice (n = 6–9/group) one-way ANOVA, Newman-Keuls test #0.05 < p < 0.1; *p < 0.05; ***p < 0.001 compared to baseline (BL). Detailed statistical analyses are presented in Supplementary Table S6.

Figure 6

M3G binding and signaling to MOR (A,B) [3H]-diprenorphine binding on membranes from MOR expressing HEK293-Glo cells (A) and [3H]-DAMGO binding on brain membranes preparations from WT mice (B). Membranes were incubated with increasing doses of DAMGO, morphine, fentanyl or M3G in assay buffer containing a fixed dose of opioid radioligand. 100% represents maximal radioligand binding in the absence of competitor. Results are presented as means ± SEM of 2 or 3 experiments. (C–E) MOR agonist-induced [35S]-GTPγS binding to brain membranes preparations from WT (C) or KO mice (D). Membranes were incubated with increasing doses of DAMGO, morphine or M3G agonists (10−9 to 10−4M) in assay buffer containing [35S]-GTPγS. Basal level (100%) represents [35S]-GTPγS binding in the absence of agonist. DAMGO, morphine and M3G significantly stimulated [35S]-GTPγS binding to membranes from WT but not KO mice. Results are presented as means ± SEM of 3–5 experiments on 3 independent membrane preparations per genotype. (E) The selective MOR antagonist CTOP inhibits DAMGO, morphine and M3G-induced [35S]-GTPγS binding to membranes from WT mice. Brain membranes from WT mice were incubated with increasing doses of the mu opioid antagonist CTOP combined with 100uM of DAMGO, morphine or M3G. Activation results are presented as means ± SEM of 6–10 experiments from 3 independent membrane preparations. (F) Effect of increasing concentrations of DAMGO, morphine, fentanyl and M3G on forskolin-stimulated cAMP production in HEK293 stably expressing GloSensor and MOR receptor. In the cases indicated, 1 µM of naloxone had been added to the cells 15 min prior to the agonist. Dose−response curves were normalised to maximal DAMGO activity. Evaluations were performed three times in duplicate for M3G, two times in duplicates for other agonists. Results are presented as means ± SEM. (G) eYFP-labelled Beta-arrestin-2 translocation to Rluc-MOR in HEK293 cells after 5 to 10 min of cells activation by DAMGO, morphine, fentanyl or M3G at 37 °C. Agonist specific BRET1 ratio were determined by subtracting BRET1 ratio of non activated cells, and normalised to maximal DAMGO-triggered effect. Presented results are means ± SEM of 2 to 4 experiments. (H–J) Dynamic mass redistribution (DMR) signals observed in HEK293-Glo-cells after activation by various concentrations of DAMGO, morphine or M3G. Baseline of buffer-treated cells has been subtracted. Evaluations were performed three times in duplicate or triplicates. Presented figures show a representative experiment.