Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia

Abstract

Analgesic effects of delta opioid receptor (DOR) -selective agonists are enhanced during persistent inflammation and arthritis. Although the underlying mechanisms are still unknown, membrane density of DOR was shown to be increased 72 h after induction of inflammation, an effect abolished in mu opioid receptor (MOR) -knockout (KO) mice [Morinville A, Cahill CM, Kieffer B, Collier B, Beaudet A (2004b) Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain. Pain 109:266-273]. In this study, we demonstrated a crucial role of MOR in DOR-mediated antihyperalgesia. Intrathecal administration of the DOR selective agonist deltorphin II failed to induce antihyperalgesic effects in MOR-KO mice, whereas it dose-dependently reversed thermal hyperalgesia in wild-type mice. The antihyperalgesic effects of deltorphin II were blocked by naltrindole but not d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) suggesting that this agonist was mainly acting through DOR. SNC80-induced antihyperalgesic effects in MOR-KO mice were also attenuated as compared with littermate controls. In contrast, kappa opioid receptor knockout did not affect deltorphin II-induced antihyperalgesia. As evaluated using mice lacking endogenous opioid peptides, the regulation of DOR's effects was also independent of beta-endorphin, enkephalins, or dynorphin opioids known to be released during persistent inflammation. We therefore conclude that DOR-mediated antihyperalgesia is dependent on MOR expression but that activation of MOR by endogenous opioids is probably not required.

Figure 1. Antihyperalgesic effect of intrathecal deltorphin II in C57BL/6 mice

A, C57BL/6 mice were injected with CFA in the plantar surface of the right hindpaw. Seventy-two hours after CFA injection, the latency to paw withdrawal (in s) was tested every 15 min (from 15 to 60 min) after intrathecal injection of saline (5 μl) or deltorphin II (0.1, 0.25, 1, 2.5 μg). Intrathecally-administered deltorphin II induced a dose-dependent relief of thermal hyperalgesia (ipsilateral hindpaw). The antihyperalgesic effect of deltorphin II peaked 15 min after the injection. −72 h indicates the baseline for the latency to paw withdrawal before CFA injection and time 0 the latency to paw withdrawal just before injection of saline or deltorphin II. Number in parenthesis indicates the number of animals in each group. B, Graphic determination of the effective dose of deltorphin II (ED₅₀) inducing a 50% relief of thermal hyperalgesia. C, No effect of deltorphin II was observed on the contralateral hindpaw. ***, p < 0.001 when compared to latency to paw withdrawal of ipsilateral hindpaw 72 h after CFA injection (PostCFA, white bar); one-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 2. Role for MOR and KOR in the regulation of DOR-mediated antihyperalgesia

The antihyperalgesic effect of deltorphin II was tested in (A) KOR-KO and (B) MOR-KO mice and in their respective littermate controls 72 h after CFA injection. A and B, Effect of intrathecal injection of deltorphin II (2.5 μg) on the latency to paw withdrawal (for the ipsilateral hindpaw) in response to a noxious heat stimulus. ***, p < 0.001 when compared to MOR^+/+ littermates, two-way ANOVA followed by Bonferroni’s multiple comparison test. Number in parenthesis indicates the number of animals in each group. C, Comparison of latencies to paw withdrawal in response to noxious heat stimulus for ipsilateral and contralateral hindpaws regarding genotypes. **, p = 0.0065, two-tailed unpaired t-test.

Figure 3. Pharmacological characterization of antihyperalgesia induced by deltorphin II

A, The antihyperalgesic effect of deltorphin II in CFA-injected C57BL/6 mice was DOR-mediated since it was completely blocked by naltrindole (n = 9) but not significantly affected by CTOP (n = 6). Naltrindole was administered subcutaneously at a dose of 10 mg/kg, 15 min prior to deltorphin II (2.5 μg) injection. CTOP (10 ng) was co-injected intrathecally with deltorphin II (2.5 μg). This dose of CTOP (10 ng; n = 6) was sufficient to fully reverse DAMGO (5 ng)-induced antihyperalgesia (n = 6), demonstrating the appropriateness of the CTOP dose. *, p < 0.05, one-way ANOVA followed by Bonferroni’s multiple comparison test. B, As for deltorphin II, antihyperalgesic effect of intrathecally administered SNC80 (45 μg), a non-peptide DOR-selective agonist, was significantly weaker in MOR-KO mice (n = 10) than in littermate controls (n = 9). Vehicle control has no effect by itself (n = 8). *, p < 0.05, one-way ANOVA followed by Bonferroni’s multiple comparison test. A and B, the effect of each treatment is expressed as %MPAHE, calculated 15 minutes post-injection.

Figure 4. Role of endogenous opioids in the regulation of DOR-mediated antihyperalgesia

A, Thermal latencies (in s) to noxious heat were recorded every 15 min following intrathecal administration of deltorphin II (2.5 μg) to control littermates (Litt.; ), pro-enkephalin-KO (pENK-KO; ○), β-endorphin-KO (β-END-KO; □), and pro-dynorphin-KO (pDYN-KO; △) mice. B, Comparative latencies to paw withdrawal (in s) in control littermates, pENK-, β-END-, and pDYN-KO mice before (0 min) and 15 min after intrathecal injection of deltorphin II (2.5 μg). Number in parenthesis indicates the number of animals in each group. #, p < 0.0001 when ipsilateral 0 min are compared with their respective ipsilateral preCFA; two-tailed unpaired t-test.

Figure 5. Effect of deltorphin II on motor functions in C57BL/6 mice

Rotarod performances were evaluated in C57BL/6 mice following intrathecal injection of deltorphin II. A, Dose-dependent induction of motor uncoordination/ataxia-like behavior induced by deltorphin II (0–2.5 μg). ***, p < 0.001 when compared to saline-injected group (○), two-way ANOVA followed by Bonferroni’s multiple comparison test. B, Blockade of deltorphin II’s effect on motor functions with the DOR-selective antagonist naltrindole (NTI; 10 mg/kg) injected subcutaneously 15 min before administration of deltorphin II (2.5 μg; ▲). Control group ( ) received an equal volume of saline subcutaneously 15 min prior to deltorphin II. Mean performance before deltorphin (or before saline/NTI) injection represents the mean performance of all mice before treatment. **, p < 0.01 when compared to saline-pretreated group, two-way ANOVA followed by Bonferroni’s multiple comparison test. Number in parenthesis indicates the number of animals in each group.

Figure 6. Effect of deltorphin II on motor functions in MOR-KO mice

Rotarod performances were evaluated following intrathecal injection of deltorphin II (2.5μg) to MOR-KO (●) and their littermate controls (MOR^+/+, ○). Mean performance before deltorphin injection corresponds to the mean performance of mice from both groups (MOR-KO and littermates) before treatment. No significant difference was observed between performances before injection of deltorphin II. Intrathecal injection of deltorphin II induced similar motor uncoordination/ataxia-like behavior (*, p < 0.05 and **, p < 0.01 when compared with the mean performance before deltorphin II injection; one-way ANOVA followed by Dunnett’s multiple comparison test) in both groups of mice (p > 0.05 when comparing MOR-KO with littermate controls, two-way ANOVA followed by Bonferroni’s multiple comparison test). Number in parenthesis indicates the number of animals in each group.