Ligand-directed c-Jun N-terminal kinase activation disrupts opioid receptor signaling

Abstract

Ligand-directed signaling has been suggested as a basis for the differences in responses evoked by otherwise receptor-selective agonists. The underlying mechanisms are not understood, yet clearer definition of this concept may be helpful in the development of novel, pathway-selective therapeutic agents. We previously showed that kappa-opioid receptor activation of JNK by one class of ligand, but not another, caused persistent receptor inactivation. In the current study, we found that the mu-opioid receptor (MOR) could be similarly inactivated by a specific ligand class including the prototypical opioid, morphine. Acute analgesic tolerance to morphine and related opioids (morphine-6-glucuronide and buprenorphine) was blocked by JNK inhibition, but not by G protein receptor kinase 3 knockout. In contrast, a second class of mu-opioids including fentanyl, methadone, and oxycodone produced acute analgesic tolerance that was blocked by G protein receptor kinase 3 knockout, but not by JNK inhibition. Acute MOR desensitization, demonstrated by reduced D-Ala(2)-Met(5)-Glyol-enkephalin-stimulated [(35)S]GTPgammaS binding to spinal cord membranes from morphine-pretreated mice, was also blocked by JNK inhibition; however, desensitization of D-Ala(2)-Met(5)-Glyol-enkephalin-stimulated [(35)S]GTPgammaS binding following fentanyl pretreatment was not blocked by JNK inhibition. JNK-mediated receptor inactivation of the kappa-opioid receptor was evident in both agonist-stimulated [(35)S]GTPgammaS binding and opioid analgesic assays; however, gene knockout of JNK 1 selectively blocked kappa-receptor inactivation, whereas deletion of JNK 2 selectively blocked MOR inactivation. These findings suggest that ligand-directed activation of JNK kinases may generally provides an alternate mode of G protein-coupled receptor regulation.

Fig. 1.

GRK3 is required for tolerance to fentanyl but not morphine. WT C57BL/6 mice and littermate GRK3^−/− mice were tested for acute analgesic tolerance to morphine and fentanyl in the warm-water (52.5 °C) tail-withdrawal assay. Data are presented as latency to tail withdrawal as a function of time after initial drug administration. Animals were injected with morphine (10 mg/kg s.c.) or fentanyl (0.3 mg/kg s.c.) at time points indicated by the arrows. GRK3^−/− mice showed significantly reduced acute analgesic tolerance to the second dose of fentanyl compared with WT controls at 30 min and 60 min after the second injection (P < 0.001) (A). In contrast, both WT and GRK3^−/− mice showed significantly reduced analgesic response to the second dose of morphine (B), indicating acute tolerance. Pretreatment of WT mice with the JNK inhibitor SP600125 (3 mg/kg, 10 mg/kg, or 30 mg/kg i.p. 60 min before opioid agent) had no effect on acute tolerance to fentanyl (C), but dose-dependently reduced analgesic tolerance to morphine compared with vehicle controls (P < 0.001) (D); n = 6–9, data analyzed by two-way ANOVA using Bonferroni post hoc tests.

Fig. 2.

JNK is required for uncoupling of MOR following morphine. (A) WT mice were injected with vehicle or SP600125 at 10 mg/kg i.p. 60 min before opioid treatment. Animals given an initial dose of morphine (10 mg/kg s.c.) followed by a challenge dose of fentanyl (0.3 mg/kg s.c.) showed reduced analgesic response to fentanyl not significantly different from tolerance following an initial dose of fentanyl (6.20 ± 0.30 s latency compared with 4.91 ± 0.37 s latency, respectively). In contrast, the increased latency of tail withdrawal in response to fentanyl following an initial dose of morphine was significantly greater in mice pretreated with SP600125 (10.22 ± 1.78 s) compared with vehicle controls (P < 0.001), and was not significantly different from the initial analgesic response to fentanyl (9.89 ± 0.50 s). (B) Animals given an initial dose of fentanyl (0.3 mg/kg s.c.) followed by a challenge dose of morphine (10 mg/kg s.c.) showed a reduced analgesic response to morphine that was insensitive to SP600125 (10 mg/kg i.p.) pretreatment. (C) DAMGO (1 μM)-stimulated [³⁵S]GTPγS binding was assayed in membranes from spinal cords of mice pretreated with saline solution, 10 mg/kg morphine, or 0.3 mg/kg fentanyl. Both morphine (P < 0.01) and fentanyl (P < 0.05) treatment significantly decreased subsequent DAMGO [³⁵S]GTPγS binding compared with binding to membranes from saline solution–treated mice. Pretreatment with SP600125 prevented the morphine-induced reduction in binding (P < 0.05), but had no effect on fentanyl-induced reduction in binding (n = 5–8, data analyzed by two-way ANOVA in A and B; by one-way ANOVA using Bonferroni post hoc tests in C).

Fig. 3.

JNK inhibition has no effect on acute tolerance to non–morphine-like opioids. Acute analgesic tolerance was assayed as before. (A) Pretreatment with SP600125 (10 mg/kg i.p.) had no effect on acute tolerance to methadone (10 mg/kg s.c.) or (B) oxycodone (3 mg/kg s.c.). In contrast, SP600125 significantly reduced acute analgesic tolerance to the partial agonist buprenorphine (3 mg/kg s.c.) compared with vehicle-treated animals (P < 0.01), but failed to completely attenuate it in WT mice (C). Buprenorphine also binds to the KOR; however, the analgesic effects of buprenorphine were not evident in MOR^−/− mice. (D) Tolerance to the principal active morphine metabolite M6G (10 mg/kg, s.c.) was completely blocked by SP600125 compared with vehicle controls (P < 0.001); n = 5–6; data analyzed by two-way ANOVA using Bonferroni post hoc tests.

Fig. 4.

MOR agonists display different internalization profiles. (A) HEK293 cells stably transfected with rMOR-GFP were treated with vehicle or 1 μM morphine sulfate (MS), buprenorphine (Bup), M6G, fentanyl (Fen), methadone (Meth), and oxycodone (Oxy) for 10, 30, and 60 min. Representative images are shown for each data set. (Scale bar: 10 μm.) Semiquantitative analysis of internalization was performed by Metamorph software and is based on three cells per plate for three plates for each data set. Dashed line represents vehicle treated controls. Fentanyl, methadone, and oxycodone treatment produced significantly more internalization of the GFP-tagged receptor than vehicle treatment (P < 0.001) (B). Data analyzed by one-way ANOVA using Bonferroni post hoc tests.

Fig. 5.

JNK1 is required for the long lasting effects of norBNI. WT mice were tested in the tail-withdrawal assay after administration of U50,488 (15 mg/kg i.p.) before (Left) and 7 d after (Right) norBNI (10 mg/kg i.p.). SP600125 significantly blocked the long-lasting antagonistic effects of norBNI on U50,488-induced analgesia compared with vehicle 15 min and 30 min after U50,488 (P < 0.05 and P < 0.01, respectively) (A). U69,593-stimulated [³⁵S]GTPγS binding was measured in spinal cord membranes from animals treated with saline solution, U50,488, and norBNI and dissected 7 d later. Only norBNI showed significant long-lasting reduction in [³⁵S]GTPγS binding (P < 0.05 vs. saline solution treatment), an effect that was significantly reversed by pretreatment with the JNK inhibitor (P < 0.01) (B). JNK1^+/+ and JNK1^−/− mice showed equivalent analgesia to U50,488 prior to norBNI (Left), but 7 d later (Right) norBNI still blocked U50,488 responses in JNK1^+/+ but not JNK1^−/− mice. (P < 0.001), indicating that the JNK1 isoform is required for norBNI antagonism (C). In contrast, JNK1^−/− animals showed normal development of acute tolerance to morphine, indicating that the JNK1 isoform is not required for morphine tolerance (D). By comparison, JNK2^−/− mice, although they show similar analgesic response to an initial dose of morphine as WT controls, do not show significant analgesic tolerance following a second challenge dose of morphine versus controls (P < 0.05) (E). JNK2^−/− mice showed similar analgesia following U50,488 (15 mg/kg i.p.) compared with WT mice. Both WT and JNK2^−/− animals injected with norBNI (10 mg/kg i.p.) and challenged with U50,488 7 d later showed significant reduction in U50,488-induced analgesia compared with the initial assay (F); n = 4–6; data analyzed by two-way ANOVA using Bonferroni post hoc tests.