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Complex formation between the vasopressin 1b receptor, β-arrestin-2, and the μ-opioid receptor underlies morphine tolerance

Nature Neuroscience volume 21, pages 820–833 (2018)Cite this article

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Abstract

Chronic morphine exposure upregulates adenylate cyclase signaling and reduces analgesic efficacy, a condition known as opioid tolerance. Nonopioid neurotransmitters can enhance morphine tolerance, but the mechanism for this is poorly understood. We show that morphine tolerance was delayed in mice lacking vasopressin 1b receptors (V1bRs) or after administration of V1bR antagonist into the rostral ventromedial medulla, where transcripts for V1bRs and μ-opioid receptors are co-localized. Vasopressin increased morphine-binding affinity in cells expressing both V1bR and μ-opioid receptors. Complex formation among V1bR, β-arrestin-2, and μ-opioid receptor resulted in vasopressin-mediated upregulation of ERK phosphorylation and adenylate cyclase sensitization. A leucine-rich segment in the V1bR C-terminus was necessary for the association with β-arrestin-2. Deletion of this leucine-rich segment increased morphine analgesia and reduced vasopressin-mediated adenylate cyclase sensitization. These findings indicate that inhibition of μ-opioid-receptor-associated V1bR provides an approach for enhancing morphine analgesia without increasing analgesic tolerance.

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Fig. 1: Responses to noxious heat stimuli are delayed and morphine analgesia is enhanced in V1b−/− mice.
Fig. 2: Pharmacological inhibition of the V1bR effectively delayed tolerance development.
Fig. 3: Co-expression of μ-receptor and V1bR in the RVM and functional interactions when co-expressed in HEK cells.
Fig. 4: AVP increased morphine-dependent AC superactivation through the β-arrestin-2–ERK pathway in HEK V1bR–μ-receptor cells.
Fig. 5: V1bR enhanced BRET between μ-receptor-Luc and β-arrestin-2–Venus.
Fig. 6: The leucine-rich segment of the V1bR C-terminal region is necessary for interaction between μ-receptor–Luc and β-arrestin-2–Venus.
Fig. 7: Genome editing of the Avpr1b gene (encoding V1bR) deleted the leucine-rich segment in the C-terminal region and enhanced morphine analgesia.
Fig. 8: Attenuated physical dependence to morphine in V1b−/− mice.

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Acknowledgements

We thank Y. Oyama for her technical assistance. This work was supported in part by research grants from the Scientific Fund of the Ministry of Education, Science, and Culture of Japan (#24590327 and #17H04323 to T.K., #23590731 to K.H., and #22590252 to Y.T.), JKA through its promotion funds from KEIRIN RACE (T.K.), and the Promotion and Mutual Aid Corporation for Private Schools of Japan (T.K.).

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Authors and Affiliations

  1. Department of Pharmacology, Division of Molecular Pharmacology, Jichi Medical University, Shimotsuke, Tochigi, Japan

    Taka-aki Koshimizu

  2. Department of Physiology and Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan

    Kenji Honda, Sachi Nagaoka-Uozumi & Yukio Takano

  3. Department of Biological Chemistry, Graduate School of Pharmaceutical Science, Kyoto University, Kyoto, Japan

    Atsuhiko Ichimura

  4. Keihanshin Consortium for Fostering the Next Generation of Global Leaders in Research (K-CONNEX), Kyoto University, Kyoto, Japan

    Atsuhiko Ichimura

  5. Department of Applied Biological Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan

    Ikuo Kimura

  6. Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

    Michio Nakaya & Hitoshi Kurose

  7. Division of Functional Genomics, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Hyogo, Japan

    Nobuya Sakai & Katsushi Shibata

  8. Department of Pharmacology, Division of Clinical Pharmacology, Jichi Medical University, Shimotsuke, Tochigi, Japan

    Kentarou Ushijima & Akio Fujimura

  9. Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

    Akira Hirasawa & Gozoh Tsujimoto

  10. Institute for Integrated Medical Sciences, Tokyo Women’s Medical University, Tokyo, Japan

    Akira Hirasawa

  11. Department of Pharmacology, National Research Institute for Child Health and Development, Tokyo, Japan

    Akito Tanoue

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Contributions

T.-a.K., K.H. and Y.T. conceived the project, designed the experiments, and wrote the paper. T.K., S.N.-U., A.I., I.K., M.N., N.S., K.S., K.U., and K.H. performed the experiments and analyzed the data. A.H., A.I., and I.K. contributed to the cell culture experiments and M.N. and H.K. contributed to the knockdown experiments. N.S. and K.S. contributed to the genome editing experiments. K.U. and A.F. measured blood concentrations of morphine. G.T. and A.T. contributed to the experiments using V1bR-deficient mice and analyzed the data. T.K. and Y.T. coordinated and directed the project.

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Correspondence to Taka-aki Koshimizu or Yukio Takano.

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Integrated supplementary information

Supplementary Figure 1 Binding sites and expression profile of V1bR in mice.

(A) Membrane fractions (0.2 mg/reaction) from whole brains of WT (n = 5) and V1b-/- (n = 3) mice were subjected to a saturation binding study using the μ-receptor-specific radioligand, [3H]DAMGO. Bmax values were 114 ± 6 and 95 ± 5 fmol/mg protein for WT (n = 5) and V1b-/- (n = 3), respectively; unpaired two-sided Wilcoxon rank sum test, P = 0.114. KD values are 0.8 ± 0.2 and 0.9 ± 0.1 nM for WT and V1b-/- mice, respectively. (B) V1bR transcripts were detected in the RVM (gray bar), but were scarce in the dorsal medulla, spinal cord and dorsal root ganglia. For positive control, pituitary tissue was included (C). The expression levels of V1bR transcripts were normalized against GAPDH (n = 5). Graphs represent the mean ± S.E.M.

Supplementary Figure 2 The V1bR antagonist SSR149415 delayed the progression of morphine tolerance in rats.

The antagonist (100 pmol) was administered into the lateral ventricle 10 min before daily morphine administration (10 mg/kg/day, s.c.) for 5 days. The tail flick test was performed 30 min after the morphine administration. Antagonist treatment had a significant effect on %MPE (In two-way ANOVA, F1,45 = 7.64. P = 0.0082 for antagonist treatment as indicated by *. F2,45 = 25.726. P < 0.0001 for time course. Significant interaction between antagonist treatment and time course was observed. F2,45 = 8.429. P = 0.0008). Graphs represent the mean ± S.E.M.

Supplementary Figure 3 Effects of V1a and/or V1b receptor antagonists on tail-flick responses.

(A) The selective V1a antagonist, d(CH2)5Tyr(Me)AVP (4.34 pmol), or saline was injected into the lateral ventricle (i.c.v.) 10 min before daily morphine administration (10 mg/kg/day, s.c.). The development of tolerance was not changed by the V1a antagonist. Tail flick tests were repeated before and 10, 30, 60, and 120 min after morphine administration. The time course of percent maximal possible effect (%MPE) was plotted after morphine injection and area under the curve was calculated. Two-way ANOVA analysis showed no effect of drug, F1,15 = 1.93, P = 0.185; significant effect of time, F2,30 = 70.11, P < 0.0001; no interaction between drug and time, F2,30 = 0.287, P = 0.756. Post hoc analysis by Tukey’s method detected significant decrease, #P < 0.0001 vs. day 1 saline and *P < 0.0001 vs. day 1 d(CH2)5Tyr(Me)AVP. (B) Daily i.c.v. administration of antagonists (dPenTyr(Me)AVP (V1a/Vb antagonist, 4.5 pmol), PhAcALVP (V1aR antagonist, 8.07 pmol) or d(CH2)5Tyr(Me)AVP (V1aR antagonist, 8.68 pmol or saline did not change morphine (10 mg/kg, s.c.) analgesia as assessed by the tail flick test. Two-way ANOVA found no effect of drugs (F3,32 = 0.6351, P = 0.5979). (C) A single administration of saline, vehicle, dPenTyr(Me)AVP (4.5 pmol), or V1b antagonist SSR149415 (10 pmol) into the lateral ventricle did not change morphine dose-response curves in the tail flick test. One-way ANOVA found no effect of antagonist treatment, when responses were compared at each morphine dose separately (F3, 23 = 0.992, P = 0.4139, F3, 23 = 0.068, P = 0.9762, and F3, 23 = 1.081, P = 0.3769 for 2.5, 5 and 10 mg/kg morphine, respectively). Graphs represent the mean ± S.E.M.

Supplementary Figure 4 AC activity in the spinal cord was similar between WT and V1b-/- mice after repeated morphine administration.

Morphine (10 mg/kg/day, s.c.) was administered daily for 5 days. On the 6th day, lumbar spinal cord was obtained for cAMP measurements. By two-way ANOVA, forskolin (F3,56 = 73.888, P < 0.0001), but not mouse genotype (F1,56 = 1.299, P = 0.259), had significant influence on cAMP levels. Graphs represent the mean ± S.E.M.

Supplementary Figure 5 Analgesic tolerance to κ-opioid agonist, U62,066, and AC activities in the RVM.

(A) WT and V1b-/- mice were injected with U62,066 (10 mg/kg i.p.) once a day for 12 days. Tail flick latencies were examined 30 min after the injections. The basal latencies on day 1 prior to drug injection were 5.0 ± 0.5 and 8.2 ± 1.0 sec for WT (n = 12) and V1b-/- (n = 11) mice, respectively (unpaired, two-sided Student’s t-test, t15 = 2.7281, P = 0.0154). Treatment periods had significant influence on tail-flick latencies (F11,251 = 6.133, P < 0.0001), but genotype did not (F1,251 = 1.584, P = 0.209) in two-way ANOVA analysis. (B) Tolerance to U62,066 had no effect on AC activity in the RVM of WT and V1b-/- mice. n = 7,7 WT mice and n = 10, 13 V1b-/- mice for saline and U62,066 treatments. Graphs represent the mean ± S.E.M.

Supplementary Figure 6 Radioligand binding studies for examining functional interactions between AVP and opioid agonists.

[3H]naloxone (1 nM) was incubated with membrane samples from HEK-μ-receptor cells (A) or from HEK-V1bR-μ-receptor cells (B) in increasing concentrations of morphine (A) or DAMGO (B) in the presence or absence of 100 nM AVP. (C) In saturation binding experiments on HEK-V1bR- μ-receptor cells, increasing concentrations of [3H]AVP (0 – 30 nM) were incubated with membrane samples in the presence of saline or 1 μM morphine. Data shown in A–C are means from four (A and B) or three (C) independent experiments. Graphs represent the mean ± S.E.M.

Supplementary Figure 7 Enhancement of AC superactivation was not detected after treatment of DAMGO plus AVP or morphine plus carbachol.

(A) DAMGO-induced AC superactivation was not influenced by 50 nM AVP. Two-way ANOVA detected that 1 μM DAMGO increased the cAMP response (F2,48 = 4.939. P = 0.0112). But no difference was found between DAMGO vs, DAMGO plus AVP (P = 0.9168), *P < 0.05 vs. control (n = 5 in each group). (B) Intracellular calcium response induced by 1 μM carbachol in HEK- μ-receptor cells. At least 20 cells were monitored in a measurement and average response was calculated. Similar results were obtained from four experiments. (C) Carbachol did not enhance morphine-induced AC superactivation in HEK-μ-receptor cells. The cells were incubated in the medium containing 1 μM morphine with or without 1 μM carbachol for 24 h. cAMP concentrations were dependent on forskolin (two-way ANOVA, F3,56 = 4.34. P = 0.0080), but not on carbachol (F1,56 = 0.086. P = 0.7706). n = 8 independent experiments. (D) Time course of ERK phosphorylation after costimulation by 25 nM AVP plus 1 μM morphine or DAMGO. Signal intensities of phosphorylated ERKs were normalized against those of unphosphorylated ERKs and data are presented as percent values relative to the maximum values. In two-way ANOVA, both drugs and time had a significant impact on the ERK phosphorylation status, F1,36 = 31.652, P < 0.0001 and F5,36 = 46.499, P < 0.0001, respectively (n = 4). Significant interaction between drugs and time was detected (F5,36 = 5.374, P < 0.001). ***P < 0.0001. Graphs represent the mean ± S.E.M.

Supplementary Figure 8 AC superactivation and cAMP levels in HEK V1bR–μ-receptor cells.

(A) The time course of the development of AC superactivation was examined after treatment of HEK–V1bR–μ-receptor cells with 100 nM AVP. Cells were collected at indicated times and stimulated with 0.3 μM forskolin. Significant increases in cAMP levels were detected after 4 h of AVP incubation (one-way ANOVA was followed by two-sided pairwise t-tests with Holm’s method for P value adjustment. F6,77 = 5.882, P < 0.0001). *P < 0.0001 vs. 0 h, (n = 12,12,12,15,6 for 0, 1, 2, 4, 24 h independent experiments). (B) AVP did not have an acute effect on cAMP levels in HEK–V1bR–μ-receptor cells. (n = 9). (C) Acute inhibition of cAMP levels by morphine was not changed by AVP in HEK–V1bR–μ-receptor cells. n = 6 independent experiments. (D) AC superactivation by 100 nM AVP was not sensitive to the PKC inhibitor Ro 31-8220 (50 nM). Two-way ANOVA, F1,33 = 2.713, P = 0.109. n = 9, 9, 9, 10. (E) AVP induced AC superactivation in the presence of 1 μM naloxone in HEK-V1bR- μ-receptor cells. For control samples, PBS was used. The cells were incubated for 24 h and forskolin (1 μM)-induced cAMP production was measured. In two-way ANOVA and Tukey’s test, no difference was detected between AVP and AVP plus naloxone (P = 0.380, n = 12). Graphs represent the mean ± S.E.M.

Supplementary Figure 9 Agonist-stimulated BRET signals and Phos-tag analysis of carboxyl-terminal V1bR mutants.

(A) Agonist increased BRET signal between μ-receptor-Luc and β-arrestin 2-Venus. In one-way ANOVA, drug treatments significantly increased the BRET signal in V1aR-transfected cells (F2,15 = 6.356, P = 0.01) and in μ-receptor-transfected cells (F2,15 = 30.65, P < 0.0001). Multiple comparisons with Tukey’s method detected significant difference; V1a-transfected cells, control vs. 1 μM DAMGO, P = 0.0222, control vs. 1 μM endomorphin, P = 0.0169; μ-receptor-transfected cells, control vs. DAMGO, P < 0.0001, control vs. endomorphin, P = 0.0002. *P < 0.05, ***P < 0.001. (n = 6 independent experiments). (B) The phosphorylated form was not detected in the deletion mutant V1bRs lacking the entire C-terminal region. Phos-tag SDS-PAGE and western blot detection of wild-type V1bR showed two bands, which correspond to the phosphorylated and unphosphorylated forms. Two consecutive cysteine residues, Cys352 and Cys353, in the V1bR C-terminus correspond to putative palmitoylation sites ‡. Residues were removed from the C-terminus up to Cys352 or Cys353, and termed V1bR-Ala351-stop or V1bR-Cys352-stop, respectively. Both of the mutant V1bRs showed a single population of unphosphorylated forms in Phos-tag analysis. The data are representative from three experiments. V1b-p indicates the phosphorylated form of V1bR. Graphs represent the mean ± S.E.M. Dots represent data from individual data points. Immunoblot in B are cropped; full image is shown in Supplementary Fig. 14. ‡ Qanbar, R. & Bouvier, M. Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function. Pharmacol. Ther. 97, 1-33 (2003).

Supplementary Figure 10 Schematic presentation of V1bR and μ-receptor-dependent signaling pathways leading to AC superactivation and morphine tolerance.

Previous studies have shown that morphine-induced AC superactivation is dependent on Gi-protein activation and is sensitive to pertussis toxin (PTX) treatment, as indicated by the signals mediated by homomeric μ-receptors (μ-R) (right hand side in light blue). However, co-expression of V1bRs and μ-receptors resulted in formation of a complex composed of V1bR, μ-receptor, and β-arrestin 2. Costimulation of these cells with AVP (brown arrows) and morphine (blue arrows) increases β-arrestin 2-dependent ERK phosphorylation and AC superactivation. Several interventions were effective at disturbing this pathway. Treatment with the V1bR antagonist, SSR149415, V1bR knockout in mice, genome editing of the V1bR C-terminal region, siRNA-mediated knockdown of β-arrestin 2 and ERK1/2, and the MEK inhibitor, U0126, suppressed AC sensitization in V1bR and μ-receptor-co-expressing cells. These results illustrate the potential usefulness of a V1bR antagonist in delaying the development of morphine tolerance without perturbing μ-receptor-mediated analgesia.

Supplementary Figure 11

Full immunoblots images of Fig. 3.

Supplementary Figure 12

Full immunoblots images of Fig. 5c.

Supplementary Figure 13 Full immunoblot images of Figs. 4e, f, and 6e.

Green squares indicate areas used the Figures.

Supplementary Figure 14

Full immunoblot and gel images of Figs. 6g, 7b, c and 9b.

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Koshimizu, Ta., Honda, K., Nagaoka-Uozumi, S. et al. Complex formation between the vasopressin 1b receptor, β-arrestin-2, and the μ-opioid receptor underlies morphine tolerance. Nat Neurosci 21, 820–833 (2018). https://doi.org/10.1038/s41593-018-0144-y

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