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. 2010 Jul 20;3(131):ra54.
doi: 10.1126/scisignal.2000807.

Increased Abundance of Opioid Receptor Heteromers After Chronic Morphine Administration

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Free PMC article

Increased Abundance of Opioid Receptor Heteromers After Chronic Morphine Administration

Achla Gupta et al. Sci Signal. .
Free PMC article

Abstract

The mu and delta types of opioid receptors form heteromers that exhibit pharmacological and functional properties distinct from those of homomeric receptors. To characterize these complexes in the brain, we generated antibodies that selectively recognize the mu-delta heteromer and blocked its in vitro signaling. With these antibodies, we showed that chronic, but not acute, morphine treatment caused an increase in the abundance of mu-delta heteromers in key areas of the central nervous system that are implicated in pain processing. Because of its distinct signaling properties, the mu-delta heteromer could be a therapeutic target in the treatment of chronic pain and addiction.

Conflict of interest statement

Conflicts of interest: None.

Figures

Fig. 1
Fig. 1
Detection of μ–δ heteromers using heteromer-selective monoclonal antibodies. (A) Receptor abundance was determined in cortical membranes from wild-type, μ knockout (KO), δ KO, or μ–δ double KO mice with monoclonal antibodies to μ–δ, μ, or δ receptors by ELISA. (B–C) Cells endogenously (B) or stably (C) coexpressing μ–δ receptors were treated without or with morphine (1 μM) for 48 hours. Heteromer abundance was determined by ELISA with μ–δ heteromer-selective antibodies. Results are means ± SEM (n = 3 experiments). *p < 0.05. (D–G) Repeated morphine treatment increased μ–δ heteromer immunoreactivity in the medial nucleus of the trapezoid body (MNTB). Immunoreactivity in μ KO or δ KO mice after morphine treatment was below the detection limits. (H) Individual neuronal profiles were outlined (n = 15–20/group) and μ–δ heteromer immunoreactivity, as assessed by mean optical density (O.D.), was determined. Morphine treatment increased μ–δ heteromer immunoreactivity in MNTB neurons from wild-type mice (p < 0.01 compared to untreated control), but not in those from μ or δ KO mice.
Fig. 2
Fig. 2
Chronic morphine treatment induces μ–δ heteromer abundance in different brain regions. (A–D) Chronic (5 days of escalating dose regime), but not acute (30 min), morphine treatment significantly increased μ–δ heteromer immunoreactivity in perineuronal net-bearing MNTB neurons detected by Wisteria floribunda agglutinin (WFA). Arrowheads indicate neurons bearing μ–δ immunoreactivity ensheated by perineuronal nets. Scale bars = 25 μm. (D) represents the mean grey-scale optical density measured within individual neurons of 2–4 mice. (E–K) Quantitative analysis of the maximal fluorescence intensity of μ–δ immunoreactivity in individual perineuronal net-bearing neurons of the MNTB after acute or chronic morphine treatment. Numbers in parentheses indicate the number of neurons. **p <0.01 (L) ELISA with membranes from the MNTB, RVM, or cortex of mice shows that a 5 day chronic escalating dose of morphine but not a single dose of morphine (acute, 10 mg/kg) or saline significantly increased μ–δ receptor abundance. Results are means ± SEM (n = 3 experiments). *p<0.05; ***p < 0.001 compared to saline. (M) ELISA with membranes from different mice brain regions shows that treatment with morphine (5 mg/kg), or NTB (0.1 mg/kg) for 9 days significantly increased μ–δ receptor abundance. Results are means ± SEM (n=3 experiments). *p<0.05; **p<0.01; ***p<0.001.
Fig. 3
Fig. 3
Chronic alkaloid treatment increases μ–δ heteromer abundance. (A) CHO cells stably expressing μ and δ receptors were treated with the indicated ligands (1 μM) for 48 hours and μ–δ heteromer abundance was determined by ELISA. Results are means ± SEM (n=3–4 experiments). **p<0.01. μ–δ abundance refers to ELISA results with the μ–δ heteromer selective antibody (B) Morphine-treated (1 μM) SK-N-SH cells were probed by ELISA using monoclonal antibodies to μ, δ, or μ–δ receptors. Results are means ± SEM (n=3–4 experiments).
Fig.4
Fig.4
μ–δ heteromer-selective antibody blocks heteromer-mediated increases in binding and signaling. (A) CHO cells coexpressing Flag-tagged μ and Myc-tagged δ receptors were pre-treated with either the μ–δ heteromer-selective antibody or monoclonal antibodies to Flag or Myc. Binding of the μ receptor agonist [3H]DAMGO (10 nM) to cells was measured in the absence or presence of δ receptor antagonist TIPPψ (10 nM). Results are means ± SEM (n=3 experiments). (B and C) Mouse cortical membranes were preincubated without or with μ–δ heteromer antibodies and G-protein activity, as assessed by [35S]GTPγS binding (expressed as G-protein activity), (B) or adenylyl cyclase activity (C) in response to DAMGO (1 μM) was determined in the absence or presence of TIPPψ (10 nM). Results are means ± SEM (n=3 experiments). (D) HEK293 cells coexpressing chimeric G16/Gi3 and μ and δ opioid receptors were preincubated without or with μ–δ heteromer antibodies, then treated with the δ opioid agonist deltorphin II (1 μM) in the absence or presence of the μ opioid antagonist CTOP (10 nM) and intracellular Ca2+ concentrations were determined. Results are means ± SEM (n=3 experiments). *p<0.05; **p<0.01; ***p<0.001

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