A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia

Abstract

Opiates such as morphine are the choice analgesic in the treatment of chronic pain. However their long-term use is limited because of the development of tolerance and dependence. Due to its importance in therapy, different strategies have been considered for making opiates such as morphine more effective, while curbing its liability to be abused. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. In particular, delta opioid receptor ligands have been useful in enhancing morphine's potency. The underlying molecular basis for these observations is not understood. We propose the modulation of receptor function by physical association between mu and delta opioid receptors as a potential mechanism. In support of this hypothesis, we show that mu-delta interacting complexes exist in live cells and native membranes and that the occupancy of delta receptors (by antagonists) is sufficient to enhance mu opioid receptor binding and signaling activity. Furthermore, delta receptor antagonists enhance morphine-mediated intrathecal analgesia. Thus, heterodimeric associations between mu-delta opioid receptors can be used as a model for the development of novel combination therapies for the treatment of chronic pain and other pathologies.

Fig. 1.

μ and δ receptor interactions in heterologous cells and endogenous tissue. (A) BRET assay shows significant energy transfer between μ and δ receptors in live cells. (Upper) Light emission was monitored for cells transfected with δLuc and CCR5YFP (long black dash) or various amounts of δLuc:μYFP; 0.1:0.8 (gray line), 0.25:2 (gray dash), and 0.5:4 (small black dash). Spectra in A expressed as BRET ratio, defined as the ratio of the area under the curve of light emission for δLuc:μYFP and the curve of light emission for δLuc alone, is shown in the Inset. (Lower) Light emission was monitored for cells coexpressing μLuc with either CCR5YFP (dash), or δYFP (black), or YFP (gray). Spectra in B expressed as BRET ratio is shown in Inset. (B) μ and δ receptor complexes can be isolated from spinal cord membranes. Solubilized spinal cord membranes from WT mice or mice lacking δ receptors (δ k/o) were subjected to immunoprecipitation by using 1 μg of anti-μ or anti-δ mAbs (μ-mAb or δ-mAb). Western blotting of immunocomplexes isolated by using anti-μ or anti-δ mAbs with δ polyclonal antibodies (Chemicon) detected δ receptors in membranes from WT mice but not from mice lacking δ receptors. Western blotting of immunocomplexes isolated by using anti-δ mAbs antibodies with μ polyclonal antibodies (gift from T. Cote) detected μ receptors in membranes from WT mice but not from mice lacking δ receptors (data not shown). (C and D) δ antagonists modulate the binding of ³H-DAMGO and ³H-morphine. (C) CHO cells stably expressing μ receptors or coexpressing μ and δ opioid receptors were incubated with increasing doses of ³H-DAMGO in the absence or presence of various ligands (10 nM). The incubations were carried out for 2 h at 37°C. Cells were washed in ice-cold 50 mM Tris·HCl, and radioactivity bound was detected as described (13). Results are mean ± SEM of three experiments in triplicate. (D) CHO cells stably expressing μ receptors, coexpressing μ and δ opioid receptors or SK-N-SH cells endogenously expressing these receptors were incubated with increasing doses of ³h-damgo or ³H-morphine in the absence or presence of various ligands (10 nM), and the radioactivity bound was detected as described (13). Results are mean ± SEM of three experiments in triplicate. ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001, Dunnett's test. NA, not available.

Fig. 2.

δ receptor ligands enhance μ receptor activity in cell lines expressing μ-δ receptors. Agonist-mediated [³⁵S]GTPγS binding in CHO cells coexpressing μ and δ receptors or in SK-N-SH cells endogenously expressing μ and δ receptors. Cells were permeabilized with 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in 50 mM Tris·HCl, pH 7.5, and were treated with indicated doses of morphine or damgo with or without 10 nM Delt II, TIPPψ, naltriben, or ICI 174,864, and the [³⁵S]GTPγS binding was measured as described (13). Basal values, determined in the absence of the μ agonist, but in the presence of δ ligands, were taken as 100%. Results are the mean ± SEM of three experiments in quadruplicate. ^**, P < 0.005; ^*, P < 0.01, Student's t test.

Fig. 3.

A variety of δ ligands enhance μ receptor signaling mediated by clinically relevant drugs. (A) Membranes from mouse spinal cords were treated with the indicated doses of damgo, morphine, fentanyl, or methadone with or without 10 nM of either TIPPψ or Delt II, and the [³⁵S]GTPγS binding was measured as described (13). Basal values, determined in the absence of μ agonist, but in the presence of δ ligands, were taken as 100%. Results are the mean ± SEM of four experiments in quadruplicate. (B) Membranes from WT mice and mice lacking δ receptors (δ k/o) were treated with 10^-7 m damgo (Left) or morphine (Right) with or without 10 nM TIPPψ (TIP), ICI174,864 (ICI), or naltriben, and [³⁵S]GTPγS binding was measured as described (13). Basal values, determined in the absence of μ agonist, but in the presence of δ ligands, were taken as 100%. Results are the mean ± SEM of three experiments in quadruplicate. ^***, P < 0.001, Student's t test.

Fig. 4.

The δ receptor antagonist enhances μ receptor activity in vitro and potentiates morphine analgesia in vivo. (A) Binding of 6 nM ³H-morphine to SK-N-SH cells endogenously expressing μ and δ receptors in the absence or presence of 10 nM TIPPψ. Specific binding (fmol/mg protein) obtained in the absence of TIPPψ is taken as 100%. Values represent the mean ± SEM of three experiments in triplicate. ^***, P < 0.001, t test. (B) Morphine (10^-7 M)-mediated [³⁵S]GTPγS binding to SK-N-SH cells endogenously expressing μ and δ receptors in the absence or presence of 10 nM TIPPψ. Basal values obtained in the absence of agonist treatment (but presence of antagonist) are taken as 100%. Results are the mean ± SEM of three experiments in quadruplicate. ^***, P < 0.001, t test. (C) Morphine (10^-9 M)-mediated inhibition of the levels of intracellular cAMP in SK-N-SH cells endogenously expressing μ and δ receptors in the absence or presence of 10 nM TIPPψ. Basal values obtained in the absence of agonist treatment (but presence of antagonist) are taken as 100%. Results are the mean ± SEM of three experiments in quadruplicate. ^***, P < 0.001, t test. (D) Morphine-mediated intrathecal analgesia in mice. Intrathecal analgesia was measured by the tail-flick assay 30 min after the injection of morphine (0.3 nmol, a dose that gives 20% maximal possible effect) in the absence or presence of 2 nmol of TIPPψ. Results are the mean ± SEM of 20 animals per group. ^**, P < 0.01, t test.