Opioid-galanin receptor heteromers mediate the dopaminergic effects of opioids

Abstract

Identifying non-addictive opioid medications is a high priority in medical sciences, but μ-opioid receptors mediate both the analgesic and addictive effects of opioids. We found a significant pharmacodynamic difference between morphine and methadone that is determined entirely by heteromerization of μ-opioid receptors with galanin Gal1 receptors, rendering a profound decrease in the potency of methadone. This was explained by methadone's weaker proficiency to activate the dopaminergic system as compared to morphine and predicted a dissociation of therapeutic versus euphoric effects of methadone, which was corroborated by a significantly lower incidence of self-report of "high" in methadone-maintained patients. These results suggest that μ-opioid-Gal1 receptor heteromers mediate the dopaminergic effects of opioids that may lead to a lower addictive liability of opioids with selective low potency for the μ-opioid-Gal1 receptor heteromer, exemplified by methadone.

Keywords: Addiction; G-protein coupled receptors; Neuroscience; Therapeutics.

Figure 1. Gal1R-dependent allosteric modulation of MOR agonists.

(A and B) Effect of the MOR antagonist CTOP and the Gal1R/Gal2R antagonist M40 on DMR induced by the MOR agonists EM1 (A) and morphine (B) in MU and MU-Gal1R cells. Values are shown as dots and the mean ± SEM (n = 5–6 triplicates/group). ^###P < 0.001 versus EM1; 1-way ANOVA with Tukey’s multiple comparisons test. (C) Effect of CTOP and M40 on MAPK activation induced by EM1 in MU-GAL cells. Values are shown as dots and the mean ± SEM n = 6–15 triplicates/group). ***P < 0.001 versus control; ^###P < 0.001 versus EM1; 1-way ANOVA with Tukey’s multiple comparisons test. (D) Effect of CTOP and M40 on internalization of MOR induced by the MOR agonist DAMGO and lack of MOR-induced internalization by the Gal1R agonist M617 in MU-GAL cells. Values are shown as dots and the mean ± SEM (n = 6 triplicates/group). ***P < 0.001 versus control; ^###P < 0.001 versus control DAMGO; 1-way ANOVA with Tukey’s multiple comparisons test. (E and F), Representative competitive inhibition experiments of [³H]DAMGO versus DAMGO in membrane preparations from MU (E) and MU-GAL (F) cells with or without M617 or M40. Values are expressed as the mean ± SEM of triplicates. See Results and Supplemental Figure 2 for the total number of experiments and statistical comparisons. Concentrations of agonists and antagonists were always 0.1 μM and 1 μM, respectively.

Figure 2. Gal1R-dependent pharmacodynamic differences of MOR agonists.

(A–E) Representative concentration-response experiments of ligand-induced BRET changes in HEK-293T cells transfected with the MOR fused to Rluc and the α subunit of the Gi1 protein fused to YFP. Values represent the mean ± SEM of triplicates. The effect of increasing concentrations of the MOR agonists morphine (A), EM1 (B), DAMGO (C), fentanyl (D), and methadone (E) were evaluated without cotransfection with Gal1R, in the absence or presence of the Gal1R/Gal2R antagonist M40 (red and green curves, respectively), or with cotransfection with Gal1R, with or without M40 (blue and purple curves, respectively). (F and G) Comparison of the E_max and E₅₀ values obtained with and without cotransfection with the Gal1R. **P < 0.01 versus transfection with MOR alone; unpaired, 2-tailed t test (F); **P < 0.01 versus transfection with MOR alone; 2-tailed Mann-Whitney U test (G). E_max and EC₅₀ values are shown as dots, presented with the mean ± SEM (F) or median with interquartile ranges (G) (n = 5–10 triplicates/group).

Figure 3. Weaker ability of methadone to stimulate the VTA-NAc dopaminergic system as compared with morphine, fentanyl, and DAMGO.

Microdialysis experiments in rats. Values represent mean dopamine concentrations as a percentage of baseline ± SEM (average of 5 samples before MOR agonist administration). The lined and white rectangles in the x axis indicate the period of MOR agonist perfusion and M40 infusion, respectively; the arrows in D and F indicate the time point of systemic administration. (A and B) Effect of intra-VTA morphine (1–10 μM) or methadone (10–300 μM) on VTA dopamine. *P < 0.05 and ***P < 0.001 versus 1 μM morphine (A); *P < 0.05 and **P < 0.01 versus 10 μM methadone (B); 1-way ANOVA with Dunnett’s multiple comparisons, comparing the average of 8 samples after MOR agonist administration (n = 7–8 animals/group). (C) Effect of intra-NAc morphine (10 μM) or methadone (300 μM) on NAc dopamine. Results were nonsignificant in both cases; paired, 2-tailed t test, comparing the average of 8 samples after MOR agonist administration versus baseline values (n = 8 animals/group). (D) Effect of systemic administration (1 mg/kg, i.p.) of morphine or methadone on VTA and NAc dopamine in the VTA and contralateral NAc. *P < 0.05; paired, 2-tailed t test, comparing the average of 5 samples after MOR administration versus baseline values (n = 6–7 animals/group). (E) Effect of intra-VTA DAMGO (10 μM) or fentanyl (10 μM) on VTA dopamine, respectively (n = 7 and 9 animals/group). **P < 0.01; paired, 2-tailed t test, comparing the average of 8 samples after MOR agonist administration versus baseline values. (F) Effect of systemic administration (0.03 mg/kg, i.p.) of fentanyl on VTA and NAc dopamine in the VTA and contralateral NAc. **P < 0.01; paired, 2-tailed t test, comparing the average of 8 samples after MOR administration versus baseline values (n = 7 animals/group). DA, dopamine.

Figure 4. Differential ability of morphine and methadone to influence metabolic activity in the basal forebrain.

Metabolic mapping using [¹⁸F]FDG PET scanning in rats. (A) Timeline of the experiment. (B) [¹⁸F]FDG uptake after administration of saline (baseline, n = 14), morphine (1 mg/kg, n = 7), or methadone (1 mg/kg, n = 7). Coronal and sagittal images (1.5 mm anterior to bregma and 1.4 mm lateral from the midline, respectively) show the average SUV_R calculated using the whole brain as a reference region. (C) Voxel-based parametric mapping analyses revealed significantly decreased metabolic activity from baseline values in a basal forebrain region that included the NAc and its projecting areas after morphine, but not methadone, treatment. Statistical parametric maps of significant decreases of [¹⁸F]FDG uptake (P < 0.05, paired t test). (D and E) VOI analyses of the frontal cortex (FCx), dorsal striatum (DS), and basal forebrain (BF) region, showing a significant differential pattern of [¹⁸F]FDG uptake after administration of morphine (D) or methadone (E). Values are shown as dots and as the median with interquartile ranges. *P < 0.05 versus the corresponding baseline value; 2-tailed Wilcoxon matched-pairs test (n = 7 animals/group).

Figure 5. Very low reporting of feeling “high” by methadone-treated subjects.

(A) Total number and proportion of patients with RLS reporting feeling “high” upon treatment with methadone or with other MOR agonists (other opioids). **P < 0.01, significantly different proportion of subjects versus patients treated with methadone; 2-sided χ² test. (B) Assessment of symptoms of craving and withdrawal (according to the SOWS) and perceived severity of methadone-associated euphoria (“high”). Both measurements (SOWS and “high”) were obtained after 14 and 84 days of methadone treatment. Values are shown as dots and as the median with interquartile ranges. **P < 0.01 versus 14 days of treatment; 2-tailed Wilcoxon matched-pairs test (n = 30). (C) Total number and proportion of patients with OUD reporting first-time nonmedical use of methadone or other MOR agonists (other opioids) with the express intent of achieving a “high” (and not for alleviating withdrawal symptoms or for other purposes; see Methods). **P < 0.01, significantly different proportion of subjects seeking a “high” with other opioids versus with methadone; 2-sided χ² test.