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, 30 (49), 16459-68

Ligand-directed Trafficking of the δ-Opioid Receptor in Vivo: Two Paths Toward Analgesic Tolerance

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Ligand-directed Trafficking of the δ-Opioid Receptor in Vivo: Two Paths Toward Analgesic Tolerance

Amynah A A Pradhan et al. J Neurosci.

Abstract

δ-Opioid receptors are G-protein-coupled receptors that regulate nociceptive and emotional responses. It has been well established that distinct agonists acting at the same G-protein-coupled receptor can engage different signaling or regulatory responses. This concept, known as biased agonism, has important biological and therapeutic implications. Ligand-biased responses are well described in cellular models, however, demonstrating the physiological relevance of biased agonism in vivo remains a major challenge. The aim of this study was to investigate the long-term consequences of ligand-biased trafficking of the δ-opioid receptor, at both the cellular and behavioral level. We used δ agonists with similar binding and analgesic properties, but high [SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)]- or low [ARM390 (N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide)]-internalization potencies. As we found previously, a single SNC80-but not ARM390-administration triggered acute desensitization of the analgesic response in mice. However, daily injections of either compound over 5 d produced full analgesic tolerance. SNC80-tolerant animals showed widespread receptor downregulation, and tolerance to analgesic, locomotor and anxiolytic effects of the agonist. Hence, internalization-dependent tolerance developed, as a result of generalized receptor degradation. In contrast, ARM390-tolerant mice showed intact receptor expression, but δ-opioid receptor coupling to Ca²+ channels was abolished in dorsal root ganglia. Concomitantly, tolerance developed for agonist-induced analgesia, but not locomotor or anxiolytic responses. Therefore, internalization-independent tolerance was produced by anatomically restricted adaptations leading to pain-specific tolerance. Hence, ligand-directed receptor trafficking of the δ-opioid receptor engages distinct adaptive responses, and this study reveals a novel aspect of biased agonism in vivo.

Figures

Figure 1.
Figure 1.
Acute SNC80, but not ARM390, produces transient receptor internalization and behavioral desensitization in DOR-eGFP mice. A, Desensitization (12 H) and recovery (24 H) of the analgesic response in the CFA tail model of inflammatory pain. Injection 1, Thermal responses in DOR-eGFP mice treated with vehicle (control), SNC80 (10 mg/kg), or ARM390 (10 mg/kg). Injection 2, Animals rechallenged 12 (left) or 24 h (right) following Injection 1. Dashed lines represent baseline thermal responses pre-CFA. For drug effects, ***p < 0.001, two-way repeated-measures ANOVA, n = 4–6 mice/group. Acute behavioral desensitization occurs for SNC80 only. B, Internalization and recovery of surface receptors 12 and 24 h after drug treatment. Brain and dorsal root ganglia were analyzed by confocal microscopy, and representative images of these regions are shown. Mean cell surface fluorescence was quantified in 3–4 sections per region per mouse. White bars, control group; gray bars, SNC80 group; black bars, ARM390 group; ***p < 0.001, one-way ANOVA, n = 3–4 mice/group. In vivo internalization is triggered by SNC80 only.
Figure 2.
Figure 2.
Chronic SNC80 and ARM390 both produce analgesic tolerance. Development of tolerance in two mouse strains and two pain models. All animals were tested every 24 h with vehicle (control), SNC80 (10 mg/kg), or ARM390 (10 mg/kg) for 5 d. Left, CFA tail, thermal responses in DOR-eGFP mice. Middle, CFA tail, thermal responses in wild-type C57BL/6J mice. Right, CFA paw, mechanical responses in wild-type C57BL/6J mice. Dashed lines represent baseline mechanical or thermal responses pre-CFA. n = 5–10 mice/group. Analgesic tolerance developed similarly for SNC80 and ARM390, independent of mouse strain or nociceptive endpoint.
Figure 3.
Figure 3.
Chronic SNC80 and ARM390 produce distinct adaptive responses at the receptor level. A, Subcellular DOR-eGFP localization. Brain and dorsal root ganglia were analyzed 24 h after the last drug treatment (day 6) by confocal microscopy, and representative images are shown. Mean cell surface fluorescence was quantified in 3–4 sections per region per mouse. White bars, control group; gray bars, SNC80 group; black bars, ARM390 group; ***p < 0.001, one-way ANOVA, n = 3–4 mice/group. Both SNC80 and ARM390 animals show surface fluorescence, but the signal is significantly reduced in the SNC80 group. B, Scatchard plot of [3H]naltrindole saturation binding performed on brain membranes. Experiments were performed in triplicate; n = 3–5 mice/group. Kd (nm) and Bmax (pmol/mg) values were comparable for control (0.13 ± 0.004 nm, 0.20 ± 0.02 pmol/mg) and ARM390 (0.10 ± 0.006 nm, 0.20 ± 0.03 pmol/mg) groups, while specific binding was undetectable in the SNC80 group. C, D, SNC80-induced [35S]GTPγS binding in brain (C) or spinal cord (D) membrane preparations. The y-axis shows mean ± SEM specific [35S]GTPγS binding expressed as percentage basal binding (i.e., absence of agonist). n = 3 mice/group. For brain, EC50 (nm) and Emax (percentage basal) values are comparable for control (466 ± 1.1 nm, 231 ± 3.3%) and ARM390-tolerant (302 ± 1.1 nm, 245 ± 3.5%) groups, while [35S]GTPγS binding was strongly attenuated (239 ± 1.3 nm, 140 ± 1.5%) in the SNC80-tolerant group. Similar results were observed in the spinal cord. EC50 and Emax values were as follows: control, 433 ± 1.2 nm, 167.4 ± 1.9%), ARM390-tolerant group (455 ± 1.2 nm, 176.2 ± 2.8%), and SNC80-tolerant group (220 ± 1.4 nm, 124.6 ± 1.4%). Chronic SNC80 produces DOR-eGFP receptor downregulation, while ARM390 leaves receptors at the cell surface.
Figure 4.
Figure 4.
Chronic SNC80 and ARM390 both produce analgesic cross-tolerance. SNC80-tolerant mice were challenged with ARM390 (10 mg/kg), and ARM390-tolerant mice were challenged with SNC80 (10 mg/kg). Graphs show tolerance and cross-tolerance data. A, CFA paw, mechanical response in DOR-eGFP mice. B, CFA tail, thermal response in C57BL/6J wild-type mice. Dashed lines represent baseline mechanical responses pre-CFA. n = 5 mice/group. Cross-tolerance occurs in both treatment groups. C, Repeated nociceptive testing produced associative tolerance. Inflammatory pain was induced in the paw of C57BL/6J mice, and 48 h later they were either tested daily for 5 d with vehicle (tested) or left in their home cage (novice). On day 6, mice were challenged with vehicle, SNC80 (10 mg/kg), or ARM390 (10 mg/kg). Dashed lines represent baseline mechanical responses pre-CFA. n = 5–6 mice/group. Antiallodynic effects of SNC80 or ARM390 were reduced in the habituated (tested) groups compared with corresponding novice drug groups (*p < 0.05, **p < 0.01), indicating that associative tolerance had developed. Nevertheless, in the habituated/tested animals, the two agonists continued to produce significant antiallodynic effects (##p < 0.01 compared with habituated vehicle controls), demonstrating that associative tolerance only partially accounts for the full tolerance and cross-tolerance observed in chronically SNC80- or ARM390-treated animals.
Figure 5.
Figure 5.
Chronic ARM390 induces analgesic tolerance only, leaving other δ-opioid receptor responses intact. A, SNC80-induced internalization in SNC80- and ARM390-tolerant animals. Striatum, hippocampus, and dorsal root ganglia were analyzed by confocal microscopy, and representative images are shown. Mean cell surface fluorescence was quantified in 3–4 sections per region per mouse. White bars, control group; gray bars, SNC80 group; black bars, ARM390 group; *p < 0.05, ***p < 0.001, one-way ANOVA, n = 3–4 mice/group. After chronic ARM390, surface δ receptors can be internalized by SNC80. B, SNC80-induced locomotor activation in SNC80- and ARM390-tolerant animals. Wild-type C57BL/6J mice were chronically treated with vehicle, SNC80, or ARM390, following which they were challenged with either vehicle or SNC80 (3 mg/kg, i.p.). For drug effects, **p < 0.01, as determined by multiple t tests with Bonferroni corrections, n = 5–6 mice/group. Control and ARM390-tolerant animals, but not SNC80-tolerant animals, showed SNC80-induced locomotor activation. C, SNC80-induced anxiolysis in SNC80- and ARM390-tolerant animals. Wild-type C57BL/6J mice were made tolerant to SNC80 or ARM390, and all groups were challenged with either vehicle or SNC80 (10 mg/kg, s.c.), and tested in the elevated plus maze. Data represent the percentage time spent in the open arms compared with total time spent in open and closed arms. For drug effects, **p < 0.01, *p < 0.05 as determined by multiple t tests with Bonferroni corrections, n = 5–6 mice/group. Control and ARM390-tolerant animals, but not SNC80-tolerant animals, showed an SNC80-induced anxiolysis. Altogether, chronic SNC80 produces generalized behavioral tolerance, while chronic ARM390 induces analgesic tolerance only.
Figure 6.
Figure 6.
Chronic SNC80 and ARM390 both reduce δ receptor-mediated inhibition of Ca2+ channels in DRGs. The inhibitory effect of SNC80 (1 μm) on voltage-dependent Ca2+ channels was measured in medium-large DRG neurons. A, B, Exemplar currents from SNC80-responsive (A) or -nonresponsive (B) DRGs. (1, baseline depolarization; 2, SNC80-induced inhibition). Calibration: 10 ms, 20 mA on the x- and y-axes, respectively. C, SNC80 inhibited Ca2+ currents in ipsilateral DRGs from CFA (V, 33 ± 11%), but not naive (N, −0.13 ± 0.87%) animals. Both chronic SNC80 (S) and ARM390 (A) treatments reduced this effect to levels seen in naive DRGs (11.7 ± 5.3 and 0.12 ± 2.4%, respectively). Cumulative data from 6–14 cells are presented showing percentage SNC80 inhibition of Ca2+ currents; *p < 0.05, ***p < 0.001.
Figure 7.
Figure 7.
Two distinct paths toward analgesic tolerance. The scheme summarizes receptor analysis at the cellular level (subcellular localization, receptor number, G-protein coupling, and Ca2+ channel activity) and behavioral data obtained throughout this study. Data highlight the distinct adaptive processes elicited by chronic treatment using either the high-internalizing agonist (SNC80, left) or the low-internalizing agonist (ARM390, right). Top, A single 24 h treatment cycle leads to restored or intact receptors, with seemingly identical functional properties both at cellular and behavioral levels. Bottom, Five 24 h treatment cycles produce highly distinct forms of tolerance. Chronic SNC80 downregulates receptors throughout the nervous system, as classically described in cellular models, leading to generalized tolerance at the behavioral level. Chronic ARM390 does not modify receptor expression at the cell surface, but decreases δ receptor-mediated Ca2+ channel responses in DRGs. Further, tolerance develops only for analgesic but not locomotor or emotional responses, indicating that in vivo adaptations occur specifically at the level of pain processing pathways.

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