Agonist-dependent development of delta opioid receptor tolerance in the colon

Abstract

The use of opioid analgesics is severely limited due to the development of intractable constipation, mediated through activation of mu opioid receptors (MOR) expressed by enteric neurons. The related delta opioid receptor (DOR) is an emerging therapeutic target for chronic pain, depression and anxiety. Whether DOR agonists also promote sustained inhibition of colonic transit is unknown. This study examined acute and chronic tolerance to SNC80 and ARM390, which were full and partial DOR agonists in neural pathways controlling colonic motility, respectively. Excitatory pathways developed acute and chronic tolerance to SNC80, whereas only chronic tolerance developed in inhibitory pathways. Both pathways remained functional after acute or chronic ARM390 exposure. Propagating colonic motor patterns were significantly reduced after acute or chronic SNC80 treatment, but not by ARM390 pre-treatment. These findings demonstrate that SNC80 has a prolonged inhibitory effect on propagating colonic motility. ARM390 had no effect on motor patterns and thus may have fewer gastrointestinal side-effects.

Keywords: Colon motility; Endocytosis; Enteric nervous system; GPCR regulation; Opioid receptor.

Fig. 1

DOR agonists have a prolonged inhibitory effect on electrically stimulated contractions of the colon. a SNC80 and ARM390 inhibited EFS-evoked contractions of the colon in a concentration-dependent manner. A single addition of the DOR agonists SNC80 (c), ARM390 (d) or DADLE (e) suppressed EFS-evoked contractions for the duration of the experimental time course (75 min). In contrast, EFS-evoked contractions were partially recovered after a 15-min recovery period following DAMGO exposure (f). Bars indicate drug exposure. Red circles represent when EFS was applied. b Quantitative analysis demonstrating the recovery of EFS-evoked contractions over time. Data points are expressed as mean ± SEM, n = 4–6 mice per treatment. Statistical comparisons for the SNC80, DADLE and DAMGO data were conducted using one-way repeated measures ANOVA followed by Dunnett’s post hoc test (**p < 0.01 and ***p < 0.001 compared to the initial response by agonist). Statistical analyses for the ARM390 data were performed using Friedman’s test followed by Dunn’s post hoc analysis

Fig. 2

DOR agonist-evoked contractions are desensitized. a, b SNC80- and c, d ARM390-evoked contractions were significantly reduced following repeated exposures to agonists. e, f In contrast, contractions evoked by the MOR agonist DAMGO were retained with subsequent DAMGO additions. Arrows indicate agonist addition. Data points represent mean ± SEM, n = 5 mice per treatment. Statistical comparison performed by one-way repeated measures ANOVA followed by Dunnett’s post hoc test (*p < 0.05 and **p < 0.01 compared to the initial addition)

Fig. 3

Myenteric excitatory motor pathways develop acute and chronic tolerance to SNC80. a, b Timeline of the acute and chronic tolerance experiments. Bath addition of the DOR agonist SNC80 (1 µM) diminished EFS-evoked contractions in colons from c acute and d chronic vehicle-treated mice. Agonist-mediated inhibition of electrically stimulated contractions was reduced in the e acute and f chronic SNC80-pretreated group, indicative of tolerance. SNC80 suppressed EFS-evoked contractions of colons after g acute and h chronic exposure to ARM390, indicating that DOR function was retained. Quantitative analysis of the SNC80-evoked reduction in EFS-evoked contractions from the i acute and j chronic agonist pre-treated groups. Circles represent where EFS was applied. Data points are expressed as mean ± SEM, n = 5–13 mice per treatment group. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test (*p < 0.05)

Fig. 4

Myenteric inhibitory motor pathways develop acute, but not chronic, tolerance to SNC80. The DOR agonist SNC80 (1 µM) evoked a robust contraction in colons following acute (a) and chronic (b) treatment with vehicle. These effects were abolished in the acute (c), but not the chronic (d) SNC80 pretreated group. Application of SNC80 also evoked a robust contraction in the acute (e) and chronic (f) ARM390-treated group. Quantitative analysis of SNC80-evoked contractions following acute (g) or chronic (h) drug treatment. Arrows represent the bath addition of SNC80 (1 µM). Data points represent the mean ± SEM, n = 5–12 mice per treatment group. Acute tolerance data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Statistical comparisons for the chronic tolerance data were performed using Kruskal–Wallis’s test followed by Dunn’s multiple comparison test (**p < 0.01)

Fig. 5

Acute administration of SNC80 has a prolonged inhibitory effect on CMMC frequency. a The number of CMMCs generated in the acute vehicle-treated group was diminished after the bath addition of SNC80 (1 µM). b CMMC frequency was significantly reduced under basal conditions in the acute SNC80-treated group compared to the vehicle-treated group. Subsequent bath application of SNC80 had no further effect on CMMC frequency. c The number of CMMCs generated in colons from the ARM390-treated group under basal conditions was similar to that in the vehicle-treated group. Application of SNC80 significantly reduced CMMC frequency, indicating that DOR retained function under these conditions. d Quantitative analysis of the changes in CMMC frequency for each treatment under basal conditions and following bath addition of SNC80. Representative CMMCs are indicated by yellow arrows. Data are expressed as mean ± SEM, n = 10 mice per treatment group. Statistical analyses were conducted using two-way repeated measures ANOVA. Dunnett’s multiple comparison test was used to compare means across treatment groups. Sidak’s multiple comparison test was used to compare means within treatment groups (*p < 0.05 and ***p < 0.001)

Fig. 6

The effects of chronic DOR activation on CMMC frequency are agonist-dependent. a The in vitro addition of SNC80 (1 µM) significantly reduced CMMC frequency in the chronic vehicle-treated group. b The number of CMMCs generated in the chronic SNC80-treated group were significantly lower under basal conditions relative to vehicle. Bath addition of SNC80 had no further effect on CMMC frequency. c CMMC frequency was similar under basal conditions in the ARM390-treated group compared to the vehicle. Subsequent bath addition of SNC80 significantly reduced CMMC frequency (d). Quantitative analysis of the changes in CMMC frequency for each treatment under basal conditions and following bath addition of SNC80. Yellow arrows indicate representative CMMCs. Data are expressed as mean ± SEM, n = 8–10 mice per treatment group. Statistical analyses were performed using two-way repeated measures ANOVA. Dunnett’s multiple comparison test was used to compare means across treatment groups. Sidak’s multiple comparison test was conducted to compare means within treatment groups

Fig. 7

DOReGFP trafficking is an agonist and time-dependent process. a In vitro treatment with SNC80 (1 µM), but not ARM390 (1 µM), for 30 min resulted in significant internalization of DOReGFP in the soma of myenteric neurons (arrowheads: internalization, arrows: no internalization). No change in DOReGFP distribution in circular muscle nerve fibers was detected. b Acute in vivo administration of both SNC80 and ARM390 (10 mg/kg, 3 h) resulted in significant redistribution of DOReGFP to punctate endosome-like structures. SNC80, but not ARM390, exposure promoted endocytosis in circular muscle nerve fibers (arrowheads). c Chronic in vivo administration of SNC80, but not ARM390 (both 10 mg/kg, 3 days), was associated with robust DOReGFP internalization in both the soma and nerve fibers of myenteric neurons. d Graph summarizing the subcellular distribution of DOReGFP in myenteric neurons under the different treatment conditions tested. e Pixel densitometry data measuring DOReGFP subcellular distribution in circular muscle nerve fibers under the different treatment conditions. Scale: 10 µm. Data are expressed as mean ± SEM, n = 3–5 mice per treatment group. Statistical comparisons performed using Kruskal–Wallis’s test followed by Dunn’s multiple comparison test (**p < 0.01, ***p < 0.001)

Fig. 8

Schematic summarizing the link between DOReGFP trafficking and the regulation of DOR agonist-mediated effects on neuromuscular transmission in the colon. Under basal conditions, DOReGFP was mainly localized to the plasma membrane and the addition of a DOR agonist produced a robust functional response. After 30 min, SNC80 promoted internalization of DOReGFP in the soma and proximal neurites, while ARM390 had no effect. Functional responses to both ARM390 and SNC80 were desensitized in inhibitory motor pathways, whereas they retained their efficacy in excitatory pathways. This demonstrates that DOR endocytosis is not a prerequisite for the desensitization of agonist-mediated responses. After an acute exposure (3 h) to SNC80, DOReGFP was significantly internalized in all compartments of the neuron. In contrast, a significant proportion of total DOReGFP remained at the plasma membrane following acute ARM390 treatment. The internalization profile correlated with the susceptibility to tolerance development in assays using muscle strips. Agonist-mediated internalization also correlated with prolonged inhibitory effects on motility. DOReGFP was significantly internalized in both the soma and nerve fibers following chronic treatment (3 days) with SNC80. However, there was a significant increase in DOReGFP associated with the plasma membrane of the soma compared to the respective acute treatment group. In addition, DOReGFP internalization was more pronounced in the nerve fibers compared to the acute SNC80-treatment group. DOReGFP was retained at the cell surface following chronic treatment with ARM390. DOR internalization was correlated with both the development of tolerance in assays using muscle strips and the sustained inhibition of colonic motility