Regulation of delta-opioid receptor trafficking via mu-opioid receptor stimulation: evidence from mu-opioid receptor knock-out mice

Abstract

We recently demonstrated that prolonged treatment with morphine increases the antinociceptive potency of the delta-opioid receptor (deltaOR) agonist deltorphin and promotes cell surface targeting of deltaORs in neurons of the dorsal horn of the rat spinal cord (Cahill et al., 2001b). In the present study we examined whether these effects were mediated selectively via muOR. Using the same intermittent treatment regimen as for morphine, we found that methadone and etorphine, but not fentanyl, enhanced [D-Ala2]-deltorphin-mediated antinociception. However, continuous delivery of fentanyl for 48 hr resulted in augmented deltaOR-mediated antinociception when compared with saline-infused animals. Time course studies confirmed that a 48 hr treatment with morphine was necessary for the establishment of enhanced deltaOR-mediated antinociception. The observed increases in deltaOR agonist potency and deltaOR plasma membrane density were reversed fully 48 hr after discontinuation of morphine injections. Wild-type C57BL/6 mice pretreated with morphine for 48 hr similarly displayed enhanced deltaOR-mediated antinociception in a tonic pain paradigm. Accordingly, the percentage of plasma membrane-associated deltaOR in the dorsal horn of the spinal cord, as assessed by immunogold electron microscopy, increased from 6.6% in naive to 12.4% in morphine-treated mice. In contrast, morphine treatment of muOR gene knock-out (KO) mice did not produce any change in deltaOR plasma membrane density. These results demonstrate that selective activation of muOR is critical for morphine-induced targeting of deltaOR to neuronal membranes, but not for basal targeting of this receptor to the cell surface.

Figure 1.

Comparative antinociceptive potencies of four μOR agonists in an acute pain paradigm. Left panels, Tail-flick latencies (in sec) were determined every 10 min subsequent to intraperitoneal administration (time 0, denoted by dotted line) of morphine (A), methadone (B), etorphine (C), and fentanyl (D) at various doses. Right panels, The percentage of the maximum possible effect (% MPE) was calculated at the time of peak antinociceptive response for each dose: 40 min for morphine (A), 30 min for methadone (B), 30 min for etorphine (C), and 20 min for fentanyl(D). The theoretical equation of the line (sigmoidal dose–response) is shown as a dotted curve (right panel). ED₅₀ values were estimated from the theoretical curve at an MPE of 50% (dotted horizontal line, right panel). Calculations were performed with Excel 97 (Microsoft) and Prism 3.02 (Graph Pad Software). Data are presented as the average ± SEM.

Figure 2.

Effect of chronic pretreatment withμOR agonists onδOR-mediated antinociception. A, Morphine, methadone, etorphine, and fentanyl were administered at 12 hr intervals for 36 hr as described in the Materials and Methods. Then 12 hr subsequent to the last injection of each opioid ligand, 10 μg of DLT was administered intrathecally, and tail-flick latencies (in sec) were recorded every 10 min. B, Each bar in the graph represents the percentage of maximal possible effect (MPE) ± SEM 20 min after the injection of DLT. The percentages of MPE for morphine (n = 4; p < 0.01), methadone (n = 6; p < 0.01), and etorphine (n = 6, p < 0.05) were significantly different from the % MPE of non-pretreated rats (n = 13; ANOVA, Tukey's MCT; denoted by an asterisk). No significant difference was found between the % MPE for fentanyl-pretreated (n = 5) and non-pretreated rats (p > 0.05; ANOVA, Tukey's MCT) or among the percentages of MPE for morphine, methadone, and etorphine (p > 0.05; ANOVA, Tukey's MCT).

Figure 3.

Continuous administration of fentanyl leads to enhanced DLT-mediated antinociception. A, Rats were implanted subcutaneously with osmotic mini-pumps for continuous delivery of 0.1 μg/hr fentanyl over 48 hr; saline controls were implanted osmotic pumps delivering 1 μl of 0.9% saline/hr for 48 hr. Pumps were removed 4 hr before antinociceptive testing. Baseline latencies were measured before and after the removal of the pumps and every 10 min subsequent to the intrathecal administration of DLT. B, Each bar in the graph represents the % MPE ± SEM calculated 20 min after the injection of DLT (n = 5 for each group). Statistical significance was determined by the means of an unpaired Student's t test (^*p < 0.05).

Figure 4.

A, Time course for the establishment of enhancedδOR-mediated antinociception. Rats were treated with morphine sulfate (MS) at 5 and 8 mg/kg (subcutaneously at 12 hr intervals); 5, 8, and 10 mg/kg (subcutaneously at 12 hr intervals); or 5, 8, 10, 15 mg/kg (subcutaneously at 12 hr intervals). Tail-flick latency responses to intrathecal injections of DLT were determined 12 hr subsequent to the last morphine injection. The data were expressed as a function of the time that had elapsed between the first morphine injection and the time of testing (i.e., MS 24, 36, and 48 hr). Saline controls were injected subcutaneously with 0.9% saline every 12 hr (4 injections) and were tested 12 hr after the last injection (Saline 48 hr). Each bar represents the % MPE ± SEM 20 min after the injection of DLT. Morphine treatment for 48 hr (p < 0.001), but not treatment for 24 or 36 hr (p > 0.05 in both cases), produced DLT-mediated antinociception that was significantly greater than that observed in saline-injected animals (denoted by an asterisk). Statistical significance was determined by ANOVA, followed by Dunnett's MCT. B, Evaluation of antinociceptive tolerance to continuous morphine treatment. Rats were pretreated or not with morphine (MS) at 5, 8, 10, and 15 mg/kg (subcutaneously every 12 hr). At 12 hr after the last morphine administration the rats were injected intraperitoneally with morphine at 1, 3, 5, or 10 mg/kg; tail-flick latency responses were determined every 10 min for 50 min. Each data point represents the % MPE ± SEM 30 min after the morphine challenge (n = 4–6 for each point). No statistical difference at any of the doses was observed between untreated (dotted) and morphine-pretreated (solid line) rats (unpaired t tests, p > 0.05). Calculations were performed with Excel 97 (Microsoft) and Prism 3.02 (Graph Pad Software).

Figure 5.

Duration of enhanced DLT-mediated antinociception. Rats were injected with 5, 8, 10, 15 mg/kg morphine (MS; subcutaneously at 12 hr intervals). DLT-mediated antinociception was determined 12 hr (n = 11), 24 hr (n = 6), 36 hr (n = 5), and 48 hr (n = 5) subsequent to the last morphine injection. The data were expressed as a function of the time that had elapsed between the last morphine injection and the time of testing (i.e., 12, 24, and 48 hr after MS). Saline controls were injected subcutaneously with 0.9% saline every 12 hr (4 injections) and were tested for DLT-mediated antinociception 12 hr after the last saline injection. Each bar represents the % MPE ± SEM 20 min after the injection of DLT. The asterisk denotes a statistically significant increase when compared with saline-treated controls (p < 0.001; ANOVA, Dunnett's MCT).

Figure 6.

Subcellular distribution ofδOR 48 hr after the last dose of the morphine pretreatment regimen. Rats (n = 3) were injected with 5, 8, 10, 15 mg/kg morphine (MS; subcutaneously at 12 hr intervals) and were processed for immunogold detection of δORs 12 and 48 hr subsequent to the last morphine injection (12 and 48 hr post-MS, respectively). Untreated rats were processed for electron microscopic detection of δORs in an analogous manner. Each bar represents the proportion of immunogold particles associated with the plasma membrane as a percentage of the total ± SEM. This percentage is not significantly different between 48 hr post-MS and untreated rats (ANOVA, Tukey's MCT; p > 0.05) but is significantly greater at 12 hr post-MS rats when compared with either 48 hr post-MS or untreated rats (ANOVA, Tukey's MCT; p < 0.01; denoted by an asterisk).

Figure 7.

Prolonged treatment with morphine leads to enhanced δOR-mediated antinociception in C57BL/6 mice. WT male and female mice were pretreated (B) or not (A) with morphine sulfate at 5, 8, 10, 15 mg/kg (subcutaneously every 12 hr). At 12 hr after the last morphine injection, saline or 5 μg of DLT was injected intrathecally. In C57BL/6 mice an intraplantar injection of formalin (2.5%) produced the biphasic nociceptive response typical of this persistent pain model (A, B). Nocifensive behaviors in female (n = 4–6 per group) and male (n = 4–7 per group) mice were assessed in 5 min intervals with a weighted score. Because nocifensive scores were not statistically different between males and females (see Table 1), pooled male and female scores are illustrated. C, Pooled area under the curve (A.U.C.) for male and female mice. The A.U.C. values for intrathecal saline in morphine and untreated animals are not significantly different. However, there is a statistically significant decrease in A.U.C. between saline-treated mice and morphine-pretreated mice intrathecally injected with DLT (ANOVA, Bonferroni MCT; p < 0.001; denoted by an asterisk). Data are presented as the average ± SEM.

Figure 8.

Subcellular localization of δOR immunoreactivity in the dorsal horn of the spinal cord of untreated versus morphine-pretreated wild-type mice. C57BL/6 WT mice were pretreated (D–F) or not (A–C) with morphine as described and were processed for immunogold detection of δORs 12 hr subsequent to the last morphine injection. In both conditions the majority of silver-intensified immunogold particles, representing δORs, is found inside dendrites (labeled D in A–F). In untreated mice a small proportion of gold particles is detected in association with the plasma membrane (denoted by arrows, A–C). However, pretreatment with morphine increases the occurrence of δORs associated with dendritic plasma membranes (arrows, D–F). At, Axon terminal. Scale bars: A–F, 0.5 μm.

Figure 10.

The presence of μOR is necessary for morphine-induced targeting of δOR to plasma membranes. A, Each bar represents the density of immunogold particles (particles/μm ²; n = 3 for each group). No statistically significant differences were observed (ANOVA; p > 0.05). B, Each bar in the graph represents the density of immunogold particles per unit length of plasma membrane. Morphine pretreatment markedly increases the density (from 0.09 to 0.18) in WT mice as compared with untreated controls (ANOVA, Bonferroni's MCT; ^*p < 0.01). No statistical difference was found between the density of immunogold particles per unit length of membrane in morphine-pretreated as compared with untreated μOR KO mice (ANOVA, Bonferroni's MCT; p > 0.05). C, The percentage of gold particles associated with the plasma membrane was increased significantly in WT mice pretreated with morphine when compared with untreated WT mice (ANOVA, Bonferroni's MCT; ^**p < 0.001). No change in the percentage of δOR associated with the plasma membrane was observed between untreated and morphine-pretreated μOR KO mice (ANOVA; p > 0.05). In all panels +/+ denotes the WT, whereas -/- represents μOR KO C57BL/6 mice. Data are presented as the average ± SEM.

Figure 9.

Immunogold electron microscopic localization of δOR in the dorsal horn of the spinal cord of untreated and morphine-pretreated μOR knock-out mice. C57BL/6 KO mice were pretreated (D–F) or not (A–C) with morphine as described and were processed for immunogold detection ofδORs 12 hr subsequent to the last morphine injection. In both untreated (A–C) and morphine-pretreated (D–F)μOR KO mice the majority of immunogold particles is found within dendrites (labeled D in A–F). Pretreatment with morphine does not change the proportion ofδORs associated with the plasma membrane. Arrows denote gold particles in association with the plasma membrane. At, Axon terminal. Scale bars: A–F, 0.5 μm.