The role of β-arrestin2 in the mechanism of morphine tolerance in the mouse and guinea pig gastrointestinal tract

Abstract

β-Arrestin2 has been reported to play an essential role in analgesic tolerance. Analgesic tolerance without concomitant tolerance to constipation is a limiting side effect of chronic morphine treatment. Because tolerance to morphine develops in the mouse ileum but not the colon, we therefore examined whether the role of β-arrestin2 in the mechanism of morphine tolerance differs in the ileum and colon. In both guinea pig and mouse, chronic in vitro exposure (2 h, 10 μM) to morphine resulted in tolerance development in the isolated ileum but not the colon. The IC(50) values for morphine-induced inhibition of electrical field stimulation contraction of guinea pig longitudinal muscle myenteric plexus shifted rightward in the ileum from 5.7 ± 0.08 (n = 9) to 5.45 ± 0.09 (n = 6) (p < 0.001) after morphine exposure. A significant shift was not observed in the colon. Similar differential tolerance was seen between the mouse ileum and the colon. However, tolerance developed in the colon from β-arrestin2 knockout mice. β-Arrestin2 and extracellular signal-regulated kinase 1/2 expression levels were determined further by Western blot analyses in guinea pig longitudinal muscle myenteric plexus. A time-dependent decrease in the expression of β-arrestin2 and extracellular signal-regulated kinase 1/2 occurred in the ileum but not the colon after 2 h of morphine (10 μM) exposure. Naloxone prevented the decrease in β-arrestin2. In the isolated ileum from guinea pigs chronically treated in vivo with morphine for 7 days, neither additional tolerance to in vitro exposure of morphine nor a decrease in β-arrestin2 occurred. We conclude that a decrease in β-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

Fig. 1.

Morphine-induced contractions in wild-type C57BL/6 and β-arrestin2 knockout mice. Bar graphs depict percentile contractions to repeated morphine (3 μM) exposures in circular muscle rings from the ileum and colon of wild-type C57BL/6 and β-arrestin2 knockout mice. The initial contraction to morphine was given a value of 100%, and each subsequent administration (second, third, and fourth) was normalized to the initial contraction (first). *, p < 0.05, paired Student's t test; n = 4–6. See Supplemental Fig. 1 for typical raw tracings from the ileum and colon.

Fig. 2.

β-Arrestin2 expression in mouse and guinea pig. Western blot analyses of β-arrestin2 (arrow) from protein samples of mouse brain and ileum and guinea pig ileum and colon LMMP preparations are shown. β-Arrestin2 was detected from the brain samples of C57BL/6 mice but only faintly seen in the mouse ileum and absent in the brain samples from the β-arrestin2 knockout mice. A robust band was present in the guinea pig colon and ileum LMMP. The specificity of the antibody was confirmed further by preabsorption with antigen peptide (Supplemental Fig. 2).

Fig. 3.

A, raw tracing of dose-response curves to morphine on EFS-induced contractions in the ileum (top) and colon (bottom) from guinea pig LMMP. An initial dose-response curve to morphine was followed by 2 h of incubation with 10 μM morphine (indicated by the gap in the trace). A dose-response curve to morphine then was repeated. B and C, dose-response curves for morphine-induced inhibition of EFS-induced contractions in the guinea pig ileum (B) and colon (C) LMMP. Open circles represent control and closed circles after 2 h of incubation with morphine (10 μM). Cumulative dose-response curves were carried out on each strip (n = 6–9 strips from at least five guinea pigs). A two-way ANOVA followed by Bonferroni's post hoc test was used to measure significance. *, p < 0.001.

Fig. 4.

Down-regulation of β-arrestin2 in the guinea pig ileum by chronic morphine exposure. A and B, protein samples were obtained from isolated LMMP from guinea pig colon and ileum that were exposed to either Krebs' solution (control) or morphine (10 μM for 2 h) and were subject to immunoblot with anti-GRK2 (A) or anti-β-arrestin2 (B) antibodies. Morphine treatment (10 μM for 2 h) did not alter the expression levels of GRK2 in either tissue but markedly reduced β-arrestin2 expression in the ileum. Equal amounts of protein loading were confirmed by the anti-GAPDH antibody. Bar graphs represent the ratio of GRK2 to GAPDH (A) or β-arrestin2 to GAPDH (B). Data represent mean ± S.E. (n = 6). *, p < 0.05 versus control sample, unpaired t test.

Fig. 5.

Expression of β-arrestin2 in guinea pig ileum neurons. A, immunohistochemical localization of β-arrestin2 immunoreactivity in a whole-mount preparation showing localization within enteric ganglia. B, myenteric neurons were isolated enzymatically from control and morphine-treated ileum LMMP. β-Arrestin2 expression was decreased significantly in morphine-treated neurons. Equal loading of neuronal samples was confirmed by anti-βIII-tubulin antibody as a neuronal marker. Results are expressed as the ratio of β-arrestin2 to β III-tubulin (bar graph). All of the experiments were performed in triplicate. Data represent mean ± S.E.M. *, p < 0.01 versus control sample, unpaired t test; n = 3. Ctl refers to the control, and Mor refers to the samples from morphine-treated tissues. C, β-arrestin2 expression from the ileum and colon LMMP treated with morphine (Mor) or morphine plus naloxone (Nal). In the presence of naloxone (10 μM), morphine does not down-regulate β-arrestin2 expression. A similar result was obtained from two separate guinea pigs.

Fig. 6.

Time course of the down-regulation of β-arrestin2 protein (A) and mRNA (B) expression by morphine in the guinea pig. A, the LMMP from the ileum and colon were treated with 10 μM morphine from 0 to 5 h, and the expression of β-arrestin2 was detected by anti-β-arrestin2 antibody at various time points. Left, expression of β-arrestin2 from the ileum and colon after 3 and 10 min of exposure to 10 μM morphine. Right, expression of β-arrestin2 after 0.5, 1, 2, and 5 h of exposure to morphine in the ileum. Significant decreases were observed at 2 h. Similar results were obtained in two separate runs. B, mRNA expression of β-arrestin2 in controls and after morphine treatment (10 μM; 2 h) in the ileum and colon LMMP preparations. Data are presented as ΔCt values calculated as the Ct value of β-arrestin2 minus the Ct value of 18S ribosomal RNA for each sample. No differences were observed in mRNA expression between the ileum and the colon after morphine treatment.

Fig. 7.

Down-regulation of phospho-ERK by chronic morphine in the guinea pig LMMP. A, Western blot analysis of phospho-ERK1/2 in the ileum and colon in the absence and presence of morphine (10 μM) treatment. Ctl, control; Mor, morphine-treated samples. Total ERK1/2 was measured in the same samples. B, bar graph representing ratios of phospho-ERK to total ERK in the ileum and colon (*, p < 0.05, paired t test; n = 3).

Fig. 8.

Tolerance and β-arrestin2 expression in LMMP from in vivo chronic morphine-treated guinea pigs. A, dose-response curves to morphine-induced inhibition of EFS-induced contractions in the ileum (left) and colon (right) from 7 day treated guinea pigs (n = 6–11). Morphine-induced inhibition of EFS contractions in the LMMP preparations were examined from saline-treated (open circles) and morphine-treated guinea pigs (closed circles). A second cumulative dose-response curve was measured after 2 h of incubation with morphine (10 μM) in chronic in vivo morphine-treated guinea pigs (closed triangles). No further shifts were observed in either ileum or colon in morphine tolerant guinea pigs. *, p < 0.05 two-way ANOVA followed by Bonferroni's post hoc. B, Western blot analyses of β-arrestin2 expression in the colon and ileum from morphine tolerant guinea pigs. Expression was measured in the absence of morphine exposure (Ctl) and after 2 h of morphine exposure (Mor). C, expression of phospho-ERK and total ERK in the colon and ileum from morphine tolerant guinea pigs in the absence of morphine exposure (Ctl) and after 2 h of morphine exposure (Mor).