Specific localization of ß-Arrestin2 in myenteric plexus of mouse gastrointestinal tract.

ß-arrestin2 is a key molecule involved in signaling and internalization of activated G protein-coupled receptors including µ-opioid receptors (MOR). Previously we have shown that decreased expression of ß-arrestin2 upon chronic morphine is associated with the development of opioid tolerance in the gastrointestinal tract. However, the localization of ß-arrestin2 within the gastrointestinal wall is not known. In this study we found that ß-arrestin2 is localized in the soma of a select group of neurons in the myenteric ganglia but not in smooth muscle. The density of ß-arestin2 was significantly higher in the ileum than the colon. We identified four variants of ß-arrestin2 in the ileum, with ARRB-005 and ARRB-013 being the most abundant. Further, the current study utilized multiple-labeling immunofluorescence to characterize the chemical coding of neurons expressing ß-arrestin2 in the murine myenteric plexus and the co-localization of MOR1 and ß-arrestin2. ß-arrestin2 co-localized with choline acetyltransferase and calretinin. In contrast, ß-arrestin2 neither co-localized with substance P, nitric oxide synthase nor calbindin. Genetic deletion of ß-arrestin2 did not affect cholinergic neuron activation by nicotine in the isolated ileum (-log M EC50: wild type = 5.8 vs. ß-arrestin2 knockout = 5.9). Our findings suggest specificity in the localization of ß-arrestin2 in the myenteric plexus within MOR1-expressing neurons and provide a relation for direct intracellular crosstalk between MOR1 receptor activation and ß-arrestin2 signaling in the myenteric neurons. ß-arrestin2 deletion does not directly alter basal enteric cholinergic neuronal function.

Differences in the characteristics of tolerance to µ-opioid receptor agonists in the colon from wild type and ß-arrestin2 knockout mice.

Drawbacks to opioid use include development of analgesic tolerance and persistent constipation. We previously reported that tolerance to morphine develops upon repeated exposure in the isolated ileum but not the isolated colon. The cellular mechanisms of antinociceptive tolerance vary among µ-opioid receptor agonists. In this study, we assess ß-arrestin2 deletion on the development of tolerance to different opioids in ileum and colon circular muscle. Tolerance was determined by assessing the ability of repeated in-vitro opioid exposure to induce contraction of the circular muscle from C57BL/6 wild type (WT) and ß-arrestin2 knockout (KO) mice. Repeated exposure every 30 min with in-between washes resulted in tolerance to all agonists in the ileum of both WT and KO mice. However, in the colon of WT mice, comparison of the contractions between the 4th exposure and 1st response was similar to DAMGO (100 ± 10%; N=5) but reduced to fentanyl (62 ± 13%; N=8) and etorphine (38 ± 4%; N=7) indicative of tolerance to fentanyl and etorphine but not DAMGO. In contrast, all agonists produced tolerance in the colon of KO: DAMGO response at the 4th exposure decreased to 52 ± 10% (N=5), fentanyl to 20 ± 5% (N=6) and etorphine 33 ± 7% (N=6). Differences in tolerance among opioid agonists in the colon suggest ligand bias. The deletion of ß-arrestin2 in colon appears to be necessary for tolerance to DAMGO but not fentanyl or etorphine. ß-arrestin2 potentially represents an important target for treating opioid-induced bowel dysfunction and warrants further exploration of its ligand bias.

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

ß-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 ß-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 ß-arrestin2 knockout mice. ß-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 ß-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 ß-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 ß-arrestin2 occurred. We conclude that a decrease in ß-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

Time course of altered sensitivity to inhibitory and excitatory agonist responses in the longitudinal muscle-myenteric plexus and analgesia in the Guinea pig after chronic morphine treatment.

Tolerance that develops after chronic morphine exposure has been proposed to be an adaptive response that develops and decays over a defined time course. The present study examined the development of tolerance to the acute hypothermic and analgesic effects of morphine and correlated the time course for the desensitization in vivo with the reduced responsiveness to DAMGO and 2-CADO and increased responsiveness to nicotine of the longitudinal muscle/myenteric plexus (LM/MP) preparation in vitro. Assessment was performed at various times after morphine or placebo pellet implantation. Morphine produced a modest hypothermic response to which no tolerance developed. However, the development of tolerance to the analgesic effect of morphine, the inhibitory effect of DAMGO and CADO on neurogenic twitches of the LM/MP and hypersensitivity to the contractile response to nicotine was observed to occur in a time-dependent manner. The alterations in sensitivity to DAMGO, nicotine, and responsiveness to morphine analgesia occurred between days 4 and 10 and returned to normal by day 14 post-implantation. In contrast, sensitivity of LM/MP preparations to 2-CADO displayed a similar time-dependent onset but the tolerance persisted beyond 14 days after implantation. These data suggest that the heterologous tolerance that develops after chronic morphine treatment is time-dependent and persistent but, ultimately returns to normal in the absence of any intervention. Furthermore, the data suggest that the basis of the adaptive phenomenon may involve multiple cellular mechanisms including the modulation of cell excitability and normal physiology but the consequences of the adaptation extend to all effects of the agonist.

Correlation of the time course of development and decay of tolerance to morphine with alterations in sodium pump protein isoform abundance.

Since the heterologous tolerance that develops after chronic morphine administration has been proposed to be an adaptive process, it follows that the time course of the change in the cellular components should coincide with the time course of the altered responsiveness. This study correlated the time course over which heterologous tolerance develops with changes in the abundance of selected proteins in the guinea-pig longitudinal muscle/myenteric plexus (LM/MP) preparation. Tissues were obtained at various times following a single surgical implantation procedure and heterologous tolerance confirmed by a significant reduction in the sensitivity of the LM/MP to inhibition of neurogenic twitches by morphine, DAMGO, and 2-CADO. Tolerance developed with a delayed onset (significant 2-5-fold reduction in sensitivity by day 4 after pellet implantation) that reached a maximum by 7 days (4-8-fold reduction in responsiveness) that was maintained through 14 days with normal sensitivity spontaneously returning by 21 days post-implantation. Dot blot analysis was used to examine the abundance of the alpha(1) and alpha(3) subunit isoforms of the Na(+)/K(+) ATPase and beta-actin over the same time course. The results showed significant decreases in abundance of the alpha(3) subunit at 4, 7, and 10 days following pellet implantation but no change in beta-actin or the alpha(1) subunit at any time period. These data support the idea that heterologous tolerance following chronic morphine exposure results from a cellular adaptive change that may involve a change in the abundance of the alpha(3) subunit isoform of the Na(+)/K(+) ATPase.