Pulmonary, Gastrointestinal and Urogenital Pharmacology
Differences in the characteristics of tolerance to μ-opioid receptor agonists in the colon from wild type and β-arrestin2 knockout mice

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Abstract

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 β-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 β-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 β-arrestin2 in colon appears to be necessary for tolerance to DAMGO but not fentanyl or etorphine. β-arrestin2 potentially represents an important target for treating opioid-induced bowel dysfunction and warrants further exploration of its ligand bias.

Introduction

Chronic use of opioids is associated with the rapid development of tolerance. Increasing the dose to compensate for the loss in efficacy raises the risk of adverse events including overdose, withdrawal and dependence. The extent of tolerance varies significantly depending on the pharmacological effect e.g. opioid tolerance develops quicker to urinary retention and respiratory depression effects compared to the analgesic effects, whereas little manifests to constipation in humans. It is for this reason that constipation is a major side effect observed in opioid-induced bowel dysfunction (Holzer et al., 2009, Thomas, 2008).

Several mechanisms have been proposed for the development of opioid tolerance depending on the ligand and tissue preparation. Classical mechanisms include decreased cell surface receptor expression, prolonged signaling resulting in desensitization, receptor phosphorylation, etc. It is however becoming increasingly clear that functional selectivity manifests with different ligands targeting the same opioid receptor (for review, see Raehal et al., 2011). Ligands that are less efficacious at promoting desensitization and internalization such as morphine produce significant antinociceptive tolerance while high efficacy agonists including etorphine and fentanyl produce less (Pawar et al., 2007, Raehal et al., 2011, Sirohi et al., 2008). In contrast, we recently reported the lack of tolerance to morphine in the colon from mice and guinea pigs: However, tolerance developed in the colon of β-arrestin2 knockout (KO) mice following repeated administration of morphine (Kang et al., 2012). Based on these findings, we compared the effects of other opioids towards tolerance development in the mouse colon. While numerous studies have focused on the effects of various opioids on antinociceptive tolerance, to our knowledge, no studies have investigated the effects of high efficacy ligands such as etorphine on tolerance in the colon. Etorphine, DAMGO and fentanyl desensitization pathways have been observed to be dependent on β-arrestin2, an integral protein which facilitates receptor desensitization, whereas morphine and levorphanol show minimal recruitment of the moiety (McPherson et al., 2010, Whistler and von Zastrow, 1998). To delineate possible differential development of tolerance among opioid agonists, we assessed the ability of repeated opioid exposure to induce contraction of isolated ileum and colon circular muscle from C57BL/6 wild-type and β-arrestin2 KO mice. In this preparation, enhanced spontaneous contractions occur as a result of suppression of inhibitory tone by μ-opioid receptors located on nitrergic and VIP-ergic myenteric neurons (Grider and Makhlouf, 1987, Iwata et al., 2007).

Our results demonstrate lack of tolerance to repeated administration of DAMGO in the colon similar to that previously observed with morphine. Partial tolerance occurs to fentanyl while significant tolerance manifests with etorphine. However, tolerance occurs to all agonists in the β-arrestin2 KO mouse colon. The differences between highly potent agonists like fentanyl and etorphine and the less potent DAMGO and morphine, point to potential ligand biased signaling. Our data demonstrate the downregulation of β-arrestin2 to be necessary for development or enhancement of tolerance to specific agonists and further illustrate differences between CNS models where tolerance is abrogated, in contrast to the colon where it is enhanced in the absence of β-arrestin2.

Section snippets

Drugs and Chemicals

Morphine (morphine sulfate pentahydrate salt), fentanyl (as citrate salt) and DAMGO ([d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate) were procured from Sigma-Aldrich Co. (St. Louis, MO), whereas etorphine (as hydrochloride salt) was acquired from the National Institute on Drug Abuse (NIDA), Bethesda, MD and CTAP (H-d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2) was procured from Tocris Biosciences (Ellisville, MO). Fentanyl and morphine were dissolved in water and etorphine and DAMGO were dissolved

Development of tolerance in circular muscle

Tolerance was determined by assessing the ability of repeated transient opioid exposure to induce contraction of the ileum and colon circular muscle ring preparations in vitro. The drug concentrations used (DAMGO — 3 μM, fentanyl — 0.1 μM, and etorphine — 0.01 μM) were based on preliminary in vitro concentration response studies performed to determine equipotent concentrations that produced significant quantifiable contractions from which tolerance could be assessed. Fig. 1A shows the

Discussion

In this study we compared the ability of three different opioid analgesics to induce tolerance in the ileum and colon of wild type and β-arrestin2 KO mice. While DAMGO, fentanyl and etorphine all produced tolerance upon repeated administration in the isolated ileum of WT and KO mice, there was differential development of tolerance in the colon. Similar to our previous findings with morphine (Kang et al., 2012, Ross et al., 2008), tolerance did not develop to DAMGO in the colon of the WT but was

Acknowledgments

This work was supported by NIH DA024009.

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