Ligand-Induced l Opioid ReceptorInternalization in Enteric NeuronsFollowing Chronic Treatment With theOpiate Fentanyl

Laura Anselmi,1 Ingrid Jaramillo,2 Michelle Palacios,1

Jennifer Huynh,1 and Catia Sternini1,2*1CURE Digestive Diseases Research Center, Digestive Diseases Division and Department of Medicine,University of California Los Angeles, David Geffen School of Medicine, Los Angeles, California2Department of Neurobiology, University of California Los Angeles, David Geffen School of Medicine,Los Angeles, California

Morphine differs from most opiates its poor ability tointernalize l opioid receptors (lORs). However, chronictreatment with morphine produces adaptational changesat the dynamin level, which enhance the efficiency ofacute morphine stimulation to promote lOR internaliza-tion in enteric neurons. This study tested the effect ofchronic treatment with fentanyl, a lOR-internalizing ago-nist, on ligand-induced endocytosis and the expression ofthe intracellular trafficking proteins, dynamin and b-arrestin, in enteric neurons using organotypic cultures ofthe guinea pig ileum. In enteric neurons from guinea pigschronically treated with fentanyl, lOR immunoreactivitywas predominantly at the cell surface after acute expo-sure to morphine with a low level of lOR translocation,slightly higher than in neurons from na€ıve animals. Thisinternalization was not due to morphine’s direct effect,because it was also observed in neurons exposed to me-dium alone. By contrast, D-Ala2-N-Me-Phe4-Gly-ol5-en-kephalin (DAMGO), a potent lOR-internalizing agonist,induced pronounced and rapid lOR endocytosis in en-teric neurons from animals chronically treated with fen-tanyl or from na€ıve animals. Chronic fentanyl treatmentdid not alter dynamin or b-arrestin expression. These find-ings indicate that prolonged activation of lORs with aninternalizing agonist such as fentanyl does not enhancethe ability of acute morphine to trigger lOR endocytosisor induce changes in intracellular trafficking proteins, asobserved with prolonged activation of lORs with a poorlyinternalizing agonist such as morphine. Cellular adapta-tions induced by chronic opiate treatment might be liganddependent and vary with the agonist efficiency to inducereceptor internalization. VC 2013 Wiley Periodicals, Inc.

Key words: G-protein-coupled receptors; endogenousopioids; tolerant and na€ıve animals

Opioids modulate a variety of biological processesby activating three classes of opioid receptors (lORs,dORs, jORs) (Kromer, 1988; Pasternak, 1988; Raynoret al., 1994; Law and Loh, 1999). lORs are expressed in

the nervous system (Ding et al., 1996), including the en-teric nervous system, the neural network innervating thegastrointestinal tract (Bagnol et al., 1997; Ho et al., 2003).lOR is the main target of opiate drugs used in humansfor pain control (Matthes et al., 1996; Kieffer, 1999;Evans, 2000; Kieffer and Gaveriaux-Ruff, 2002).Unfortunately, the development of side effects, includingtolerance and opioid bowel dysfunction (OBD) dimin-ishes their usefulness in patients requiring chronic paintreatment (Williams et al., 2001; Christie, 2008; Morganand Christie, 2011; Brock et al., 2012). OBD is a seriouscondition characterized by abdominal pain and severeconstipation mediated by lOR in enteric neurons.Whereas patients develop tolerance to the opiate analgesiceffect, tolerance to GI adverse effects does not developover time, and OBD symptoms worsen with increasinganalgesic doses with a negative impact on the quality oflife (Panchal et al., 2007; Brock et al., 2012).

lOR–ligand interaction promotes phosphorylation,internalization, and recycling, which contribute todesensitization and resensitization, mechanisms regulatingcellular function (Bohm et al., 1997; Sternini, 2001;Gainetdinov et al., 2004; Martini and Whistler, 2007).Opiates differ in their ability to induce lOR desensitiza-tion and internalization (Sternini, 2001; Martini andWhistler, 2007). High-efficacy opiates such as etorphineand fentanyl and endogenous opioids induce rapidlOR endocytosis in enteric neurons (Sternini et al.,

Contract grant sponsor: National Institutes of Health; Contract grant

numbers: DK54155 (to C.S.); contract grant number: 41301 (Morphol-

ogy and Cell Imaging Core; to C.S.).

*Correspondence to: Catia Sternini, MD, CURE, Digestive Diseases

Research Center, Bldg. 115, Room 223, VAGLAHS, 11301 Wilshire

Blvd., Los Angeles, CA 90073. E-mail: csternin@ucla.edu

Received 11 December 2012; Revised 11 January 2013; Accepted 14

January 2013

Published online 29 March 2013 in Wiley Online Library

(wileyonlinelibrary.com). DOI: 10.1002/jnr.23214

VC 2013 Wiley Periodicals, Inc.

Journal of Neuroscience Research 91:854–860 (2013)

1996; Minnis et al., 2003). By contrast, morphine is apoor internalizing agonist and does not induce detectableinternalization (Sternini et al., 1996; Keith et al., 1998;Minnis et al., 2003). Morphine’s inefficiency to internal-ize activated-lORs could be due to its low efficacy forinducing receptor phosphorylation, b-arrestin recruit-ment, and desensitization (Yu et al., 1997; Kovoor et al.,1998; Whistler and von Zastrow, 1999; Alvarez et al.,2002). However, other studies have shown that morphinepromotes lOR phosphorylation and desensitization with-out triggering internalization (Zhang et al., 1996; Yuet al., 1997; Borgland et al., 2003; Arttamangkul et al.,2008) and that overexpression of intracellular proteinsinvolved in receptor trafficking increases morphine’s abil-ity to induce receptor internalization (Zhang et al., 1998;Whistler et al., 1999). This agonist-selective internaliza-tion may reflect a differential ability of agonists to regulatebiological effects mediated by the lOR, including theinduction of tolerance that is inversely correlated withendocytosis effectiveness (Paronis and Holtzman, 1992;Duttaroy and Yoburn, 1995; Martini and Whistler,2007).

Chronic opiates induce cellular adaptations, includ-ing changes in the expression of proteins implicated in re-ceptor trafficking, which might vary depending on ligandpotency, efficacy, and efficiency to induce lOR endocy-tosis (Paronis and Holtzman, 1992; Duttaroy and Yoburn,1995; Martini and Whistler, 2007). We have shown thatprolonged activation of lORs with morphine reversesthe resistance of morphine-activated lORs to undergointernalization in enteric neurons (Patierno et al., 2011),which depends on the increased expression of dynamin, aprotein required for lOR internalization (von Zastrowet al., 2003). In this study, we tested the hypothesis thatcellular adaptations induced by prolonged activation oflOR depend on the internalizing efficiency of lOR ago-nists. Therefore, we investigated whether chronic expo-sure to fentanyl, an internalizing lOR agonist, enhancesmorphine’s efficiency to induce lOR internalization inenteric neurons and alters the expression of dynamin andb-arrestin, intracellular proteins regulating receptortrafficking.

MATERIALS AND METHODS

Male albino Porcellus guinea pigs (150–250 g; Hartley; CharlesRiver Laboratory, San Diego, CA) were used. Care and han-dling of the animals were in accordance with all NationalInstitutes of Health recommendations for the humane use ofanimals. All experimental procedures were reviewed andapproved by the Animal Committee at UCLA, and all effortswere made to minimize the number of animals used. Guineapigs received subcutaneous injection of saline or fentanyl(0.24 mg/kg; 35 and 26 animals per group, respectively) twiceper day for 4 days (Schulz et al., 1985) for chronic treatment.The following drugs were used: fentanyl (Baxter, Deerfield,IL), which was dissolved in 0.9% sodium chloride, and tetro-dotoxin (TTX; EMD, Biosciences, CA) to block transmitterrelease.

Organotypic Cultures of the Ileum andImmunohistochemistry

Guinea pigs were anesthetized with an overdose of iso-fluorane and euthanized by decapitation 2 hr after the lastinjection, and the distal ileum was rapidly removed. For thepreparation of organotypic cultures, the distal ileum was dis-sected and washed in sterile Krebs solution (in mM: KCl, 5.9;NaCl, 118; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 22.7;NaH2PO4, 1.4; glucose, 5; fungizone, 2.5 lg/ml; penicillin,100 IU/ml; and streptomycin, 100 lg/ml), bubbled with 95%O2:5% CO2, pH 7.4, at 37�C (Minnis et al., 2003). Tissuewas incubated in either Krebs alone (unstimulated) or Krebscontaining saturating doses of morphine (100 lM) orDAMGO (10 lM) for 1 hr at 4�C to allow ligand–receptorbinding. These doses were selected on the basis of previousstudies (Minnis et al., 2003). After incubation, the ileum wasopened along the mesentery, pinned flat, and transferred toligand-free medium (Dulbecco’s modified Eagle’s medium,containing 10% v/v fetal bovine serum, penicillin, streptomy-cin, and fungizone) at 37�C for 30 min to allow receptorinternalization. In some experiments, tissues were preincu-bated for 90 min in Krebs containing TTX 1027 M at 37�Cto block neuronal depolarization. Organotypic cultures werefixed in 0.1 M phosphate-buffered 4% paraformaldehyde, pH7.4, for 2 hr at room temperature and washed (Patierno et al.,2011). The longitudinal muscle with the myenteric plexusattached was separated and processed as whole mounts forimmunohistochemistry as previously described using the indi-rect immunofluorescence technique (Ho et al., 2003). Thelocation of the lOR was identified using a well-characterizedlOR antibody (Incstar Science, Technology and Research,Stillwater, MN; McConalogue et al., 1999; Minnis et al.,2003). In brief, whole-mount preparations were incubated in5% normal donkey serum with 0.5% Triton X-100 for 60min. Tissue was transferred to the same solution containingthe lOR antibody (1:2,000) for 48 hr at 4�C. Tissue waswashed and incubated with Alexa Fluor 488 affinity-purifieddonkey anti-rabbit (1:1,000; Invitrogen Molecular Probes,Eugene, OR) for 2 hr at room temperature.

lOR immunoreactivity distribution was analyzed with aZeiss 510 Meta laser scanning confocal microscope with a 363PlanApo 1.4 numerical aperture objective (Carl Zeiss, Thorn-wood, NY). Images were adjusted for brightness and contrast inAdobe Photoshop 7.0 (Adobe Systems, Mountain View, CA).The level of lOR internalization was quantified in Image J;typically a single confocal image included the nucleus and alarge area of cytoplasm. For each data point, internalization wasquantified for 35–60 neurons.

Dynamin and b-Arrestin Expression

Strips of longitudinal muscle-myenteric plexus prepara-tions (LMMPs) were homogenized in 1 ml ice-cold lysis buffer[5 mM Pipes/Tris buffer, pH 7.4, with 2 mM ethylenediamine-tetraacetic acid and ethylene glycol-bis(b-aminoethyl ether)-N,N,N0,N0-tetraacetic acid] and centrifuged at 14,000 rpm for20 min at 4�C as described by Patierno et al. (2011). The super-natant proteins were size separated by sodium dodecyl sulfate-polyacrylamide gel and transferred onto Immobilon membranes

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(Millipore, Billerica, MA). Membranes were incubated withmouse antibody to dynamin 2 (1:1,000; Transduction Labora-tories, San Diego, CA), which is ubiquitous and shares highhomology with dynamin 1, more specific for the central nerv-ous system or rabbit antiserum to b-arrestin 1 (1:75,000; kindlyprovided by Robert Lefkowitz, Duke University, Durham,NC), which shares high homology with b-arrestin 2, followedby Alexa 488 anti-mouse or anti-rabbit immunoglobulin (IgG)at 1:1,000 dilution for 1 hr at room temperature.

Electrically Induced Neurogenic Contractions of theIleum In Vitro

To evaluate the level of tolerance induced by chronicfentanyl treatment, we measured the neurogenic contractionsof the ileum in response to electrical field stimulation inLMMPs of the ileum from animals chronically treated with fen-tanyl or saline (12–14 animals for each group) prepared asdescribed by Sternini et al. (2000). Isometric contractions wererecorded with a force-displacement transducer. After a 45-minequilibration period, strips were stimulated with square wavepulses (0.5 msec) of supramaximal amplitude (60 V) at a fre-quency of 0.1 Hz. Concentration–response curves for the in-hibitory effects of morphine (1029 M to 1026 M) onelectrically induced contractions in preparations from animalstreated with saline or chronic fentanyl were constructed in anoncumulative fashion. Strips were washed for three 5-minperiods after each agonist administration, and contractions wereallowed to return to baseline before any subsequent pharmaco-logical treatment.

Statistical Analysis

For statistical analysis, we used a one-way ANOVA, fol-lowed by the post hoc Bonferroni test or a paired Student’s t-test (GraphPad Prism 5.0); significance was set at P< 0.05, andvariability is reported as 6SEM.

RESULTS

Effect of Morphine and DAMGO on EntericNeurons of the Ileum In Vitro

In organotypic cultures from animals treated with sa-line (na€ıve), lOR immunoreactivity (IR) was mostly con-fined to the plasma membrane of unstimulated (Fig. 1A)and morphine (100 lM)-stimulated (Fig. 1B) enteric neu-rons, with a low level of lOR internalization as indicatedby the occasional punctate immunofluorescence in thecytosol observed in a small proportion of neurons. By con-trast, DAMGO (10 lM) stimulation induced abundanttranslocation of lOR-IR into the cytoplasm (Fig. 1C),confirming previous results from our group (Minnis et al.,2003). In unstimulated and morphine-stimulated neuronsfrom animals chronically treated with fentanyl, lOR-IRwas located predominantly at the plasma membrane, with alow level of lOR internalization, which could be detectedin most neurons as sparse, punctate immunofluorescence inthe cytoplasm (Fig. 1D,E). DAMGO induced strong lORinternalization in neurons from fentanyl-treated guinea pigs(Fig. 1F), which was comparable to DAMGO-inducedinternalization observed in neurons from na€ıve animals(Fig. 1C).

Fig. 1. Localization of lOR immunoreactivity (IR) in neurons fromanimals chronically treated with saline (A–C) or fentanyl (D–F).lOR-IR is confined at the cell surface (arrows) of unstimulated (A)and morphine-stimulated (B) neurons from saline-treated animals andis localized predominantly at the cell surface (arrows) with low levelsof lOR-IR in the cytosol (arrowheads) of unstimulated (D) and

morphine-stimulated (E) neurons from animals treated chronicallywith fentanyl. By contrast, receptor internalization is pronounced inDAMGO-stimulated neurons from both saline- and chronic fentanyl-treated animals, as indicated by the prominent translocation of lOR-IR in the cytoplasm. Scale bars 5 5 lm in A (applies to A,B,D,E); 4lm for C,F.

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Quantification analysis showed that the proportionof lOR endocytosis in unstimulated enteric neuronsfrom na€ıve animals was similar to that in the morphine-stimulated neurons (34.3% 6 1.6% vs. 33.0% 6 2.6%,P 5 NS) and was just below the threshold level previouslyreported for normal neurons (Patierno et al., 2004),whereas DAMGO induced pronounced lOR internaliza-tion (65.0% 6 1.4%, P< 0.005 vs. unstimulated and mor-phine-stimulated; Fig. 2A). The levels of lORendocytosis in unstimulated and morphine-stimulated en-teric neurons from fentanyl-treated animals were alsocomparable (45.0% 6 2.3% vs. 44.6% 6 2.3%, P 5 NS)but slightly, though significantly, higher than in na€ıveanimals (P< 0.05 for both unstimulated and morphinestimulated). This internalization was about 10%higher than the normal threshold (Patierno et al., 2004),

suggesting that it was due to experimental manipulation.However, it could not be due to a direct effect of mor-phine on lOR, because it was comparable to the level ofinternalization observed in neurons exposed only to me-dium. Furthermore, TTX pretreatment to block neuronaldepolarization did not affect the translocation of lOR-IRin any of these conditions (data not shown), indicatingthat it was not due to endogenously released opioids.Because fentanyl is an internalizing lOR agonist, it islikely that the continued treatment induced baselineinternalization in enteric neurons in the absence of acuteagonist stimulation. By contrast, the levels of OR endo-cytosis were significantly higher in DAMGO-stimulatedneurons from animals chronically treated with fentanyl(72.6% 6 0.9%, P< 0.005 vs. unstimulated and morphinestimulated; Fig. 2B). Pretreatment with TTX did notaffect DAMGO-induced lOR internalization, indicatingthat it was due to a direct interaction of DAMGO withthe receptor and not to the activation of the receptor byendogenous opioids.

Effect of Chronic Fentanyl on Dynamin and b-Arrestin Expression

Because dynamin and b-arrestin mediate receptorinternalization of activated lORs (Whistler and von Zas-trow, 1999; von Zastrow, 2003), we investigated theexpression of these proteins using Western blots inLMMP preparations from both saline- and chronic fen-tanyl-treated animals. There was no change in the proteinlevels of either dynamin 2 or b-arrestin 1 in LMMPsfrom distal ileum of saline- or fentanyl-treated animals(Fig. 3). This differs from data obtained from LMMPpreparations from animals chronically treated with mor-phine, which induced increased expression of dynaminbut not b-arrestin, suggesting different cellular responsesto chronic stimulation with different lOR agonists(Patierno et al., 2011).

Effect of Chronic Fentanyl on TwitchContractions in Electrically Stimulated LMMPPreparations

Morphine inhibited electrically induced twitch con-tractions of LMMP preparations from both saline- andchronic fentanyl-treated animals in a concentration-de-pendent manner (Fig. 4). Morphine caused a rightwardshift in the sigmoidal concentration–response curve fromanimals chronically treated with fentanyl, which is con-sistent with these animals being tolerant to lOR agonists.

DISCUSSION

Chronic activation of lORs induces a variety of cellularadaptations at the receptor level and within receptor-asso-ciated signal transduction pathways, which are mecha-nisms underlying the development of tolerance andadverse side effects. We have previously shown that pro-longed treatment with morphine induced upregulation ofintracellular dynamin that enhances the efficiency of acutemorphine stimulation to induce rapid lOR

Fig. 2. Quantification of lOR immunoreactivity in the cytoplasm ofenteric neurons from animals chronically treated with saline (A; na€ıve)and fentanyl (B; chronic). The proportion of lOR-IR translocated inthe cytosol of neurons incubated with medium (Krebs; unstimulated)was comparable to that of the morphine-stimulated neurons in bothna€ıve (A) and chronically treated (B) animals. The level of lOR-IRin the cytosol of both unstimulated and morphine-stimulated neuronsfrom animals chronically treated with fentanyl was higher (�10%)compared with neurons from saline-treated animals. The levels oflOR-IR internalized following DAMGO stimulation is significantlyhigher than in unstimulated and morphine-stimulated neurons fromboth na€ıve and chronically treated animals. Fluorescence density valuesare expressed as mean 6 SEM. ***P< 0.005.

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internalization in enteric neurons (Patierno et al., 2011).By contrast, the present study shows that chronic treat-ment with fentanyl did not alter the expression of intra-cellular trafficking proteins, including dynamin, and didnot improve the efficiency of acute morphine to inducelOR endocytosis. The low level of lOR translocationobserved in morphine-stimulated neurons from animalschronically treated with fentanyl was slightly (�10%)above the normal threshold, suggesting that it was due toexperimental manipulation. However, it was comparableto the level of internalization observed in unstimulatedneurons, thus ruling out a direct effect of morphine onlORs. Furthermore, it was not modified by treatment toblock neuronal depolarization, arguing against an effect ofendogenous opioids. It is reasonable to suggest that thebaseline of lOR translocation is induced by the continu-ous stimulation with fentanyl, which is an internalizingagonist (Hashimoto et al., 2006; Martini and Whistler,2007). By contrast, the magnitude of DAMGO-inducedlOR internalization in enteric neurons from animalschronically treated with fentanyl was pronounced despitethe development of tolerance. Tolerance was supportedby the reduction of opiate inhibitory effect on neurogenicmuscle contraction, which is consistent with diminishedcellular responsiveness, a typical side effect of prolongedlOR activation (Chavkin and Goldstein, 1984).

Ligand-induced receptor internalization is an impor-tant mechanism that regulates receptor-mediated functions(Sternini, 2001; Martini and Whistler, 2007; Dang andChristie, 2012). Receptor internalization contributes to thediminution of cellular response by removing the receptorsfrom the surface. Internalization also contributes to the re-covery of cellular response by allowing the ligand–receptor

complexes to traffic inside of the cell and either to recycleor to undergo degradation. Morphine, a poor internalizingopiate, has higher propensity to induce opioid tolerancethan internalizing opiates (Duttaroy and Yoburn, 1995), soit has been suggested that ligand-induced lOR endocytosisserves as a protective mechanism from the development oftolerance (Eisinger et al., 2002; Koch et al., 2005; Martiniand Whistler, 2007). The possibility that the resistance ofmorphine-activated lORs to undergo internalization is theinitial trigger for the cellular adaptations induced by pro-longed activation of lOR has gained substantial support(Martini and Whistler, 2007). However, other parameters,including the different affinity of agonists to interact withG proteins, as well as the different potency, efficacy, andhalf-life of opiates, might also play a role (Martini andWhistler, 2007; Dang and Christie, 2012). Furthermore,differences in the internalizing properties of morphine alsodepend on the cell system. Indeed, morphine fails toinduce lOR endocytosis in heterologous cells and highlydifferentiated neurons in the central and peripheral nervoussystems (Keith et al., 1996; Sternini et al., 1996; Haber-stock-Debic et al., 2005). By contrast, morphine promotesrapid lOR endocytosis in embryonic striatal neurons incultures (Haberstock-Debic et al., 2005) as well as in adultenteric neurons in vivo following chronic treatment withmorphine (Patierno et al., 2011). Furthermore, it has beenproposed that differences in the intracellular concentrationsof proteins regulating GPCR trafficking might influencethe ability of opioids to drive lOR endocytosis (Bohn

Fig. 4. Morphine (1 nM to 1 lM) induces a dose-dependent inhibi-tion of electrically induced twitch contractions of longitudinal muscle-myenteric plexus preparations from animals treated with saline (opencircles) or chronic fentanyl (solid circle). The chronic treatment withfentanyl induces a parallel right shift of the inhibitory curve withoutreduction of the maximum response, indicative of tolerance.*P< 0.05.

Fig. 3. Quantification of dynamin 2 and b-arrestin 1 immunoreactiv-ity in longitudinal muscle-myenteric plexus preparations of the guineapig distal ileum from animals treated with saline (open bars) and ani-mals chronically treated with fentanyl (solid bars) measured by West-ern blot. Results represent the percentage increase of dynamin 2 orb-arrestin 1 compared with tissue from animals treated with saline.Representative immunoblots are shown on top the histograms.

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et al., 2004). This is consistent with findings in heterol-ogous cells showing morphine-induced lOR endocytosisin the presence of GRKs or b-arrestin overexpression(Whistler and von Zastrow, 1998; Zhang et al., 1998). Anincreased expression of dynamin is likely to be the mecha-nism underlying the enhanced efficiency of morphine totrigger lOR endocytosis in enteric neurons from animalschronically treated with morphine (Patierno et al., 2011).This is supported by the inhibition of morphine-inducedlOR internalization by pretreatment with dynasore, adynamin inhibitor (Macia et al., 2006). The lack of increasein dynamin expression in enteric neurons from animalschronically treated with fentanyl might be the basis for thefailure of morphine to induce lOR internalizationobserved in the present study. Intracellular trafficking pro-teins might influence the ability of poorly internalizinglOR agonists such as morphine to induce receptor inter-nalization efficiently, and it is likely that they differ accord-ing to the cell type and stimulation condition.

The high efficiency of DAMGO to induce lORinternalization is not affected by chronic treatment withfentanyl, as it was not affected by chronic stimulationwith morphine (Patierno et al., 2011). This is in agree-ment with findings in the central nervous system showingthat neurons that developed tolerance to morphine-induced analgesia in the spinal cord undergo the samelevel of lOR endocytosis in response to DAMGO asna€ıve neurons (Trafton and Basbaum, 2004). DAMGO-induced lOR endocytosis is due to a direct action ofDAMGO on the receptor and is independent of endoge-nous opioids release by enteric neurons in response tochronic opioid treatment, because it was not affected byblocking neuronal depolarization. Together these findingssuggest that receptor trafficking in neurons induced byhighly efficient internalizing agonists is not impaired byprolonged receptor activation independently of the typeof agonist used chronically.

In summary, chronic stimulation of lORs with fen-tanyl does not induce the adaptational changes at thedynamin levels, which promote internalization of mor-phine-activated lOR in enteric neurons. This supportsthe concept that the internalizing efficacy of the ligandaffects cellular adaptations induced by prolonged receptoractivation. This observation is of relevance in that recep-tor internalization contributes to the regulation of recep-tor-mediated functions by participating in the attenuationand recovery of cellular response.

ACKNOWLEDGMENTS

The authors thank Dr. Nicholas Brecha for insightfulcomments on the manuscript.

REFERENCES

Alvarez VA, Arttamangkul S, Dang V, Salem A, Whistler JL, Von Zas-

trow M, Grandy DK, Williams JT. 2002. Mu-opioid receptors: ligand-

dependent activation of potassium conductance, desensitization, and

internalization. J Neurosci 22:5769–5776.

Arttamangkul S, Quillinan N, Low MJ, von Zastrow M, Pintar J, Wil-

liams JT. 2008. Differential activation and trafficking of micro-opioid

receptors in brain slices. Mol Pharmacol 74:972–979.

Bagnol D, Mansour A, Akil H, Watson SJ. 1997. Cellular localization

and distribution of the cloned mu and kappa opioid receptors in rat

gastrointestinal tract. Neuroscience 81:579–591.

Bohm S, Grady EF, Bunnett NW. 1997. Mechanisms attenuating signal-

ing by G protein-coupled receptors. Biochem J 322:1–18.

Bohn LM, Dykstra LA, Lefkowitz RJ, Caron MG, Barak LS. 2004. Rel-

ative opioid efficacy is determined by the complements of the G pro-

tein-coupled receptor desensitization machinery. Mol Pharmacol

66:106–112.

Borgland SL, Connor M, Osborne PB, Furness JB, Christie MJ. 2003.

Opioid agonists have different efficacy profiles for G protein activation,

rapid desensitization, and endocytosis of mu-opioid receptors. J Biol

Chem 278:18776–18784.

Brock C, Olesen SS, Olesen AE, Frokjaer JB, Andresen T, Drewes AM.

2012. Opioid-induced bowel dysfunction: pathophysiology and man-

agement. Drugs 72:1847–1865.

Chavkin C, Goldstein A. 1984. Opioid receptor reserve in normal and

morphine-tolerant guinea pig ileum myenteric plexus. Proc Natl Acad

Sci U S A 81:7253–7257.

Christie MJ. 2008. Cellular neuroadaptations to chronic opioids: toler-

ance, withdrawal and addiction. Br J Pharmacol 154:384–396.

Dang VC, Christie MJ. 2012. Mechanisms of rapid opioid receptor

desensitization, resensitization and tolerance in brain neurons. Br J

Pharmacol 165:1704–1716.

Ding YQ, Kaneko T, Nomura S, Mizuno N. 1996. Immunohistochemi-

cal localization of mu-opioid receptors in the central nervous system of

the rat. J Comp Neurol 367:375–402.

Duttaroy A, Yoburn BC. 1995. The effect of intrinsic efficacy on opioid

tolerance. Anesthesiology 82:1226–1236.

Eisinger DA, Ammer H, Schulz R. 2002. Chronic morphine treatment

inhibits opioid receptor desensitization and internalization. J Neurosci

22:10192–10200.

Evans CJ. 2000. Agonist selective mu-opioid receptor trafficking in rat

central nervous system. Mol Psychiatry 5:121.

Gainetdinov RR, Premont RT, Bohn LM, Lefkowitz RJ, Caron MG.

2004. Desensitization of G protein-coupled receptors and neuronal

functions. Annu Rev Neurosci 27:107–144.

Haberstock-Debic H, Kim KA, Yu YJ, von Zastrow M. 2005. Morphine

promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors

in striatal neurons. J Neurosci 25:7847–7857.

Hashimoto T, Saito Y, Yamada K, Hara N, Kirihara Y, Tsuchiya M.

2006. Enhancement of morphine analgesic effect with induction of

mu-opioid receptor endocytosis in rats. Anesthesiology 105:574–580.

Ho A, Lievore A, Patierno S, Kohlmeier SE, Tonini M, Sternini C. 2003.

Neurochemically distinct classes of myenteric neurons express the l-

opioid receptor in the guinea pig ileum. J Comp Neurol 458:404–411.

Keith DE, Murray SR, Zaki PA, Chu PC, Lissin DV, Kag L, Evans CJ,

von Zastrow M. 1996. Morphine activates opioid receptors without

causing their rapid internalization. J Biol Chem 271:19021–19024.

Keith DE, Anton B, Murray SR, Zaki PA, Chu PC, Liissin DV, Mon-

teillet-Agius G, Stewart PL, Evans CJ, von Zastrow M. 1998. l-Opioid

receptor internalization: opiate drugs have differential effects on a con-

served endocytic mechanism in vitro and in mammalian brain. Mol

Pharmacol 53:377–384.

Kieffer BL. 1999. Opioids: first lessons from knockout mice. Trends

Pharmacol Sci 20:19–26.

Kieffer BL, Gaveriaux-Ruff C. 2002. Exploring the opioid system by

gene knockout. Prog Neurobiol 66:285–306.

Koch T, Widera A, Bartzsch K, Schulz S, Brandenburg LO, Wundrack

N, Beyer A, Grecksch G, Hollt V. 2005. Receptor endocytosis counter-

acts the development of opioid tolerance. Mol Pharmacol 67:280–287.

Fentanyl and lOR Endocytosis 859

Journal of Neuroscience Research

Kovoor A, Celver JP, Wu A, Chavkin C. 1998. Agonist induced homol-

ogous desensitization of mu-opioid receptors mediated by G protein-

coupled receptor kinases is dependent on agonist efficacy. Mol Pharma-

col 54:704–711.

Kromer W. 1988. Endogenous and exogenous opioids in the control of

gastrointestinal motility and secretion. Pharmacol Rev 40:121–162.

Law PY, Loh HH. 1999. Regulation of opioid receptor activities. J Phar-

macol Exp Ther 289:607–624.

Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T.

2006. Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell

10:839–850.

Martini L, Whistler JL. 2007. The role of mu opioid receptor desensitiza-

tion and endocytosis in morphine tolerance and dependence. Curr

Opin Neurobiol 17:556–564.

Matthes HW, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen

I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J,

Roques BP, Kieffer BL. 1996. Loss of morphine-induced analgesia,

reward effect and withdrawal symptoms in mice lacking the mu-

opioid-receptor gene. Nature 383:819–823.

McConalogue K, Grady EF, Minnis J, Balestra B, Tonini M, Brecha

NC, Bunnett NW, Sternini C. 1999. Activation and internalization

of the l opioid receptor by the newly described endogenous ago-

nists, endomorphin-1 and endomorphin-2. Neuroscience 90:

1051–1059.

Minnis J, Patierno S, Kohlmeier SE, Brecha N, Tonini M, Sternini C.

2003. Ligand-induced l opioid receptor endocytosis and recycling in

enteric neurons. Neuroscience 119:33–42.

Morgan MM, Christie MJ. 2011. Analysis of opioid efficacy, tolerance,

addiction and dependence from cell culture to human. Br J Pharmacol

164:1322–1334.

Panchal SJ, Muller-Schwefe P, Wurzelmann JI. 2007. Opioid-induced

bowel dysfunction: prevalence, pathophysiology and burden. Int J Clin

Pract 61:1181–1187.

Paronis CA, Holtzman SG. 1992. Development of tolerance to the anal-

gesic activity of mu agonists after continuous infusion of morphine, me-

peridine or fentanyl in rats. J Pharmacol Exp Ther 262:1–9.

Pasternak GW. 1988. The opiate receptors. Totowa, NJ: Humana.

Patierno S, Raybould HE, Sternini C. 2004. Abdominal surgery induces

l opioid receptor endocytosis in enteric neurons of the guinea pig il-

eum. Neuroscience 123:101–109.

Patierno S, Anselmi L, Jaramillo I, Scott D, Garcia R, Sternini C. 2011.

Morphine induces mu opioid receptor endocytosis in guinea pig enteric

neurons following prolonged receptor activation. Gastroenterology

140:618–626.

Raynor K, Kong H, Yasuda K, Chen Y, Yu L, Bell GI, Reisine T.

1994. Pharmacological characterization of the clones j, d and l opioid

receptors. Mol Pharmacol 45:330–334.

Schulz R, Seidl E, Herz A. 1985. Opioid dependence in the guinea-pig

myenteric plexus is controlled by non-tolerant and tolerant opioid

receptors. Eur J Pharmacol 110:335–341.

Sternini C. 2001. Receptors and transmission in the brain-gut axis:

potential for novel therapies. III. l opioid receptors in the enteric nerv-

ous system. Am J Physiol 281:G8–G15.

Sternini C, Spann M, Anton B, Keith DE Jr, Bunnett NW, von Zastrow

M, Evans C, Brecha NC. 1996. Agonist-selective endocytosis of l opioid

receptor by neurons in vivo. Proc Natl Acad Sci U S A 93:9241–9246.

Sternini C, Brecha NC, Minnis J, D’Agostino G, Balestra B, Fiori E,

Tonini M. 2000. Role of agonist-dependent receptor internalization in

the regulation of l opioid receptors. Neuroscience 98:233–241.

Trafton JA, Basbaum AI. 2004. [d-Ala2,N-MePhe4,Gly-ol5]enkephalin-

induced internalization of the micro opioid receptor in the spinal cord

of morphine tolerant rats. Neuroscience 125:541–543.

von Zastrow M. 2003. Mechanisms regulating membrane trafficking of G

protein-coupled receptors in the endocytic pathway. Life Sci 74:217–224.

von Zastrow M, Svingos A, Haberstock-Debic H, Evans C. 2003. Regu-

lated endocytosis of opioid receptors: cellular mechanisms and proposed

roles in physiological adaptation to opiate drugs. Curr Opin Neurobiol

13:348–353.

Whistler JL, von Zastrow M. 1998. Morphine-activated opioid receptors elude

desensitization by b-arrestin. Proc Natl Acad Sci U S A 95:9914–9919.

Whistler JL, von Zastrow M. 1999. Dissociation of functional roles of

dynamin in receptor-mediated endocytosis and mitogenic signal trans-

duction. J Biol Chem 274:24575–24578.

Whistler JL, Chuang H-H, Chu P, Jan LY, von Zastrow M. 1999. Functional

dissociation of l opioid receptor signaling and endocytosis: implications for

the biology of opiate tolerance and addiction. Neuron 23:737–746.

Williams JT, Christie MJ, Manzoni O. 2001. Cellular and synaptic adap-

tations mediating opioid dependence. Physiol Rev 81:299–343.

Yu Y, Zhang L, Yin X, Sun H, Uhl GR, Wang JB. 1997. Mu opioid

receptor phosphorylation, desensitization, and ligand efficacy. J Biol

Chem 272:28869–28874.

Zhang J, Ferguson SSG, Barak LS, Menard L, Caron MG. 1996. Dyna-

min and beta-arrestin reveal distinct mechanisms for G protein-coupled

receptor internalization. J Biol Chem 271:18302–18305.

Zhang J, Ferguson SSG, Barak LS, Bodduluri SB, Laporte SA, Law P-Y,

Caron MG. 1998. Role of G protein-coupled receptor kinase in ago-

nist-specific regulation of l-opioid receptor responsiveness. Proc Natl

Acad Sci U S A 95:7157–7162.

860 Anselmi et al.

Journal of Neuroscience Research