- Email: [email protected]
Ligand-induced μ opioid receptor endocytosis and recycling in enteric neurons
Neuroscience 119 (2003) 33– 42
LIGAND-INDUCED OPIOID RECEPTOR RECYCLING IN ENTERIC NEURONS J. G. MINNIS,a,b S. PATIERNO,a,b S. E. KOHLMEIER,a,b N. C. BRECHA,a,b,c,d M. TONINIe AND a,b,c * C. STERNINI
ENDOCYTOSIS
AND
acidification, but not by the protein synthesis inhibitor, cycloheximide. This study shows that native OR in enteric neurons undergoes ligand-selective endocytosis, which is primarily clathrin-mediated, and recycles following endosomal acidification. Following recycling, OR is activated and internalized by DAMGO indicating that recycled receptors are functional. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
a CURE Digestive Diseases Research Center, Building 115, Veterans Administration Greater Los Angeles Healthcare System, Digestive Diseases Division, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA b Department of Medicine, University of California, Los Angeles, CA 90095, USA
Key words: guinea-pig ileum, gastrointestinal motility, G protein-coupled receptors, receptor trafficking.
c
Department of Neurobiology, University of California, Los Angeles, CA 90095, USA
d
Opioid Research Center, University of California, Los Angeles, CA 90095, USA
G protein-coupled receptors transduce information from extracellular stimuli into intracellular second messengers via coupling to heterotrimeric G proteins (Bohm et al., 1997; Ferguson, 2001; Trowbridge et al., 1993). Signal transduction induced by ligand-receptor interaction is followed by a cascade of events, including phosphorylation, receptor endocytosis, intracellular sorting and recycling. The presence of receptors at the plasma membrane is critical for cellular activation, and agonist-induced receptor endocytosis participates to the regulation of receptor-mediated signal transduction by removing receptors from the cell surface. Receptor endocytosis also provides an indication of receptor activation by allowing the visualization of the receptor in endosomes thus serving as a means to identify sites of ligand release and neuronal circuits activated by a ligand. Receptor endocytosis and recycling are important processes because they contribute to desensitization and resensitization, thus controlling cellular responsiveness to stimulation. The opioid receptor (OR) is a G protein-coupled receptor functionally coupled to several effector pathways, including inhibition of cyclic AMP formation and calcium current, increase in K⫹ current, and activation of mitogenactivated protein (Dhawan et al., 1996; North, 1993). OR is the main target of opiates, which are commonly used drugs for pain treatment, but it is also activated by opioids (Minami and Satoh, 1995; Reisine and Bell, 1993; Simon, 1991). ORs are expressed by central and peripheral neurons, and have been implicated in many physiological processes, including analgesia, motor activity and gastrointestinal transit (Kromer, 1988, 1989; Minami and Satoh, 1995; Reisine and Bell, 1993; Simon, 1991). In the intestine, ORs are primarily expressed by Dogiel type I myenteric neurons (Sternini et al., 1996; Ho et al., 2003), which comprise both motor neurons and interneurons (Furness et al., 1993; Furness and Costa, 1987). Enteric neurons are synaptically connected to form microcircuits that are capable of mediating reflex activity and control multiple
e
Department of Physiological and Pharmacological Sciences, University of Pavia, Pavia, Italy
Abstract—Immunohistochemistry and confocal microscopy were used to investigate endocytosis and recycling of the native opioid receptor (OR) in enteric neurons. Isolated segments of the guinea-pig ileum were exposed to increasing concentrations of OR agonists at 4 °C to allow ligand binding and warming to 37 °C for 0 min (baseline) to 6 h in ligand-free medium to allow receptor internalization and recycling. The endogenous ligand, [Met]enkephalin, and [D-Ala2,MePhe4,Gly-ol5] enkephalin (DAMGO), an opioid analog, and the alkaloids, etorphine and fentanyl, induced rapid internalization of OR immunoreactivity in enteric neurons, whereas morphine did not. OR internalization was prevented by OR antagonists. Basal levels of OR immunoreactivity in the cytoplasm were 10.52ⴞ2.05%. DAMGO (1 nM– 100 M) induced a concentration-dependent increase of OR immunofluorescence density in the cytoplasm to a maximum of 84.37ⴞ2.26%. Translocation of OR immunoreactivity in the cytoplasm was detected at 2 min, reached the maximum at 15–30 min, remained at similar levels for 2 h, began decreasing at 4 h, and was at baseline values at 6 h. A second exposure to DAMGO (100 nM) following recovery of internalized OR immunoreactivity at the cell surface induced a translocation of OR immunoreactivity in the cytoplasm comparable to the one observed following the first exposure (46.89ⴞ3.11% versus 43.31ⴞ3.80%). OR internalization was prevented by hyperosmolar sucrose, phenylarsine oxide or potassium depletion, which inhibit clathrin-mediated endocytosis. OR recycling was prevented by pre-treatment with bafilomycin A1, an acidotropic agent that inhibits endosomal *Correspondence to: C. Sternini, CURE Digestive Diseases Research Center, Building 115, Room 224, Veterans Administration Greater Los Angeles Healthcare System, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA. Tel: ⫹1-310-312-9477; fax: ⫹1-310-268-4615. E-mail address: [email protected] (C. Sternini). Abbreviations: CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2; DAMGO, [D-Ala2,MePhe4,Gly-ol5] enkephalin; DMEM, Dulbecco’s Modified Eagle medium; DMSO, dymethylsulfoxide; HEPES, (N-[2hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]); OR, opioid receptor; PAO, phenylarsine oxide; PB, phosphate buffer.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00135-0
33
34
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
functions, including motility, secretion, absorption and blood flow (Furness and Costa, 1987; Gershon et al., 1994). OR distribution matches that of the opioid enkephalin (Furness et al., 1983). In addition, enkephalin and OR immunoreactivities colocalize in myenteric neurons (Ho et al., 2003). ORs mediate the opioid inhibition of electrically induced acetylcholine release, resulting in inhibition of muscle contraction (Kromer, 1988). OR also appears to mediate opioid-induced inhibition of compliance of the intestinal-wall resistance, which is likely the result of a direct inhibition of inhibitory motor neurons and interneurons, and would partly account for the excitatory opioid effect on smooth muscle (Bauer et al., 1991; Tonini et al., 1985; Waterman et al., 1992). OR undergoes endocytosis in response to exogenously administered selective OR agonists in neurons and in OR-expressing transfected cells (Burford et al., 1998; Gaudriault et al., 1997; Keith et al., 1998; McConalogue et al., 1999; Sternini et al., 1996), and to endogenously released opioids in a stimulus-dependent manner, thus providing a means to quantify opioid release in response to stimulation (Sternini et al., 2000). By contrast, morphine, a high-affinity OR agonist (Reisine and Pasternak, 1996), does not induce receptor endocytosis (Burford et al., 1998; Gaudriault et al., 1997; Keith et al., 1998; Sternini et al., 2000, 1996). Agonist-selective OR internalization might play an important role in mediating physiological responses to opiate analgesics and the development of drug tolerance (Whistler and von Zastrow, 1998; Zhang et al., 1998). The aims of the present study were to 1) quantify ligand-induced internalization of native OR in enteric neurons using organotypic cultures with immunohistochemistry and confocal microscopy, 2) determine the role of clathrin in OR endocytosis, 3) examine OR recycling using inhibitors of protein synthesis and endosomal acidification, and 4) examine whether native ORs that have recycled to the surface are capable of binding OR ligands and undergo a second cycle of internalization.
EXPERIMENTAL PROCEDURES Experimental animals Animal care and procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experimental procedures were reviewed and approved by the appropriate Animal Use Committee of the institutions where the experiments were performed. Because in vitro preparations were used throughout the study, discomfort was reduced to a minimum. The number of animals used was also kept to the minimum necessary for a meaningful interpretation of the data. Male albino, Harlan Porcellus guinea-pigs (Hartley; 250 – 350 g; Harlan Laboratories, San Diego, CA, USA) were used.
Organotypic cultures of the ileum Guinea-pigs were killed with an overdose of sodium pentobarbital (100 mg/kg; i.p.). The distal ileum was dissected and placed 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 g/ml; penicillin, 100 IU/ml; and streptomycin, 100 g/ ml), bubbled with 95% O2, 5% CO2, pH 7.4, at 37 °C, washed and
then incubated in culture medium (Dulbecco’s Modified Eagle medium [DMEM]) containing 10% v/v fetal bovine serum, penicillin, streptomycin and Fungizone at 37 °C with 95% O2, 5% CO2 for 30 min, as described (McConalogue et al., 1999; Sternini et al., 2000). The ileum was incubated in oxygenated DMEM with fetal bovine serum, penicillin/streptomycin and Fungizone at 37 °C, exposed to opiates (etorphine, fentanyl, morphine) or opioids (Met-enkephalin or [D-Ala2,MePhe4,Gly-ol5] enkephalin [DAMGO]) (1 nM-100 M) for 1 h at 4 °C for equilibrium binding of the agonist to the receptor, washed and then incubated in agonist-free medium at 37 °C for 0 – 6 h for internalization and recycling. Specificity was assessed by pretreating the tissues with the opioid antagonist, naloxone (1 M) or the selective OR antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) (1 M), prior to OR agonist.
Immunohistochemistry Organotypic cultures were fixed in 0.1-M phosphate-buffered (PB) 4% paraformaldehyde for 2 h at 4 °C. The longitudinal muscle with the myenteric plexus attached was separated and processed as whole mount for immunohistochemistry with a commercially available OR antibody (Incstar Science, Technology and Research, Stillwater, MN, USA) directed to the C-terminal region (384 –398) of rat OR that has been characterized (McConalogue et al., 1999; Sternini et al., 2000). Briefly, whole-mount preparations were incubated in 5% normal goat serum in 0.5% Triton X-100/PB for 60 min, incubated in the same solution with OR antibody (1:4000) for 48 h at 4 °C, washed and incubated with affinitypurified donkey anti-rabbit immunoglobulin G conjugated to fluorescein isothiocyanate (Jackson Immunoresearch Laboratories, West Grove, PA, USA) (1:100) for 2 h at room temperature. OR distribution was analyzed with a Zeiss 410 laser scanning confocal microscope equipped with a krypton/argon laser and attached to a Zeiss Axiovert 100 microscope with a 100⫻ PlanApo 1.4 na objective (Carl Zeiss Inc., Thornwood, NY, USA). Images of 512⫻512 pixels were collected at a magnification zoom of 1.5⫻. Typically, 10 –20 optical sections were taken at 0.5– 0.75-m intervals through the cells. Images were processed and labeled using Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA, USA).
Quantitative analysis The level of OR internalization was quantified using an NIH quantification program: receptor internalization was quantified using single confocal images that include the nucleus and a large area of cytoplasm. Density values of immunofluorescence were kept fixed. A line was drawn around the outside of the neuron and total neuronal fluorescence (surface plus cytoplasm) was measured as the number of pixels with fixed intensity. In order to measure surface and cytoplasmic receptor separately, a second line was drawn inside the cell membrane, 0.5 m from the first line, and the number of pixels at the same fluorescence density was measured. The percentage of fluorescence present in the cytoplasm was then calculated from the second measure (immunoreactivity in the cytoplasm only) divided by the first measure (total immunoreactivity). For each data point, internalization was quantified for 40 –50 neurons from three to five animals.
Statistical analysis Fluorescence density values are shown as mean⫾S.E.M. We compared means using one way analysis of variance and the corresponding post hoc t-tests. Statistical significance was assessed using the Tukey–Fisher LSD criterion. A value of P⬍0.05 was considered significant.
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
35
Fig. 1. Single confocal microscopic images showing OR immunoreactivity in enteric neurons in baseline and stimulated conditions. Organotypic cultures were incubated with different OR ligands for 1 h at 4 °C, washed, incubated in ligand-free medium at 37 °C for 0 –30 min, fixed and processed for immunohistochemistry. OR immunoreactivity is at the plasma membrane of the soma (arrow) and neurite (arrowhead) in baseline conditions (control) and in endosomes in the soma and neurites of neurons 30 min at 37 °C following agonist stimulation with DAMGO (1 M), enkephalin (10 M), etorphine (10 nM) and fentanyl (10 M). OR immunoreactivity is at the cell surface (arrows) in neurons at 37 °C for 30 min following exposure to morphine (100 M). Calibration bars⫽10 m.
Drug treatments The effects of inhibitors of endocytosis on OR internalization were evaluated by exposing the organotypic cultures to hyperosmolar sucrose pretreatment or potassium depletion that block the formation of clathrin-coated pits (Hansen et al., 1993; Heuser and Anderson, 1989; Larkin et al., 1983) or to pre-treatment with phenylarsine oxide (PAO) that cross-links proteins with sulfur groups therefore blocking endocytosis of macromolecules (Frost et al., 1985). Isolated specimens of the guinea-pig ileum were exposed to the following treatments. 1) Pre-incubation with 0.45-M sucrose in PB for 30 min at 37 °C (the same concentration of sucrose was added to all subsequent solutions throughout the experiment), then exposure to OR ligands; controls were not exposed to hypertonic sucrose solution. 2) Pre-incubation with 80-M PAO (Fisher Scientific Int. USA) for 5 min at 37 °C; following washing and recover for 30 – 60 min at 37 °C, tissues were exposed to OR agonists; controls included tissue treated with PAO without opioid stimulation to assess the effect of this drug on OR immunoreactivity and on cell viability. 3) Preincubation for 5 min in hypotonic medium (DMEM/water, 1:1), followed by incubation in isotonic K⫹-free buffer (20-mM sodium-HEPES, 100-mM NaCl and 2-mM glutamine) for various times (10 – 60 min), then
exposure to OR agonists; controls were incubated in the same buffer supplemented with KCl. To determine whether OR undergoes recycling, specimens were treated with cycloheximide, a protein synthesis inhibitor, prior to agonist stimulation (Grady et al., 1995). Organotypic cultures were preincubated with 70 M of cycloheximide (Sigma, St. Louis, MO, USA), a dose that has been shown to cause inhibition of protein synthesis for 60 min at 37 °C before agonist stimulation. This drug was also added to all subsequent solutions throughout the experiment. To examine the relationship between endosome acidification and receptor trafficking, the acidotropic agent, bafilomycin A1 (Sigma, St. Louis, MO, USA) (1 M in dymethylsulfoxide [DMSO]), a specific inhibitor of vacuolar-type H⫹-ATP, was used prior to ligand stimulation. DMSO at the same concentration (1: 100) was also used for control specimens (Yoshimori et al., 1991).
RESULTS In basal conditions, OR immunoreactivity was primarily concentrated at the plasma membrane of enteric neurons and processes (Fig. 1). There was a translocation of OR immunoreactivity from the plasma membrane to the cyto-
36
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
Fig. 2. Prevention of OR endocytosis by OR antagonist. Single confocal images showing OR internalization in a neuron from an organotypic culture stimulated with etorphine (100 nM) and warmed up at 37 °C for 10 min in ligand-free medium, and at the cell surface in a neuron from an organotypic culture pretreated with the opioid antagonist naloxone (10 M) before exposure to 100-nM etorphine (etorphine⫹Naloxone). Arrow points to the cell-surface staining. Calibration bar⫽5 m.
plasm following stimulation with DAMGO, enkephalin, etorphine, and fentanyl, whereas OR immunoreactivity remained confined to the cell surface in neurons exposed to morphine at any concentration tested (up to 1 mM) (Fig. 1). OR internalization was prevented by the opioid antagonist, naloxone (Fig. 2) or the OR selective antagonist, CTOP (not shown), confirming that the translocation of OR immunoreactivity in response to OR agonists is due to the activation of the OR on the cell surface. OR immunoreactivity was observed in vesicles in the cytoplasm as early as 2 min (Fig. 3) following warming up at 37 °C, and by 30 min OR immunoreactivity was in cyto-
Fig. 3. OR immunoreactivity in enteric neurons in baseline conditions and after DAMGO stimulation at different times (2 min– 4 h). OR immunoreactivity is at the cell surface (arrow) in baseline conditions (time 0) and in endosomes in neurons exposed to DAMGO (1 M) and warmed up to 37 °C in ligand-free medium for 2 min, 2 h and 4 h. Single confocal microscopic images. Calibration bar⫽5 m.
Fig. 4. Quantification of OR immunofluorescence. Diagram showing the levels of OR internalization in control neurons, and following exposure to increasing concentrations of DAMGO (1 nM–100 M) and warmed up at 37 °C in a ligand-free medium for 30 min. OR internalization is expressed as percent of immunoreactivity translocated into the cytoplasm. ** P⬍0.05; * P⬍0.0001 compared with controls.
plasmic and perinuclear locations (Figs. 1 and 2). OR immunoreactivity was still in intracellular compartment at 2 and 4 h (Fig. 3) and by 6 h it was mostly back at cell surface (Figs. 10 and 11). Quantification of OR internalization was performed in neurons exposed to increasing concentrations of DAMGO (1 nM–100 M) and warmed up to 37 °C for 30 min in ligand-free medium (Fig. 4). The basal level of OR immunoreactivity in the cytoplasm in baseline conditions was 10.52⫾2.05% of total cellular immunoreactivity. The level of OR immunoreactivity in the cytoplasm was significantly higher compared with baseline at concentrations of 1-nM DAMGO (23.66⫾1.99%, P⬍0.05 versus control). At 10-nM DAMGO, OR immunoreactivity in the cytoplasm was 40.20⫾2.85% (P⬍0.0001 versus control). OR internalization increased with increasing concentrations of DAMGO and reached a maximum of 84.37⫾2.26% of total cellular immunoreactivity at the highest concentrations (100 M) (Fig. 4). This level of internalization was higher, but not significantly different from that observed at one and 10 M (71.13⫾4.13% and 78.19⫾3.55%, respectively, P⬍0.0001 versus baseline). The time-course experiment, in which organotypic cultures were exposed to 1-M DAMGO and processed for immunohistochemistry following incubation in ligand-free medium at 37 °C for 30 min to 6 h (Fig. 5), showed that the levels of OR immunoreactivity in the cytoplasm at 30 min was about 78.15⫾4.00%, remained at a similar level of internalization for 2 h (74.11⫾4.00%) (P⬍0.0001 versus controls), began decreasing at 4 h (55.46⫾6.00%) but was still significantly higher than in controls (P⬍0.0005), and returned to values comparable to controls at 6 h (20.45⫾6.00%, P⫽NS versus controls) (Fig. 5). To determine whether the OR is functional once it has returned to the cell surface, organotypic cultures were
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
Fig. 5. Quantification of OR immunofluorescence at different times. Diagram showing the time course of OR internalization induced by exposure to 1-M DAMGO and warmed at 37 °C in a ligand-free medium for 30 min to 6 h. OR internalization is expressed as percent of immunoreactivity translocated into the cytoplasm. * P⬍0.0001; ** P⬍0.0005 compared with controls. The levels of OR internalization at 6 h are not significantly different from those in controls.
exposed to 100-nM DAMGO for 1 h at 4 °C and were fixed and processed for immunohistochemistry (control), or transferred to ligand-free medium at 37 °C for 30 min or 6 h (times required for the receptor to reach maximum internalization or to return to the cell surface, respectively), equilibrated a second time at 4 °C with 100-nM DAMGO and then incubated in
37
ligand-free medium at 37 °C for an additional 30 min to allow receptors to internalize for the second time. The translocation of OR immunoreactivity in the cytoplasm following the second exposure to DAMGO was similar to the one observed after the first exposure, whereas OR immunoreactivity was predominantly located at the cell surface at 6 h following the first exposure as in baseline conditions (Fig. 6). Quantitative analysis confirmed that the levels of OR immunoreactivity in the cytoplasm following the first or second exposure to DAMGO were comparable (43.31⫾3.80% versus 46.89⫾ 3.11%, respectively) (Fig. 7), and were significantly greater (P⬍0.0005) than the levels of internalization following exposure to DAMGO, washing and warming at 37 °C in ligandfree medium for 6 h (14.51⫾2.49% versus 11.52⫾2.51% in control). The robust OR internalization induced by OR ligands was prevented by pretreatment with hyperosmolar sucrose (Fig. 8), with PAO (Fig. 9 top panel) or by potassium depletion (Fig. 9 bottom panel). Indeed, OR immunoreactivity was confined at the cell surface following these pharmacological treatment prior to exposure to etorphine (Fig. 8) or fentanyl (Fig. 9) as in control neurons. These findings support a clathrin-mediated mechanism of OR endocytosis. When organotypic cultures were pretreated with bafilomycin A1, an acidotropic agent that inhibits endosomal acidification, before exposure to OR ligand, OR immunoreactivity was in endosomes at 30 min as well as at 6 h following warming at 37 °C (Fig. 10), whereas in control specimens not pre-treated with bafilomycin A1, OR immunoreactivity at 6 h was predominantly at the cell surface as in control neurons that were not exposed to etorphine (Fig. 10). Finally, in neurons from
Fig. 6. Effects of two consecutive exposures to DAMGO on OR internalization. OR immunoreactivity is at the cell surface (arrows) in neurons from organotypic cultures exposed to DAMGO (100 nM) and warmed up to 37 °C in a ligand-free medium for 0 min (time 0) and 6 h; OR immunoreactivity is in endosomes in neurons following first exposure to DAMGO (100 nM) and warming up to 37 °C for 30 min in a ligand-free medium and following second exposure to DAMGO for 1 h at 4 °C followed by 30 min at 37 °C in a ligand-free medium 6 h after the first exposure. Note that the internalization induced by the first exposure is comparable to the internalization induced by the second exposure following receptor recycling. Calibration bar⫽5 m.
38
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
Fig. 7. Quantification of OR immunofluorescence following two consecutive exposures to DAMGO. Diagram showing the levels of OR internalization induced by first exposure to 100-nM DAMGO for 1 h at 4 °C followed by warming at 37 °C for 0 min in a ligand-free medium (control), 30 min (30 m) or 6 h (6 hr), and by the second exposure to DAMGO for an additional 1 h at 4 °C followed by 30 min at 37 °C in a ligand-free medium 6 h following the first exposure (6 hr 30 m). * P⬍0.0005 compared with controls and 6 h (6 hr) following the first exposure.
organotypic cultures treated with the protein synthesis inhibitor, cycloheximide, OR immunoreactivity was in endosomes 15 min following fentanyl stimulation and warming at 37 °C, whereas it was confined at the cell surface at 6 h as in neurons that were not pre-treated with cycloheximide or were not stimulated (Fig. 11). The findings that OR recycling was prevented by pre-treatment with bafilomycin A1 (Fig. 10), but not by the protein synthesis inhibitor, cycloheximide (Fig. 11), indicate that the reappearance of the receptor at the surface requires endosomal acidification and it represents recycling and not new receptor synthesis.
DISCUSSION This study shows that OR undergoes specific and ligandselective endocytosis in a concentration-dependent manner in the soma and neurites of enteric neurons. OR
endocytoses primarily via a clathrin-mediated pathway and recycles to the cell surface within 6 h of receptor internalization following endosomal acidification. Furthermore, ORs internalize once they return to the cell surface, since the level of receptor internalization induced by a second exposure to ligand following receptor recycling was comparable to that induced by the first ligand exposure. Receptor internalization was agonist-induced, since it was prevented by the addition of the opioid receptor antagonist, naloxone, or the OR selective antagonist, CTOP. There was an average baseline level of 10.52% of OR immunoreactivity in the cytoplasm of OR enteric neurons. This could be due to constitutive receptor internalization, which is not directly induced by agonist exposure. Alternatively, this cytoplasmic receptor immunoreactivity could be degraded receptor or newly synthesized receptor. OR internalization is induced by the opioid, enkephalin, and its analog, DAMGO, as well as by the opiates etorphine and fentanyl, but not by morphine, extending previous observations in neurons of the enteric and CNS in vivo and in OR-expressing transfected cell lines (Arden et al., 1995; Burford et al., 1998; Gaudriault et al., 1997; Keith et al., 1998, 1996; McConalogue et al., 1999; Sternini, 2001; Sternini et al., 2000, 1996; Whistler et al., 1999; Zhang et al., 1998). Morphine is a high-affinity OR agonist, that activates the OR through the same signaling pathways as other opiates (Nestler et al., 1993; Raynor et al., 1994). Morphine did not induce detectable receptor internalization even at concentrations far in excess of those necessary to inhibit cAMP production, analgesia and complete abolition of electrically induced muscle twitch (Childers, 1988; Porreca and Burks, 1983; Raynor et al., 1994; Sternini et al., 2000). Even though morphine has been reported to act as a partial agonist in some assays (Sim et al., 1996), the lack of detectable OR endocytosis following morphine stimulation does not appear to be related to its potency (Keith et al., 1998; Whistler et al., 1999). Morphine’s failure to induce internalization is accompanied by the inability of this compound to induce desensitization, receptor phosphorylation, and translocation of  arrestins (Zhang et al., 1998). However, morphine induces OR phosphorylation with -arrestin translocation and receptor sequestration when G protein kinase 2 is overexpressed (Zhang et al., 1998). This observation sug-
Fig. 8. Confocal images showing the effect of hyperosmolar sucrose on etorphine-induced OR internalization. Organotypic cultures were incubated with etorphine (500 nM) for 1 h at 4 °C, washed, and incubated in etorphine-free medium at 37 °C for 0 or 15 min. OR immunoreactivity is at the cell surface (arrows) in neurons incubated for 0 min (time 0) and 15 min in the presence of 0.45-M sucrose solution. OR immunoreactivity is in endosomes in neurons incubated for 15 min in the absence of hyperosmolar solution (15 min). Calibration bar⫽10 m.
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
39
Fig. 9. Confocal images showing the effect of PAO (top panel) and potassium deprivation (bottom panel) on fentanyl-induced OR internalization. Organotypic cultures were incubated with fentanyl (10 M) for 1 h at 4 °C, washed, and incubated in fentanyl-free medium at 37 °C for 0 or 15 min. OR immunoreactivity is in endosomes in neurons exposed to fentanyl and incubated for 15 min in the absence of PAO or in medium not depleted of potassium (15 min top and bottom panels). OR immunoreactivity is at the cell surface (arrows) in neurons exposed to fentanyl and incubated for 0 min (time 0) and 15 min in the presence of 80-M PAO, 15 min (PAO) or in neurons exposed to fentanyl in medium not depleted of potassium for 0 min (time 0) or to fentanyl in K⫹-free medium for 15 min (K⫹ free). Calibration bars⫽10 m.
gests that alterations in the trafficking machinery can affect the ability of a ligand to induce receptor endocytosis, which might represent a novel property that distinguishes OR agonists. The dissociation of OR signaling and endocytosis, and differential OR trafficking and desensitization properties support the hypothesis that OR ligands differ in their ability to regulate the biological effects mediated by the OR (Whistler et al., 1999). In this study, we have used enkephalins as endogenous opioids, since enkephalins are likely to be the main OR agonists in the enteric nervous system. Enkephalins and dynorphins are expressed by myenteric neurons (Furness et al., 1983; Steele and Costa, 1990). Enkephalins bind to the ORs with higher affinity than the endogenous opioid dynorphins, which preferentially activate k receptors (Chavkin and Goldstein, 1981; Corbett et al., 1993; Lord et al., 1977; Waterfield et al., 1977). On the other hands, endomorphins, which have powerful OR biological activity with the highest affinity and selectivity for OR than any other known opioid peptides, are expressed in the nervous system (Zadina et al., 1999), but they have not been reported in the enteric nervous system. Endomorphins interact with ORs as shown by their inhibition of neurogenic cholinergic twitch contractions in the guinea-pig ileum and their ability to induce receptor endocytosis (McConalogue et al., 1999; Zadina et al., 1997). Ligand-induced OR endocytosis is rapid, occurring within 2 min, and concentration-dependent. Quantification
analysis of the levels of receptor internalization induced by different concentrations of the OR-selective agonist, DAMGO, showed that the threshold concentration of ligand that induced a significant internalization of OR immunoreactivity is 1 nM, a concentration that induces about 20% decrease of electrically stimulated muscle twitch in the longitudinal muscle–myenteric plexus preparation of the guinea-pig ileum (McConalogue et al., 1999; Sternini et al., 2000). The potency value of DAMGO in inhibiting twitch contraction by 50% in this functional assay is less than 10 nM (ranging from 8.0 – 8.31) (Corbett et al., 1985; Galligan, 1993; McConalogue et al., 1999), a concentration that induced over 40% OR internalization. DAMGO is a OR full agonist in that is capable of suppressing twitch contractions. Indeed, 100% inhibition of twitch contraction is observed at 1-M DAMGO. In the present study, 1-M DAMGO resulted in greater than 70% internalization of OR immunoreactivity at 30 min compared with baseline values. The levels of OR internalization 30 min and 2 h after endocytosis initiated were comparable (about 78% and 74%, respectively). Four hours following internalization, a portion of OR begins to return to the cell surface, but the levels of internalization returned to baseline values only at 6 h. OR immunoreactivity recovery at the cell surface is due to recycling, since it is not affected by pretreatment with the protein-synthesis inhibitor, cycloheximide, whereas it is prevented by treatment with an acidotropic agent that prevented endosomal acidification, a re-
40
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
quired step in receptor recycling. Finally, recycled OR are rapidly available for activation by agonists and internalization as indicated by the experiment in which tissue was exposed to the same concentration of DAMGO for the second time after the receptor has recovered at the cell surface. This indicates that OR is functional once it is returned at the cell surface. OR appears to internalize primarily through a clathrindependent pathway, since conditions interfering with the formation of clathrin pits or the internalization of macromolecules markedly reduced or prevented ligand-induced internalization. This extends previous observations in OR transfected cells (Keith et al., 1998), showing that native receptors follow a similar internalization pathway. Furthermore, clathrin-mediated mechanisms have been suggested for the internalization of other G protein-coupled receptors; examples include neurokinin, gastrin-releasing peptide and -adrenergic receptors (Grady et al., 1996, 1995; Keith et al., 1996; Slice et al., 1994; von Zastrow and Kobilka, 1992). Nevertheless, we cannot exclude the involvement of a clathrin-independent pathway in the OR internalization, as it has been demonstrated for the cholecystokinin receptor that is internalized by a caveolae-dependent pathway in transfected cells (Roettger et al., 1995). Fig. 10. Confocal images showing the effect of bafilomycin treatment on etorphine-induced OR internalization and recycling. Organotypic cultures were incubated with etorphine (100 nM) for 1 h at 4 °C, washed, and incubated in etorphine-free medium at 37 °C for 0– 6 h. OR immunoreactivity is in endosomes in neurons exposed to etorphine and incubated for 30 min with DMSO or 6 h in the presence of 1-M bafilomycin in DMSO. OR immunoreactivity is at the cell surface (arrow) in neurons exposed to etorphine and incubated for 0 min in the presence of 1-M bafilomycin in DMSO (time 0) and 6 h in solution containing DMSO only without bafilomycin. Calibration bar⫽5 m.
CONCLUSIONS This study shows that ligand-selective endocytosis of native ORs in enteric neurons is primarily clathrin-mediated and that internalized receptors recycle to the cell surface upon endosomal acidification. OR can repetitively cycle between the cell surface and the cytoplasm of enteric neurons, and previous internalization of OR does not
Fig. 11. Confocal images showing the effect of cycloheximide treatment on fentanyl-induced OR internalization and recycling. Organotypic cultures were incubated with fentanyl (10 M) for 1 h at 4 °C, washed, and incubated in fentanyl-free medium at 37 °C for 0 – 6 h. OR immunoreactivity is in endosomes in neurons incubated for 15 min (cycloheximide) with fentanyl in the presence of cycloheximide. OR immunoreactivity is at the cell surface (arrow) in neurons exposed to fentanyl and incubated for 0 min in the presence of 70-M cycloheximide (time 0) and 6 h in the absence or presence of cycloheximide (bottom panel). Calibration bar⫽10 m.
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
diminish the level of ligand-induced receptor translocation from the plasma membrane to the cytoplasm. Receptor endocytosis and recycling are regulatory processes that are associated with functional diminution of receptor-mediated signaling and with resensitization (Bohm et al., 1997), but might also influence biological and adaptive responses mediated by activated receptors (Whistler et al., 1999). In the myenteric plexus, OR-bearing neurons have a similar distribution as enkephalin-immunoreactive neurons, and colocalization of OR and enkephalin immunoreactivities has been demonstrated in a subpopulation of myenteric neurons (Ho et al., 2003). Furthermore, release of endogenous opioids in vitro (Sternini et al., 2000) and in vivo (S. Patierno et al., unpublished observations) induces OR internalization. Thus, endocytosis and recycling of OR are likely to modulate opioid- and opiatemediated neurotransmission. Indeed, receptor trafficking might play an important role in the pharmacological effects of opiates, potent analgesics whose clinical use is limited by the development of many side effects, including tolerance, respiratory depression and profound impairment of gastrointestinal transit. Acknowledgements—This research was supported by the National Institutes of Health grants DK54155 (C.S.), DK 41301 (Morphology/Imaging subsection of CURE Center Grant, C.S.), VA Career Scientist Funds and DA 05010 (Antibody Core subsection of Center for Study of Opioid Receptors and Drugs of Abuse, N.C.B.), and Italian Funds (FAR 2001, M.T.).
REFERENCES Arden JR, Segredo V, Wang Z, Lameh J, Sadee W (1995) Phosphorylation and agonist-specific intracellular trafficking of an epitopetagged -opioid receptor expressed in HEK293 cells. J Neurochem 65:1636 –1645. Bauer AJ, Sarr MG, Szurszewski JH (1991) Opioids inhibit neuromuscular transmission in circular muscle of human and baboon jejunum. Gastroenterology 101:970 –976. Bohm S, Grady EF, Bunnett NW (1997) Mechanisms attenuating signaling by G protein-coupled receptors. Biochem J 322:1–18. Burford NT, Tolbert LM, Sadee W (1998) Specific G protein activation and -opioid receptor internalization caused by morphine, DAMGO and endomorphin 1. Eur J Pharmacol 342:123–126. Chavkin C, Goldstein A (1981) Demonstration of a specific dynorphin receptor in guinea pig ileum myenteric plexus. Nature 291:591– 593. Childers SR (1988) Opiate-inhibited adenylate cyclase in rat brain membranes depleted of Gs-stimulated adenylate cyclase. J Neurochem 50:543–553. Corbett AD, Kosterlitz HW, McKnight AT, Paterson SJ, Robson LE (1985) Pre-incubation of guinea-pig myenteric plexus with -funaltrexamine: discrepancy between binding assays and bioassays. Br J Pharmacol 85:665–673. Corbett AD, Paterson SJ, Kosterlitz HW (1993) Selectivity of ligands for opioid receptors. In: Handbook of experimental pharmacology, opioids I (Herz A, ed), pp 645–679. New York: Springer. Dhawan BN, Cesselin F, Raghubir R, Reisine T, Bradley PB, Portoghese PS, Hamon M (1996) International Union of Pharmacology: XII. Classification of opioid receptors. Pharmacol Rev 48:567–592. Ferguson SS (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1–24. Frost SC, Lane MD (1985) Evidence for the involvement of vicinal
41
sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes. J Biol Chem 260:2646 –2652. Furness JB, Bornstein JC, Pompolo S, Young HM, Kunze WA, Yuan SY, Kelly H (1993) The nerve circuits for motility control in the gastrointestinal tract. J Smooth Muscle Res 29:143–144. Furness JB, Costa M (1987) The enteric nervous system. New York: Churchill Livingstone. Furness JB, Costa M, Miller RJ (1983) Distribution and projections of nerves with enkephalin-like immunoreactivity in the guinea-pig small intestine. Neuroscience 8:644 –653. Galligan JJ (1993) Differential inhibition of cholinergic and noncholinergic neurogenic contractions by mu opioid and alpha-2 adrenergic agonists in guinea pig ileum. J Pharmacol Exp Ther 264:375–383. Gaudriault G, Nouel D, Dal Farra C, Beaudet A, Vincent JP (1997) Receptor-induced internalization of selective peptidic and ␦ opioid ligands. J Biol Chem 272:2880 –2888. Gershon MD, Kirchgessner AL, Wade PR (1994) Functional anatomy of the enteric nervous system. In: Physiology of the gastrointestinal tract, Vol. 1 (Johnson LR, ed), pp 381– 422. New York: Raven Press. Grady EF, Gamp PD, Jones E, Baluk P, McDonald DM, Payan DG, Bunnett NW (1996) Endocytosis and recycling of neurokinin 1 receptors in enteric neurons. Neuroscience 79:1239 –1254. Grady EF, Garland AM, Gamp PD, Lovett M, Payan DG, Bunnett NW (1995) Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor. Mol Biol Cell 6:509 – 524. Hansen SH, Sandvig K, van Deurs B (1993) Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification. J Cell Biol 121:61–72. Heuser JE, Anderson RG (1989) Hypertonic media inhibit receptormediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol 108:389 –400. Ho A, Lievore A, Patierno S, Kohlmeier SE, Tonini M, Sternini C (2003) Neurochemically distinct classes of myenteric neurons express the -opioid receptor in the guinea pig ileum. J Comp Neurol 458:404 – 411. Keith DE, Anton B, Murray SR, Zaki PA, Chu PC, Liissin DV, Monteillet-Agius G, Stewart PL, Evans CJ, von Zastrow M (1998) -opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in mammalian brain. Mol Pharmacol 53:377–384. 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. Kromer W (1988) Endogenous and exogenous opioids in the control of gastrointestinal motility and secretion. Pharmacol Rev 40:121–162. Kromer W (1989) The current status of opioid research on gastrointestinal motility. Life Sci 44:579 –589. Larkin JM, Brown MS, Goldstein J, Anderson RGW (1983) Depletion of intracellular potassium arrests coated pit formation and receptormediated endocytosis in fibroblasts. Cell 33:273–285. Lord JAH, Waterfield AA, Hughes J, Kosterlitz HW (1977) Endogenous opioid peptides: multiple agonists and receptors. Nature 267:495– 499. McConalogue K, Grady EF, Minnis J, Balestra B, Tonini M, Brecha NC, Bunnett NW, Sternini C (1999) Activation and internalization of the opioid receptor by the newly described endogenous agonists, endomorphin-1 and endomorphin-2. Neuroscience 90:1051–1059. Minami M, Satoh M (1995) Molecular biology of the opioid receptors: structures, functions and distributions. Neurosci Res 23:121–145. Nestler EJ, Hope BT, Widnell KL (1993) Drug addiction: a model for the molecular basis of neural plasticity. Neuron 11:995–1006. North R (1993) Opioid actions on membrane ion channels. In: Opioids I, Vol. 104 (Herz A, ed), pp 773–797. Berlin: Springer-Verlag. Porreca F, Burks TF (1983) Affinity of normorphine for its pharmacologic receptor in the naive and morphine-tolerant guinea-pig isolated ileum. J Pharmacol Exp Ther 225:688 –693.
42
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
Raynor K, Kong H, Yasuda K, Chen Y, Yu L, Bell GI, Reisine T (1994) Pharmacological characterization of the clones k, ⌬ and opioid receptors. Mol Pharmacol 45:330 –334. Reisine T, Bell GI (1993) Molecular biology of opioid receptors. Trends Neurosci 16:506 –510. Reisine T, Pasternak G (1996) Opioid analgesics and antagonists. In: Goodman and Gilman’s The pharmacological basis of therapeutics (Hardman JGL, Limbird LE, eds), pp 521–555. New York: McGrawHill. Roettger BF, Rentsch RU, Pinon D, Holicky E, Hadac E, Larkin JM, Miller LJ (1995) Dual pathways of internalization of the cholecystokinin receptor. J Cell Biol 128:1029 –1041. Sim LJ, Selley DE, Dworkin SI, Childers SR (1996) Effects of chronic morphine administration on opioid receptor-stimulated [35]GTPgammaS autoradiography in rat brain. J Neurosci 16:2684 –2692. Simon EJ (1991) Opioid receptors and endogenous opioid peptides. Med Res Rev 11:357–374. Slice LW, Wong HC, Sternini C, Grady EF, Bunnett NW, Walsh JH (1994) The conserved NPXnY motif present in the gastrin-releasing peptide receptor is not a general sequestration sequence. J Biol Chem 269:21755–21761. Steele PA, Costa M (1990) Opioid-like immunoreactive neurons in secretomotor pathways of the guinea-pig ileum. Neuroscience 38: 771–786. Sternini C (2001) Receptors and transmission in the brain-gut axis: potential for novel therapies: III. Opioid receptors in the enteric nervous system. Am J Physiol 281:G8 –G15. 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 opioid receptors. Neuroscience 98:233–241. Sternini C, Spann M, Anton B, Keith DE Jr, Bunnett NW, von Zastrow M, Evans C, Brecha NC (1996) Agonist-selective endocytosis of opioid receptor by neurons in vivo. Proc Natl Acad Sci USA 93: 9241–9246. Tonini M, Onori L, Perucca E, Manzo L, De Ponti F, Crema A (1985)
Depression by morphine of the excitability of intrinsic inhibitory neurons in the guinea pig colon. Eur J Pharmacol 115:317–320. Trowbridge IS, Collawn JF, Hopkins CR (1993) Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol 9:129 –161. von Zastrow M, Kobilka BK (1992) Ligand-regulated internalization and recycling of human  2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem 267:3530 –3538. Waterfield AA, Smokcum RWJ, Hughes JH, Kosterlitz HW, Henderson G (1977) In vitro pharmacology of the opioid peptides, enkephalins and dynorphins. Eur J Pharmacol 43:107–116. Waterman SA, Costa M, Tonini M (1992) Modulation of peristalsis in the guinea-pig isolated small intestine by exogenous and endogenous opioids. Br J Pharmacol 106:1004 –1010. Whistler JL, Chuang H-H, Chu P, Jan LY, von Zastrow M (1999) Functional dissociation of opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction. Neuron 23:737–746. Whistler JL, von Zastrow M (1998) Morphine-activated opioid receptors elude desensitization by -arrestin. Proc Natl Acad Sci USA 95:9914 –9919. Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y (1991) Bafilomycin A1, a specific inhibitor of vacuolar-type H(⫹)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem 266:17707–17712. Zadina JE, Hackler L, Ge L-J, Kastin A (1997) A potent and selective endogenous agonist for the -opiate receptor. Nature 386:499 –502. Zadina JE, Martin-Schild S, Gerall AA, Kastin AJ, Hackler L, Ge LJ, Zhang X (1999) Endomorphins: novel endogenous mu-opiate receptor agonists in regions of high mu-opiate receptor density. Ann NY Acad Sci 897:136 –144. Zhang J, Ferguson SSG, Barak LS, Bodduluri SB, Laporte SA, Law P-Y, Caron MG (1998) Role of G protein-coupled receptor kinase in agonist-specific regulation of -opioid receptor responsiveness. Proc Natl Acad Sci USA 95:7157–7162.
(Accepted 31 December 2002)
LIGAND-INDUCED OPIOID RECEPTOR RECYCLING IN ENTERIC NEURONS J. G. MINNIS,a,b S. PATIERNO,a,b S. E. KOHLMEIER,a,b N. C. BRECHA,a,b,c,d M. TONINIe AND a,b,c * C. STERNINI
ENDOCYTOSIS
AND
acidification, but not by the protein synthesis inhibitor, cycloheximide. This study shows that native OR in enteric neurons undergoes ligand-selective endocytosis, which is primarily clathrin-mediated, and recycles following endosomal acidification. Following recycling, OR is activated and internalized by DAMGO indicating that recycled receptors are functional. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
a CURE Digestive Diseases Research Center, Building 115, Veterans Administration Greater Los Angeles Healthcare System, Digestive Diseases Division, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA b Department of Medicine, University of California, Los Angeles, CA 90095, USA
Key words: guinea-pig ileum, gastrointestinal motility, G protein-coupled receptors, receptor trafficking.
c
Department of Neurobiology, University of California, Los Angeles, CA 90095, USA
d
Opioid Research Center, University of California, Los Angeles, CA 90095, USA
G protein-coupled receptors transduce information from extracellular stimuli into intracellular second messengers via coupling to heterotrimeric G proteins (Bohm et al., 1997; Ferguson, 2001; Trowbridge et al., 1993). Signal transduction induced by ligand-receptor interaction is followed by a cascade of events, including phosphorylation, receptor endocytosis, intracellular sorting and recycling. The presence of receptors at the plasma membrane is critical for cellular activation, and agonist-induced receptor endocytosis participates to the regulation of receptor-mediated signal transduction by removing receptors from the cell surface. Receptor endocytosis also provides an indication of receptor activation by allowing the visualization of the receptor in endosomes thus serving as a means to identify sites of ligand release and neuronal circuits activated by a ligand. Receptor endocytosis and recycling are important processes because they contribute to desensitization and resensitization, thus controlling cellular responsiveness to stimulation. The opioid receptor (OR) is a G protein-coupled receptor functionally coupled to several effector pathways, including inhibition of cyclic AMP formation and calcium current, increase in K⫹ current, and activation of mitogenactivated protein (Dhawan et al., 1996; North, 1993). OR is the main target of opiates, which are commonly used drugs for pain treatment, but it is also activated by opioids (Minami and Satoh, 1995; Reisine and Bell, 1993; Simon, 1991). ORs are expressed by central and peripheral neurons, and have been implicated in many physiological processes, including analgesia, motor activity and gastrointestinal transit (Kromer, 1988, 1989; Minami and Satoh, 1995; Reisine and Bell, 1993; Simon, 1991). In the intestine, ORs are primarily expressed by Dogiel type I myenteric neurons (Sternini et al., 1996; Ho et al., 2003), which comprise both motor neurons and interneurons (Furness et al., 1993; Furness and Costa, 1987). Enteric neurons are synaptically connected to form microcircuits that are capable of mediating reflex activity and control multiple
e
Department of Physiological and Pharmacological Sciences, University of Pavia, Pavia, Italy
Abstract—Immunohistochemistry and confocal microscopy were used to investigate endocytosis and recycling of the native opioid receptor (OR) in enteric neurons. Isolated segments of the guinea-pig ileum were exposed to increasing concentrations of OR agonists at 4 °C to allow ligand binding and warming to 37 °C for 0 min (baseline) to 6 h in ligand-free medium to allow receptor internalization and recycling. The endogenous ligand, [Met]enkephalin, and [D-Ala2,MePhe4,Gly-ol5] enkephalin (DAMGO), an opioid analog, and the alkaloids, etorphine and fentanyl, induced rapid internalization of OR immunoreactivity in enteric neurons, whereas morphine did not. OR internalization was prevented by OR antagonists. Basal levels of OR immunoreactivity in the cytoplasm were 10.52ⴞ2.05%. DAMGO (1 nM– 100 M) induced a concentration-dependent increase of OR immunofluorescence density in the cytoplasm to a maximum of 84.37ⴞ2.26%. Translocation of OR immunoreactivity in the cytoplasm was detected at 2 min, reached the maximum at 15–30 min, remained at similar levels for 2 h, began decreasing at 4 h, and was at baseline values at 6 h. A second exposure to DAMGO (100 nM) following recovery of internalized OR immunoreactivity at the cell surface induced a translocation of OR immunoreactivity in the cytoplasm comparable to the one observed following the first exposure (46.89ⴞ3.11% versus 43.31ⴞ3.80%). OR internalization was prevented by hyperosmolar sucrose, phenylarsine oxide or potassium depletion, which inhibit clathrin-mediated endocytosis. OR recycling was prevented by pre-treatment with bafilomycin A1, an acidotropic agent that inhibits endosomal *Correspondence to: C. Sternini, CURE Digestive Diseases Research Center, Building 115, Room 224, Veterans Administration Greater Los Angeles Healthcare System, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA. Tel: ⫹1-310-312-9477; fax: ⫹1-310-268-4615. E-mail address: [email protected] (C. Sternini). Abbreviations: CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2; DAMGO, [D-Ala2,MePhe4,Gly-ol5] enkephalin; DMEM, Dulbecco’s Modified Eagle medium; DMSO, dymethylsulfoxide; HEPES, (N-[2hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]); OR, opioid receptor; PAO, phenylarsine oxide; PB, phosphate buffer.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00135-0
33
34
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
functions, including motility, secretion, absorption and blood flow (Furness and Costa, 1987; Gershon et al., 1994). OR distribution matches that of the opioid enkephalin (Furness et al., 1983). In addition, enkephalin and OR immunoreactivities colocalize in myenteric neurons (Ho et al., 2003). ORs mediate the opioid inhibition of electrically induced acetylcholine release, resulting in inhibition of muscle contraction (Kromer, 1988). OR also appears to mediate opioid-induced inhibition of compliance of the intestinal-wall resistance, which is likely the result of a direct inhibition of inhibitory motor neurons and interneurons, and would partly account for the excitatory opioid effect on smooth muscle (Bauer et al., 1991; Tonini et al., 1985; Waterman et al., 1992). OR undergoes endocytosis in response to exogenously administered selective OR agonists in neurons and in OR-expressing transfected cells (Burford et al., 1998; Gaudriault et al., 1997; Keith et al., 1998; McConalogue et al., 1999; Sternini et al., 1996), and to endogenously released opioids in a stimulus-dependent manner, thus providing a means to quantify opioid release in response to stimulation (Sternini et al., 2000). By contrast, morphine, a high-affinity OR agonist (Reisine and Pasternak, 1996), does not induce receptor endocytosis (Burford et al., 1998; Gaudriault et al., 1997; Keith et al., 1998; Sternini et al., 2000, 1996). Agonist-selective OR internalization might play an important role in mediating physiological responses to opiate analgesics and the development of drug tolerance (Whistler and von Zastrow, 1998; Zhang et al., 1998). The aims of the present study were to 1) quantify ligand-induced internalization of native OR in enteric neurons using organotypic cultures with immunohistochemistry and confocal microscopy, 2) determine the role of clathrin in OR endocytosis, 3) examine OR recycling using inhibitors of protein synthesis and endosomal acidification, and 4) examine whether native ORs that have recycled to the surface are capable of binding OR ligands and undergo a second cycle of internalization.
EXPERIMENTAL PROCEDURES Experimental animals Animal care and procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experimental procedures were reviewed and approved by the appropriate Animal Use Committee of the institutions where the experiments were performed. Because in vitro preparations were used throughout the study, discomfort was reduced to a minimum. The number of animals used was also kept to the minimum necessary for a meaningful interpretation of the data. Male albino, Harlan Porcellus guinea-pigs (Hartley; 250 – 350 g; Harlan Laboratories, San Diego, CA, USA) were used.
Organotypic cultures of the ileum Guinea-pigs were killed with an overdose of sodium pentobarbital (100 mg/kg; i.p.). The distal ileum was dissected and placed 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 g/ml; penicillin, 100 IU/ml; and streptomycin, 100 g/ ml), bubbled with 95% O2, 5% CO2, pH 7.4, at 37 °C, washed and
then incubated in culture medium (Dulbecco’s Modified Eagle medium [DMEM]) containing 10% v/v fetal bovine serum, penicillin, streptomycin and Fungizone at 37 °C with 95% O2, 5% CO2 for 30 min, as described (McConalogue et al., 1999; Sternini et al., 2000). The ileum was incubated in oxygenated DMEM with fetal bovine serum, penicillin/streptomycin and Fungizone at 37 °C, exposed to opiates (etorphine, fentanyl, morphine) or opioids (Met-enkephalin or [D-Ala2,MePhe4,Gly-ol5] enkephalin [DAMGO]) (1 nM-100 M) for 1 h at 4 °C for equilibrium binding of the agonist to the receptor, washed and then incubated in agonist-free medium at 37 °C for 0 – 6 h for internalization and recycling. Specificity was assessed by pretreating the tissues with the opioid antagonist, naloxone (1 M) or the selective OR antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) (1 M), prior to OR agonist.
Immunohistochemistry Organotypic cultures were fixed in 0.1-M phosphate-buffered (PB) 4% paraformaldehyde for 2 h at 4 °C. The longitudinal muscle with the myenteric plexus attached was separated and processed as whole mount for immunohistochemistry with a commercially available OR antibody (Incstar Science, Technology and Research, Stillwater, MN, USA) directed to the C-terminal region (384 –398) of rat OR that has been characterized (McConalogue et al., 1999; Sternini et al., 2000). Briefly, whole-mount preparations were incubated in 5% normal goat serum in 0.5% Triton X-100/PB for 60 min, incubated in the same solution with OR antibody (1:4000) for 48 h at 4 °C, washed and incubated with affinitypurified donkey anti-rabbit immunoglobulin G conjugated to fluorescein isothiocyanate (Jackson Immunoresearch Laboratories, West Grove, PA, USA) (1:100) for 2 h at room temperature. OR distribution was analyzed with a Zeiss 410 laser scanning confocal microscope equipped with a krypton/argon laser and attached to a Zeiss Axiovert 100 microscope with a 100⫻ PlanApo 1.4 na objective (Carl Zeiss Inc., Thornwood, NY, USA). Images of 512⫻512 pixels were collected at a magnification zoom of 1.5⫻. Typically, 10 –20 optical sections were taken at 0.5– 0.75-m intervals through the cells. Images were processed and labeled using Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA, USA).
Quantitative analysis The level of OR internalization was quantified using an NIH quantification program: receptor internalization was quantified using single confocal images that include the nucleus and a large area of cytoplasm. Density values of immunofluorescence were kept fixed. A line was drawn around the outside of the neuron and total neuronal fluorescence (surface plus cytoplasm) was measured as the number of pixels with fixed intensity. In order to measure surface and cytoplasmic receptor separately, a second line was drawn inside the cell membrane, 0.5 m from the first line, and the number of pixels at the same fluorescence density was measured. The percentage of fluorescence present in the cytoplasm was then calculated from the second measure (immunoreactivity in the cytoplasm only) divided by the first measure (total immunoreactivity). For each data point, internalization was quantified for 40 –50 neurons from three to five animals.
Statistical analysis Fluorescence density values are shown as mean⫾S.E.M. We compared means using one way analysis of variance and the corresponding post hoc t-tests. Statistical significance was assessed using the Tukey–Fisher LSD criterion. A value of P⬍0.05 was considered significant.
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
35
Fig. 1. Single confocal microscopic images showing OR immunoreactivity in enteric neurons in baseline and stimulated conditions. Organotypic cultures were incubated with different OR ligands for 1 h at 4 °C, washed, incubated in ligand-free medium at 37 °C for 0 –30 min, fixed and processed for immunohistochemistry. OR immunoreactivity is at the plasma membrane of the soma (arrow) and neurite (arrowhead) in baseline conditions (control) and in endosomes in the soma and neurites of neurons 30 min at 37 °C following agonist stimulation with DAMGO (1 M), enkephalin (10 M), etorphine (10 nM) and fentanyl (10 M). OR immunoreactivity is at the cell surface (arrows) in neurons at 37 °C for 30 min following exposure to morphine (100 M). Calibration bars⫽10 m.
Drug treatments The effects of inhibitors of endocytosis on OR internalization were evaluated by exposing the organotypic cultures to hyperosmolar sucrose pretreatment or potassium depletion that block the formation of clathrin-coated pits (Hansen et al., 1993; Heuser and Anderson, 1989; Larkin et al., 1983) or to pre-treatment with phenylarsine oxide (PAO) that cross-links proteins with sulfur groups therefore blocking endocytosis of macromolecules (Frost et al., 1985). Isolated specimens of the guinea-pig ileum were exposed to the following treatments. 1) Pre-incubation with 0.45-M sucrose in PB for 30 min at 37 °C (the same concentration of sucrose was added to all subsequent solutions throughout the experiment), then exposure to OR ligands; controls were not exposed to hypertonic sucrose solution. 2) Pre-incubation with 80-M PAO (Fisher Scientific Int. USA) for 5 min at 37 °C; following washing and recover for 30 – 60 min at 37 °C, tissues were exposed to OR agonists; controls included tissue treated with PAO without opioid stimulation to assess the effect of this drug on OR immunoreactivity and on cell viability. 3) Preincubation for 5 min in hypotonic medium (DMEM/water, 1:1), followed by incubation in isotonic K⫹-free buffer (20-mM sodium-HEPES, 100-mM NaCl and 2-mM glutamine) for various times (10 – 60 min), then
exposure to OR agonists; controls were incubated in the same buffer supplemented with KCl. To determine whether OR undergoes recycling, specimens were treated with cycloheximide, a protein synthesis inhibitor, prior to agonist stimulation (Grady et al., 1995). Organotypic cultures were preincubated with 70 M of cycloheximide (Sigma, St. Louis, MO, USA), a dose that has been shown to cause inhibition of protein synthesis for 60 min at 37 °C before agonist stimulation. This drug was also added to all subsequent solutions throughout the experiment. To examine the relationship between endosome acidification and receptor trafficking, the acidotropic agent, bafilomycin A1 (Sigma, St. Louis, MO, USA) (1 M in dymethylsulfoxide [DMSO]), a specific inhibitor of vacuolar-type H⫹-ATP, was used prior to ligand stimulation. DMSO at the same concentration (1: 100) was also used for control specimens (Yoshimori et al., 1991).
RESULTS In basal conditions, OR immunoreactivity was primarily concentrated at the plasma membrane of enteric neurons and processes (Fig. 1). There was a translocation of OR immunoreactivity from the plasma membrane to the cyto-
36
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
Fig. 2. Prevention of OR endocytosis by OR antagonist. Single confocal images showing OR internalization in a neuron from an organotypic culture stimulated with etorphine (100 nM) and warmed up at 37 °C for 10 min in ligand-free medium, and at the cell surface in a neuron from an organotypic culture pretreated with the opioid antagonist naloxone (10 M) before exposure to 100-nM etorphine (etorphine⫹Naloxone). Arrow points to the cell-surface staining. Calibration bar⫽5 m.
plasm following stimulation with DAMGO, enkephalin, etorphine, and fentanyl, whereas OR immunoreactivity remained confined to the cell surface in neurons exposed to morphine at any concentration tested (up to 1 mM) (Fig. 1). OR internalization was prevented by the opioid antagonist, naloxone (Fig. 2) or the OR selective antagonist, CTOP (not shown), confirming that the translocation of OR immunoreactivity in response to OR agonists is due to the activation of the OR on the cell surface. OR immunoreactivity was observed in vesicles in the cytoplasm as early as 2 min (Fig. 3) following warming up at 37 °C, and by 30 min OR immunoreactivity was in cyto-
Fig. 3. OR immunoreactivity in enteric neurons in baseline conditions and after DAMGO stimulation at different times (2 min– 4 h). OR immunoreactivity is at the cell surface (arrow) in baseline conditions (time 0) and in endosomes in neurons exposed to DAMGO (1 M) and warmed up to 37 °C in ligand-free medium for 2 min, 2 h and 4 h. Single confocal microscopic images. Calibration bar⫽5 m.
Fig. 4. Quantification of OR immunofluorescence. Diagram showing the levels of OR internalization in control neurons, and following exposure to increasing concentrations of DAMGO (1 nM–100 M) and warmed up at 37 °C in a ligand-free medium for 30 min. OR internalization is expressed as percent of immunoreactivity translocated into the cytoplasm. ** P⬍0.05; * P⬍0.0001 compared with controls.
plasmic and perinuclear locations (Figs. 1 and 2). OR immunoreactivity was still in intracellular compartment at 2 and 4 h (Fig. 3) and by 6 h it was mostly back at cell surface (Figs. 10 and 11). Quantification of OR internalization was performed in neurons exposed to increasing concentrations of DAMGO (1 nM–100 M) and warmed up to 37 °C for 30 min in ligand-free medium (Fig. 4). The basal level of OR immunoreactivity in the cytoplasm in baseline conditions was 10.52⫾2.05% of total cellular immunoreactivity. The level of OR immunoreactivity in the cytoplasm was significantly higher compared with baseline at concentrations of 1-nM DAMGO (23.66⫾1.99%, P⬍0.05 versus control). At 10-nM DAMGO, OR immunoreactivity in the cytoplasm was 40.20⫾2.85% (P⬍0.0001 versus control). OR internalization increased with increasing concentrations of DAMGO and reached a maximum of 84.37⫾2.26% of total cellular immunoreactivity at the highest concentrations (100 M) (Fig. 4). This level of internalization was higher, but not significantly different from that observed at one and 10 M (71.13⫾4.13% and 78.19⫾3.55%, respectively, P⬍0.0001 versus baseline). The time-course experiment, in which organotypic cultures were exposed to 1-M DAMGO and processed for immunohistochemistry following incubation in ligand-free medium at 37 °C for 30 min to 6 h (Fig. 5), showed that the levels of OR immunoreactivity in the cytoplasm at 30 min was about 78.15⫾4.00%, remained at a similar level of internalization for 2 h (74.11⫾4.00%) (P⬍0.0001 versus controls), began decreasing at 4 h (55.46⫾6.00%) but was still significantly higher than in controls (P⬍0.0005), and returned to values comparable to controls at 6 h (20.45⫾6.00%, P⫽NS versus controls) (Fig. 5). To determine whether the OR is functional once it has returned to the cell surface, organotypic cultures were
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
Fig. 5. Quantification of OR immunofluorescence at different times. Diagram showing the time course of OR internalization induced by exposure to 1-M DAMGO and warmed at 37 °C in a ligand-free medium for 30 min to 6 h. OR internalization is expressed as percent of immunoreactivity translocated into the cytoplasm. * P⬍0.0001; ** P⬍0.0005 compared with controls. The levels of OR internalization at 6 h are not significantly different from those in controls.
exposed to 100-nM DAMGO for 1 h at 4 °C and were fixed and processed for immunohistochemistry (control), or transferred to ligand-free medium at 37 °C for 30 min or 6 h (times required for the receptor to reach maximum internalization or to return to the cell surface, respectively), equilibrated a second time at 4 °C with 100-nM DAMGO and then incubated in
37
ligand-free medium at 37 °C for an additional 30 min to allow receptors to internalize for the second time. The translocation of OR immunoreactivity in the cytoplasm following the second exposure to DAMGO was similar to the one observed after the first exposure, whereas OR immunoreactivity was predominantly located at the cell surface at 6 h following the first exposure as in baseline conditions (Fig. 6). Quantitative analysis confirmed that the levels of OR immunoreactivity in the cytoplasm following the first or second exposure to DAMGO were comparable (43.31⫾3.80% versus 46.89⫾ 3.11%, respectively) (Fig. 7), and were significantly greater (P⬍0.0005) than the levels of internalization following exposure to DAMGO, washing and warming at 37 °C in ligandfree medium for 6 h (14.51⫾2.49% versus 11.52⫾2.51% in control). The robust OR internalization induced by OR ligands was prevented by pretreatment with hyperosmolar sucrose (Fig. 8), with PAO (Fig. 9 top panel) or by potassium depletion (Fig. 9 bottom panel). Indeed, OR immunoreactivity was confined at the cell surface following these pharmacological treatment prior to exposure to etorphine (Fig. 8) or fentanyl (Fig. 9) as in control neurons. These findings support a clathrin-mediated mechanism of OR endocytosis. When organotypic cultures were pretreated with bafilomycin A1, an acidotropic agent that inhibits endosomal acidification, before exposure to OR ligand, OR immunoreactivity was in endosomes at 30 min as well as at 6 h following warming at 37 °C (Fig. 10), whereas in control specimens not pre-treated with bafilomycin A1, OR immunoreactivity at 6 h was predominantly at the cell surface as in control neurons that were not exposed to etorphine (Fig. 10). Finally, in neurons from
Fig. 6. Effects of two consecutive exposures to DAMGO on OR internalization. OR immunoreactivity is at the cell surface (arrows) in neurons from organotypic cultures exposed to DAMGO (100 nM) and warmed up to 37 °C in a ligand-free medium for 0 min (time 0) and 6 h; OR immunoreactivity is in endosomes in neurons following first exposure to DAMGO (100 nM) and warming up to 37 °C for 30 min in a ligand-free medium and following second exposure to DAMGO for 1 h at 4 °C followed by 30 min at 37 °C in a ligand-free medium 6 h after the first exposure. Note that the internalization induced by the first exposure is comparable to the internalization induced by the second exposure following receptor recycling. Calibration bar⫽5 m.
38
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
Fig. 7. Quantification of OR immunofluorescence following two consecutive exposures to DAMGO. Diagram showing the levels of OR internalization induced by first exposure to 100-nM DAMGO for 1 h at 4 °C followed by warming at 37 °C for 0 min in a ligand-free medium (control), 30 min (30 m) or 6 h (6 hr), and by the second exposure to DAMGO for an additional 1 h at 4 °C followed by 30 min at 37 °C in a ligand-free medium 6 h following the first exposure (6 hr 30 m). * P⬍0.0005 compared with controls and 6 h (6 hr) following the first exposure.
organotypic cultures treated with the protein synthesis inhibitor, cycloheximide, OR immunoreactivity was in endosomes 15 min following fentanyl stimulation and warming at 37 °C, whereas it was confined at the cell surface at 6 h as in neurons that were not pre-treated with cycloheximide or were not stimulated (Fig. 11). The findings that OR recycling was prevented by pre-treatment with bafilomycin A1 (Fig. 10), but not by the protein synthesis inhibitor, cycloheximide (Fig. 11), indicate that the reappearance of the receptor at the surface requires endosomal acidification and it represents recycling and not new receptor synthesis.
DISCUSSION This study shows that OR undergoes specific and ligandselective endocytosis in a concentration-dependent manner in the soma and neurites of enteric neurons. OR
endocytoses primarily via a clathrin-mediated pathway and recycles to the cell surface within 6 h of receptor internalization following endosomal acidification. Furthermore, ORs internalize once they return to the cell surface, since the level of receptor internalization induced by a second exposure to ligand following receptor recycling was comparable to that induced by the first ligand exposure. Receptor internalization was agonist-induced, since it was prevented by the addition of the opioid receptor antagonist, naloxone, or the OR selective antagonist, CTOP. There was an average baseline level of 10.52% of OR immunoreactivity in the cytoplasm of OR enteric neurons. This could be due to constitutive receptor internalization, which is not directly induced by agonist exposure. Alternatively, this cytoplasmic receptor immunoreactivity could be degraded receptor or newly synthesized receptor. OR internalization is induced by the opioid, enkephalin, and its analog, DAMGO, as well as by the opiates etorphine and fentanyl, but not by morphine, extending previous observations in neurons of the enteric and CNS in vivo and in OR-expressing transfected cell lines (Arden et al., 1995; Burford et al., 1998; Gaudriault et al., 1997; Keith et al., 1998, 1996; McConalogue et al., 1999; Sternini, 2001; Sternini et al., 2000, 1996; Whistler et al., 1999; Zhang et al., 1998). Morphine is a high-affinity OR agonist, that activates the OR through the same signaling pathways as other opiates (Nestler et al., 1993; Raynor et al., 1994). Morphine did not induce detectable receptor internalization even at concentrations far in excess of those necessary to inhibit cAMP production, analgesia and complete abolition of electrically induced muscle twitch (Childers, 1988; Porreca and Burks, 1983; Raynor et al., 1994; Sternini et al., 2000). Even though morphine has been reported to act as a partial agonist in some assays (Sim et al., 1996), the lack of detectable OR endocytosis following morphine stimulation does not appear to be related to its potency (Keith et al., 1998; Whistler et al., 1999). Morphine’s failure to induce internalization is accompanied by the inability of this compound to induce desensitization, receptor phosphorylation, and translocation of  arrestins (Zhang et al., 1998). However, morphine induces OR phosphorylation with -arrestin translocation and receptor sequestration when G protein kinase 2 is overexpressed (Zhang et al., 1998). This observation sug-
Fig. 8. Confocal images showing the effect of hyperosmolar sucrose on etorphine-induced OR internalization. Organotypic cultures were incubated with etorphine (500 nM) for 1 h at 4 °C, washed, and incubated in etorphine-free medium at 37 °C for 0 or 15 min. OR immunoreactivity is at the cell surface (arrows) in neurons incubated for 0 min (time 0) and 15 min in the presence of 0.45-M sucrose solution. OR immunoreactivity is in endosomes in neurons incubated for 15 min in the absence of hyperosmolar solution (15 min). Calibration bar⫽10 m.
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
39
Fig. 9. Confocal images showing the effect of PAO (top panel) and potassium deprivation (bottom panel) on fentanyl-induced OR internalization. Organotypic cultures were incubated with fentanyl (10 M) for 1 h at 4 °C, washed, and incubated in fentanyl-free medium at 37 °C for 0 or 15 min. OR immunoreactivity is in endosomes in neurons exposed to fentanyl and incubated for 15 min in the absence of PAO or in medium not depleted of potassium (15 min top and bottom panels). OR immunoreactivity is at the cell surface (arrows) in neurons exposed to fentanyl and incubated for 0 min (time 0) and 15 min in the presence of 80-M PAO, 15 min (PAO) or in neurons exposed to fentanyl in medium not depleted of potassium for 0 min (time 0) or to fentanyl in K⫹-free medium for 15 min (K⫹ free). Calibration bars⫽10 m.
gests that alterations in the trafficking machinery can affect the ability of a ligand to induce receptor endocytosis, which might represent a novel property that distinguishes OR agonists. The dissociation of OR signaling and endocytosis, and differential OR trafficking and desensitization properties support the hypothesis that OR ligands differ in their ability to regulate the biological effects mediated by the OR (Whistler et al., 1999). In this study, we have used enkephalins as endogenous opioids, since enkephalins are likely to be the main OR agonists in the enteric nervous system. Enkephalins and dynorphins are expressed by myenteric neurons (Furness et al., 1983; Steele and Costa, 1990). Enkephalins bind to the ORs with higher affinity than the endogenous opioid dynorphins, which preferentially activate k receptors (Chavkin and Goldstein, 1981; Corbett et al., 1993; Lord et al., 1977; Waterfield et al., 1977). On the other hands, endomorphins, which have powerful OR biological activity with the highest affinity and selectivity for OR than any other known opioid peptides, are expressed in the nervous system (Zadina et al., 1999), but they have not been reported in the enteric nervous system. Endomorphins interact with ORs as shown by their inhibition of neurogenic cholinergic twitch contractions in the guinea-pig ileum and their ability to induce receptor endocytosis (McConalogue et al., 1999; Zadina et al., 1997). Ligand-induced OR endocytosis is rapid, occurring within 2 min, and concentration-dependent. Quantification
analysis of the levels of receptor internalization induced by different concentrations of the OR-selective agonist, DAMGO, showed that the threshold concentration of ligand that induced a significant internalization of OR immunoreactivity is 1 nM, a concentration that induces about 20% decrease of electrically stimulated muscle twitch in the longitudinal muscle–myenteric plexus preparation of the guinea-pig ileum (McConalogue et al., 1999; Sternini et al., 2000). The potency value of DAMGO in inhibiting twitch contraction by 50% in this functional assay is less than 10 nM (ranging from 8.0 – 8.31) (Corbett et al., 1985; Galligan, 1993; McConalogue et al., 1999), a concentration that induced over 40% OR internalization. DAMGO is a OR full agonist in that is capable of suppressing twitch contractions. Indeed, 100% inhibition of twitch contraction is observed at 1-M DAMGO. In the present study, 1-M DAMGO resulted in greater than 70% internalization of OR immunoreactivity at 30 min compared with baseline values. The levels of OR internalization 30 min and 2 h after endocytosis initiated were comparable (about 78% and 74%, respectively). Four hours following internalization, a portion of OR begins to return to the cell surface, but the levels of internalization returned to baseline values only at 6 h. OR immunoreactivity recovery at the cell surface is due to recycling, since it is not affected by pretreatment with the protein-synthesis inhibitor, cycloheximide, whereas it is prevented by treatment with an acidotropic agent that prevented endosomal acidification, a re-
40
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
quired step in receptor recycling. Finally, recycled OR are rapidly available for activation by agonists and internalization as indicated by the experiment in which tissue was exposed to the same concentration of DAMGO for the second time after the receptor has recovered at the cell surface. This indicates that OR is functional once it is returned at the cell surface. OR appears to internalize primarily through a clathrindependent pathway, since conditions interfering with the formation of clathrin pits or the internalization of macromolecules markedly reduced or prevented ligand-induced internalization. This extends previous observations in OR transfected cells (Keith et al., 1998), showing that native receptors follow a similar internalization pathway. Furthermore, clathrin-mediated mechanisms have been suggested for the internalization of other G protein-coupled receptors; examples include neurokinin, gastrin-releasing peptide and -adrenergic receptors (Grady et al., 1996, 1995; Keith et al., 1996; Slice et al., 1994; von Zastrow and Kobilka, 1992). Nevertheless, we cannot exclude the involvement of a clathrin-independent pathway in the OR internalization, as it has been demonstrated for the cholecystokinin receptor that is internalized by a caveolae-dependent pathway in transfected cells (Roettger et al., 1995). Fig. 10. Confocal images showing the effect of bafilomycin treatment on etorphine-induced OR internalization and recycling. Organotypic cultures were incubated with etorphine (100 nM) for 1 h at 4 °C, washed, and incubated in etorphine-free medium at 37 °C for 0– 6 h. OR immunoreactivity is in endosomes in neurons exposed to etorphine and incubated for 30 min with DMSO or 6 h in the presence of 1-M bafilomycin in DMSO. OR immunoreactivity is at the cell surface (arrow) in neurons exposed to etorphine and incubated for 0 min in the presence of 1-M bafilomycin in DMSO (time 0) and 6 h in solution containing DMSO only without bafilomycin. Calibration bar⫽5 m.
CONCLUSIONS This study shows that ligand-selective endocytosis of native ORs in enteric neurons is primarily clathrin-mediated and that internalized receptors recycle to the cell surface upon endosomal acidification. OR can repetitively cycle between the cell surface and the cytoplasm of enteric neurons, and previous internalization of OR does not
Fig. 11. Confocal images showing the effect of cycloheximide treatment on fentanyl-induced OR internalization and recycling. Organotypic cultures were incubated with fentanyl (10 M) for 1 h at 4 °C, washed, and incubated in fentanyl-free medium at 37 °C for 0 – 6 h. OR immunoreactivity is in endosomes in neurons incubated for 15 min (cycloheximide) with fentanyl in the presence of cycloheximide. OR immunoreactivity is at the cell surface (arrow) in neurons exposed to fentanyl and incubated for 0 min in the presence of 70-M cycloheximide (time 0) and 6 h in the absence or presence of cycloheximide (bottom panel). Calibration bar⫽10 m.
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
diminish the level of ligand-induced receptor translocation from the plasma membrane to the cytoplasm. Receptor endocytosis and recycling are regulatory processes that are associated with functional diminution of receptor-mediated signaling and with resensitization (Bohm et al., 1997), but might also influence biological and adaptive responses mediated by activated receptors (Whistler et al., 1999). In the myenteric plexus, OR-bearing neurons have a similar distribution as enkephalin-immunoreactive neurons, and colocalization of OR and enkephalin immunoreactivities has been demonstrated in a subpopulation of myenteric neurons (Ho et al., 2003). Furthermore, release of endogenous opioids in vitro (Sternini et al., 2000) and in vivo (S. Patierno et al., unpublished observations) induces OR internalization. Thus, endocytosis and recycling of OR are likely to modulate opioid- and opiatemediated neurotransmission. Indeed, receptor trafficking might play an important role in the pharmacological effects of opiates, potent analgesics whose clinical use is limited by the development of many side effects, including tolerance, respiratory depression and profound impairment of gastrointestinal transit. Acknowledgements—This research was supported by the National Institutes of Health grants DK54155 (C.S.), DK 41301 (Morphology/Imaging subsection of CURE Center Grant, C.S.), VA Career Scientist Funds and DA 05010 (Antibody Core subsection of Center for Study of Opioid Receptors and Drugs of Abuse, N.C.B.), and Italian Funds (FAR 2001, M.T.).
REFERENCES Arden JR, Segredo V, Wang Z, Lameh J, Sadee W (1995) Phosphorylation and agonist-specific intracellular trafficking of an epitopetagged -opioid receptor expressed in HEK293 cells. J Neurochem 65:1636 –1645. Bauer AJ, Sarr MG, Szurszewski JH (1991) Opioids inhibit neuromuscular transmission in circular muscle of human and baboon jejunum. Gastroenterology 101:970 –976. Bohm S, Grady EF, Bunnett NW (1997) Mechanisms attenuating signaling by G protein-coupled receptors. Biochem J 322:1–18. Burford NT, Tolbert LM, Sadee W (1998) Specific G protein activation and -opioid receptor internalization caused by morphine, DAMGO and endomorphin 1. Eur J Pharmacol 342:123–126. Chavkin C, Goldstein A (1981) Demonstration of a specific dynorphin receptor in guinea pig ileum myenteric plexus. Nature 291:591– 593. Childers SR (1988) Opiate-inhibited adenylate cyclase in rat brain membranes depleted of Gs-stimulated adenylate cyclase. J Neurochem 50:543–553. Corbett AD, Kosterlitz HW, McKnight AT, Paterson SJ, Robson LE (1985) Pre-incubation of guinea-pig myenteric plexus with -funaltrexamine: discrepancy between binding assays and bioassays. Br J Pharmacol 85:665–673. Corbett AD, Paterson SJ, Kosterlitz HW (1993) Selectivity of ligands for opioid receptors. In: Handbook of experimental pharmacology, opioids I (Herz A, ed), pp 645–679. New York: Springer. Dhawan BN, Cesselin F, Raghubir R, Reisine T, Bradley PB, Portoghese PS, Hamon M (1996) International Union of Pharmacology: XII. Classification of opioid receptors. Pharmacol Rev 48:567–592. Ferguson SS (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1–24. Frost SC, Lane MD (1985) Evidence for the involvement of vicinal
41
sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes. J Biol Chem 260:2646 –2652. Furness JB, Bornstein JC, Pompolo S, Young HM, Kunze WA, Yuan SY, Kelly H (1993) The nerve circuits for motility control in the gastrointestinal tract. J Smooth Muscle Res 29:143–144. Furness JB, Costa M (1987) The enteric nervous system. New York: Churchill Livingstone. Furness JB, Costa M, Miller RJ (1983) Distribution and projections of nerves with enkephalin-like immunoreactivity in the guinea-pig small intestine. Neuroscience 8:644 –653. Galligan JJ (1993) Differential inhibition of cholinergic and noncholinergic neurogenic contractions by mu opioid and alpha-2 adrenergic agonists in guinea pig ileum. J Pharmacol Exp Ther 264:375–383. Gaudriault G, Nouel D, Dal Farra C, Beaudet A, Vincent JP (1997) Receptor-induced internalization of selective peptidic and ␦ opioid ligands. J Biol Chem 272:2880 –2888. Gershon MD, Kirchgessner AL, Wade PR (1994) Functional anatomy of the enteric nervous system. In: Physiology of the gastrointestinal tract, Vol. 1 (Johnson LR, ed), pp 381– 422. New York: Raven Press. Grady EF, Gamp PD, Jones E, Baluk P, McDonald DM, Payan DG, Bunnett NW (1996) Endocytosis and recycling of neurokinin 1 receptors in enteric neurons. Neuroscience 79:1239 –1254. Grady EF, Garland AM, Gamp PD, Lovett M, Payan DG, Bunnett NW (1995) Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor. Mol Biol Cell 6:509 – 524. Hansen SH, Sandvig K, van Deurs B (1993) Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification. J Cell Biol 121:61–72. Heuser JE, Anderson RG (1989) Hypertonic media inhibit receptormediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol 108:389 –400. Ho A, Lievore A, Patierno S, Kohlmeier SE, Tonini M, Sternini C (2003) Neurochemically distinct classes of myenteric neurons express the -opioid receptor in the guinea pig ileum. J Comp Neurol 458:404 – 411. Keith DE, Anton B, Murray SR, Zaki PA, Chu PC, Liissin DV, Monteillet-Agius G, Stewart PL, Evans CJ, von Zastrow M (1998) -opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in mammalian brain. Mol Pharmacol 53:377–384. 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. Kromer W (1988) Endogenous and exogenous opioids in the control of gastrointestinal motility and secretion. Pharmacol Rev 40:121–162. Kromer W (1989) The current status of opioid research on gastrointestinal motility. Life Sci 44:579 –589. Larkin JM, Brown MS, Goldstein J, Anderson RGW (1983) Depletion of intracellular potassium arrests coated pit formation and receptormediated endocytosis in fibroblasts. Cell 33:273–285. Lord JAH, Waterfield AA, Hughes J, Kosterlitz HW (1977) Endogenous opioid peptides: multiple agonists and receptors. Nature 267:495– 499. McConalogue K, Grady EF, Minnis J, Balestra B, Tonini M, Brecha NC, Bunnett NW, Sternini C (1999) Activation and internalization of the opioid receptor by the newly described endogenous agonists, endomorphin-1 and endomorphin-2. Neuroscience 90:1051–1059. Minami M, Satoh M (1995) Molecular biology of the opioid receptors: structures, functions and distributions. Neurosci Res 23:121–145. Nestler EJ, Hope BT, Widnell KL (1993) Drug addiction: a model for the molecular basis of neural plasticity. Neuron 11:995–1006. North R (1993) Opioid actions on membrane ion channels. In: Opioids I, Vol. 104 (Herz A, ed), pp 773–797. Berlin: Springer-Verlag. Porreca F, Burks TF (1983) Affinity of normorphine for its pharmacologic receptor in the naive and morphine-tolerant guinea-pig isolated ileum. J Pharmacol Exp Ther 225:688 –693.
42
J. G. Minnis et al. / Neuroscience 119 (2003) 33– 42
Raynor K, Kong H, Yasuda K, Chen Y, Yu L, Bell GI, Reisine T (1994) Pharmacological characterization of the clones k, ⌬ and opioid receptors. Mol Pharmacol 45:330 –334. Reisine T, Bell GI (1993) Molecular biology of opioid receptors. Trends Neurosci 16:506 –510. Reisine T, Pasternak G (1996) Opioid analgesics and antagonists. In: Goodman and Gilman’s The pharmacological basis of therapeutics (Hardman JGL, Limbird LE, eds), pp 521–555. New York: McGrawHill. Roettger BF, Rentsch RU, Pinon D, Holicky E, Hadac E, Larkin JM, Miller LJ (1995) Dual pathways of internalization of the cholecystokinin receptor. J Cell Biol 128:1029 –1041. Sim LJ, Selley DE, Dworkin SI, Childers SR (1996) Effects of chronic morphine administration on opioid receptor-stimulated [35]GTPgammaS autoradiography in rat brain. J Neurosci 16:2684 –2692. Simon EJ (1991) Opioid receptors and endogenous opioid peptides. Med Res Rev 11:357–374. Slice LW, Wong HC, Sternini C, Grady EF, Bunnett NW, Walsh JH (1994) The conserved NPXnY motif present in the gastrin-releasing peptide receptor is not a general sequestration sequence. J Biol Chem 269:21755–21761. Steele PA, Costa M (1990) Opioid-like immunoreactive neurons in secretomotor pathways of the guinea-pig ileum. Neuroscience 38: 771–786. Sternini C (2001) Receptors and transmission in the brain-gut axis: potential for novel therapies: III. Opioid receptors in the enteric nervous system. Am J Physiol 281:G8 –G15. 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 opioid receptors. Neuroscience 98:233–241. Sternini C, Spann M, Anton B, Keith DE Jr, Bunnett NW, von Zastrow M, Evans C, Brecha NC (1996) Agonist-selective endocytosis of opioid receptor by neurons in vivo. Proc Natl Acad Sci USA 93: 9241–9246. Tonini M, Onori L, Perucca E, Manzo L, De Ponti F, Crema A (1985)
Depression by morphine of the excitability of intrinsic inhibitory neurons in the guinea pig colon. Eur J Pharmacol 115:317–320. Trowbridge IS, Collawn JF, Hopkins CR (1993) Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol 9:129 –161. von Zastrow M, Kobilka BK (1992) Ligand-regulated internalization and recycling of human  2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem 267:3530 –3538. Waterfield AA, Smokcum RWJ, Hughes JH, Kosterlitz HW, Henderson G (1977) In vitro pharmacology of the opioid peptides, enkephalins and dynorphins. Eur J Pharmacol 43:107–116. Waterman SA, Costa M, Tonini M (1992) Modulation of peristalsis in the guinea-pig isolated small intestine by exogenous and endogenous opioids. Br J Pharmacol 106:1004 –1010. Whistler JL, Chuang H-H, Chu P, Jan LY, von Zastrow M (1999) Functional dissociation of opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction. Neuron 23:737–746. Whistler JL, von Zastrow M (1998) Morphine-activated opioid receptors elude desensitization by -arrestin. Proc Natl Acad Sci USA 95:9914 –9919. Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y (1991) Bafilomycin A1, a specific inhibitor of vacuolar-type H(⫹)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem 266:17707–17712. Zadina JE, Hackler L, Ge L-J, Kastin A (1997) A potent and selective endogenous agonist for the -opiate receptor. Nature 386:499 –502. Zadina JE, Martin-Schild S, Gerall AA, Kastin AJ, Hackler L, Ge LJ, Zhang X (1999) Endomorphins: novel endogenous mu-opiate receptor agonists in regions of high mu-opiate receptor density. Ann NY Acad Sci 897:136 –144. Zhang J, Ferguson SSG, Barak LS, Bodduluri SB, Laporte SA, Law P-Y, Caron MG (1998) Role of G protein-coupled receptor kinase in agonist-specific regulation of -opioid receptor responsiveness. Proc Natl Acad Sci USA 95:7157–7162.
(Accepted 31 December 2002)