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CELLULAR AND MOLECULAR

Different Regulation of Human -Opioid Receptors by SNC-80 [(+)-4-[(R)--((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide] and Endogenous Enkephalins

Laboratoire de Pharmacologie Moléculaire de la Tolérance aux opiacés-Centre Hospitalier et Universitaire, Caen Cedex, France

Received December 8, 2003; accepted April 12, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Among the different mechanisms underlying opioid tolerance, receptor desensitization would represent a major cellular adaptation process in which the role of receptor internalization is still a matter of debate. In the present study, we examined desensitization of the human -opioid receptor (hDOR) produced by endogenous opioid peptides Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) and Met-enkephalin (Tyr-Gly-Gly-Phe-Met), and the contribution of internalization in this process. Results obtained with natural peptides were compared with those produced by a synthetic opioid agonist, SNC-80 [(+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide). After a 30-min treatment, we observed a different regulation of hDOR between agonists. SNC-80 produced a stronger and faster desensitization and was associated with a loss of opioid binding sites by 50%. SNC-80 also caused a marked hDOR down-regulation by 30% as observed by Western blot. Immunocytochemistry revealed that SNC-80 induced a complete redistribution of hDOR from cell surface into intracellular compartments, whereas a partial internalization was visualized upon enkephalin exposure. In constrast, a stronger hDOR recycling and resensitization were measured after enkephalin treatment compared with SNC-80. These data strongly suggested a differential sorting of the internalized receptors caused by enkephalins and SNC-80 that was further confirmed by chloroquine as a lysosomal degradation blocker and monensin as a recycling endosome inhibitor. Finally, by preventing hDOR internalization with 0.5 M sucrose, we demonstrated that hDOR internalization contributes partially to desensitization. In conclusion, hDOR desensitization depends both on its internalization and its sorting either to the recycling pathway or to lysosomes.

Opioid agonists also differ in their ability to promote desensitization as illustrated by the works of Reisine's group (Blake et al., 1997a,b; Bot et al., 1997). For example, when activated by [D-Pen²,D-Pen⁵]-enkephalin (DPDPE), -opioid receptor desensitization was characterized by both a reduction of the maximal inhibition on cAMP accumulation and a right-shift of the dose-response curve. Under the same conditions of pretreatment, morphine was shown to produce sensitization rather than desensitization (Bot et al., 1997). In our laboratory, we also described a differential desensitization of the human -opioid receptors (hDORs) by peptidic [DPDPE and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH₂)] and alkaloid (etorphine) agonists (Allouche et al., 1999a). After a 30-min period of pretreatment, no inhibition of cAMP accumulation was detected with peptidic agonists, whereas a moderate desensitization by 50% was measured upon etorphine exposure. We proposed that -receptor desensitization relies on the chemical nature of agonist (peptidic versus alkaloid). We extend our study to other opioid agonists and investigate the regulation of hDOR by enkephalins (Leuand Met-enkephalin), known as nonselective endogenous peptides (for review, see Reisine and Pasternak, 1996), and by SNC-80, a nonpeptidic -selective agonist (Calderon et al., 1994). The choice of the two enkephalins was based on a previous study showing an increase of Met-enkephalin extracellular levels in brain of chronic morphine-treated rats (Mas Nieto et al., 2002). These data suggested that beside the sustained receptor activation by morphine, up-regulation of endogenous opioid system played a role in the development of tolerance by promoting opioid receptor desensitization. Moreover, to our knowledge, relatively few studies have been conducted to evaluate the ability of the natural enkephalins to induce desensitization of these receptors.

After the pharmacological characterization of agonists in the SK-N-BE cell line that endogenously expresses only hDOR (Polastron et al., 1994), we performed a detailed comparison on desensitization, internalization, resensitization, and recycling of opioid receptors upon enkephalin and SNC-80 exposure. The quantification of -receptor level was achieved by binding studies, and their functionality was measured on the inhibition of adenylyl cyclase; receptor internalization was visualized by immunocytochemical experiments. Our results showed that hDORs were more rapidly desensitized by SNC-80 than by enkephalins. In the presence of SNC-80, hDOR internalization via clathrin-coated pits was followed by their degradation in lysosomes, whereas in the presence of enkephalins, the opioid receptors were internalized and sorted to recycling endosomes. By using hypertonic sucrose solution, we also showed that opioid receptors internalization contributed partially to desensitization whatever the agonist.

	Materials and Methods

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Cell Culture and Transfection. The human neuroblastoma cell line SK-N-BE, endogenously expressing only hDOR (Polastron et al., 1994), was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, and 1% (v/v) antibiotic-antimycotic solution (100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin B) at 37°C in a water-saturated atmosphere containing 5% CO₂.

To study the localization of opioid receptors by immunocytochemical experiments, we transfected SK-N-BE cells with the cDNA encoding for the FLAG-tagged hDOR or with the empty plasmid (pcDNA3). Transfections were performed by electroporation (150,000 cells/µg DNA, 250 V, 350 µF), and selection of transfected cells was started 48 h later using 0.5 mg/ml geneticin (G-418; Sigma-Aldrich, St. Louis, MO). Stable transfected cells (FLAG-tagged hDOR) were obtained without clonal selection after 2 months in a culture medium supplemented with G-418. Cells overexpressed hDOR by 20 fold (1000-2500 fmol/mg of protein for transfected cells versus 50-150 fmol/mg of protein for untransfected cells).

Competitive Binding Studies. SK-N-BE and FLAG-tagged hDOR-transfected cells were harvested in 10 mM Tris-HCl/1 mM EDTA, pH 7.4, sonicated, and ultracentrifuged at 100,000g for 30 min at 4°C. The resulting membrane pellet was resuspended in 50 mM Tris-HCl, pH 7.4, to a protein concentration of 4 mg/ml determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard. Competitive binding experiments were carried out in triplicate using 170 to 200 µg of crude membrane fraction, 2 nM (for wild-type SK-N-BE cells and pcDNA3 transfected cells) or 10 nM (for FLAG-tagged hDOR-transfected cells) [³H]diprenorphine (total binding) in the presence of 10 µM etorphine (nonspecific binding) or in the presence of various concentrations of agonist (10^-10-10^-5 M) in a final volume of 1 ml. After 30-min incubation at 37°C, samples were filtered onto glass fiber discs (Whatman GF/B; Whatman, Maidstone, UK) and washed twice with 3 ml of ice-cold 10 mM Tris-HCl, pH 7.4. Bound radioactivity was measured after addition of 3 ml of scintillation cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA). Inhibition binding data were analyzed for one- and two-site models using Radlig Software, and K_i values were calculated from the equation K_i = IC₅₀/1 + [L]/K_d (Cheng and Prusoff, 1973). For calculation, we used K_d values of 0.7 and 1.3 nM [³H]diprenorphine for hDOR in wild-type SK-N-BE and in the FLAG-tagged hDOR-transfected cells, respectively (Marie et al., 2003a).

Saturation Binding Studies on Attached Cells. Cells were seeded in 24-well plates at a density of 100,000 cells/well and were allowed to grow for 48 h. SK-N-BE were pretreated or not with 500 nM SNC-80 (30 min) or 10 µM Leu- and Met-enkephalin (120 min) in DMEM/20 mM Hepes. When inhibitors of lysosomal degradation or of recycling endosome were used, cells were pretreated, respectively, with 200 µM chloroquine (5 min) or 50 µM monensin (30 min) before agonist pretreatment. To block hDOR sequestration via clathrin-coated pits, cells were pretreated with 0.5 M sucrose (30 min) before agonist treatment. In recycling experiments, SK-N-BE cells were washed with DMEM/20 mM Hepes to remove ligands and were left for 30 min in agonist-free medium. Before binding experiments, cells were washed with DMEM/20 mM Hepes for 5 min and then incubated for 30 min at 37°C with appropriate concentrations of [³H]diprenorphine (0.05-1.75 nM) in a 0.3-ml final volume of 50 mM Tris-HCl/1% BSA (w/v), pH 7.4. Total and nonspecific binding were determined in the absence or in the presence of 20 µM levorphanol, respectively. The medium was rapidly removed and cells were harvested in 200 µl of 1 N NaOH and placed into vials in the presence of 3 ml of scintillation cocktail (PerkinElmer Life and Analytical Sciences). The vials were counted for radioactivity in a scintillation counter. Scatchard analysis was performed using Radlig software to calculate K_d and B_max values.

cAMP Assay. Cells were seeded in 24-well plates at a density of 120,000 cells/well in a culture medium supplemented with 0.6 µCi of [³H]adenine and incubated overnight. In desensitization experiments, wild-type SK-N-BE cell line and FLAG-tagged hDOR-transfected cells were incubated or not with different agonists (500 nM SNC-80 and 10 µM Leu- and Met-enkephalin) for various times in DMEM/20 mM Hepes. At the end of the preincubation, the accumulation of cAMP was determined without rinsing cells to avoid overshoot of adenylyl cyclase. When inhibitors of lysosomal degradation (chloroquine), recycling endosome (monensin), or internalization (sucrose) were used, SK-N-BE cells were pretreated as described in saturation binding studies section. In resensitization experiments, agonists were removed, cells were washed with DMEM/20 mM Hepes, and replaced in agonist-free medium. Inhibition of cAMP accumulation was determined in the presence of 1 mM 3-isobutyl-1-methylxanthine (IBMX) and 40 µM forskolin (FSK) and in the presence or in the absence of different agonists for 5 min at 37°C. The basal cAMP level was determined in the presence of 1 mM IBMX, in the absence of both FSK and any agonist, and then subtracted for calculation. The reaction was stopped by addition of 250 µl of 5% (w/v) trichloracetic acid, and the separation of [³H]cAMP was realized by chromatography on acid alumina columns (Allouche et al., 1996).

Immunocytochemistry. SK-N-BE cells overexpressing FLAG-tagged hDOR were grown on glass coverslips in culture medium containing 0.5 mg/ml G-418 until 50% confluence. When inhibitors of lysosomal degradation (chloroquine), recycling endosome (monensin), or internalization (sucrose) were used, FLAG-tagged hDOR cells were pretreated as described above. Then, cells were exposed or not to different agonists for various times. The medium was rapidly removed, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% (w/v) paraformaldehyde for 15 min. Cell permeabilization was carried out in the presence of 0.1% (w/v) saponin/PBS. After blocking nonspecific sites in 1% (w/v) BSA/PBS, anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) was added in the blocking buffer at the final concentration of 5 µg/ml for at least 1 h at room temperature. Cells were rinsed thoroughly with PBS and stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Sigma-Aldrich). After numerous washes, glass coverslips were mounted, and images were acquired using a charge-coupled device camera coupled to a fluorescence microscope with a 63x objective (Carl Zeiss, Thornwood, NY).

Western Blot Analysis. SK-N-BE cells were treated or not with 500 nM SNC-80 (30 min) or 10 µM Leu- and Met-enkephalin (120 min) in DMEM/20 mM Hepes. When the lysosomal inhibitor was used, cells were exposed to 200 µM chloroquine for 5 min before agonist treatment. The medium was removed, cells were harvested by centrifugation, and solubilized in lysis buffer [0.1% (v/v) Nonidet P-40/50 mM Tris-HCl, pH 7.4] followed by sonication. Protein concentrations were determined as described in Lowry et al. (1951), and 10% (w/v) acrylamide gels were loaded with 20 µg of proteins. Proteins were transferred onto nitrocellulose membranes for 45 min at a constant voltage (15 V). Using anti-mDOR polyclonal antibody (Oncogene), hDOR were first detected then blots were reprobed with anti-actin monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Revelation was performed using chemiluminescence reagent kit (PerkinElmer Life and Analytical Sciences). Quantification of immunoreactivity bands was realized by densitometry using Vilber Lourmat software.

	Results

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Competitive Binding Studies. In a first series of experiments realized in SK-N-BE cells, we determined the affinity of the two endogenous opioid agonists, Leu- and Met-enkephalin, and the nonpeptidic -selective agonist SNC-80. Competitive binding experiments showed significant displacements of [³H]diprenorphine by nanomolar agonist concentrations and biphasic curves, whatever the ligand used. The three different agonists bind hDOR with a high affinity, and comparison of K_i1 values showed a significantly higher capacity of SNC-80 and Leu-enkephalin than Met-enkephalin in competing with the radioligand (Table 1).

Inhibition of cAMP Accumulation by SNC-80 and Enkephalins. Functional studies were performed to examine the inhibitory effects of SNC-80 and enkephalins over a large range of concentrations (0.1 nM-10 µM) on the FSK-stimulated cAMP accumulation in SK-N-BE cells. When adenylyl cyclase was stimulated by 40 µM FSK, we found that all the three agonists inhibited cAMP accumulation in a concentration-dependent manner with similar IC₅₀ values (Table 2). An identical and maximal inhibition of cAMP accumulation by 40% was obtained at 500 nM for SNC-80 and at 10 µM for enkephalins. These differences in agonist concentrations producing the maximal cAMP inhibition are explained by a steeper dose-response curve when using SNC-80 compared with enkephalins. This would result from a stronger G proteins activation by the -selective agonist. To compare hDOR regulation by the three opioid agonists, concentrations producing almost identical inhibition of adenylyl cyclase were retained for all further experiments. When naloxone, the prototypic opioid antagonist, was used in a large excess (1 mM), we observed a total blockade of enkephalin-induced adenylyl cyclase inhibition, whereas a partial reduction of the maximal inhibition induced by SNC-80 was noticed (data not shown). We previously reported such a partial antagonism between naloxone and deltorphin I in the SK-N-BE cells (Allouche et al., 2000). These results suggest that naloxone, SNC-80, and deltorphin I would share binding sites that are not completely overlapping. However, when the high-affinity -selective antagonist naltrindole (10 µM) was used, no further SNC-80-induced cAMP inhibition was measured (data not shown).

hDOR Desensitization. We next examined the ability of SNC-80 and enkephalins to desensitize the hDOR-mediated inhibition of cAMP accumulation. SK-N-BE cells were challenged for various times (0, 5, 10, 15, 30, 60, and 120 min) in the presence of the maximal effective concentration of each agonist. At the end of the pretreatment, cAMP inhibition was determined by the addition of IBMX and forskolin for 5 min at 37°C without any rinsing. The maximal inhibition induced by the different agonists was considered as 100% in naive cells. As soon as 5 min, hDOR desensitization was significantly more important for SNC-80 than for enkephalins (Fig. 1). The difference of desensitization between agonists still persisted until 30 min because the SNC-80-induced cAMP inhibition was reduced by 95 ± 6%, whereas during the same period, a modest desensitization by 50% was measured for enkephalins. To obtain an identical and almost total hDOR desensitization, SK-N-BE cells were exposed for 30 and 120 min in the presence of SNC-80 and enkephalins, respectively, because no significant difference in desensitization was noted between 30- and 60-min enkephalin pretreatment (data not shown). The decrease in inhibition of cAMP accumulation clearly reflected hDOR desensitization and not opioid agonist degradation because addition of freshly prepared agonist in the preincubation medium after 30- (30 + agonist) or 120- min (120 + Leu or Met) exposure could not restore the initial inhibitory action of agonists. Moreover, preincubation of SKN-BE cells for various times (10, 30 min or 24 h) at different concentrations (10- to 100-fold the K_i of the inhibitor) of RB3020, described as a dual enkephalin-degrading enzyme inhibitor (Chen et al., 2000), did not significantly affect enkephalins-mediated desensitization (ANOVA) (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 1. Differential efficacy of SNC-80 and enkephalins to induce desensitization of hDOR-mediated inhibition of cAMP accumulation. SKN-BE cells were exposed to 500 nM SNC-80 and 10 µM enkephalin for various times. At the end of the exposure, we determined the ability of each agonist to inhibit cAMP accumulation. Agonist-induced inhibition of cAMP accumulation was taken as 100% in naive cells, and results are presented as the percentage of control. The 30+ agonist represents 30-min agonist exposure followed by addition of fresh agonist, and 120+ Leu or Met represents 120-min enkephalin exposure followed by addition of fresh Leu- or Met-enkephalin. Data are means ± S.E.M. of three to eight different experiments performed in triplicate.

Quantification of Opioid Receptor Number after Agonist Exposure. To evaluate a putative correlation between desensitization and opioid receptor number, we performed binding experiments using a radiolabeled antagonist rather than agonist to avoid internalization. First, we used a non-permeable derivative of [³H][3',5'-³H-Tyr]-Tic(CH₂-NH) Cha-Phe-OH, but its low specific activity did not allow an accurate measurement of receptors level. Binding experiments were then conducted with [³H]diprenorphine, a lipophilic antagonist, that crosses cellular membranes and binds cell surface but also to intracellular opioid receptors. Thus, reduction of [³H]diprenorphine binding would represent receptor internalization followed or not by receptor degradation. Decrease in pH within endosomes was proposed to dissociate certain ligands from their receptors (Krueger et al., 1997) and perhaps a similar event occurs preventing interactions between [³H]diprenorphine and -opioid receptors. To explore this possibility, binding assays were done in acidic (pH 5) or neutral buffer (pH 7.4). Decrease in pH results in a significant impairment of [³H]diprenorphine binding, suggesting that opioid receptors internalized in acidified vesicles are not necessary detected by this approach (data not shown). Opioid receptors degradation was instead investigated by Western blot experiments using anti-DOR antibody.

Conditions of agonists pretreatment were similar to those used in functional experiments (30 min for SNC-80 and 120 min for enkephalins) and B_max values were determined by Scatchard analysis in binding experiments using [³H]diprenorphine. Data presented in Fig. 2 correspond to the reduction of the opioid binding sites compared with naive cells in which the total number of receptors (100 fmol/mg of protein) was considered as 100%. SNC-80 and enkephalins were able to promote a similar decrease by 45% in opioid receptors number after 30-min exposure. When enkephalins treatment was prolonged until 120 min, no further significant reduction of hDOR level was observed (Fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2. hDOR down-regulation and recycling. SK-N-BE cells were exposed or not to SNC-80, Leu- or Met-enkephalins for 30 or 120 min. At the end of these periods, we determined either the number of opioid receptors () or cells left for an additional 30 min in agonist-free medium at 37°C (recycling) (). Bmax values were obtained from binding experiments using [3H]diprenorphine, and data are expressed as a loss of opioid binding sites compared with naive cells. Data are means ± S.E.M. of three experiments performed in triplicate. *, significantly different from 30- and 120-min pretreatment with Leuand Met-enkephalin; p < 0.05, ANOVA followed by Bonferroni-Dunn test.

Decrease of opioid receptors was next assessed on whole cell lysate by immunoblot experiments using anti-DOR antibody. We previously demonstrated that this antibody recognized a band at 50 kDa (Marie et al., 2003a), corresponding to the hDOR molecular weight in the SK-N-BE cell line (Fig. 3A), but not in human fibroblasts, known to express no opioid receptor (data not shown). SK-N-BE cells were exposed or not (Cont) to SNC-80 for 30 min or to enkephalins for 120 min, and then we determined the immunoreactivity level of both hDOR and actin by densitometric analysis. As shown in Fig. 3A, a significant reduction by 30% of the ratio hDOR immunoreactivity/actin immunoreactivity was only observed in SNC-80-pretreated cells (SNC), whereas no modification was evidenced upon enkephalin (Leu and Met) pretreatment.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Quantification of hDOR down-regulation by Western blot. SK-N-BE cells were pretreated (B, ) or not (A, ) with 200 µM chloroquine followed by DMEM/Hepes (Cont), SNC-80 (SNC), or enkephalins (Leu, Met) exposure. Proteins obtained from whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted first with anti-DOR polyclonal antibody, deshybrided, and second blotted with anti-actin monoclonal antibody. Levels of hDOR and actin were quantified by densitometric analysis, and results are presented as percentage of control; 100% represents the ratio of hDOR immunoreactivity (IR)/actin IR in naive cells. Results are means ± S.E.M. (n = 4). ***, significantly different from control and SNC with chloroquine pretreatment; p < 0.001, ANOVA followed by Bonferroni-Dun test.

hDOR Internalization. Complementary experiments were conducted to visualize the hDOR redistribution after agonist exposure. For this purpose, we used SK-N-BE stably overexpressing the FLAG-tagged hDOR, and the visualization was achieved by immunofluorescence experiments using an anti-FLAG monoclonal antibody. Control experiments showed that expression of the FLAG-tagged hDOR did not change the affinity of the receptor for the different agonists, or their ability to inhibit the cAMP accumulation or their propensity to desensitize the opioid receptors (data not shown).

In pcDNA3 or in the FLAG-tagged hDOR-transfected cells (Fig. 4, secondary antibody), no labeling was detected when only the FITC-conjugated secondary antibody was used. In untreated cells, a bright staining restricted to the cell surface was observed, suggesting that most of the FLAG-tagged receptors were localized at the plasma membrane (Fig. 4, Cont); this was further confirmed by confocal microscopy (data not shown). Whereas after 30-min SNC-80 exposure a complete hDOR internalization was visualized, the residual cell surface staining in enkephalins-pretreated cells strongly suggested that these agonists differentially regulated the hDOR in terms of sequestration (Fig. 4, SNC-30, Leu-30, and Met-30). The agonist-induced internalization was antagonized by an excess of the -selective antagonist naltrindole (10 µM), which by itself is devoid of any effect on hDOR internalization (Fig. 4, NTI), demonstrating that hDOR endocytosis was due to their specific activation by agonists (Fig. 4, NTI + SNC-30, NTI + Leu-30, and NTI + Met 30).

View larger version (51K): [in this window] [in a new window]   Fig. 4. FLAG-tagged hDOR internalization. SKN-BE cells were transfected with the FLAG-tagged hDOR. Using the anti-FLAG M2 antibody, we visualized the distribution of opioid receptors by fluorescence microscopy (with a Zeiss 63x objective). Fluorescence background was determined using only FITC-conjugated secondary antibody (secondary antibody). Cells were treated for 30 min (SNC-30, Leu-30, and Met-30) or not (Cont) as described under Materials and Methods. The -selective antagonist naltrindole was used alone (NTI) or in combination with agonists (NTI + SNC-30, NTI + Leu-30, and NTI + Met-30).

hDOR Recycling and Resensitization. To compare hDOR recycling and resensitization after agonists pretreatment, it was necessary to prolong the preincubation time until 120 min for enkephalins to obtain a similar pattern of FLAG-tagged hDOR internalization as observed in SNC-80 pretreated cells (Fig. 5A, Leu-120 and Met-120 versus SNC-30). After their internalization, GPCR can be recycled to plasma membrane via recycling endosomes or degraded into lysosomes (for review, see Ferguson, 2001).

View larger version (48K): [in this window] [in a new window]   Fig. 5. Differential efficacy of SNC-80 and enkephalins to induce hDOR recycling and resensitization. SK-N-BE cells were exposed for 30 min to SNC-80 (SNC-30) and for 120 min to enkephalins (Leu-120 and Met-120) to induce internalization or desensitization. For recycling or resensitization experiments, agonists were removed, and cells were placed in agonist-free medium for 30 min (SNC-30 + 30, Leu-120 + 30, and Met-120 + 30). A, FLAG-tagged hDOR internalization and recycling experiments visualized by fluorescence microscopy using anti-FLAG monoclonal antibody. B, inhibition of [3H]cAMP accumulation measured as described under Materials and Methods. Opioid agonist-induced inhibition was referred as 100% in naive cells. Data are means ± S.E.M. of three to four different experiments performed in triplicate. **, SNC-30 + 30 pretreated cells significantly different from Leu-120 + 30 and Met-120 + 30; p < 0.01, ANOVA followed by Bonferroni-Dunn test.

First, to investigate hDOR trafficking after their internalization, recycling experiments were carried out and opioid receptor level was quantified by binding studies in SK-N-BE cells. After pretreatment for 30 or 120 min with SNC-80 and enkephalins, respectively, cells were washed to eliminate the ligand and left in agonist-free medium for 30 min at 37°C then opioid receptor number was determined. Scatchard analysis revealed an almost total opioid binding sites recovery in enkephalin-pretreated cells, suggesting an effective recycling process (103.2 ± 2.3 and 88.0 ± 3.0% for Leu- and Met-enkephalins, respectively) (Fig. 2). In contrast, no significant opioid receptors recycling was measured in SNC-80-pretreated cells (52.9 ± 5.9 versus 40.0 ± 4.7%, respectively) (Fig. 2). To corroborate these binding data, immunocytochemistry experiments were performed in SK-N-BE expressing the FLAG-tagged hDOR. As depicted in Fig. 5A, the tagged opioid receptor failed to recycle after 30 min in SNC-80-free medium as shown by a strong vesicular labeling (SNC-30 + 30), whereas we visualized an obvious reappearance of the immunofluorescence at the plasma membrane in cells preexposed to enkephalins (Leu-120 + 30 and Met-120 + 30).

Second, cAMP assays were further conducted in these recycling conditions to determine the functionality of the hDOR. To induce a similar and an almost total desensitization, SK-N-BE cells were challenged for 30 or 120 min in the presence of SNC-80 and enkephalins, respectively (Fig. 5B). Then, agonists were removed, cells were placed in DMEM/Hepes for 30 to 120 min at 37°C, and the inhibition of [³H]cAMP accumulation was measured after addition of fresh agonists. After a 30-min period in enkephalins-free medium, we observed an important resensitization by 75%, whereas in SNC-80-pretreated cells a significantly weaker resensitization was obtained (43.6 ± 1.2% for SNC-80 versus 76.3 ± 3.2 and 76.9 ± 2.0% for Leu- and Met-enkephalin, respectively) (Fig. 5B). When cells were left in agonist-free medium until 120 min, no further resensitization could be measured regardless of the agonist (data not shown).

Effects of Chloroquine on hDOR Sequestration. The weak hDOR resensitization and recycling and the decrease of hDOR immunoreactivity detected on Western blot associated with the absence of [³H]diprenorphine binding site recovery suggested that after SNC-80 exposure, the opioid receptors could be targeted to lysosomes and degraded. To test this hypothesis, we examined the effect of chloroquine, known to inhibit mDOR (Tsao and Von Zastrow, 2000), and hDOR (Marie et al., 2003a) degradation in lysosomes by elevating the intraluminal pH. This inhibitor was used in immunoblot, immunocytochemical, binding, and functional studies. After ensuring that chloroquine pretreatment was devoid of any effect on opioid receptors level, we demonstrated that this inhibitor of lysosomal proteases significantly decreased the loss of opioid binding sites induced by 30 min SNC-80 exposure (26.6 ± 4.6 versus 57.7 ± 0.5% with and without chloroquine pretreatment, respectively) (Fig. 6A). Conversely, 200 µM chloroquine failed to affect the loss of [³H]diprenorphine binding sites promoted by a 120-min pretreatment with Met-enkephalin (46.4 ± 5.8 versus 42.6 ± 5.8% with and without chloroquine pretreatment, respectively) (Fig. 6A). In Western blot experiments, chloroquine was also shown to completely block the reduction of -receptor immunoreactivity promoted by SNC-80 pretreatment (Fig. 3B).

View larger version (44K): [in this window] [in a new window]   Fig. 6. Effect of chloroquine on hDOR down-regulation and sequestration. A, cells pretreated () or not () for 5 min with 200 µM chloroquine and then exposed or not for 30 min to SNC-80 or for 120 min to Met-enkephalin. Opioid receptors number was determined by using [3H]diprenorphine, and results are expressed as a loss of opioid binding sites compared with naive cells. Data are means ± S.E.M. of three experiments performed in triplicate. **, significantly different from 30-min SNC-80 without chloroquine pretreatment; p < 0.01, Student's t test. B, SK-N-BE cells stably expressing the FLAG-tagged hDOR pretreated or not with 200 µM chloroquine and then exposed (SNC-30, Leu-120, and Met-120) or not (Cont) to the different agonists. The tagged-receptor was visualized by fluorescence microscopy using anti-FLAG monoclonal antibody.

We examined the chloroquine effect on the FLAG-tagged hDOR internalization induced by the various opioid agonists. In naive cells, the immunofluorescence labeling was restricted to the plasma membrane in both chloroquine-treated and untreated cells (Fig. 6B, Cont). After sustained activation of -receptors by SNC-80 and enkephalins, FLAG-tagged hDOR was detected in intracytoplasmic vesicles (Fig. 6B, SNC-30, Leu-120, and Met-120). When cells were preexposed to chloroquine and then to SNC-80, the hDOR immunolabeling manifested as greater vesicles with more important fluorescence intensity than in chloroquine-untreated cells. When -receptors were activated for 120 min by enkephalins, no marked difference in the number and the size of vesicles between untreated and chloroquine-pretreated cells could be detected (Fig. 6B).

Then, chloroquine effects were tested on hDOR desensitization. Cells were pretreated or not with 200 µM chloroquine and then exposed to SNC-80 or enkephalins for 30 or 120 min, respectively. Whatever the agonist, no significant differences in the extent of desensitization were observed between chloroquine-treated and untreated cells (Fig. 7).

View larger version (19K): [in this window] [in a new window]   Fig. 7. Effect of chloroquine on hDOR desensitization. SK-N-BE cells were pretreated or not with 200 µM chloroquine and then exposed or not for 30 min to SNC-80 or for 120 min to Leuor Met-enkephalin. Agonist-induced inhibition of cAMP accumulation was referred as 100% in naive cells treated or not with chloroquine. Data are means ± S.E.M. of three different experiments performed in triplicate.

Effects of Monensin on hDOR Desensitization and Sequestration. Resensitization and recycling (binding and immunocytochemical experiments) patterns obtained after enkephalins pretreatment strongly argued that hDOR would be internalized and sorted toward recycling endosomes. To test this hypothesis, monensin, known as a recycling endosomes inhibitor, was used in desensitization and internalization experiments. In a first set of experiments, SK-N-BE cells were pretreated or not for 30 min in the presence of 50 µM monensin. In naive cells, we did not observe any significant effect of monensin on the inhibitory action of the different opioid agonists (data not shown). To avoid a maximal desensitization, cells were exposed for 10 min to SNC-80 and for 30 min to enkephalins. Monensin significantly increased enkephalins-induced desensitization from 50 to 80%. In contrast, a similar hDOR desensitization (60%) promoted by SNC-80 was obtained in the absence or in the presence of monensin pretreatment (Fig. 8A).

View larger version (44K): [in this window] [in a new window]   Fig. 8. Effect of monensin on hDOR desensitization and internalization. Cells were pretreated () or not () for 30 min with 50 µM monensin and then exposed or not (Cont) for 10 min to SNC-80 (SNC-10), 30 min to Leu-(Leu-30), or 30 min to Met-enkephalins (Met-30). A, inhibition [3H]cAMP accumulation measured as described under Materials and Methods. Opioid agonist-induced inhibition was referred as 100% in naive cells. Data are means ± S.E.M. of three experiments performed in triplicate. *, significantly different from 30-min enkephalin without monensin pretreatment; p < 0.05, Student's t test. B, FLAG-tagged hDOR visualized by fluorescence microscopy.

When FLAG-tagged hDOR-transfected cells were pretreated for 30 min with monensin, we noticed a slight increase of intracellular immunolabeling compared with untreated cells (Fig. 8B, Cont). This could be due to a basal internalization and recycling of hDOR. In monensin-untreated cells, we observed both intracytoplasmic and cell surface immunolabeling after a 30-min period of enkephalin exposure (Fig. 8B, Leu-30 and Met-30). When recycling endosomes were blocked by monensin, we observed 1) an absence of plasma membrane immunofluorescence and 2) an increase of intracytoplasmic vesicles both in number and size (Fig. 8B, Leu-30 and Met-30). Conversely, no effect of monensin could be observed on the hDOR redistribution promoted by 10-min pretreatment with SNC-80 (Fig. 8B, SNC-10).

Effects of Hypertonic Sucrose Solution on hDOR Down-Regulation and Internalization. GPCR sequestration is achieved by clathrin-coated pits or by the less well described pathway involving the caveolin (for review, see Ferguson, 2001). Although there is no obvious proof for caveolin-mediated opioid receptors endocytosis, we studied the clathrin-associated pathway by using hypertonic sucrose solution as demonstrated for the 2-adrenergic receptor (Pippig et al., 1995).

To determine the biological importance of hDOR sequestration in the desensitization process, functional experiments were conducted on SK-N-BE cells with or without 0.5 M sucrose pretreatment. First, binding and immunocytochemical experiments were done to ensure the efficiency of this treatment. When cells were exposed for 30 min in the presence of 0.5 M sucrose, we observed no modifications of either the opioid receptors level (68.8 ± 13.1 fmol/mg of protein for sucrose-pretreated cells versus 74.5 ± 10.0 fmol/mg of protein for untreated cells (n = 8), Student's t test, p = 0.25), or the affinity of receptor for [³H]diprenorphine. Although after opioid agonists exposure we were able to measure a loss of opioid binding sites to the same extent as in Fig. 2, sucrose pretreatment almost abolished hDOR disappearance induced by SNC-80 (12.6 ± 5.2% in sucrose-pretreated cells versus 59.3 ± 4.4% in sucrose-untreated cells), Leu-enkephalin (1.3 ± 5.2% in sucrose-pretreated cells versus 37.2 ± 7.0% in sucrose-untreated cells), and Met-enkephalin (-3.3 ± 8.7% in sucrose-pretreated cells versus 36.8 ± 2.8% in sucrose-untreated cells) (Fig. 9A).

View larger version (44K): [in this window] [in a new window]   Fig. 9. Effect of hypertonic sucrose on hDOR down-regulation and internalization. Cells were pretreated () or not () for 30 min with 0.5 M sucrose and then exposed or not (Cont) for 30 min to SNC-80 (SNC-30) or for 120 min to enkephalins (Leu-120 and Met-120). A, number of opioid receptors determined by using [3H]diprenorphine. Results are expressed as a loss of opioid binding sites compared with naive cells. Data are means ± S.E.M. of three experiments performed in triplicate. *, significantly different from sucrose-untreated cells; p < 0.05, Student's t test. B, FLAG-tagged hDOR visualized by fluorescence microscopy.

These data were further corroborated by immunofluorescence experiments performed in SK-N-BE transfected cells showing that 0.5 M sucrose effectively impaired SNC-80- and enkephalin-induced endocytosis (Fig. 9B).

Effects of Hypertonic Sucrose Solution on hDOR Desensitization. After ensuring that hypertonic sucrose pretreatment did not modify inhibition of cAMP accumulation produced by the different agonists in naive cells, we examined the consequences of the endocytosis impairment on hDOR desensitization. In sucrose-untreated SK-N-BE cells, agonist exposures were done to promote a profound and similar desensitization by 90% (Fig. 10). When cells were pretreated by hypertonic sucrose solution before challenging with agonists, we observed an important reduction of hDOR desensitization promoted by SNC-80 (94 ± 1% in sucrose-untreated cells versus 33 ± 2% in sucrose-pretreated cells), Leu-enkephalin (92 ± 5% in sucrose-untreated cells versus 48 ± 2% in sucrose-pretreated cells), and Met-enkephalin (94 ± 4% in sucrose-untreated cells versus 39 ± 1% in sucrose-pretreated cells) (Fig. 10).

View larger version (14K): [in this window] [in a new window]   Fig. 10. Effect of hypertonic sucrose on hDOR desensitization. Cells were pretreated () or not () for 30 min with 0.5 M sucrose and then exposed or not (naive cells) for 30 min to SNC-80 or for 120 min to enkephalins. Inhibition of [3H]cAMP accumulation was measured as described under Materials and Methods. Opioid agonist-induced inhibition was referred to as 100% in naive cells pretreated or not with 0.5 M sucrose. Data are means ± S.E.M. of three experiments performed in triplicate. **, significantly different from agonist-pretreated cells without sucrose pretreatment; p < 0.01, ANOVA followed by Bonferroni-Dunn test.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The aim of the present study was to confirm or invalidate the hypothesis of a differential hDOR regulation by alkaloid and peptidic agonists. This idea was based on our previous works performed on the SK-N-BE cell line using DPDPE and deltorphin I, two peptidic opioid agonists; etorphine, an alkaloid agonist related to morphine; and AR-M1000390 [N,N-diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide], a nonpeptidic ligand derived from SNC-80 (Allouche et al., 1999a,b; Hasbi et al., 2000; Marie et al., 2003a,b).

For the present study, Leu- and Met-enkephalin, two endogenous peptides, and a nonpeptidic opioid agonist, SNC-80, were chosen. After a classical pharmacological characterization, which allowed us to determine the concentration of agonist producing similar inhibition of cAMP accumulation, we showed that the nonpeptidic agonist SNC-80 was able to induce a strong desensitization after a short pretreatment (30 min). This decrease in the ability of hDOR to inhibit adenylyl cyclase was comparable with the desensitization promoted by the two peptidic agonists DPDPE and deltorphin I (Allouche et al., 1999a) and by a nonpeptidic agonist AR-M1000390 (Marie et al., 2003b). In contrast, a slower desensitization was observed after exposure with enkephalins, because it was necessary to prolong time pretreatment until 120 min to obtain the same desensitization rate as with 30-min exposure to SNC-80. We also ensured that the weak potency of enkephalins to promote hDOR desensitization was not due to a peptidic agonist degradation as demonstrated by using a dual enkephalin-degrading enzyme inhibitor and by addition of freshly prepared agonists after pretreatment. The desensitization kinetics obtained with endogenous peptides was rather similar to that previously reported with the alkaloid agonist etorphine (Namir et al., 1997; Allouche et al., 1999a). According to previous data (Allouche et al., 1999a) and the present study, we can reasonably rule out the hypothesis of a differential hDOR desensitization by peptidic and alkaloid agonists. But, when examining the various agonists used, it seems that the rapid and important receptor desensitization was observed for DPDPE, deltorphin I, ARM1000390, and SNC-80, all described as -selective agonists (for reviews, see Calderon et al., 1994; Reisine and Pasternak, 1996; Wei et al., 2000). In contrast, nonselective opioid agonists (etorphine, Leu- and Met-enkephalin) (for review, see Reisine and Pasternak, 1996) induced moderate desensitization after a 30-min period. Our results are not consistent with the data obtained on the mDOR by Bot et al. (1997) who did not observe a clear-cut difference between -selective and -nonselective agonists. Such discrepancies could be related to the different cellular models and the different expression level of opioid receptors.

Why would different opioid agonists induce differential regulation of the same receptor? As evidenced by Akil's group, -selective and -nonselective ligands bind on distinct regions of the receptor. For example, the transmembrane domain VI seems to be exclusively required for interactions with -selective ligands (Meng et al., 1996). Thus, it is tempting to speculate that the binding of -selective and -nonselective agonists would induce different conformational changes of the hDOR leading to different exposure of putative phosphorylation residues (to explain the differential desensitization) and/or to interactions with different intracellular partners (to explain the differential G protein coupling).

The second part of this work was to establish the relationships between hDOR desensitization and intracellular trafficking. Whereas all opioid agonists were able to promote -receptor sequestration but after different times of pretreatment, a major divergence in hDOR trafficking was evidenced when opioid receptors were activated either by SNC-80 or enkephalins. Activation by endogenous enkephalins drove hDOR toward the recycling pathway and receptors undergo a weak and progressive desensitization. This was demonstrated by an efficient resensitization and recycling, and by the use of monensin. In contrast, SNC-80-induced hDOR sequestration is followed by its sorting to lysosomes. This was indicated by the weak resensitization and recycling, and especially by opioid receptor down-regulation detected both in binding and Western blot experiments. By interfering with degradation using chloroquine, we confirmed the irreversible hDOR sorting to the lysosomal compartment. When blocking hDOR degradation, we were unable to modify their desensitization. Down-regulation of opioid receptor is generally observed for more prolonged agonist exposure as described previously (Malatynska et al., 1996). Using Chinese hamster ovary cells, they showed that SNC-80 promoted 50 to 60% hDOR down-regulation but after a 15- to 24-h pretreatment. However, as observed in the present study, they found a good correlation between loss of opioid receptors and desensitization measured on the inhibition of adenylyl cyclase. In SKN-BE cells, a rapid hDOR down-regulation after DPDPE and deltorphin I exposure associated with a rapid and profound desensitization was demonstrated previously (Marie et al., 2003a). Although a similar loss of opioid receptors is observed after exposure with SNC-80, DPDPE, and deltorphin I, we cannot exclude the participation of different molecular mechanisms as suggested for SNC-80 and DPDPE by Okura et al. (2000). Nevertheless, all these data favor the idea that reduction of active opioid receptors from the cell surface would potentiate their desensitization. In addition to the lysosomal sorting of hDOR, the sequestration of opioid receptors would effectively participate in desensitization as shown by the effects of hypertonic sucrose. In other words, the maximum inhibition of cAMP accumulation requires the full population of cell surface receptors and the rapid hDOR sequestration would promote desensitization. This step is common for all the agonists used in the study, and the second factor determining the desensitization rate is the fate of the internalized receptor (discussed above). In spite of opioid receptor recycling, enkephalins also induce hDOR desensitization but with a delay compared with SNC-80. This is probably due to the difference of kinetics between internalization and recycling processes. This latter step, which includes dephosphorylation of the internalized receptors into endosomes, would represent a limiting factor of GPCR recycling (Pan et al., 2003). So, after 120 min of pretreatment, the majority of the opioid receptors would be localized into endosomes. This is probably the case because the withdrawal of enkephalins produced a total recovery of receptors associated with an important resensitization after a 30-min period in agonist-free medium.

What is the molecular signal that allows receptor recycling rather than its lysosomal degradation? Partners of GPCR such as arrestins were identified as modulators of GPCR trafficking (Oakley et al., 1999, 2001) and could target the receptor to degradation. Strong interactions between arrestin and hDOR activated by -selective agonists would prevent receptor dephosphorylation and its subsequent recycling. The role of arrestins in the differential hDOR trafficking is under investigation. Contribution of other proteins such as G protein-coupled receptor-associated sorting protein, whose role in opioid receptors recycling was recently identified (Whistler et al., 2002), also should be explored. This molecular signal also could be an intrinsic property of the receptor. The presence of a specific sequence acting as a switch for recycling could be present in the amino acid sequence of hDOR. Such a signal was found for the 2-adrenergic receptor (Gage et al., 2001) as well as in the MOR (Tanowitz and von Zastrow, 2003). Concerning the hDOR, such a sequence has not been characterized yet, but we can imagine that binding of nonselective opioid agonists would expose such signal in the intracellular loops, allowing receptor recycling.

A new hypothesis, largely based on Von Zastrow's work, has recently emerged on the role of opioid receptor sequestration in tolerance. Indeed, µ-opioid receptors internalization was shown to reduce morphine tolerance, probably by promoting receptor recycling and resensitization (He et al., 2002). In the present study, we also found that the weak hDOR desensitization promoted by enkephalins is associated with receptor sequestration and recycling in contrast to SNC-80 that promotes -receptor internalization and degradation. Thus, our in vitro data suggest that endogenous enkephalins would be poorer inducers of tolerance than SNC-80. Even if it is difficult to compare in vivo data because distinct experimental conditions are generally used (e.g., doses, routes of administration, duration of treatment), it seems that the natural enkephalins induce antinociceptive effects without producing significant tolerance. Indeed, no tolerance was observed after impairment of endogenous enkephalins catabolism by RB101 (Noble et al., 1992). In contrast, acute or chronic administration of SNC-80 was shown to promote an important tolerance in rhesus monkeys (Brandt et al., 2001).

In conclusion, we demonstrated that hDOR desensitization is complex: 1) it depends on the selectivity of the agonist; 2) opioid receptor internalization would serve as a regulatory mechanism to modulate hDOR responsiveness; and 3) differential sorting either to recycling endosomes or lysosomes would be crucial during sustained activation to maintain or not an active receptor population at the cell surface.

	Acknowledgements

 
We thank Prof. M. C. Fournie-Zaluski (INSERM U266, Centre National de la Recherche Scientifique FRE2463, Université de Paris V) for providing the dual enkephalin-degrading enzyme inhibitor RB3020; Prof. M. Bouvier (Université de Montréal, Canada) for generously providing pcDNA3-FLAG-tagged-hDOR; and Prof. A Borsodi and Dr. G. Toth for providing the derivative of [³H][3',5'-³HTyr]-Tic(CH₂-NH)Cha-Phe-OH. We are also grateful to Dr. B. Sola (UPRES-EA 2128, Université de Caen) for critical review of the manuscript. N. Marie is a recipient of a fellowship from the Ministère de l'Éducation Nationale, de la Recherche et de la Technologie and from the Fondation pour la Recherche Médicale.

	Footnotes

 
DOI: 10.1124/jpet.103.063958.

ABBREVIATIONS: GPCR, G protein-coupled receptor; MOR, µ-opioid receptor; DPDPE, [D-Pen²,D-Pen⁵]-enkephalin; deltorphin I, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH₂; hDOR, human -opioid receptor; Leu-enkephalin, Tyr-Gly-Gly-Phe-Leu; Met-enkephalin, Tyr-Gly-Gly-Phe-Met; SNC-80, (+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; IBMX, 3-isobutyl-1-methylxanthine; FSK, forskolin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; mDOR, mouse -opioid receptor; ANOVA, analysis of variance; NTI, naltrindole; RB3020, 2-(3-[(1-amino-ethyl)-hydroxy-phosphinyl]-2-biphenyl-4-ylmethyl-propionylamino)-propionic acid; RB101, N-{(R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-1-oxopropyl}-L-phenylalanine benzyl ester.

Address correspondence to: Dr. Stéphane Allouche, Laboratoire de Biochimie A, avenue Côte de Nacre, CHU de Caen, 14033 Caen Cedex, France. E-mail: allouche-s{at}chu-caen.fr

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