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INFLAMMATION AND IMMUNOPHARMACOLOGY

The Expression of - and -Opioid Receptor Is Enhanced during Intestinal Inflammation in Mice

Department of Anesthesiology, Institut Municipal Investigació Mèdica, Hospital Universitario del Mar, Universistat Autònoma de Barcelona, Barcelona, Spain (O.P., M.M.P.); and Department of Immunology, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain (J.R.P.)

Received January 29, 2003; accepted April 16, 2003.

	Abstract

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In the gut, µ-, -, and -opioid receptors are present in the submucous and myenteric plexi and in enterocytes. Using pharmacological methods, our group has shown that intestinal inflammation enhances the antitransit and antisecretory effects of systemic opioids. The aim of the present study was to evaluate whether the enhanced antisecretory effects of and -agonists were associated with an increased transcription and/or expression of these receptors at central (brain and spinal cord) and/or peripheral sites (gut); we also evaluated the expression of - and -opioid receptors in dissected sections of the gut containing the myenteric (MP/LM) or submucous (SP/M) plexi. The mRNA and protein levels of both opioid receptors were determined using a reverse-transcriptase polymerase chain reaction and immunoprecipitation/Western blot, respectively. Intestinal inflammation significantly augmented the transcription of -opioid receptors in the spinal cord (34%) and in the whole gut (102%). Also increased mRNA and protein levels of -opioid receptors in the MP/LM and SP/M preparations. The -opioid receptors gene transcription was not altered by inflammation, whereas -opioid receptors protein levels were significantly enhanced in the SP/M preparation. No changes in gene transcription or protein levels for - and -opioid receptors could be demonstrated in the brain. These results suggest that local transcriptional and post-transcriptional changes of the - and -opioid receptors genes could be responsible for the enhanced antisecretory effects of - and -opioid agonists during intestinal inflammation.

In the central nervous system, µ-, -, and -opioid receptors are found in the superficial layers of the dorsal horn of the spinal cord and in the brain, where particularly dense concentrations are present in the cortex, limbic structures, thalamic nuclei, and olfactory bulb (Mansour et al., 1994). In the intestine, µ-, -, and -opioid receptors are widely distributed in the myenteric and submucous plexi of rats and pigs (Bagnol et al., 1997; Townsend and Brown, 2002), whereas lower densities of -opioid receptors have been demonstrated in rat enterocytes (Nano et al., 2000). In mice, we have previously reported that µ-opioid receptors are present in both intestinal plexi, but the specific localization of - and -opioid receptors in the different anatomical structures of the gut has not been established. In mice, we have also shown that - ([D-Pen²,D-Pen⁵]-enkephalin) and -opioid receptors (U-50488H) agonists decrease intestinal permeability in the small intestine and that the effect is increased during croton oil-induced intestinal inflammation (Valle et al., 2001). Moreover, other investigators have reported that -opioid receptor agonists suppress neurogenic secretion evoked by several inflammatory mediators and that the antinociceptive effects of -opioid receptor agonists are enhanced during colonic inflammation (Sengupta et al., 1999; Poonyachoti and Brown, 2001). In these studies, intestinal inflammation increased the effects of opioids by a peripheral mechanism, suggesting an up-regulation of intestinal opioid receptors. Recently, using the model of croton oil-induced intestinal inflammation, we have demonstrated an increased transcription and expression of intestinal µ-opioid receptors during peripheral inflammation (Pol et al., 2001).

The aim of the present investigation was to evaluate whether the greater antisecretory potency of [D-Pen²,D-Pen⁵]-enkephalin and U-50488H observed during inflammation is associated with an increased transcription and/or expression of the - and -opioid receptors located in the gut (whole gut and MP/LM, SP/M preparations) and in the central nervous system (brain and spinal cord). The expression of - and -opioid receptors mRNA and their protein levels was determined by RT-PCR and immunoprecipitation/Western blot, respectively. The present experiments provide for the first time information regarding the expression of - and -opioid receptors in basal conditions and during intestinal inflammation in mice.

	Materials and Methods

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Animals. Male Swiss CD-1 mice weighing 25 to 30 g were used in all experiments. The study protocol was approved by the local Committee of Animal Use and Care of the Institution in accordance with the International Association for the Study of Pain guidelines on ethical standards for investigations in animals. Mice were housed under 12-h light/dark conditions in a room with controlled temperature (22°C) and humidity (66%). Animals had free access to food and water and were used after a minimum of four days acclimatization to the housing conditions. All experiments were conducted between 9:00 AM and 2:00 PM.

Intestinal Inflammation. Intestinal inflammation was induced by the intragastric administration of two 0.05-ml doses of the irritant agent croton oil administered 24 h apart, while control animals received the same volume of intragastric saline. Before the administration of croton oil or saline, animals were fasted for 18 h and had free access to water. Controls and animals with intestinal inflammation were sacrificed 5 days after the first dose of saline or croton oil, respectively. Morphological changes induced by croton oil have been previously reported by our group (Puig and Pol, 1998) and were established by optical microscopy. In brief, a clear disruption of the mucosa with a massive infiltration of lymphocytes within the submucosa was observed in animals treated with croton oil but not in saline-treated animals. The greatest morphological inflammatory changes after treatment with croton oil were observed in the jejunum.

Tissue Isolation and Total RNA Extraction. For RT-PCR, tissues were prepared in two ways. Intestine (jejunum), spinal cord (entire), and whole-brain samples from animals with and without intestinal inflammation were excised, placed in sterile microfuge tubes, snap frozen in liquid nitrogen, and stored at –80°C until assay. For the isolation of jejunum, we collected 10 cm of the small intestine, starting 2 cm distal to the ligament of Treitz. The dissection of the gut was performed by placing segments of jejunum in ice-chilled phosphate-buffered saline (PBS), and the gut was opened longitudinally to expose the mucosal side, which was then pinned to a silicone elastomer-coated Petri dish. The submucosal plexus together with the mucosa (SP/M) were separated with forceps from the remaining layers (circular muscle layers, myenteric plexus and longitudinal muscle; MP/LM), and both sections were frozen in liquid nitrogen and stored at –80°C (Fletcher et al., 2001). All dissections were performed under a stereomicroscope at 4°C. In all samples, total RNA was extracted with TRIzol (Invitrogen, Renfrewshire, England), and the amount of the purified RNA (A₂₆₀/A₂₈₀ ratio was 1.9) was determined by spectrophotometry.

Reverse Transcriptase-Polymerase Chain Reaction. The expression of - and -opioid receptors mRNA in the different tissue samples (whole intestine, dissected MP/LM and SP/M, spinal cord, and brain) was assessed by RT-PCR with -actin as an internal standard. In all experiments, 1 µg of total RNA was reverse transcribed into cDNA using SuperScript II RNase H^– reverse transcriptase (Invitrogen, Remfrewshire, UK) in a final volume of 10 µl. Afterward, 1 or 4 µl of the reaction mixture was used as a template for the PCR reaction of -actin or - and -opioid receptors, respectively. Negative controls were performed for PCR; in these samples, all the components were included, except RT, to show that the PCR products were a result of RNA and not of genomic DNA amplification. Animals without intestinal inflammation (receiving intragastric saline) were used as controls. Two primers that anneal to different exons: bases 395 to 415; 5'-GCTGTGCTCTCCATTGATAC-3'; sense and bases 875 to 895; 5'-GATGTCCACCAGCGTCCAGAC-3'; antisense primers of the gene receptor sequence were used to detect -opioid receptors expression (Buzas and Cox, 1997). This primer pair bind to sequences in different exons and produces a 500-bp -opioid receptor fragment. PCR using a PerkinElmer 2400 Thermal Cycler was initially set at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s. The last primer extension step was at 72°C for 7 min. The primers used to generate the -opioid receptors were: 5'-CAGCTCTTGGTTCCCCAACTG-3' and 5'-TGCAAGGAGCATTCAATGACATC-3', corresponding to bases 263 to 283 and 801 to 823, respectively. These primers anneal to different exons of the gene -opioid receptors sequence and produce a PCR product of 560 bp (Winkler and Spanagel, 1998). After an initial denaturation of 5 min at 94°C, PCR conditions were 35 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 1 min followed by an extensional at 72°C for 10 min. Reverse transcribed products of -actin were measured for normalization the data, using the following primers: 5'-TCATGAAGTGTGACGTTGACATCCGT-3' and 5'-CCTAGAAGCATTTGCGGTGCACGATG-3', which anneal to different exons of the -actin gene sequence and generate a 289-bp PCR product (Pol et al., 2001). The PCR conditions were 23 cycles at 94°C for 30 s, 55°C for 40 s, and 72°C for 90 s followed by a final extension at 72°C for 10 min.

The PCR products were resolved on 1% agarose gels (Sigma-Aldrich, St. Louis, Mo) containing ethidium bromide and visualized under UV light. The image was digitized using a Gel Doc 2000 (Bio-Rad Laboratories, Hercules, CA) and the optical density of the bands determined using the Diversity database program. During digitalization, the color saturation was checked to ensure that the image was not oversaturated. Results are expressed as the ratio of the optical density of the band of target (- or -opioid receptors) to the housekeeping gene (-actin).

Membrane Preparation and Protein Extraction. Samples were minced with scissors and homogenized (Ultra-Turf, T8.01; Ika Werke, Staufen, Germany) in ice-cold buffer (4°C) containing: 50 mM Tris-HCl and 0.32 M sucrose, pH 7.5. The homogenate was centrifuged at 1,000g at 4°C for 10 min. The pellet was discarded, and the supernatant was centrifuged at 20,000g for 20 min; the new pellet was resuspended in buffer and centrifuged again at 20,000g for an additional 20 min. The final pellet was diluted in Tris buffer to a final protein concentration of 3 µg/µl (Bradford, 1976). Membranes were solubilized in a buffer containing 62.5 mM Tris-HCl, 2.3% SDS, 10% glycerol, and 5% -mercaptoethanol, adjusted to a pH 6.8. After a 3-h incubation at room temperature, the samples were boiled for 5 min and stored at –20°C until use. In these experiments, a similar efficiency in protein extraction was obtained between controls and inflamed tissues.

Immunoprecipitation and Western Blotting. Because - and -opioid receptors proteins are expressed at low levels in the mouse intestine, especially in the MP/LM and SP/M preparations, an immunoprecipitation assay was used. The optimal amount of - and -opioid receptor antibodies and the tissue proteins concentration used in the immunoprecipitation assay were determined in preliminarily experiments. Immunoprecipitation was performed in all samples (intestine, MP/LM and SP/M sections, spinal cord, and brain) of animals with and without intestinal inflammation. In the immunoprecipitation, 25 µg of each polyclonal antibody anti--opioid receptor (against sequences in the N-terminus of the -opioid receptors protein; Chemicon International, Inc., Temecula, CA) or anti--opioid receptor (against sequences in the N-terminus of the -opioid receptors protein; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated with 200 µl of resuspended protein A-Sepharose (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) for 1 h at 4°C. Afterward, samples were centrifuged at 1.000 rpm for 5 min at 4°C, and pellets washed four times with PBS-bovine serum albumin 1%. Immunocomplexes were obtained by incubation of 100 µl of the protein A-Sepharose linked to the antibody with 60 µg of tissue protein (for each sample) or PBS (as a control without protein) overnight at 4°C. After washing the pellets four times with PBS buffer, they were resuspended in 50 µl of Laemmli SDS buffer and heated at 100°C for 5 min. Finally, 20 µl of each sample were separated on a 10% SDS-polyacrylamide gel electrophoresis at 100 V during 4 h (Amersham Biosciences, Piscataway, NJ).

To confirm that immunoprecipitated samples contained specific proteins for - or -opioid receptors antibodies, we performed a Western blot immunoassay. Gels were transferred (Mini-Trans-Blot electrophoretic transfer cell; Bio-Rad) to nitrocellulose membranes (Amersham Biosciences) by the application of 100 V (200–300 mA) for 2 h. Membranes were first blocked with nonfat dry milk in PBS overnight at 4°C; then they were incubated with antibodies anti--opioid receptors (against sequences in the C-terminus of the -opioid receptors protein; Santa Cruz Biotechnology; 1:100) or anti--opioid receptors (against sequences in the C-terminus of the -opioid receptors protein; Santa Cruz Biotechnology; 1:100) for 1 h at room temperature and then overnight at 4°C. After removal of the antibody, membranes were washed with PBS and then incubated with a universal secondary antibody conjugated with biotin at a 1:200 dilution (AB600; The Binding Site Ltd., Birmingham, UK) for 1 h at room temperature. The secondary antiserum was removed and the membranes washed again and incubated in streptavidin peroxidase (IC019; The Binding Site Ltd.; 1:100) for 1 h at room temperature. A substrate solution containing 0.05% of 3–3'-diaminobenzidine and 100 µl of hydrogen peroxidase in PBS was then added. Negative controls for the Western blot assay with all components except the first antibody were also used.

After characterization of the specific immunoreactive bands for proteins and antibodies to - or -opioid receptors by Western blot in samples from brain, spinal cord, and intestine, the method for detecting - and -opioid receptors proteins was simplified. In this case, 15 µl of each sample (obtained by immunoprecipitation) was separated in SDS-polyacrylamide gel electrophoresis, and proteins were detected by silver staining. Images were digitalized and the intensity of the bands measured by using the Diversity database program.

Statistics. Data are expressed as a group mean ± S.E.M. Statistical analysis for significant differences between two groups was obtained by Student's t test. When multiple groups were compared, one- or two-way ANOVA was used, followed by a Student-Newman-Keuls test, whenever applicable. A value of p < 0.05 was considered significant.

	Results

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Transcription of -Opioid Receptors in Mice with Intestinal Inflammation. The mRNA levels of -opioid receptors were determined in the intestine, spinal cord, and brain of mice with intestinal inflammation. Figure 1A shows the separation of the -opioid receptors (upper gel) and -actin cDNA (lower gel) by gel electrophoresis of a representative experiment. For each tissue (intestine, spinal cord, and brain), the first and second lanes show the PCR products obtained form controls and animals with intestinal inflammation, respectively. In the figure, the left lane contains a sample in which RNA was not reverse-transcribed (blank); no bands were detected in this sample. -Opioid receptors transcripts (500 bp) are expressed at relatively low levels in the intestine when compared with the spinal cord and brain. Under the same experimental conditions, the amplification of -actin transcripts (289 bp) did not show an induction of the housekeeping gene and thus -actin mRNA was used as an internal control.

View larger version (18K): [in this window] [in a new window]   Fig. 1. A, gel electrophoresis of -opioid receptor (upper gel) and -actin (lower gel) mRNA levels in intestine, spinal cord, and brain from animals with (croton oil) and without intestinal inflammation (saline). The figure shows the PCR products of a representative experiment obtained from samples of intestine (saline in lane 2; croton oil in lane 3), spinal cord (saline in lane 4; croton oil in lane 5), and brain (saline in lane 6; croton oil in lane 7); lane 1 is a sample in which RNA was not reverse-transcribed (blank). The band of 500 bp is a product of the -opioid receptors, and the band of 289 bp is -actin mRNA. Inflammation produced a significant increase in the expression of -opioid receptor mRNA in the gut and spinal cord (upper gel), whereas the expression of -actin remained unaltered in all experimental conditions (lower gel). B, is a graphical representation of the ratios of the optical density of the bands of -opioid receptor to -actin in intestine, spinal cord, and brain samples from animals with and without intestinal inflammation (saline). Different letters (a, b, c, and d) indicate significant differences between groups (p < 0.05, Student-Newman-Keuls test). Each column represents the mean ± S.E.M. from four samples of independent experiments.

The quantification of the results is shown in Fig. 1B, where the ratios of optical density of the bands of target to the housekeeping gene are graphically represented. The statistical analysis of the -opioid receptors transcript levels by a two-way ANOVA revealed a significant effect of the treatment (saline or croton oil; p < 0.007), tissue (p < 0.001), and their interaction (p < 0.031). In control conditions, -opioid receptors mRNA showed a higher expression in the brain and the spinal cord compared with the gut. Intestinal inflammation induced a significant increase in -opioid receptors mRNA levels in the gut (102%) and spinal cord (34%) but not in the brain (p < 0.05, Student-Newman-Keuls test). Thus, the inflammatory stimulus was able to induce an increase in -opioid receptors mRNA levels both locally (in the gut) as well as in the spinal cord, where integration of sensory information occurs.

-Opioid receptors mRNA levels were also determined in two dissected portions of the intestine, one containing the myenteric (MP/LM) and the other the submucous plexus (SP/M). In the absence of inflammation, -opioid receptors mRNA could be detected in both preparations, and the relative levels were approximately two times higher in the SP/M than in the MP/LM preparations (Table 1). During inflammation, -opioid receptors mRNA levels were similarly increased in both preparations (P < 0.05, Student-Newman-Keuls test). Each experiment was repeated in samples obtained from four different animals (saline or croton oil) and showed similar results.

View this table: [in this window] [in a new window]   TABLE 1 Levels of -OR mRNA (OD of -OR/-actin) in the dissected MP/LM and SP/M sections from the intestine of mice with and without intestinal inflammation (saline) Data are shown as mean values ± S.E.M. of four animals. Different letters (a, b, and c) indicate statistical differences between the groups (p < 0.05; Student-Newman-Keuls, test). MP/LM: circular muscle layers, myenteric plexus, and longitudinal muscle; SP/M: submucosal plexus and mucosa.

Expression of -Opioid Receptors during Intestinal Inflammation. In these experiments, we investigated whether the changes observed in the -opioid receptors mRNA levels were associated with an increase in the expression of -opioid receptors protein. Using immunoprecipitation with a -opioid receptors antibody, we evaluated the following samples (obtained from animals with and without intestinal inflammation): whole intestine, dissected portions of MP/LM and SP/M, and samples of spinal cord and brain. Figure 2A shows a gel electrophoresis of -opioid receptor protein from a representative experiment in samples of whole intestine, spinal cord, and brain. For each tissue, the first and second bands were obtained from saline and croton oil-treated animals. The gel shows two bands, one at approximately 110 kDa (antibody without protein) and another at 68 kDa corresponding to the -opioid receptor protein. In the figure, the first lane indicates the molecular mass marker and the second lane the immunoprecipitate without protein. To corroborate that the 68-kDa band was the -opioid receptor protein, we used another -opioid receptor antibody in a Western blot. Figure 2B illustrates a representative immunoblot experiment using a brain sample (lane 3) showing a single band of approximately 68 kDa and confirming the specificity of the results. In this figure, lane 1 is the molecular mass marker, lane 2 is a sample without protein, and lane 4 is a brain sample which immunostaining was carried out without the primary antibody. When Western blot experiments were performed with intestine and spinal cord samples, a single band at 68 kDa was also obtained.

View larger version (16K): [in this window] [in a new window]   Fig. 2. A, gel electrophoresis of -opioid receptor proteins from a representative experiment performed by the immunoprecipitation of -opioid receptors antibody with protein samples from whole intestine, spinal cord, and brain. For each tissue, the first and second bands were obtained from saline and croton oil-treated animals. Lane 1 is the molecular mass marker, and lane 2 is a control where the immunoprecipitation was performed without protein. The gel shows two bands, one at approximately 110 kDa and other at 68 kDa. Equal amounts of samples (15 µl/lane) from saline and croton oil-treated animals were loaded into the gels. B, immunoblot of a representative experiment using a brain sample (lane 3) or a sample without protein (lane 2). Lanes 1 and 4 show the molecular mass marker and a brain sample in which immunostaining was performed without the primary antibody, respectively. A single immunoreactive band of approximately 68 kDa was detected in the brain sample. Immunoreactivity was completely abolished when staining was performed in the sample without protein (lane 2) or in the brain sample immunostained in the absence of the first antibody (lane 4). The molecular mass standards used were: myosin (233 kDa), -galactosidase (135 kDa), bovine serum albumin (112 kDa), and ovalbumin (53 kDa) (Bio-Rad Laboratories). All data were reproducible for four independent experiments. C, is a graphical representation of the optical density (arbitrary units) of the bands of -opioid receptors in intestine, spinal cord and brain samples from animals with and without intestinal inflammation (saline). Different letters (a,b,c) indicate significant differences between groups (p < 0.05, Student-Newman-Keuls test). Each column represents the mean ± S.E.M. of four different samples obtained from independent experiments.

The data related to -opioid receptor protein levels in the different tissues (Fig. 2A) were quantified by densitometry and expressed as the optical density (arbitrary units) of the bands of -opioid receptor protein. A two-way ANOVA revealed a significant effect of the treatment (saline or croton oil; p < 0.001), type of tissue (p < 0.05), and their interaction (p < 0.023). The effects of the treatment and of the interaction could be explained by the fact that intestinal inflammation induces a significant increase (p < 0.001) in the peripheral but not in the central levels of -opioid receptor proteins (Fig. 2C; Student-Newman-Keuls test). In the absence of inflammation, the detected levels of -opioid receptor protein were significantly higher in the brain and spinal cord than in the whole intestine (p < 0.01; Student-Newman-Keuls test).

-Opioid receptor protein levels were also measured in the dissected MP/LM and SP/M from control animals and mice with intestinal inflammation. The levels of -opioid receptor protein in these preparations were very low, and results of a representative experiment are shown in Fig. 3A. In these experiments, we obtained similar bands to the whole intestine at 110 kDa (antibody) and 68 kDa (-opioid receptors protein). The quantification of the results indicates (Fig. 3B) that in the absence of inflammation -opioid receptor protein levels in the SP/M are higher than in the MP/LM. The results also show that inflammation induces a significant increase in -opioid receptor protein expression in both preparations (P < 0.05, Student-Newman-Keuls test). Each experiment was repeated in four different animals with similar results.

View larger version (15K): [in this window] [in a new window]   Fig. 3. A, electrophoresis gel of -opioid receptor proteins from a representative experiment of the dissected MP/LM and SP/M sections from animals with and without intestinal inflammation. For each intestinal section, the first and second bands were obtained from saline and croton oil-treated animals. Lane 1 is the molecular mass marker and lane 2 is a control which immunoprecipitation was performed without protein. The gel shows two bands, one at approximately 110 kDa and other at 68 kDa. Equal amounts of samples (15 µl/lane) from saline and croton oil-treated animals were loaded into the gels. The molecular mass standards used were: myosin (233 kDa), -galactosidase (135 kDa), bovine serum albumin (112 kDa), and ovalbumin (53 kDa) (Bio-Rad Laboratories). All data were reproducible for four independent experiments. B, is a graphical representation of the optical density (arbitrary units) of bands of -opioid receptors in samples of MP/LM and SP/M sections obtained from control animals (saline) and mice with intestinal inflammation (inflammation). Different letters (a, b, and c) indicate significant differences between groups (p < 0.05, Student-Newman-Keuls test). Each column represents the mean ± S.E.M. of four different samples obtained from independent experiments.

Transcription of -Opioid Receptor in Mice with Intestinal Inflammation. The expression of -opioid receptor mRNA was evaluated in the whole intestine, the spinal cord, and the brain of mice with and without intestinal inflammation (semiquantitative RT-PCR). Figure 4A shows a gel electrophoresis of -opioid receptor (560 bp) and -actin (289 bp) cDNA levels of a representative experiment; for each tissue, the first lane was obtained from saline-treated animals and the second from animals with intestinal inflammation. In the figure, the left lane contains a sample, which RNA was not reverse-transcribed (blank); no bands were detected in this sample. The expression of -opioid receptor mRNA was higher in brain and spinal cord when compared with the intestine (p < 0.05, Student-Newman-Keuls test). -Opioid receptor mRNA levels were quantified using the ratio of the optical density of -opioid receptor to -actin mRNAs (Fig. 4B), and the data were analyzed by two-way ANOVA. The results revealed a significant effect of the type of tissue sample (p < 0.001) but not of the treatment (saline or croton oil) or their interaction. Thus, intestinal inflammation does not increase -opioid receptor mRNA levels in any of the tissues evaluated.

View larger version (16K): [in this window] [in a new window]   Fig. 4. A, gel electrophoresis of -opioid receptors (upper gel) and -actin (lower gel) mRNA levels in whole intestine, spinal cord and brain from animals with (croton oil) and without intestinal inflammation (saline). The figure shows the PCR products of a representative experiment obtained from samples of intestine (saline in lane 2; croton oil in lane 3), spinal cord (saline in lane 4; croton oil in lane 5), and brain (saline in lane 6; croton oil in lane 7); lane 1 is a sample which RNA was not reverse-transcribed (blank). The band of 560 bp is a product of the -opioid receptors and the band of 289 bp is -actin mRNA (lower gel). Inflammation does not alter the expression of -opioid receptors mRNA in any of the tissues evaluated, and the expression of -actin remained unaltered in all experimental conditions. B, is a graphical representation of the ratios of the optical density bands of -opioid receptor to -actin, from intestine, spinal cord, and brain samples of animals with and without intestinal inflammation (saline). Different letters (a and b) indicate significant differences between groups (p < 0.05, Student-Newman-Keuls test). Each column represents the mean ± S.E.M. from four different samples from independent experiments. C, a representative gel electrophoresis of -opioid receptor mRNA (560 bp) in the MP/LM and SP/M sections from mice with and without intestinal inflammation. In this gel, 20 µl of each cDNA were loaded. The figure shows the PCR products from a single experiment obtained with samples of the MP/LM section from saline and croton oil-treated animals (lanes 2 and 3) and of the SP/M section from saline (lane 4) and mice treated with croton oil (lane 5). Lane 1 is a sample which RNA was not reverse-transcribed (blank). This experiment was performed four additional times with similar results.

When the dissected portions of the intestine were studied (Fig. 4C), significantly higher levels of -opioid receptor mRNA were obtained in the SP/M than in the MP/LM preparations (Table 2). The levels of the -opioid receptor transcripts were not altered during inflammation.

View this table: [in this window] [in a new window]   TABLE 2 Levels of -OR mRNA (OD of -OR/-actin) in the dissected MP/LM and SP/M sections from the intestine of mice with and without intestinal inflammation (saline) Data are shown as mean values ± S.E.M. of four animals. Different letters (a and b) indicate statistical differences between groups (p < 0.05; Student-Newman-Keuls test). MP/LM: circular muscle layers, myenteric plexus, and longitudinal muscle; SP/M: submucosal plexus and mucosa.

Expression of -Opioid Receptors during Intestinal Inflammation. The evaluation of the levels of -opioid receptor protein in the different tissues (intestine, spinal cord, and brain) obtained from animals with and without intestinal inflammation was performed by immunoprecipitation. The resulting gel showed two bands, one at approximately 110 kDa (antibody) and other at 72 kDa corresponding to the -opioid receptor protein (corroborated by using another -opioid receptor antibody in a Western blot). Data obtained after quantification by optical density is shown in Table 3. A two-way ANOVA demonstrated a significant effect of the treatment (p < 0.001) and the type of tissue (p < 0.041) but not of their interaction (p < 0.067). Thus, intestinal inflammation induced a significant increase (p < 0.05) in the peripheral (gut) but not in central levels of -opioid receptor protein. In the absence of inflammation, the expression of -opioid receptor protein in the gut was lower than in the spinal cord and brain (p < 0.01; Student-Newman-Keuls test).

View this table: [in this window] [in a new window]   TABLE 3 Levels of -OR proteins (OD, arbitrary units) in the intestine, spinal cord, and brain from mice with and without intestinal inflammation Data are shown as mean values ± S.E.M. of four animals. Different letters (a, b, and c) indicate significant changes between groups (p < 0.05; Student-Newman-Keuls test).

The levels of -opioid receptor protein in the dissected preparations of the intestine (MP/LM and SP/M) were also evaluated (Fig. 5A), and the results are shown in Fig. 5B. In saline-treated animals, there was a similar expression of -opioid receptor protein levels in both preparations; however during inflammation, a significant increase in the SP/M but not in the MP/LM was observed (p < 0.05, Student-Newman-Keuls test). These results suggest that intestinal inflammation does not induce a de novo synthesis of -opioid receptor protein but may induce either post-transcriptional or translational changes in the -opioid receptors gene.

View larger version (18K): [in this window] [in a new window]   Fig. 5. A, a representative electrophoresis gel of -opioid receptor proteins from MP/LM (saline lane 3 and croton oil lane 4) and SP/M sections (saline lane 5 and croton oil lane 6). Lane 1 is the molecular mass marker, and lane 2 is a sample where immunoprecipitation was performed without protein. Two bands of about 110 (antibody anti--opioid receptors) and 72 kDa (-opioid receptors) were detected in the gel. Equal amounts of sample (15 µl/lane) from saline and croton oil-treated animals were loaded into the gels. The molecular mass standards used were: myosin (233 kDa), -galactosidase (135 kDa), bovine serum albumin (112 kDa), and ovalbumin (53 kDa) (Bio-Rad Laboratories). All data were reproducible for four independent experiments. B, is a graphical representation of the ratio of the optical density (arbitrary units) of the bands of -opioid receptors on samples of MP/LM and SP/M sections from animals with and without intestinal inflammation (saline). A different letter (a and b) indicates significant differences between groups (p < 0.05, Student-Newman-Keuls test). Each column represents the mean ± S.E.M. of four different samples obtained from independent experiments.

	Discussion

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The present investigation demonstrates for the first time that intestinal inflammation increases the transcription of - but not -opioid receptors, both at the site of injury (gut) and in the spinal cord. Intestinal inflammation also enhances the protein levels of - and -opioid receptors in the gut. Similar results have been obtained by different investigations in a rat paw model of peripheral inflammation. In these studies, an increased number of - and -opioid receptor proteins was demonstrated on peripheral nerve terminals (Stein, 1995) together with a decreased - and -opioid receptor immunoreactivity in the dorsal root ganglia (Ji et al., 1995; Zhang et al., 1998). Moreover, an increased axonal transport of both opioid receptor proteins from the dorsal root ganglia to the periphery could be demonstrated (Ji et al., 1995). In the spinal cord, up- and down-regulation/s of - and -opioid receptors have been reported in the literature (Ji et al., 1995; Cahill et al., 2003), whereas both - and -opioid receptor mRNA levels were shown to be increased in animals with peripheral inflammation (Maekawa et al., 1995; Cahill et al., 2003). At present, there are no studies in the literature evaluating mRNA levels of - and -opioid receptors in the dorsal root ganglia of animals with peripheral inflammation. A recent study has demonstrated that proinflammatory substances increase trafficking of intracellular -opioid receptors to the plasma membrane of dorsal root ganglia neurons, suggesting that early stages of inflammation may affect the cellular localization of preformed -opioid receptors (Bao et al., 2003).

The differences in -opioid receptor proteins and -opioid receptor transcripts observed in the two models of inflammation could be related to the distinct models/organ systems of peripheral inflammation. Moreover, the duration, type, and severity of the inflammatory response, as well as the administration pattern of the inflammatory agent, could be important factors that determine changes in the expression of opioid receptor mRNA.

In the gut, the enhanced transcription and the expression of the -opioid receptor protein induced by intestinal inflammation, was similarly observed in both dissected preparations (SP/M and MP/LM). This finding would explain the enhanced effects of -agonists on the inhibition of intestinal function (transit and permeability) observed 5 days after croton oil-induced intestinal inflammation (Puig and Pol, 1998; Valle et al., 2001). The precise mechanisms implicated in the increased transcription of -opioid receptors during inflammation are not yet elucidated. In cell cultures, the expression of the -opioid receptor gene has been reported to increase in the presence of retinoic acid, nerve growth factor, O-tetradecanoylphorbol-13-acetate, and phytohemagglutinin (Woltje et al., 2000; Sun and Loh, 2002; Wei and Loh, 2002) among others. In contrast, a reduction of -opioid receptor mRNA has been observed in the presence of cAMP analogs (Gylys et al., 1997). Some of the suggested sites that control -opioid receptor gene transcription include the activation of the Ap1, Ap2, Sp1, Ets-1, and Ikaros transcription factors (Woltje et al., 2000; Sun and Loh, 2002; Wei and Loh, 2002), but many other transcription factor-binding sites in the -opioid receptor gene are likely to be present. An appealing possibility would be that interleukins (interleukin-1 and others) would increase the expression of nerve growth factor, and induce the -opioid receptor gene by interaction with either the nerve growth factor-IB transcription factor or the nerve factor-B binding sites.

The -opioid receptor gene transcription in the intestine was not altered by inflammation, whereas -opioid receptors protein expression was significantly augmented in the whole gut due to an increased expression in the SP/M preparation (submucous plexus plus mucosa). The increased receptor protein levels in the SP/M (but not MP/LM) without changes in their mRNA expression, suggest post-transcriptional and/or post-translational changes of the -opioid receptor gene. An activation of a pre-existing pool of "reserve or silent" receptors and/or a decrease in receptor degradation could be postulated. Since the submucous plexus is mainly involved in the control of intestinal secretion and permeability, these results could explain the increased antisecretory potency of -opioids observed 5 days after the induction of intestinal inflammation.

Recently, the -opioid receptor gene transcription has been reported to be negatively regulated by nitric oxide (Park et al., 2002) and positively and negatively regulated by retinoic acid (Hu et al., 2002). The stimulatory and repressive effects of retinoic acid on the -opioid receptor gene expression are mediated by the Sp1 and Ikaros transcription factors, respectively (Li et al., 2002). In addition, a post-transcriptional regulation of this gene was demonstrated by the characterization of three mRNA variants of the -opioid receptors in different tissues in mice. These results suggest different levels of regulation for the -opioid receptor gene expression, i.e., alternative splicing and differential control for translation (Wei et al., 2000; Wei and Loh, 2002). Nevertheless, there is no data available regarding the conditions that may generate a specific splicing variant for -opioid receptors (Wei et al., 2000).

In the spinal cord, a significant increase in the expression of -opioid (but not ) receptor mRNA was observed, whereas the -opioid receptor protein levels remained unchanged. The increase in -opioid receptor mRNA in the spinal cord (34%) was less pronounced than in the gut (102%), indicating that distinct biochemical changes occur in the central and peripheral nervous system after intestinal inflammation. The differences in expression could be explained by the extent and intensity of the intestinal inflammation induced by croton oil, which can be considered moderate according to the morphological studies. Most probably, the increased transcription of -opioid receptors in the spinal cord is a consequence of the increased excitability of the peripheral nerves induced by the inflammatory mediators (Richardson and Vasko, 2002).

No variations in gene transcription or protein levels for - or -opioid receptors could be demonstrated in whole-brain samples; however, such changes cannot be excluded from the present investigation since they may occur in specific areas of the brain (hypothalamus for example) and/or be of a lesser magnitude. If no increase in the expression of cerebral -opioid receptors occurs during intestinal inflammation, however, we could then explain the finding that the inhibitory effects of centrally (i.c.v.) -agonists on the gut are unaltered during intestinal inflammation. Similarly, the antinociceptive effects of the supraspinal administration of -opioid receptor agonists were unchanged 4 days after the induction of hind paw inflammation (Hurley and Hammond, 2000).

Our results also show a higher expression in the gut of -actin than the expression of - and -opioid receptors mRNA. Possibly, the important number of non-neural cells present in the intestinal preparations used in the study might contribute to enhance the expression of -actin in these tissues.

When the protein levels of - and -opioid receptors were evaluated, a unique band was obtained for each protein (68 kDa for the -opioid receptor and 72 for the -opioid receptor), and their specificity was demonstrated by Western blot. The band-size for the - and -opioid receptor proteins is consistent with the results obtained in rodents by other investigators, which describe molecular masses of 43 to 125 kDa for the -opioid receptor (Sánchez-Blázquez et al., 1997; Cahill et al., 2001; Cichewicz et al., 2001) and between 43 to 70 kDa for the -opioid receptor (Joshi et al., 2000; Cichewicz et al., 2001). In our experiments, the detected bands of 68 and 72 kDa could correspond to the monomeric receptors ( and ), which presumably represent different post-translational maturation forms (glycosylation) of these receptors.

In mice intestine without inflammation, we are the first group to report the expression of - and -opioid receptor mRNA and their respective receptor proteins. Our results also show, that both - and -opioid receptor transcripts are primarily expressed in the submucous rather than in the myenteric plexus. The differential expression of these receptors in mice seems to differ from the rat intestine, where -opioid receptors are more abundant in the myenteric than submucosal plexi (Bagnol et al., 1997). Our results cannot discriminate if the opioid receptors detected in the SP/M preparation are located in the submucous plexus, the enterocytes, or in immune cells such as lymphocytes since our samples contained all these structures. A population of -opioid receptors in rat enterocytes has been demonstrated, although their physiological relevance in the modulation of intestinal permeability is controversial (Dashwood et al., 1986; Nano et al., 2000). In conclusion, the present report shows that intestinal inflammation increases the transcription and translation of -opioid receptors in the myenteric and submucosal preparations of the gut. For the -opioid receptors, no changes were observed in mRNA levels, but an increased expression of -opioid receptor proteins in the submucosal preparation could be demonstrated, suggesting that post-transcriptional and/or post-translational changes occur during inflammation. An increased transcription of -opioid receptors was also observed in the spinal cord of animals with intestinal inflammation, without changes in protein expression. No changes in gene transcription or protein levels for - and -opioid receptors could be demonstrated in the brain samples after inflammation. These results suggest that local transcriptional and post-transcriptional changes of the - and -opioid receptor genes could be responsible for the enhanced antisecretory effects of - and -opioid agonists during chronic intestinal inflammation.

	Acknowledgements

 
We thank Sergi Leánez for his excellent technical assistance.

	Footnotes

 
This work was supported by grants from FIS (00/0658) and CICYT (PM980155), Madrid and Generalitat de Catalunya (2001SGR00409), Barcelona, Spain. Part of these results have been presented as a communication to the 31th Annual Meeting Society for Neuroscience (San Diego, CA) on November 10–15, 2001.

DOI: 10.1124/jpet.103.049346.

ABBREVIATIONS: MP/LM, circular muscle layers-myenteric plexus-longitudinal muscle; SP/M, submucosal plexus-mucosa; RT, reverse transcriptase; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; bp, base pair; ANOVA, analysis of variance; U-50488H, (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide.

Address correspondence to: Dr. Olga Pol, Anesthesiology Research Unit, Institut Municipal Investigació Mèdica, Doctor Aiguader, 80, 08003 Barcelona, Spain. E-mail: opol{at}imim.es

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