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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Characterization of Specific Opioid Binding Sites in Neural Membranes from the Myenteric Plexus of Porcine Small Intestine

Department of Veterinary Pathobiology, College of Veterinary Medicine (D.T., D.R.B.), and Department of Medicinal Chemistry, College of Pharmacy (P.S.P.), University of Minnesota Academic Health Center, Minneapolis, Minnesota

Received July 31, 2003; accepted October 9, 2003.

	Abstract

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- and -Opioid receptors (OPRs), but not µ-OPRs, are expressed in the myenteric plexus of the porcine distal small intestine. In a subpopulation of myenteric neurons, - and -OPRs seem to be colocalized and may functionally interact. In this study, radioligand binding was used to characterize myenteric OPR populations in detail. The nonselective OPR antagonist [³H]diprenorphine bound to a single, high-affinity site in myenteric neural membrane homogenates. Naloxone displaced 65 and 59% of [³H]diprenorphine binding from this site in Na⁺-free Tris and Krebs-HEPES buffers, respectively. Naltrexone-derived - and -OPR antagonists, including naltriben, 7-benzylidenenaltrexone, nor-binaltorphimine, and 5'-guanidinonaltrindole, displaced [³H]diprenorphine from two distinct binding sites to levels similar to that of naloxone. The selective -OPR ligands Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (TIPP), [D-Pen²,D-Pen⁵]enkephalin (DPDPE), [D-Ala², Glu⁴]deltorphin II, and (+)-4-[(R)-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl-3-methoxybenzyl)-N,N-diethylbenzamide (SNC-80) and the -OPR agonist (D-(5,7,8)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxoaspiro-(4,5)dec-8-yl) benzeneacetamide (U-69,593) displaced [³H]diprenorphine from three independent binding sites; these included high-affinity - and -OPR sites, and a residual binding site. Residual [³H]diprenorphine binding was displaced by the selective -OPR antagonist nor-binaltorphimine after saturation of and sites, respectively, with DPDPE and U-69,593. The residual binding site displayed low affinity for - and -OPR agonists and TIPP, as well as moderate affinity for naltrexone-derived ligands, properties reminiscent of -/-OPR heterodimers.

In studies of opioid receptors in the central nervous system, µ-, -, and -OPR types seem to manifest additional heterogeneity (Gutstein and Akil, 2001). In the case of -OPRs, for example, subtype-selective agonists, the absence of cross-tolerance between these agonists, and subtype-selective antagonists of both competitive and noncompetitive natures have been used in behavioral studies to distinguish putative ₁- and ₂-OPR subtypes (Zaki et al., 1996). The molecular basis of these subtypes is unknown; however, the presence of OPR mRNA splice variants (Abbadie et al., 2001) and evidence that these receptors form homo- and heterodimeric associations in recombinant systems (Jordan and Devi, 1999; Ramsay et al., 2002) are among the variables that may contribute to their distinctive pharmacological profiles in vivo. Indeed, a recent pharmacological study suggests that ₁-OPRs in the mouse spinal cord may represent -/-OPR heterodimers (Portoghese and Lunzer, 2003), a conclusion supported by the colocalization of - and -OPR immunoreactivities in a population of murine spinal axons (Wessendorf and Dooyema, 2001). Although these limited pharmacological and immunohistochemical approaches suggest that OPR heterodimers are expressed in vivo, definitive proof is lacking.

The existence of OPR heterodimers has been documented in recombinant systems with cloned OPRs detected with fluorescent or immunological tags. Because this technique is not feasible in studies of native OPRs, alternative methods must be used for the detection of the receptor complexes in tissues, a critical step in determining the physiological significance of these OPR associations. Ligand binding studies performed in recombinant systems containing heterodimeric OPRs reveal a mix of the binding characteristics of each receptor constituent (Maggio et al., 1993; Jordan and Devi, 1999). Membrane homogenates from native tissues are often heterogeneous, a fact that complicates the analysis of binding assays; these membrane preparations may contain both monomeric and heterodimeric receptor populations, from separate cells or distinct domains from within a single cell.

Neural membrane preparations from the myenteric plexus of the porcine small intestine express specific - and -OPR binding sites, but relatively few µ-OPR sites (Townsend and Brown, 2002). Immunoreactivities for - and -OPRs, but not µ-OPRs are expressed in myenteric neurons and are colocalized in some cells (Poonyachoti et al., 2001, 2002). Moreover, neurogenic contractions of a smooth muscle-myenteric plexus preparation from the porcine ileum are inhibited by agonists acting selectively at either -or -OPRs, although the actions of a -OPR agonist can be prevented by nor-binaltorphimine (nor-BNI), a selective -OPR antagonist (Poonyachoti et al., 2001). These results suggest that some myenteric neurons may express functional - and -OPRs and that some of these receptors might be in association. In the present study, we characterized in detail the - and -OPR binding sites in myenteric neural membranes. The binding affinities of selective OPR ligands in displacing the poorly selective opioid antagonist [³H]diprenorphine (DPN) from these sites were determined and compared in Na⁺-free and physiologically relevant solutions. In addition, we examined a residual nor-BNI binding site that was revealed after agonist saturation of and sites.

	Materials and Methods

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Radioligands, Drugs, and Reagents. [³H]Saxitoxin (STX; 14.9 Ci/mmol) and [³H]diprenorphine [DPN; (5,7)-17-(cyclopropylmethyl)-4,5-epoxy-18,19-dihydro-3-hydroxy-6-methoxy-,-dimethyl-6,14-ethenomorphinan-7-methanol; 70 Ci/mmol] were obtained from Amersham Biosciences Inc. (Piscataway, NJ) and PerkinElmer Life Sciences (Boston, MA), respectively. Both radioligands were diluted to the desired concentration in 5 mM HCl and stored at -20°C until use. Naloxone, tetrodotoxin, D-(5,7,8)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxoaspiro-(4,5)dec-8-yl)benzeneacetamide (U-69,593), and (+)-4-[(R)-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl-3-methoxybenzyl)-N,N-diethylbenzamide (SNC-80) were obtained from Sigma-Aldrich (St. Louis, MO). [D-Pen²,D-Pen⁵]enkephalin (DPDPE), [D-Ala²,Glu⁴]deltorphin II, and Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (TIPP) were obtained from Bachem (Torrance, CA). Naltriben (NTB), 7-benzylidenenaltrexone (BNTX), naltrindole (NTI), 5'-guanidinonaltrindole (GNTI), and nor-BNI were synthesized in the laboratory of P.S.P. as reported previously (Portoghese et al., 1988, 1991; Jones et al., 1998). All other reagents were obtained from Fisher Scientific (Chicago, IL).

Tissue Isolation. Intestinal segments were obtained from 30 weaned outbred Yorkshire pigs of each sex (6-10 weeks of age; 10-18 kg body weight) who received food and water ad libitum. Animals were sedated with an intramuscular injection of tiletamine hydrochloride-zolazepam (8 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA) in combination with xylazine (3 mg/kg; Phoenix Pharmaceuticals Inc., St. Joseph, MO). The animals were subsequently euthanized by barbiturate overdose. All procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee. A midline laparotomy was performed to expose the intestine, and a section of distal small intestine was resected that extended approximately 1.5 m orad from the ileocecal junction. Intestinal segments were removed rapidly and placed in an ice-cold, oxygenated physiological salt solution (composition 143 mM Na⁺, 128.7 mM Cl^-, 4.7 mM K⁺, 2.5 mM Ca²⁺, 0.5 mM Mg²⁺, 25.0 mM HCO₃^-, 1.0 mM H₂PO₄^2-, and 11 mM D-glucose; pH 7.4). Subsequent tissue dissections were performed at 4°C.

Isolation of Neurally Enriched Myenteric Membranes. Intestinal segments were opened along the antimesenteric border after removal of any mesenteric attachments. The smooth muscle layers containing the myenteric and deep muscular plexuses were carefully separated from the overlying submucosa, diced into 5 x 5-mm² pieces, and stored at -70°C. This preparation was later thawed and diluted in either Na⁺-free Tris buffer (50 mM Tris in water) or Krebs-HEPES buffer (composition 143 mM Na⁺, 130.2 mM Cl^-, 4.8 mM K⁺, 2.5 mM Ca²⁺, 1.2 mM Mg²⁺, and 25.0 mM HEPES); the pH of both buffers was corrected to 7.4 by the addition of 6 M HCl. The P2 membrane fractions were isolated as described previously in either Tris or Krebs-HEPES buffer (Townsend and Brown, 2002) from smooth muscle-myenteric plexus homogenates; the P2 membrane fraction was enriched in specific [³H]STX binding sites (see below) and is henceforth referred to as the "neural membrane" fraction. After isolation, membrane fractions were stored at -70°C until binding assays were performed. Protein concentrations were determined with a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL).

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting. Equivalent amounts of crude membrane proteins were resolved by nonreducing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred to Immobilon membranes (Millipore Corporation, Billerica, MA) and blocked with 5% milk in wash solution (10 mM Tris and 150 mM NaCl and 0.05% Tween 20). Blots were incubated for 4 h with an antiserum raised in goats against human growth-associated protein (GAP)-43, a neuronal marker (Santa Cruz Biotechnology Inc., Santa Cruz, CA) that was diluted 1:1000 in the blocking solution. After blots were washed three times (for 15, 5, and 5 min, respectively) with wash solution, they were incubated in horseradish peroxidase-conjugated anti-goat IgG (1:5000) for 2 h. After one 15 min and four 5-min washes, the blots were developed using an enhanced chemiluminescence detection system (Amersham, Piscataway, NJ).

Radioligand Binding Assays. Neural membranes were thawed on ice just before each experiment and diluted to a final concentration of 500 µg/ml in either Tris or Krebs-HEPES buffer. Actual protein concentrations were determined from single aliquots of the diluted membrane fraction used in each assay. Specific binding sites for the neuronal Na⁺ channel blocker STX were detected through the binding of 1 nM [³H]STX to membranes in the absence and presence of 1 µM of unlabeled tetrodotoxin. Saturation analyses of specific OPR binding sites were performed using the poorly selective OPR antagonist DPN (0.03-3 nM); nonspecific binding was determined in the presence of 1 µM unlabeled naloxone. The binding affinities of various OPR ligands were determined by their displacement of 1 nM [³H]DPN from specific binding sites. All displacement assays were initiated by the addition of membranes to tubes containing radioligand in the absence or in combination with an unlabeled ligand. To ensure that equilibrium conditions were achieved, assays were allowed to incubate for 60 min at room temperature before rapid filtration of unbound ligands through glass fiber filters using a 24-sample cell harvester (Brandel, Inc., Gaithersburg, MD). Glass fiber filters were then washed twice with 4 ml of cold Tris or Krebs-HEPES buffer and subsequently submerged in scintillation fluid for approximately 12 h before being counted.

Data Analysis. Specific radioligand binding was determined in saturation analyses by calculating the difference between [³H]DPN or [³H]STX binding in the presence and absence of 1 µM naloxone or tetrodotoxin, respectively. The resulting data were averaged at each radioligand concentration and analyzed by nonlinear regression. Data obtained in [³H]DPN displacement studies were also averaged and analyzed by nonlinear regression, and K_i values for unlabeled displacing ligands were calculated by the method of Cheng and Prusoff (1973). In all cases, a single binding site model was chosen, unless a two-site model gave a significantly better fit by F test (p < 0.05). All nonlinear regression analyses were performed using the Prism statistical software package (version 3.0c; GraphPad Software Inc., San Diego, CA).

	Results

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Neural Enrichment of Membranes Isolated in Na⁺-Free Tris Buffer or Krebs-HEPES Buffer. Specific binding sites for the neuronal Na⁺ channel ligand [³H]STX were detected in crude membrane preparations incubated in either Na⁺-free Tris or Na⁺-replete Krebs-HEPES buffer. The magnitude of specific binding by 1 nM [³H]STX was significantly reduced with membranes isolated in Krebs-HEPES buffer compared with those incubated in Tris buffer (26.7 ± 4.6 and 83.5 ± 14.1 fmol/mg protein ± S.E.M. respectively; Fig. 1). The reduction in specific [³H]STX binding in a physiologically appropriate medium is consistent with the marked decrease in affinity of STX for neuronal Na⁺ channels in the presence of Na⁺ ions (Rhoden and Goldin, 1979).

View larger version (21K): [in this window] [in a new window]   Fig. 1. A, specific [3H]STX binding to crude myenteric neural membrane fractions. Data represent the mean ± S.E.M. of 40 replicate experiments with membranes incubated in Tris buffer from 27 pigs and 29 replicate experiments with membranes incubated in Krebs-HEPES buffer from 14 pigs. B, representative Western blots of GAP-43 immunoreactive protein bands from myenteric neural membranes isolated in either Tris or Krebs-HEPES buffer. Blot is representative of three experiments performed with membranes from three different pigs.

To confirm that membrane isolates incubated in the two buffer solutions were equally enriched in neural elements, immunoreactivity to the neural marker GAP-43 was quantified with membrane fractions by Western blotting (Coggins and Zwiers, 1991). Membranes isolated in both buffers yielded immunoreactive bands of approximately 48 kDa, the predicted molecular mass of GAP-43. These bands were absent in the presence of the blocking peptide, confirming the specificity of the anti-GAP-43 antiserum used. There was no significant difference in the relative density of GAP-43 immunoreactive bands with membranes incubated in Tris or Krebs-HEPES buffer (Fig. 1).

Saturation Analysis of Specific [³H]Diprenorphine Binding Sites in Myenteric Neural Membranes. The OPR antagonist [³H]DPN bound to a single specific binding site with high affinity in neural membranes incubated in either Tris or Krebs-HEPES buffer. The density of the [³H]DPN binding sites were significantly greater in crude membrane fractions isolated in Tris buffer than those isolated in Krebs-HEPES buffer (Fig. 2; Table 1). This difference in B_max was eliminated by an additional high speed (45,000g) centrifugation of the membrane fraction isolated in Krebs-HEPES buffer; the density of specific [³H]DPN binding sites in membranes reconstituted in Krebs-HEPES buffer was similar to that of membranes reconstituted in Tris (Fig. 2C; B_max = 106.4 ± 16.7 and 95.8 ± 16.3 fmol/mg protein, respectively); both binding site densities were not significantly different from the [³H]DPN binding density of membranes isolated in Tris buffer (Table 1). However, the density of specific [³H]STX binding sites in membrane fractions subjected to additional centrifugation did not change significantly (B_max = 26.7 ± 4.6 and 31.9 ± 7.3 fmol/mg protein before and after centrifugation, respectively).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Saturation analyses of high-affinity [3H]DPN binding in myenteric neural membranes incubated in either Tris (A) or Krebs-HEPES buffer (B). Specific (SB; ), nonspecific (NSB; ), and total (TB; ) [3H]DPN binding is shown. Ordinate indicates binding site density (Bmax) in femtomoles per milligram of protein, and abscissa indicates radioligand concentration. The bold line represents the results of a nonlinear regression analysis on SB data. The lighter solid and dashed lines represent the best fit of nonlinear and linear functions to TB and NSB data, respectively. The results of these regression analyses are presented in Table 1. Each point represents the mean ± S.E of 5 to 11 replicate experiments in membranes from 4 to 11 pigs. C, specific [3H]DPN binding in myenteric neuronal membranes isolated in Krebs-HEPES before centrifugation () or after additional centrifugation and reconstitution of membranes in either Tris () or Krebs-HEPES buffer (). Data shown represent the mean ± S.E.M. in the difference in radioligand binding observed in the presence and absence of 1 µM naloxone of four replicate experiments with membranes from four pigs.

Displacement of Specific [³H]Diprenorphine Binding in Myenteric Neural Membranes by Selective OPR Antagonists. We have reported previously that -opioid binding sites predominate in myenteric neural membranes from the porcine ileum, although -opioid binding sites are expressed as well (Townsend and Brown, 2002). To further assess the pharmacological identity of these OPR populations, the displacement of [³H]DPN binding by highly selective - and -OPR antagonists was examined. Naltrexonederived antagonists that selectively bind to either - or -OPRs displaced [³H]DPN to levels similar to that of 1 µM naloxone (naloxone displaced 65 ± 5 and 59 ± 5% of total [³H]DPN binding in Tris and Krebs-HEPES buffers, respectively). In myenteric membranes incubated in either buffer, the -OPR antagonist naltriben displaced [³H]DPN from two distinct binding sites (Fig. 3A; Table 2). The related -OPR antagonist BNTX also seemed to displace [³H]DPN from two sites in membranes incubated in Tris buffer, but these sites were not distinguishable in membranes incubated in Krebs-HEPES buffer (Fig. 3B; Table 2). Like BNTX, the naltrexonederived -OPR antagonists nor-BNI and GNTI failed to show selectivity for either of these binding sites, displacing [³H]DPN from two distinct sites in only one of the buffers examined (Fig. 4, A and B; Table 2).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Displacement of 1 nM [3H]DPN binding from myenteric neural membranes by NTB (A), BNTX (B), and TIPP (C). Radioligand displacement assays were performed with membranes incubated in either Tris () or Krebs-HEPES () buffer. The ordinate indicates the percentage of total [3H]DPN binding remaining relative to the amount determined in the absence of the unlabeled displacing ligand, and the abscissa indicates the log10 molar concentration of each displacing ligand. Bold dashed and solid lines indicate the results of the nonlinear regression of the displacement assays in Tris and Krebs-HEPES buffers, respectively. The results of these regression analyses are presented in Table 2. Lighter dashed and solid lines indicate the percentage of [3H]DPN binding remaining after incubation of membranes with 1 µM naloxone in Tris or Krebs-HEPES buffers, respectively. Each point represents the mean ± S.E. of three to nine replicate experiments with membranes from three to seven pigs.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Displacement of 1 nM [3H]DPN binding from myenteric neural membranes by nor-BNI (A) or GNTI (B). Assays were performed with membranes incubated in either Tris () or Krebs-HEPES () buffers. The ordinate indicates the percentage of total [3H]DPN binding remaining relative to the amount determined in the absence of unlabeled displacing ligands, and the abscissa indicates the log10 molar concentration of each displacing ligand. Bold dashed and solid lines indicate the results of the nonlinear regression of the displacement assays in Tris and Krebs-HEPES buffers, respectively. The results of these regression analyses are presented in Table 2. Lighter dashed and solid lines indicate the percentage of [3H]DPN binding remaining after incubation of membranes with 1 µM naloxone in Tris or Krebs-HEPES buffers, respectively. Each point represents the mean ± S.E. of three to six replicate experiments with membranes from three to six pigs.

In contrast, the peptidic -OPR antagonist TIPP displaced [³H]DPN binding from a single site, which was identical in both buffers. Furthermore, a significant amount of specific (i.e., naloxone-displaceable) [³H]DPN binding remained even in the presence of TIPP at very high concentrations (Fig. 3C; Table 2).

Displacement of Specific [³H]Diprenorphine Binding in Myenteric Neural Membranes by Selective OPR Agonists. The affinities of the -OPR agonist DPDPE and the -OPR agonist U-69,593 for specific [³H]DPN binding sites in myenteric neural membranes were significantly lower in Krebs-HEPES buffer than in Tris buffer (Fig. 5, C and D; Table 3). In contrast, the affinities of the -OPR agonists deltorphin II and SNC-80 were not significantly different between these buffer conditions (Fig. 5, A and B; Table 3). All agonists examined displaced only a portion of the specific [³H]DPN binding with high affinity. Relatively high (>30 µM) concentrations of the -OPR agonist U-69,593 displaced [³H]DPN from an additional specific binding site in membranes bathed in the Tris buffer. When data were fitted to a two site model, with the total displacement constrained to specific [³H]DPN binding (i.e., 65% of total binding in Tris buffer), high- and low-affinity sites were defined with respective K_i values of 3.4 nM (95% CI = 0.8-15.7 nM) and 18.0 µM (7.9-41.0 µM).

View larger version (27K): [in this window] [in a new window]   Fig. 5. Displacement of 1 nM [3H]DPN binding from myenteric neural membranes by [D-Ala2]deltorphin II (A), SNC-80 (B), DPDPE (C), or U-69,593 (D). Assays were performed with membranes incubated in either Tris () or Krebs-HEPES () buffers. The ordinate indicates the percentage of total [3H]DPN binding remaining relative to the amount determined in the absence of displacing, unlabeled ligand, and the abscissa indicates the log10 molar concentration of each displacing ligand. Bold dashed and solid lines indicate the results of the nonlinear regression of the displacement assays in Tris and Krebs-HEPES buffers, respectively. The results of these regression analyses are presented in Table 3. Lighter dashed and solid lines indicate the percentage of [3H]DPN binding remaining after incubation of membranes with 1 µM naloxone in Tris or Krebs-HEPES buffers, respectively. Each point represents the mean ± S.E. of three to five replicate experiments with membranes from three to five pigs.

Characterization of Residual [³H] Diprenorphine Binding after Saturation of - and -OPR Agonist-Displaceable [³H]Diprenorphine Binding Sites. The displacement of [³H]DPN by the -OPR agonist U-69,593 was examined in the presence of a saturating concentration of the -OPR agonist DPDPE in myenteric neural membranes. In membranes bathed in either Tris and Krebs-HEPES buffer containing 10 µM DPDPE, U-69,593 displaced [³H]DPN from a single binding site with affinities and at magnitudes similar to that measured in the absence of DPDPE (Fig. 6A; Table 4). Similarly, DPDPE displaced [³H]DPN binding from a single site in membranes bathed in either Tris and Krebs-HEPES buffer and its binding did not vary in the presence of 10 µM U-69,593 (Fig. 6B; Table 4).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Displacement of 1 nM [3H]DPN binding from myenteric neural membranes by saturating combinations of - and -OPR ligands. A, displacement of specific [3H]DPN binding by U-69,593 in the presence of 10 µM DPDPE. B, displacement of specific [3H]DPN binding by DPDPE in the presence of 10 µM U-69,593. C, displacement of specific [3H]DPN binding by nor-BNI in the presence of both 10 µM DPDPE and 10 µM U-69,593. Assays were performed with membranes incubated in either Tris () or Krebs-HEPES () buffers. The abscissa indicates the percentage of total [3H]DPN binding remaining relative to the amount determined in the absence of any unlabeled ligand. The abscissa indicates the log10 molar concentration of each displacing ligand. Bold dashed and solid lines indicate the results of the nonlinear regression of the radioligand displacement assays in membranes incubated in Tris and Krebs-HEPES buffer, respectively. The results of these regression analyses are presented in Table 4. Lighter dashed and solid lines indicate the percentage of [3H]DPN binding remaining after incubation of membranes with 1 µM naloxone in Tris or Krebs-HEPES, respectively. Each point represents the mean ± S.E. of three to five replicate experiments with membranes from two to four pigs.

View this table: [in this window] [in a new window]   TABLE 4 Binding parameters for the -opioid ligand DPDPE or the -opioid ligands U-69,593 and nor-BNI in displacing [3H]DPN from its specific binding sites in myenteric neural membranes DPDPE or U-69,593 was present at a saturating concentration of 10 µM. Results were obtained from nonlinear regression analyses of [3H]DPN displacement assays. Data are expressed as mean values (95% confidence interval) obtained in experiments with 3-5 replicate experiments, using membranes isolated from 2-4 pigs.

After saturation of both the DPDPE and U-69,593 binding sites, a small amount of specific [³H]DPN binding remained. In membranes incubated in Krebs-HEPES buffer, the -OPR antagonist nor-BNI displaced this remaining [³H]DPN binding with an affinity similar to that of the low-affinity binding site observed in the absence of agonist receptor occupancy (Fig. 6C; Table 4).

	Discussion

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Using a technique similar to that presently used in preparing myenteric neural membranes from the porcine small intestine, Allescher et al. (1989) established a crude membrane preparation of the myenteric plexus from the canine small intestine that was enriched in neuronal membranes (i.e., [³H]saxitoxin binding sites) but contained only of a relatively small amount of smooth muscle plasma membranes. The present isolation procedure yielded a similar distribution of neurally enriched membrane fractions from the porcine myenteric plexus (Townsend and Brown, 2002). In preparations of the myenteric plexus enriched in neural membranes from either the canine or porcine small intestine, specific binding sites for [³H]DPN were detected and binding of this radioligand could be displaced by the prototypic OPR antagonist naloxone. These results strongly suggest that OPRs are expressed in crude membrane fractions from the myenteric plexus. Although we cannot exclude the possibility that opioid binding sites are expressed on the membranes of contaminating intestinal smooth muscle cells as well, the lack of detectable [³H]DPN binding to purified canine smooth muscle membranes (Allescher et al., 1989) and absence of OPR immunoreactivity on intestinal smooth muscle cells in the porcine ileum (Poonyachoti et al., 2001) indicate that intestinal myocytes are not a significant source of OPR binding sites. Crude membrane fractions were used in these studies because they were highly enriched in both specific [³H]STX and [³H]DPN binding sites and it is possible that further purification steps might disrupt any noncovalent protein-protein interactions that may exist in the neural membranes. Because [³H]DPN is an OPR antagonist ligand, its affinity for specific binding sites and those of other OPR antagonists used in this study did not change significantly in the absence or presence of physiologically relevant concentrations of Na⁺ and other ions. However, membranes incubated in Krebs-HEPES buffer manifested lower specific [³H]DPN binding than those maintained in Na⁺-free Tris buffer. This difference in binding site density was eliminated in membranes subjected to an additional centrifugation step. This observation could be due to a disruption of noncovalent associations of nonmembrane proteins present in the fractions isolated in Krebs-HEPES buffer, which may be dependent on divalent cations present within this buffer. However, there was no similar alteration in the density of specific binding sites for [³H]STX in membranes subjected to additional centrifugation. Additional experiments will be needed to determine whether this phenomenon represents an artifact associated with the membrane isolation procedure or an unmasking of cryptic opioid binding sites.

Naltrexone-derived - and -OPR antagonists and naloxone maximally displaced [³H]DPN by a similar magnitude, indicating that this portion of [³H]DPN binding was to specific opioid sites. Previous immunocytochemical studies suggest that these sites are presynaptic because of the abundance of - and -OPR-like immunoreactivities in nerve fibers within the myenteric plexus and intestinal smooth muscle, and the cytoplasmic localization of these receptor immunoreactivities in myenteric neurons (Brown et al., 1998; Poonyachoti et al., 2001, 2002). The naltrexone-derived -OPR-selective antagonists BNTX and NTB preferentially bind to the -OPR with 83- and 1000-fold greater affinity than to the -OPR, respectively (Raynor et al., 1994). These ligands are structurally related to the -OPR antagonists GNTI and nor-BNI, which bind, respectively, to -OPRs with 390- and 2400-fold greater affinity than to -OPRs (Raynor et al., 1994; Jones and Portoghese, 2000). These antagonists displaced [³H]DPN from two distinct binding sites under some conditions. It is not clear whether these binding sites represent two distinct receptor entities or different isoforms of a single receptor. However, the -OPR antagonists displaced specific [³H]DPN binding with affinities similar to their affinities for recombinant -OPRs (Raynor et al., 1994; Clark et al., 1997), whereas the low-affinity site was characteristic of the binding to these ligands to the -OPR (Raynor et al., 1994) or to -/-OPR heterodimers (Jordan and Devi, 1999). Parallel results were obtained with the -OPR selective antagonists (Jones and Portoghese, 2000; Remmers et al., 1999). In contrast, the peptidic -OPR antagonist TIPP partially displaced [³H]DPN from a single specific binding site with an affinity similar to its affinity for recombinant -OPRs (Martin et al., 2002). Its apparent interaction with a single [³H]DPN binding site in myenteric neural membranes might be attributable to its relatively higher selectivity for -OPRs than the other -OPR antagonists (Nevin et al., 1993) or to the pharmacological characteristics of the low-affinity site that may only bind neutral antagonists, of which TIPP is not (Martin et al., 2002).

Both - and -OPR agonists displaced [³H]DPN from single high-affinity binding sites in myenteric neural membranes. As expected for agonists, the displacement curves of these ligands were shifted rightward in membranes incubated in Na⁺-replete Krebs-HEPES buffer relative to those determined in membranes bathed in Na⁺-free Tris. Agonist displacement, like that produced by TIPP, accounted for only about two-thirds of the specific [³H]DPN binding. Thus, a significant amount of specific [³H]DPN binding remained even in the presence of very high agonist concentrations. The extensive overlap of the predicted OPR binding domains for DPN and the other OPR ligands supports the hypothesis that these ligands interact competitively (Pogozheva et al., 1998; Filizola et al., 1999). Noncompetitive interactions between the displacing ligand and [³H]DPN may result in the partial displacement of [³H]DPN from its specific binding site, if the affinity of the receptor for the radioligand is significantly reduced. Alternatively, the reported association of OPRs offers the possibility that both competitive and noncompetitive ligand interactions occur through the interaction of these receptors. As with the case of -OPR antagonists, ligands acting as -OPR agonists displaced [³H]DPN with affinities similar to their affinities for recombinant -OPR (Clark et al., 1997). The order of affinities of these agonists in Krebs-HEPES buffer, i.e., SNC-80 > DPDPE > deltorphin II, differed from that determined in a functional assay of their actions in a porcine smooth muscle-myenteric plexus preparation in which the order of agonist potencies for inhibiting field-stimulated muscle contractions was deltorphin II > SNC-80 > DPDPE (Poonyachoti et al., 2001). This discrepancy between receptor affinities and potencies likely reflects differences in the efficacies of these three agonists at myenteric -OPRs. Indeed, although deltorphin II exhibited the lowest relative affinity in displacing [³H]DPN from specific binding sites, it produced the greatest inhibitory action (Poonyachoti et al., 2001). The -OPR agonist U-69,593 displaced [³H]DPN with a relatively lower affinity than reported previously for its binding to the cloned -OPR (Remmers et al., 1999). This lower affinity estimate for U-69,593 is consistent with a previous determination of its affinity based on a saturation binding analysis in porcine myenteric neural membranes (Townsend and Brown, 2002). The appearance of the additional U-69,593 binding site in membranes bathed in Tris buffer may represent the displacement of [³H]DPN by this ligand from a site different from the -OPR.

The observation that saturation of the -OPR binding site with DPDPE has little effect on the ability of U-69,593 to displace [³H]DPN and vice versa suggests that binding sites for these two ligands represent two independent receptor subpopulations. Furthermore, in the presence of saturating concentrations of DPDPE and U-69,593, it seems that nor-BNI displaces [³H]DPN from a third subpopulation of specific binding sites with relatively low affinity. From these data, we hypothesize that myenteric neurons in the porcine small intestine express both - and -OPRs as well as an additional receptor subpopulation capable of binding [³H]DPN that possesses some unique features. First, these latter receptors display low affinity for - or -OPR agonists. Second, the peptidic -OPR antagonist TIPP does not seem to bind these receptors even at very high concentrations. On the other hand, naltrexone-derived - and -OPR antagonists displace [³H]DPN from this receptor population with moderately high affinity. Recombinant -/-OPR heterodimers have many of these same properties (Jordan and Devi, 1999). Indeed, it is tempting to speculate that this small subpopulation of [³H]DPN binding sites represents -/-OPR heterodimers, especially when taken together with previous functional and immunohistochemical data from the porcine small intestine that were described above (see Introduction). Although the present study provides additional evidence in support of the hypothesis that -/-OPR heterodimers are expressed in myenteric neurons, additional investigations with porcine distal small intestine preparations may provide further details on the existence and pharmacological characteristics of OPR heterodimers, their endogenous ligands, and their role in the modulation of synaptic transmission in the enteric and central nervous systems.

	Acknowledgements

 
We acknowledge the able technical assistance of Melanie Townsend in the design or execution of some experiments.

	Footnotes

 
This study was funded in part by National Institutes of Health Grants RO1 DA-10200 (to D.R.B.) and R37 DA01533 (to P.S.P.). D.T. was a predoctoral trainee supported by National Institutes of Health/National Institute on Drug Abuse Training Grant T32 DA-07234.

DOI: 10.1124/jpet.103.058016.

ABBREVIATIONS: OPR, opioid receptor; nor-BNI, nor-binaltorphimine; DPN, diprenorphine; STX, saxitoxin; U-69,593, (+)-(5,7,8)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide; SNC-80, (+)-4-[(R)-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl-3-methoxybenzyl)-N,N-diethylbenzamide; DPDPE, [d-Pen²,d-Pen⁵]enkephalin; TIPP, Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH; NTB, naltriben; BNTX, 7-benzylidenenaltrexone; NTI, naltrindole; GNTI, 5'-guanidinonaltrindole; CI, confidence interval; GAP-43, growth-associated protein-43.

¹ Current address: Department of Physiology, University of Michigan, 1301 E. Catherine St., Ann Arbor, MI 48109-0622.

Address correspondence to: Dr. David R. Brown, Department of Veterinary Pathobiology, University of Minnesota, 1988 Fitch Ave., St. Paul, MN 55108-6010. E-mail: brown013{at}umn.edu

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