Predominance of -Opioid-Binding Sites in the Porcine Enteric Nervous System

The antidiarrheal and constipating actions of opioids are mediated in part by enteric neurons, which lie within the wall of the small intestine and colon, but the differential expression of specific, high-affinity opioid-binding sites in ganglionated plexuses within functionally distinct intestinal segments has not been examined. We determined the saturation binding characteristics under Na -free conditions of the nonselective opioid receptor (OPR) ligand [H][(5 ,7 )-17-(cyclopropylmethyl)-4,5-epoxy-18,19-dihydro-3-hydroxy-6-methoxy, -dimethyl-6,14-ethenomorphinan-7-methanol] (diprenorphine) and the respective -, -, and -OPR ligands [H]naltrindole, D-(5 ,7 ,8 )( )-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxoaspiro-(4,5)dec-8-yl]benzeneacetamide ([H]U-69,593), and [H][D-Ala,N-Me-Phe,Gly-ol]enkephalin (DAMGO) in neuronal membranes isolated from myenteric and submucosal plexuses of porcine small intestine and colon. Naloxone-displaceable [H]diprenorphine-binding sites (KD values ranging from 0.2–0.5 nM and Bmax 50–95 fmol/mg of protein) were found in both subregions from all gut segments examined. High-affinity [H]naltrindole sites (KD 60–140 pmol) were at highest densities (approximately 60 fmol/mg of protein) in submucosal plexus of the ileum and distal colon myenteric plexus and were at lowest densities (8–9 fmol/mg of protein) in the submucosal plexuses of cecum and distal colon. [H]U-69,593 sites (KD 0.3–4 nM) were present only in the myenteric plexuses of all segments examined, with highest densities in cecum and proximal colon (44–47 fmol/mg of protein). [H]DAMGO-binding sites were expressed at relatively low densities in the enteric plexuses of all gut regions. These results indicate that -OPRs predominate in the porcine enteric nervous system with a more circumscribed expression of and -OPRs. Opium has been employed in the treatment of diarrheal disease for centuries. Indeed, the opiate alkaloids morphine and codeine and the peripherally selective opioid loperamide continue to be among the most effective antidiarrheal agents in clinical use (Schiller, 1995). In addition, the endopeptidase inhibitor racecadotril has been found to effectively arrest acute diarrhea in children and adults by a naloxone-sensitive mechanism (Matheson and Noble, 2000). The gastroenterologic use of opioids has recently been extended to the alleviation of diarrhea and visceral pain in irritable bowel syndrome (Corazziari, 1999). In addition to their therapeutic effects, opioids produce severe constipation during their prolonged use in pain management (Mancini and Bruera, 1998). The actions of opioids are thought to stem from their ability to decrease both intestinal propulsion and mucosal anion secretion (De Luca and Coupar, 1996). The intestinal effects of opioids are mediated by opioid receptors (OPRs) expressed in the central nervous system and by neurons in the myenteric and submucosal ganglionated plexuses of the small intestine and colon. Stimulation of enteric OPRs by opioid agonists is associated with neuronal hyperpolarization or reduced neurotransmitter release due to the G protein-coupled activation of K channels or inhibition of N-type Ca channel gating in myenteric and submucosal neurons (Cherubini and North, 1985; Mihara and North, 1986; Surprenant et al., 1990). Both inhibitory and excitatory enteric neurons may express OPRs (De Luca and Coupar, 1996). Immunoreactivities for at least some of the cognate endogenous ligands for these receptors, dynorphin and the enkephalins, are expressed in neurons and nerve fibers in either the myenteric or submucosal plexuses along the length of the intestinal tract (Kromer, 1990). Despite the neurobiological and clinical significance of intestinal OPRs, there have been no studies designed to examine the regional distribution, densities, and pharmacological characteristics of OPRs in structurally and functionally distinct intestinal segments. In the rat intestine, for example, OPR mRNA expression has been determined by ribonuclease protection assays (Fickel et al., 1997) or reverse transcriptase-polymerase chain reactions (Wittert et al., 1996). The neuronal localization of OPR proteins has been examined also by immunohistochemical approaches (Bagnol et al., This study was funded in part by National Institutes of Health Grant R01 DA-10200. D.T. was a predoctoral trainee supported by National Institutes of Health/National Institute on Drug Abuse Training Grant T32 DA-07234. ABBREVIATIONS: OPR, opioid receptor; DAMGO, [D-Ala,N-Me-Phe,Gly-ol]-enkephalin; diprenorphine, (5 ,7 )-17-(cyclopropylmethyl)-4,5epoxy-18,19-dihydro-3-hydroxy-6-methoxy, -dimethyl-6,14-ethenomorphinan-7-methanol; NTI, naltrindole; STX, saxitoxin; U-69,593, ( )(5 ,7 , 8 )-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide; CI, confidence interval. 0022-3565/02/3003-900–909$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 300, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4340/964819 JPET 300:900–909, 2002 Printed in U.S.A. 900 at A PE T Jornals on M ay 0, 2017 jpet.asjournals.org D ow nladed from 1997). However, these studies do not provide information about the pharmacological characteristics and functional identity of enteric OPRs. Quantitative receptor autoradiography has been used in attempts to ascertain both the distribution and ligand specificity of opioid-binding sites in the rodent and porcine intestinal tract, but this approach has been limited by poor cellular resolution of binding sites and inadequate quantitation of OPR densities within the intestinal wall (Nishimura et al., 1986; James et al., 1987; Quito et al., 1991). Radioligand binding to sites in cell membrane homogenates remains the most direct and quantitative means for measuring the pharmacological characteristics and densities of OPRs in tissues. In the intestine, especially in neuronally enriched fractions, ligand-binding assays to OPRs require significant amounts of starting materials. This problem has been overcome by reducing regional specificity (Creese and Snyder, 1975), pooling membranes from several animals (Monferini et al., 1981), or selecting large animal species as tissue donors (Allescher et al., 1989). The latter approach has been limited thus far to a characterization of OPRs in the canine ileum through the displacement of the nonselective OPR ligand [H][(5 ,7 )-17-(cyclopropylmethyl)-4,5-epoxy18,19-dihydro-3-hydroxy-6-methoxy, -dimethyl-6,14-ethenomorphinan-7-methanol] (diprenorphine) by type-selective OPR ligands (Ahmad et al., 1989; Allescher et al., 1989). In this study, we addressed the hypothesis that multiple OPRs are expressed in the submucosal and myenteric plexuses within the small intestine and colon. The pig was used as a tissue donor because the porcine intestine is considered to be a homolog of the human intestine (Almond, 1996). In the porcine small intestine, -OPR immunoreactivity has been localized in myenteric and submucosal neurons, and opioid agonists inhibit neurogenic contractions of intestinal smooth muscle and mucosal ion transport (Quito and Brown, 1991; Brown et al., 1998; Poonyachoti et al., 2001a,b). Saturation analyses of high-affinity, specific opioid binding were performed using radiolabeled ligands with high selectivity for each OPR type, with neuronal membranes isolated from both the myenteric and submucosal plexuses along the length of the porcine intestine. Materials and Methods Radioligands, Drugs, and Reagents. The radioligands [H]saxitoxin (STX; 14.9 Ci/mmol) and [H]diprenorphine (70 Ci/ mmol) were obtained from Amersham Biosciences (Arlington Heights, IL); [H]naltrindole (NTI; 33 Ci/mmol) and [H]DAMGO (54.5 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA); and [H]U-69,593 (65 Ci/mmol) was obtained from both commercial sources. All radioligands were diluted to the desired concentration in 5 mM HCl and stored at 20°C until use. Naloxone, tetrodotoxin, and other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). 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) that were not fasted before sacrifice. Animals Fig. 1. A and C, specific binding of 1 nM [H]saxitoxin in membrane fractions isolated from ileal submucosal (A) and cecal myenteric (C) plexuses. Nonspecific binding was determined in the presence of 1 M tetrodotoxin. B and D, specific [H]diprenorphine binding in membrane fractions that were enriched in [H]saxitoxinbinding sites from ileal submucosal (B) and cecal myenteric (D) plexuses. Bars represent the specific binding mean S.E.M. obtained in experiments using 2 to 10 replicates, using membranes isolated from two to five pigs (saxitoxin binding), or 9 to 11 replicates, using membranes isolated from four to six pigs (diprenorphine binding). PNS, postnuclear supernatant; S, supernatant; P, pellet; and Mic, microsomal fraction. Opioid Receptors in the Enteric Nervous System 901 at A PE T Jornals on M ay 0, 2017 jpet.asjournals.org D ow nladed from were sedated with an intramuscular injection of tiletamine hydrochloride-zolazepam (Telazol, 8 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA), in combination with xylazine (8 mg/kg). The animals were subsequently euthanized by barbiturate overdose in accordance with approved University of Minnesota Animal Care Committee protocols. A midline laparotomy was performed to expose the intestine. Intestinal segments were collected as follows: jejunum, from the ligament of Trietz caudad for approximately 1.5 m; ileum/distal jejunum, from the ileocecal junction orad approximately 1.5 m; cecum, entire cecum; proximal colon, approximately 50 cm of the spiral (ascending) colon; and distal colon, from the rectum to approximately 50 cm orad. All segments were removed rapidly and placed in an ice-cold physiological salt solution (composition: 153 mM Na , 3 mM K , 139 mM Cl , 0.7 mM Mg , 1.5 mM Ca , 19.6 mM HCO3 , 0.29 mM HPO4 , 1.3 mM H2PO4 2 , and 11 mM D-glucose, pH 7.4). Subsequent tissue dissections were performed at 4°C. The mesenteric attachments were removed from all segments, which then were opened along the antimesenteric surface. The longitudinal and circular muscle layers were carefully separated from the underlying submucosa. These muscle layers were then diced into 5 5-mm cubes and stored at 70°C until neuronal membranes were isolated. After removal of smooth muscle layers, each intestinal segment was rotated to expose the mucosa. The antimesenteric lymphoid tissue was excised from the segments of distal jejunum/ileum; Peyer’s patches in the proximal jejunum were not excised, because they comprised only a small percentage of the overall submucosal tissue. The intestinal mucosa was removed by gently scraping the mucosal surface with a razor blade. The submucosa prepared in this manner was inspected by secondary immunofluorescence for the presence of both submucosal plexuses with a primary antibody directed toward the neuronal marker PGP 9.5. The submucosa contained the internal submucosal plexus; the outer submucosal plexus often adhered to the circular muscle layer and thus, was partially included in both the myenteric and submucosal preparations. Isolation of Neuronally Enriched Membranes. The isolation of neuronally enriched fractions was performed as described previously (Hildebrand et al., 1993). Submucosal membranes were isolated the same day on which they were harvested. Submucosal scrapings were diluted 1:10 in 50 mM Tris HCl (pH was adjusted to 7.4 with NaOH) and homogenized using a Polytron (Brinkmann Instruments, Westburg, NY; 25,000 rpm; three 8-s bursts). The homogenate was centrifuged at 800g for 10 min, and the resulting pellet, containing nuclei and extracellular debris, was discarded. The postnuclear supernatant (PNS) was then subjected to centrifugation at 4,000g for 10 min. The pellet (P1) resulting from this procedure was also discarded; the supernatant (S1) was centrifuged at 48,000g for 15 min. The final microsomal-synaptosomal pellet (P2) was resuspended in 50 mM Tris and stored at 70°C until use in radioligand binding assays. In some cases, additional pellet (P3), supernatant (S2 and S3), and microsomal (Mic1 and Mic2) fractions were examined in [H]STXor [H]diprenorphine binding assays. Myenteric neuronal membranes were isolated no more than 72 h after tissue collection using a similar technique (Kostka et al., 1987). Minced tissue was diluted in 50 mM Tris HCl and homogenized as described above. The homogenate was then centrifuged at 800g for 10 min. The resulting PNS was centrifuged at 2500g for 10 min to yield a P1 fraction, and a second supernatant (S1) was subjected to centrifugation at 10,000g for 10 min to obtain the P2 fraction. As in the case of the submucosal plexus fractions, additional pellet, supernatant, and microsomal fractions were prepared to examine their radioligand binding characteristics. Neuronal enrichment of myenteric and submucosal P2 fractions was confirmed by [H]STX binding at a radioligand concentration of 1 nM; nonspecific binding was determined by measuring [H]STX binding in the presence of 1 M tetrodotoxin. Protein concentrations were determined using the BCA protein assay (Pierce Chemical, Rockford, IL). Radioligand Binding Assays. Isolated membranes were thawed on the day of the experiment and diluted, with 50 mM Tris, to a concentration of approximately 500 g/ml. The actual protein concentration was determined from an aliquot of the diluted membrane used in the assay. Saturation analyses of OPR binding were performed using six concentrations of labeled ligand within the ranges indicated below. Nonspecific binding of OPR radioligands was determined by the binding of the labeled ligand in the presence of 1 M unlabeled naloxone. The nonselective OPR antagonist diprenorphine possesses nearly equal affinities for all three OPR types, and therefore, [H]diprenorphine (0.03–3 nM) was used to determine the density of total OPRs. Other ligands used in these studies included [H]NTI (0.003–0.3 nM), a selective -OPR antagonist; [H]DAMGO (0.03–10 nM), a highly selective -OPR agonist; and [H]U-69,593 (0.1–10 nM), a selective -OPR agonist. All radioligand binding experiments were performed in a final volume of 500 l with approximately 250 g of protein. Binding assays were conducted at room temperature for 60 min to insure that equilibrium was achieved. Incubations were terminated by rapid vacuum filtration through GF/B glass fiber filters (Brandel Inc., Gaithersburg, MD) using a 24-well Brandel cell harvester. Filters were washed twice with 4 ml of 50 mM Tris (pH 7.4). Filters were then submerged in Ecolite scintillation fluid (ICN Pharmaceuticals, Costa Mesa, CA). After a 12-h incubation period, the radioactivity of the samples was assessed by liquid scintillation spectroscopy. Data Analysis. Specific opioid binding was calculated as the difference between binding in the presence (nonspecific) and absence (total) of 1 M naloxone. The percentage of specific radioligand binding relative to total binding was also calculated. The integrity of each membrane preparation was determined by the density of both [H]STXand [H]diprenorphine-specific binding sites. Tissue preparations (approximately 23% of the total examined) in which 50% of total [H]STX or [H]diprenorphine binding was, respectively displaced by tetrodotoxin or naloxone were omitted from the data analysis. Our results suggest that this screening procedure is effective at removing persistently low outliers, with the assumption that the receptor proteins in these discarded tissues were extensively degraded during the isolation procedure. Individual data points deviating more than two standard deviations from the mean ( 5% of points) were also excluded from the analysis. Nonlinear regressions were used to determine the means and 95% confidence intervals (CI) for Bmax and KD of each radioligand in saturation analyses using the Prism 2.0 software program (GraphPad, San Diego, CA).