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Background & Aims: The μ opioid receptor (μOR) undergoes rapid endocytosis after acute stimulation with opioids and most opiates, but not with morphine. We investigated whether prolonged activation of μOR affects morphine's ability to induce receptor endocytosis in enteric neurons., Methods: We compared the effects of morphine, a poor μOR-internalizing opiate, and (D-Ala2,MePhe4,Gly-ol5) enkephalin (DAMGO), a potent μOR-internalizing agonist, on μOR trafficking in enteric neurons and on the expression of dynamin and β-arrestin immunoreactivity in the ileum of guinea pigs rendered tolerant by chronic administration of morphine., Results: Morphine (100 μmol/L) strongly induced endocytosis of μOR in tolerant but not naive neurons (55.7% ± 9.3% vs 24.2% ± 7.3%; P < .001) whereas DAMGO (10 μmol/L) strongly induced internalization of μOR in neurons from tolerant and naive animals (63.6% ± 8.4% and 66.5% ± 3.6%). Morphine- or DAMGO-induced μOR endocytosis resulted from direct interactions between the ligand and the μOR because endocytosis was not affected by tetrodotoxin, a blocker of endogenous neurotransmitter release. Ligand-induced μOR internalization was inhibited by pretreatment with the dynamin inhibitor, dynasore. Chronic morphine administration resulted in a significant increase and translocation of dynamin immunoreactivity from the intracellular pool to the plasma membrane, but did not affect β-arrestin immunoreactivity., Conclusions: Chronic activation of μORs increases the ability of morphine to induce μOR endocytosis in enteric neurons, which depends on the level and cellular localization of dynamin, a regulatory protein that has an important role in receptor-mediated signal transduction in cells., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Release of 5-hydroxytryptamine (5-HT) from mucosal enterochromaffin cells and activation of 5-HT(3) receptors (5-HT(3)Rs) on neurons in the gut wall is important in the response of the gut to the luminal environment. Intestinal inflammation is associated with increased levels of mucosal 5-HT. The aims of the study were to determine the following: (1) if 5-HT(3)R undergoes ligand-induced internalization in myenteric neurons, and (2) the effect of long-term increase of mucosal 5-HT on 5-HT(3)Rs., Methods: Acute effects of exogenous 5-HT or endogenous release of 5-HT by luminal glucose on cellular localization of 5-HT(3)Rs was determined by immunohistochemistry and confocal microscopy. Treatment with the serotonin re-uptake inhibitor, fluoxetine, for 6 days (20 mg/kg daily orally) was used to increase mucosal 5-HT chronically in rats. Net ileal fluid movement was measured in anesthetized rats by the weight change of a 2.5% agarose cylinder., Results: Acute increases in 5-HT induced by exogenous or endogenous 5-HT decreased 5-HT(3)R immunoreactivity at the neuronal cell membrane by 70% and 60%, respectively. Chronic fluoxetine treatment increased mucosal levels of 5-HT and decreased membrane 5-HT(3)R immunoreactivity by 27%. Net fluid absorption was decreased by a 5-HT(3)R agonist or by luminal glucose; this was attenuated 88% and 99%, respectively, by fluoxetine treatment., Conclusions: Long-term increase in 5-HT in the intestinal mucosa results in increased 5-HT(3)R internalization in myenteric neurons. Chronic changes in mucosal 5-HT may alter gastrointestinal secretory and motor function via ongoing loss of receptor from neuronal membrane, causing a mismatch between luminal content and absorption.

Background & Aims: The neurokinin 1 receptors (NK(1)Rs) and substance P (SP) have been implicated in the stress and/or pain pathways involved in chronic pain conditions. Here we examined the participation of NK(1)Rs in sustained visceral hyperalgesia observed in rats exposed to chronic psychological stress., Methods: Male Wistar rats were exposed to daily 1-hour water avoidance stress (WA) or sham WA for 10 consecutive days. We tested intraperitoneal or intrathecal injection of the NK(1)R antagonist SR140333 on the visceromotor reflex to colorectal distention in both groups at day 11. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry were used to assess the expression of NK(1)Rs and/or SP in samples of colon, spinal cord, and dorsal root ganglia., Results: Both intraperitoneal and intrathecal SR140333 injection diminished the enhanced visceromotor reflex to colorectal distention at day 11 in stressed rats but did not affect the response in control animals. Real-time polymerase chain reaction and Western blotting demonstrated stress-induced up-regulation of spinal NK(1)Rs. Immunohistochemistry showed an increased number of NK(1)R-expressing neurons in the laminae I of the dorsal horn in stressed rats. The expression of NK(1)Rs was decreased in colon from stressed rats compared with control. The expression of SP gene precursor in dorsal root ganglia was unchanged in stressed rats compared with controls., Conclusions: Stress-induced increased NK(1)R expression on spinal neurons and the inhibitory effect of intrathecal NK(1)R antagonist on visceral hyperalgesia support the key contribution of spinal NK(1)Rs in the molecular pathways involved in the maintenance of visceral hyperalgesia observed after chronic WA.

Background & Aims: The 5-hydroxytryptamine 7 (5-HT7) receptors mediate intestinal smooth muscle relaxation. In this study, we evaluated the expression of 5-HT7 receptors in the guinea pig ileum and their role in peristalsis and accommodation of the circular muscle., Methods: We used immunohistochemistry and confocal microscopy with whole tissue and cultured myenteric neurons. Peristalsis was induced by delivering a solution into the oral end of an isolated ileal segment. The effect of the selective 5-HT7 receptor antagonist SB-269970 (100 nmol/L) on peristaltic activity was evaluated at 30, 60, and 90 minutes and compared with control., Results: 5-HT7 receptor immunoreactivity was localized to numerous myenteric neurons, a few submucosal neurons, and a few smooth muscle cells of the ileum. In enteric cultured neurons, 5-HT7 receptor immunoreactivity was observed in subpopulations of after hyperpolarizing neurons and descending neurons as identified by neuron-specific nuclear protein or calbindin and neuronal nitric oxide synthase or vasoactive intestinal peptide antibodies, respectively. SB-269970 significantly increased the threshold pressure by 33.3% +/- 2.2% (P < .001) and by 27.2% +/- 1.6% (P < .05) at 60 and 90 minutes, respectively, without modifying the threshold volume. The accommodation significantly decreased by 27.5% both at 60 and 90 minutes (P < .05)., Conclusions: Our results indicate that endogenous 5-HT is involved in the modulation of circular muscle accommodation during the preparatory phase of peristalsis via the activation of 5-HT7 receptors expressed by neurons in addition to smooth muscle cells. Overstimulation of these receptors leading to an exaggerated accommodation of circular muscle might contribute to abdominal symptoms in functional bowel disorders.

Background & Aims: We tested the hypothesis that N-methyl-D-aspartate (NMDA) receptors mediate surgery-induced opioid release in enteric neurons., Methods: We used mu opioid receptor (muOR) internalization as a measure of opioid release with immunohistochemistry and confocal microscopy. MuOR internalization was quantified in enteric neurons from nondenervated and denervated ileal segments of guinea pig after abdominal laparotomy with and without pretreatment with NMDA-receptor antagonists acting at different recognition sites (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,b] cyclohepten-5,10-imine (MK-801) or (D) 2-amino-5-phosphopenoic acid (AP-5) at .5, 1 mg/kg; 8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazinol [4,5-]quinoline-5-oxide choline (MRZ 2/576) or 8-chloro-1,4-dioxo-1,2,3,4-tetrahydropyridazinol [4,5-]quinoline choline salt (MRZ 2/596) at .3, 1 mg/kg, or with an antagonist for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (1, 3 mg/kg). To determine whether NMDA stimulation induces opioid release, (1) ilea were exposed to NMDA (100 micromol/L) and D-serine (10 micromol/L) with or without the antagonist MK-801 or AP-5 (50 micromol/L); and (2) neuromuscular preparations of the ileum were stimulated electrically (20 Hz, 20 min) with or without MK-801 or AP-5 (50 micromol/L)., Results: MuOR endocytosis induced by abdominal laparotomy was inhibited significantly by NMDA-receptor antagonists in nondenervated and denervated ileal segments, but not by the AMPA-receptor antagonist. MuOR endocytosis in neurons exposed to NMDA or electrical stimulation was prevented by NMDA-R antagonists., Conclusions: Abdominal laparotomy evokes local release of glutamate that results in endogenous opioid release through the activation of peripheral NMDA receptors. This suggests an interaction between the glutamatergic and opioid systems in response to the noxious and perhaps mechanosensory stimulation of surgery.

Background & Aims: Functional effects mediated via the 5-hydroxytryptamine3 receptor (5-HT3R) can be elicited from both extrinsic and intrinsic neurons innervating the gastrointestinal (GI) tract. Clinically, 5-HT3 antagonists are important in the treatment of emesis and have been used for the treatment of symptoms in functional bowel disease. The aim of the present study was to elucidate the cellular sites of 5-HT3R expression in the rat GI tract using immunohistochemistry., Methods: Immunohistochemistry was performed in fixed cryostat sections and whole mounts of stomach and intestine of fasted rats, using an affinity-purified antibody directed to a 19-amino acid sequence of the cytoplasmic loop of the 5-HT3R., Results: 5-HT3R immunoreactivity was localized to numerous neurons of the myenteric and submucosal plexus, concentrated primarily near the neuronal plasma membrane, and to fibers in the circular and longitudinal muscles, submucosa, and mucosa. 5-HT3R immunoreactivity was also expressed by interstitial cells of Cajal and a few endocrine cells. Numerous 5-HT3R-positive myenteric neurons were cholinergic, and few neurons coexpressed VIP or SP immunoreactivity. Fibers immunoreactive for 5-HT3R in the duodenal but not ileal mucosa were markedly reduced by subdiaphragmatic vagotomy or chemical denervation of vagal afferents., Conclusions: These findings indicate that 5-HT3Rs are expressed by distinct cells in the GI tract, including functionally distinct classes of neurons, interstitial cells of Cajal, and endocrine cells. The effects of serotonin mediated by 5-HT3Rs involve the activation of neuronal and nonneuronal pathways.

Background & Aims: In the intestine, tachykinins regulate motility by participating in neuromuscular and neuro-neuronal transmission. The aim of this study was to test the hypothesis that colonic propulsion is regulated by an interplay between tachykinergic and cholinergic transmission., Methods: Propulsion was elicited by intraluminal distention of a thin rubber balloon, which traveled from the oral to the anal end of guinea pig isolated distal colon segments. The overall contribution of endogenous tachykinins to colonic propulsion was examined by blocking NK1, NK2, and NK3 receptors simultaneously., Results: NK2-receptor blockade by MEN 11420 inhibited propulsion, whereas blockade of NK(1) by SR 140333 or of NK3 receptors by SR 142801 had minor effects on motility. Blockade of muscarinic or nicotinic receptors by hyoscine or hexamethonium decelerated peristalsis up to propulsion arrest. In the presence of partial muscarinic receptor blockade, the NK1-receptor antagonist SR 140333 and the NK2-receptor antagonist MEN 11420 markedly inhibited propulsion. Propulsion was also inhibited by the NK3-receptor antagonist SR 142801 in the presence of partial nicotinic receptor blockade. The simultaneous administration of the 3 tachykinin antagonists inhibited propulsion by 50%., Conclusions: This study demonstrates the existence of an interplay between tachykinergic and cholinergic pathways during peristalsis and the importance of endogenous tachykinins acting at multiple receptor sites in the control of colonic propulsion.

Background & Aims: Two distinct receptors, cholecystokinin (CCK)-A and CCK-B, mediate CCK effects in the digestive system. The aim of this study was to elucidate the cellular sites of expression of CCK-A receptor in the rat stomach and small intestine., Methods: We developed and characterized an antibody to the N-terminal region (LDQPQPSKEWQSA) of rat CCK-A receptor and used it for localization studies with immunohistochemistry., Results: Specificity of the antiserum was demonstrated by (1) detection of a broad band at 85-95 kilodaltons in Western blots of membranes from CCK-A receptor CHO-transfected cells; (2) cell surface staining of CCK-A receptor-transfected cells, (3) translocation of CCK-A receptor immunostaining in CCK-A receptor-transfected cells after exposure to CCK; and (4) abolition of tissue immunostaining by preadsorbtion of the antibody with the peptide used for immunization. CCK-A receptor immunoreactivity was localized to myenteric neurons and to fibers in the muscle and mucosa. In the stomach, myenteric neurons and mucosal fibers were abundant. Many CCK-A receptor myenteric neurons contained the inhibitory transmitter vasoactive intestinal polypeptide, and some were immunoreactive for the excitatory transmitter substance P. Subdiaphragmatic vagotomy reduced the density of CCK-A receptor fibers in the gastric mucosa by approximately 50%, whereas celiac/superior mesenteric ganglionectomy had no detectable effect on fiber density., Conclusions: CCK-A receptor is expressed in functionally distinct neurons of the gastrointestinal tract. CCK-A receptor may mediate reflexes stimulated by CCK through the release of other transmitters from neurons bearing the receptor.

Background & Aims: Transforming growth factor alpha (TGF-alpha), a member of the epidermal growth factor family, has been proposed to mediate protection against mucosal injury and promote healing of the gastrointestinal mucosa. TGF-alpha acts via a plasma membrane receptor, which is distributed throughout the digestive system with the highest density in epithelia. The aim of this study was to investigate the pattern of TGF-alpha binding sites in the normal and inflamed rabbit colon., Methods: The immune complex/formalin model of acute colitis and tissue section receptor autoradiography were used. Inflammation was characterized by cellular infiltration, edema, and necrosis. TGF-alpha binding relative density was determined by densitometry on film autoradiograms., Results: The normal colon had a low to moderate density of specific TGF-alpha binding sites in the mucosa and external muscle. TGF-alpha binding density was significantly increased in the mucosa at 4 hours and remained higher than normal for up to 48 hours. The density of binding sites in the mucosa and the inflammatory index returned to near normal values at 96 hours, when colitis had subsided., Conclusions: The increase in TGF-alpha binding in the mucosa during experimental colitis supports the hypothesis that members of the epidermal growth factor family play a role in inflammation, perhaps acting as mediators of mucosal protection and repair.

The possibility that chronic uremia renders the gastric mucosa more susceptible to acid injury was investigated. A rat model of chronic renal failure was induced by subtotal nephrectomy. [H+] back-diffusion across the mucosa, following intragastric perfusion of 0.15N HCl or 15% ethanol in 0.15N HCl, was significantly greater in uremic than in sham-operated rats. Gastric mucous gel thickness and transmural potential difference were significantly lower in rats with renal insufficiency. Furthermore, a significantly greater acidification rate of the surface epithelial cells was found in uremic rats than in sham-operated rats during superfusion with pH 1.7 buffer. Intragastric administration of acidified ethanol or aspirin solutions markedly increased gastric mucosal blood flow (68% and 89% respectively) in the sham-operated group producing mild injury, in contrast to uremic rats, where a lesser increase in mucosal blood flow (7% and 14% respectively) was associated with more pronounced mucosal injury. It was concluded that enhanced susceptibility to acid injury in uremia is due to a reduction of function of pre-epithelial, epithelial, and postepithelial elements of the gastric mucosal barrier.

Transforming growth factor alpha (TGF-alpha) interacts with the same plasma membrane receptor as epidermal growth factor and is likely to play a role in proliferative and trophic processes of gastrointestinal tissues. The distribution of receptor binding sites for TGF-alpha was examined in the canine gastrointestinal tract (distal esophagus, stomach fundus, descending duodenum, jejunum, ileum, transverse colon) by tissue section autoradiography. 125I-TGF-alpha yielded a labeling pattern comparable to that of 125I-epidermal growth factor. Specific binding sites were particularly abundant in the mucosa in each region, with the highest concentration in the esophagus, colon, and stomach, as assessed by computer assisted densitometry. The density of binding sites was moderate in the stomach muscularis mucosae, low in the external muscle layer, and very low to undetectable in the submucosa throughout the gastrointestinal tract. In most cases, the greatest density within the individual regions was detected in the area characterized by the highest proliferative rate. Lymphoid aggregates were not labeled. In conclusion, TGF-alpha receptor binding sites are present throughout the gastrointestinal tract with differential patterns in the various regions; they are principally distributed to the mucosa and predominantly located to proliferative cell areas. These results are consistent with a role of this factor in regional regulation of proliferation and differentiation in the gut.

The sensory neuropeptides, substance P and calcitonin gene-related peptide, have been implicated in inflammatory reactions in several tissues. An immune-complex model of colitis was used in rabbits to determine the colonic content (nmol/g protein) of immunoreactive substance P and calcitonin gene-related peptide at various times after induction of inflammation to assess changes in these neuropeptides during the inflammatory response. Calcitonin gene-related peptide content was decreased by 66% 4 hours after induction of inflammation and reached a maximum of 80% at 48 hours. The substance P content was decreased at 8 hours, with a maximum decrease of 64% at 48 hours. Substance P decrease was detected in the muscle layer. The amounts of substance P in the mucosal/submucosal layer extracts were too low to allow accurate measurements. Calcitonin gene-related peptide decreased both in the muscle and the mucosal-submucosal layers. Immunohistochemical analysis showed that calcitonin gene-related peptide and substance P innervation patterns were comparable in normal and inflamed colon, even though there appeared to be a decrease in density and intensity of the staining, particularly for calcitonin gene-related peptide at 48 hours. The early decrease of calcitonin gene-related peptide and substance P during the time course of colitis might be due to release from nerve terminals of the gut during the inflammatory response. The profound changes in colonic calcitonin gene-related peptide and substance P content during colitis may have important implications during inflammation and subsequent tissue repair and may also lead to disturbances in gut motility.

The distribution and characterization of calcitonin gene-related peptide immunoreactivity in the digestive system of normal, capsaicin-treated, and littermate control rats were studied by radioimmunoassay, chromatography, and immunohistochemistry. The highest concentrations of calcitonin gene-related peptide immunoreactivity were found in the stomach (45 +/- 2.8 pmol/g wet wt, nonsecretory region; 38.7 +/- 4.4 pmol/g wet wt, secretory region) and rectum (30.9 +/- 1.6 pmol/g wet wt). Significant amounts of peptide were also found in the other regions of the gut and in the pancreas. Neonatal treatment with capsaicin, which causes a permanent degeneration of most of the small-diameter sensory neurons, reduced calcitonin gene-related peptide content by greater than 95% in the esophagus and stomach, by 60% in the pancreas, and by less than 50% in the intestine, when compared with littermate controls. Separation of extracts from the gut, pancreas, and brain by chromatography gave major peaks corresponding to the predicted rat calcitonin gene-related peptide and small unidentified peaks, which presumably arise from metabolism of the peptide. Immunohistochemical studies demonstrated that in the esophagus and stomach, calcitonin gene-related peptide immunoreactivity is restricted to nerve fibers, whereas in the intestine it is localized in both nerve fibers and enteric ganglion cells. In capsaicin-treated rats there was a virtually complete elimination of calcitonin gene-related peptide immunoreactive fibers innervating the esophagus and stomach, whereas in the small and large intestine there was a dramatic reduction and often a complete elimination of those associated with blood vessels and a slighter reduction of the nonvascular immunoreactive fibers. The results of this study indicate that calcitonin gene-related peptide immunoreactive nerve fibers innervating the rat digestive system originate from both intrinsic (enteric) and extrinsic (presumably sensory) sources and that both the intrinsic and extrinsic components appear to contain a substance that corresponds to the predicted calcitonin gene-related peptide.

Calcitonin gene-related peptide-like immunoreactivity has been localized in endocrinelike or paracrinelike cells and nerve fibers of the rat pancreas. Immunoreactive cells with short and thick, elongated processes were mainly distributed at the periphery of the islets of Langerhans and occasionally disseminated among the acini. Positive varicose fibers were observed in both the endocrine and exocrine parenchyma as well as around blood vessels. Treatment with the small-diameter sensory fiber neurotoxin capsaicin resulted in virtually complete elimination of calcitonin gene-related peptide immunoreactive fibers innervating the parenchyma and blood vessels, suggesting an extrinsic, sensory origin of these fibers. The sympathetic neurotoxin 6-hydroxydopamine did not affect the immunoreactive staining. The results of this study support the possibility that a distinct cell population within the endocrine pancreas of the rat contains calcitonin gene-related peptide, and that this peptide plays both an endocrine/paracrine role, as well as a modulator role in the regulation of pancreatic function.

The cellular localization of substance P/neurokinin A-encoding preprotachykinin mRNAs in the rat enteric nervous system was studied by means of in situ hybridization histochemistry using 35S- or 3H-labeled single-stranded ribonucleic acid (RNA) probes which recognize all three preprotachykinin mRNA species, alpha, beta, and gamma. Substance P/neurokinin A-encoding mRNAs are expressed in neurons within the myenteric plexus of the esophagus and stomach, being more numerous in the latter, and in ganglion cells distributed to both the myenteric and submucosal plexuses of the intestine. Specificity of the hybridization was demonstrated by the lack of specific signal above background in sections incubated with a sense RNA probe or pretreated with ribonuclease A before hybridization. Ribonucleic acid blot hybridization analysis of RNA extracts from both the muscle layer-myenteric plexus and submucosal layer preparations of the duodenum demonstrated a single band of hybridization at 1.3 kb. Solution hybridization-nuclease protection assays showed multiple preprotachykinin-encoding transcripts in these RNA extracts, with an abundance level of gamma-mRNA greater than beta-mRNA much greater than alpha-mRNA, which is similar to that observed in the rat brain. Our results indicate that the preprotachykinin gene encoding the tachykinin peptides, substance P and neurokinin A, is transcribed in a population of enteric neurons that have a regional distribution comparable to the previously described tachykinin-like immunoreactive neurons, suggesting that specific mRNAs and the posttranslationally processed peptides are localized in the same structures.

We studied the effect of bombesin (9 ng/kg X min for 30 min by intravenous infusion) on serum immunoreactive trypsin in healthy subjects and in chronic pancreatitis patients. Bombesin administration caused a marked and significant increase of serum immunoreactive trypsin concentration in healthy subjects. The increase occurred in the first 15 min after the beginning of bombesin infusion and persisted for the duration of the study (2 h). In patients with chronic pancreatitis, the increase was much less pronounced. In these patients, the integrated immunoreactive trypsin response to bombesin was significantly correlated with bicarbonate, lipase, and chymotrypsin outputs into the duodenum. The response of serum immunoreactive trypsin to bombesin stimulation seems to vary according to the degree of pancreatic exocrine dysfunction and to reflect the functional capacity of acinar cell mass.