Rat Gastrointestinal Tract Research Articles

SAFETY ASSESSMENT OF GENETICALLY MODIFIED POTATO SPUNTA: DEGRADATION OF DNA IN GASTROINTESTINAL TRACT AND CARRYOVER TO RAT ORGANS

Diet groups for albino rats were (group 1) control basal, (group 2) control plus 30% freeze-dried nongenetically modified potatoes Spunta, (group 3) control plus 30% freeze-dried Spunta genetically modified potato (GMP) G2 and (group 4) control plus 30% freeze-dried GMP G3. After feeding ad libitum for 30 days, no statistical differences were found in food intake, daily body weight gain and feed efficiency. However, there was a small but significant difference in final body weight between the control (group 1) and experimental treatments (groups 2-4). DNA ingested in feed was partially resistant to the mechanical, chemical and enzymatic activities of the rat gastrointestinal (GI) tract (GIT) and was not completely degraded. Modified and nonmodified potato Spunta DNA survival was comparable during feed passage in the rat GIT. Modified constructs from GMP lines G2 and G3 fragment DNA were not detectable in tissue samples.

Distribution, transition, adhesion and release of insulin loaded nanoparticles in the gut of rats

The purpose of this work was to investigate distribution, transition, bioadhesion and release behaviors of insulin loaded pH-sensitive nanoparticles in the gut of rats, as well as the effects of viscosity agent on them. Insulin was labeled with fluorescein isothiocyanate (FITC). The FITC-insulin solution and FITC-insulin nanoparticle aqueous dispersions with or without hydropropylmethylcellulose (HPMC, 0.2%, 0.4%, or 0.8% (w/v)) were orally administered to rats, respectively. The amounts of FITC-insulin in both the lumen content and the intestinal mucosa were quantified by a spectrofluorimeter. The release profiles in the gut were plotted by the percentages of FITC-insulin released versus time. FITC-insulin nanoparticle aqueous dispersion showed similar stomach but lower intestine empty rates, and enhanced intestinal mucosa adhesion in comparison with FITC-insulin solution. Addition of the HPMC reduced the stomach and intestine empty rates, enhanced the adhesion of FITC-insulin to the intestine mucosa. The release of FITC-insulin from nanoparticles in the gut showed an S-shape profile, and addition of HPMC prolonged the release half-life from 0.77 to 1.51 h. It was concluded that the behaviors of pH-sensitive nanoparticles tested in gastrointestinal tract of rats and the addition of HPMC were favorable to the absorption of the drug loaded.

Genetic Modification of Somatic Gut Mucosa

Genetic Modification of Somatic Gut Mucosa

Epithelial cell proliferation is promoted by the histamine H 3 receptor agonist (R)-α-methylhistamine throughout the rat gastrointestinal tract

The temporal effect of (R)-α-methylhistamine on epithelial cell proliferation throughout the rat gastrointestinal tract was investigated. (R)-α-methylhistamine was administered at 100 mg/kg orally and the rats were sacrificed 1, 24, 48, 72 and 144 h later. All the animals received 5-bromo-2′-deoxyuridine, (BrdU), 200 mg/kg i.p., 2 h before sacrifice. Gastrointestinal tissue was processed for histology and immunohistochemistry. (R)-α-methylhistamine caused a progressive increase in mucosal thickness of gastric fundus, distal small intestine and distal colon. Statistically significant differences from control values were found between 48 and 72 h after (R)-α-methylhistamine. (R)-α-methylhistamine significantly increased the number of BrdU-positive cells in the gastric fundus and antrum, intermediate and distal small intestine and distal colon. Peak effects were observed between 1 and 24 h after (R)-α-methylhistamine administration. Proliferating cell number and mucosal thickness were comparable to those of control rats at 144 h. (R)-α-methylhistamine exerts a long lasting growth-promoting effect on the stomach, distal small intestine and distal colon. Present data support a role of histamine H 3 receptors in the normal regulation of cell cycle in epithelial tissue.

Release behaviour and biocompatibility of drug-loaded pH sensitive particles

The purpose of this work was to investigate the physical properties of drug-loaded poly(methacrylic acid-g-ethylene glycol) {P(MAA-g-EG)} particles, their biocompatibility with the gastrointestinal tract of rats and also the effects of these particles on the tight junctions of the rat intestinal epithelium. Model drugs such as diltiazem HCl, diclofenac Na, ciprofloxacin HCl and isoniazid were used in this study. P(MAA-g-EG) particles were prepared by free radical solution polymerization of methacrylic acid (MAA) and poly(ethylene glycol) (PEG). The loading efficiency of the model drugs in the particles and in vitro release profiles were investigated in pH 7.4 phosphate buffer and in gradually pH changing buffers (pH 1.2, 5.8, 6.8 and 7.4). The stability of free particles and drug-loaded particles was established by Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). In conclusion, P(MAA-g-EG) particles controlled the release rate of small molecular weight model drugs according to the pH of the medium. Stability of those particles loaded with drugs did not change in accelerated stability conditions. Histopathological results indicated that loading drugs to the particles prevented cell and tissue damage after 20 h. Free particles showed no change of tight junctions after 2 and 10 h. The results of TEM showed that increasing the amount of P(MAA-g-EG) particles from 100 to 385 mg clearly opened the tight junction, but with serious epithelial cell disruption.

Adaptive changes of the enterochromaffin and gastrin cells in the rat gastrointestinal tract following subtotal colectomy

Objective. Colectomized patients often have diarrhoea and increased gastric acid secretion. Although serotonin influences gastrointestinal (GI) motility and secretion, GI serotonin-producing enterochromaffin (EC) cells have not been investigated after colectomy, nor have the antral gastrin cells. The aim of this experimental study was to investigate the GI tract in rats 8 weeks after subtotal colectomy, with particular emphasis on the frequency and distribution of EC and gastrin cells. Material andmethods. Immunohistochemical techniques were used to identify the two endocrine cell types. Results. The colectomized animals had diarrhoea. Body-weight was lower and the small intestine shorter in the colectomized animals compared with sham-operated and untreated controls. In the two surgically treated groups, the antral mucosa was thinner and the small intestinal mucosa was thicker compared with that of the untreated rats, whereas the thickness of the rectum of the colectomized rats was increased compared with that of the control groups. In the colectomized animals, the number of EC cells was increased in the small intestine and rectum, whereas the numbers of both EC and gastrin cells were decreased in the antrum. Conclusions. The results indicate that colectomy exerts a significant influence on the GI mucosa and on the endocrine cell systems studied. An increased number of EC cells can result in alterations in motility and secretion, which may be important in the pathogenesis of the diarrhoea that often occurs after colectomy.

A new method for visualization of gut mucosal cells, describing the enterochromaffin cell in the rat gastrointestinal tract

Objective. Enterochromaffin (EC) cells in the gastrointestinal tract have an important function as regulators of secretion, motility and sensation. The EC cell has traditionally been described as bottle-shaped, with basally located stores of serotonin. Stimuli acting on the apical membrane trigger serotonin release, which in turn activates the subepithelial sensory nerve terminals. To better describe the appearance of EC cells, we developed a new method for visualization of mucosal cells. Material and methods. The stomach, small intestine and large intestine were excised from Sprague-Dawley rats and then fixed in formalin. The organs were everted and filled with pronase solution. Single cells and aggregates of formalin-fixed mucosal cells were collected by scraping the mucosa off the muscularis mucosa. EC cells were visualized by staining for immunoreactivity against serotonin. Results. EC cells with luminal extensions and very long (up to 80 µM) basally located axon-like extensions, sometimes connecting to neuron-like structures, were found. Other EC cells had no or only short and blunt basal extensions. Dividing serotonin-containing EC cells were also seen. Conclusions. These findings could make an important contribution towards furthering our understanding of EC cell function in gastrointestinal physiology. This new method can also readily be used to give better visualization of the morphology of other mucosal cells.

Effects of acute renal failure on the pharmacokinetics of DA‐7867, a new oxazolidinone, in rats

The pharmacokinetic parameters of DA-7867 were compared after intravenous and oral administration at a dose of 10 mg/kg to control rats and rats with acute renal failure induced by uranyl nitrate (rats with U-ARF). After intravenous administration in rats with U-ARF, the time-averaged total body clearance (Cl) was significantly faster (2.45 versus 0.932 ml/min/kg) than controls due to significantly faster nonrenal clearance (2.25 versus 0.855 ml/min/kg) in rats with U-ARF. The faster nonrenal clearance could be due to significantly greater gastrointestinal (including biliary) excretion; the amount of unchanged DA-7867 recovered from the entire gastrointestinal tract at 24 h was significantly greater (30.3% versus 9.38% of intravenous dose) in rats with U-ARF. In rats with U-ARF, the Vss was significantly larger (1420 ml/kg compared with 580 ml/kg), but this was not due to a difference in plasma protein binding; the values were comparable between the two groups of rats. After oral administration to rats with U-ARF, the total area under the plasma concentration-time from time zero to time infinity (AUC) of DA-7867 was significantly smaller than the controls (2560 microg min/ml versus 7440 microg min/ml), and this was not due mainly to a decrease in absorption from the gastrointestinal tract in rats with U-ARF.

Neostigmine, but not Metoclopramide, Abolishes Ethosuximide-Induced Functional Gastrointestinal Disturbances

Ethosuximide is a drug used for the treatment of absence seizures. Its prolonged application leads to gastrointestinal (GI) disturbances. The purpose of the present study was to determine the severity and nature of the disturbances, as well as the influence exerted upon them by neostigmine methylsulfate and metoclopramide. The drug-induced alterations, occurring in the rat GI tract, were registered by X-ray contrast examination. In vitro experiments were carried out on isolated GI smooth muscle (SM) preparations. The latter revealed that the drug hyperpolarized the SM tissues and inhibited their contractile activity. X-rays of ethosuximide-treated rats showed GI hypotonia, disturbed peristalsis and decreased evacuation activity. The inhibition of the GI functions was associated with hyperpolarization of SM and a reduction in Ca²⁺ influx, ensuring spontaneous contractile activity. The application of neostigmine methylsulfate significantly removed ethosuximide-induced functional GI disturbances in rats treated for 15 days with ethosuximide.

Mitogenic effects of both amidated and glycine-extended gastrin-releasing peptide in defunctioned and azoxymethane-treated rat colon in vivo

Although there is abundant evidence that gastrin-releasing peptide acts as a mitogen in various carcinoma cell lines, the effect of administration of gastrin-releasing peptide on the colorectal mucosa in vivo has not been reported. The aims of this study were to determine whether continuous infusion of gastrin-releasing peptide stimulated proliferation or accelerated carcinogenesis in the rat gastrointestinal tract and other organs. The possible requirement for C-terminal amidation for mitogenic activity in vivo was also investigated. Proliferation was measured in the colon by metaphase index and by immunostaining for the proliferation marker Ki-67, and in other tissues by immunostaining alone. Acceleration of colorectal carcinogenesis was assessed by counting aberrant crypt foci after treatment with the carcinogen azoxymethane. Defunctioning of the rectum reduced both the proliferative index and the crypt height of the rectal mucosa of untreated rats. Treatment with amidated or glycine-extended gastrin-releasing peptide for 4 weeks using implanted mini-osmotic pumps resulted in a two- to three-fold increase in proliferation, and an increase in crypt height, in the defunctioned rectal mucosa ( p < 0.001), with smaller but significant increases in the caecum and distal colon. No changes in proliferation were detected in lung, pancreas or gastric mucosa. The numbers of aberrant crypt foci in the mid-colon, distal colon and rectum following treatment with azoxymethane were also significantly increased by infusion with amidated or glycine-extended gastrin-releasing peptide. We conclude that administration of gastrin-releasing peptide to mature rats stimulates proliferation and accelerates carcinogenesis in the colorectal mucosa, and that C-terminal amidation is not essential for either effect. Gastrin-releasing peptides could thus potentially act as promoters of colorectal carcinogenesis.

Comparison of opioid receptor distributions in the rat ileum

The cellular expression patterns of μ-, δ- and κ-opioid receptors in the rat ileum were examined using fluorescence immunohistochemistry. Double-labelling was used to examine cellular receptor co-localisation as a pre-requisite for intracellular molecular interactions, such as heterodimerisation. Tissues were stained as whole-mount preparations. Strong, broadly distributed immunoreactivity (ir) was observed for each receptor in the myenteric and submucous plexuses. Although intracellular μ- and δ-ir patterns differed in ganglion neurons, μ/δ co-expression was extensive in these cells. μ/δ co-expression was also observed in interstitial cells, which were diffusely distributed in submucous plexus preparations but generally located adjacent to myenteric plexus structures. Punctate κ-ir was seen broadly in nerve fibres in both plexuses, suggesting localisation in varicosities. Neuronal μ/κ co-localisation was not apparent, although κ-ir fibres were often apposed against μ-ir cells. μ/κ co-localisation was detected in interstitial cells in submucous plexus preparations. Similarities in μ and δ expression patterns might reflect similar functional properties previously detected for these receptors. This study indicates that the rat gastrointestinal tract might provide a useful tool for the future study of molecular interactions between opioid receptor types.

Diamine oxidase, a plasma biomarker in rats to GI tract toxicity of oral fluorouracil anti-cancer drugs

Diamine oxidase (DAO; EC 1.4.3.6), which catabolizes a variety of substrates including histamine and diamines, is the degradative enzyme of the catabolic pathway of polyamines found in high activity in the mature upper villus cells of the rat intestinal mucosa [Luk, G.D., Bayless, T.M., Baylin, S.B., 1983. Plasma post-heparin diamine oxidase. Sensitive provocative test for quantitating length of acute intestinal mucosal injury in the rat. J. Clin. Invest. 71, 1308–1315; Wolvekamp, M.C.J., de Bruin, R.W.F., 1994. Diamine oxidase: an overview of historical, biochemical and functional aspects. Dig. Dis. 12, 2–14]. Rats were given 1-week repeated oral administration of anti-cancer drugs S-1, containing FT + CDHP + Oxo, and FCD, containing FT + CDHP, and the ameliorating effect of Oxo on the rat gastrointestinal (GI) tract toxicity from 5-FU was evaluated by measuring plasma DAO activity which is related to the enzyme located in the rat intestinal mucosa. Plasma DAO activity in the FCD-treated group was significantly less than that in the S-1-treated group while the jejunal mucosal area in the FCD group was significantly smaller than that in the S-1 group. In addition the histopathological findings in the FCD group showed villus atrophy in the jejunal mucosa which was not observed in the S-1 group. The degree of these findings correlated with the plasma DAO levels. Therefore, the protective effect of Oxo on 5-FU-induced GI tract toxicity was clarified by measuring plasma DAO activity in rats. In summary, DAO is a very sensitive plasma biomarker and will be useful for the quantitative evaluation of the small intestinal mucosal lesions induced by the anti-cancer drug, 5-FU, in rats.

Immunoelectron microscopic study of polyamines in the gastrointestinal tract of rat

Polyamines (PAs) are ubiquitous polycationic metabolites in the eukaryotic and prokaryotic cells and are believed to be intimately involved in the regulation of DNA, RNA, and protein biosynthesis. However, the subcellular localization of PAs has not yet been fully elucidated in a variety of cell types. In the present study, a pre-embedding indirect immunoperoxidase approach was used to define the fine structural localization of PAs in the gastrointestinal tract of rat, which was fixed with glutaraldehyde and the monoclonal antibody ASPM-29 specific for spermine (Spm) and spermidine (Spd). Examination by a transmission electron microscopy showed that the peroxidase end products were commonly and predominantly localized in the free and attached ribosomes of the rough endoplasmic reticulum (rER) in the active protein- or peptide-secreting cells, and in rapidly proliferating cells including the gastric chief cells, mucous neck cells, and intestinal crypt cells. The nuclei, mitochondria, and secretory vesicles were devoid of PAs. Of note is the new finding that PAs are also located even on the small number of ribosomes in the cytoplasm of the parietal cells and of the villus-tip cells, because these were the cell types that were found to be almost PA-negative at the light microscopic level. These results seem to be completely consistent with those recently obtained for rat neurons. Thus, the present study generalized the subcellular localization of PAs on the ribosomes, and demonstrated that PAs are one of the components of biologically active ribosomes, possibly in any type of cell, that are closely involved in the translation processes of protein biosynthesis.

Toxicokinetics of 14C‐endosulfan in male Sprague–Dawley rats following oral administration of single or repeated doses

Endosulfan (ES), an organochlorine (OC) insecticide that belongs to the cyclodiene group, is one of the most commonly used pesticides to control pests in vegetables, cotton, and fruits. The toxicokinetics of 14C-endosulfan following oral administration of a single dose of 5 mg/kg body weight was investigated in male Sprague-Dawley rats. Three rats were sacrificed 30 min, 1 h, 2 h, 4 h, and 8 h after dosing. 14C-endosulfan radioactivity was detected in all tissues at each time point. In a separate experiment urine and feces were collected for 96 h. The total radioactivity recovered in the excreta for 4 days was 106.8% +/- 26.2%, with fecal elimination the major route of elimination route (94.4% +/- 21.4%). The cumulative excretion in the urine for 4 days was 12.4% +/- 4.8%. Radioactivity 8 h after administration was highest in gastrointestinal (GI) tract tissue (20.28 +/- 16.35 mg ES eq./L) and lowest in muscle (0.18 +/- 0.06 mg ES eq./L). The toxicokinetic parameters obtained from 14C-endosulfan-derived radioactivity in blood were distribution half-life (T1/2 x) = 31 min and terminal elimination half-life (T1/2 y) = 193 h. Blood concentration reached its maximum (Cmax) of 0.36 +/- 0.08 mg ES eq./L 2 h after the oral dose. Endosulfan was rapidly absorbed into the GI tract in rats, with an absorption rate constant (ka) of 3.07 h(-1).

Expression and immunolocalization of aquaporin‐7 in rat gastrointestinal tract

In the gastrointestinal tract of mammals, water can either be secreted with digestive juices or absorbed by the small and large intestine. Transcellular water movement can be mediated by the transmembrane protein family of AQPs (aquaporins), as has also been recently identified in the gastrointestinal tract. However, the localization, expression and functioning of AQPs in the gastrointestinal tract have not been completely characterized. For the present study, we investigated: (1) the expression of AQP7 in some portions of rat gastrointestinal tract by semiquantitative reverse transcriptase-PCR and by immunoblotting and (2) the cellular and subcellular localization of AQP7 by immunohistochemistry. AQP7 mRNA and proteins were highly expressed in the small intestine, weakly in the caecum, colon and rectum and were absent in the stomach. Immunoblotting analysis using rat gastrointestinal tract membrane fractions showed two major bands corresponding to a molecular mass of approx. 34 and 40 kDa for the AQP7 protein. No bands were observed when the anti-AQP7 antibody was preadsorbed with the immunizing peptide. Immunohistochemistry revealed strong AQP7 labelling in the surface epithelial cells of duodenum, jejunum, ileum, caecum, colon and rectum, whereas weak or no labelling was observed in the crypt cells. The labelling was manifest particularly in the apical membrane but intracellular staining was also observed. The results indicate that AQP7 is present in the small and large intestine. The higher expression of AQP7 protein at the apical pole of the superficial epithelial cells suggests its involvement in rapid fluid movement through the villus epithelium.

Effects of cholecystokinin-receptor antagonists on Fos-like immunoreactivity stimulated by sulfated cholecystokinin-8 in neurons of the myenteric plexus and hindbrain of rats

To evaluate the role of cholecystokinin (CCK)-receptor antagonists in the activation of enteric and hindbrain neurons by sulfated CCK-8. 81 male Sprague-Dawley rats. Rats were allocated to 10 groups (5 to 22 rats/group). Each rat received 2 IP injections (15 minutes between injections). The first injection consisted of a specific CCK2-receptor (CCK2R) antagonist (L365,260; 150, 500, or 1,000 microg/kg), a specific CCK1-receptor (CCK1R) antagonist (devazepide; 150 microg/kg), or 1% dimethyl sulfoxide (DMSO [ie, vehicle]), and the second injection consisted of sulfated CCK-8 (10 microg/kg) or saline (0.9% NaCl) solution. Rats were anesthetized and perfused with 500 mL of Krebs saline solution, and the myenteric plexuses of the duodenum and jejunum were collected. Rats were then perfused with 500 mL of phosphate-buffered 4% formaldehyde solution; rats were then euthanatized, and the hindbrain of each was harvested. Tissues were stained by use of a diaminobenzidine reaction enhanced with nickel to reveal Fos-like immunoreactivity (Fos-LI), a marker of neuronal activation, in the aforementioned neurons. Sulfated CCK-8 significantly increased Fos-LI in the myenteric and hindbrain neurons, compared with values for the DMSO injections. All dosages of L365,260 failed to attenuate this increase; however, injection of devazepide attenuated the increase in Fos-LI. Analysis of the results of this study reveals that sulfated CCK-8 activates myenteric and hindbrain neurons of rats primarily through CCK1 R. It provides evidence that CCK2R are lacking or not functional in the gastrointestinal tract of rats.

Transmural gradient of leukocyte-endothelial interaction in the rat gastrointestinal tract

Gastrointestinal injury usually starts in the superficial mucosa. We investigated whether leukocyte-endothelial interactions were greater in the gastrointestinal mucosa than the submucosa and muscularis in control tissue and after upregulation of adhesion molecules with endotoxin and after chemical insult with nonsteroidal anti-inflammatory drugs. Inactin-anesthetized rats were given either endotoxin, flurbiprofen, or nitric oxide (NO)-flurbiprofen, after which ICAM-1 and P-selectin expression was measured with the dual-label antibody technique. Leukocyte-endothelial interactions in the different gastric layers were assessed after endotoxin using intravital microscopy. Endotoxin caused a two- to threefold increase in ICAM-1 expression in the stomach and duodenum. There was, however, a gradient in expression across the gut wall with the level of expression in the superficial mucosa (per g) being only 10-25% of that in the deeper layers in both control and endotoxin-treated animals. Constituitive expression of P-selectin in control animals was barely detectable. Endotoxin caused a modest increase in mucosal P-selectin but a very significant increase in the deeper layers. Flurbiprofen caused a slight upregulation of ICAM-1 in the gastric mucosa and duodenum, whereas NO-flurbiprofen had no affect on expression. Intravital microscopy revealed no adhesion and virtually no leukocyte rolling in the vessels of the gastric mucosa despite endotoxin treatment. There was, however, some adhesion and significant leukocyte rolling in the submucosa and muscularis. Thus the superficial gastric and duodenal mucosal microcirculations have a much lower density of ICAM-1 and P-selectin and less leukocyte-endothelial interactions than occurs in the deeper layers of the gut wall even during stimulated upregulation with endotoxin.

The Chemical Speciation of Uranium in Water Does Not Influence Its Absorption from the Gastrointestinal Tract of Rats

Studies of the chemical speciation of uranium in water can enhance the knowledge of the mechanisms of its absorption from the gastrointestinal tract and its storage in the body. They can also help to improve the dosimetric models recommended by the International Commission on Radiological Protection (ICRP). The aim of this work was to assess the influence of uranium speciation on its absorption from the gastrointestinal tract by using both computer speciation modeling and direct measurement of the fractional absorption in vivo in rats after ingestion of five different samples of contaminated water. Preliminary ex vivo studies with human saliva and gastric juice showed that 90% of uranium was recovered with the natural components of the fluid studied. The computer studies of uranium speciation among the electrolytes of these fluids showed that under the set conditions, the chemical species changed in a broadly similar manner under the influence of fluid composition and pH. In vivo studies in rats validated these observations by indicating an average fractional absorption of about 0.4% for each of five different water samples. It is concluded that the chemical form of uranium in the water ingested did not influence its absorption into the body.

Galanin receptors in the rat gastrointestinal tract

Galanin functions are mediated by three distinct G-protein-coupled receptors, galanin receptor 1 (GalR1), GalR2 and GalR3, which activate different intracellular signaling pathways. Here, we quantified mRNA levels of GalR1, GalR2 and GalR3 in the gastrointestinal tract using real time RT-PCR. GalR1 and GalR2 mRNAs were detected in all segments with the highest levels in the large intestine and stomach, respectively. GalR3 mRNA levels were quite low and mostly confined to the colon. We also investigated the effect of galanin 1-16, which has high affinity for GalR1 and GalR2 and low affinity for GalR3 on depolarization-evoked Ca2+ increases in rat cultured myenteric neurons using Ca2+-imaging. Intracellular Ca2+ changes in myenteric neurons were monitored using the Ca2+ sensitive dye, fluo-4, and confocal microscopy. Galanin 1-16 (1 microM) markedly inhibited the K+-evoked Ca2+ increases in myenteric neurons. In summary, the differential distribution of GalRs supports the hypothesis that the complex effects of galanin in the gastrointestinal tract result from the activation of multiple receptor subtypes. Furthermore, this study confirms the presence of functional GalRs and suggests that galanin modulates transmitter release from myenteric neurons through inhibition of voltage-dependent calcium channels involving a G(i/o)-coupled GalR.

Bombesin receptor subtype-3 is expressed by the enteric nervous system and by interstitial cells of Cajal in the rat gastrointestinal tract

Bombesin receptor subtype-3 (BRS-3), a G-protein-coupled orphan receptor, shares 47% and 55% homology with other known mammalian bombesin receptors. Despite the molecular characterization of BRS-3, its function remains unclear as a consequence of its low affinity for bombesin and the absence of an identified natural ligand. Although the other mammalian bombesin receptors are widely distributed in the gut and central nervous system, expression of BRS-3 in the gastrointestinal tract has not been previously described. We report the expression of BRS-3 mRNA and protein in the tunica muscularis of the rat gastrointestinal tract. The mRNA expression pattern was studied by reverse transcription followed by quantitative polymerase chain reaction. To identify the cellular sites of expression of BRS-3, we performed immunocytochemistry by using a N-terminus-specific affinity-purified antiserum. BRS-3 was found to be widely expressed in the rat gastrointestinal tract at both the mRNA and protein levels. BRS-3-like immunoreactivity (BRS-3-LI) was localized in neurons of the myenteric and submucosal ganglia, being primarily concentrated near the neuronal plasma membrane, and in fibers distributed in the longitudinal and circular muscle layers. In addition, BRS-3-LI was observed in the cell bodies and processes of c-kit+ interstitial cells of Cajal. These data have functional applications for the effects mediated by the activation of BRS-3 on gut motility through distinct neuronal and non-neuronal pathways.