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• W2021959823 abstract "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: 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. The enterochromaffin (EC) cells of the enteric mucosa are the main source of 5-hydroxytryptamine (5-HT) in the body.1Erspamer V. Occurrence of indolealkylamines in nature.in: Erspamer V. Handbook of experimental pharmacology 5-hydroxytryptamine and related indolealkylamines. Springer-Verlag, New York1966: 132-181Google Scholar, 2Gershon M.D. Review article roles played by 5-hydroxytryptamine in the physiology of the bowel.Aliment Pharmacol Ther. 1999; 13: 15-30PubMed Google Scholar They respond to mechanical and chemical stimulation by releasing 5-HT into the gut wall,3Bülbring E. Lin R.C.Y. The effect of intraluminal application of 5-hydroxytryptamine and 5-hydroxytryptophan on peristalsis, the local production of 5-hydroxytryptamine and its release in relation to intraluminal pressure and propulsive activity.J Physiol (Lond). 1958; 140: 381-407Google Scholar, 4Bülbring E. Crema A. The release of 5-hydroxytryptamine in relation to pressure exerted on the intestinal mucosa.J Physiol (Lond). 1959; 146: 18-28Google Scholar, 5Kim M. Cooke H.J. Javed N.H. Carey H.V. Christofi F. Raybould H.E. D-glucose releases 5-hydroxytryptamine from human BON cells as a model of enterochromaffin cells.Gastroenterology. 2001; 121: 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 6Raybould H.E. Glatzle J. Robin C. Meyer J.H. Phan T. Wong H. Sternini C. Expression of 5-HT3 receptors by extrinsic duodenal afferents contribute to intestinal inhibition of gastric emptying.Am J Physiol. 2003; 284: G367-G372Crossref PubMed Scopus (149) Google Scholar, 7Fukumoto S. Tatewaki M. Yamada T. Fujimiya M. Mantyh C. Voss M. Eubanks S. Harris M. Pappas T.N. Takahashi T. Short-chain fatty acids stimulate colonic transit via intraluminal 5-HT release in rats.Am J Physiol. 2003; 284: R1269-R1276Crossref PubMed Google Scholar where 5-HT exerts neurocrine/paracrine functions and stimulates the nerve endings of neurons with after hyperpolarizing (AH)-type electrophysiologic behavior and multipolar Dogiel type II morphology (AH neurons), initiating peristaltic activity.8Grider J.R. Foxx-Orenstein A.E. Jin J.G. 5-hydroxytryptamine4 receptor agonists initiate the peristaltic reflex in human, rat, and guinea pig intestine.Gastroenterology. 1998; 115: 370-380Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar Small amounts of 5-HT also are synthesized and stored by a class of descending interneurons, which represent about 2% of all myenteric neurons and project anally, partly to inhibitory myenteric and submucosal neurons.9Costa M. Brookes S.J. Steele P.A. Gibbins I. Burcher E. Kandiah C.J. Neurochemical classification of myenteric neurons in the guinea-pig ileum.Neuroscience. 1996; 75: 949-967Crossref PubMed Scopus (446) Google Scholar, 10Furness J.B. Types of neurons in the enteric nervous system.J Auton Nerv Syst. 2000; 81: 87-96Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar In the gut, 5-HT activates different receptor subtypes including 5-HT1, 5-HT2, 5-HT3, 5-HT4, and 5-HT7 receptors expressed by neurons, smooth muscle cells, and enterocytes.11De Ponti F. Tonini M. Irritable bowel syndrome new agents targeting serotonin receptor subtypes.Drugs. 2001; 61: 317-332Crossref PubMed Scopus (154) Google Scholar, 12De Ponti F. Pharmacology of serotonin what a clinician should know.Gut. 2004; 53: 1520-1535Crossref PubMed Scopus (114) Google Scholar After mucosal stroking, 5-HT released by EC cells binds to receptors located on AH neurons, including 5-HT4/5-HT1P receptors. Indeed, mucosal application of selective 5-HT4 receptor agonists triggers peristalsis in human, rat, and guinea pig intestine,8Grider J.R. Foxx-Orenstein A.E. Jin J.G. 5-hydroxytryptamine4 receptor agonists initiate the peristaltic reflex in human, rat, and guinea pig intestine.Gastroenterology. 1998; 115: 370-380Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 13Gershon M.D. Serotonin and its implication for the management of irritable bowel syndrome.Rev Gastroenterol Disord. 2003; 3: S25-S34PubMed Google Scholar whereas intraluminal administration of a 5-HT3 receptor antagonist blocks the facilitatory effect of intraluminally applied 5-HT on peristalsis in the guinea pig isolated ileum.14Tuladhar B.R. Kaisar M. Naylor R.J. Evidence for a 5-HT3 receptor involvement in the facilitation of peristalsis on mucosal application of 5-HT in the guinea pig isolated ileum.Br J Pharmacol. 1997; 122: 1174-1178Crossref PubMed Scopus (54) Google Scholar The latter evidence suggests that 5-HT3 receptors also could be present on AH neurons. 5-HT7 receptors, previously classified as 5-HT1–like receptors,15Saxena P.R. De Vries P. Villalon C.M. 5-HT1-like receptors a time to bid goodbye.Trends Pharmacol Sci. 1998; 19: 311-316Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar are the most recently discovered members of the 5-HT receptor family.16Eglen R.M. Jasper J.R. Chang D.J. Martin G.R. The 5-HT7 receptor orphan found.Trends Pharmacol Sci. 1997; 18: 104-107Abstract Full Text PDF PubMed Scopus (167) Google Scholar They are coupled positively to adenylyl cyclase,17Bard J.A. Zgombick J. Adham N. Vaysse P. Branchek T.A. Weinshank R.L. Cloning of a novel human serotonin receptor (5-HT7) positively linked to adenylate cyclase.J Biol Chem. 1993; 268: 23422-23426Abstract Full Text PDF PubMed Google Scholar they have been cloned from several species,18Plassat J.L. Amlaiky N. Hen R. Molecular cloning of a mammalian serotonin receptor that activates adenylate cyclase.Mol Pharmacol. 1993; 44: 229-236PubMed Google Scholar, 19Tsou A.P. Kosaka A. Bach C. Zuppan P. Yee C. Tom L. Alvarez R. Ramsey S. Bonhaus D.W. Stefanich E. Cloning and expression of a 5-hydroxytryptamine7 receptor positively coupled to adenylyl cyclase.J Neurochem. 1994; 63: 456-464Crossref PubMed Scopus (177) Google Scholar, 20Bhalla P. Saxena P.R. Sharma H.S. Molecular cloning and tissue distribution of mRNA encoding porcine 5-HT7 receptor and its comparison with the structure of other species.Mol Cell Biochem. 2002; 238: 81-88Crossref PubMed Scopus (33) Google Scholar and have a receptor-binding profile consistent across species. 5-HT7 receptor messenger RNA has been reported in the central nervous system and periphery.17Bard J.A. Zgombick J. Adham N. Vaysse P. Branchek T.A. Weinshank R.L. Cloning of a novel human serotonin receptor (5-HT7) positively linked to adenylate cyclase.J Biol Chem. 1993; 268: 23422-23426Abstract Full Text PDF PubMed Google Scholar, 21Shen Y. Monsma Jr, F.J. Metcalf M.A. Jose P.A. Hamblin M.W. Sibley D.R. Molecular cloning and expression of a 5-hydroxytryptamine7 serotonin receptor subtype.J Biol Chem. 1993; 268: 18200-18204Abstract Full Text PDF PubMed Google Scholar, 22Ruat M. Traiffort E. Leurs R. Tardivel-Lacombe J. Diaz J. Arrang J.M. Schwartz J.C. Molecular cloning, characterization, and localization of a high-affinity serotonin receptor (5-HT7) activating cAMP formation.Proc Natl Acad Sci U S A. 1993; 90: 8547-8551Crossref PubMed Scopus (564) Google Scholar, 23To Z.P. Bonhaus D.W. Eglen R.M. Jakeman L.B. Characterization and distribution of putative 5-HT7 receptors in guinea-pig brain.Br J Pharmacol. 1995; 115: 107-116Crossref PubMed Scopus (232) Google Scholar In the gut, it has been reported that 5-HT induces muscle relaxation by a direct effect on 5-HT7 receptors on smooth muscle cells in the guinea pig ileum,24Carter D. Champney M. Hwang B. Eglen R.M. Characterization of a postjunctional 5-HT receptor mediating relaxation of guinea-pig isolated ileum.Eur J Pharmacol. 1995; 280: 243-250Crossref PubMed Scopus (90) Google Scholar canine stomach,25Janssen P. Prins N.H. Meulemans A.L. Lefebvre R.A. Pharmacological characterization of the 5-HT receptors mediating contraction and relaxation of canine isolated proximal stomach smooth muscle.Br J Pharmacol. 2002; 136: 321-329Crossref PubMed Scopus (41) Google Scholar and human colon.26Prins N.H. Briejer M.R. Van Bergen P.J. Akkermans L.M. Schuurkes J.A. Evidence for 5-HT7 receptors mediating relaxation of human colonic circular smooth muscle.Br J Pharmacol. 1999; 128: 849-852Crossref PubMed Scopus (88) Google Scholar However, it is not known whether 5-HT also affects muscle relaxation indirectly through the activation of neuronal 5-HT7 receptors. Our aims were to determine whether 5-HT7 receptors are expressed by enteric neurons using immunohistochemistry and whether endogenous 5-HT modulates peristalsis by activating these receptors using a selective 5-HT7 receptor antagonist, SB-269970,27Lovell P.J. Bromidge S.M. Dabbs S. Duckworth D.M. Forbes I.T. Jennings A.J. King F.D. Middlemiss D.N. Rahman S.K. Saunders D.V. Collin L.L. Hagan J.J. Riley G.J. Thomas D.R. A novel, potent, and selective 5-HT7 antagonist (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)pyrrolidine-1-sulfonyl) phenol (SB-269970).J Med Chem. 2000; 43: 342-345Crossref PubMed Scopus (239) Google Scholar in intact isolated segments of guinea pig ileum. Male albino Harlan Porcellus guinea pigs (Hartley; 250–450 g; G. Bettinardi; Momo, Novara, Italy, and Harlan Labs, San Diego, CA) were used. Animal care and handling were in accordance with the European Union Directive 86/609 and National Institutes of Health recommendations for the humane use of animals. Experimental procedures were reviewed and approved by the Animal Use Committee of the University of Pavia and the Animal Committees at UCLA. For the functional experiments and the preparations of cultured myenteric neurons, animals were killed by stunning followed by severing of the carotid arteries and the spinal cord. For the morphologic experiments with whole mounts and cryostat sections, animals were anesthetized deeply with intraperitoneal sodium pentobarbital (Nembutal, 100 mg/kg; Abbott Laboratories, Chicago, IL). The number of animals used was kept to the minimum necessary for a meaningful interpretation of data. The distal ileum was dissected, opened up along the mesentery, pinned flat on wax, and fixed in 4% paraformaldehyde in .1 mol/L phosphate-buffered solution, pH 7.4 (PB) for 2 hours at room temperature and then stored in PB with .1% g sodium azide at 4°C before processing for immunohistochemistry as whole mounts or placed in 25% sucrose in PB at 4°C for cryoprotection. Tissues were cut at 12-μm thickness using a cryostat perpendicularly to the lumen and collected onto gelatin-coated slides.28Pham T. Guerrini S. Wong H. Reeve Jr, J. Sternini C. Distribution of galanin receptor 1 immunoreactivity in the rat stomach and small intestine.J Comp Neurol. 2002; 450: 292-302Crossref PubMed Scopus (48) Google Scholar, 29Ho A. Lievore A. Patierno S. Kohlmeier S.E. Tonini M. Sternini C. Neurochemically distinct classes of myenteric neurons express the m-opioid receptor in the guinea pig ileum.J Comp Neurol. 2003; 458: 404-411Crossref PubMed Scopus (31) Google Scholar Fixed whole mounts or cryostat sections of the guinea pig ileum were washed in PB, incubated in 10% normal donkey serum for 1 hour at room temperature to minimize the background, processed for immunofluorescence using rabbit polyclonal 5-HT7 receptor antibody (1:100; Sigma Chemical, St. Louis, MO) for 3 days (whole-mounts) or overnight (cryostat sections) at 4°C, followed by 2 hours of incubation at room temperature with affinity-purified donkey anti-rabbit immunoglobulin (Ig)G coupled with Alexa-fluorescein isothiocyanate (Jackson Immunolabs, West Grove, PA; 1:1000) and coverslipped with 90% glycerol containing 2% potassium iodide in .1 mol/L PB. Primary and secondary antibodies were diluted in .5% Triton X-100 (Sigma Chemical) in .1 mol/L PB. Tissues were examined with a Zeiss Axioplan 2 research microscope for fluorescence with an axiocam color digital camera fluorescence microscope (Carl Zeiss Inc., Thornwood, NY), equipped with fluorescein isothiocyanate and Red-X cubes and with confocal microscopy (see later). Myenteric neuron cultures were isolated from the guinea pig small intestine according to established protocols.30Van den Berghe P. Tack J. Coulie B. Andrioli A. Bellon E. Janssens J. Synaptic transmission induces transient Ca2+ concentration changes in cultured myenteric neurones.Neurogastroenterol Motil. 2000; 12: 117-124Crossref PubMed Scopus (33) Google Scholar, 31Jessen K.R. Saffrey M.J. Burnstock G. The enteric nervous system in tissue culture I. Cell types and their interactions in explants of the myenteric and submucous plexuses from guinea pig, rabbit and rat.Brain Res. 1983; 262: 17-35Crossref PubMed Scopus (95) Google Scholar, 32Hanani M. Xia Y. Wood J. Myenteric ganglia from the adult guinea-pig small intestine in tissue culture.Neurogastroenterol Motil. 1994; 6: 103-118Crossref PubMed Scopus (38) Google Scholar, 33De Giorgio R. Bovara M. Barbara G. Canossa M. Sarnelli G. De Ponti F. Stanghellini V. Tonini M. Cappello S. Pagnotta E. Nobile-Orazio E. Corinaldesi R. Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility.Gastroenterology. 2003; 125: 70-79Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Briefly, the longitudinal muscle and myenteric plexus were stripped from the circular muscle and mucosa and digested in an enzymatic solution containing 1 mg/mL protease and 1.25 mg/mL collagenase (both from Sigma Chemical). After 30 minutes of incubation at 37°C, the suspension was placed on ice and centrifuged at 200g. Ganglia were picked and plated in culture dishes, where they adhered to the cover-glass bottom. After about 7–10 days of plating, cultured neurons developed network-like structures closely resembling ganglionated plexuses. The culture medium was changed every 2 days and consisted of Medium 199 enriched with 10% fetal calf serum and 50 ng/mL of nerve growth factor 7s. Antibiotics (penicillin, 100 IU/mL; streptomycin, 100 μg/mL; gentamicin, 50 μg/mL) were added to the medium; the glucose concentration was 30 mmol/L. All products were obtained from Gibco BRL Laboratories (Gaithersburg, MD). The culture chambers were kept in an incubator at 37°C and were gassed continuously with a mixture of 95% O2 and 5% CO2. Cultured myenteric neurons were fixed in 4% paraformaldehyde solution in .1 mol/L phosphate-buffered saline (PB, pH 7.4) (Gibco BRL Laboratories) for 30 minutes at 4°C and washed with PBS. Fixed cells were washed for 10 minutes at room temperature with buffer containing 3% bovine serum albumin (Sigma Chemical) in a .1 mol/L PB solution with .1% Triton X-100 and then incubated for 30 minutes at room temperature in PBS–bovine serum albumin 3%. Fixed cells were incubated for 1 hour at 37°C with rabbit polyclonal 5-HT7 receptor antibody (1:100), washed (3 times in PB, 5 min each), and incubated for 1 hour at 37°C with either Alexa488- or Alexa633-conjugated goat anti-rabbit IgG antibody (1:500). For double-labeling immunohistochemistry, cells were incubated with a mixture containing rabbit polyclonal 5-HT7 receptor antibody and either mouse monoclonal antibody to human neuron-specific enolase (NSE, 1:500; DakoCytomation, Glostrup, Denmark), a neuronal marker; to calcium-binding protein (Calbindin D-28k, 1:1000; Swant, Bellinzona, Switzerland) or to neuron-specific nuclear protein (NeuN, 1:100; Chemicon International, Temecula, CA), markers for intrinsic sensory neurons34Chiocchetti R. Poole D.P. Kimura H. Aimi Y. Robbins H.L. Castelucci P. Furness J.B. Evidence that two forms of choline acetyltransferase are differentially expressed in subclasses of enteric neurons.Cell Tissue Res. 2003; 311: 11-22Crossref PubMed Scopus (86) Google Scholar, 35Brody K.M. Costa M. Brookes S.J.H. NeuN immunoreactivity marks primary afferent neurons in the guinea-pig submucous plexus.Proc Aust Neurosci Soc. 2002; 13: 105Google Scholar; to vasoactive intestinal peptide (VIP; 1:400; Acris Antibodies GmbH, Hiddenhausen, Germany); or to neuronal nitric oxide synthase (nNOS; 1:100, Alexis Biochemicals Vinci, Firenze, Italy), markers for descending neurons. Subsequently, neurons were washed (3 times in PB, 5 min each) and incubated for 1 hour at 37°C with a solution of either Alexa488- or Alexa633-conjugated goat anti-rabbit IgG antibody (1:500) and Alexa633- or Alexa488-conjugated goat anti-mouse IgG antibody (1:500). All antibodies were diluted in PB–bovine serum albumin 3%. All the secondary antibodies were obtained from Molecular Probes Inc. (Eugene, OR). Coverslips were mounted on glass slides using a drop of Mowiol (Calbiochem, Darmstadt, Germany). Appropriate controls for the double-labeling technique were performed to determine that the primary antibodies did not cross-react when mixed together and that the secondary antibodies recognized the appropriate antigen-antibody complexes.33De Giorgio R. Bovara M. Barbara G. Canossa M. Sarnelli G. De Ponti F. Stanghellini V. Tonini M. Cappello S. Pagnotta E. Nobile-Orazio E. Corinaldesi R. Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility.Gastroenterology. 2003; 125: 70-79Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar For quantitative purposes, first a number of myenteric neurons expressing NSE, NeuN, Calbindin D-28k, nNOS, or VIP was evaluated in microscopic fields; then the proportion of NSE, NeuN, Calbindin D-28k, nNOS, or VIP neurons also immunoreactive for 5-HT7 receptor was calculated and expressed as a percentage. Second, a number of cells expressing 5-HT7 receptors was evaluated; then the proportion of 5-HT neurons also expressing NSE, NeuN, Calbindin D-28k, nNOS, or VIP was calculated and expressed as a percentage. At least 5 randomly nonoverlapping selected fields from 5 dishes were counted blindly for each double-labeling experiment. Cultured myenteric neurons were analyzed with confocal laser scanning microscopy (Leica TCS-SP system mounted on a Leica DMIRBE inverted microscope, Wetzlar, Germany). An Ar/Vis laser at 500/530 nm and 650/720 nm was used to excite Alexa488 and Alexa633 fluorescence, respectively. Whole-mount preparations and cryostat sections were analyzed with a Zeiss 510 Meta laser scanning confocal microscope equipped with both HeCd and argon lasers and attached to a Zeiss Axioplan 2 microscope (Carl Zeiss Inc., Thornwood, NY). Optical sections (.5 μm) were recorded using a 63× oil-immersion objective or a 100× Plan Apo 1.4 objective. Images were processed and labeled using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA). Portions of the ileum about 10 cm proximal to the ileocecal junction were excised, flushed of luminal contents, and mounted in an organ bath containing standard Tyrode solution kept at 37°C and oxygenated with a mixture of 95% O2 and 5% CO2. Peristalsis was studied with a constant intraluminal perfusion system as detailed previously.36Tonini M. Frigo G. Lecchini S. D’Angelo L. Crema A. Hyoscine-resistant peristalsis in guinea-pig ileum.Eur J Pharmacol. 1981; 71: 375-381Crossref PubMed Scopus (62) Google Scholar, 37Sternini C. Anselmi L. Guerrini S. Cervio E. Pham T. Balestra B. Vicini R. Baiardi P. D’Agostino G.L. Tonini M. Role of galanin receptor 1 in peristaltic activity in the guinea pig ileum.Neuroscience. 2004; 125: 103-112Crossref PubMed Scopus (24) Google Scholar Briefly, ileal segments (approximately 7–8 cm in length) were secured horizontally in an organ bath containing 100 mL of Tyrode solution. Peristalsis was elicited by delivering Tyrode solution into the oral end of the intestinal lumen; the infusion rate was .75 mL · min−1. Longitudinal muscle contraction was recorded with an isotonic transducer (load, 1 g). Two transducers measured aboral pressure; 1 recorded at high sensitivity and 1 recorded at low sensitivity. Each peristaltic wave caused the expulsion of fluid from the aboral end of the intestine via a 1-way valve. The volume expelled at the end of each wave of contraction was collected and measured. The expelled fluid activated a photocell system that switched off the infusion pump. The arrest of the inflow, controlled by a timer, was maintained for 10 seconds so as not to force the contracted organ during propulsion and to allow a period of rest between 2 peristaltic waves. Two phases of peristalsis can be identified: the preparatory phase, when the intestine is being filled with fluid, causing the longitudinal muscle to contract, and the emptying phase, when the circular muscle contracts and forces fluid from the aboral end.38Kosterlitz H.W. Lees G.M. Pharmacological analysis of intrinsic intestinal reflexes.Pharmacol Rev. 1964; 16: 301-339PubMed Google Scholar The following parameters were measured: (1) longitudinal muscle contraction during the preparatory phase of peristalsis, (2) peak pressure (ie, maximum ejection pressure) that reflects the anally propagated wave of contraction of the circular muscle responsible for the clearance of the intraluminal content, (3) threshold pressure, (4) residual pressure, (5) threshold volume, (6) residual volume, and (7) compliance. The threshold pressure is the pressure at the beginning of peristalsis, whereas the residual pressure corresponds to the intraluminal pressure after completion of peristalsis. The threshold volume required to trigger the emptying phase was measured as the volume infused into the intestine plus the residual volume remaining at the end of the previous peristaltic wave. The residual volume, which remained in the intestine after a peristaltic wave, was estimated by deducting the expelled volume from that infused into the lumen during the preparatory (filling) phase. The accommodation of the circular muscle during the preparatory phase (change in volume/change in pressure) was defined as the change in intraluminal pressure in response to a given change in intraluminal volume. This value reflects the resistance of the intestinal wall to infused fluid. It can be calculated from the inverse of the slope of the preparatory phase of the intraluminal pressure trace39Waterman S.A. Costa M. Tonini M. Modulation of peristalsis in the guinea-pig isolated small intestine by exogenous and endogenous opioids.Br J Pharmacol. 1992; 106: 1004-1010Crossref PubMed Scopus (58) Google Scholar as follows: average compliance = (threshold volume − residual volume)/(threshold pressure − residual pressure) = ΔV/ΔP, where threshold volume − residual volume corresponds to the volume infused during the preparatory phase. Peristaltic activity was studied in 10-minute cycles; each cycle of peristaltic activity was followed by at least 10 minutes of rest. After a stable control cycle, 100 nmol/L of SB-269970 (ie, a concentration 100 times as high as its inhibition constant value)27Lovell P.J. Bromidge S.M. Dabbs S. Duckworth D.M. Forbes I.T. Jennings A.J. King F.D. Middlemiss D.N. Rahman S.K. Saunders D.V. Collin L.L. Hagan J.J. Riley G.J. Thomas D.R. A novel, potent, and selective 5-HT7 antagonist (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)pyrrolidine-1-sulfonyl) phenol (SB-269970).J Med Chem. 2000; 43: 342-345Crossref PubMed Scopus (239) Google Scholar was administered and its effect on peristaltic activity was evaluated after 30, 60, and 90 minutes of contact and compared with control. Signals were recorded as described earlier using a PowerLab data acquisition system (Analogue Digital Instruments, Crowborough, UK) and analyzed using PowerLab Chart v4.1.1 software. The composition of the Tyrode solution was as follows (in mmol/L): NaCl 136.9, KCl 2.7, CaCl2 1.8, MgCl2 · 6H2O 1.04, NaHCO3 11.9, NaH2PO4 · H2O .4, and glucose 5.5. (R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol hydrochloride (SB-269970) (Sigma Chemical) was dissolved in deionized water. Data are expressed as means ± SEM of experiments using n guinea pigs. Each measurement was made with a separate segment of tissue. The effect of SB-269970 was measured 30, 60, and 90 minutes after administration. One-way analysis of variance for repeated measures was used for statistical analysis. A P value of less than .05 was considered significant. In whole-mount preparations and cryostat sections, 5-HT7 receptor immunoreactivity was identified in cell bodies of myenteric (Figure 1A) and submucosal neurons and in few smooth muscle cells (Figure 1B), but not in nerve terminals in muscle layers. In both myenteric (Figure 2) and submucosal neurons (Figure 3), 5-HT7 receptors colocalized with neurons expressing NeuN immunoreactivity, indicating they are AH neurons. However, submucosal neurons immunoreactive for 5-HT7 receptors were quite sparse and typically only 1 neuron could be visualized in 1 or 2 ganglia in each preparation. 5-HT7 receptor myenteric neurons were more abundant and about 3–5 myenteric neurons could be identified in at least 2 or 3 ganglia per preparation.Figure 2(A) 5-HT7 receptor and (B) NeuN immunoreactivity in neurons of the guinea pig ileum myenteric plexus. (C) Simultaneous localization of 5-HT7 receptor and NeuN immunoreactivities. Arrows indicate myenteric neurons containing both 5-HT7 receptor and NeuN immunoreactivities. Confocal images of cryostat sections. Single optical sections. Calibration bar: 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3(A) 5-HT7 receptor and (B) NeuN immunoreactivity in neurons of the guinea pig ileum submucosal plexus. (C) Simultaneous localization of 5-HT7 receptor and NeuN immunoreactivities in a submucosal neuron. Confocal images of cryostat sections. Single optical sections. Calibration bar: 5 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In cultured myenteric neurons, 5-HT7 receptor immunoreactivity was observed in more than 50% of neurons as identified by NSE (Figure 4), where it was localized predominantly at the cell surface. Double-labeling immunohistochemistry in cultured neurons showed that 5-HT7 immunoreactivity was expressed by a subpopulation of neurons containing NeuN or calbindin immunoreactivity, indicating they are AH neurons (Figure 5). 5-HT7 receptor immuno" @default.
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• W2021959823 date "2005-11-01" @default.
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• W2021959823 title "5-HT7 Receptors Modulate Peristalsis and Accommodation in the Guinea Pig Ileum" @default.
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