Tunica Muscularis Mucosae Research Articles

Modeling of culture conditions by culture system, glucose and propionic acid and their impact on metabolic profile in IPEC-J2.

The microbiological environment and their corresponding secreted metabolite spectrum are an essential modulator of the enterocyte function, effecting the whole organism. Intestinal porcine jejunal epithelial cell line (IPEC-J2) is an established in vitro model for differentiation of enterocytes in different cell culture models. An improved oxygen supply seems to be the main reason for differentiation in an air-liquid-interface culture, but this has not yet been conclusively clarified. In this context, the nutrition of the cell and its influence on the metabolism is also of crucial importance. The interest in short-chain fatty acids (SCFAs) has grown steadily in recent years due to their clinical relevance in certain diseases such as multiple sclerosis and other inflammatory diseases, but not much is known of FFAR2 and FFAR3 (free fatty acid receptor 2 and 3) in pigs. We want to address the questions: 1. about the distribution of FFAR2 and FFAR3 in vivo and in vitro in sus scrofa 2. whether there is an influence of propionic acid, glucose content and cultivation on metabolism of enterocytes? The morphological analysis of FFAR2 and FFAR3 in vivo was investigated through immunostaining of frozen sections of the porcine gut segments jejunum, ileum and colon. Both receptors are expressed along the gut and were found in the smooth muscle cells of the tunica muscularis and lamina muscularis mucosae. Furthermore, a high expression of FFAR2 and a low expression of FFAR3 in the enteric nerve system was also observed in jejunum, ileum and colon of sus scrofa. In addition, FFAR2 and FFAR3 within the vessels was investigated. FFAR3 showed a strong expression on endothelial cells of veins and lymphatic vessels but was not detectable on arteries. Furthermore, we demonstrate for the first time, FFAR2 and FFAR3 in IPEC-J2 cells on RNA- and protein level, as well as with confocal microscopy. In addition, ENO1 and NDUFA4 were investigated on RNA-level in IPEC-J2 cells as 2 important genes, which play an essential role in metabolism. Here, NDUFA4 is detected in the model animal sus scrofa as well as in the porcine cell line IPEC-J2. A potential impact of propionic acid and/or glucose and/or cultivation method on the metabolism of the cells was tested with the Seahorse analyzer. Here, a significant higher ECAR was observed in the SMC than in the OCR. In summary, we were able to show that the cultivation system appears to have a greater influence than the medium composition or nutrition of the cells. However, this can be modulated by incubation time or combination of different SCFAs.

Effects of chronic ethanol consumption on rat upper gastrointestinal system: Functional and histologic findings

The purpose of the present study was to determine the effect of chronic alcohol consumption on reactivity of esophageal tunica muscularis mucosae (TMM) and lower esophageal sphincter (LES) smooth muscle. Six male rats in alcohol-fed group received ethanol (7.2% v/v) in a modified liquid diet for 4 weeks. Two control groups were used; six rats in the standard diet-fed group received rat chow and water for 4 weeks. Six rats in sucrose-fed group were given sucrose and received a liquid diet. The smooth muscle reactivity of TMM and LES strips from ethanol-fed and control animals was evaluated in organ chambers. Also histologic study was undertaken to show effects of chronic alcohol consumption. Maximum contractile responses of TMM to KCl and carbachol were decreased in the ethanol-fed group compared to the control groups. Relaxant responses to serotonin were decreased in the ethanol-fed group compared to the control groups. In TMM, isoproterenol- and papaverine-induced relaxant responses were similar in the ethanol-fed and control groups. In LES smooth muscle, relaxant responses to papaverine or isoproterenol were similar in the control groups and the ethanol-fed group. There was no change in agonist potency among the groups. The relaxation response elicited by nicotine and sodium nitroprusside (SNP) or contractile response elicited by carbachol and 80 mM KCl was decreased with maximum responses and pD2 values, in the ethanol-fed group compared to that of the control groups in LES. Decreased nNOS immunoreactivity in myenteric plexus was found in alcohol-exposed group compared to control groups. Our findings suggest that chronic alcohol consumption impairs relaxant and contractile responses of both TMM and LES smooth muscle and it may contribute to gastroesophageal reflux commonly seen after alcohol binges.

VB20B7, a novel 5-HT-ergic agent with gastrokinetic activity. I. Interaction with 5-HT3 and 5-HT4 receptors.

This study describes the in-vitro interaction of the gastrokinetic agent 2[1-(4-piperonyl)piperazinyl]benzothiazole (VB20B7) with the 5-hydroxytryptamine 5-HT3 and 5-HT4 receptor subtypes, using functional as well as radioligand binding studies. The benzamide derivative cisapride was used as a comparison. In radioligand binding assays VB20B7 showed, like cisapride, a weak affinity at 5-HT3 receptors from rat cerebral cortex. The new compound lacked any affinity at other 5-HT receptors or at dopaminergic D2 receptors, whereas cisapride showed high affinity for the 5-HT4 receptors from guinea-pig hippocampus and moderate affinity at dopaminergic D2 receptors. In the non-stimulated guinea-pig ileum, the concentration-response curves to the specific 5-HT3 agonist 2-Me-5-HT and to 5-HT were shifted to the right by VB20B7. In the rat oesophagus tunica muscularis mucosae preparation (TMM), VB20B7 was evaluated for its activity at 5-HT4 receptors. VB20B7 behaved as a 5-HT4 receptor agonist, inducing a concentration-dependent relaxation of the preparation precontracted with carbachol. In this preparation, VB20B7 and cisapride were able to stimulate adenylate cyclase activity, an effect probably mediated through activation of 5-HT4 receptors, as can be inferred from the blockade by the 5-HT4 antagonist, tropisetron, of the enhanced cAMP formation. However, consistent with the lack of affinity at central 5-HT4 receptors, VB20B7 did not stimulate cAMP formation in guinea-pig hippocampal slices. VB20B7 also caused an increase in the twitch response of the transmurally stimulated guinea-pig ileum, although at a concentration higher than cisapride. This effect was blocked by desensitization of the 5-HT4 receptor with 5-MeOT and also by the 5-HT4 receptor antagonist tropisetron. Both VB20B7 and cisapride increased the K(+)-evoked acetylcholine release in this preparation. The results show that VB20B7 possesses affinity for 5-HT4 receptors located in the rat TMM and guinea-pig ileum preparations, but is devoid of affinity at central 5-HT4 receptors. In addition, VB20B7 shows low to moderate affinity at both central and peripheral (enteric) 5-HT3 receptors. The interaction of VB20B7 with the peripheral 5-HT4 and 5-HT3 receptors may be relevant for the gastrokinetic effects of the new compound.

Effects of ginger constituents on the gastrointestinal tract: role of cholinergic M3 and serotonergic 5-HT3 and 5-HT4 receptors.

The herbal drug ginger (Zingiber officinale Roscoe) may be effective for treating nausea, vomiting, and gastric hypomotility. In these conditions, cholinergic M (3) receptors and serotonergic 5-HT (3) and 5-HT (4) receptors are involved. The major chemical constituents of ginger are [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol. We studied the interaction of [6]-gingerol, [8]-gingerol, [10]-gingerol (racemates), and [6]-shogaol with guinea pig M (3) receptors, guinea pig 5-HT (3) receptors, and rat 5-HT (4) receptors. In whole segments of guinea pig ileum (bioassay for contractile M (3) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol slightly but significantly depressed the maximal carbachol response at an antagonist concentration of 10 µM. In the guinea pig myenteric plexus preparation (bioassay for contractile 5-HT (3) receptors), 5-HT maximal responses were depressed by [10]-gingerol from 93 ± 3 % to 65 ± 6 % at an antagonist concentration of 3 µM and to 48 ± 3 % at an antagonist concentration of 5 µM following desensitization of 5-HT (4) receptors and blockade of 5-HT (1) and 5-HT (2) receptors. [6]-Shogaol (3 µM) induced depression to 61 ± 3 %. In rat esophageal tunica muscularis mucosae (bioassay for relaxant 5-HT (4) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol (2-6.3 µM) showed no agonist effects. The maximal 5-HT response remained unaffected in the presence of the compounds. It is concluded that the efficiency of ginger in reducing nausea and vomiting may be based on a weak inhibitory effect of gingerols and shogaols at M (3) and 5-HT (3) receptors. 5-HT (4) receptors, which play a role in gastroduodenal motility, appear not to be involved in the action of these compounds.

Tissue dependent differences in G-protein coupled receptor kinases associated with 5-HT 4 receptor desensitization in the rat gastro-intestinal tract

Desensitization of 5-HT 4 receptors is regulated by G-protein coupled receptor kinases (GRKs). However, the specific GRK(s) that regulates the desensitization of 5-HT 4 receptors in the in vivo setting is unknown. We investigated the in situ expression of 5-HT 4 receptors and the GRKs in the rat gastrointestinal tract using immunohistochemistry and their interaction using coimmunoprecipitation. 5-HT 4 receptors were expressed in the tunica muscularis mucosae of the oesophagus, longitudinal muscle, myenteric plexus, circular muscle, submucosal plexus and muscularis mucosae of both the proximal and distal colon. GRK2 was expressed in longitudinal muscle and occasionally in myenteric plexus whilst GRK5 showed limited expression in the nerve endings of the myenteric plexus and submucosal plexus of the colon. GRK3 was expressed in the tunica muscularis mucosae of the oesophagus, circular muscle, submucosal plexus and muscularis mucosae of the colon. GRK6 was expressed in the tunica muscularis mucosae of the oesophagus, longitudinal muscle, circular muscle, and muscularis mucosae of the colon. Stimulation of tunica muscularis mucosae of the oesophagus and distal colon using the 5-HT 4 receptor agonist, tegaserod, followed by analysis of the 5-HT 4 receptor antibody immunoprecipitate, revealed the coimmunoprecipitation of GRK6 with 5-HT 4 receptors in the tunica muscularis mucosae of oesophagus while GRK2 and GRK6 were coimmunoprecipitated with 5-HT 4 receptors in the distal colon. This study indicates that GRK6 may be involved in the regulation of the desensitization of 5-HT 4 receptors in the rat oesophagus whilst GRK2 and GRK6 may be involved in regulation of the desensitization of 5-HT 4 receptors in the distal colon.

In vitro and in vivo pharmacological characterization of PF-01354082, a novel partial agonist selective for the 5-HT 4 receptor

The pharmacological profile of PF-01354082, a selective 5-HT 4 receptor partial agonist, was investigated. PF-01354082 displayed high affinity for human 5-HT 4d and dog 5-HT 4h receptors in binding studies, having Ki values of 2.0 nM and 4.2 nM, respectively. By contrast, PF-01354082 did not show significant affinity for several other 5-HT receptors (5-HT 1A, 5-HT 1B, 5-HT 1D, 5-HT 2A, 5-HT 2B, 5-HT 2C, 5-HT 3A, and 5-HT 7) or the dopamine D 2long receptor. Functional assays using either cells expressing human recombinant 5-HT 4d receptors or rat tunica muscularis mucosae demonstrated that PF-01354082 exhibited partial agonist activity at the 5-HT 4 receptor. The effects of PF-01354082 on in vitro receptor binding, ion channel activity, and sites of uptake were further investigated. PF-01354082 did not show biologically relevant binding activity at concentrations up to 10 µM except for binding to the 5-HT 4e receptor. Furthermore, PF-01354082 decreased I HERG current by only 11% at a concentration of 300 µM, indicating that the compound had greater than 150,000-fold selectivity for the human 5-HT 4d receptor over hERG channels. An in vivo study using a gastric motility model in conscious dogs demonstrated that oral administration of PF-01354082 resulted in marked and sustained stimulation of gastric motility in a dose-dependent manner. These results indicate that PF-01354082 is an orally active, highly selective, partial agonist of the human 5-HT 4 receptor that is expected to exert a favorable effect on gastrointestinal motor disorders with reduced adverse effects mediated by other related receptors.

Does the Expression of Fascin-1 and Tumor Subclassification Help to Assess the Risk of Recurrence and Progression in T1 Urothelial Urinary Bladder Carcinoma?

Introduction: To evaluate the prognostic value of T1 subclassification and fascin-1 expression in T1 human urothelial cell carcinoma of the bladder. Materials and Methods: In a prospective study with 105 consecutive patients, T1 tumors were subclassified into 2 groups according to the depth of tumor invasion. The tunica muscularis mucosae was used as a landmark. The expression of fascin-1 was examined by using an anti-fascin-1 mouse monoclonal antibody and was evaluated semiquantitatively for both intensity and distribution. The patients were followed up for 27.3 ± 13.7 months. Results: The T1 tumor subclassification was feasible in 99 patients (94%). T1a tumor was detected in 77 patients (73%), T1b tumor in 22 patients (21%). An invasive tumor was found in 5 patients (4.8%) during the restaging transurethral resection of the bladder. The risk of understaging in patients with T1b tumor was 18%. There was not a significant difference in time to the recurrence in the T1a and the T1b group. The progression-free survival rates were significantly different between both groups (p = 0.0034). No correlation was found between fascin-1 positivity and the depth of tumor invasion. Fascin-1 positivity did not correlate with recurrence or the progression-free intervals. In the multivariate analysis, only the extent of lamina propria invasion was an independent predictor of the tumor progression. The fascin positivity was not an independent prognostic factor relating to the risk of recurrence or progression. Conclusion: The finding of T1b tumor was connected with a significantly higher risk of progression and understaging. The fascin-1 expression did not correlate with the depth of tumor invasion or with the tumor recurrence or progression.

Fibrosing Gastrointestinal Leiomyositis as a Cause of Chronic Intestinal Pseudo-Obstruction in an 8-Month-Old Dog

An 8-month-old, female, mixed-breed dog presented to the Iowa State University Veterinary Teaching Hospital with a 1-month history of vomiting and diarrhea. An exploratory laparotomy was performed revealing markedly distended and fluid-filled small and large intestines that were not obstructed. The clinical condition of the dog did not improve subsequent to exploratory surgery, and it was euthanized. At necropsy, both the small and large intestines were distended (approximately 4 cm in diameter) and fluid-filled, and the wall was thin. The abdominal cavity contained approximately 500 ml of a brownish clear fluid. Microscopic lesions of the intestines were confined to the intestinal tunica muscularis and muscularis mucosae and consisted of locally extensive-to-diffuse replacement of the smooth muscle by fibrous tissue and multifocal infiltration by a moderately dense mononuclear inflammatory infiltrate. A unique finding was the presence of similar microscopic lesions in the tunica muscularis of the urinary bladder and stomach.

Pyrrolizidine Esters and Amides as 5-HT4 Receptor Agonists and Antagonists

A series of pyrrolizidine esters, amides, and ureas was prepared and tested for 5-HT(4) and 5-HT(3) receptor binding, 5-HT(4) receptor agonism in the rat tunica muscularis mucosae (TMM) assay, and for 5-HT(3) receptor-mediated functional antagonism in the Bezold-Jarisch reflex assay. Several pyrrolizidine derivatives were identified with high affinity for the 5-HT(4) receptor, including benzamide 12a (SC-53116), a potent and selective 5-HT(4) partial agonist that exhibits efficacy in promoting antral contractions and activity in promoting gastric emptying in canine models. Also discovered were 5-HT(4) receptor antagonists, including imidazopyridine amide 12h (SC-53606), which is a potent and selective 5-HT(4) receptor antagonist with a pA(2) value of 8.13 in the rat TMM assay. N-Methyl indole ester 13d was identified as a potent 5-HT(4) antagonist with a pA(2) value of 8.93. High selectivity was observed for these pyrrolizidine derivatives versus other monoamine receptors, including 5-HT(1), 5-HT(2), D(1), D(2), alpha(1), alpha(2), and beta receptors.

New benzimidazole derivatives: selective and orally active 5-HT 3 receptor antagonists

The synthesis of new 5-HT 3 receptor antagonists is an interesting field of research because of their wide therapeutic use. The aim of this work is to functionally characterise a new series of benzimidazole derivatives previously described. These compounds bind to 5-HT 3 receptors and have been evaluated using in vitro (rat tunica muscularis mucosae) and in vivo tests (Bezold–Jarisch reflex in rat and gastrointestinal motility and spontaneous motility in mice). Ondansetron and 1-[4-amino-5-chloro-2-(3,5-dimethoxyphenil)methyloxy]-3-[1-[2-methylsulfonylamino]piperidin-4-yl]propan-1-one hydrochloride (RS 39604) were used as well known 5-HT 3 and 5-HT 4 receptor antagonists. These benzimidazole derivatives have proved to be 5-HT 3 receptor antagonists. Interestingly, they are as active as ondansetron when they are intraperitoneally (i.p.) or orally (p.o.) administered and, in mice, they seem to induce fewer behavioural changes at similar effective doses than does ondansetron. The present results confirm the usefulness of the previously proposed pharmacophore and justify the interest in these new benzimidazole derivatives.

Monochloramine directly modulates Ca 2+-activated K + channels in rabbit colonic muscularis mucosae

Background & Aims: Mesenteric ischemia, infection, and inflammatory bowel disease may eventuate in severe colitis, complicated by toxic megacolon with impending intestinal perforation. Monochloramine (NH 2Cl) is a membrane-permeant oxidant generated during colitis by the large amount of ambient luminal NH 3 in the colon. Reactive oxygen metabolites can modulate smooth muscle ion channels and thereby affect colonic motility, which is markedly impaired in colitis. Methods: Effects of NH 2Cl on ionic currents in the innermost smooth muscle layer of the colon, the tunica muscularis mucosae, were examined using the patch clamp technique. Membrane potential in whole tissue strips was measured using high-resistance microelectrodes. Results: Whole cell voltage clamp experiments showed that NH 2Cl (3-30 μmol/L) enhanced outward currents in a dose-dependent manner, increasing currents more than 8-fold at a test potential of +30 mV. Tail current analysis showed that the currents enhanced by NH 2Cl were K + currents. Inhibition by tetraethylammonium and iberiotoxin suggested that these currents represented activation of large-conductance, Ca 2+-activated K + channels. The membrane-impermeant oxidant taurine monochloramine, however, had no effect on whole cell currents. Single-channel studies in inside-out patches showed that NH 2Cl increased open probability of a 257-pS channel in symmetrical (140 mmol/L) K +. In the presence of NH 2Cl, the steady-state voltage dependence of activation was shifted by −22 mV to the left with no change in the single-channel amplitude. The sulfhydryl alkylating agent N-ethylmaleimide prevented NH 2Cl-induced channel activation. NH 2Cl also hyperpolarized intact muscle strips, an effect blocked by iberiotoxin. Conclusions: NH 2Cl, at concentrations expected to be found during colitis, may contribute to smooth muscle dysfunction by a direct oxidant effect on maxi K + channels. GASTROENTEROLOGY 1999;117:906-917

2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides with selective affinity for the 5-HT(4) receptor: synthesis and structure-affinity and structure-activity relationships of a new series of partial agonist and antagonist derivatives.

A series of 2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxamide derivatives bearing a piperazine moiety was synthesized. Their in vitro 5-HT(4), 5-HT(3), and D(2) receptors affinities were evaluated by radioligand binding assay. For selected compounds functional studies at the 5-HT(4) receptor were made by using precontracted (by carbachol) preparations of rat esophageal tunica muscularis mucosae (TMM). The influence of the 3-substituent of the benzimidazole ring, the 4-substituent of the piperazine moiety, and the alkylene spacer was studied. Compounds with an ethyl or a cyclopropyl substituent in the 3-position of the benzimidazole ring showed moderate to high affinity (K(i) = 6.7-75.4 nM) for the 5-HT(4) receptor with selectivity over 5-HT(3) and D(2) receptors and moderate antagonist activity (pK(b) = 6.19-7.73). Compounds with an isopropyl substituent in the 3-benzimidazole position exhibited moderate and selective 5-HT(4) affinity (K(i) >/= 38.9 nM) and a partial agonist activity (5a, i.a. = 0.94) higher than that of the reference compound BIMU 8 (i.a. = 0.70). This reversal of the pharmacological activity due only to a small structural difference might confirm the existence of two binding sites on the 5-HT(4) receptor. In the alkylene spacer, a two-methylene chain is favorable to optimize the affinity and the antagonist or the partial agonist activity. In the ethyl and cyclopropyl series, 5-HT(4) antagonist activity seems to be unrelated to the size of the 4-substituent of the piperazine moiety, whereas a methyl group is optimal for high partial agonist activity in the isopropyl series; however, the presence of a butyl substituent is a favorable pattern for 5-HT(4) antagonism and even causes a reversal of the pharmacological profile in the isopropyl series (5h, pK(b) = 7.94). N-Butyl quaternization of 5a led to an improvement in affinity for the 5-HT(4) receptor and mantained the high partial agonist activity (5r, K(i) = 66.3 nM, i.a. = 0.93).

Identification of SK-951, a Novel Benzofuran Derivative, as an Agonist to 5-HT4 Receptors

The pharmacological profile of SK-951 ((—)4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl) ethyl]-5-chloro-2,3-dihydro-2-methylbenzo[b]furan-7-carboxamide hemifumarate) was identified in relation to serotonin 5-HT3 and 5-HT4 receptors by the receptor binding assay and functional studies. The receptor binding assay showed that SK-951 bound to the 5-HT3 receptor with a high affinity, to the 5-HT4 receptor with relatively higher affinity and to the muscarinic M2 receptor with a low affinity, but not to dopamine D1 and D2 and serotonin 5-HT1 and 5-HT2 and muscarinic M1 and M3 receptors. SK-951 caused relaxations of tunica muscularis mucosae preparations from rat esophagus which were precontracted with carbachol, and the effects were antagonized by GR113808, a selective 5-HT4 antagonist. In the longitudinal muscle with myenteric plexus (LMMP) preparations from guinea pig ileum, SK-951 enhanced the electrically-stimulated contraction of preparations in which the 5-HT1, 5-HT2 and 5-HT3 receptors were blocked, and it enhanced the electrically-stimulated release of [3H]acetylcholine (ACh). These effects of SK-$51 were antagonized by GR113808. SK-951 inhibited the 5-HT3 receptor-mediated contractions. These results indicate that SK-951 possesses properties of an agonist for the 5-HT4 receptor and an antagonist for the 5-HT3 receptor. Thus, SK-951 is a new and potent 5-HT4-receptor agonist and causes contractions of guinea pig ileum mediated by enhancement of ACh release via the 5-HT4 receptor.

The response of rat colonic mucosa to 5-hydroxytryptamine in health and following restraint stress

The present study characterized the rat colonic secretory response to 5-hydroxytryptamine (5-HT) and determined alterations in this response following stress. 5-HT stimulated rat colonic short-circuit current in a concentration-dependent fashion (p D 2=5.19). This response was subject to desensitization and was mimicked by the indolealkylamines with a rank order potency of 5-HT≈α-methyl-5-HT>5-carboxytryptamine≈5-methoxytryptamine. 2-Methyl-5-HT was a partial agonist. The colonic response to 5-HT was unaltered by methysergide (10 μM), ritanserin (0.1 μM), ondansetron (1 μM) or clozapine (10 μM), but was antagonized by the 5-HT 4 receptor antagonists SB204070 (p D 2′=9.32), GR113808 (p K b=8.56), DAU6285 (p K b=6.07) and SDZ205557 (p K b=6.80). The response of colonic epithelial and oesophageal tunica muscularis mucosae to 5-HT is therefore mediated by a similar 5-HT 4 receptor. Following wrap restraint stress, the colonic response to 5-HT became bimodal. Half of the preparations were hyper-responsive, while the rest were hypo-responsive to 5-HT. This 5-HT 4 receptor may therefore be involved in stress related changes in fluid transport.

Serotonin 5-HT4 agonist activity of a series of meso-azanoradamantane benzamides

A series of meso-amino(methyl)azanoradamantane benzamides has been prepared and evaluated for 5-HT4 agonism activity in the rat tunica muscularis mucosae (TMM) assay. Compound 8i is the most potent 5-HT4 agonist in the series, with an EC50 of 217 nM.

Post-synaptic 5-HT 4 receptor modulation of tachykinergic excitation of rat oesophageal tunica muscularis mucosae

Interaction between the 5-HT 4 receptor and cholinergic-dependent and -independent contraction of the rat oesophageal muscularis mucosae was determined. Substance P- (in the presence of atropine) and carbachol-precontracted tissue was relaxed by tryptamines and the substituted benzamides with the following rank order of potency: 5-HT>5-methoxytryptamine>cisapride>( R)-zacopride>lintopride>metoclopramide, consistent with 5-HT 4 receptor activation. The response to 5-HT was not antagonized by tetrodotoxin, methysergide or ondansetron, but was shifted to the right by GR113808 ([1-[2-[methylsulphonyl)amino]ethyl]-4-piperidinyl]methyl-1- H-indole-3-carboxylate) in substance P- and carbachol-precontracted tissue, confirming 5-HT 4-mediated relaxation. This study shows for the first time that although 5-HT 4 receptors are involved in the modulation of cholinergic neurotransmission they can also act independently of this system modulating tachykinergic responsiveness. © 1997 Elsevier Science B.V. All rights reserved.

Beta 3-adrenoceptors mediating relaxation of the oesophageal tunica muscularis mucosae and distal colon in the rat: comparative pharmacology and their desensitization by BRL 37344.

1. In the rat isolated distal colon and oesophagus, relaxant responses to (-)-isoprenaline were poorly antagonized by propranolol (0.1 microM), suggesting an involvement of beta 3-adrenoceptors. 2. In the presence of propranolol the rank order of potency for the agonist-induced relaxations was similar in the two tissues. 3. BRL 37344 was a partial agonist relative to isoprenaline in the colon but when a single maximal concentration of BRL 37344 (1 microM) was added, a full relaxation was observed. 4. BRL 37344 desensitized responses to isoprenaline in both the colon and oesophagus. 5. (+/-)-Cyanopindolol (1.0 microM) antagonized the relaxatory responses to both isoprenaline and BRL 37344 in the distal colon and oesophagus and reduced the desensitizing effect of BRL 37344. 6. The present study demonstrates that the beta-adrenoceptors mediating relaxation in both the rat isolated distal colon and oesophagus are of the beta 3-adrenoceptor subtype. Desensitization appears to result from the binding of BRL 37344 to the beta 3-adrenoceptor in a manner which is blockable by cyanopindolol.

CD68+ Reactive histiocytosis complicating early gastric cancer

A peculiar form of reactive, non-granulomatous proliferation of CD68+ histiocytes was demonstrated, for the first time, in the submucosal layer just beneath the IIa-type early gastric adenocarcinoma that focally invaded submucosal lymphatics. The histiocytic cells possessed plump eosinophilic and often foamy cytoplasm and eccentric small nuclei with apparent nucleoli. They contained periodic acid-Schiff-reactive granules, and were further immunoreactive for lysozyme and alpha 1-antitrypsin. The infiltration was ill-defined, and partly involved the tunica muscularis mucosae and the proper muscle layer. Markers of epithelial cells, Langerhans' histiocytes, smooth muscle cells and myofibroblasts were all negative.

Effect of Serotonin (5-HT)3-Receptor Antagonists YM060, YM114 (KAE-393), Ondansetron and Granisetron on 5-HT4 Receptors and Gastric Emptying in Rodents

We investigated the effects of YM060 {(R)-5-[(1-methyl-3-indolyl)carbonyl]-4,5,6,7-tetrahydro-1//-benzimidazole hydrochloride} and YM114 (KAE-393) {(R)-5-[(2,3-dihydro-1-indolyl)-carbonyl]-4,5,6,7-tetrahydro-lH-benzimidazole hydrochloride} on 5-HT4 receptors and gastric emptying in normal and cisplatin-treated rats and compared results with those for ondansetron and granisetron. YM060, YM114, ondansetron and granisetron dose-dependently inhibited the specific binding of [3H]-GR113808 {[[l-[(2-methylsulphonyl)amino]ethyl]-4-piperidin-yl]methyl 1-methyl-lH-indole-3-carboxylate} in guinea pig striatum, with pKi values of 5.53, 5.13, 5.21 and 5.63, respectively. According to the pKi values reported in 5-HT3-receptor binding of [3H]GR65630 to rat cortical membranes, the affinity of YM060, YM114, ondansetron and granisetron for 5-HT4 receptors was approximately 5, 5, 3.5 and 3.5 log units lower than that for 5-HT3 receptors, respectively. In the guinea pig longitudinal muscle with myenteric plexus and rat esophageal tunica muscularis mucosae, YM060 and YM114 showed neither 5-HT4-agonistic nor antagonistic properties. Although ondansetron produced concentration-dependent increases in the magnitude of the twitch response in longitudinal muscle, it did not possess 5-HT3- and 5-HT4-agonistic activity. Granisetron antagonized 5-HT-induced relaxation of the rat esophagus with an apparent pA2 value of 5.39. Intravenous YM060, YM114, ondansetron and granisetron significantly enhanced gastric emptying of glass beads and improved cisplatin-induced slowing of gastric emptying in rats. These results indicate that the selectivity of YM060 and YM114 for 5-HT3 receptors is higher than that of ondansetron and granisetron and that these 5-HT3 antagonists have gastroprokinetic activity in normal and cisplatin-treated rats without affecting 5-HT4 receptors.

In vitro characterizat of the non-peptide tachykinin NK 1 and NK 2-receptor antagonists, SR140333 and SR48968 in different rat and guinea-pig intestinal segments

We investigated the potent non-peptide tachykinin receptor antagonists SR140333 and SR48968 for their ability to prevent the contraction of isolated intestinal tissues elicited by the non-selective agonists substance P (SP) and neurokinin A (NKA), or by [Sar 9,Met(O 2) 11]SP and [β-Ala 8]NKA-(4–10) that are selective agonist for NK 1 and NK 2 receptors,respectively. In guinea-pig ileum, containing mainly NK 1-receptors: SR140333 caused a pseudo-irreversible blockade of contractions induced by either SP (K B, 0.01 nM) or [Sar 9, Met(O 2) 11]SP (K B, 0.03 nM); SR140333 but not SR48968, dose-dependently (IC 50, 0.06 nM) antagonized the contractions elicited by capsaicin. In rat duodenum, containing mainly NK 2 receptors, SR48968 caused a parallel rightward shift of the concentration-response curves of [β-Ala 8]NKA-(4–10) (pA 2, 9.5), but not of NKA. In rat esophageal tunica muscularis mucosae, SR48968 and non-competitively antagonized [β-Ala 8]NKA-(4–10) and NKA. SR48968 and SR140333 thus appear to be potent tachykinin receptor antagonists, selective for intestinal receptors respectively of the NK 2 and NK 1 type. The results also suggest that rat esophagus might contain a NK 2-receptor subtype different from that of rat duodenum.