# Activation of PAR-2 increases colonic permeability by MLCK activation and INF-c-dependent

Nicolas Cenac, Rafael Garcia Villar, Laurent Ferrier, Jean Fioramonti, Lionel Bueno

► To cite this version:

Nicolas Cenac, Rafael Garcia Villar, Laurent Ferrier, Jean Fioramonti, Lionel Bueno. Activation of PAR-2 increases colonic permeability by MLCK activation and INF-c-dependent. 19th International Symposium on Gastrointestinal Motility, Oct 2003, Barcelone, Spain. 2003, Neurogastroenterol. Motil. 15 (5). [hal-01600598](https://hal.science/hal-01600598)

HAL Id: hal-01600598

https://hal.science/hal-01600598

Submitted on 4 Jun 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution - ShareAlike 4.0 International License
INTERNATIONAL GROUP for the study of
NEUROGASTROENTEROLOGY & MOTIL

■ Committees

■ Symposium

• Call For Abstracts

• Abstract Submission

• Travel

• Registration

• Host City

• Convention Center

■ Awards

■ Announcements

International
Symposium on
Gastrointestinal
Motility

<figure><img src="image_2.png" /></figure>

19TH

2003

BARCELONA, SPAIN
OCTOBER 5-8

Abstract Instructions

Abstract Selection

All abstracts will be reviewed and ranked on the basis of
scientific merit by experts. The Symposium Secretariat will
remove all text identifying the authors before sending the
abstracts to the reviewers. Abstracts will not be considered
or will be removed from the program if they have been
published as an article before October 1, 2003.

Instructions for Authors

**Title.** The title should be brief, but long enough to identify the nature of the study. The title should be in upper case (capitals) and extend to no more than two lines.
Abbreviations must not be used in the title.

*Authors*. Omit degrees and titles. Initials should precede the last name. Underline the name of the author who will present the abstract if it is accepted. Indicate name and location of the institution where the work was done.

*Structure*. Organize the body of the abstract as follows:

a) The aim of the study

b) A brief description of the methods.

c) A summary of the results. **A simple table or a graph may be used if they fit within the form.**

d) A statement of the conclusions reached.

*Abbreviations*. Generally accepted abbreviations may be used. Other abbreviations must be defined between parentheses after the first use of the word.

*Drugs*. Use non-proprietary (generic) names of drugs. Do not capitalize these names.

A. Typing Instructions: electronic submission

1. Please adhere precisely to the typing instructions. The maximum number of characters allowed are 1.600. If you do not respect this limit and try to send the abstract form through the website it will appear an error message and
stimulation of gastric vagal afferents following postprandial gastric distension. We have previously demonstrated in dogs that TLESRs can be inhibited by the synthetic cannabinoid (CB) receptor agonist, WIN 55, 212-2, probably through a central site of action on CB1 receptors (CBR1). The aim of the present study was to extend these observations using the endocannabinoid, anandamide, and to further investigate the potential for a peripheral site of action of cannabinoids in TLESR inhibition. TLESR inhibition was studied in dog using Dentsleeve manometry. Anandamide (2.9 µmol/kg; n = 4) produced a reduction of TLESRs by 43 ± 9%. In addition, the esophageal acid exposure was reduced by 64 ± 10% and the latency time to the occurrence of the first TLESR was increased by 44 ± 14%. To address the question of a possible peripheral localisation of CBR1 in vagal afferents, ligations of the vagus nerve were performed below the diaphragm in dogs. An accumulation of CBR1 immunoreactivity was observed both proximally and distally to the ligatures, suggesting both anterograde and retrograde transport of CBR1. From the present results it can be concluded that anandamide potently inhibits TLESRs and that CBR1 are transported to peripheral vagal nerve terminals, raising the possibility that endocannabinoids can have an inhibitory effect on TLESRs through both a peripheral and central site of action.

426
**Activation of PAR-2 increases colonic permeability by MLCK activation and INF-γ-dependent and -independent pathways**

N. CENAC, R. GARCIA VILLAR, L. FERRIER, J. FIORAMONTI & L. BUENO

INRA Neuro-Gastroenterology & Nutrition Unit

Protease-activated receptors (PAR)-2 activated by the peptide agonist SLIGRL (100 µg/mouse) mediate inflammation and increase epithelial paracellular permeability in mice colon, whereas at 5 µg/mouse, SLIGRL increases permeability but does not produce inflammation. As MLCK (myosin light chain kinase)-induced phosphorylation of MLC increases epithelial permeability, we investigated the implication of MLCK in PAR-2-mediated permeability increase in inflammatory and non-inflammatory conditions. Firstly, 3 groups of 8 Swiss mice were infused intracolonically (50 µL) with SLIGRL (5 µg, group 1; 100 µg, group 2,) or saline (group 3); 4 hrs later colons were collected for western-blot assay of MLCK, MLC and phosphorylated-MLC (p-MLC). Secondly, 6 groups of 8 B6 *ifng*<sup>-/-</sup> (IFNγ-KO) and 6 groups of 8 C57BL/6J (controls) mice were infused with SLIGRL or saline as above; 3 groups of B6 *ifng*<sup>-/-</sup> mice and 3 of C57BL/6J received ML-7 (MLCK inhibitor, 2 mg/kg, ip), and the other groups its vehicle. Colonic paracellular permeability was measured by infusing 51Cr-EDTA into the colon (0.7 µCi; 0.25 ml/h; 2 hrs), and expressed as % of radioactivity recovered outside the colon. In Swiss mice MLC expression decreased and p-MLC increased after both doses of SLIGRL; MLCK was not affected. In C57BL/6J mice, permeability dose-dependently increased after SLIGRL, but while ML-7 abolished the effect of the low dose of SLIGRL it only decreased that of the high dose. In B6 *ifng*<sup>-/-</sup> mice, the increase in permeability was similar for both doses of SLIGRL and prevented by ML-7. Our study reveals for the first time (i) that a non-inflammatory dose of SLIGRL increased paracellular permeability via MLC phosphorylation by MLCK independently of IFNγ secretion; (ii) that the increase in paracellular permeability mediated by the inflammatory dose of SLIGRL involves a INFγ-dependent mechanism.

<table><thead><tr><th>SLIGRL (µg)</th><th>0</th><th>0</th><th>5</th><th>5</th><th>100</th><th>100</th></tr></thead><tbody><tr><td>ML-7 (mg/kg)</td><td>-</td><td>2</td><td>-</td><td>2</td><td>-</td><td>2</td></tr><tr><td>C57BL/6J</td><td>1.4 ± 0.2</td><td>1.3 ± 0.1</td><td>3.3 ± 0.2*</td><td>1.7 ± 0.2</td><td>6.6 ± 0.6*</td><td>4.0 ± 0.6*</td></tr><tr><td>B6 <i>ifng</i><sup>-/-</sup></td><td>1.2 ± 0.1</td><td>1.2 ± 0.2</td><td>3.0 ± 0.1*</td><td>1.4 ± 0.2</td><td>3.4 ± 0.2*</td><td>1.7 ± 0.3</td></tr></tbody></table>

Data are mean ± SEM (n = 8) in % of 51Cr-EDTA permeability; *P ≤ 0.05; compared to control values.

430
**The effects of prokineticin 2, [NLE13]-motilin and Ghrelin on mouse isolated gastrointestinal tissue**

A. BASSIL,* N. B. DASS,† C. D. MURRAY,† G. J. SANGER* & A. I. MUIR‡
*GlaxoSmithKline, †Department of Physiology, St Marks Hospital, Harrow, UK
‡Discovery Research, GlaxosmithKline

**Aim.** Prokineticin 2 (PK2) and its receptors have been detected in the mammalian gastrointestinal (GI) tract, where PK2 may directly contract smooth muscle (Li et al. 2001 Mol Pharmacol 59, 692; Lin et al. 2002 J Biol Chem 277, 19276). This study sought to determine the ability of PK2 to modulate electrically-stimulated, nerve-mediated contractions in mouse isolated stomach and colon which was compared with the GI prokinetic agents metoclopramide, [Nle¹³]-motilin and ghrelin, the latter facilitating cholinergic function in, respectively, rabbit (Van Assche et al. 1997 Eur J Pharmacol 337, 267) and mouse (Murray et al. 2002 Br J Pharmacol 136, 18P) isolated stomach preparations. **Methods.** Gastric fundus and/or distal colonic circular muscle preparations from adult male C57BLK6/J mice (20–30 g) were tensioned for isometric recording in tissue baths and electrically field stimulated (EFS) for 30 s every 150 s (fundus) or 10 s every 110 s (colon) at 5 Hz (0.5 ms, max-effective voltage) to evoke tetrodotoxin-sensitive responses. Human PK2 has been shown to have nmol L⁻¹ affinity for both the human (Lin et al. 2002) and rodent receptors for PK1 and 2. **Results.** Metoclopramide (10–100 µmol L⁻¹), previously shown to facilitate EFS-evoked contractions in the mouse gastric fundus (Murray et al. 2002) had no effect on baseline muscle tension or the nerve-evoked response in the distal colon. PK2 (0.001 nmol L⁻¹-0.1 µmol L⁻¹) and [Nle¹³]-motilin (0.1 nmol L⁻¹-1 µmol L⁻¹) had no significant effects on baseline muscle tension or the nerve-evoked contractions in gastric fundus (n = 3–7, P > 0.07 and n = 6–8, P > 0.08, each concentration, respectively) or distal colon (n = 3, P > 0.27 and n = 4–6, P > 0.37, each concentration). Similarly, rat ghrelin (0.1–100 µmol L⁻¹), previously shown to facilitate EFS-evoked contractions in the mouse gastric fundus (Murray et al. 2002) had no significant effect on the baseline muscle tension or the nerve-evoked response in the distal colon (n = 5–8, P > 0.05, each concentration). **Discussion.** In contrast to metoclopramide or ghrelin, neither PK2 nor [Nle¹³]-motilin facilitated EFS-evoked contractions in mouse isolated stomach and are thus unlikely to exert gastric prokinetic activity in this species. None of these agents exerted similar excitatory activity in the mouse colon, at least when using these parameters of EFS. The lack of response to [Nle¹³]-motilin is consistent with the reported absence of a motilin receptor in rodents (Hill et al. 2002 Gastroenterol 122, 447). The data with PK2 does not reflect the functional effect the name suggests.

438
**Distinct role for vip receptor subtypes and intracellular signalling pathways in mediating relaxation of human stomach**

I. TATTOLI, C. SEVERI, V. D. CORLETO & G. F. DELLE

Dept. Clinical Sciences, University "La Sapienza", Roma

VIP relaxes smooth muscle by interacting with two subtypes of receptors: VIP2 coupled to adenylate cyclase and NPR-C (natriuretic peptide clearance receptor) coupled, through eNOS, to guanylate cyclase. The relative contribution of each receptor subtype in inducing gastric relaxation remains to be elucidated, species and regional differences being reported. **Aim.** To investigate in human stomach smooth muscle, obtained separately from fundus and antrum, the action of VIP in terms of biological activity, receptor subtypes expression and relative associated signalling pathways. **Results.** VIP caused a dose-dependent relaxation of both muscle strips and isolated cells (SMC) presenting an higher efficacy and potency on antrum (D50:0.53 ± 0.17 nmol L⁻¹) than fundus (D50:3.4 ± 1.4 nmol L⁻¹). RT-PCR products analysis showed that both VIP2 and NPR-C are expressed on SMC of the two gastric regions with a greater expression of VIP2 in the antrum. Forskolin (1 µmol L⁻¹), a direct activator of adenylate cyclase, induced a greater relaxation (P < 0.05) of antrum (42.3 ± 5.8%) than fundus SMC (23.9 ± 3.1%) whereas sodium nitroprusside (10 nmol L⁻¹), a direct activator of guanylate cyclase, relaxed more the fundus (16.2 ± 2.1%) than the antrum (10.9 ± 1.5%)
Continued

<table><thead><tr><td>Number</td><td>Date</td><td>Main Author</td><td>Institution</td><td>Abstract Title</td></tr></thead><tbody><tr><td>224</td><td>03/04/2003</td><td>De Maeyer, Joris</td><td>Heymans Institute of Pharmacology, Ghent University, Ghent/Johnson &amp; Johnson Pharmaceutical Research and Development, Beerse, Belgium</td><td>Organ specificity of 5-HT4 receptor agonists in porcine stomach and atrium?</td></tr><tr><td>229</td><td>03/04/2003</td><td>Van Nassauw, Luc</td><td>Laboratory of Cell Biology &amp; Histology, University of Antwerp</td><td>Presence of the P2X1 receptor in the guinea-pig gastrointestinal tract</td></tr><tr><td>236</td><td>03/04/2003</td><td>Christinane, Löken</td><td>Solvay Pharmaceuticals GmbH, PH-REB</td><td>Effects of neuromedin u on the gastrointestinal tract of rats and guinea-pigs</td></tr><tr><td>395</td><td>04/04/2003</td><td>Bisschops, Raf</td><td>University of Leuven</td><td>Ghrelin induces CA2+ transients via release from intracellular stores in a subset of myenteric plexus neurones.</td></tr><tr><td>399</td><td>04/04/2003</td><td>De Breucker, Sabine</td><td>J&amp;J Pharmaceutical Research and Development, a division of Janssen Pharmaceutica NV</td><td>Development of a 3H- gaba binding assay in recombinant GABABR1a/R2 receptor expressing cells</td></tr><tr><td>402</td><td>04/04/2003</td><td>Mitselos, Anna</td><td>Gastroenterological Research, Gasthuisberg O&amp;N. University of Leuven</td><td>Comparison of motilin antagonists confirms the role of the c-terminal domain of motilin in causing complete homologous desensitization.</td></tr><tr><td>404</td><td>04/04/2003</td><td>Jensen, Jorgen</td><td>Gastrointestinal Biology, Integrative Pharmacology, AstraZeneca R&amp;D Molndal</td><td>Vagal transport of cb1 receptors and inhibitory effects of an endocannabinoid on transient lower esophageal sphincter relaxations</td></tr><tr><td>426</td><td>04/04/2003</td><td>Cenac, Nicolas</td><td>INRA Neuro-Gastroenterology &amp; Nutrition Unit</td><td>Activation of PAR-2 increases colonic permeability by MLCK activation and IFN-gamma-dependent and -independent pathways</td></tr><tr><td>430</td><td>04/04/2003</td><td>Bassil, Anna</td><td>GlaxoSmithKline</td><td>The effects of prokineticin 2, [NLE13]-motilin and ghrelin on mouse isolated gastrointestinal tissue</td></tr><tr><td>438</td><td>04/04/2003</td><td>Tattoli, Ivan</td><td>Dept. Clinical Sciences, University "La Sapienza", Roma</td><td>Distinct role for VIP receptor subtypes and intracellular signalling pathways in mediating relaxation of human stomach</td></tr><tr><td>461</td><td>04/04/2003</td><td>Bassil K, A</td><td>Dept. GI Research, Neurology &amp; GI Cedd, Glaxosmithkline</td><td>The effects of prokineticin 2, [NLE13]-motilin and ghrelin on mouse isolated gastrointestinal tissue</td></tr><tr><td>470</td><td>04/04/2003</td><td>Edholm, Therese</td><td>Dept. of Gastroenterology. Karolinska hospital</td><td>Ghrelin stimulates interdigestive migrating myoelectrical complex in the rat small: involvement of cholinergic neurons.</td></tr><tr><td>478</td><td>04/04/2003</td><td>Shea-Donohue, Terez</td><td>USDA-ARS-NRFL</td><td>Role of enteric nerves in helminth infection-induced downregulation of protease-activated receptor (PAR) responses</td></tr><tr><td>483</td><td>04/04/2003</td><td>Muinuddin, Ahmad</td><td>Department of Physiology, University of Toronto</td><td>Distinct expression of M3 muscarinic receptors in feline lower esophageal sphincter smooth muscle</td></tr><tr><td>498</td><td>04/04/2003</td><td>Ito, Moriko</td><td>Novartis Pharma AG, Novartis Institute for Biomedical Research Basel</td><td>Distribution of 5-HT4 receptor splice variants in the human and non-human primate gastro-intestinal tract</td></tr><tr><td>499</td><td>04/04/2003</td><td>Näslund, Erik</td><td>Dept of Surgery, Karolinska Institutet Danderyd Hospital</td><td>Circulating levels of ghrelin, motilin and GH after food intake in different phases of the migrating motor complex in man</td></tr><tr><td>524</td><td>05/04/2003</td><td>Czimmer, Jozsef</td><td>UCLA/CURE: Digestive Diseases Research Center</td><td>Distinct neural pathways in modulation of gastric emptying by CRF2 selective and non-selective CRF receptor agonist peptides in rats</td></tr></tbody></table>