Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jun 28, 2013; 19(24): 3747-3760
Published online Jun 28, 2013. doi: 10.3748/wjg.v19.i24.3747
Predominant mucosal expression of 5-HT4(+h) receptor splice variants in pig stomach and colon
Evelien KV Priem, Joris H De Maeyer, Mado Vandewoestyne, Dieter Deforce, Romain A Lefebvre
Evelien KV Priem, Romain A Lefebvre, Heymans Institute of Pharmacology, Ghent University, B-9000 Ghent, Belgium
Joris H De Maeyer, Shire-Movetis NV, B-2300 Turnhout, Belgium
Mado Vandewoestyne, Dieter Deforce, Laboratory for Pharmaceutical Biotechnology, Ghent University, B-9000 Ghent, Belgium
Author contributions: Priem EKV performed the experiments; Lefebvre RA designed the study; Priem EKV and Lefebvre RA interpreted the data and wrote the manuscript; De Maeyer JH, Vandewoestyne M and Deforce D provided technical support for this work and approved the final submitted version.
Supported by Grant G.0061.08 from the Fund for Scientific Research Flanders
Correspondence to: Romain A Lefebvre, Professor, Heymans Institute of Pharmacology, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium. romain.lefebvre@ugent.be
Telephone: +32-9-3323373 Fax: +32-9-3324988
Received: August 17, 2012
Revised: December 3, 2012
Accepted: December 15, 2012
Published online: June 28, 2013
Processing time: 315 Days and 3.8 Hours
Abstract

AIM: To investigate cellular 5-HT4(-h/+h) receptor distribution, particularly in the epithelial layer, by laser microdissection and polymerase chain reaction (PCR) in porcine gastrointestinal (GI) tissues.

METHODS: A stepwise approach was used to evaluate RNA quality and to study cell-specific 5-HT4 receptor mRNA expression in the porcine gastric fundus and colon descendens. After freezing, staining and laser microdissection and pressure catapulting (LMPC), RNA quality was evaluated by the Experion automated electrophoresis system. 5-HT4 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions were examined by endpoint reverse transcription (RT)-PCR in mucosal and muscle-myenteric plexus (MMP) tissue fractions, in mucosal and MMP parts of hematoxylin and eosin (HE) stained tissue sections and in microdissected patches of the epithelial and circular smooth muscle cell layer in these sections. Pig gastric fundus tissue sections were also stained immunohistochemically (IHC) for enterochromaffin cells (EC cells; MAB352); these cells were isolated by LMPC and examined by endpoint RT-PCR.

RESULTS: After HE staining, the epithelial and circular smooth muscle cell layer of pig colon descendens and the epithelial cell layer of gastric fundus were identified morphologically and isolated by LMPC. EC cells of pig gastric fundus were successfully stained by IHC and isolated by LMPC. Freezing, HE and IHC staining, and LMPC had no influence on RNA quality. 5-HT4 receptor and GAPDH mRNA expressions were detected in mucosa and MMP tissue fractions, and in mucosal and MMP parts of HE stained tissue sections of pig colon descendens and gastric fundus. In the mucosa tissue fractions of both GI regions, the expression of h-exon containing receptor [5-HT4(+h) receptor] mRNA was significantly higher (P < 0.01) compared to 5-HT4(-h) receptor expression, and a similar trend was obtained in the mucosal part of HE stained tissue sections. Large microdissected patches of the epithelial and circular smooth muscle cell layer of pig colon descendens and of the epithelial cell layer of pig gastric fundus, also showed 5-HT4 receptor and GAPDH mRNA expression. No 5-HT4 receptor mRNA expression was detected in gastric LMPC-isolated EC cells from IHC stained tissues, which cells were positive for GAPDH.

CONCLUSION: Porcine GI mucosa predominantly expresses 5-HT4(+h) receptor splice variants, suggesting their contribution to the 5-HT4 receptor-mediated mucosal effects of 5-HT.

Keywords: 5-hydroxytryptamine 4 receptors; Pig; Gastric fundus; Colon descendens; Epithelium; Smooth muscle; Laser microdissection and pressure catapulting