Pulmonary, Gastrointestinal and Urogenital Pharmacology
Molecular cloning and characterization of dog TRPA1 and AITC stimulate the gastrointestinal motility through TRPA1 in conscious dogs

https://doi.org/10.1016/j.ejphar.2009.06.038Get rights and content

Abstract

Transient receptor potential ankyrin1 (TRPA1) is a non-selective cation channel activated by cold stimuli under 17 °C, mechanosensation, and pungent irritants such as allyl isothiocyanates (AITC) and cinnamaldehyde (CA). In this study, we cloned the dog orthologue of TRPA1 for the first time and induced its heterologous expression in HEK293 cells to investigate its functional properties using a fluorescence imaging plate reader-based Ca2+ influx assay. Moreover, we examined the effect of AITC on gastrointestinal motility in dogs. At the amino acid level, the sequence of dog TRPA1 was 82–83% identical to that of human, mouse, and rat orthologues. TRPA1 is strongly expressed in the brain, cerebellum, stomach, pancreas, and small and large intestine of dogs. Like other mammalian orthologues, TRPA1 agonists, including AITC, CA, allicin, and diallyl disulfide, evoked a concentration-dependent increase in intracellular Ca2+ influx in dog TRPA1-expressing cells. AITC stimulated gastric antrum and jejunum motility and induced the occurrence of giant migrating contractions in the colon of fasted dogs. The effects of AITC were inhibited by ruthenium red, a TRPA1 antagonist. These results indicate that AITC stimulated the gastrointestinal motility through TRPA1 in conscious dogs.

Introduction

Transient receptor potential (TRP) channels are non-selective cation channels that respond to a variety of chemical and physical stimuli. To date, seven TRP families have been identified: TRPC, TRPV, TRPM, TRPP, TRPML, TRPA, and TRPN (Cortright et al., 2007, Levine and Alessandri-Haber, 2007, Montell, 2005, Moran et al., 2004). The transient receptor potential ankyrin 1 (TRPA1) channel is known to be activated by cold stimuli under 17 °C, mechanical stimuli, and by pungent irritants such as allyl isothiocyanates (AITC) and cinnamaldehyde (CA) (Bandell et al., 2004, Bautista et al., 2005, Lee et al., 2008, Obata et al., 2005, Tominaga and Caterina, 2004, Zhang et al., 2008). TRPA1 is known to be expressed in sensory neurons, suggesting that it functions as an environmental sensor. In fact, TRPA1 agonists cause nociception, and TRPA1 knock-out mice lack sensitivity to chemical agonists (Bang et al., 2007, Bautista et al., 2006, Katsura et al., 2006, Kwan et al., 2006).

TRPA1 was recently shown to be functionally expressed in the gastrointestinal tract as well as in sensory neurons (Purhonen et al., 2008, Stokes et al., 2006). Stimulation of enterochromaffin (EC) cells with TRPA1 agonists was found to enhance serotonin (5-hydroxytryptamine: 5-HT) secretion (Nozawa et al., 2009), and stimulation of the mouse intestinal endocrine cell line STC-1 with TRPA1 agonists was found to enhance cholecystokinin secretion (Purhonen et al., 2008). While TRPA1 agonists are known to have a contractile effect in isolated mouse intestine (Penuelas et al., 2007), the in vivo effects of these agonists on gastrointestinal motility have not been well studied.

Dogs are useful animal models for studying gastrointestinal motility because the anatomy, physiology, and biochemistry of the canine gastrointestinal tract are strongly similar to that in humans (Kararli, 1995). To date, however, no studies have investigated the activity of dog TRPA1.

Here, to show the effects of TRPA1 agonist on gastrointestinal motility and identify the functional properties of dog TRPA1, dog TRPA1 was cloned for the first time and transfected into human embryo kidney 293 (HEK293) cells. The in vivo effects of TRPA1 agonist on canine gastrointestinal motility were examined using AITC.

Section snippets

In silico cloning and analysis of dog TRPA1 genome structure

The amino acid sequence of human TRPA1 (GenBank Accession No. NP_015628.2) was set to query, and a tblastn was performed using the NCBI BLAST search engine. We identified a nucleotide sequence (GenBank Accession No. XM_544123.2) which showed high homology to human TRPA1. Chromosomal localization of the gene was examined with NCBI Map Viewer.

Polymerase chain reaction (PCR)-based cloning and analysis of dog TRPA1 mRNA

We designed oligonucleotide primers based on the found sequence and amplified the cDNA of dog TRPA1 from dog small intestinal mRNA purchased from Clontech

Molecular cloning of dog TRPA1

Dog TRPA1 cDNA cloned from dog small intestine tissue samples was used to deduce the amino acid sequence of dog TRPA1 (Fig. 1). Sequence analysis revealed that dog TRPA1 has an open reading frame of 3354 bp and encodes 1118 amino acid residues. A comparative analysis of amino acid homology among different mammalian TRPA1 orthologues showed that dog TRPA1 was highly homologous to human (83.0%), mouse (82.2%), and rat (82.2%) TRPA1 (Fig. 2A and B).

Tissue distribution of dog TRPA1

Reverse transcription-PCR analysis was performed

Discussion

In the present study, we investigated the effects of TRPA1 agonist on gastrointestinal motility in dogs because they are known to be useful animals for these types of studies (Kararli, 1995). Few studies have investigated dog TRPA1, and here we cloned dog TRPA1 for the first time and deduced its amino acid sequence. Dog TRPA1 was found to be highly conserved, along with other mammalian orthologues, and showed tissues distribution similar to that of other species (Stokes et al., 2006). These

Acknowledgments

We would like to thank T. Koizumi, M. Yamano, Y. Takinami, R. Takezawa, Y. Takemoto, T. Goto, H. Tanaka, H. Okada, H. Kamada, and M. Okada for their excellent technical assistance and advice.

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    These authors have contributed equally to this work.

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