Elsevier

Neuropharmacology

Volume 43, Issue 8, December 2002, Pages 1313-1323
Neuropharmacology

P2 receptors in the murine gastrointestinal tract

https://doi.org/10.1016/S0028-3908(02)00294-0Get rights and content

Abstract

The actions of adenosine, adenosine 5′-triphosphate (ATP), 2-methylthio adenosine diphosphate ADP (2-MeSADP), 2-methylthio ATP (2-MeSATP), α,β-methylene ATP (α,β-meATP) and uridine triphosphate (UTP) on isolated segments of mouse stomach (fundus), duodenum, ileum and colon were investigated. The localization of P2Y1, P2Y2, P2Y4, P2X1 and P2X2 receptors and neuronal nitric oxide synthase (NOS) were examined immunohistochemically, and P2Y1 mRNA was examined with in situ hybridization. The order of potency for relaxation of longitudinal muscle of all regions was: 2-MeSADP≥2-MeSATP>α,β-meATP>ATP=UTP=adenosine. This is suggestive of P2Y1-mediated relaxation and perhaps a further P2Y receptor subtype sensitive to α,β-meATP. As ATP and UTP are equipotent, the presence of a P2Y2 receptor is indicated. ATP responses were inhibited by the P2Y1-selective antagonist MRS 2179, and suramin. P2Y1 receptors were visualized immunohistochemically in the smooth muscle of the ileum and in a subpopulation for myenteric neurones, which also stained for NOS. P2Y1 mRNA was localized in neurones in both myenteric and submucosal ganglia in the ileum. Taken together, these results suggest that ATP was acting on non-adrenergic, non-cholinergic inhibitory neurons, which release both nitric oxide (NO) and ATP. Reduced relaxations to 2-MeSADP by tetrodotoxin and Nω-nitro-l-arginine methyl ester, are consistent with this possibility. Adenosine acts via P1 receptors to relax smooth muscle of the mouse gut. Segments of mouse colon (in contrast to the stomach and small intestine) were contracted by nucleotides with the potency order: 2-MeSATP>α,βmeATP>ATP; the contractions showed no desensitization and were antagonized by suramin and PPADS, consistent with responses mediated by P2X2 receptors. Immunoreactivity to P2X2 receptors was demonstrated on both longitudinal and circular muscle of the colon, but not in the other regions of the gut, except for a small subpopulation of myenteric neurones. In summary, neuronal P2Y1 receptors appear to mediate relaxation, largely through NO in all regions of the mouse gut, and to a lesser extent by P2Y1, P2Y2 and a novel P2Y receptor subtype responsive to α,β-meATP in smooth muscle, while P2X2 receptors mediate contraction of colonic smooth muscle.

Introduction

Purine nucleotides and nucleosides have long been known to have effects on the gastrointestinal (GI) tract, influencing both motor and secretory functions. In 1970, Burnstock and colleagues proposed that adenosine 5′-triphosphate (ATP) was a transmitter involved in non-adrenergic, non-cholinergic (NANC) nerve-mediated responses of smooth muscle in the gut (Burnstock et al., 1970). Since then evidence has accumulated in support of the hypothesis that ATP is a NANC transmitter in the enteric nervous system (Burnstock, 2001). High concentrations of ATP in subpopulations of myenteric neurons in different regions of the gut of the guinea-pig, rabbit and rat have been reported (Crowe and Burnstock, 1981, Belai and Burnstock, 1994). In addition, after application of an appropriate stimulus, ATP is released from enteric nerves (Burnstock et al., 1978, McConalogue et al., 1996) and the subsequent activation of specific receptors on enteric neurons and muscle cells may evoke either excitation or inhibition of smooth muscle function (see Burnstock, 2001).

Burnstock (1978) proposed a formal classification of receptors for adenosine and ATP, collectively called purinoceptors. Receptors selective for adenosine and adenosine monophosphate were designated as P1-purinoceptors and those selective for ATP and adenosine diphosphate (ADP) called P2-purinoceptors. This classification set the stage for further subdivisions of P1 receptors, into A1, A2A, A2B and A3 and of P2 receptors into P2X and P2Y families (Burnstock and Kennedy, 1985, Abbracchio and Burnstock, 1994, Ralevic and Burnstock, 1998). P2X receptors are ionotropic while P2Y receptors are G protein-coupled, each with their own subtypes, P2X1–7 and P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12 and P2Y13 (see King et al., 2000, Khakh et al., 2001, Nicholas, 2001). At present there are few selective agonists and antagonists that discriminate clearly between families of P2X and P2Y receptors, or between subtypes of receptors within each family, although 2-methylthio ADP (2-MeSADP) and MRS 2179 have been claimed to be selective agonists and antagonists, respectively, for the P2Y1 receptor (Camaioni et al., 1998). The use of characteristic agonist potency profiles and effective antagonists, immunostaining and in situ hybridization for receptor subtypes can be used to help identify specific P2 receptor subtypes.

Both adenosine and ATP receptors have been suggested to play important roles in the modulation of motility in the GI tract. Adenosine can directly activate receptors located on smooth muscle (Nicholls et al., 1996, Kadowaki et al., 2000) or act prejunctionally, suppressing the release of excitatory neurotransmitters such as acetylcholine (ACh) and substance P (Moneta et al., 1997, Kadowaki et al., 2000). In most species, ATP activates P2 receptors on smooth muscle to produce relaxation of smooth muscle in the GI tract, although there are some examples where ATP also produces contraction of smooth muscle of some regions of the gut (Burnstock, 2001).

Although there is good evidence to suggest that purines can influence motility in the GI tract, a clear characterization of the different subtypes of P2 receptors involved has not yet been established. This is certainly true of the mouse GI tract, where there is very little information concerning the activity of purine compounds and the receptors they activate. The present investigation was carried out to characterize P2 receptors in the mouse gut, using pharmacological and morphological approaches. To this end, we studied the effect of purinoceptor agonists and antagonists on the longitudinal muscle of the stomach fundus, duodenum, ileum and colon. Further, the presence of P2Y1 receptor mRNA was investigated in the ileum by in situ hybridization and P2Y1, P2Y2, P2Y4 and P2X2 receptors and nitric oxide synthase (NOS) were studied with immunohistochemical methods in the ileum, and P2X1 and P2X2 receptors were studied in the colon. This study was carried out on the GI tract of the mouse, partly because only scarce information is available concerning the role of purinergic transmission in the gut of this species, but also because the increasing availability of P2 receptor knockout mice makes such control information desirable.

Section snippets

Animals

Principles of good laboratory animal care were followed and animal experimentation was in compliance with specific national (UK) laws and regulations. Adult male mice (strain C57/BL10) weighing 20–28 g, were killed by exposure to an increasing concentration of carbon dioxide and death was ensured by cervical dislocation according to Home Office (UK) regulations covering Schedule One procedures. After the abdominal cavity had been opened, the gut was rapidly removed and placed in a beaker with a

Relaxation

Adenosine, ATP, 2-MeSATP, 2-MeSADP, α,β-meATP and UTP relaxed all regions of the CCh-contracted mouse GI tract. The order of potency for the stomach fundus and colon was: 2-MeSADP=2-MeSATP>α,β-meATP>UTP=ATP=adenosine (Fig. 1, Fig. 4, respectively). The order of potency for the duodenum and ileum was: 2-MeSADP=2-MeSATP>α,β-meATP>UTP=ATP=adenosine (Fig. 2, Fig. 3, respectively). As none of the concentration–response curves from any of the regions of the GI tract reached a maximum response, pD2

Discussion

This study has characterized multiple P2 receptors on the longitudinal muscle of the mouse GI tract. The predominant effect of stimulation of these receptors is relaxation, with the exception of the colon longitudinal muscle, which also possesses a contractile P2 receptor.

In the mouse stomach fundus, duodenum, ileum and colon, 2-MeSADP, 2-MeSATP, α,β-meATP, ATP, UTP and adenosine all relaxed the longitudinal muscle in a concentration-dependent manner. 2-MeSADP and 2-MeSATP were equipotent and

Acknowledgements

The authors are very grateful to Dr C. Orphanides for excellent editorial assistance. Also to Miss A. Fahey for technical assistance with some of the immunohistochemical staining.

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