Postnatal development of P2 receptors in the murine gastrointestinal tract
Introduction
Purine nucleotides and nucleosides play an important role in the modulation of both secretory and motor functions in the gastrointestinal (GI) tract. In 1970, Burnstock and colleagues proposed adenosine 5′-triphosphate (ATP) as a transmitter involved in non-adrenergic, non-cholinergic (NANC), nerve-mediated responses of smooth muscle in the gastrointestinal tract (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, 2001a).
Burnstock (1978) proposed that receptors selective for adenosine and adenosine monophosphate (AMP) be designated P1-purinoceptors and those selective for ATP and adenosine diphosphate (ADP) designated as P2-purinoceptors. P1 receptors have been further subdivided into A1, A2A, A2B and A3. P2 receptors have been divided into two families (Abbracchio and Burnstock, 1994), currently seven mammalian P2X ionotropic ligand-gated ion channel receptors, P2X1–7 and eight P2Y metabotropic G protein-coupled receptors P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14 have been identified (Khakh et al., 2001, Abbracchio et al., 2003).
Both adenosine and ATP receptors participate in the regulation of GI motility. Adenosine can directly activate receptors located on smooth muscle (Nicholls et al., 1996, Kadowaki et al., 2000), or act prejunctionally to suppress the release of acetylcholine and substance P (Moneta et al., 1997, Kadowaki et al., 2000).
In most species, ATP acts via P2 receptors to produce relaxations of smooth muscle in the GI tract, although there are reports of ATP causing contraction of smooth muscle in some regions of the gut (Burnstock, 2001a). Recent pharmacological and morphological evidence has been provided for the presence of P2Y1 receptors on myenteric and submucosal NANC inhibitory neurons mediating relaxation of the longitudinal muscle in the mouse gut, via nitric oxide (NO) and ATP acting on P2Y receptors located on smooth muscle (Giaroni et al., 2002). In the mouse gut, ATP can produce relaxation via P2Y1 receptors, but also via P2Y2 receptors and an unidentified α,β-methylene ATP (α,β-meATP)-sensitive P2Y receptor, located on smooth muscle. Activation of P2X2 receptors mediates contraction of the mouse colon smooth muscle (Giaroni et al., 2002).
Ontogenetic studies of the development of purinergic effects in the GI tract have demonstrated that these are present during prenatal development (Burnstock, 2001b). NANC responses have been demonstrated in the rat stomach (Ito et al., 1988) and in the mouse and rabbit small intestine (Gershon and Thompson, 1973) during foetal development. In addition, intense quinacrine fluorescence, which is indicative of high levels of ATP, has been detected in enteric neurons of the rabbit stomach and ileum before birth (Crowe and Burnstock, 1981).
There are reports indicating that developmental changes occur in purinergic signalling in the small intestine after birth. In the rat duodenum, changes in purinergic signalling have been observed in the third postnatal week, corresponding to the weaning period (Furukawa and Nomoto, 1989, Nicholls et al., 1990, Irie et al., 1994, Hourani, 1999). ATP has been shown to produce relaxation of the longitudinal muscle in the neonatal rat duodenum via P2Y receptors, which were fully developed by day 25 (Brownhill et al., 1997). In the same study, P2Y receptors mediating contraction have been described in the muscularis mucosae before day 20, but subsequently this effect was mediated by P2X receptors.
In the present study, we examined the presence and function of P2 receptors in the neonatal mouse gut during postnatal development, using pharmacological, morphological and biochemical 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 obtained from animals between days 3 and 12 after birth and at day 20, which corresponds to the weaning period. The presence and localization of P2Y1, P2Y2, P2Y4, P2X1, P2X2 and P2X3 receptors was studied with immunohistochemical methods in the colon and ileum between days 1 and 20. Further, the levels of expression of P2Y1, P2X1 and P2X2 receptors were investigated by Western immunoblotting in the colon between days 1 and 20. This study was carried out on the GI tract of the mouse, partly because only scarce information is available concerning the postnatal development of purinergic transmission in the gut of this species, but also because the later development of the mouse, being born physiologically very immature, makes such information of particular interest. The increasing availability of P2 receptor knockout mice also make such control information desirable.
Section snippets
Animals
Principles of good laboratory animal care were followed and animal experimentation was in compliance with specific national (U.K.) laws and regulations. Neonatal male and female mice (1–12 days old; strain C57/BL10) were killed by cervical dislocation, whereas 20 days old and adult mice were killed by 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
Stomach
2-MeSADP, α,β-meATP, UTP, ATP and adenosine relaxed the CCh-contracted mouse stomach fundus at day 3 at high concentrations of agonists ( > 1 μM) and at lower concentrations (10–100 nM) from day 6. The order of potency for 2-MeSADP was: 3 days = 6 days < 8 days < 20 days = adult (significant difference between groups P < 0.05 or less) (Fig. 1a). Concentration–response curves to 2-MeSADP in 8–12-day-old animals were significantly inhibited (P < 0.05) by MRS 2179 (1 μM) (Fig. 1b). The potency order for α,β-meATP was:
Discussion
This study has investigated the changing roles of multiple P2 receptor subtypes on the longitudinal muscle of the mouse GI tract during postnatal development. A striking feature of postnatal development is that a relaxant response mediated by P2Y1 receptors gradually develops along the length of the GI tract from the first week after birth, detectable in the stomach from day 3 and in the duodenum from day 6, while in the ileum and colon relaxation responses could not be detected until days 8
Acknowledgements
The authors are very grateful to Dr H.Z. Ruan and Mr T. Robson for technical assistance with the immunohistochemical staining.
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