Region-specific distribution of the P2Y4 receptor in enteric glial cells and interstitial cells of Cajal within the guinea-pig gastrointestinal tract
Introduction
Adenine and uridine nucleotides are present in all types of cells and are released in response to various stimuli. Once in the extracellular space, they are able to activate membrane proteins, which are categorized as P2 receptors, comprising ionotropic P2X and metabotropic P2Y receptors. P2Y receptors are characterized by seven putative transmembrane domains typical of G protein-coupled receptors. At present, 15 heptahelical proteins have been associated with the P2Y receptor family. However, only eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) are accepted as clearly defined, distinct, nucleotide receptors. P2Y receptors can be broadly subdivided into Gq-coupled subtypes (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11) and Gi-coupled subtypes (P2Y12, P2Y13 and P2Y14) (for review, see King and Burnstock, 2002, Burnstock and Knight, 2004).
The P2Y4 receptor was cloned first from human placenta (Communi et al., 1995) and from genomic human DNA (Nguyen et al., 1995), followed by cloning of rat (Bogdanov et al., 1998, Webb et al., 1998) and mouse (Lazarowski et al., 2001, Suarez-Huerta et al., 2001) orthologs. The human P2Y4 receptor is a selective UTP receptor, whereas the rodent ones are equipotently activated by UTP and ATP. P2Y4 mRNA has been detected in the murine stomach and intestine (Suarez-Huerta et al., 2001, Robaye et al., 2003), in murine colonic crypts (Matos et al., 2005), and in rat (Christofi et al., 2004) and guinea-pig (Cooke et al., 2004) colonic submucosa. Pharmacological studies have provided evidence that the P2Y4 receptor is a dominant nucleotide-sensitive regulator of salt and fluid transport in the intestine (Cressman et al., 1999, Robaye et al., 2003, Christofi et al., 2004, Ghanem et al., 2005, Matos et al., 2005). Although some morphological studies have been performed to reveal the distribution of the P2Y4 receptor in the gut, the distribution of this receptor in the gastrointestinal (GI) tract remains obscure. In archival, paraffin-embedded human bowel tissue, P2Y4 immunoreactivity (IR) has been localized in enteric neurons and in a subpopulation of epithelial cells (Clunes et al., 2002). P2Y4 immunostaining has also been observed in a few submucosal enteric neurons in the rat (Christofi et al., 2004) and guinea-pig (Cooke et al., 2004) distal colon. A more recent study reported that the P2Y4 receptor was expressed in enteric glial cells (EGCs) and in some interstitial cells of Cajal (ICCs) in the rat distal colon (Van Nassauw et al., 2005). However, an immunocytochemical study attempting to determine the distribution pattern of P2Y4 receptors in the murine GI tract failed to demonstrate IR for this purinoceptor along the mouse gut (Giaroni et al., 2002).
Given the current lack of knowledge about the GI expression of the P2Y4 receptor, the present study was performed to disclose the distribution of the P2Y4 receptor in the GI tract to better understand how this receptor is involved in intrinsic control of GI functions and to provide a more detailed morphological substrate for the pharmacological data.
Section snippets
Materials and methods
Tissue was obtained from adult guinea-pigs (n = 5, weight = ca. 350 g) of both genders (Charles River Laboratories, Brussels, Belgium). All animals had free access to water and complete rodent diet and were kept in a 12-h/12-h light/dark cycle. The animals were sacrificed by cranial concussion. All procedures were approved by the local ethics committee of the University of Antwerp.
Different regions of the guinea-pig GI tract (esophagus, gastric fundus and corpus, ileum, cecum, proximal and distal
Results
In the guinea-pig GI tract, P2Y4 IR was observed in each GI segment under study. Control immunostainings, in which the primary antibody was omitted or preabsorbed, did not yield immunolabelling. Interference control stainings showed no linking of secondary antibodies with primary antibodies used in previous steps. In the esophagus, immunostaining for the P2Y4 receptor was only observed in the cell membrane of striated muscle cells (Fig. 1). No other cell types expressed P2Y4 IR. In general,
Discussion
Previous studies in mouse and rat indicate that ATP and UTP modulate electrolyte transport in the small and large intestine, and that these responses are mediated by P2Y receptors, including the P2Y4 receptor (Cressman et al., 1999, Robaye et al., 2003, Christofi et al., 2004, Ghanem et al., 2005, Matos et al., 2005). Although P2Y4 expression has been demonstrated in the GI tract (mouse: Suarez-Huerta et al., 2001, Robaye et al., 2003, Matos et al., 2005, Christofi et al., 2004, Cooke et al.,
Acknowledgements
This study was supported by the Interuniversity Pole of Attraction Programme of the Federal Services for Scientific, Technical and Cultural Affairs (IUPA-P5/20), a travel grant of the University of Naples Federico II, a concerted research project (GOA-2004/2007) and a small research project (BOF-UA-KP-2005) granted by the Special Research Fund of the University of Antwerp. J.-M. Vanderwinden is a senior research associate of the National Fund for Scientific Research (Belgium).
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