Elsevier

Autonomic Neuroscience

Volumes 126–127, 30 June 2006, Pages 299-306
Autonomic Neuroscience

Region-specific distribution of the P2Y4 receptor in enteric glial cells and interstitial cells of Cajal within the guinea-pig gastrointestinal tract

https://doi.org/10.1016/j.autneu.2006.02.018Get rights and content

Abstract

Although there is pharmacological evidence to assume that the P2Y4 receptor is a regulator of epithelial ion transport, no detailed data about its distribution within the gut are available. Therefore, this study, using whole mounts and cryosections, aimed to reveal the expression pattern of P2Y4 along the entire guinea-pig gastrointestinal tract. P2Y4 immunoreactivity was absent from enteric neurons but present in enteric glial cells of the stomach, small and large intestine. In the esophagus, P2Y4 appeared to be exclusively located within striated muscle cells. P2Y4 showed also a region dependency regarding its presence in different subpopulations of interstitial cells of Cajal: in myenteric interstitial cells of Cajal in the stomach and ileum; in some intramuscular interstitial cells in the stomach and cecum; in some deep muscular plexus interstitial cells in the ileum; and in some submucosal surface interstitial cells in the colon. These results and the knowledge that P2Y4 activation causes intracellular Ca2+ recruitment led us to suggest that P2Y4 in enteric glia plays a modulatory role in intercellular Ca2+ waves, while P2Y4 in interstitial cells of Cajal modulates intracellular Ca2+ oscillations.

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).

References (40)

  • R.J. Phillips et al.

    Quantification of neurons in the myenteric plexus: an evaluation of putative pan-neuronal markers

    J. Neurosci. Methods

    (2004)
  • N. Suarez-Huerta et al.

    Molecular cloning and characterization of the mouse P2Y4 nucleotide receptor

    Eur. J. Pharmacol.

    (2001)
  • M. Weick et al.

    P2 receptors in satellite glial cells in trigeminal ganglia of mice

    Neuroscience

    (2003)
  • Y.D. Bogdanov et al.

    Molecular cloning and characterization of rat P2Y4 nucleotide receptor

    Br. J. Pharmacol.

    (1998)
  • A.J. Burns et al.

    Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry

    Cell Tissue Res.

    (1997)
  • J. Cabarrocas et al.

    Role of enteric glial cells in inflammatory bowel disease

    Glia

    (2003)
  • F.L. Christofi et al.

    Mechanically evoked reflex electrogenic chloride secretion in rat distal colon is triggered by endogenous nucleotides acting at P2Y1, P2Y2, and P2Y4 receptors

    J. Comp. Neurol.

    (2004)
  • M.T. Clunes et al.

    Immunohistochemical localization of the P2Y4 receptor in human bowel

    J. Physiol.

    (2002)
  • H.J. Cooke et al.

    Mechanical stimulation releases nucleotides that activate P2Y1 receptors to trigger neural reflex chloride secretion in guinea pig distal colon

    J. Comp. Neurol.

    (2004)
  • J.E. Fries et al.

    Identification of P2Y receptor subtypes in human Müller glial cells by physiology, single cell RT-PCR, and immunohistochemistry

    Investig. Ophthalmol. Vis. Sci.

    (2005)
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