Pulmonary, Gastrointestinal and Urogenital Pharmacology
Differential recruitment of high affinity A1 and A2A adenosine receptors in the control of colonic neuromuscular function in experimental colitis

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Abstract

This study investigated the expression of A1 and A2A receptors in the rat colonic neuromuscular compartment, and characterized their roles in the control of motility during inflammation. Colitis was induced by 2,4-dinitrobenzenesulfonic acid. A1, A2A receptors, and ecto-5′-nucleotidase (CD73, adenosine producing enzyme) mRNA expression was examined by RT-PCR. The effects of DPCPX (A1 receptor antagonist), CCPA (A1 receptor agonist), 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (A2A receptor antagonist), 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (A2A receptor agonist), AOPCP (CD73 inhibitor) were tested on electrically or carbachol-evoked contractions in colonic longitudinal muscle preparations. In normal colon, RT-PCR revealed the presence of A1 receptors, A2A receptors and CD73, and an increased expression of A2A receptors and CD73 was detected in inflamed tissues. In normal colon, DPCPX or 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol enhanced electrically-induced contractions, while in inflamed preparations the effect of DPCPX no longer occurred. In normal colon, CCPA or 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride decreased electrically-induced contractions. Under inflammation, 4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride reduced electrically evoked contractions with higher efficacy, while the inhibition by CCPA remained unchanged. A1 and A2A receptor ligands did not affect carbachol-induced contractions. AOPCP enhanced electrically-induced contractions and prevented the contractile effects of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol, without interfering with DPCPX, both in normal and inflamed colons. These results indicate that, in normal colon, both A1 and A2A receptors contribute to the inhibitory control of motor functions at neuronal level. Under bowel inflammation, A1 receptor loses its modulating actions, while the recruitment of A2A receptor by CD73-dependent endogenous adenosine drives an enhanced inhibitory control of colonic neuromotility.

Introduction

Crohn's disease and ulcerative colitis are chronic, relapsing inflammatory bowel diseases arising from an inappropriate inflammatory response against intestinal microbes in a genetically susceptible host (Abraham and Cho, 2009). Inflammatory bowel diseases are characterized by recurrent and serious inflammation of the enteric mucosa with significant alterations of gastrointestinal functions, as a consequence of marked changes in the enteric nervous system (Lomax et al., 2005, Antonioli et al., 2008a). Indeed, the exposure of enteric neurons to inflammatory mediators contributes to alterations in the physiological functions of the intestinal tract, leading to the abdominal symptoms observed in patients with inflammatory bowel diseases (Lomax et al., 2006). However, most of the mechanisms through which intestinal inflammation can affect bowel neuromuscular activities remain unknown.

Several lines of evidence suggest that adenosine takes part in the modulation of enteric immune and inflammatory responses through the recruitment of four receptors: A1, A2A, A2B and A3 (Haskó and Cronstein, 2004, Antonioli et al., 2008b). In particular, once released at sites of inflammation, adenosine plays a prominent role in maintaining tissue integrity by modulation of immune functions, interfering with the biosynthesis of proinflammatory cytokines and inhibiting neutrophil adhesion and oxidant activity (Antonioli et al., 2008b).

Besides the regulation of immune/inflammatory response, adenosine seems to play a role also in the modulation of intestinal motility. Under normal conditions, this nucleoside can regulate digestive motility through the control of neurotransmitter release via A1 or A2A receptors located on enteric nerves (Kadowaki et al., 2000, Storr et al., 2002, Duarte-Araújo et al., 2004, Zizzo et al., 2009). Recently, Fornai et al. (2009) characterized the role played by A1 and A2A receptors in the control of motor functions in normal human colon, showing that these receptors are involved in the inhibitory actions of endogenous adenosine on the excitatory cholinergic motility.

An involvement of adenosine in the pathophysiology of gut dysmotility associated with intestinal inflammation has been hypothesized, but supporting data are scarce and often conflicting (Antonioli et al., 2008b). Kadowaki et al., 2000, Kadowaki et al., 2003 observed that A1 receptors contribute to the alteration of colonic propulsion in rat models of mesenteric ischemia-reperfusion and post-operative ileus. By contrast, De Man et al. (2003) reported a significant loss of inhibitory control by A1 receptors on bowel motility during intestinal inflammation, thus suggesting that the regulatory activity of this receptor pathway may vary depending on different pathological conditions. Scarce information is available about the involvement of A2A receptors in the enteric motor disturbances associated with bowel inflammation. In this regard, we previously observed that A2A receptors exert an inhibitory control of colonic motility during experimental colitis (Antonioli et al., 2006). However, the possibility that high affinity A1 and A2A receptors could play differential roles in gut dysmotility associated with bowel inflammation has not been investigated.

Based on these considerations, the present study was designed to investigate the expression of A1 and A2A receptors in the neuromuscular compartment of rat colon and to characterize their functional roles in the control of colonic neuromuscular activity in the presence of experimental colitis.

Section snippets

Animals

Albino male Sprague–Dawley rats, 200–250 g body weight, were used throughout the study. The animals were fed standard laboratory chow and tap water ad libitum and were not employed for at least one week after their delivery to the laboratory. They were housed, three in a cage, in temperature-controlled rooms on a 12-h light cycle at 22–24 °C and 50–60% humidity. Their care and handling were in accordance with the provisions of the European Community Council Directive 86-609, recognized and

RT-PCR

RT-PCR revealed the expression of mRNA coding for A1 and A2A receptors, and ecto-5′-nucleotidase in colonic neuromuscular tissues dissected from normal or DNBS-treated animals (Fig. 1). The densitometric analysis indicated no significant differences in A1 receptor mRNA expression under both conditions, while a significant increase in A2A receptor and ecto-5′-nucleotidase mRNA expression was observed in colonic tissues dissected from rats with colitis (Fig. 1).

Contractile activity of colonic longitudinal smooth muscle

During the equilibration period in

Discussion

The present study was designed to examine the expression of the high affinity A1 and A2A adenosine receptors in the colonic neuromuscular compartment, as well as to characterize the effects of A1 and A2A receptor ligands on colonic motility, both under normal conditions and in the presence of experimental colitis. For this purpose, our experiments were performed in rats with DNBS-induced colitis, a useful model for investigating changes in enteric motility associated with colonic inflammation.

Acknowledgment

This work was supported by a grant from the Italian Ministry of Education, University and Research (COFIN 2003, project no. 2003052707_002).

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