Altered adenosine signalling in the presence of bowel inflammation: role of A2B receptors in the control of dolonic motility

Introduction: Under physiological conditions adenosine A2B receptors (A2BR) regulate immune responses, intestinal secretive/absorptive functions and colonic motility. However, the role played by these receptors in the pathophysiology of enteric dysmotility associated with intestinal inflammation is unknown. This study investigates the expression of A2BR in rat colon and characterizes their role in the control of colonic motility in the setting of experimental colitis. Methods: Colitis was induced by intrarectal 2,4-dinitrobenzenesulfonic acid (DNBS) in Sprague-Dawley rats. After six days, A2BR expression and localization in the colonic neuromuscular layer were examined by RT-PCR and immunofluorescence. Colonic longitudinal muscle strips (LMS) were suspended in Krebs solution containing guanethidine, L-Nω-nitroarginine methylester, GR-159897 and SB-218795 (to block adrenergic, nitrergic and tachykininergic pathways), and connected to isotonic transducers. The effects of MRS 1754 (MRS, 0.001-1 μM; A2BR antagonist) were assayed on contractile responses evoked by electrical stimulation (ES; 0.5 ms, 28 V, 10 Hz), carbachol (1 μM) or substance P (1 μM). Results: RT-PCR revealed the presence of A2BR mRNA in normal colon, and their expression pattern did not vary in inflamed tissues. Immunofluorescence showed a predominant localization of A2BR both in the longitudinal muscle layer and myenteric plexus; in the presence of colitis, A2BR expression was increased at muscular level, but reduced in myenteric ganglia. Upon blockade of NK1 receptors with L-732,138 (10 μM), MRS enhanced the ES-induced atropine-sensitive cholinergic contractions in normal LMS (+36% at 0.01 μM), while being almost ineffective in inflamed tissues (+11.8% at 0.01 μM). In the presence of atropine, ES elicited L-732,138-sensitive tachykininergic contractions, which were unaffected by MRS in normal tissues, while they were reduced in inflamed LMS (-50.3% at 0.01 μM). Upon incubation with tetrodotoxin, MRS enhanced carbachol-induced contractions in normal LMS (+24.5% at 0.01 μM), while it was ineffective in the presence of colitis. By contrast, MRS did not affect the contractile responses evoked by substance P in normal tissues, while exerting an inhibitory effect in LMS from inflamed rats (-30.8% at 0.01 μM). Conclusions: In normal colon, endogenous adenosine exerts an inhibitory control on excitatory cholinergic, but not tachykininergic, motility via A2BR located mainly at the muscular level. Bowel inflammation leads to a rearrangement of A2BR expression in the neuromuscular layer. In this setting, the inhibitory modulation of A2BR on cholinergic motility is lost, while an enhancing control of A2BR on tachykininergic motility comes into play. These functional changes could contribute to the altered pattern of intestinal motility usually associated with inflammatory bowel diseases.